The Interactive Fly

Zygotically transcribed genes

INDEX

• Genes coding for Neuromuscular Junctional proteins
• Biology of the Presynapse
• Synaptic Proteins
• Vesicles in the Presynapse
• Homeostasis and plasticity at the presynapse
• Distinguishing tonic and phasic synaptic function
• Biology of the Periactive Zone
• Biology of the Postsynapse
• Signaling between Pre- and Post-synapse
• The Extracellular Matrix
• Disease Models
• Miscellaneous

Presynaptic Active Zone Five evolutionarily conserved proteins - RIM (Drosophila Rab3 interacting molecule) , Munc13 (Drosophila unc-13), RIM-BP (Drosophila Rim-binding protein), &alpha-liprin (Drosophila Liprin-α), and ELKS (Drosophila Bruchpilot) proteins - form the core of active zones. SYD-1 is a Rho GAP that is essential for synapse assembly in invertebrates but its functional homolog is unknown in vertebrates. RIM, Munc13, and RIM-BP are multidomain proteins composed of a string of identifiable modules, whereas α-liprin and ELKS exhibit a simpler structure. The five core active zone proteins form a single large protein complex that docks and primes synaptic vesicles, recruits Ca2+ channels to the docked and primed vesicles, tethers the vesicles and Ca2+ channels to synaptic cell-adhesion molecules, and mediates synaptic plasticity (Südhof, 2013). Imaging of developing Drosophila glutamatergic synapses revealed that the Unc13B isoform was recruited to nascent active zones by the scaffolding proteins Syd-1 and Liprin-α, and Unc13A is positioned by Bruchpilot and Rim-binding protein complexes at maturing active zones (Bohme, 2016).

Postsynapse of the larval neuromuscular junction (A) At the postsynaptic membrane, shown at the bottom, Discs large (Dlg) localizes to spectrin-actin complexes. Homophilic adhesion between Fasciclin 2 (Fas2) transmembrane proteins links the presynaptic and postsynaptic sides, with the intracellular C-terminal domains anchored to the first and second PDZ domains of Dlg. The adducin Hu-li tai shao (Hts) is in a complex with Dlg at the postsynaptic membrane, though the interaction may not be direct. Hts also binds to the lipid Phosphatidylinositol 4,5-bisphosphate (PIP2) via the MARCKS-homology domain. (B) Hts promotes the accumulation of par-1 and camkII transcripts in the muscle cytoplasm through an as of yet identified mechanism. PAR-1 and CaMKII phosphorylate Dlg. Phosphorylation disrupts Dlg postsynaptic targeting. (C) Phosphorylation translocates Hts away from the postsynaptic membrane and hinders Hts' ability to regulate Dlg localization, presumably through the control of PAR-1 and CaMKII at the transcriptional level. Phosphorylation of the MARCKS-homology domain also inhibits Hts' ability to bind to PIP2 (Wang, T., 2014). Neuropilin and tolloid-like (Neto) engages the iGluR complexes extrajunctionally and together they traffic and cluster at the synapses, opposite from the active zones marked by T-bars. Neto and the essential iGluR subunits are limiting for formation of functional iGluR complexes at the NMJ and for growth of synaptic structures (Kim, 2014).

The schizophrenia susceptibility gene dysbindin controls synaptic homeostasis

The molecular mechanisms that achieve homeostatic stabilization of neural function remain largely unknown. To better understand how neural function is stabilized during development and throughout life, an electrophysiology-based forward genetic screen was used, and the function of more than 250 neuronally expressed genes was assessed for a role in the homeostatic modulation of synaptic transmission in Drosophila. This screen ruled out the involvement of numerous synaptic proteins and identified a critical function for dysbindin, a gene linked to schizophrenia in humans. dysbindin was found to be required presynaptically for the retrograde, homeostatic modulation of neurotransmission, and functions in a dose-dependent manner downstream or independently of calcium influx. Thus, dysbindin is essential for adaptive neural plasticity and may link altered homeostatic signaling with a complex neurological disease (Dickman, 2009).

At glutamatergic synapses of species ranging from Drosophila to human, disruption of postsynaptic neurotransmitter receptor function can be precisely offset by an increase in presynaptic neurotransmitter release to homeostatically maintain normal postsynaptic excitation. The Drosophila neuromuscular junction (NMJ) is a glutamatergic synapse that is used as a model for this form of homeostatic signaling in the nervous system. Efficient homeostatic modulation of presynaptic release at the Drosophila NMJ can occur in ten min following bath application of philanthotoxin-433 (PhTx; a polyamine toxin present in the venom sac of the solitary digger wasp Philanthus triangulum), which persistently and specifically inhibits postsynaptic glutamate receptors (Dickman, 2009).

This study has systematically screened for mutations that block the rapid, PhTx-dependent induction of synaptic homeostasis. Mutations in 276 genes were screened electrophysiologically. For each mutant, an average value was calculated for the amplitude of both the spontaneous miniature excitatory junctional potential (mEJP) and evoked excitatory junctional potential (EJP) following treatment of the dissected neuromuscular preparation with PhTx for 10 min. 14 mutants were isolated with average EJP amplitudes more than two standard deviations smaller than the distribution mean. From these candidates, 7 mutants were identified that block synaptic homeostasis without an obvious effect on NMJ morphology or baseline synaptic transmission. It is concluded that the molecular mechanisms of synaptic homeostasis can be genetically separated from the mechanisms responsible for normal neuromuscular development and baseline synaptic transmission (Dickman, 2009).

A fraction of the mutants assayed (19.5%) are previously published genetic lesions. This allows ruling out of the involvement of numerous genes and associated biochemical processes. Mutations that disrupt RNA-interference/micro-RNA processing, retrograde trans-synaptic signaling, synaptic transmission, active zone assembly, synaptic vesicle endocytosis and mitochondria all showed reliable homeostatic compensation. Therefore, synaptic homeostasis is a robust phenomenon, unperturbed by a broad spectrum of synaptic mutations. In addition, significant homeostatic compensation in synaptojanin and endophilin mutants argues against the involvement of synaptic vesicle endocytosis and indicates that the size of the recycling synaptic vesicle pool is not a limiting factor for synaptic homeostasis. These data also emphasize the importance and specificity of those identified mutations that do block synaptic homeostasis. These include four ion channels, two of which are of unknown function, and two calcium-binding proteins of unknown function. Thus, homeostatic signaling at the NMJ may include previously unexplored mechanisms of synaptic modulation (Dickman, 2009).

One mutation that was identified with a specific defect in homeostatic compensation is a transposon insertion that resides in the Drosophila homologue of dysbindin (CG6856). The DTNBP1 (dysbindin) locus is linked with schizophrenia in humans. A transposon insertion was identified within the dysbindin locus (pBac^e01028, referred to as dysb¹, that showed a complete absence of homeostatic compensation following application of PhTx. A similar effect was observed when dysb¹ was placed in trans to a deficiency that uncovers the dysb locus, indicating that the dysb¹ mutant was a strong loss of function or null mutation. No significant change in baseline synaptic transmission was observed in dysb¹ mutant animals (0.5 mM extracellular calcium). Thus, under these recording conditions, this mutation disrupted synaptic homeostasis without altering baseline neurotransmission. As a control, synaptic homeostasis was normal in animals in which the pBac^e01028 transposon was precisely excised (Dickman, 2009).

The dysb gene is ubiquitously expressed in Drosophila embryos. Therefore, a dysbindin transgene was generated and expressed in the dysb¹ mutant. Presynaptic expression of dysb fully restored homeostatic compensation in the dysb¹ mutant background, whereas muscle-specific expression of dysb did not. Thus, Dysbindin is necessary presynaptically for the rapid induction of synaptic homeostasis (Dickman, 2009).

It was next asked whether Dysbindin is also required for the sustained expression of synaptic homeostasis. Double mutant animals were generated harboring both the dysb¹ mutation and a mutation in a gene encoding a postsynaptic glutamate receptor (GluRIIA). GluRIIA mutant animals normally show robust homeostatic compensation. However, homeostatic compensation was blocked in GluRIIA; dysb¹ double mutant animals. Thus, dysbindin was also necessary for the sustained expression of synaptic homeostasis over several days of larval development (Dickman, 2009).

Synapse morphology was qualitatively normal in dysb mutants including both the shape of the presynaptic nerve terminal and the levels, localization and organization of synaptic markers including futsch-positive microtubules, synapsin and synaptotagmin. Bouton number and active zone density are also normal in dysb mutants. Thus, the disruption of synaptic homeostasis in dysb¹ mutants is not a secondary consequence of altered or impaired NMJ development (Dickman, 2009).

In the vertebrate nervous system, Dysbindin is associated with synaptic vesicles. The localization was examined of a Venus-tagged dysb transgene (ven-dysb) that rescues the dysb¹ mutant. Ven-Dysb showed extensive overlap with synaptic vesicle associated proteins when expressed in neurons. Thus, Dysbindin functions presynaptically, potentially at or near the synaptic vesicle pool (Dickman, 2009).

To further define the function of Dysbindin, baseline synaptic transmission in the dysb mutant was investigated in greater detail. At 0.5 mM extracellular calcium, synaptic transmission in dysb¹ mutant animals was indistinguishable from wild type. However, when extracellular calcium was reduced, baseline synaptic transmission was significantly impaired in dysb compared to wild type and this defect was rescued by presynaptic expression of dysb. Thus, there is an alteration of the calcium dependence of synaptic transmission in the dysb mutant. Indeed, at reduced extracellular calcium, both paired-pulse facilitation and facilitation that occurs during a prolonged stimulus train were increased in dysb mutants (Dickman, 2009).

In vertebrates, the levels of dysb expression correlate with parallel changes in extracellular glutamate concentration. Therefore, whether dysb overexpression might increase presynaptic release was tested. In wild-type animals overexpressing dysb in neurons, synaptic transmission is normal at low extracellular calcium (0.2 and 0.3 mM Ca²⁺) but was enhanced at relatively higher extracellular calcium (0.5 mM Ca²⁺). The complementary effects of dysb loss-of-function and overexpression confirm that Dysbindin has an important influence on calcium-dependent vesicle release (Dickman, 2009).

The presynaptic Ca_V2.1 calcium channel, encoded by cacophony (cac), is required for synaptic vesicle release at the Drosophila NMJ. cac mutations decrease presynaptic calcium influx and also block synaptic homeostasis. Genetic interaction between dysb and cac was tested during synaptic homeostasis. Because homozygous cac and dysb mutations individually block synaptic homeostasis, analysis of double mutant combinations would not be informative. An analysis of heterozygous mutant combinations and gene overexpression were examined. Synaptic homeostasis was suppressed by a heterozygous mutation in cac. However, this suppression was not enhanced by the presence of a heterozygous mutation in dysb. In addition, neuronal overexpression of cac did not restore homeostatic compensation in dysb mutant animals and the enhancement of presynaptic release caused by neuronal dysb overexpression still occurs in a heterozygous cac mutant background. Thus, Dysbindin may function downstream or independently of Cac during synaptic homeostasis (Dickman, 2009).

To further explore the relationship between Dysbindin and Cac, it was asked whether dysb mutations might directly influence presynaptic calcium influx. The spatially averaged calcium signal in dysb¹ was indistinguishable from wild type, indicating no difference in presynaptic calcium influx. Thus, Dysbindin appears to function downstream or independently of calcium influx to control synaptic homeostasis (Dickman, 2009).

Through a systematic electrophysiological analysis of more than 250 mutants this study could rule out the involvement of numerous synaptic proteins and biochemical processes in the mechanisms of synaptic homeostasis and demonstrate that this phenomenon is separable from the molecular mechanisms that specify structural and functional synapse development. Dysbindin is therefore identified as an essential presynaptic component within a homeostatic signaling system that regulates and stabilizes synaptic efficacy. Dysbindin functions downstream or independently of the presynaptic Ca_V2.1 calcium channel in the mechanisms of synaptic homeostasis (Dickman, 2009).

Emerging lines of evidence suggest that glutamate hypofunction could be related to the etiology of schizophrenia. Likewise, reduced levels of dysbindin expression were associated with schizophrenia. The sandy mouse, which lacks Dysbindin, has a decreased rate of vesicle release (~30% decrease), a correlated decrease in vesicle pool size and an increased thickness of the postsynaptic density. This study confirms a modest, facilitatory function for Dysbindin during baseline transmission. However, numerous mutations with similar or more severe defects in baseline transmission show normal synaptic homeostasis. By contrast, loss of Dysbindin completely blocks the adaptive, homeostatic modulation of vesicle release, suggesting that the potential contribution of dysbindin mutations to schizophrenia may be derived from altered homeostatic plasticity as opposed to decreased baseline glutamatergic transmission (Dickman, 2009).

Distinct roles of Drosophila cacophony and Dmca1D Ca(2+) channels in synaptic homeostasis: genetic interactions with slowpoke Ca(2+) -activated BK channels in presynaptic excitability and postsynaptic response

Ca(2+) influx through voltage-activated Ca(2+) channels and its feedback regulation by Ca(2+) -activated K(+) (BK) channels is critical in Ca(2+) -dependent cellular processes, including synaptic transmission, growth and homeostasis. This study report differential roles of cacophony (CaV 2) and Dmca1D (CaV 1) Ca(2+) channels in synaptic transmission and in synaptic homeostatic regulations induced by slowpoke (slo) BK channel mutations. At Drosophila larval neuromuscular junctions (NMJs), a well-established homeostatic mechanism of transmitter release enhancement is triggered by experimentally suppressing postsynaptic receptor response. In contrast, a distinct homeostatic adjustment is induced by slo mutations. To compensate for the loss of BK channel control presynaptic Sh K(+) current is upregulated to suppress transmitter release, coupled with a reduction in quantal size. This study demonstrated contrasting effects of cac and Dmca1D channels in decreasing transmitter release and muscle excitability, respectively, consistent with their predominant pre- vs. postsynaptic localization. Antibody staining indicated reduced postsynaptic GluRII receptor subunit density and altered ratio of GluRII A and B subunits in slo NMJs, leading to quantal size reduction. Such slo-triggered modifications were suppressed in cac;;slo larvae, correlated with a quantal size reversion to normal in double mutants, indicating a role of cac Ca(2+) channels in slo-triggered homeostatic processes. In Dmca1D;slo double mutants, the quantal size and quantal content were not drastically different from those of slo, although Dmca1D suppressed the slo-induced satellite bouton overgrowth. Taken together, cac and Dmca1D Ca(2+) channels differentially contribute to functional and structural aspects of slo-induced synaptic modifications (Lee, 2014).

Synapse formation is tightly associated with neuronal excitability. This study found striking synaptic overgrowth caused by Drosophila K(+)-channel mutations of the seizure and slowpoke genes, encoding Erg and Ca(2+)-activated large-conductance (BK) channels, respectively. These mutants display two distinct patterns of "satellite" budding from larval motor terminus synaptic boutons. Double-mutant analysis indicates that BK and Erg K(+) channels interact with separate sets of synaptic proteins to affect distinct growth steps. Post-synaptic L-type Ca(2+) channels, Dmca1D, and PSD-95-like scaffold protein, Discs large, are required for satellite budding induced by slowpoke and seizure mutations. Pre-synaptic cacophony Ca(2+) channels and the NCAM-like adhesion molecule, Fasciclin II, take part in a maturation step that is partially arrested by seizure mutations. Importantly, slowpoke and seizure satellites were both suppressed by rutabaga mutations that disrupt Ca(2+)/CaM-dependent adenylyl cyclase, demonstrating a convergence of K(+) channels of different functional categories in regulation of excitability-dependent Ca(2+) influx for triggering cAMP-mediated growth plasticity (Lee, 2014).

Distinct satellite patterns induced by sloand sei mutations support the notion that the two K+ channels act on separate growth steps in concert with localized molecular partners. Double-mutant analysis leads to a minimal model involving functional interactions of Slo and Sei K+ channels with distinct assemblies of pre- and post-synaptic regulators in the sequential steps of synaptic growth and differentiation. Expression of slomutant phenotypes depends on scaffold protein, Dlg, and post-synaptic Dmca1D Ca2+ channels, both of which appear to be important for initial budding of satellites. Double-mutant analysis reveals a tight association between Sei, but not Slo, K+ channels and adhesion molecule, FasII, and pre-synaptic Cac Ca2+ and Para Na+ channels in initial satellite formation as well as the ensuing process. In the same vein, manipulations of pre-synaptic cAMP affect only sei-induced satellite formation, whereas slo satellites are more susceptible to modulations in post-synaptic cAMP signaling (Lee, 2014).

Whereas these pre- and post-synaptic molecules can contribute to the initial growth of satellites in sloand sei mutants, they may also be important for further differentiation and stabilization of such intermediate structures. The stabilized satellites could accumulate over time and would facilitate their capture in fixed preparations. Immunohistochemical and electron-microscopic analyses has indicated that the majority of sloand sei satellites are well differentiated in molecular composition and ultrastructure. As live imaging studies have demonstrated, differentiation of early 'ghost boutons' occurs at a slow rate, taking hours to days. Consistently, preliminary live imaging indicates type B and M satellites abundant in mutants as stable structures with no active morphological changes over the observation period up to 1 h, during which new satellites were sighted budding from primary boutons after high K+ stimulation. Thus, the synaptic differentiation process involving Slo or Sei K+ channels and their interacting partners may occur at a slower time scale (Lee, 2014).

The results demonstrate a more profound influence of post-synaptic molecules on initial induction of satellite formation and major pre-synaptic contribution in subsequent steps. This picture is in line with potential retrograde signaling during the sequential growth process. Recent studies at Drosophila larval NMJs have revealed significant contributions of retrograde factors, such as bone morphogenetic protein, to synaptic development and function. It will be important to examine whether and how these factors take part in particular steps of the proposed sequential growth process (Lee, 2014).

There has been emerging evidence for colocalization of post-synaptic BK channels with L-type Ca2+ channels and with PSD-95 scaffold protein at vertebrate synapses. This genetic analysis thus demonstrates the functional significance of the homologous post-synaptic macromolecular association (Slo BK, Dmca1D/L-type Ca2+ channels, and Dlg/PSD-95) in synaptic growth at the Drosophila NMJ. Whether interactions among these players are also important for regulation of synaptic transmission awaits further investigations (Lee, 2014).

It has been shown that sei^ts1 mutants display increased spontaneous activities in the giant-fiber neuron and enhanced synaptic growth at larval NMJs when exposed to high temperature. However, this study observed synaptic overgrowth even at room temperature in sei^ts2 and, to a lesser extent, sei^ts2/sei^ts1. DNA sequencing predicts truncated versus full-length polypeptides in the sei^ts1 and sei^ts2 alleles, respectively, which could explain the observed allele-dependent differences. Notably, altered pre-synaptic Ca2+ and cAMP regulation drastically suppressed sei phenotypes, but was ineffective on slo-induced overgrowth, suggesting significant interactions between Erg K+ channels and these pre-synaptic components, although pre-synaptic interaction of the Sei K+ channel with the Ca2+/cAMP pathway has not been well established in Drosophila. DNA sequence analysis suggests a putative cyclic nucleotide-binding domain in Sei K+ channels, similar to Eag that belongs to the same K+-channel family. Whether cAMP-dependent modification of sei phenotypes is related to the action of this putative domain should be further investigated in future studies (Lee, 2014).

Multimeric assembly of K+ channels, including Sei Erg and Slo BK, has been implicated in regulating the channel properties. Indeed, sei^ts2/+ and slo/+ larvae, presumably containing a mixture of mutated and WT subunits in their Erg and BK channels, display dominant mutational effects on satellite formation and associated synaptic growth. Importantly, pre-synaptic expression of a mutated sei transgene (UAS-sei^ts2) in WT led to a similar, but less extreme, phenotype, confirming the pre-synaptic action of sei^ts2 and its dominant effects in multimeric Sei channels (Lee, 2014).

It is interesting to ask whether simply reducing the amount of Sei and Slo channel proteins may produce phenotypes similar to heterozygous sei^ts2/+ and slo/+ animals. The RNA interference (RNAi) technique was used to test this possibility, using multiple combinations of GAL4 drivers and UAS-slo/sei-RNAi constructs, with Dicer-2 to facilitate RNA interference in some combinations. However, none of these combinations caused characteristic behavioral and physiological abnormalities of sei and slo. Only marginal and inconsistent synaptic growth phenotypes was observed among these combinations. For instance, the expression of sloand sei RNAi in motoneurons with the driver C164-GAL4 led to a slightly elevated satellite frequency, but the pan-neuronal driver C155-GAL4 produced even less overgrowth. Bouton formation was enhanced in these GAL4-UAS-RNAi combinations but not significantly above the elevated levels intrinsic to individual GAL4 and RNAi lines (Lee, 2014).

The results suggest that dysfunctions induced by RNAi knockdown may not reproduce all aspects of mutant phenotypes. A match in protein levels or altered protein properties may be required to produce the phenotype of interest. At this time, the efficiency of these RNAi lines has not been documented. Since it was not possible to measure the levels of Slo and Sei proteins because of a lack of appropriate antibodies, it is not possible to determine the levels of each RNAi knockdown. The sloand sei mutations induced by a chemical mutagen, ethyl methanesulfonate, may affect the properties and/or the amount of the gene product. For example, sei^ts2 mutants carry a point mutation near the pore domain of the channels, and thus may act as neomorphs that confer dominant effects in the heterozygote, a property difficult to be mimicked by RNAi knockdown (Lee, 2014).

These results point out the critical role of cAMP signaling in the expression of both sloand sei mutant phenotypes and further highlight the profound functional consequences of altered excitability in neuronal plasticity. Activation of rut AC by activity-dependent accumulation of intracellular Ca2+ is pivotal in several forms of synaptic plasticity. For instance, in the Aplysia siphon-gill withdrawal reflex model, sensitizing stimuli increase cAMP levels and subsequently enhance transmission efficacy at sensorimotor synapses, and repeated conditioning induces sensory varicosity growth. Similarly, cAMP-dependent activation of protein kinase A in hippocampal slices is required for late-phase LTP that involves formation of new dendritic spines (Lee, 2014).

At Drosophila larval NMJs, altered cAMP metabolism in rut and dnc mutants impairs synaptic transmission stability and post-tetanic potentiation. In addition, fewer docked vesicles and retarded reserve pool mobilization have been documented in these mutants, indicating vesicle targeting and cycling defects. Thus, it will be interesting to examine the possibility that suppression of sloand sei satellites by rut is associated with alterations in membrane recycling. Such studies can be facilitated by relevant mutations, such as shibire defective in Dynamin, which is responsible for vesicle pinch-off, or drp1 (Dynamin-related protein 1) defective in reserve pool mobilization (Lee, 2014).

In summary, these observations reveal distinct patterns of satellite formation induced by sei and slomutations affecting two separate categories of K+ channels, which are apparently regulated by pre- and post-synaptic Ca2+/cAMP signaling, respectively. Together with previous studies, convergence on the Ca2+/CaM-activated cAMP synthesis by rut AC in the regulation of synaptic growth induced by a variety of K+ channel mutations further establishes a central role of rut AC in activity-dependent plasticity of synaptic function and growth (Lee, 2014).

Separation of presynaptic Ca_V2 and Ca_V1 channel function in synaptic vesicle exo- and endocytosis by the membrane anchored Ca²⁺ pump PMCA

Synaptic vesicle (SV) release, recycling, and plastic changes of release probability co-occur side by side within nerve terminals and rely on local Ca²⁺ signals with different temporal and spatial profiles. The mechanisms that guarantee separate regulation of these vital presynaptic functions during action potential (AP)-triggered presynaptic Ca²⁺ entry remain unclear. Combining Drosophila genetics with electrophysiology and imaging reveals the localization of two different voltage-gated calcium channels at the presynaptic terminals of glutamatergic neuromuscular synapses (the Drosophila Ca_v2 homolog, Dmca1A or cacophony, and the Ca_v1 homolog, Dmca1D) but with spatial and functional separation. Ca_v2 within active zones is required for AP-triggered neurotransmitter release. By contrast, Ca_v1 localizes predominantly around active zones and contributes substantially to AP-evoked Ca²⁺ influx but has a small impact on release. Instead, L-type calcium currents through Ca_v1 fine-tune short-term plasticity and facilitate SV recycling. Separate control of SV exo- and endocytosis by AP-triggered presynaptic Ca²⁺ influx through different channels demands efficient measures to protect the neurotransmitter release machinery against Ca_v1-mediated Ca²⁺ influx. The plasma membrane Ca²⁺ ATPase (PMCA) resides in between active zones and isolates Ca_v2-triggered release from Ca_v1-mediated dynamic regulation of recycling and short-term plasticity, two processes which Ca_v2 may also contribute to. As L-type Ca_v1 channels also localize next to PQ-type Ca_v2 channels within axon terminals of some central mammalian synapses, it is proposed that Ca_v2, Ca_v1, and PMCA act as a conserved functional triad that enables separate control of SV release and recycling rates in presynaptic terminals (Krick, 2021).

Neuronal network function critically depends on the tight control of synaptic vesicle (SV) release probability at chemical synapses over wide ranges of activity regimes. At the same time, synaptic gain remains adjustable to render network function flexible. To maintain synapse function over time, SV recycling rates must be matched to vastly different activity patterns and synaptic gains. While SV release and recycling as well as their plasticity-related adjustments all include Ca²⁺-dependent steps, they operate in parallel but on different time scales. A tight spatial and temporal coordination of presynaptic Ca²⁺ signals and their effectors is thus needed for both the induction of changes in synaptic strength and the maintenance of robust synapse function. However, the mechanisms that effectively separate Ca²⁺ signals in time and space (e.g., through different voltage-gated calcium channels [VGCCs]) to allocate these to different presynaptic functions are not well understood (Krick, 2021).

SV release probability depends on the sensitivity of the vesicular Ca²⁺ sensor and the positioning of VGCCs inside active zones (AZs). Various mechanisms that can tune release probability by modulating their precise localization or kinetic properties have been uncovered. Irrespective of such modulation, efficient Ca²⁺-triggered SV release through presynaptic VGCCs (mainly Ca_V2.1 and Ca_V2.2 in vertebrates) remains spatially restricted to a few hundred nanometers due to the limited abundance and brief opening of the channels and the presence of endogenous Ca²⁺ buffers. It is thus conceivable that Ca²⁺ signals originating within presynaptic terminals but outside AZs are engaged to tune SV recycling and plastic changes according to changes in activity (Krick, 2021).

Apart from the need for fast activating and inactivating Ca_V2 channels (Drosophila Cacophony) for SV release, other types of VGCCs have been implicated in presynaptic plasticity. In GABAergic synapses, pharmacological blockade of Ca_V1 channels does not affect AP-induced SV release but converts posttetanic potentiation into synaptic depression. In hippocampal CA3 mossy fiber boutons or in synapses of the lateral amygdala, Ca_V2.3 and Ca_V1.2 channels are required for presynaptic long-term plasticity but are unable to trigger SV release (Krick, 2021).

Differential functions of Ca_V2 and Ca_V1 channels in neurotransmitter release versus other Ca²⁺-dependent presynaptic processes can hardly be explained just by different coupling distances to SVs, since there are also situations where loose coupling is predominant. Moreover, compared with Ca_V2.1 and Ca_V2.2, Ca_V1 channels display higher conductances, suggesting that additional mechanisms are required to allocate Ca_V1-related Ca²⁺ signals to specific presynaptic functions while avoiding interference with SV release. SV recycling also includes regulation by presynaptic Ca²⁺ signals but operates mostly at different subsynaptic sites and at slower time scales than Ca²⁺-triggered SV release. It is hypothesized that activity-dependent regulation of SV recycling employs Ca_V1-dependent Ca²⁺ entry and that active mechanisms exist to regulate the relative contributions of Ca_V2 and Ca_V1 channels to SV release versus recycling. These hypotheses are addressed at the Drosophila larval neuromuscular junction (NMJ), an established model for glutamatergic synapse function (Krick, 2021).

Neuronal network function critically depends on the tight control of synaptic vesicle (SV) release probability at chemical synapses over wide ranges of activity regimes. At the same time, synaptic gain remains adjustable to render network function flexible. To maintain synapse function over time, SV recycling rates must be matched to vastly different activity patterns and synaptic gains. While SV release and recycling as well as their plasticity-related adjustments all include Ca²⁺-dependent steps, they operate in parallel but on different time scales. A tight spatial and temporal coordination of presynaptic Ca²⁺ signals and their effectors is thus needed for both the induction of changes in synaptic strength and the maintenance of robust synapse function. However, the mechanisms that effectively separate Ca²⁺ signals in time and space (e.g., through different voltage-gated calcium channels [VGCCs]) to allocate these to different presynaptic functions are not well understood (Krick, 2021).

SV release probability depends on the sensitivity of the vesicular Ca²⁺ sensor and the positioning of VGCCs inside active zones (AZs). Various mechanisms that can tune release probability by modulating their precise localization or kinetic properties have been uncovered. Irrespective of such modulation, efficient Ca²⁺-triggered SV release through presynaptic VGCCs (mainly Ca_V2.1 and Ca_V2.2 in vertebrates) remains spatially restricted to a few hundred nanometers due to the limited abundance and brief opening of the channels and the presence of endogenous Ca²⁺ buffers. It is thus conceivable that Ca²⁺ signals originating within presynaptic terminals but outside AZs are engaged to tune SV recycling and plastic changes according to changes in activity (Krick, 2021).

Apart from the need for fast activating and inactivating Ca_V2 channels for SV release, other types of VGCCs have been implicated in presynaptic plasticity. In GABAergic synapses, pharmacological blockade of Ca_V1 channels does not affect AP-induced SV release but converts posttetanic potentiation into synaptic depression. In hippocampal CA3 mossy fiber boutons or in synapses of the lateral amygdala, Ca_V2.3 and Ca_V^1.2 channels are required for presynaptic long-term plasticity but are unable to trigger SV release (Krick, 2021).

Differential functions of Ca_V2 and Ca_V1 channels in neurotransmitter release versus other Ca²⁺-dependent presynaptic processes can hardly be explained just by different coupling distances to SVs, since there are also situations where loose coupling is predominant. Moreover, compared with Ca_V2.1 and Ca_V2.2, Ca_V1 channels display higher conductances, suggesting that additional mechanisms are required to allocate Ca_V1-related Ca²⁺ signals to specific presynaptic functions while avoiding interference with SV release. SV recycling also includes regulation by presynaptic Ca²⁺ signals but operates mostly at different subsynaptic sites and at slower time scales than Ca²⁺-triggered SV release. It is hypothesized that activity-dependent regulation of SV recycling employs Ca_V1-dependent Ca²⁺ entry and that active mechanisms exist to regulate the relative contributions of Ca_V2 and Ca_V1 channels to SV release versus recycling. These hypotheses are addressed at the Drosophila larval neuromuscular junction (NMJ), an established model for glutamatergic synapse function (Krick, 2021).

The data show strict functional separation of AP-triggered neurotransmitter release by Ca_v2 and activity-dependent modulation of SV recycling and short-term plasticity by Ca_v1 VGCCs. Although task sharing and partial redundancy among Ca_v2 isoforms is known for mammalian synapses, and the dynamic regulation of their relative abundance within AZs can add to synaptic plasticity, insight into mechanisms that allow for the separate regulation of different aspects of presynaptic function by Ca_v2 and Ca_v1 channels is currently sparse (Krick, 2021).

Ultrastructural support for the coexistence of Ca_v2 and Ca_v1 channels has been obtained in rat hippocampal neurons, where Ca_v2 localizes to AZs and Ca_v1 outside AZs, largely as is found for Drosophila. Moreover, pharmacological data in mammals indicate that Ca_v1 and Ca_v2 VGCCs separately control SV release and synaptic plasticity. In synapses of the amygdala, Ca_v1 is not required for SV release but for presynaptic forms of LTP; in GABAergic basket cells, Ca_v1 is not required for evoked release but for posttetanic potentiation; and at mouse neuromuscular synapses, anatomical and physiological data indicate the presence of both presynaptic Ca_v1 and Ca_v2 channels, but again with little contribution of Ca_v1 to evoked SV release. Therefore, studies of different synapse types in various species support the idea that multiple fundamental aspects of presynaptic function are executed in parallel on the basis of spatially separated VGCCs with different kinetics and conductances. This study provides a mechanism for functional separation in the small space of the axon terminal (Krick, 2021).

The fast activation and inactivation kinetics of Ca_v2 channels in the AZ seem well suited for tight excitation-release coupling, and Ca_v2 activation mediates release mostly in an all or none fashion, though dynamic modulation of channel-SV coupling to adjust release probability is reported. By contrast, Ca_v1 channels typically have larger single-channel conductances and slower inactivation kinetics, suggesting that they are well suited to cope with the need for relatively high Ca²⁺ and the slow time course of endocytic vesicle retrieval (Krick, 2021).

Endocytosis regulation by activity-dependent Ca²⁺ influx is discussed for mammalian and invertebrate synapses. At the Drosophila NMJ, separate Ca²⁺ entry routes for differential exo- and endocytosis regulation have been postulated, and the SV-associated calcium channel Flower has been suggested to contribute to this function. This study identified Ca_v1 channels within the periphery of AZs as a distinct entry route for Ca²⁺-dependent augmentation of SV endocytosis. Although the precise underlying mechanisms remain to be investigated, an attractive hypothesis is that Ca_v1 may serve as an activity-dependent switch to direct recycling into different SV pools. In basket cells, Ca_v1 mediated Ca²⁺ influx has been speculated to mobilize vesicles into the releasable pool to maintain synaptic transmission during high-frequency bursting. Similarly, at the mouse NMJ, pharmacological blockade of L-type Ca_v1 channels decreases FM2-10 loading and quantal release upon high-frequency stimulation. This is in line with the findings of increased synaptic depression, reduced SV reacidification, decreased FM1-43 uptake, and reduced PSC recovery after RRP depletion upon reduction of presynaptic Ca_v1 function. However, the effects of Ca_v1-kd manifest within a few seconds. Unless recycling and SV reformation are ultrafast, this seems too fast for SV reuse. In cultured hippocampal neurons, for example, SVs are not reused during the first 200 APs, irrespective of stimulation frequency between 5 and 40 Hz. However, given that endocytic proteins can also function in release site clearance, reduced endocytosis in Ca_v1-kd may increase synaptic depression and decrease recovery from RRP depletion indirectly as a result of reduced release site clearance. Additional effects of Ca_v1 channels on other steps in the SV cycle, such as SV priming, can also not be excluded (Krick, 2021).

For the mouse NMJ, it has been inferred that Ca_v1 activity directs recycled SVs into a high-probability release pool. Ultrastructural analysis of Drosophila synapses has also revealed two different recycling modes, one that depends on external Ca²⁺ and directs recycled SVs to AZs and another one that does not depend on external Ca²⁺ and replenishes other SV pools. Taken together, peri-AZ localization of presynaptic Ca_v1 channels as found in hippocampus and at the Drosophila NMJ may provide a common control mechanism to direct SV recycling to different pools in an activity-dependent manner. Protection of AZs by the peri-AZ PMCA provides a mechanism to maintain mean quantal content, and thus coding reliability, in the face of Ca²⁺-mediated endocytosis regulation (Krick, 2021).

As in many mammalian neurons, in Drosophila motoneurons, Ca_v1 channels localize also to dendrites to boost excitatory synaptic input. Therefore, cooperative functions of Ca_v1 channels in different subneuronal compartments coordinate firing and SV recycling rates. Moreover, as in spinal motoneurons, Drosophila Ca_v1 channel function is modulated by biogenic amines, thus providing means for integrative regulation of motoneuron excitability and SV recycling rates in the context of internal state and behavioral demands (Krick, 2021).

This study shows that 1) axon terminal Ca_v1 segregates into the peri-AZ compartment to augment SV endocytosis, and 2) PMCA, rather than directly acting on Ca²⁺ entering through Ca_v2, actively controls Ca_v1-dependent Ca²⁺ changes, thereby enabling side-by-side Ca²⁺ domains with profiles that meet the different requirements for SV release and recycling. This is consistent with reports on spatially restricted expression and/or regulation of PMCA in small T lymphocytes as a means to steer Ca²⁺-dependent processes specifically within cellular microdomains. In consequence, this study proposes to expand the concept of controlling release probability by presynaptic Ca²⁺ buffering systems after nanodomain collapse, which has been scrutinized in many studies, with the idea of nanodomain protection from presynaptic Ca²⁺ signals originating outside the AZ (Krick, 2021).

PMCAs have high Ca²⁺ affinity and can accelerate Ca²⁺ clearance on millisecond timescales. While isolating AZs from Ca²⁺ influx through Ca_v1, PMCA otherwise does not affect the spatiotemporal properties of AZ Ca²⁺ nanodomains, because transmission amplitudes are not altered by PMCA-kd in the absence of Ca_v1 channels. Instead, it ensures stable release probability in the face of presynaptic Ca²⁺ signals that augment SV recycling, shape APs, and control synaptic plasticity. In contrast to soluble Ca²⁺ buffers and fixed ones in the AZ, the membrane-bound peri-AZ PMCA can be regulated on short time scales (e.g., by downstream effectors of Ca²⁺ and phospholipids). In addition, release from autoinhibition by binding of Ca²⁺/calmodulin, which is conserved across phyla, provides a molecular memory due to the slow time course of calmodulin release, allowing PMCA to persist in a preactivated state and to respond instantaneously to the next Ca²⁺ signal. Therefore, PMCA-mediated control of SV release probability is likely adjusted by the local activity at the synaptic terminal. The data show that changes in PMCA-dependent AZ protection largely impact SV release probability by allowing or preventing functional coupling of Ca_v1 channels with readily releasable SVs. It is proposed that the distant localization of Ca_v1 channels and PMCA in between AZs enables effective and versatile regulation of synaptic strength on a short time scale. In fact, theoretical considerations and recent studies on Ca_v2.1 dynamic coupling in hippocampal synapses and on differential spacing of Ca_v2 channels in cerebellar synapses suggest that modulation of SV release probability favors loose coupling of VGCCs to SV. Thus, regulation of presynaptic PMCA activity emerges as an effective means to dynamically regulate plasticity and SV recycling rates downstream of Ca_v1 (Krick, 2021).

The N-ethylmaleimide-sensitive factor and dysbindin interact to modulate synaptic plasticity

Dysbindin is a schizophrenia susceptibility factor and subunit of the biogenesis of lysosome-related organelles complex 1 (BLOC-1) required for lysosome-related organelle biogenesis, and in neurons, synaptic vesicle assembly, neurotransmission, and plasticity. Protein networks, or interactomes, downstream of dysbindin/BLOC-1 remain partially explored despite their potential to illuminate neurodevelopmental disorder mechanisms. This study consisted of a proteome-wide search for polypeptides whose cellular content is sensitive to dysbindin/BLOC-1 loss of function. Components of the vesicle fusion machinery were identified as factors downregulated in dysbindin/BLOC-1 deficiency in neuroectodermal cells and iPSC-derived human neurons, among them the N-ethylmaleimide-sensitive factor (NSF). Human dysbindin/BLOC-1 coprecipitates with NSF and vice versa, and both proteins colocalized in a Drosophila model synapse. To test the hypothesis that NSF and dysbindin/BLOC-1 participate in a pathway-regulating synaptic function, the role for NSF was studied in dysbindin/BLOC-1-dependent synaptic homeostatic plasticity in Drosophila. As previously described, this study found that mutations in dysbindin precluded homeostatic synaptic plasticity elicited by acute blockage of postsynaptic receptors. This dysbindin mutant phenotype is fully rescued by presynaptic expression of either dysbindin or Drosophila NSF. However, neither reduction of NSF alone or in combination with dysbindin haploinsufficiency impaired homeostatic synaptic plasticity. These results demonstrate that dysbindin/BLOC-1 expression defects result in altered cellular content of proteins of the vesicle fusion apparatus and therefore influence synaptic plasticity (Gokhale, 2015).

Dysbindin associates with seven other polypeptides to form the biogenesis of lysosome-related organelles complex 1. Null mutations in mouse dysbindin reduce the expression of other BLOC-1 subunit mRNAs and polypeptides. This suggests that dysbindin genetic downregulation could elicit multiple alterations of protein content in cells. This study identified 224 proteins whose content was modified by dysbindin/BLOC-1 partial loss of function using unbiased quantitative mass spectrometry. The screen prominently identified components of the N-ethylmaleimide-sensitive factor (NSF)-dependent vesicle fusion machinery. Focus was placed on NSF, a catalytic component of the fusion machinery, and it was asked whether NSF participates in dysbindin/BLOC-1-dependent synaptic mechanisms. Drosophila presynaptic plasticity produced by the inhibition of postsynaptic receptors was used as an assay. As previously, it was observed that mutations in fly dysbindin precluded the establishment of homeostatic synaptic plasticity, a phenotype that is rescued by presynaptic expression of dysbindin. Neuron-specific expression of dNSF1, the gene encoding Drosophila NSF, by itself does not modulate this form of plasticity, yet NSF1 expression at the synapse of dysbindin mutants rescued homeostatic synaptic plasticity defects to the same extent as dysbindin re-expression in the presynaptic compartment. These results demonstrate that partial dysbindin/BLOC-1 loss of function alters the cellular content of proteins that specifically have roles in synaptic mechanisms (Gokhale, 2015).

Genetic polymorphisms associated with schizophrenia mostly reside in noncoding regions modifying gene and/or protein levels rather protein sequence. The question of how widespread the effects are of a single mutation or polymorphism across the proteome has been poorly explored. This study addressed this question by modeling a partial reduction in the cellular content of dysbindin/BLOC-1 using shRNAs against BLOC-1 complex subunits. 224 proteins were whose content is affected by a partial loss of function of dysbindin/BLOC-1 and focused on an interactome centered around a schizophrenia susceptibility gene, dysbindin, and NSF, a component of the membrane fusion machinery that localizes to the synapse and was previously implicated in schizophrenia mechanisms. Functional outcomes of the dysbindin/BLOC-1 and NSF association were confirmed using a Drosophila synaptic adaptive response. The results demonstrate that dysbindin/BLOC-1 expression defects induce multiple downstream quantitative protein expression traits associated with the vesicle fusion apparatus, which influence synaptic plasticity in an invertebrate model synapse (Gokhale, 2015).

A proteomic search prominently highlights the following components of the vesicle fusion apparatus: munc18, tomosyn, NSF; and the SNAREs syntaxin 7, syntaxin 17, SNAP23, SNAP25, SNAP 29, and VAMP7. Importantly, most of the aforementioned vesicle fusion machinery components have been implicated by genomic and postmortem studies in several neurodevelopmental disorders, including schizophrenia, intellectual disability, and autism spectrum disorder. The current strategy is validated by the identification of proteins previously known to be downregulated in null alleles of BLOC-1 subunits and/or known to interact with BLOC-1. These proteins include subunits of the BLOC-1 complex and the SNARE VAMP7. This study further authenticated these fusion machinery components as part of a dysbindin/BLOC-1 network by (1) coimmunoprecipitation of a fusion machinery component with dysbindin/BLOC-1 subunits and/or (2) downregulation of a fusion machinery component after genetic or shRNA-mediated reduction of dysbindin/BLOC-1 subunits. NSF was studied since it is a hub of protein-protein interactions with components of the fusion machinery, and is a catalytic activity that is required for the resolution of fusion reaction products and other protein-protein complexes. NSF was found to associate with dysbindin and BLOC-1 subunits in neuroblastoma cells in culture. However, efforts to document the association of NSF and dysbindin-BLOC-1 by immunoprecipitation with NSF antibodies were unsuccessful in brain. This outcome occurred regardless of whether NSF was immunoprecipitated from brain homogenates or cross-linked synaptosomal lysates from adult mouse brain. This negative result is attributed to the high abundance of NSF in brain compared with dysbindin/BLOC-1. Reverse immunoprecipitations with dysbindin/BLOC-1 antibodies were not possible, as none of the available antibodies were suitable for immunoprecipitation. Since most of the associations between NSF and dysbindin/BLOC-1 are detected in the presence of the cross-linker DSP in cell lines, it is likely that the biochemical interactions between NSF and dysbindin/BLOC-1 are indirect. However, NSF cellular levels are decreased following shRNA-mediated or genomic reduction of BLOC-1 complex members, arguing in favor of a functional outcome of this association. No NSF downregulation phenotype was detected in hippocampal extracts of Bloc1s8sdy/sdy mice at days 7 or 50 of postnatal development. This suggests that NSF phenotypes may be anatomically restricted either to a region of the hippocampus or to an earlier and transient developmental stage. However, this reduced NSF trait is robustly and reversibly induced by genetic disruption of the dysbindin/BLOC-1 complex or by downregulation of dysbindin/BLOC-1 subunits in neuroblastoma and human embryonic kidney cells, neuroectodermal cells, and iPSC-derived human neurons (Gokhale, 2015).

These studies indicate that the functional outcome of NSF reduction in BLOC-1 loss of function become evident only when the synapse is challenged. Constitutive secretion in Drosophila or mammalian non-neuronal cells is unaffected, as are spontaneous and evoked neurotransmission at the Drosophila neuromuscular junction. However, a requirement for NSF in BLOC-1 loss-of-function phenotypes can be localized to a presynaptic homeostatic mechanism, which is engaged when postsynaptic receptors are blocked with philanthotoxin. After a brief incubation with philanthotoxin, the resultant reduction in postsynaptic signal transduction rapidly induces a compensatory increase in quantal content, a response known as homeostatic synaptic plasticity. This adaptive compensatory mechanism is precluded by dysbindin mutations, and can be rescued by presynaptic expression of dysbindin. However, it was possible to rescue this phenotype in the dysbindin mutants to the exact same extent through presynaptic expression NSF. The observation that RNAi downregulation or overexpression of NSF in the neuromuscular junction does not interfere with homeostatic synaptic plasticity argues that the NSF is not an obligate component downstream of dysbindin/BLOC-1 in a linear pathway, but rather is an adaptive response to network perturbation induced by a dysbindin mutant allele. This hypothesis predicts that transheterozygotic reduction of NSF and Dysbindin should impair plasticity, a result that is at odds with the finding that plasticity is normal in dysb1-/+;UAS-NSF RNAi. It is believe that this may be a consequence of a modest reduction of dysbindin polypeptide in dysb1-/+ animals, which was predicted to be ~25% (Gokhale, 2015).

How does the BLOC-1-NSF interaction affect synaptic mechanisms? A model integrating these findings has to consider three key elements. First, BLOC-1 subunits reside at endosomes as well as on synaptic vesicles in presynaptic terminals in neurons. Second, BLOC-1 binds monomeric SNAREs rather than tetrahelical SNARE bundles in vitro. Finally, NSF and SNAREs bind to dysbindin/BLOC-1, yet they do not seem to form a ternary complex. Thus, it is proposed that BLOC-1 bound to a single SNARE (perhaps for SNARE sorting into vesicles) is resolved by NSF, making SNAREs permissive for vesicle fusion. Therefore, when dysbindin and NSF levels are reduced by hypomorphic mutations in the fly or as a quantitative expression trait in humans, SNARE-dependent mechanisms might be impaired due to defective SNARE sorting, a consequence of the reduced levels of BLOC-1 complex and, additionally, by decreased NSF content that would impair the resolution of remaining SNARE-BLOC-1 complexes. Thus, noncoding polymorphisms in several genes and their quantitative expression traits may converge to impair synaptic mechanisms. It is proposed that unbiased identification of quantitative traits across the proteome of neurodevelopmental deficiency models is a simple approach to unravel mechanisms of complex neurodevelopmental disorders (Gokhale, 2015).

Dysbindin links presynaptic proteasome function to homeostatic recruitment of low release probability vesicles

This study explores the relationship between presynaptic homeostatic plasticity and proteasome function at the Drosophila neuromuscular junction. First, it was demonstrated that the induction of homeostatic plasticity is blocked after presynaptic proteasome perturbation. Proteasome inhibition potentiates release under baseline conditions but not during homeostatic plasticity, suggesting that proteasomal degradation and homeostatic plasticity modulate a common pool of vesicles. The vesicles that are regulated by proteasome function and recruited during homeostatic plasticity are highly EGTA sensitive, implying looser Ca2+ influx-release coupling. Similar to homeostatic plasticity, proteasome perturbation enhances presynaptic Ca2+ influx, readily-releasable vesicle pool size, and does not potentiate release after loss of specific homeostatic plasticity genes, including the schizophrenia-susceptibility gene dysbindin. Finally, genetic evidence is provided that Dysbindin levels regulate the access to EGTA-sensitive vesicles. Together, these data suggest that presynaptic protein degradation opposes the release of low-release probability vesicles that are potentiated during homeostatic plasticity and whose access is controlled by dysbindin (Wentzel, 2018).

At the Drosophila NMJ, PHP can be rapidly induced within minutes in the presence of the protein synthesis inhibitor cyclohexamide, suggesting that the acute induction of PHP does not require synthesis of new proteins. In contrast, protein degradation has not been studied in the context of PHP at this synapse. The ubiquitin-proteasome system (UPS) is a major protein degradation pathway. At the Drosophila NMJ it has been shown that all components of the UPS are present at presynaptic terminals and that acute proteasome inhibition causes a rapid strengthening of neurotransmission There is accumulating evidence for links between neural activity and UPS-mediated degradation of presynaptic proteins in mice and rats. However, only two presynaptic proteins -- Rab3-interacting molecule (RIM: Jiang, 2010; Lazarevic, 2011; Yao, 2007) and Dunc-13/munc-13 (Speese, 2003) -- as well as one E3-ligase (SCRAPPER Yao, 2007) have so far been implicated in UPS-dependent control of presynaptic protein turnover and release. Thus, the molecular pathways underlying the regulation of presynaptic release through protein degradation remain enigmatic (Wentzel, 2018).

PHP at the Drosophila NMJ requires high-release probability (p_r) vesicles that are 'tightly coupled' to Ca2+ channels through rim-binding protein. The distance between Ca2+ channels and Ca2+ sensors of exocytosis, typically referred to as 'coupling distance', is a major factor determining the p_r of synaptic vesicles. However, despite their implication in short-term plasticity, little is known about how vesicles that are differentially coupled to Ca2+ influx are modulated during synaptic plasticity (Wentzel, 2018).

This study explored the relationship between PHP and presynaptic proteasome function at the Drosophila NMJ and provides evidence for links between presynaptic protein degradation and homeostatic potentiation of loosely-coupled synaptic vesicles (Wentzel, 2018).

Rapid effects of proteasome perturbation were observed on neurotransmitter release and the abundance of ubiquitinated proteins on the minute time scale. These data suggest that the proteasomal degradation rate is relatively high under baseline conditions, consistent with previous work on local protein degradation in the presynaptic or postsynaptic compartment, and considerably faster than the average neuron-wide turnover rates of synaptic proteins (2-5 days). Such rapid degradation rates could in turn allow for potent modulation of protein abundance and/or ubiquitination through regulation of UPS function during PHP (Wentzel, 2018).

Perturbation of proteasome function has diverse effects on cellular physiology, such as altering the levels of free ubiquitin and mono-ubiquitinated proteins, activating macroautophagy or upregulation of lysosomal enzyme levels. The observed phenotypes could thus be due to indirect effects of impaired proteasome function on synaptic physiology. Even if it is not possible to rule out this possibility, several lines of evidence argue against a major contribution of indirect effects. First, acute or prolonged proteasome perturbation does not affect release at synapses that express PHP. Second, genetic evidence is provided that proteasome perturbation-induced changes in release are blocked in two PHP mutants. Third, no major changes were detected in synaptic morphology or synaptic development upon prolonged proteasome perturbation. Fourth, interfering with proteasome function does not impair neurotransmitter release, but rather results in a net increase in release. Taken together, these data suggest that proteasome function opposes release by degrading proteins under baseline conditions, and that normal degradation of these proteins is required for PHP (Wentzel, 2018).

The genetic data imply that not all proteins required for PHP are regulated through UPS-dependent degradation under the experimental conditions (pharmacological proteasome perturbation for 15 min) used in this study, because release can be potentiated upon brief proteasome inhibition in most PHP mutants. This observation is consistent with a recent study demonstrating that the abundance of most synaptic proteins does not change after prolonged pharmacological proteasome perturbation in cultured mouse hippocampal neurons. Based on the current observation that PHP is blocked in cut-up mutants, which were recently shown to have a defect in proteasome trafficking, it is conceivable that proteasome mobility and/or recruitment are modulated during PHP (Wentzel, 2018).

Evidence is provided that proteasome perturbation and homeostatic signaling recruit EGTA-sensitive vesicles with a lower p_r in addition to vesicles with higher p_r. Previous work revealed that tightly-coupled, high-p_r vesicles are required for PHP. PHP therefore likely involves vesicle pools with different p_r . Homeostatic regulation of EGTA-sensitive, loosely-coupled vesicles depends on dysbindin, whereas tightly-coupled vesicles are controlled by RIM-binding protein. Together, these results imply that PHP involves two genetically separable populations of vesicles with different p_r (Wentzel, 2018).

Vesicle pools with different p_r and release kinetics have been observed at various synapses, and these pools might be differentially regulated during synaptic plasticity. This study provides evidence that dysbindin is required for the recruitment of EGTA-sensitive vesicles during PHP. It will be exciting to investigate the roles of other presynaptic proteins that have been implicated in the release of EGTA-sensitive vesicles, such as Tomosyn, in the context of proteasome degradation and PHP. Interestingly, a recent study at the mouse NMJ observed accelerated release kinetics of 'slow' synaptic vesicles during PHP. Thus, homeostatic potentiation of vesicles with lower p_r /slower release kinetics may be an evolutionarily conserved mechanism (Wentzel, 2018).

Which mechanisms could potentiate the release of low-p_r vesicles? This study revealed that presynaptic proteasome perturbation results in enhanced presynaptic Ca2+ influx, independent of major changes in Ca2+ buffering and/or extrusion. Therefore changes in presynaptic Ca2+ influx are considered as a possible mechanism underlying the increase in low-p_r vesicle release. Proteasome perturbation also increased RRP size. Earlier work revealed that changes in presynaptic Ca2+ influx modulate release in part by altering apparent RRP size. The increase in RRP size or EGTA sensitivity upon proteasome inhibition may therefore be in part a secondary consequence of enhanced presynaptic Ca2+ influx. However, several observations, such as the increased amplitude of the fast recovery phase after pool depletion or the slowing of EPSC decay kinetics upon presynaptic proteasome perturbation, which are not seen after increasing [Ca2+]e, indicate that the increase in release is not caused by presynaptic Ca2+ influx alone. Together, these data imply that a combination of increased presynaptic Ca2+ influx and RRP size underlie the enhancement of release after proteasome or glutamate receptor perturbation. Which molecular mechanisms may link UPS function to the modulation of presynaptic Ca2+ influx or RRP size? Genetic data suggest that dysbindin functions independently of presynaptic Ca2+ influx. Interestingly, rim mutants were shown to have a defect in homeostatic RRP size modulation, but unchanged homeostatic control of presynaptic Ca2+ influx. It is therefore speculated that RIM and Dysbindin may be involved in proteasome-dependent RRP size regulation. How do these observations relate to mammalian synapses? At cultured hippocampal rat synapses, proteasome inhibition augments recycling vesicle pool size or prevents a decrease in RRP size induced by prolonged (4h) depolarization. Moreover, several studies at mammalian synapses suggest that UPS-dependent regulation of RIM abundance regulates neurotransmitter release during baseline synaptic transmission and synaptic plasticity. Finally, there is evidence that ubiquitination acutely regulates release on the minute time scale at cultured hippocampal rat synapses (Rinetti, 2010). Together, these studies suggest that rapid, UPS-dependent control of RRP size may be evolutionarily conserved. Future studies will further elucidate the molecular signaling pathways relating UPS function to neurotransmitter release during baseline synaptic transmission and homeostatic plasticity (Wentzel, 2018).

Genetic analysis in Drosophila reveals a role for the mitochondrial protein p32 in synaptic transmission

Mitochondria located within neuronal presynaptic terminals have been shown to play important roles in the release of chemical neurotransmitters. In the present study, a genetic screen for synaptic transmission mutants of Drosophila has identified the first mutation in a Drosophila homolog of the mitochondrial protein P32. Although P32 is highly conserved and has been studied extensively, its physiological role in mitochondria remains unknown and it has not previously been implicated in neural function. The Drosophila P32 mutant, referred to as dp32^EC1, exhibited a temperature-sensitive (TS) paralytic behavioral phenotype. Moreover, electrophysiological analysis at adult neuromuscular synapses revealed a TS reduction in the amplitude of excitatory postsynaptic currents (EPSC) and indicated that dP32 functions in neurotransmitter release. These studies are the first to address P32 function in Drosophila and expand the knowledge of mitochondrial proteins contributing to synaptic transmission (Lutas, 2012).

A genetic screen for synaptic transmission mutants in Drosophila isolated a new mutation in a Drosophila homolog of the mitochondrial protein P32, which represents the first P32 mutation in a multicellular organism. Although P32 is highly conserved and has been studied extensively, its physiological function in mitochondria remains unknown. This new mutant, referred to as dP32^EC1, exhibited a temperature-sensitive (TS) paralytic behavioral phenotype. Moreover, electrophysiological analysis at adult neuromuscular synapses revealed a TS reduction in neurotransmitter release, indicating that dP32 serves an important function in synaptic transmission. Immunocytochemical analysis has shown that dP32 is located within presynaptic mitochondria, which are known to be important in ATP production and calcium signaling at synapses. Furthermore, the basic molecular and structural organization of synapses appears to be normal in the dP32 mutant, suggesting a direct role for this protein in synaptic function. At the molecular level, biochemical studies indicated conserved homomultimeric interactions of dP32 subunits. Finally, assessment of presynaptic mitochondrial function was examined in the dP32 mutant through measurement of ATP levels and imaging studies of mitochondrial membrane potential and presynaptic calcium. This work indicated that mitochondrial ATP production and membrane potential in the dP32 mutant resembled wild-type, whereas the mutant exhibited a TS increase in both resting and evoked presynaptic calcium concentration. Taken together, the preceding findings reveal a role for dP32 in synaptic transmission and mitochondrial regulation of presynaptic calcium (Lutas, 2012).

Mitochondrial localization of P32 proteins involves an N-terminal targeting domain that is cleaved from the mature targeted protein. Comparison of Drosophila and vertebrate P32 sequences indicates conservation of the proteolytic cleavage site in dP32. In the present study, an equivalent targeting function for the N-terminal domain of dP32 was demonstrated through its ability to mediate mitochondrial targeting. When the first 71 amino acids of dP32, including the proteolytic cleavage site, was fused to GCaMP3, this fusion protein (mito-GCaMP3) was efficiently targeted to mitochondria. Although only modest sequence conservation was observed between the N-terminal domains of dP32 and vertebrate P32 proteins, previous studies suggest that mitochondrial targeting domains vary in amino acid sequence but often share an amphipathic helical structure (Lutas, 2012).

Structural studies have established that P32 is a homotrimer in which monomers are arranged around a central pore in a donut-like structure. In the present study, homomultimerization of dP32 subunits was demonstrated in co-immunoprecipitation experiments. The trimeric structure of P32 exhibits a highly asymmetric charge distribution that creates a concentration of negatively charged residues along one side of the donut, raising the possibility that P32 may participate in calcium binding within the mitochondrial matrix. Notably, five residues that are spatially clustered to form a pocket on the negatively charged side of human P32, Glu-89, Leu-231, Asp-232, Glu-264, and Tyr-268, are identical in the Drosophila protein. Further genetic analysis may address the importance of these clustered residues in dP32 function at synapses (Lutas, 2012).

Several possible mechanisms of dP32 function in mitochondria and synaptic transmission were considered and investigated in this paper, most notably possible roles in supporting mitochondrial membrane potential, ATP production, and presynaptic calcium signaling. Among these, the observations favor a function for dP32 in mitochondrial mechanisms regulating presynaptic calcium. Although neurotransmitter release was reduced at restrictive temperatures in dP32^EC1, the presynaptic calcium concentration was increased both at rest and in response to synaptic stimulation. It is of interest to consider why the increase in presynaptic calcium in dP32^EC1 is TS in what appears to be a complete loss-of-function mutant. Previous studies at Drosophila larval neuromuscular synapses at elevated temperatures have observed a TS increase in resting cytosolic calcium and associated inhibition of neurotransmitter release. This calcium increase was enhanced by pharmacological inhibition of presynaptic calcium clearance mechanisms or genetic removal of presynaptic mitochondria, but it remained dependent on temperature. The present findings may reflect a similar TS process involving calcium-dependent inhibition of neurotransmitter release and dP32-dependent mitochondrial mechanisms. Efforts to further address these mechanisms were pursued by employing a calcium indicator targeted to the mitochondrial matrix, mito-GCaMP3. Although this approach was successful for imaging mitochondrial calcium transients elicited by motor axon stimulation in both WT and dP32^EC1 at 20°C, robust calcium transients could not be observed in either genotype when the temperature was increased to the restrictive temperatures of 33° or 36° (Lutas, 2012).

The preceding observations suggest that sustained elevation of presynaptic calcium in the dP32 mutant may lead to reduced neurotransmitter release. Such a calcium-dependent mechanism has been reported previously in the squid giant synapse and attributed to calcium-dependent adaptation of the neurotransmitter release apparatus. Understanding the precise mechanism by which loss of dP32 impairs neurotransmitter release will require further investigation. One interesting question is how the absence of dP32 in the mitochondrial matrix leads to increased presynaptic calcium and whether this reflects the putative calcium binding capacity of this protein. Finally, while the present study is focused on the newly discovered role for P32 in neurotransmitter release, the resulting research materials are expected to facilitate in vivo analysis of P32 function in a broad range of biological processes (Lutas, 2012).

The Bruchpilot cytomatrix determines the size of the readily releasable pool of synaptic vesicles

Synaptic vesicles (SVs) fuse at a specialized membrane domain called the active zone (AZ), covered by a conserved cytomatrix. How exactly cytomatrix components intersect with SV release remains insufficiently understood. Previous studies have shown that loss of the Drosophila ELKS family protein Bruchpilot (BRP) eliminates the cytomatrix (T bar) and declusters Ca2⁺ channels. This paper explores additional functions of the cytomatrix, starting with the biochemical identification of two BRP isoforms. Both isoforms alternated in a circular array and are important for proper T-bar formation. Basal transmission is decreased in isoform-specific mutants, attributable to a reduction in the size of the readily releasable pool (RRP) of SVs. A corresponding reduction was found in the number of SVs docked close to the remaining cytomatrix. It is proposed that the macromolecular architecture created by the alternating pattern of the BRP isoforms determines the number of Ca2⁺ channel-coupled SV release slots available per AZ and thereby sets the size of the RRP (Matkovic, 2013).

An elaborate protein cytomatrix covering the AZ membrane is meant to facilitate and control the SV release process. Quantitative analysis of neurotransmitter release has provided evidence that the number of SV release sites per AZ might be fixed. Although these sites are thought to be located in close proximity to presynaptic Ca²⁺ channels, ultrastructural and molecular information is largely missing here. Potentially, specific interactions between SVs and certain cytomatrix components might be involved. This study provides evidence that the BRP-based cytomatrix plays a role in defining the number of readily releasable SVs, possibly by offering morphological and molecular-determined 'release slots' (Matkovic, 2013).

Previous studies have characterized the role of BRP based on null alleles, which result in a complete absence of AZ cytomatrix (T bar), partially declustered Ca²⁺ channels, and likely as a direct consequence, reduced vesicular release probability. In contrast, in the analysis of BRP isoform-specific mutants, the current study neither observed any Ca²⁺ channel clustering deficits nor changes in vesicular release probability (Matkovic, 2013).

Previous studies have found a binding site between the intracellular C terminus of the Cac Ca²⁺ channel and an N-terminal stretch of BRP, which is unique to BRP-190 (Fouquet, 2009). That solely losing BRP-190 is not sufficient to affect Ca²⁺ channel clustering could possibly be explained by the presence of redundant binding sites within BRP-170. Ca²⁺ channel clustering might well be a collective feature of the cytomatrix, and Ca²⁺ channels likely use multiple simultaneous interactions with several cytomatrix proteins to anchor within the AZ membrane (Matkovic, 2013).

In fact, RIM-binding protein family proteins at rodent and Drosophila AZs bind Ca²⁺ channels, and loss of the only RIM-binding protein in Drosophila results in partial loss of Ca²⁺ channels from AZs. RIM-binding protein levels at AZ were slightly but significantly reduced in the BRP isoform mutants. Clearly, it remains a possibility that RIM-binding protein is a major scaffold determinant of the release slots and that e.g., subtle mislocalizations of RIM-binding protein might in part contribute to the BRP isoform mutant phenotype. The brp-null phenotype can now be interpreted as a 'catastrophic event' in which a complete loss of this large scaffold protein leads to a severe decrease of cytomatrix avidity (potentially mediated via a loss of RIM-binding protein) below a critical level, resulting in a 'collapse' of the normal cytomatrix architecture. Thus, functionalities associated with discrete regions of BRP and RIM-binding protein can apparently be masked when the BRP-based AZ scaffold is completely eliminated (Matkovic, 2013).

The distal cytomatrix in brp^nude is bare of SVs in EM, and SV replenishment is defective, resulting in short-term depression (and not facilitation as in brp nulls). However, no change of short-term plasticity could be detected in the brp isoform alleles with the same analyses, consistent with neither a change in Ca²⁺ channel clustering nor in SV clustering at the distal cytomatrix. Nevertheless, a basal release deficit was observed, which can be explained by a reduction in the size of the readily releasable vesicle pool, assigning an additional function to the BRP cytomatrix (Matkovic, 2013).

Release-ready SVs are meant to be molecularly and positionally primed for release. Important factors are the equipment with or the attachment to the proteins of the core release machinery and the localization of the SV in proximity to the Ca²⁺ source. At the Drosophila NMJ, SV release is insensitive to slow Ca²⁺ buffers such as EGT; therefore, SVs are thought to be spatially tightly coupled to Ca²⁺ channels (nanodomain coupling; Eggermann, 2012). Since Ca²⁺ channels are found localized directly underneath the T-bar pedestal composed of the N-terminal region of BRP (Fouquet, 2009), release-ready SVs might well correspond to the SVs that were found docked at the pedestal of the T bar and thus in very close proximity to the Ca²⁺ channels. This in turn is in agreement with BRP itself being important for defining the number of release-ready SVs determined by electrophysiology and EM (Matkovic, 2013).

Light microscopic inspection of an AB directed against the C terminus of BRP, common to both isoforms, with 50-nm STED resolution, typically revealed approximately five dots arranged as a circle or regular pentagon. Both isoforms were labelled individually, and it was found that (1) both isoforms seem to localize with their C termini similarly toward the distal edge of the cytomatrix and (2) both isoforms typically form an identical number of dots per AZ similar to the number of dots observed with the BRP^C-Term AB recognizing both isoforms. Thus, the BRP isoforms seem to be arranged in neighboring but not overlapping clusters, forming a circular array. Consistent with both BRP isoforms not overlapping in space, there was neither efficient co-IP between them nor did elimination of one isoform substantially interfere with the AZ localization of the respective other isoform. Thus, BRP-190 and -170 seem to form discrete oligomers. The alternating pattern of BRP-190 and -170 appears to set a typical cytomatrix size, as both isoform mutants had a reduced T-bar width in EM and a reduced mean number of BRP dots per AZ. As this corresponded with a similar reduction in the number of SVs in the RRP, this AZ architecture could set a typical number of Ca²⁺ nanodomain-coupled RRP slots possibly located between BRP clusters. However, beyond providing a discrete morphological architecture, the two BRP isoforms described in this study might harbor additional functionalities. The brp^Δ190 phenotype was more pronounced than the brp^Δ170, leaving the possibility that the highly conserved N terminus of BRP-190 promotes release by further mechanisms going beyond the points analyzed in this study. Future analysis will also have to address whether localization and regulation of additional cytomatrix and release components, such as RIM-binding protein, Unc-13 family proteins, or RIM, contribute to the formation of release slots as well (Matkovic, 2013).

Ultimately, functional differences between individual synaptic sites must be defined by variances in their molecular organization. Functional features of a synapse can be extracted electrophysiologically. Thereby, the number of Ca²⁺ channels was recently identified as a major determinant of the release probability of single vesicles, P_vr, in rat calyces (Sheng, 2012). Furthermore, AZ size seems to scale with the overall likelihood of release from a given AZ (Holderith, 2012). The current results suggest that the BRP-based cytomatrix should be a general determinant of the release likelihood per AZ by establishing P_vr, through Ca²⁺ channel clustering, as shown previously, and, as shown in this study, by determining the size of the RRP. The genetic results show that the cytomatrix can, in principle, control the RRP size independent of Ca²⁺ channel clustering. A coupled increase in the size of the T-bar cytomatrix together with increasing SV release was previously observed at NMJs compensating for loss of the glutamate receptor subunit glurIIA. Moreover, an increase in the number of release-ready SVs together with an increase in the amount of BRP was recently described as part of a homeostatic presynaptic response after pharmacological block of postsynaptic GluRIIA (Weyhersmuller, 2011). In line with this scenario, it was recently shown that lack of acetylation of BRP in elp3 mutants led to an increase in the complexity of the AZ cytomatrix along with an increase in RRP size (Miskiewicz, 2011). Furthermore, in vivo imaging of synaptic transmission with single synapse resolution revealed that the likelihood of release correlates with the amount of BRP present at an individual AZ (Peled, 2011). This cytomatrix size-SV release scaling might be a general principle, as a correlation between the amount of SV exocytosis, measured by an optical assay, and the amount of the AZ protein Bassoon at individual synapses of cultured rat hippocampal neurons has also been observed (Matz, 2010). The current results suggest that not only the mere size, but also the distinct architecture of the cytomatrix influence release at individual synapses through determining RRP size (Matkovic, 2013).

Estimation of the readily releasable synaptic vesicle pool at the Drosophila larval neuromuscular junction

Presynaptic boutons at nerve terminals are densely packed with synaptic vesicles, specialized organelles for rapid and regulated neurotransmitter secretion. Upon depolarization of the nerve terminal, synaptic vesicles fuse at specializations called active zones that are localized at discrete compartments in the plasma membrane to initiate synaptic transmission. A small proportion of synaptic vesicles are docked and primed for immediate fusion upon synaptic stimulation, which together comprise the readily releasable pool. The size of the readily releasable pool is an important property of synapses, which influences release probability and can dynamically change during various forms of plasticity. A detailed protocol is described for estimating the readily releasable pool at a model glutamatergic synapse, the Drosophila neuromuscular junction. This synapse is experimentally robust and amenable to sophisticated genetic, imaging, electrophysiological, and pharmacological approaches. The experimental design, electrophysiological recording procedure, and quantitative analysis that is necessary to determine the readily releasable pool size is described in this paper. This technique requires the use of a two-electrode voltage-clamp recording configuration in elevated external Ca(2+) with high frequency stimulation. This assay is sused to measure the readily releasable pool size and reveal that a form of homeostatic plasticity modulates this pool with synapse-specific and compartmentalized precision. This powerful approach can be utilized to illuminate the dynamics of synaptic vesicle trafficking and plasticity and determine how synaptic function adapts and deteriorates during states of altered development, stress and neuromuscular disease (Goel, 2019).

Pathogenic Huntington alters BMP signaling and synaptic growth through local disruptions of endosomal compartments

Huntington's disease (HD) is a neurodegenerative disorder caused by expansion of a polyglutamine (polyQ) stretch within the Huntingtin (Htt) protein. Pathogenic Htt disrupts multiple neuronal processes, including gene expression, axonal trafficking, proteasome and mitochondrial activity, and intracellular vesicle trafficking. However, the primary pathogenic mechanism and subcellular site of action for mutant Htt are still unclear. Using a Drosophila HD model, this study found that pathogenic Htt expression leads to a profound overgrowth of synaptic connections that directly correlates with the levels of Htt at nerve terminals. Branches of the same nerve containing different levels of Htt show distinct phenotypes, indicating Htt acts locally to disrupt synaptic growth. The effects of pathogenic Htt on synaptic growth arise from defective synaptic endosomal trafficking, leading to expansion of a recycling endosomal signaling compartment containing Sorting Nexin 16, and a reduction in late endosomes containing Rab11. The disruption of endosomal compartments leads to elevated BMP signaling within nerve terminals, driving excessive synaptic growth. Blocking aberrant signaling from endosomes or reducing BMP activity (see Wishful thinking) ameliorates the severity of HD pathology and improves viability. Pathogenic Htt is present largely in a non-aggregated form at synapses, indicating cytosolic forms of the protein are likely to be the toxic species that disrupt endosomal signaling. These data indicate that pathogenic Htt acts locally at nerve terminals to alter trafficking between endosomal compartments, leading to defects in synaptic structure that correlate with pathogenesis and lethality in the Drosophila HD model (Akbergenova, 2017).

Nitric oxide-mediated posttranslational modifications control neurotransmitter release by modulating complexin farnesylation and enhancing its clamping ability

Nitric oxide (NO) regulates neuronal function and thus is critical for tuning neuronal communication. Mechanisms by which NO modulates protein function and interaction include posttranslational modifications (PTMs) such as S-nitrosylation. Importantly, cross signaling between S-nitrosylation and prenylation can have major regulatory potential. However, the exact protein targets and resulting changes in function remain elusive. This study interrogated the role of NO-dependent PTMs and farnesylation in synaptic transmission. NO was found to compromise synaptic function at the Drosophila neuromuscular junction (NMJ) in a cGMP-independent manner. NO suppressed release and reduced the size of available vesicle pools, which was reversed by glutathione (GSH) and occluded by genetic up-regulation of GSH-generating and de-nitrosylating glutamate-cysteine-ligase and S-nitroso-glutathione reductase activities. Enhanced nitrergic activity led to S-nitrosylation of the fusion-clamp protein complexin (cpx) and altered its membrane association and interactions with active zone (AZ) and soluble N-ethyl-maleimide-sensitive fusion protein Attachment Protein Receptor (SNARE) proteins. Furthermore, genetic and pharmacological suppression of farnesylation and a nitrosylation mimetic mutant of cpx induced identical physiological and localization phenotypes as caused by NO. Together, these data provide evidence for a novel physiological nitrergic molecular switch involving S-nitrosylation, which reversibly suppresses farnesylation and thereby enhances the net-clamping function of cpx. These data illustrate a new mechanistic signaling pathway by which regulation of farnesylation can fine-tune synaptic release (Robinson, 2018).

Throughout the central nervous system (CNS), the volume transmitter nitric oxide (NO) has been implicated in controlling synaptic function by multiple mechanisms, including modulation of transmitter release, plasticity, or neuronal excitability. NO-mediated posttranslational modifications (PTMs) in particular have become increasingly recognized as regulators of specific target proteins. S-nitrosylation is a nonenzymatic and reversible PTM resulting in the addition of a NO group to a cysteine (Cys) thiol/sulfhydryl group, leading to the generation of S-nitrosothiols (SNOs). In spite of the large number of SNO-proteins thus far identified, the functional outcomes and mechanisms of the underlying specificity of S-nitrosylation in terms of target proteins and Cys residues within these proteins are not clear (Robinson, 2018).

Synaptic transmitter release is controlled by multiple signaling proteins and involves a cascade of signaling steps. This process requires the assembly of the soluble N-ethyl-maleimide-sensitive fusion protein Attachment Protein Receptor (SNARE) complex and associated proteins, the majority of which can be regulated to modulate synapse function. Regulatory mechanisms include phosphorylation of SNARE proteins as well as SNARE-binding proteins such as Complexin (Cpx), which have been reported at different synapses such as the Drosophila neuromuscular junction (NMJ) or in the rat CNS (Robinson, 2018).

Several contrasting effects on transmitter release are induced by NO-mediated PTMs. Other forms of protein modification to modulate cellular signaling include prenylation, an attachment of a farnesyl or geranyl-geranyl moiety to a Cys residue in proteins harboring a C-terminal CAAX prenylation motif. This process renders proteins attached to endomembrane/endoplasmic reticulum (ER) and Golgi structures until further processing, as shown for Rab GTPases. Farnesylation also regulates mouse cpx 3/4 and Drosophila Cpx function. The Cys within CAAX motifs can also undergo S-nitrosylation, which interferes with the farnesylation signaling; however, direct evidence in a physiological environment is lacking. Cpx function has been studied in many different systems and there is controversy regarding its fusion-clamp activity. Cpx supports Ca2+-triggered exocytosis but also exhibits a clamping function. Analysis of mouse cpx double-knockout neurons lacking cpx 1 and 2 found only a facilitating function for cpx on release, and different D. melanogaster and Caenorhabditis elegans cpx mutant lines exhibit altered phenotypes in clamping or priming/fusion function, illustrating the controversial actions of Cpx (Robinson, 2018).

This study investigated the effects of NO on synaptic transmission and found that NO reduces Ca2+-triggered release as well as the size of the functional vesicle pool, which was reversed by glutathione (GSH) signaling. At the same time, spontaneous release rates were negatively affected by NO. It was confirmed that cpx is S-nitrosylated and that NO changes the synaptic localization of cpx, as also seen following genetic and pharmacological inhibition of farnesylation. Thus, it is proposed that the function of cpx is regulated by S-nitrosylation of Cys within the CAAX motif to prevent farnesylation. This increases cpx-SNARE-protein interactions, thereby rendering cpx with a dominant clamping function, which suppresses both spontaneous and evoked release (Robinson, 2018).

NO regulates a multitude of physiological and pathological pathways in neuronal function via generation of cGMP, thiol-nitrosylation, and 3-nitrotyrosination in health and disease. This study shows by employing biochemical and genetic tools in Drosophila, mouse, and HEK cells that NO can S-nitrosylate Cpx and modulate (in a cGMP-independent manner) neurotransmitter release at the NMJ by interfering with its prenylation status, thereby affecting the localization and function of this fusion-clamp protein. These nitrergic effects are reversed by GSH application or overexpression of GSH-liberating and de-nitrosylating enzymes (GCLm/c, GSNOR). GSH is the major endogenous scavenger for the NO moiety by the formation of S-nitrosoglutathione (GSNO) and consequently reduces protein-SNO levels via trans- and de-nitrosylation. The suppression of NOS activity facilitates synaptic function and the data support the notion that endogenous or exogenous NO enhances S-nitrosylation, reduces cpx farnesylation, and diminishes release (Robinson, 2018).

Of the numerous synaptic molecules involved in release, Cpx in particular has been implicated in the regulation of both evoked and spontaneous release due to its fusion-clamp activity. Despite the seemingly simple structure of Cpx, its physiological function is highly controversial, as this small SNARE-complex binding protein can both facilitate but also diminish fast Ca2+-dependent and spontaneous release, depending on the system studied. In addition, there are different mammalian isoforms of Cpx (1-4), which differ in their C-terminal region, with only cpx 3/4 containing the CAAX prenylation motif. Farnesylation in general determines protein membrane association and protein-protein interactions, and some cpx isoforms, such as muscpx 3/4 and Dmcpx 7A, are regulated in this manner. However, muscpx 1/2 does not possess a CAAX motif, suggesting differential regulatory pathways to modulate cpx function. In Drosophila, there are alternative splice variants resulting from a single cpx gene, but the predominant isoform contains the CAAX motif (Dmcpx 7A), implicating the importance of this signaling molecule. The other splice isoform (Dmcpx 7B) lacks the CAAX motif and is expressed at about 1,000-fold lower levels at the larval stage [15], thus making Dmcpx 7A the dominant isoform to be regulated by farnesylation. However, the lack of Dmcpx 7B phosphorylation by PKA induces similar phenotypes as seen in the current experiments when assessed following an induction of activity-dependent potentiation of mEJC frequency, which also may involve cpx-synaptotagmin 1 interactions (Robinson, 2018).

Interestingly, both depletion and excessive levels of cpx suppress Ca2+-dependent and -independent exocytosis. Cpx may promote SNARE complex assembly and simultaneously block completion of fusion by retaining it in a highly fusogenic state. Ca2+-dependent fusion is promoted below a concentration of 100 nM of cpx, whereas above 200 nM, it exhibits a clamping function resulting in a bell-shaped response curve. Previous work suggests that synaptotagmin 1, once bound to Ca2+, relieves the cpx block and allows fusion. Another study reported that selective competition between cpx and synaptotagmin 1 for SNARE binding allows regulation of release. The data are in agreement with the latter findings, as reduced synaptotagmin 1-Cpx interactions was observed following the block of farnesylation, indicating fewer synaptotagmin molecules binding to the SNARE complex to displace Cpx. This limited replacement of Cpx by synaptotagmin has been implicated in biochemical studies showing that local excess of Cpx inhibits release, presumably by outcompeting synaptotagmin binding. Thus, synaptotagmin-SNARE binding is strongly dependent upon the local concentration of Cpx. Alternatively, and this possibility cannot be excluded, the modulation of Cpx may simply alter its binding to the SNARE complex without directly displacing synaptotagmin, but interpretation of the data from the current assays (PLA, co-localization) would not allow distinguishing between these possibilities (Robinson, 2018).

The data are compatible with the idea that Cpx binds to the SNARE complex, facilitates assembly, and then exerts its clamping function by preventing full fusion due to SNARE complex stabilization and subsequent increased energy barrier to allow fusion. The current model could provide an explanation of how Cpx can be regulated to signal downstream to modulate transmitter release. So far, there are no data available, apart from mutation studies, as to how Cpx function can be altered. This study provides data indicating a physiologically relevant mechanism to adjust Cpx function, possibly to the requirements of the neuron to adjust synaptic transmission. This likely occurs due to Cys S-nitrosylation and suppression of farnesylation, allowing greater amounts of hydrophilic Cpx, not bound to endomembranes, to be available for binding with the SNARE complex in an altered configuration. This cross signaling between nitrosylation/farnesylation has been proposed to act as a molecular switch to modulate Ras activity. The current data show that enhanced nitrergic activity and blocking farnesylation, either genetically (CpxΔX) or pharmacologically, alters the localization of Cpx at the Drosophila NMJ and that of GFP-CAAX in HEK cells. Furthermore, by using a nitroso-mimetic cpx mutant, this study found enhanced co-localization of Cpx with the AZ protein Brp, implying a localization-function relationship. This consequently increases the net-clamping function because of elevated local concentrations of Cpx. Dmcpx specifically exhibits a strong clamping function, as shown following overexpression in hippocampal neurons, which causes suppression of evoked and spontaneous release accompanied by a reduction of the release probability or reduced vesicle fusion efficiency in in vitro assays (Robinson, 2018).

Two independent studies eliminating the CAAX motif in Dmcpx (cpx⁵⁷² and cpx¹²⁵⁷) investigated localization-function interactions and showed disagreeing effects on both release and cpx localization. In particular, it has also been shown that the truncated Cpx (cpx⁵⁷², lacking the last 25 amino acids) does not co-localize with Brp. Interestingly, this mutant causes a strong decrease in C-terminal hydrophobicity and a modest physiological response (increased mini frequency, decreased evoked amplitudes equivalent to a loss of clamping and loss of fusion function) relative to the total knock-out (KO). In contrast, the cpx mutant with single amino acid deletion (cpx¹²⁷⁵) causes no effect on evoked but identical effects on the frequency of spontaneous release, suggesting a lack of clamping but no lack of fusion function. In addition, this mutant now co-localizes with the AZ at the NMJ. These two studies indicate that the different mutations cause contrasting electrophysiological and morphological phenotypes, indicating that it is due to the nature of the mutation (lack of the last 25 amino acids versus 1 amino acid), which highlights the importance of a functional C-terminus. More recent studies have shown that deletions of the final amino acids (6 or 12 residues) completely abolished the membrane binding of Cpx-1, impairing its inhibitory function and confirming the requirement of an intact C-terminus for inhibition of release. This study used an endogenous Cpx with intact hydrophobic C-terminus, allowing physiological membrane binding. This is essential for inhibitory function, as the C-terminus is required for selective binding to highly curved membranes, such as those of vesicles. Thus, as different approaches are used to alter farnesylation, and a single a single amino acid mutant cpx (Dmcpx 7AC140W) was created, leaving the C-terminus intact, these studies were performed under conditions of endogenous regulation of Cpx function and thus provide new functional data on Cpx signaling. Importantly, the data show that this regulation alters cpx function, and this is the first study to provide an explanation for the differential effects observed using cpx mutants or even Cpx protein fragments in mammals, worm, and fly in various cross-species rescue experiments (Robinson, 2018).

The data are in agreement with a model that non-farnesylated hydrophilic and soluble cytosolic Cpx binds to the vesicular membrane via its C-terminal interactions, thereby exerting its inhibitory effect. When proteins are farnesylated, they are likely tethered to endomembranes, other than vesicle membranes. It has to be distinguished between Cpx interaction with the vesicle membrane as a result of the hydrophobic C-terminus, allowing Cpx to become in close proximity to the AZ, and Cpx endomembrane binding following farnesylation, which prevents Cpx interactions with the AZ. However, in the current, SNO modification may enhance the binding to other proteins (e.g., SNAREs), thereby augmenting the effects. These additional interactions with unknown binding partners may affect proper Cpx function and explain some of the discrepancies seen in studies using other genetically altered Cpxs (Robinson, 2018).

In summary, this study provides new data to illustrate a potential mechanism to regulate cpx function in a physiological environment, and it was shown that NO acts as an endogenous signaling molecule that regulates synaptic membrane targeting of Cpx, a pathway that may reconcile some of the controversial findings regarding Cpx function. It is suggested that increased S-nitrosylation and consequent lack of farnesylation leads to enhanced cytosolic levels of a soluble hydrophilic Cpx and less endomembrane-bound fractions, because farnesylation-incompetent proteins remain in the cytosol. These novel observations advance understanding of similar nitrergic regulation of farnesylation that may be relevant for mammalian cpx-dependent synaptic transmission at the retina ribbon synapse and other brain regions. Finally, this work has broader implications for physiological or pathological regulation of the prenylation pathway not only during neurodegeneration and aging, when enhanced S-nitrosylation might contribute to abnormal farnesylation signaling, but also in other biological systems in which nitrergic activity and prenylation have important regulatory functions such as in cardio-vasculature or cancer signaling (Robinson, 2018).

Determinants of synapse diversity revealed by super-resolution quantal transmission and active zone imaging

Neural circuit function depends on the pattern of synaptic connections between neurons and the strength of those connections. Synaptic strength is determined by both postsynaptic sensitivity to neurotransmitter and the presynaptic probability of action potential evoked transmitter release (P(r)). Whereas morphology and neurotransmitter receptor number indicate postsynaptic sensitivity, presynaptic indicators and the mechanism that sets P(r) remain to be defined. To address this, this study developed QuaSOR, a super-resolution method for determining P(r) from quantal synaptic transmission imaging at hundreds of glutamatergic synapses at a time. P(r) was mapped onto super-resolution 3D molecular reconstructions of the presynaptic active zones (AZs) of the same synapses at the Drosophila larval neuromuscular junction (NMJ). P(r) varies greatly between synapses made by a single axon, the contribution of key AZ proteins to P(r) diversity was quantified; one of these, Complexin, was found to suppress spontaneous and evoked transmission differentially, thereby generating a spatial and quantitative mismatch between release modes. Transmission is thus regulated by the balance and nanoscale distribution of release-enhancing and suppressing presynaptic proteins to generate high signal-to-noise evoked transmission (Newman, 2022).

The operation of neural circuits depends on the synaptic connections between neurons. To understand how neural circuits process and store information, one needs to understand the molecular mechanisms that govern the synaptic transmission and distribute synaptic weights across large numbers of connections. While determinants of postsynaptic strength (e.g. dendritic spine size, postsynaptic scaffold size, number of postsynaptic receptors) are well characterized, the presynaptic determinants are not as clear. The relationship between synapse morphology and presynaptic action potential (AP)-evoked neurotransmitter release probability (Pr) is weak as is the dependence of Pr on specific elements of the transmitter release apparatus, the active zone (AZ) (Newman, 2022).

To understand how presynaptic machinery governs quantal transmission, one needs to measure Pr at identified synapses whose molecular constituents and organization can be analyzed directly. Three approaches have been used to measure transmission at multiple identified synapses. Postsynaptic quantal (i.e. single synaptic vesicle resolution) imaging with Ca2+ indicators detects flux through ionotropic receptors as a proxy for the excitatory postsynaptic response, biosensors detect released neurotransmitters, and presynaptic synaptopHluorins detect vesicle fusion3. However, the diffraction-limited nature of these imaging paradigms makes it difficult to assign transmission events to particular synapses when AZs are densely arrayed (Newman, 2022).

To overcome these limitations, a combination of super-resolution imaging modalities were developed to precisely relate quantal transmission to synaptic architecture at the glutamatergic model synapse of the Drosophila NMJ. The logic of stochastic single-molecule super-resolution localization microscopy was used to develop Quantal Synaptic Optical Reconstruction (“QuaSOR”), analogous to recent super-resolution imaging of transmission in neuronal culture with synaptopHluorin and iGluSnFR. QuaSOR resolved both action potential evoked and spontaneous quantal transmission events to individual synapses, even in regions where the synapses are crowded. QuaSOR allowed maping locations of quantal transmission, quantifing Pr using failure analysis, and measuring the frequency of spontaneous transmission (Fs) at hundreds of synapses simultaneously throughout the NMJ, under physiological conditions. QuaSOR analysis was followed by super-resolution molecular imaging of presynaptic AZ proteins, enabling spatial averaging of protein and transmission localizations that revealed new aspects of synaptic release mechanisms (Newman, 2022).

Pr was found to have a high power dependence on the quantity of the presynaptic voltage-gated Ca2+ channel Cacophony (Cac), consistent with the power dependence of quantal content on Ca2+. Pr also had a strong dependence on the scaffolding protein Bruchpilot (Brp), which organizes the AZ and anchors synaptic vesicles near the site of release. However, Cac and Brp together accounted for only a minor fraction of the variance in Pr, indicating that other important factors control and diversify AP-evoked release. A clue about one additional contributor came from an observation that evoked and spontaneous transmission modes are mismatched spatially and quantitatively. This led to an investigation of Complexin (Cpx), whose Drosophila homolog is a powerful inhibitor of spontaneous transmitter release and which contains subdomains that both facilitate and inhibit evoked release. As the Cpx/Brp ratio increased, Pr declined. When Cpx was knocked down, the mismatch between spontaneous and evoked transmission disappeared. Additionally, Pr was higher compared to control synapses with the same Brp content. It is concluded that the interplay between release-promoting Cac and Brp and release-suppressing Cpx sets presynaptic transmission strength, generates synapse-to-synapse diversity, and enhances quantal signal-to-noise by suppressing spontaneous release at the site of maximal evoked release. The results demonstrate how super-resolution structure/function imaging can reveal the mechanisms of regulation of synaptic function (Newman, 2022).

To understand the mechanisms that regulate synaptic strength and generate synapse diversity, this study set out to develop a new set of super-resolution imaging tools that together would allow relaying quantal transmission to presynaptic molecular composition in an intact model synapse. Imaging of Ca2+ influx through ionotropic glutamate receptors, with a postsynaptically targeted reporter, provided a quantal-resolution proxy for the excitatory postsynaptic current (EPSC), and QuaSOR analysis increased spatial resolution sufficiently to resolve synapses even in dense areas of the Drosophila NMJ. QuaSOR makes it possible to determine Pr directly by failure analysis under physiological Ca2+, i.e. at physiological Pr, avoiding reliance on estimation based on the ratio between evoked and spontaneous EPSC amplitudes (problematic in view of the finding that the sites of evoked and spontaneous transmission are segregated within the synapse), fits of amplitude distributions or analysis of variance. Post-hoc super-resolution presynaptic axon reconstructions enabled correlation of transmission to the molecular composition and nano-architecture of the presynaptic AZ for thousands of synapses (Newman, 2022).

Earlier work suggested that, despite a common history of activity and postsynaptic target, transmission varies greatly between the synapses of a single Ib motor axon. QuaSOR assignment of transmission events to identified synapses showed this to be the case across thousands of synapses and revealed that the heterogeneity is even greater than previously thought, with Pr ranging over at least 100-fold, from <0.005 to 0.6. Half of the synapses are very weak (Pr < 0.02) and AP-evoked transmission is dominated by a small fraction of higher Pr synapses during low levels of activity. This large pool of low-Pr synapses could operate as a reserve that would be recruited to sustain transmission during long, high-frequency AP bursts, such as occur during locomotion (Newman, 2022).

Previous studies at the NMJ demonstrated a positive relationship between Pr and both Cac and Brp. The ability to relate quantal transmission to multi-color 3D-STORM clarifies the nature of this relationship, by showing that Pr increases with the ~5th power of Cac, both in wildtype synapses and in synapses of a rab3 mutant whose AZs are enlarged, consistent with the power-dependence of release on Ca2+. Cac and Brp levels were also correlated with one another, consistent with Brp recruiting Cac to the AZ46. Although they are strong determinants, Cac and Brp only account for a fraction of the variance of Pr, indicating that other factors are at play. When AZs were expanded by the rab3 mutant to include more Brp and Cac, Pr increased to higher values, while maintaining the shallow Fs-Pr relation, the displacement of spontaneous transmission to locations outside the sites of evoked and the high power dependence of Pr on Cac. These observations are consistent with a mechanism that tunes Pr by regulating the size of the Brp scaffold and the number of Cac channels (Newman, 2022).

In considering other potential regulators of presynaptic strength, it is necessary to take into account an almost complete lack of correspondence between evoked and spontaneous transmission in WT animals. Most startlingly was a complete suppression of spontaneous transmission at the site of maximal evoked transmission. This segregation is only possible to detect with these analysis tools and agrees with evidence from the use-dependent block that spontaneous and evoked release activate distinct populations of glutamate receptors in hippocampal neurons and the Drosophila NMJ. The observations reveal that this separation arises not only from synapse specialization, as proposed in earlier studies but from physical segregation of evoked and spontaneous transmission within the synapse. This spatial mismatch is remarkably consistent with recent iGluSnFR mapping of spontaneous and evoked transmission events in cultured hippocampal synapses, suggesting that segregation of transmission modes within the synapse may be a general phenomenon (Newman, 2022).

It was considered that a factor that regulates both spontaneous and evoked release could be responsible for their spatial mismatch. Cpx has been shown to regulate both spontaneous and evoked release in complicated and contradictory ways. In vitro, Cpx interacts with the coiled-coiled domains of the SNARE complex to inhibit fusion and is displaced by Ca2+-bound synaptotagmin to trigger AP-evoked release. The mammalian isoforms of Cpx contain both fusogenic and inhibitory domains. Pan-neuronal removal of Cpx in Drosophila reduces postsynaptic response amplitude, suggesting that Cpx promotes evoked release. In contrast, expression of Drosophila Cpx in mammalian neurons suppresses evoked release. Cpx may also adjust the relationship between release and internal Ca2+ concentration through its role as an adapter that helps determine the composition of the release apparatus. Cpx is broadly distributed in the axon, enriched at the AZ and most densely concentrated in the Brp annular core. As the Cpx/Brp ratio within the AZ core rises, the Pr of Ib synapses decreases. This suggests that Cpx in the AZ core, which is positioned to interact with SNARE complexes, inhibits evoked release. Consistent with this relationship, Cpx knockdown increases the dependence of Pr on Brp so that at equivalent Brp levels Pr is higher when Cpx is knocked down and low Brp synapses with no detected transmission events become active (Newman, 2022).

Knockdown of Cpx increased Fs by ~11-fold at Ib synapses and ~66-fold at Is synapses, indicating that Cpx suppresses spontaneous transmission more strongly than evoked transmission. In light of this and of the findings that: (a) Cpx density is highest in the Brp annular core, where Cac is also located, and where AP-evoked vesicle fusion is therefore expected to take place, (b) spontaneous transmission is suppressed at the site of maximal evoked transmission, (c) spontaneous and evoked transmission are poorly correlated, and (d) knockdown of Cpx eliminates the spatial and quantitative mismatch between spontaneous and evoked transmission. It is proposed that Cpx within the AZ core partly suppresses evoked release and completely suppresses spontaneous release. This differential suppression can preserve vesicles that are docked near Ca2+ channels in a state that is ready for release when the AP arrives, yielding a higher signal-to-noise for AP-evoked transmission over background spontaneous transmission (Newman, 2022).

It is striking how knockdown of Cpx converts the relationship between Pr and Fs to near 1:1 and the spatial relationship of spontaneous and evoked transmission to coincident. This suggests that spontaneous and evoked release rates are, after all, governed by common factors. Brp levels were reduced in the CpxKD, possibly reflecting a compensatory mechanism that keeps the Pr of Ib synapses at near WT levels, as shown in recent focal extracellular recordings from Ib boutons. While Cpx in the Brp annular core suppresses Pr, this study found that higher bulk Cpx around the AZ is associated with higher Pr. This bulk Cpx likely reflects prenylated Cpx that is associated with endosomes and synaptic vesicles, which links vesicles to Brp69, and so may reflect higher vesicle content (Newman, 2022).

Together, QuaSOR and super-resolution molecular imaging of AZs reveals that the balance between the quantity and nanoscale localizations of Cac, Brp, and Cpx contribute to a wide diversity in release dynamics for synapses that otherwise share common pre-post pairing and activity history. This heterogeneity could serve to maintain a deep pool of reserve synapses upon which the system can draw under diverse physiological demands (Newman, 2022).

Ca2+ and cAMP open differentially dilating synaptic fusion pores

Neuronal dense-core vesicles (DCVs) contain neuropeptides and much larger proteins that affect synaptic growth and plasticity. Rather than using full collapse exocytosis that commonly mediates peptide hormone release by endocrine cells, DCVs at the Drosophila neuromuscular junction release their contents via fusion pores formed by kiss-and-run exocytosis. This study used fluorogen-activating protein (FAP) imaging to reveal the permeability range of synaptic DCV fusion pores and then shows that this constraint is circumvented by cAMP-induced extra fusions with dilating pores that result in DCV emptying. These Ca2+-independent full fusions require PKA-R2, a PKA phosphorylation site on Complexin and the acute presynaptic function of Rugose, the homolog of mammalian neurobeachin, a PKA-R2 anchor implicated in learning and autism. Therefore, localized Ca2+-independent cAMP signaling opens dilating fusion pores to release large cargoes that cannot pass through the narrower fusion pores that mediate spontaneous and activity-dependent neuropeptide release. These results imply that the fusion pore is a variable filter that differentially sets the composition of proteins released at the synapse by independent exocytosis triggers responsible for routine peptidergic transmission (Ca2+) and synaptic development (cAMP) (Bulgari, 2023).

Molecular and organizational diversity intersect to generate functional synaptic heterogeneity within and between excitatory neuronal subtypes

Synaptic heterogeneity is a hallmark of complex nervous systems that enables reliable and responsive communication in neural circuits. This study investigated the contributions of voltage-gated calcium channels (VGCCs) to synaptic heterogeneity at two closely related Drosophila glutamatergic motor neurons, one low- and one high-Pr. VGCC levels are highly predictive of heterogeneous release probability among individual active zones (AZs) of low- or high-Pr inputs, but not between neuronal subtypes. Underlying organizational differences in the AZ cytomatrix, VGCC composition, and a more compact arrangement of VGCCs alter the relationship between VGCC levels and Pr at AZs of low- vs. high-Pr inputs, explaining this apparent paradox. It was further found that the CAST/ELKS AZ scaffolding protein Bruchpilot differentially regulates VGCC levels at low- and high-Pr AZs following acute glutamate receptor inhibition, indicating that synapse-specific organization also impacts adaptive plasticity. These findings reveal intersecting levels of molecular and spatial diversity with context-specific effects on heterogeneity in synaptic strength and plasticity (Medeiros, 2023).

Spontaneous neurotransmission at evocable synapses predicts their responsiveness to action potentials

Synaptic transmission relies on presynaptic neurotransmitter (NT) release from synaptic vesicles (SVs) and on NT detection by postsynaptic receptors. Transmission exists in two principal modes: action-potential (AP) evoked and AP-independent, "spontaneous" transmission. AP-evoked neurotransmission is considered the primary mode of inter-neuronal communication, whereas spontaneous transmission is required for neuronal development, homeostasis, and plasticity. While some synapses appear dedicated to spontaneous transmission only, all AP-responsive synapses also engage spontaneously, but whether this encodes functional information regarding their excitability is unknown. This study reports on functional interdependence of both transmission modes at individual synaptic contacts of Drosophila larval neuromuscular junctions (NMJs) which were identified by the presynaptic scaffolding protein Bruchpilot (BRP) and whose activities were quantified using the genetically encoded Ca(2+) indicator GCaMP. Consistent with the role of BRP in organizing the AP-dependent release machinery (voltage-dependent Ca(2+) channels and SV fusion machinery), most active BRP-positive synapses (>85%) responded to APs. At these synapses, the level of spontaneous activity was a predictor for their responsiveness to AP-stimulation. AP-stimulation resulted in cross-depletion of spontaneous activity and both transmission modes were affected by the non-specific Ca(2+) channel blocker cadmium and engaged overlapping postsynaptic receptors. Thus, by using overlapping machinery, spontaneous transmission is a continuous, stimulus independent predictor for the AP-responsiveness of individual synapses (Grasskamp, 2023).

Interactive nanocluster compaction of the ELKS scaffold and Cacophony Ca(2+) channels drives sustained active zone potentiation

At presynaptic active zones (AZs), conserved scaffold protein architectures control synaptic vesicle (SV) release by defining the nanoscale distribution and density of voltage-gated Ca(2+) channels (VGCCs). While AZs can potentiate SV release in the minutes range, an understanding of how AZ scaffold components and VGCCs engage into potentiation is lacking. This study establish dynamic, intravital single-molecule imaging of endogenously tagged proteins at Drosophila AZs undergoing presynaptic homeostatic potentiation. During potentiation, the numbers of α1 VGCC subunit Cacophony (Cac) increased per AZ, while their mobility decreased and nanoscale distribution compacted. These dynamic Cac changes depended on the interaction between Cac channel's intracellular carboxyl terminus and the membrane-close amino-terminal region of the ELKS-family protein Bruchpilot, whose distribution compacted drastically. The Cac-ELKS/Bruchpilot interaction was also needed for sustained AZ potentiation. This single-molecule analysis illustrates how the AZ scaffold couples to VGCC nanoscale distribution and dynamics to establish a state of sustained potentiation (Ghelani, 2023).

Genome-wide analysis reveals novel regulators of synaptic maintenance in Drosophila

Maintaining synaptic communication is required to preserve nervous system function as an organism ages. While much work has been accomplished to understand synapse formation and development, Relatively little is understood regarding maintaining synaptic integrity throughout aging. To better understand the mechanisms responsible for maintaining synaptic structure and function, an unbiased forward genetic screen was performed to identify genes required for synapse maintenance of adult Drosophila neuromuscular junctions. Using flight behavior as a screening tool, this study evaluated flight ability in 198 lines from the Drosophila Genetic Reference Panel to identify single nucleotide polymorphisms (SNPs) that are associated with a progressive loss of flight ability with age. Among the many candidate genes identified from this screen, this study focussed on 10 genes with clear human homologs harboring SNPs that are most highly associated with synaptic maintenance. Functional validation of these genes using mutant alleles revealed a progressive loss of synaptic structural integrity. Tissue-specific knockdown of these genes using RNA interference (RNAi) uncovered important roles for these genes in either presynaptic motor neurons, postsynaptic muscles, or associated glial cells, highlighting the importance of each component of tripartite synapses. These results offer greater insight into the mechanisms responsible for maintaining structural and functional integrity of synapses with age (Sidisky, 2023).

Periplocoside P affects synaptic transmission at the neuromuscular junction and reduces synaptic excitability in Drosophila melanogaster by inhibiting V-ATPase

Periplocoside P (PSP) is a major component of Periploca sepium Bunge known for its potent insecticidal activity. V-Type adenosine triphosphatase (V-ATPase), which is widely distributed in the cytoplasmic membranes and organelles of eukaryotic cells, plays a crucial role in synaptic excitability conduction. Previous research has shown that PSP targets the apical membrane of goblet cells in the insect midgut. However, the effects of PSP on synaptic transmission at the neuromuscular junction are often overlooked.The bioassay revealed that Drosophila adults with different genetic backgrounds showed varying levels of susceptibility to PSP in the order: para^ts1  > para^ts1 ;DSC1^-/-  ≊ w¹¹¹⁸  > DSC1^-/-. Intracellular electrode recording demonstrated that PSP, similar to bafilomycin A1, had an impact on the amplitude of the excitatory junction potential (EJP) and accelerated excitability decay. Furthermore, the alteration in EJP amplitude is concentration-dependent. Another surprising discovery was that the knockout DSC1 channel showed insensitivity to PSP. These findings confirm that PSP can influence synaptic transmission at the neuromuscular junction of Drosophila larvae by targeting V-ATPase. These results provide a basis for investigating the mechanism of action of PSP and its potential application in designing novel insecticides (Luo, 2023).

Kinetochore proteins have a post-mitotic function in neurodevelopment

The kinetochore is a complex of proteins, broadly conserved from yeast to man, that resides at the centromere and is essential for chromosome segregation in dividing cells. There are no known functions of the core complex outside of the centromere. This study shows that the proteins of the kinetochore have an essential post-mitotic function in neurodevelopment. At the embryonic neuromuscular junction of Drosophila melanogaster, mutation or knockdown of many kinetochore components cause neurites to overgrow and prevent formation of normal synaptic boutons. Kinetochore proteins were detected in synapses and axons in Drosophila. In post-mitotic cultured hippocampal neurons, knockdown of mis12 increased the filopodia-like protrusions in this region. It is concluded that the proteins of the kinetochore are repurposed to sculpt developing synapses and dendrites and thereby contribute to the correct development of neuronal circuits in both invertebrates and mammals (Zhao, 2019).

Although there is no precedent for core kinetochore proteins functioning outside of chromosome mechanics, several lines of evidence argue that the observed Drosophila phenotypes in neurons are not secondary to chromosome segregation defects. First, errors of kinetochore assembly are lethal during cell division, arresting at the spindle assembly checkpoint, and this would have prevented motor neurons from forming; this study saw no loss of motor neurons in mis12 mutants. Second, were aneuploid or polyploid cells sometimes to escape that lethality, they would do so rarely, randomly, and with heterogeneity in the chromosomes lost. This study, however, detect synaptic phenotypes consistently at the NMJs of all mutant embryos examined. Third, the selective nature of the NMJ defect is difficult to reconcile with chromosomal aberrations: muscles are correctly patterned and have the correct complement; motor neurons are born and target consistently and appropriately to their muscles; and synaptic specializations form with appropriate components, despite the failure to form large boutons. Fourth, Ndc80 immunoreactivity is present at the embryonic NMJ and elsewhere in the nervous system; tagged kinetochore proteins, expressed under control of their normal promoters, were detected outside of nuclei in synaptic and axonal regions. In mammalian neurons, although only mis12 has been examined so far, the function of mis12 is clearly post-mitotic. It was detected by western blot in post-mitotic neurons, and the knockdown of mis12 in post-mitotic hippocampal neurons altered the morphology of hippocampal dendrites. A similar post-mitotic requirement for the proteins of the kinetochore has also been demonstrated in C. elegans, where the degradation of kinetochore proteins selectively in post-mitotic and differentiated sensory neurons disrupts their morphogenesis. Interestingly, there is precedent for the repurposing of other mitotic proteins, such as those of the anaphase-promoting complex, for post-mitotic functions in synaptogenesis (Zhao, 2019).

This study was fortunate to find the synaptogenic role of mis12 in screen: had the maternal contribution been less, the motor neurons might not have formed, and had the contribution persisted into late-stage embryos, it might have been sufficient fully to support synaptogenesis. The observation that at least eight kinetochore components, including representatives of each of the kinetochore subcomplexes, give rise to the same phenotype at the NMJ suggests that they are functioning in a complex very much similar to that at the centromere. This supposition is further strengthened by the observations that, in several cases examined, the proper localization of one component was altered in a genetic background mutant for another component (Zhao, 2019).

Although some alleles and RNAi lines did not have a phenotype, their role may have been obscured by the persistence of sufficient maternal contribution, as in the case of Cenp-C, or poor efficacy of the RNAi line. This is particularly true for Nuf2, which was detectable in larval axons although its RNAi line lacked a phenotype. It remains to be determined if the complete complement of kinetochore proteins or only mis12 function in the development of hippocampal dendrites (Zhao, 2019).

In light of the knockdown phenotype of ndc80, the microtubule-binding subunit, at the embryonic NMJ and its presence in sparse puncta at that synapse, one parsimonious hypothesis is that the kinetochore proteins interact with neuronal microtubules akin to their function at the centromere. Microtubule dynamics are crucial to the formation of both synapses and dendrites, and this is consistent with the observation of significant overextension of both synaptic branches and sensory dendrites in Drosophila and alterations in dendritic morphology in hippocampal neurons. At the embryonic NMJ and in larval nerves, where individual puncta of kinetochore proteins could be resolved, the puncta were sparse, with just one or two per bouton. The axonal puncta in larval nerves were not as bright as those in nearby glial nuclei. Whereas multiple kinetochores and microtubule + ends are present at each centromere, the dim axonal puncta may represent individual kinetochore-like complexes at individual + ends, and this would account for the difficulty of imaging the proteins outside of the densely synaptic neuropil of the ventral nerve cord. In axons, the + ends of axonal microtubules are oriented toward growth cones and synapses, and while microtubules are splayed in growth cones, they are replaced during synaptogenesis by more stable bundles. When microtubules are not appropriately stabilized, synapse formation is perturbed, giving rise to abnormal extensions of axons and improper bouton formation. The phenotypes now reported suggest that this developmental transition requires a kinetochore-like complex. Hippocampal dendrites contain microtubules of both polarities and the increased filopodia-like protrusions that appear upon the knockdown of mis12 may arise from misorganization or overgrowth of dendritic microtubules. Future studies will need to clarify how the kinetochore influences microtubule organization. Analysis of kinetochore mutations has thus uncovered a previously unknown mechanism that appears to co-opt the fundamental mitotic functions of the ancient kinetochore complex for non-mitotic functions in both invertebrate and vertebrate neurodevelopment. A deeper understanding of these synaptogenic functions should therefore illuminate a process central to the accurate wiring of the brain (Zhao, 2019).

Secreted C-type lectin regulation of neuromuscular junction synaptic vesicle dynamics modulates coordinated movement

The synaptic cleft manifests enriched glycosylation, with structured glycans coordinating signaling between presynaptic and postsynaptic cells. Glycosylated signaling ligands orchestrating communication are tightly regulated by secreted glycan-binding lectins. Using the Drosophila neuromuscular junction (NMJ) as a model glutamatergic synapse, this study identified a new Ca2+-binding (C-type) lectin, Lectin-galC1 (LGC1), which modulates presynaptic function and neurotransmission strength. LGC1 is enriched in motoneuron presynaptic boutons and secreted into the NMJ extracellular synaptomatrix. LGC1 limits locomotor peristalsis and coordinated movement speed, with a specific requirement for synaptic function, but not NMJ architecture. LGC1 controls neurotransmission strength by limiting presynaptic active zone (AZ) and postsynaptic glutamate receptor (GluR) aligned synapse number, reducing both spontaneous and stimulation-evoked synaptic vesicle (SV) release, and capping SV cycling rate. During high-frequency stimulation (HFS), mutants have faster synaptic depression and impaired recovery while replenishing depleted SV pools. Although LGC1 removal increases the number of glutamatergic synapses, it was found that LGC1-null mutants exhibit decreased SV density within presynaptic boutons, particularly SV pools at presynaptic active zones. Thus, LGC1 regulates NMJ neurotransmission to modulate coordinated movement (Bhimreddy, 2021).

Synapse assembly and subsequent neurotransmission strength are dependent on secreted bidirectional signaling pathways, which communicate between presynaptic and postsynaptic partners to coordinate synaptic development, function and plasticity. Both synaptic cleft and the surrounding extracellular perisynaptic space (the 'synaptomatrix') are characterized by heavy glycosylation, which has been shown over the last half-century with extensive lectin imaging. Synaptic signals are both mediated by, and regulated via, secreted glycoproteins (GPs), proteoglycans (PGs) and endogenous glycan-binding lectins. Disruption of these synaptic glycan-binding and modifying mechanisms is linked to numerous heritable neurological diseases, which include autism spectrum disorder (ASD) and intellectual disability (ID) states such as fragile X syndrome (FXS), movement and degeneration disorders such as hereditary spastic paraplegias (HSPs), and most broadly, the rapidly expanding congenital disorders of glycosylation. Secreted lectins play important roles in synaptic processes, including the secreted Ca2+-binding (C-type) lectins first identified by Kurt Drickamer. For example, the Drosophila Mind-the-Gap (MTG) patterns synaptomatrix glycans, regulates glutamate receptor (GluR) clustering, and modulates trans-synaptic signaling to control neurotransmission strength (Bhimreddy, 2021).

This study has identified a second C-type lectin, Lectin-galC1 (LGC1), in a whole-genome transgenic Drosophila RNAi screen for genes regulating synapse structure and function at the neuromuscular junction (NMJ). LGC1 is one of 33 C-type lectins encoded by the Drosophila genome, which are classified by their binding to extracellular β-galactoside glycans via a single characteristic Ca2+-dependent carbohydrate-binding domain (CBD). Similar to the many other lectins encoded by the Drosophila genome, LGC1 exists within a gene cluster with two other duplicated C-type lectin gene homologs. However, these two neighbors, with homologies of 59% and 24% respectively, show both differential expression and function. Previous LGC1 studies have revealed roles in development and cellular defense, showing enhanced expression in imaginal discs during pupation, binding to surface polysaccharide chains on gram-negative bacteria, and upregulation in response to injury due to an upstream nuclear factor κB-binding site, which is a common regulator of immune defense genes. Consistent with other glycosylation mechanisms, LGC1 has a pleiotropic role in neurotransmission (Scott and Panin, 2014). The current RNAi screen identification of LGC1 indicated critical roles in the neuromusculature, and specifically at NMJ synapses, with increased neurotransmission strength following LGC1 knockdown (Dani, 2012b). The goal of the current study was to pursue this lead by generating selective tools to test LGC1 requirements in coordinated movement and NMJ neurotransmission mechanisms (Bhimreddy, 2021).

This study generated a LGC1 loss-of-function null allele using CRISPR/Cas9 genome editing by creating a frame-shift near the beginning of the coding sequence. To characterize neuromuscular roles, this null was combined with a characterized LGC1 deficiency. A LGC1::GFP fusion and specific LGC1 antibody were generated to reveal LGC1 protein at the NMJ, in presynaptic boutons and secreted into the extracellular synaptomatrix. Consistent with this NMJ localization, it was found that loss of LGC1 caused faster locomotory muscle peristalsis and much more rapid coordinated movement. Previously, LGC1 RNAi was used to show increased NMJ excitatory junction current (EJC) transmission, but no change in synaptic architecture (Dani, 2012b). The current study confirmed these preliminary findings in null mutants employing confocal imaging and two-electrode voltage-clamp (TEVC) electrophysiology. Although the loss of LGC1 did not alter NMJ architecture, it did increase synapse number. Consistently, LGC1 mutants showed higher spontaneous synaptic vesicle (SV) fusion rates as well as elevated motor nerve stimulation-evoked EJC amplitudes. To test mechanisms, FM1-43 dye imaging was used to reveal faster SV cycling dynamics with smaller SV pools in the mutants. With high-frequency stimulation (HFS), faster depression and slower recovery were found. Both confocal and electron microscopy revealed smaller membrane-associated SV pools at mutant active zones, consistent with faster activity-dependent SV cycling. Taken together, these results indicate that LGC1 plays a role in presynaptic SV release dynamics to cap NMJ neurotransmission strength and thus limit motor function (Bhimreddy, 2021).

Anterograde and retrograde signals secreted into the NMJ synaptomatrix by both motor neuron and muscle cells orchestrate intercellular communication at the synapse. The signaling ligands, their receptors and other molecules in the synaptomatrix environment are all very highly glycosylated, and mutation of many genes affecting glycosylation has strong effects on both coordinated movement and NMJ synaptic function. Glycan-binding lectins interact with the NMJ synaptomatrix at many levels, to effectively modulate NMJ neurotransmission strength. In the extracellular space, lectins serve to regulate the distribution of glycans (as scaffolds, co-receptors and signaling platforms), thereby directing secreted and transmembrane protein. For example, the secreted C-type lectin Mind-the-Gap (MTG) was extensively characterized at the Drosophila NMJ. MTG regulates glycan distribution, secreted trans-synaptic signaling ligands, synaptic position-specific (PS) integrins and the postsynaptic glutamate receptor (GluR) field, as well as the downstream internal Drosophila Pix–Pak–Dock signaling cascade and the critical DLG synaptic scaffold. These secreted MTG C-type lectin roles controlling postsynaptic function suggest that similar C-type lectin roles may govern presynaptic function (Bhimreddy, 2021).

In a previous systematic transgenic RNAi screen for NMJ modulator genes, a new secreted C-type lectin was uncovered that strongly controls neurotransmission strength (Dani, 2012b). Lectin-galC1 (LGC1) is an ideal candidate for modulation of synaptic function due to its characterized Ca2+-binding (C-type) and glycan-binding properties as a secreted protein. To create a LGC1-specific null mutation, CRISPR/Cas9 homology-directed DNA repair was used to create a targeted frame-shift mutation. This LGC1 null mutant was paired with a deletion removing the C-type lectin gene cluster containing LGC1 to characterize a new specific LGC1 antibody and to show that LGC1 is locally secreted at the NMJ. Then the effects were studied of LGC1 loss on coordinated movement, which intimately depends on both NMJ synaptic architecture and function. Consistent with the initial discovery of increased NMJ neurotransmission following LGC1 RNAi knockdown (Dani, 2012b), and earlier studies demonstrating LGC1 Ca2+-dependent binding functions, this study reports that LGC1-null mutants exhibit accelerated movement. LGC1 loss increases both the rate of locomotion and the speed to complete complex coordinated behaviors when challenged. These findings suggest LGC1 acting locally at the NMJ is important for modulating locomotion and, particularly, complex coordinated motor function (Bhimreddy, 2021).

LGC1 is secreted at the NMJ, as demonstrated using non-permeabilized labeling, and concentrated within the extracellular synaptomatrix surrounding synaptic boutons; a space characterized by striking glycan accumulation. LGC1-null mutants have absolutely no alterations in NMJ structure. This is surprising, since NMJ overelaboration is a common feature of glycosylation mutations. For example, mutations in the key phosphomannomutase type 2 (PMM2) glycosylation enzyme strongly alter synaptomatrix pauci-mannose glycans to cause significant NMJ architectural expansion, including more synaptic branching and supernumerary boutons. Similarly, loss of UDP-sugar precursors, which are required for synaptic glycosylation also results in obvious NMJ structural overelaboration, with both increased branching and synaptic bouton number (Jumbo-Lucioni, 2016). However, synaptic structure and function are independently regulated, with separable glycan roles. Consistently, LGC1-null mutants exhibit an elevated number of synapses within otherwise normal NMJ boutons, thereby exhibiting greater synaptic density. LGC1 nulls have more presynaptic BRP-positive active zones (AZs) and more apposed postsynaptic GluR fields, suggesting that a higher level of synaptic connectivity contributes to elevated neurotransmission strength (Bhimreddy, 2021).

LGC1 limits NMJ function by reducing SV fusion and evoked transmission. An elevated LGC1 EJC amplitude reflects an increase in number of released vesicles (quantal content) in a more mobile readily releasable pool (RRP). Since spontaneous mEJC amplitude does not change in LGC1 nulls, an increase in postsynaptic GluR number or function is ruled out as a contributing factor. In addition to increased synapse density, it was hypothesized that there would be higher SV fusion probability from altered SV cycling in LGC1 mutants. FM1-43 dye imaging shows that LGC1 loss results in a smaller cycling SV pool with higher cycling rate. A smaller SV pool could negate the effects of increased synapse number and SV fusion probability. However, most SVs do not participate detectably in transmission cycling, and smaller SV pools can show high transmission due to increased synaptic density and SV cycling rates. Moreover, altered SV endocytosis–exocytosis cycling efficacy can cause altered synaptic depression/recovery with high-frequency stimulation (HFS). Stimulation frequency can change SV cycling dynamics, with higher frequencies triggering differential SV release and recovery mechanisms. Consequently, NMJ function (depression and recovery) was assayed during varying levels of frequency stimulation, to stress neurotransmission machinery, and thus gain insight into the LGC1 mutant SV cycling mechanisms (Bhimreddy, 2021).

In LGC1 nulls, HFS causes more rapid fatigue and slower recovery. Consistent with this, smaller SV cycling pools with higher turnover rates can increase basal transmission strength while impairing transmission maintenance during HFS demand. Thus, the smaller cycling SV pool in LGC1 mutants can maintain elevated basal transmission while exhibiting impaired replenishment of SV pools and maintenance of transmission during HFS. Other glutamatergic synapse classes maintain a small cycling RRP for high probability SV release during basal stimulation conditions, yet are unable to maintain transmission under conditions of greater demand. Likewise, LGC1 nulls exhibit reduced membrane-associated SV pools at both the light microscope and electron microscope levels, including lower SV density at AZs. Other Drosophila mutants with increased NMJ neurotransmission can similarly show presynaptic SV depletion in TEM ultrastructural studies. At the AZ, SVs near the presynaptic membrane make up the RRP and linked cycling pools, but internal SV pools can be held in 'reserve' or serve other functions apart from neurotransmitter release. Overall, these findings are consistent with LGC1 mutants exhibiting a smaller and faster SV cycling pool, with altered SV cycle dynamics during and following HFS stress (Bhimreddy, 2021).

Similar to what is seen with LGC1 mutants, the loss of a number of C-type lectin domain (CTLD) proteins causes synaptic defects. For example, Mincle-deficient mice suppress TNF induction, which lowers glutamate release from central synapses, resulting in a direct modulation of neurotransmission strength. Although LGC1 appears to have the opposite effect, the Drosophila TNF homolog (Eiger) downregulates excitatory amino acid transporters 1 and 2 (EAAT1/2), and the EAAT1 mutation impairs locomotory peristaltic movement. This show'that LGC1 limits locomotory peristaltic movement, suggesting a possible relationship between LGC1, Eiger and EAAT1 may be a useful avenue to explore in future studies. Another CTLD protein is Caenorhabditis elegans CLEC-38, which has been shown to regulate presynaptic organization and mediate changes in the integral SV protein Synaptobrevin. As the vesicle SNARE (V-SNARE), Synaptobrevin drives SV fusion with the presynaptic membrane AZ to directly mediate neurotransmitter release. Since LGC1 mutants display neurotransmission defects comparable to CLEC-38 mutants, such as similar increased SV fusion and impaired SV cycling, the exploration of downstream LGC1 interactions and Synaptobrevin regulation in the presynaptic terminal could offer more insight into the mechanisms by which LGC1 produces neurotransmission changes (Bhimreddy, 2021).

In conclusion, this study provides insight into the roles of a novel C-type lectin at a glutamatergic neuromuscular synapse. Beyond the previously characterized roles in development and immunity, this study found LGC1 secreted at the NMJ synapse modulates presynaptic function. Along with acting to limit synapse number and neurotransmission strength, LGC1 also regulates SV cycling to increase SV availability during basal levels of use, but decrease SV availability during high levels of use. In resting basal conditions, LGC1 caps NMJ neurotransmission strength to limit coordinated motor function. With intense stimulation, LGC1 loss causes more fatigue and decreases the recovery rate, indicating impaired activity-dependent SV cycling. This study revealed functional defects with electrophysiology, and visualized SVs with both FM1-43 dye and TEM electron microscopy imaging. In future studies, it is planned to dissect LGC1 mechanisms by focusing on SV pools and trafficking, as well as testing the possible involvement in signaling via Eiger, EAAT1 and downstream SV Synaptobrevin changes. This work indicates that LGC1-dependent signaling fine-tunes the multiple presynaptic pools that exchange SVs differentially based on activity levels and release demand to control SV release probability. Beyond this LGC1 mechanism, this new model will allow further exploration of SV cycling-dependent NMJ function in neuromuscular disease states (Bhimreddy, 2021).

The ankyrin repeat domain controls presynaptic localization of Drosophila Ankyrin2 and is essential for synaptic stability

The structural integrity of synaptic connections critically depends on the interaction between synaptic cell adhesion molecules (CAMs) and the underlying actin and microtubule cytoskeleton. This interaction is mediated by giant Ankyrins, that act as specialized adaptors to establish and maintain axonal and synaptic compartments. In Drosophila, two giant isoforms of Ankyrin2 (Ank2) control synapse stability and organization at the larval neuromuscular junction (NMJ). Both Ank2-L and Ank2-XL are highly abundant in motoneuron axons and within the presynaptic terminal, where they control synaptic CAMs distribution and organization of microtubules. This study addresses the role of the conserved N-terminal ankyrin repeat domain (ARD) for subcellular localization and function of these giant Ankyrins in vivo. A P[acman] based rescue approach was used to generate deletions of ARD subdomains, that contain putative binding sites of interacting transmembrane proteins. Specific subdomains control synaptic but not axonal localization of Ank2-L. These domains contain binding sites to L1-family member CAMs, and these regions are necessary for the organization of synaptic CAMs and for the control of synaptic stability. In contrast, presynaptic Ank2-XL localization only partially depends on the ARD but strictly requires the presynaptic presence of Ank2-L demonstrating a critical co-dependence of the two isoforms at the NMJ. Ank2-XL dependent control of microtubule organization correlates with presynaptic abundance of the protein and is thus only partially affected by ARD deletions. Together, these data provides novel insights into the synaptic targeting of giant Ankyrins with relevance for the control of synaptic plasticity and maintenance (Weber, 2019).

Giant ankyrins serve as central organizing adaptor molecules in both invertebrate and vertebrate neurons. This study provides first insights into the control of synaptic localization and function of giant Ankyrins by the N-terminal ankyrin repeat domain in vivo. By selectively deleting subdomains of the ARD of ank2 this study unravel critical requirements of specific regions of the ARD for the synaptic localization of the neuronal specific giant isoforms Ank2-L and Ank2-XL. The data demonstrate that the N-terminal domain controls not only synaptic targeting of individual isoforms but also contributes to synaptic localization of the alternative isoform. The functional requirements of Ank2-L and Ank2-XL for synaptic stability and microtubule organization clearly correlate with ARD-dependent regulation of protein abundance at the presynaptic terminal, with individual subdomains providing unique functional features (Weber, 2019).

The N-terminal ARD mediates membrane-association of Ankyrins and is essential for the subcellular localization and organization of transmembrane binding partners. Prior work largely focused on the organizational roles of giant Ankyrins at the AIS and nodes of Ranvier in vertebrate neurons. In vivo analysis of ARD deletions now revealed a critical importance of this domain for the localization of Ank2-L at the presynaptic terminal of motoneurons. The synaptic localization of Ank2-L depends on AnkD2-4 (repeats 7-24) while the first domain (repeats 1-6) only had a minor impact on protein abundance. Interestingly, axonal targeting of Ank2-L was only partially affected by these deletions. These results indicate that separate and distinctive mechanisms exist in vivo that enable selective localization of giant Ankyrins in axons and within the presynaptic motoneuron terminal. As similar localization defects were observed already at earlier stages of development it argues that AnkD2-4 are essential for initial synaptic localization. Previous work demonstrated that a fusion protein comprising the specific C-terminal tail domain of Ank2-L efficiently localizes to both axonal and synaptic areas. The demonstration that synaptic localization of full length Ank2-L requires an intact ARD indicates that subcellular distribution is precisely regulated and not achieved by passive distribution within the neuron. Of particular interest is the observation that the first six ankyrin repeats are dispensable for synaptic localization of Ank2-L in the ank2^ΔL mutant background. The recent characterization of the structure of the human ARD demonstrated distinct and independent binding sites for voltage-gated sodium channels (Nav1.2) and for L1-family CAMs (Neurofascin) in vitro and in cultured neurons. Interestingly, in cultured hippocampal neurons the L1-family CAM binding sites within AnkD2 (ankyrin repeats 8-9 and 11-13) are essential for clustering of the 270 kDa AnkG isoform at the AIS. In contrast, AnkG lacking the Nav1.2 binding site within AnkD1 (ankyrin repeats 2-6) efficiently localized to the AIS but failed to cluster Nav channels at the AIS. Thus, similar to the situation at the vertebrate AIS, the synaptic localization of Drosophila Ank2-L largely depends on interactions with L1-family CAMs or alternative transmembrane proteins that occupy binding sites within the central and C-terminal part of the ARD. This localized L1-CAM-Ankyrin complex can then serve as an assembly platform for additional molecules like voltage-gated sodium channels that acquired Ankyrin-binding capacities during chordate evolution to determine the physiological properties of specific subcellular neuronal compartments. While the AnkD1 of Drosophila ank2 had the least impact on synaptic stability phenotypes it will be interesting to identify putative binding proteins that may provide specific functional properties as absence of this domain resulted in decreased survival and locomotion of adult flies (Weber, 2019).

In contrast to Ank2-L, the deletion of individual parts of the ARD had smaller effects on synaptic targeting of Ank2-XL in the ank2^ΔXL mutant background. In this study, only the deletion of AnkD3 (repeats 13-18) resulted in a significant reduction of axonal and synaptic localization. However, the analysis revealed a striking dependence of Ank2-XL on the presence of the intact ARD of Ank2-L. Impairments of Ank2-L localization, including the minor effects caused by deletion of AnkD1, resulted in a severe reduction of synaptic Ank2-XL levels. This shows that Ank2-XL localizes via Ank2-L to the presynaptic terminal consistent with prior observations. Interestingly, deletions of parts of the ARD of Ank2-XL also significantly reduced synaptic Ank2-L levels indicating a critical co-dependence of Ank2-L and Ank2-XL at the NMJ. This interaction may depend on direct interactions between the two Ankyrin isoforms, potentially mediated within the ARD. However, it is equally possible that incorporation of mutated Ankyrins with altered binding properties resulted in a disruption of the larger Ankyrin-Spectrin scaffold that in turn leads to reduced targeting of the other isoform. Indeed, in vertebrate axons it has been demonstrated that different affinities of specific giant Ankyrins control the isoform-specific incorporation into selective axonal compartments like the nodes of Ranvier (Weber, 2019).

At a functional level, a strong correlation was observed between synaptic Ank2-L level and the control of synaptic stability. At all muscle groups analyzed it was observed that deletion of AnkD1 domain had the mildest impact on synaptic stability consistent with the least impairments of synaptic Ank2-L localization. Importantly, the analysis of synaptic stability at muscle 4 also revealed that AnkD3 is most critical for synaptic maintenance. Despite an almost identical reduction in Ank2-L level compared to AnkD2 and 4 the rescue construct lacking the third domain was unable to restore any synaptic stability of ank2^ΔL mutant animals. Interestingly, this assay also highlighted the specificity of the individual ankyrin repeats within the ARD. The exchange of second domain sequences with analogous sequences of the first domain failed to restore both Ank2-L localization and the synaptic stability phenotype of ank2ΔL mutants. The failure to support synaptic stability is at least in part due to a failure to organize and stabilize synaptic cell adhesion molecules. This analysis demonstrated that AnkD2 and 3 are critical for normal clustering of the NCAM homolog Fas II and of the L1 CAM homolog Nrg. This is consistent with prior observations that Ank2-L supports synaptic organization of both Fas II and Nrg and the co-dependence of Ank2-L and these CAMs at the synapse. This function is evolutionary conserved as mutations in the second and third binding site of vertebrate AnkG failed to restore clustering of the L1 CAM family members Neurofascin or Nr-CAM at the AIS of hippocampal neurons (Weber, 2019).

In contrast to the clear requirements of the ARD for presynaptic targeting of Ank2-L the same mutations only partially affected localization of Ank2-XL. Consistently, only mild disruptions were observed of the presynaptic microtubule cytoskeleton and of the synaptic bouton organization compared to the ank2^ΔXL mutation. The effects were most severe when deleting AnkD2 despite the fact that Ank2-XL protein levels were more compromised in AnkD3 mutants. As the large C-terminal repeat region of Ank2-XL is essential for the interaction with microtubules these results indicate that inappropriate ARD-dependent complex assembly may influence the functional properties of Ank2-XL at the NMJ (Weber, 2019).

Together, this in vivo analysis of the ARD of Drosophila giant Ankyrins uncovered a structural basis for presynaptic localization and provided a genetic basis for the identification of regulatory mechanisms controlling structural synaptic plasticity via the selective Ankyrin complex assembly (Weber, 2019).

The ubiquitin ligase Ariadne-1 regulates neurotransmitter release via ubiquitination of NSF

Ariadne-1 (Ari-1) is an E3 ubiquitin-ligase essential for neuronal development, but whose neuronal substrates are yet to be identified. To search for putative Ari-1 substrates, this study used an in vivo ubiquitin biotinylation strategy coupled to quantitative proteomics of Drosophila heads. Sixteen candidates were identified that met the established criteria: a significant change of at least two-fold increase on ubiquitination, with at least two unique peptides identified. Amongst those candidates, Comatose (Comt), the homologue of the N-ethylmaleimide sensitive factor (NSF), which is involved in neurotransmitter release, was identified. Using a pulldown approach that relies on the overexpression and stringent isolation of a GFP-fused construct, Comt/NSF was validated to be an ubiquitination substrate of Ari-1 in fly neurons, resulting in the preferential monoubiquitination of Comt/NSF. The possible functional relevance of this modification was tested using Ari-1 loss of function mutants that displayed a lower rate of spontaneous neurotransmitter release due to failures at the pre-synaptic side. By contrast, evoked release in Ari-1 mutants was enhanced compared to controls in a Ca(2+) dependent manner without modifications in the number of active zones, indicating that the probability of release per synapse is increased in these mutants. This phenotype distinction between spontaneous versus evoked release suggests that NSF activity may discriminate between these two types of vesicle fusion. These results thus provide a mechanism to regulate NSF activity in the synapse through Ari-1-dependent ubiquitination (Ramirez, 2021).

Neurotransmitter release is mediated by a set of protein-protein interactions that include the N-ethylmaleimide sensitive factor (NSF), soluble NSF attachment proteins (SNAPs), and SNAP receptors (SNAREs). These proteins assemble into a tripartite complex in order to elicit synaptic vesicle fusion, which is formed by one synaptic vesicle membrane SNARE protein (v-SNARE), Synaptobrevin, and two plasma membrane SNARE proteins (t-SNAREs), Syntaxin and the 25-kDa synaptosome-associated protein. Following vesicle fusion, the tripartite SNARE complex disassembles by the activities of NSF and SNAPs. Free t-SNAREs from the plasma membrane can then participate in new priming reactions, while the v-SNAREs can be incorporated into recycled synaptic vesicles. These interactions, also routinely used for intracellular vesicle trafficking in all cell types, are conserved across species, including Drosophila (Ramirez, 2021).

Deviations on the rate of neurotransmitter release are at the origin of multiple neural diseases, including Parkinson's disease. Under physiological conditions, the Leucine-rich repeat Serine/Threonine-protein kinase 2 (LRRK2) phosphorylates NSF to enhance its ATPase activity, which facilitates the disassembly of the SNARE complex. However, the most common Parkinson's disease mutation in LRRK2 causes an excess of kinase activity that interferes with the vesicle recycling. Similarly, α-Synuclein, another Parkinson's disease protein, alters neurotransmitter release by preventing the v-SNARE vesicle-associated membrane protein (VAMP)-2, also known as Synaptobrevin-2, from joining the SNARE complex cycle. Correct neural functioning, therefore, requires delicate regulation in vesicle trafficking. This regulation can be achieved by posttranslational modifications, such as ubiquitination. In fact, ubiquitination of certain proteins can affect their activity or life span. At the presynaptic side, for example, increased neurotransmitter release correlates with decreased protein ubiquitination. Similarly, acute pharmacological proteasomal inhibition causes rapid strengthening of neurotransmission (Ramirez, 2021).

Ariadne 1 is an E3 ubiquitin-ligase, first identified in Drosophila, from a conserved gene family defined by two C3HC4 Ring fingers separated by a C6HC in-Between-Rings domain (the RBR motif). Ari-1 had been described to be essential for neuronal development, and its mutants reported to exhibit reduced eye rhabdomere surface and endoplasmic reticulum, as well as aberrant axonal pathfinding. However, despite its importance, no neuronal substrates have been reported so far. Only three Ari-1 substrates have been postulated, either in cultured cells or in vitro, while three Parkin substrates were reported to interact with Ari-1 in COS-1 cells. For this reason, with the aim to identify neuronal Ari-1 substrates, advantage was taken of two methodologies. The first one, the ^bioUb strategy, allows the identification of hundreds of ubiquitinated proteins from neuronal tissues. The system relies on the overexpression of a tagged ubiquitin that bears a 16 amino acid long biotinylatable peptide, which can be biotinylated by the Escherichia coli biotin holoenzyme synthetase enzyme (BirA) in neurons in vivo. Remarkably, this approach can be efficiently applied to identify neuronal E3 ligase substrates. In contrast, the second methodology favors the isolation of GFP-tagged proteins under denaturing conditions to further characterize their ubiquitination pattern under the presence or absence of an E3 ligase (Ramirez, 2021).

This study has combined the ^bioUb strategy with the overexpression of Ari-1 and identified 16 putative neuronal substrates of Ari-1. Among those, focus was placed on Comatose (Comt), the fly NSF orthologue, due to its relevance in normal and pathological function at the synapse. By the isolation of GFP-tagged Comt from Drosophila photoreceptor neurons overexpressing Ari-1, this study confirmed Comt/NSF as an Ari-1 ubiquitin substrate and showed that it is mostly monoubiquitinated. Furthermore, Ari-1 loss-of-function mutants displayed lower rate of spontaneous neurotransmitter release, but enhanced evoked release, due to failures at the presynaptic side. These defects in the mutants are compatible with a deregulation of Com/NSF activity. Altogether, these data show that Ari-1 regulates neurotransmitter release by controlling Comt/NSF activity through ubiquitination (Ramirez, 2021).

This study identified sixteen novel putative substrates of Ari-1 in Drosophila photoreceptor neurons in vivo by means of an unbiased proteomic approach. Remarkably, despite Ari-1 being recently shown to regulate the positioning of the cell nucleus in muscles via a direct interaction with Parkin (Tan, 2018), as well as to interact with some Parkin substrates, there is no overlap between the substrates identified for Ari-1 and those previously identified for Parkin in flies. Taken together, the available data suggest that the 16 targets identified in this study are specifically regulated by Ari-1 in Drosophila photoreceptor neurons and that this E3 ligase has a wide functional repertoire (Ramirez, 2021).

This study focused on Comt, an ATPase required for the maintenance of the neurotransmitter release. Ubiquitination of proteins involved in vesicle trafficking and neurotransmitter release had been previously reported. Similarly, the importance of the ubiquitination machinery for the proper neuronal function has also been demonstrated. The alterations produced on synaptic transmission by ubiquitination are typically attributed to an acute control of synaptic protein turnover. However, many of these presynaptic proteins have been reported to be mainly mono- or di-ubiquitinated, a type of ubiquitin modification that is not usually associated with protein degradation. In line with this, the results showed that Comt/NSF is preferentially monoubiquitinated by Ari-1/ARIH1, suggesting that Ari-1/ARIH1 could be regulating Comt/NSF activity, rather than its life span or expression levels (Ramirez, 2021).

Ari-1 mutations result in abnormal synaptic function at the larval stage, a result consistent with a regulatory function of NSF. All mutant alleles examined exhibit a reduced frequency of spontaneous synaptic release. In addition, ari-1² mutants exhibit a large calcium-dependent evoked release. Analysis of the mechanism for enhanced evoked release in ari-1² suggests that the primary defect consists in an increased probability of vesicle fusion in response to calcium entry in the presynaptic side. First, by comparing the amplitude and time course of spontaneously occurring postsynaptic events in mutant and control animals, the possibility of a postsynaptic modification was ruled out. Since no significant differences were found, it is concluded that the receptor field size and kinetic properties of postsynaptic receptors are normal in the mutant (Ramirez, 2021).

The ari-1 functional defects could result from alterations of synaptic transmission during development; therefore, the number of synaptic contacts was quantified, assuming that most release sites occur within varicosities. No significant difference was observed between mutant and control. Although this study does not include electron microscopy quantification of synaptic vesicles, th confocal microscopy and electrophysiology data point toward an upregulation of release from single synapses (Ramirez, 2021).

The failure analysis from single varicosities represents direct evidence that, at relatively low calcium concentrations, mutant terminals release more quanta than controls in response to an action potential. Whether increased quantal release could be explained on the basis of more release sites being concentrated on mutant terminals was examined. The focal recordings using saturating calcium concentrations argue against this possibility. When mutant terminals are exposed to high calcium, in order to increase the likelihood that all active zones within the bouton will release a quantum, EJP amplitudes in the mutant are indistinguishable from that of controls. These data suggest that the number of release sites in mutant and control terminals is similar and favor the hypothesis that, at physiological calcium concentrations, the probability of vesicle fusion upon calcium entry is increased in the mutant (Ramirez, 2021).

It was found that ari-1 mutants have opposite effects on spontaneous and evoked release. Classically, the two modes of vesicular release have been considered to represent a single exocytotic process that functions at different rates depending on the Ca2+ concentration. However, recent work challenges this idea and supports the alternative model where spontaneous and evoked response might come from different vesicles pools. Several experimental evidences indicate that both forms of release may represent separate fusion pathways. Employing a state-of-the-art optical imaging in larval NMJ, it has been shown that evoked and spontaneous release can be segregated across active zones. Thus, three types of active zones could be defined: those that only release vesicles in response to a rise of intracellular calcium (evoked release), a second population that only participates in spontaneous release, and a third small proportion (around 4%) that participates in both evoked and spontaneous release. This result advocates for a different molecular and spatial segregation of both modes of release (Ramirez, 2021).

Differential content or activity of regulatory SNARE binding proteins could discriminate between spontaneous and evoked release. It has been shown that the presence of the Vamp-7 isoform could participate in this differential release. Vamp-7 preferentially labels vesicles unresponsive to stimulation, and it colocalizes only partially with the endogenous synaptic vesicle glycoprotein Sv2 and the vesicular glutamate transporter Vglut1, suggesting that this vesicle pool does not support evoked transmitter release. Recently, it was shown that the double knockout mouse for Synaptobrevin genes, syb1 and syb2, results in a total block of evoked release, while spontaneous release was increased in both frequency and quantal size without changes in the number of docked vesicles at the active zone, confirming the idea that evoked and spontaneous releases are differentially regulated. Interestingly, Vamp-7 was found by MS to be less ubiquitinated when Ari-1 is overexpressed, suggesting that Ari-1 mutants could be favoring evoked release through NSF and reducing spontaneous release through Vamp7. Thus, Ari-1 could be acting as a repressor and activator of evoked and spontaneous release, respectively. All together, these results evidence a new layer of complexity over the actual fine-tuning of synaptic transmission. A physiological regulatory mechanism for both types of release has been recently demonstrated for inhibitory synapses at the trapezoid body, an important brain area in auditory integration. In this nucleus, activation of metabotropic glutamate receptor mGluR1 differentially modulates both spontaneous and evoked release in both GABAergic and Glycynergic synapses (Ramirez, 2021).

Early functional studies of NSF employing the fly thermo-sensitive mutant allele comt^ts17, have reported a reversible reduction of synaptic transmission. Consistent with a role of NSF on SNARE dissociation, this inhibition parallels an increase in the number of synaptic vesicles at the presynaptic terminal. At this point, it can only be speculated how specifically Ari-1/ARIH1 regulates Comt/NSF activity within the presynaptic terminal. Opposite to the role of NSF mutant comt^ts17, which impairs SNARE complex disassembly, a change that enhances NSF functionality due to the lack of its ubiquitination would favor the dissociation of the so-called trans-SNARE complex. Further, this would build up the number of SNARE complexes assembled per vesicle, thus increasing the efficiency of fusion machinery in a Ca2+-dependent manner. Consistent with this interpretation, it has been shown that fast release of a synaptic vesicle requires at least three SNARE complexes, whereas slower release may occur with fewer complexes (Ramirez, 2021).

Interestingly, some of the additional putative substrates identified are also related to synapse physiology and neurotransmitter release. PPO1 is an enzyme with L-DOPA monooxygenase activity, hence, may be involved in the metabolism of dopamine neurotransmitter. Similarly, GstO3 is involved in glutathione metabolism, another type of neurotransmitter. Vha44 and Vha68-1 are components of the vacuolar proton-pump ATPase, whose mutations have been reported to impair neurotransmitter release. Vha44 has also been described as an enhancer of Tau-induced neurotoxicity, and CG15117, orthologue of human GUSB, has been associated with neuropathological abnormalities. The long recovery time from paralysis observed in comt⁶ ari2/comt6 females could result from the role of Ari-1 in the ubiquitination of these additional substrates, in addition to the role of Comt in tissues other than the nervous system (Ramirez, 2021).

The data reported in this study may be relevant in the context of Parkinson's disease. It should be noted that most Parkinson's-related genes encode proteins involved in vesicle recycling and neurotransmitter release at the synapse. Thus, the kinase LRRK2 phosphorylates NSF to enhance its ATPase activity upon the SNARE complex and facilitate its disassembly. Pathological mutations in this protein, such as G2019S, cause an excess of kinase activity that interferes with vesicle recycling. Deregulated synaptic aggregates of α-Synuclein may target VAMP-2 hampering the formation of the SNARE complex. Parkin is a structural relative of Ari-1, based on their common Cysteine rich C₃HC₄ motif, which is also at the origin of some forms of Parkinson's disease. All these genes and their corresponding mechanisms of activity sustain the scenario in which several types of Parkinson's disease seem to result from a defective activity of the synapse. In this context, the role of Ari-1/ARIH1 emerges as a mechanism to regulate a key component of the SNARE complex, Comt/NSF. Conceivably, Ari-1/ARIH1 may become a suitable target for either diagnosis or pharmacological treatment of Parkinson's and related diseases (Ramirez, 2021). Regulation of excitation-contraction coupling at the Drosophila neuromuscular junction

Larval muscle contraction force increases with stimulation frequency and duration, revealing substantial plasticity between 5 and 40 Hz. Fictive contraction recordings demonstrate endogenous motoneuron burst frequencies consistent with the neuromuscular system operating within the range of greatest plasticity. Genetic and pharmacological manipulation of critical components of pre- and post-synaptic Ca(2+) regulation significantly impact the strength and time-course of muscle contractions. A screen for modulators of the excitation-contraction machinery identified a FMRFa peptide, TPAEDFMRFa, and its associated signaling pathway that dramatically increases muscle performance. Drosophila serves as an excellent model for dissecting components of the excitation-contraction coupling machinery. This study developed and used a force transducer system to characterize excitation-contraction coupling at Drosophila larval neuromuscular junctions (NMJs), examining how specific neuronal and muscle manipulations disrupt muscle contractility. Muscle contraction force increased with motoneuron stimulation frequency and duration, showing considerable plasticity between 5-40 Hz and saturating above 50 Hz. Endogenous recordings of fictive contractions revealed average motoneuron burst frequencies of 20-30 Hz, consistent with the system operating within this plastic range of contractility. Temperature was also a key factor in muscle contractility, as force was enhanced at lower temperatures and dramatically reduced with increasing temperatures. Pharmacological and genetic manipulations of critical components of Ca(2+) regulation in both pre- and post-synaptic compartments impacted the strength and time-course of muscle contractions. A screen for modulators of muscle contractility led to identification and characterization of the molecular and cellular pathway by which the FMRFa peptide, TPAEDFMRFa, increases muscle performance. These findings indicate Drosophila NMJs provide a robust system to correlate synaptic dysfunction, regulation, and modulation, to alterations in excitation-contraction coupling (Ormerod, 2021).

The Putative Drosophila TMEM184B Ortholog Tmep Ensures Proper Locomotion by Restraining Ectopic Firing at the Neuromuscular Junction

TMEM184B is a putative seven-pass membrane protein that promotes axon degeneration after injury. TMEM184B mutation causes aberrant neuromuscular architecture and sensory and motor behavioral defects in mice. The mechanism through which TMEM184B causes neuromuscular defects is unknown. This study employed Drosophila melanogaster to investigate the function of the closely related gene, Tmep (CG12004), at the neuromuscular junction. Tmep was shown to be required for full adult viability and efficient larval locomotion. Tmep mutant larvae have a reduced body contraction rate compared to controls, with stronger deficits in females. In recordings from body wall muscles, Tmep mutants show substantial hyperexcitability, with many postsynaptic potentials fired in response to a single stimulation, consistent with a role for Tmep in restraining synaptic excitability. Additional branches and satellite boutons at Tmep mutant neuromuscular junctions are consistent with an activity-dependent synaptic overgrowth. Tmep is expressed in endosomes and synaptic vesicles within motor neurons, suggesting a possible role in synaptic membrane trafficking. Using RNAi knockdown, this study show that Tmep is required in motor neurons for proper larval locomotion and excitability, and that its reduction increases levels of presynaptic calcium. Locomotor defects can be rescued by presynaptic knockdown of endoplasmic reticulum calcium channels or by reducing evoked release probability, further suggesting that excess synaptic activity drives behavioral deficiencies. This work establishes a critical function for Tmep in the regulation of synaptic transmission and locomotor behavior (Cho, 2022).

Activity-induced synaptic structural modifications by Akt

The activity-dependent regulation of synaptic structures plays a key role in synaptic development and plasticity; however, the signaling mechanisms involved remain largely unknown. The serine/threonine protein kinase Akt, a downstream effector of phosphoinositide 3-kinase (PI3K), plays a pivotal role in a wide range of physiological functions. This study focused on the importance of Akt in rapid synaptic structural changes after stimulation at the Drosophila neuromuscular junction, a well-studied model synapse. Compared with wild-type larvae, akt mutants showed significantly reduced muscle size and an increased number of boutons per area, suggesting that Akt is required for proper pre- and postsynaptic growth. In addition, the level of cysteine string protein (CSP) was significantly increased, and its distribution was different in akt mutants. After high K(+) single stimulation, the CSP level of akt mutant NMJs increased dramatically compared with that of wild-type NMJs. Interestingly, ghost boutons without postsynaptic specialization were found in akt mutant NMJs, and the number of these boutons was significantly increased by patterned stimulation. In contrast, the postsynaptic change in the subsynaptic reticulum (SSR) in the akt mutant occurred independent of stimulation. These results suggest that Akt functions in both pre- and postsynaptic growth and differentiation, and in particular, presynaptic action occurs in an activity-dependent manner (Kim, 2022).

Intellectual disability-associated disruption of O-GlcNAc cycling impairs habituation learning in Drosophila

O-GlcNAcylation is a reversible co-/post-translational modification involved in a multitude of cellular processes. The addition and removal of the O-GlcNAc modification is controlled by two conserved enzymes, O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (OGA). Mutations in OGT have recently been discovered to cause a novel Congenital Disorder of Glycosylation (OGT-CDG) that is characterized by intellectual disability. The mechanisms by which OGT-CDG mutations affect cognition remain unclear. This study manipulated O-GlcNAc transferase and O-GlcNAc hydrolase activity in Drosophila and demonstrated an important role of O-GlcNAcylation in habituation learning and synaptic development at the larval neuromuscular junction. Introduction of patient-specific missense mutations into Drosophila O-GlcNAc transferase using CRISPR/Cas9 gene editing leads to deficits in locomotor function and habituation learning. The habituation deficit can be corrected by blocking O-GlcNAc hydrolysis, indicating that OGT-CDG mutations affect cognition-relevant habituation via reduced protein O-GlcNAcylation. This study establishes a critical role for O-GlcNAc cycling and disrupted O-GlcNAc transferase activity in cognitive dysfunction, and suggests that blocking O-GlcNAc hydrolysis is a potential strategy to treat OGT-CDG (Fenckova, 2022).

Crucial Roles of Ubiquitin Carboxy-Terminal Hydrolase L1 in Motor Neuronal Health by Drosophila Model

Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) plays an important role in the ubiquitin-proteasome system and is distributed mostly in the brain. Previous studies have shown that mutated forms or reduction of UCH-L1 are related to neurodegenerative disorders, but the mechanisms of pathogenesis are still not well understood. To study its roles in motor neuronal health, the Drosophila model in which dUCH, a homolog of human UCH-L1, was specifically knocked down in motor neurons. RThe reduction of Drosophila ubiquitin carboxyl-terminal hydrolase (dUCH) in motor neurons induced excessive reactive oxygen species production and multiple aging-like phenotypes, including locomotive defects, muscle degeneration, enhanced apoptosis, and shortened longevity. In addition, there is a decrease in the density of the synaptic active zone and glutamate receptor area at the neuromuscular junction. Interestingly, all these defects were rescued by vitamin C treatment, suggesting a close association with oxidative stress. Strikingly, the knockdown of dUCH at motor neurons exhibited aberrant morphology and function of mitochondria, such as mitochondrial DNA (mtDNA) depletion, an increase in mitochondrial size, and overexpression of antioxidant enzymes. This research indicates a new, possible pathogenesis of dUCH deficiency in the ventral nerve cord and peripheral nervous systems, which starts with abnormal mitochondria, leading to oxidative stress and accumulation aging-like defects in general. Taken together, by using the Drosophila model, these findings strongly emphasize how the UCH-L1 shortage affects motor neurons and further demonstrate the crucial roles of UCH-L1 in neuronal health (Huynh, 2022).

Stochastic Properties of Spontaneous Synaptic Transmission at Individual Active Zones

Using postsynaptically tethered calcium sensor GCaMP, this study investigated spontaneous synaptic transmission at individual active zones (AZs) at the Drosophila (both sexes) neuromuscular junction. Optical monitoring of GCaMP events coupled with focal electrical recordings of synaptic currents revealed "hot spots" of spontaneous transmission, which corresponded to transient states of elevated activity at selected AZs. The elevated spontaneous activity had two temporal components, one at a timescale of minutes and the other at a subsecond timescale. To investigate the mechanisms of elevated activity, focused was first placed on the protein Complexin, which binds the SNARE protein complex and serves to clamp spontaneous fusion. Overexpression of Drosophila complexin largely abolished the high-activity states of AZs, while complexin deletion drastically promoted it. How presynaptic Ca(2+) transients affect the states of elevated activity was investiged at individual AZs. Ca(2+) influx was blocked or promoted pharmacologically, and also Ca(2+) release was promoted from internal stores. These experiments coupled with computations revealed that Ca(2+) transients can trigger bursts of spontaneous events from individual AZs or AZ clusters at a subsecond timescale. Together, these results demonstrated that spontaneous transmission is highly heterogeneous, with transient hot spots being regulated by the SNARE machinery and Ca(2+) (Astacio, 2022).

Abnormal larval neuromuscular junction morphology and physiology in Drosophila prickle isoform mutants with known axonal transport defects and adult seizure behavior. J Neurogenet: 1-9

Previous studies have demonstrated the striking mutational effects of the Drosophila planar cell polarity gene prickle (pk) on larval motor axon microtubule-mediated vesicular transport and on adult epileptic behavior associated with neuronal circuit hyperexcitability. Mutant alleles of the prickle-prickle (pk(pk)) and prickle-spiny-legs (pk(sple)) isoforms (hereafter referred to as pk and sple alleles, respectively) exhibit differential phenotypes. While both pk and sple affect larval motor axon transport, only sple confers motor circuit and behavior hyperexcitability. However, mutations in the two isoforms apparently counteract to ameliorate adult motor circuit and behavioral hyperexcitability in heteroallelic pk/pk/pk(sple) flies. The consequences of altered axonal transport was further investigated in the development and function of the larval neuromuscular junction (NMJ). Robust dominant phenotypes were uncovered in both pk and sple alleles, including synaptic terminal overgrowth (as revealed by anti-HRP and -Dlg immunostaining) and poor vesicle release synchronicity (as indicated by synaptic bouton focal recording). However, recessive alteration of synaptic transmission was observed only in pk/pk larvae, i.e. increased excitatory junctional potential (EJP) amplitude in pk/pk but not in pk/+ or sple/sple. Interestingly, for motor terminal excitability sustained by presynaptic Ca(2+) channels, both pk and sple exerted strong effects to produce prolonged depolarization. Notably, only sple acted dominantly whereas pk/+ appeared normal, but was able to suppress the sple phenotypes, i.e. pk/sple appeared normal. Thesd observations contrast the differential roles of the pk and sple isoforms and highlight their distinct, variable phenotypic expression in the various structural and functional aspects of the larval NMJ (Ueda, 2022).

Loss of NF1 in Drosophila larvae causes tactile hypersensitivity and impaired synaptic transmission at the neuromuscular junction

Autism Spectrum Disorder (ASD) is a neurodevelopmental condition in which the mechanisms underlying its core symptomatology are largely unknown. Studying animal models of monogenic syndromes associated with ASD, such as neurofibromatosis type 1 (NF1), can offer insights into its aetiology. This study shows that loss of function of the Drosophila NF1 ortholog results in tactile hypersensitivity following brief mechanical stimulation in the larva (mixed sexes), paralleling the sensory abnormalities observed in individuals with ASD. Mutant larvae also exhibit synaptic transmission deficits at the glutamatergic neuromuscular junction (NMJ), with increased spontaneous but reduced evoked release. While the latter is homeostatically compensated for by a postsynaptic increase in input resistance, the former is consistent with neuronal hyperexcitability. Indeed, diminished expression of NF1 specifically within central cholinergic neurons induces both excessive neuronal firing and tactile hypersensitivity, suggesting the two may be linked. Furthermore, both impaired synaptic transmission and behavioural deficits are fully rescued via knockdown of Ras proteins. These findings validate NF1(-/-) Drosophila as a tractable model of ASD with the potential to elucidate important pathophysiological mechanisms (Dyson, 2022).

Nanoscaled RIM clustering at presynaptic active zones revealed by endogenous tagging

Chemical synaptic transmission involves neurotransmitter release from presynaptic active zones (AZs). The AZ protein Rab-3-interacting molecule (RIM) is important for normal Ca(2+)-triggered release. However, its precise localization within AZs of the glutamatergic neuromuscular junctions of Drosophila melanogaster remains elusive. CRISPR/Cas9-assisted genome engineering of the rim locus was used to incorporate small epitope tags for targeted super-resolution imaging. A V5-tag, derived from simian virus 5, and an HA-tag, derived from human influenza virus, were N-terminally fused to the RIM Zinc finger. Whereas both variants are expressed in co-localization with the core AZ scaffold Bruchpilot, electrophysiological characterization reveals that AP-evoked synaptic release is disturbed in rim^V5-Znf but not in rim^HA-Znf. In addition, rim^HA-Znf synapses show intact presynaptic homeostatic potentiation. Combining super-resolution localization microscopy and hierarchical clustering, ∼10 RIM(HA-Znf) subclusters were identified with ∼13 nm diameter per AZ that are compacted and increased in numbers in presynaptic homeostatic potentiation (Mrestani, 2023).

Neuroligin 2 governs synaptic morphology and function through RACK1-cofilin signaling in Drosophila

Neuroligins are transmembrane cell adhesion proteins well-known for their genetic links to autism spectrum disorders. Neuroligins can function by regulating the actin cytoskeleton, however the factors and mechanisms involved are still largely unknown. Here, using the Drosophila neuromuscular junction as a model, it was revealed that F-Actin assembly at the Drosophila NMJ is controlled through Cofilin signaling mediated by an interaction between DNlg2 and RACK1, factors not previously known to work together. The deletion of DNlg2 displays disrupted RACK1-Cofilin signaling pathway with diminished actin cytoskeleton proteo-stasis at the terminal of the NMJ, aberrant NMJ structure, reduced synaptic transmission, and abnormal locomotion at the third-instar larval stage. Overexpression of wildtype and activated Cofilin in muscles are sufficient to rescue the morphological and physiological defects in dnlg2 mutants, while inactivated Cofilin is not. Since the DNlg2 paralog DNlg1 is known to regulate F-actin assembly mainly via a specific interaction with WAVE complex, the present work suggests that the orchestration of F-actin by Neuroligins is a diverse and complex process critical for neural connectivity (Sun, 2023).

Gbb glutathionylation promotes its proteasome-mediated degradation to inhibit synapse growth

Glutathionylation is a posttranslational modification involved in various molecular and cellular processes. However, it remains unknown whether and how glutathionylation regulates nervous system development. To identify critical regulators of synapse growth and development, this study performed an RNAi screen and found that postsynaptic knockdown of glutathione transferase omega 1 (GstO1) caused significantly more synaptic boutons at the Drosophila neuromuscular junctions. Genetic and biochemical analysis revealed an increased level of glass boat bottom (Gbb), the Drosophila homolog of mammalian bone morphogenetic protein (BMP), in GstO1 mutants. Further experiments showed that GstO1 is a critical regulator of Gbb glutathionylation at cysteines 354 and 420, which promoted its degradation via the proteasome pathway. Moreover, the E3 ligase Ctrip negatively regulated the Gbb protein level by preferentially binding to glutathionylated Gbb. These results unveil a novel regulatory mechanism in which glutathionylation of Gbb facilitates its ubiquitin-mediated degradation. Taken together, these findings shed new light on the crosstalk between glutathionylation and ubiquitination of Gbb in synapse development (Hossain, 2023).

Loss of the extracellular matrix protein Perlecan disrupts axonal and synaptic stability during Drosophila development

Heparan sulfate proteoglycans (HSPGs) form essential components of the extracellular matrix (ECM) and basement membrane (BM) and have both structural and signaling roles. Perlecan is a secreted ECM-localized HSPG that contributes to tissue integrity and cell-cell communication. Although a core component of the ECM, the role of Perlecan in neuronal structure and function is less understood. This study identified a role for Drosophila Perlecan in the maintenance of larval motoneuron axonal and synaptic stability. Loss of Perlecan causes alterations in the axonal cytoskeleton, followed by axonal breakage and synaptic retraction of neuromuscular junctions. These phenotypes are not prevented by blocking Wallerian degeneration and are independent of Perlecan's role in Wingless signaling. Expression of Perlecan solely in motoneurons cannot rescue synaptic retraction phenotypes. Similarly, removing Perlecan specifically from neurons, glia, or muscle does not cause synaptic retraction, indicating the protein is secreted from multiple cell types and functions non-cell autonomously. Within the peripheral nervous system, Perlecan predominantly localizes to the neural lamella, a specialized ECM surrounding nerve bundles. Indeed, the neural lamella is disrupted in the absence of Perlecan, with axons occasionally exiting their usual boundary in the nerve bundle. In addition, entire nerve bundles degenerate in a temporally coordinated manner across individual hemi-segments throughout larval development. These observations indicate disruption of neural lamella ECM function triggers axonal destabilization and synaptic retraction of motoneurons, revealing a role for Perlecan in axonal and synaptic integrity during nervous system development (Guss, 2023).

Role of DSC1 in Drosophila melanogaster synaptic activities in response to haedoxan A

Drosophila sodium channel 1 (DSC1) encodes a voltage-gated divalent cation channel that mediates neuronal excitability in insects. Previous research revealed that DSC1 knockout Drosophila melanogaster conferred different susceptibility to insecticides, which indicated the vital regulation of stress. Haedoxan A (HA) is a lignan compound isolated from Phryma leptostachya, and HA was found to have excellent insecticidal activity and is worthy of further study as a botanical insecticide. This study performed bioassay and electrophysiological experiments to test the biological and neural changes in the larval Drosophila with/without DSC1 knockout in response to HA. Bioassay results showed that knockout of DSC1 reduced the sensitivity to HA in both w¹¹¹⁸ (a common wild-type strain in the laboratory) and parats1 (a pyrethroid-resistant strain) larvae. Except for parats1 DSC1^-/-, electrophysiology results implicated that HA delayed the decay rate and increased the frequency of para(ts1) , and DSC1^-/- strains. Moreover, the neuromuscular synapse excitatory activities of para^ts1DSC1^-/- larvae were more sensitive to HA than DSC1^-/- larvae, which further confirmed the functional contribution of DSC1 to neuronal excitability. Collectively, these results indicated that the DSC1 channel not only regulated the insecticidal activity of HA, but also maintained the stability of neural circuits through functional interaction with voltage-gated sodium channels. Therefore, this study provides useful information for elucidating the regulatory mechanism of DSC1 in the neural system of insects involving the action of HA derived from P. leptostachya (Qie, 2023).

Activity-dependent global downscaling of evoked neurotransmitter release across glutamatergic inputs in Drosophila

Within mammalian brain circuits, activity-dependent synaptic adaptations such as synaptic scaling stabilise neuronal activity in the face of perturbations. It remains unclear whether activity-dependent uniform scaling also operates within peripheral circuits. This study tested for such scaling in a Drosophila larval neuromuscular circuit, where the muscle receives synaptic inputs from different motoneurons. Motoneuron-specific genetic manipulations were employed to increase the activity of only one motoneuron and recordings of postsynaptic currents from inputs formed by the different motoneurons. An adaptation was detected which caused uniform downscaling of evoked neurotransmitter release across all inputs through decreases in release probabilities. This 'presynaptic downscaling' maintained the relative differences in neurotransmitter release across all inputs around a homeostatic set point, caused a compensatory decrease in synaptic drive to the muscle affording robust and stable muscle activity, and was induced within hours. Presynaptic downscaling was associated with an activity-dependent increase in Drosophila vesicular glutamate transporter (DVGLUT) expression. Activity-dependent uniform scaling can therefore manifest also on the presynaptic side to produce robust and stable circuit outputs. Within brain circuits, uniform downscaling on the postsynaptic side is implicated in sleep- and memory-related processes. These results suggest that evaluation of such processes might be broadened to include uniform downscaling on the presynaptic side (Karunanithi, 2020).

FOXO Regulates Neuromuscular Junction Homeostasis During Drosophila Aging

The transcription factor Foxo is a known regulator of lifespan extension and tissue homeostasis. It has been linked to the maintenance of neuronal processes across many species and has been shown to promote youthful characteristics by regulating cytoskeletal flexibility and synaptic plasticity at the neuromuscular junction (NMJ). However, the role of foxo in aging neuromuscular junction function has yet to be determined. This study profiled adult Drosophila foxo- null mutant abdominal ventral longitudinal muscles and found that young mutants exhibited morphological profiles similar to those of aged wild-type flies, such as larger bouton areas and shorter terminal branches. Changes to the axonal cytoskeleton and an accumulation of late endosomes were also observed in foxo null mutants and motor neuron-specific foxo knockdown flies, similar to those of aged wild-types. Motor neuron-specific overexpression of foxo can delay age-dependent changes to NMJ morphology, suggesting foxo is responsible for maintaining NMJ integrity during aging. Through genetic screening, several downstream factors mediated through foxo-regulated NMJ homeostasis were identified, including genes involved in the MAPK pathway. Interestingly, the phosphorylation of p38 was increased in the motor neuron-specific foxo knockdown flies, suggesting foxo acts as a suppressor of p38/MAPK activation. This work reveals that foxo is a key regulator for NMJ homeostasis, and it may maintain NMJ integrity by repressing MAPK signaling (Birnbaum, 2020).

Active zone compaction correlates with presynaptic homeostatic potentiation

Neurotransmitter release is stabilized by homeostatic plasticity. Presynaptic homeostatic potentiation (PHP) operates on timescales ranging from minute- to life-long adaptations and likely involves reorganization of presynaptic active zones (AZs). At Drosophila melanogaster neuromuscular junctions, earlier work ascribed AZ enlargement by incorporating more Bruchpilot (Brp) scaffold protein a role in PHP. This study use localization microscopy (direct stochastic optical reconstruction microscopy [dSTORM]) and hierarchical density-based spatial clustering of applications with noise (HDBSCAN) to study AZ plasticity during PHP at the synaptic mesoscale. This study found compaction of individual AZs in acute philanthotoxin-induced and chronic genetically induced PHP but unchanged copy numbers of AZ proteins. Compaction even occurs at the level of Brp subclusters, which move toward AZ centers, and in Rab3 interacting molecule (RIM)-binding protein (RBP) subclusters. Furthermore, correlative confocal and dSTORM imaging reveals how AZ compaction in PHP translates into apparent increases in AZ area and Brp protein content, as implied earlier (Mretiani, 2021).

A comparison of three different methods of eliciting rapid activity-dependent synaptic plasticity at the Drosophila NMJ

The Drosophila NMJ is a system of choice for investigating the mechanisms underlying the structural and functional modifications evoked during activity-dependent synaptic plasticity. Because fly genetics allows considerable versatility, many strategies can be employed to elicit this activity. This study compared three different stimulation methods for eliciting activity-dependent changes in structure and function at the Drosophila NMJ. It was found that the method using patterned stimulations driven by a K+-rich solution creates robust structural modifications but reduces muscle viability, as assessed by resting potential and membrane resistance. It is argued that, using this method, electrophysiological studies that consider the frequency of events, rather than their amplitude, are the only reliable studies. These results were contrasted with the expression of CsChrimson channels and red-light stimulation at the NMJ, as well as with the expression of TRPA channels and temperature stimulation. With both these methods reliable modifications were observed of synaptic structures, along with consistent changes in electrophysiological properties. Indeed, a rapid appearance was observed of immature boutons that lack postsynaptic differentiation, and a potentiation of spontaneous neurotransmission frequency. Surprisingly, a patterned application of temperature changes alone is sufficient to provoke both structural and functional plasticity. In this context, temperature-dependent TRPA channel activation induces additional structural plasticity but no further increase in the frequency of spontaneous neurotransmission, suggesting an uncoupling of these mechanisms (Maldonado-Diaz, 2021).

Imidacloprid Impairs Glutamatergic Synaptic Plasticity and Desensitizes Mechanosensitive, Nociceptive, and Photogenic Response of Drosophila melanogaster by Mediating Oxidative Stress, Which Could Be Rescued by Osthole

Imidacloprid (IMD) is a widely used neonicotinoid-targeting insect nicotine acetylcholine receptors (nAChRs). However, off-target effects raise environmental concerns, including the IMD's impairment of the memory of honeybees and rodents. Although the down-regulation of inotropic glutamate receptor (iGluR) was proposed as the cause, whether IMD directly manipulates the activation or inhibition of iGluR is unknown. Using electrophysiological recording on fruit fly neuromuscular junction (NMJ), this study found that IMD of 0.125 and 12.5 mg/L did not activate glutamate receptors nor inhibit the glutamate-triggered depolarization of the glutamatergic synapse. However, chronic IMD treatment attenuated short-term facilitation (STF) of NMJ by more than 20%. Moreover, by behavioral assays, it was found that IMD desensitized the fruit flies' response to mechanosensitive, nociceptive, and photogenic stimuli. Finally, the treatment of the antioxidant osthole rescued the chronic IMD-induced phenotypes. It was clarified that IMD is neither agonist nor antagonist of glutamate receptors, but chronic treatment with environmental-relevant concentrations impairs glutamatergic plasticity of the NMJ of fruit flies and interferes with the sensory response by mediating oxidative stress (Liu, 2022).

Endogenous tagging of Unc-13 reveals nanoscale reorganization at active zones during presynaptic homeostatic potentiation

Neurotransmitter release at presynaptic active zones (AZs) requires concerted protein interactions within a dense 3D nano-hemisphere. Among the complex protein meshwork the (M)unc-13 family member Unc-13 of Drosophila melanogaster is essential for docking of synaptic vesicles and transmitter release. This study employed minos-mediated integration cassette (MiMIC)-based gene editing using GFSTF (EGFP-FlAsH-StrepII-TEV-3xFlag) to endogenously tag all annotated Drosophila Unc-13 isoforms enabling visualization of endogenous Unc-13 expression within the central and peripheral nervous system. Electrophysiological characterization using two-electrode voltage clamp (TEVC) reveals that evoked and spontaneous synaptic transmission remain unaffected in unc-13 (GFSTF) 3rd instar larvae and acute presynaptic homeostatic potentiation (PHP) can be induced at control levels. Furthermore, multi-color structured-illumination shows precise co-localization of Unc-13(GFSTF), Bruchpilot, and GluRIIA-receptor subunits within the synaptic mesoscale. Localization microscopy in combination with HDBSCAN algorithms detect Unc-13(GFSTF) subclusters that move toward the AZ center during PHP with unaltered Unc-13(GFSTF) protein levels (Dannhauser, 2023).

Synaptic development is controlled in the periactive zones of Drosophila synapses

A cell-adhesion molecule Fasciclin 2 (FAS2), which is required for synaptic growth and Still life (SIF), an activator of RAC, were found to localize in the surrounding region of the active zone, defining the periactive zone in Drosophila neuromuscular synapses. BetaPS integrin and discs large (DLG), both involved in synaptic development, also decorated the zone. However, Shibire (SHI), the Drosophila dynamin that regulates endocytosis, was found in the distinct region. Mutant analyses showed that sif genetically interacted with Fas2 in synaptic growth and that the proper localization of SIF required FAS2, suggesting that they are components in related signaling pathways that locally function in the periactive zones. It is proposed that neurotransmission and synaptic growth are primarily regulated in segregated subcellular spaces, active zones and periactive zones, respectively (Sone, 2000).

This study characterized the periactive zone in three respects. Initially, the periactive zone was defined on the basis of the distribution patterns of SIF and FAS2, both clearly surrounding the electron-dense region marked with anti-DPAK antibody. The concentric staining patterns that represent pairs of the periactive zone and electron-dense region were often separated from the adjacent pairs and therefore suggested that these two regions constitute a structural unit in the NMJ. Secondly, the biochemical properties of the molecules found in the periactive zone characterize this zone. Cell adhesion molecules, FAS2, and integrin, were found on the plasma membrane, and DLG and SIF were found inside the membrane of the zone, although βPS integrin and DLG were more widely distributed, at least on the postsynaptic side. These findings suggest that the periactive zone may link the information from cell adhesion molecules to the intracellular signaling pathways. Finally, the role for the periactive zone was suggested by the genetic evidence for the molecules localized in the zone. The mutant analyses in this and the previous studies have shown that all these molecules are involved in synaptic development. In Fas2 null mutants, neuromuscular synapses fail to grow and eventually retract. In the mutants affecting βPS integrin, the growth of larval NMJ is increased or decreased dependent on the alleles. The mutation in dlg caused the reduction in size of the subsynaptic reticulum in postsynapses and the increase in the number of active zones in presynapses. Also, the sif mutation exhibited a modulatory effect on synaptic growth when combined with the Fas2 mutation as shown in this report. Taken together, all gene products localized in the peri-active zone participate in the synaptic development. These findings provide genetic evidence suggesting that the periactive zone serves as a membrane domain for the structural control of synaptic terminals (Sone, 2000).

In addition, the data indicate that the periactive zone is distinct from the zone for endocytosis recently reported. Staining for SHI, which is in a donut-like pattern, does not overlap with most of the FAS2-positive region, and is nearly within the hole of the FAS2 rings. This observation suggests that endocytosis actively occurs near or within the electron-dense region, which is consistent with the previous finding that one type of endocytosis occurs near the active zone in the Drosophila optic lobe synapses. Although it is possible that endocytosis occurs in the periactive zone at some frequency, these data suggest that the major role for the zone is directed to events other than shi-dependent endocytosis (Sone, 2000).

Genetic data suggested that FAS2 and SIF are components in the related signaling pathways that locally function in the periactive zone. The altered localization of SIF in the Fas2 mutants suggests that a certain level of FAS2 expression is required to maintain the configuration of the intracellular molecules including SIF in the periactive zone. Further evidence that shows the functional interaction between SIF and FAS2 was also obtained by mutant analyses. While each of the sif and Fas2 mutations causes the reduction in bouton number, the sif and Fas2 double mutations exhibited a suppressive genetic interaction in synaptic growth. This suppression suggests that the two pathways for FAS2 and SIF signals are both involved in synaptic development and the balance between the pathways is important for the regulation of synaptic growth. These notions were supported by the exacerbated reduction of the bouton number when SIF was overexpressed in the Fas2^e76 background (Sone, 2000).

It has been shown that FAS2 mediates synaptic stabilization, the varying extent of which seems to cause the increase or decrease in synaptic growth. The current data may be therefore consistent with the idea that SIF acts to regulate synaptic stabilization that is mediated by FAS2 or other adhesion molecules. Moreover, SIF may modulate synaptic growth in an inhibitory manner when the FAS2-mediated synapse stabilization is reduced. It may be further noteworthy that the Fas2^e76 sif^ES11 double mutants exhibit low viability and are difficult to maintain as a stock even as heterozygotes. This observation may be also interpreted as indicating that eliminating the doses of both sif and Fas2 impairs a regulatory cascade that is established by the balance of the two signaling pathways (Sone, 2000).

In summary, the cell adhesion molecule FAS2 and intracellular signaling molecule SIF interact with each other, both controlling synaptic development in the periactive zone. This finding enforces the idea that the periactive zone is the functional membrane domain where various types of proteins constitute signaling networks or protein complexes that control synapse formation. This notion is further supported by the fact that DLG regulates the localization of FAS2 in the periactive zone. In addition, FAS2, in turn, may function to organize the arrangement of the molecules including SIF in the periactive zones (Sone, 2000).

An in vitro assay shows that SIF catalyzes the guanine- nucleotide exchange reaction for mammalian RAC1. This is consistent with the previous observation that SIF induces ruffling membranes in human KB cells, as does the constitutive active form of RAC1. In addition, Drosophila RHO family G proteins are more than 85% identical in amino acid sequences to the corresponding mammalian G proteins. These lines of evidence suggest that SIF activates Drosophila RAC in the periactive zones. As SIF contains multiple domains that potentially mediate interaction with other molecules, RAC and several other molecules may be recruited to make a protein complex in the zone. In mice, TIAM1 and STEF have been identified as GEFs that specifically activate RAC1, and both are highly related to SIF in domain organization and amino acid sequence in several domains, indicating that these two proteins are likely to be the mouse orthologs of SIF. Interestingly, both Tiam1 and Stef are expressed in the brain, and Tiam1 expression is observed in the adult hippocampus. It would be important therefore to examine whether TIAM1 and STEF are localized in the synaptic terminals (Sone, 2000).

RAC is well known as a regulator of the actin-based cytoskeleton and cell adhesion in various cells. RAC is also implicated in the structural changes of nerve terminals including growth cones and dendrites. Therefore, in the periactive zones, activated RAC may locally regulate the processes of the structural change in the synaptic terminals, which include reorganization of the actin-based cytoskeleton and cell adhesion (Sone, 2000).

Previous studies have shown that RAC acts in the neurite outgrowth of neuroblastoma cells that depends on the signal from integrin on the cell surface. The mammalian SIF homolog TIAM1, which functions as a RAC GEF, recruits integrin to specific adhesive contacts at the cell periphery. Moreover, expression of TIAM1 increases cadherin-mediated cell adhesion in epithelial MDCK cells. Therefore, there appear to be signaling links between the RAC and cell-adhesion molecules. This study shows that SIF activates RAC, sif genetically interacts with Fas2 in synaptic growth and the SIF localization was perturbed in the Fas2 mutants. Taken together, these data suggest that the SIF-RAC pathway is linked to the cell-adhesion molecule FAS2 in close vicinity in the periactive zone (Sone, 2000).

This study has indicated the periactive zone as a region for the control of synaptic development. The periactive zone surrounds the active zone, which is the site for vesicle exocytosis or neurotransmission. This concentric organization suggests that the two zones specialized for the different cellular functions constitute an elemental unit for the presynaptic structure. Investigation of how these zones are incorporated into the synaptic bouton during development will be interesting but remains to be carried out (Sone, 2000).

The segregated distribution of the two zones suggests that the mechanisms controlling synaptic development and neurotransmission may be separable. This view was supported by the mutant analyses for FAS2 and SIF; both mutations affect structural properties of synapses without changing basic electrophysiological functions. In the NMJs of Fas2 mutants, the bouton number is decreased or increased depending on the alleles but the total synaptic strength is maintained at the normal level (Sone, 2000).

Functional strength of the synapse is regulated only through the activity of a transcription factor, cAMP-response-element- binding protein (CREB), which functions independently of FAS2. Also in sif mutants, basic electrophysiological properties of NMJs are normal. These observations clearly contrast with the mutant phenotypes for the proteins controlling vesicle exocytosis: synaptotagmin, cysteine string protein, n-synaptobrevin and syntaxin 1A. All these mutants show the impaired EJPs. Taken together, these results indicate that synaptic development and neurotransmission are genetically separable phenomena and are regulated by independent pathways. It is proposed that these genetically separable phenomena are spatially segregated into the two zones on the presynaptic plasma membrane, although the possibility that the two zones interact with each other cannot be excluded (Sone, 2000).

Thia study identified new members to be added to the periactive zone proteins. In these studies, Drosophila highwire protein and its C. elegans homolog, RPM-1, were demonstrated to function in the growth or structural development of synapses, and highwire was found to localize in the periactive zone. Discovery of these proteins further enforces the current view and highlights the importance of periactive zones for synaptic growth and stability (Sone, 2000).

In the nervous system, various cell-adhesion molecules and signaling molecules may provide characteristic attributes for each synapse. In addition to the molecules described in this article, cadherins and catenins are likely to play such a role in synapses. Notably, N-cadherin and SIF are localized laterally to the active zones in the neuron-neuronal synapses of the mammalian and Drosophila brain, respectively. This suggests that the presence of the specialized area surrounding the active zone is a general feature among synapses. Further examination of signaling pathways that function locally in the periactive zone would provide insights as to how synapses grow or retract during development and plasticity (Sone, 2000).

Sphingolipids regulate neuromuscular synapse structure and function in Drosophila

Sphingolipids are found in abundance at synapses and have been implicated in regulation of synapse structure, function and degeneration. Serine Palmitoyl-transferase (SPT) is the first enzymatic step for synthesis of sphingolipids. Analysis of the Drosophila larval neuromuscular junction revealed mutations in the SPT enzyme subunit, lace/SPTLC2 resulted in deficits in synaptic structure and function. Although NMJ length is normal in lace mutants, the number of boutons per NMJ is reduced to approximately 50% of the wild type number. Synaptic boutons in lace mutants are much larger but show little perturbation to the general ultrastructure. Electrophysiological analysis of lace mutant synapses revealed strong synaptic transmission coupled with predominance of depression over facilitation. The structural and functional phenotypes of lace mirrored aspects of Basigin (Bsg), a small Ig-domain adhesion molecule also known to regulate synaptic structure and function. Mutant combinations of lace and Bsg generated large synaptic boutons, while lace mutants showed abnormal accumulation of Bsg at synapses, suggesting that Bsg requires sphingolipid to regulate structure of the synapse. This data points to a role for sphingolipids in the regulation and fine-tuning of synaptic structure and function while sphingolipid regulation of synaptic structure may be mediated via the activity of Bsg (West, 2018).

A prominent enrichment of wglycosphingolipids has been demonstrated within synaptic structures in the mammalian brain. To date understanding of the role of these enigmatic lipids in synapse structure and function has yet to be fully elucidated. Sphingolipids are major lipid components of the plasma and endomembrane system and have been implicated in many forms of neuropathy and neurodegeneration. Sphingolipids are proposed to generate structure in membranes due to their rigidity and association with cholesterol. They are also known to be potent signalling molecules regulating processes such as apoptosis, proliferation, migration and responses to oxidative stress (West, 2018).

Numerous neurological and neurodegenerative conditions are directly attributable to the inability to synthesise or catabolise sphingolipids. The failure to synthesise all or particular sphingolipids gives rise to a number of neurological conditions such as infant-onset symptomatic epilepsy (loss of GM3 ganglioside synthesis, bovine spinal muscular atrophy (loss of 3- ketohydrosphingosine reductase and hereditary sensory and autonomic neuropathy type 1 (HSAN1; recessive and dominant mutations in serine palmitoyl transferase subunit 1 (SPTLC1). Conversely failure to catabolise sphingolipids in the lysosome generates a subset of lysosomal storage diseases/disorders (LSD's) known as sphingolipidoses, of which there are approximately 14 identified separate genetic conditions. Sphingolipids are now suggested to have a prominent role in the onset and progression of Alzheimer's disease while the production after bacterial infection of autoimmune antibodies to gangliosides present at the neuromuscular synapse is likely to cause the dramatic and often lethal paralysis seen in Guillain-Barrê and Miller-Fisher syndromes. The presence of sphingolipids at the synapse is further attested by the ability of tetanus and botulinal toxins to effect their entry to synapses via co-attachment to synaptic glycosphingolipids (West, 2018).

While the presence of sphingolipids (in particular, glycosphingolipids) at the synapse is well established, little is known about their functional or structural role in the operational life of the synapse. Some in vitro studies have addressed the role of sphingolipids at synapses in the context of sphingolipid/cholesterol microdomains and indicate roles in the function and localisation of neurotransmitter receptors and synaptic exocytosis. The prominence of sphingolipids in neurological disease suggests that absence or accumulation of sphingolipids can exert an influence in synaptic function and indicates an inappropriately large gap in knowledge regarding the actions of these lipids at the synapse. In the above outlined context, roles for sphingolipids in synapse structure and function remain to be determined. To this end, this study has undertaken an analysis of sphingolipid function at a model synapse, the third instar neuromuscular junction of Drosophila. Mutations were analyzed in SPT2/SPTLC2 (Serine Palmitoyltransferase, Long Chain Base Subunit 2), which encodes an essential subunit of the Serine Palmitoyltransferase (SPT) heterodimer necessary for the initial step in sphingolipid synthesis, for defects in neuromuscular synapse structure. Evidence is presented to suggest that sphingolipids are essential for synaptic structure and function, and structural regulation may be mediated partially through function of the Ig domain adhesion protein Basigin/CD147 (Bsg) (West, 2018).

The enrichment of sphingolipids at synapses has been long known. Assigning functions for these enigmatic lipids at the synapse has remained problematic. Ablation of gangliosides in mouse has identified subtle defects in neurotransmission while loss of G3- ganglioside synthesis results in an infantile onset epilepsy, the mechanism for which remains obscure. A specific role for sphingosine has been identified in promoting SNARE protein fusion and synaptic exocytosis (West, 2018).

Many sphingolipid species present in the outer leaflet of the plasma membrane are found in association with cholesterol as 'lipid rafts'. Neurons receive supplementary cholesterol from glia which is essential for supporting synapse maturation and additional synaptogenesis suggesting cholesterol, and potentially lipid rafts, are rate limiting for these processes. Depletion of both cholesterol and sphingolipids together has been shown to reduce and enlarge dendritic spines with eventual loss of synapses in hippocampal neurons in culture possibly due to reduced association with lipid rafts of synapse structure promoting proteins such as Post-Synaptic Density protein 95 (PSD95). This study reduced synthesis of sphingolipids with a mutation in SPTLC2, and examined the development of neuromuscular synapses in the Drosophila larval preparation. This approach has allowed study of the genetic depletion of sphingolipids at an identified synapse in vivo and investigate a role for sphingolipids in the regulation of synaptic structure and activity. As part of this study, a potential role was identified for the Ig domain cell adhesion protein Bsg in sphingolipid dependent regulation of synaptic structure (West, 2018).

The enrichment of sphingolipids at synapses has been long known. Assigning functions for these enigmatic lipids at the synapse has remained problematic. Ablation of gangliosides in mouse has identified subtle defects in neurotransmission while loss of G3-ganglioside synthesis results in an infantile onset epilepsy, the mechanism for which remains obscure. A specific role for sphingosine has been identified in promoting SNARE protein fusion and synaptic exocytosis. Many sphingolipid species present in the outer leaflet of the plasma membrane are found in association with cholesterol as 'lipid rafts'. Neurons receive supplementary cholesterol from glia which is essential for supporting synapse maturation and additional synaptogenesis suggesting cholesterol, and potentially lipid rafts, are rate limiting for these processes. Depletion of both cholesterol and sphingolipids together has been shown to reduce and enlarge dendritic spines with eventual loss of synapses in hippocampal neurons in culture possibly due to reduced association with lipid rafts of synapse structure promoting proteins such as Post-Synaptic Density protein 95 (PSD95). This study has reduced synthesis of sphingolipids with a mutation in SPTLC2, and examined the development of neuromuscular synapses in the Drosophila larval preparation. This approach has allowed study of the genetic depletion of sphingolipids at an identified synapse in vivo and investigate a role for sphingolipids in the regulation of synaptic structure and activity. As part of this study, a potential role has been identified for the Ig domain cell adhesion protein Bsg in sphingolipid dependent regulation of synaptic structure (West, 2018).

On examination of sphingolipid deficient synapses, a disruption to the normal synaptic structure was observed. Synaptic boutons were enlarged and the overall numbers of boutons reduced by ~50% while the length of the neuromuscular synapse remained indistinguishable from wildtype. This phenotype is highly reminiscent of the reduction of synapse number, but increase in synapse size observed in hippocampal neurons in culture depleted for lipid rafts. Nevertheless, beyond the structural deficit of the synapse, the ultrastructure of the synapse was remarkably intact, suggesting a role in fine-tuning of synaptic properties (West, 2018).

Synapses depleted for sphingolipids were capable of greater growth when combined with the synaptic overgrowth mutation highwire (hiw). The data suggests the mutations in lace, encoding a pyridoxal phosphate-dependent transferase, and sphingolipid depletion decouples bouton structure from normal synaptic length. Large boutons are observed in mutants of mothers against dpp (mad), thick veins (tkv), saxophone (sax) medea (med), and glass-bottom-boat (gbb), components of the TGF-β pathway that is known to regulate synaptic growth. However these mutations reduce synaptic length by ~50% and ultrastructural synaptic defects such as non-plasma membrane attached active zones (T-bars), large endosomal vesicles and ripples in pre-synaptic peri-active membranes are observed. One obvious ultrastructural defect that is present in sphingolipid depleted synapses is enlarged mitochondria. Enlarged mitochondria are observed in a number of sensory neuropathies and it is of interest that dominant mutations in SPTLC1 and SPTLC2 that generate aberrant sphingolipids give rise to Hereditary and Sensory Neuropathy Type 1 (HSAN1) where enlarged mitochondria are often observed. This may be attributable to a recognised role for sphingolipids in mitochondrial fission (West, 2018).

To dissect the spatial requirement for sphingolipid regulation of synapse structure, the lace mutant was rescued with a rescue transgene, expressed globally, pre- or post-synaptically. It was possible to rescue synaptic bouton size and number with a global expression of the rescue transgene, but no aspects of the phenotype could be rescued with a pre-synaptic expression. Perturbed NMJ morphology could also be rescued by glial or post-synaptic expression of lace, however post-synaptic muscle expression generated a partial rescue, with an excess of 'satellite' boutons, a phenotype normally associated with integrin dysfunction or endocytic defects. Previous data feeding lace mutant larvae with sphingosine, the product of the SPT enzyme, partially rescued lace mutant associated phenotypes. Taken together with the current analysis, there is a strong suggestion that sphingolipid precursors such as sphingosine may be able to act non-cell autonomously, and traffic between cells to support synapse structure and function, but not when supplied from the nervous system (West, 2018).

Analysis of EJP and miniEJP characteristics at the 3rd instar larval NMJ reveals mutations in lace produce, at the Ca2+/Mg2+ concentrations that were used, a small but significant increase in synaptic strength, accompanied by a change in short-term plasticity, with synaptic depression predominating over synaptic facilitation. NMJs with high-quantal content EJPs normally show synaptic depression during paired or short-train repetitive stimulation, while those with a low basal quantal content show synaptic facilitation (Lnenicka, 2000; Lnenicka, 2006). Further analysis is required, for instance using a range of Ca2+ concentrations, to establish whether this apparent change in synaptic plasticity is commensurate with a greater basal synaptic strength in the lace mutant larvae, or whether it represents a specific effect of the mutation, disrupting the normal link between mechanisms that couple basal quantal content to short-term synaptic plasticity. In vitro and in vivo analysis has suggested a role for sphingolipids in synaptic vesicle endocytosis and exocytosis in addition to a role in neurotransmitter distribution. No evident defects were observed in neurotransmitter receptor distribution. Interestingly, ablation of major subsets of gangliosides and subsequent synaptic function at the NMJ in a mouse model reveals a more pronounced run-down of neurotransmitter release upon sustained stimulation, consistent with the data presented in this study. It is not possible, however, to directly attribute the apparent deficit in synaptic facilitation observed in lace mutants to exoF or endocytosis, at this point (West, 2018).

A phenotypic similarity at the larval neuromuscular synapse is noted between the lace mutants and mutations in the small Ig domain adhesion protein Basigin/CD147. Bsg is a glycoprotein localised in the plasma membrane that is known to genetically interact with integrins during development of the Drosophila eye. In Bsg mutants, synaptic boutons at the larval neuromuscular junction are enlarged in size and reduced in number with a modest reduction in synaptic span. Bsg has previously been localised to sphingolipid enriched lipid rafts in invading epithelial breast cells, and this study observed that Bsg is abundant in the lipid raft associated membrane fraction, co-sedimenting with syntaxin, a known component of lipid rafts. It cannot be said at this juncture if Bsg function is directly regulated by sphingolipids. Indeed, recruitment of Bsg to lipid rafts can be critical for the recruitment of other protein factors such as claudin-5 in retinal vascular epithelial cells. However, given the genetic interaction between Bsg and lace, with bsg;lace transheterozygous double mutants phenocopying both lace and bsg mutants, the data suggests Bsg and sphingolipids genetically interact to regulate synaptic structure. This interaction is interpreted as indirect; the loss of sphingolipid generated in the lace mutant affecting Bsg function to regulate synapse structure and function (West, 2018).

Synaptic sphingolipids have previously been implicated in synaptic vesicle release, endocytosis, neurotransmitter receptor localisation and maintenance of synaptic activity. However other roles at the synapse for these enigmatic lipids remain elusive. Two potential functions for sphingolipid at the synapse are suggested by this study. Mitochondrial uptake of Ca2+ shapes Ca2+ dependent responses. The enlarged mitochondria observed in lace mutants may impinge on Ca2+ uptake to affect synaptic facilitation. A further deficit in Ca2+ handling at the synapse is suggested by the recent finding that Bsg is an obligatory subunit of plasma membrane Ca2+-ATPases (PMCAs). PMCAs extrude Ca2+ to the extracellular space, and knock-out of Bsg considerably affects Ca2+ handling by PMCAs. Sphingolipid deficient synapses in the lace mutant have deficits in Bsg function which may in turn have an effect on Ca2+ dynamics via PMCA function (West, 2018).

Ablation of sphingolipid synthesis at a Drosophila model synapse supports a role for sphingolipids in maintenance of synaptic activity and regulation of synaptic structure. The analysis also points to sphingolipid dependent regulation of synaptic structure via function of the small Ig-domain protein Bsg. The precise regulation of synapse structure and function is a potent mechanism underlying synaptic plasticity and it is suggested that the presence of sphingolipids at synapse may partially reflect this function (West, 2018).

Parvalbumin expression affects synaptic development and physiology at the Drosophila larval NMJ

Presynaptic Ca(2+) appears to play multiple roles in synaptic development and physiology. This study examined the effect of buffering presynaptic Ca(2+) by expressing parvalbumin (PV) in Drosophila neurons, which do not normally express PV. The studies were performed on the identified Ib terminal that innervates muscle fiber 5. The volume-averaged, residual Ca(2+) resulting from single action potentials (APs) and AP trains was measured using the fluorescent Ca(2+) indicator, OGB-1. PV reduced the amplitude and decay time constant (tau) for single-AP Ca(2+) transients. For AP trains, there was a reduction in the rate of rise and decay of [Ca(2+)]i but the plateau [Ca(2+)]i was not affected. Electrophysiological recordings from muscle fiber 5 showed a reduction in paired-pulse facilitation, particularly the F1 component; this was likely due to the reduction in residual Ca(2+). These synapses also showed reduced synaptic enhancement during AP trains, presumably due to less buildup of synaptic facilitation. The transmitter release for single APs was increased for the PV-expressing terminals and this may have been a homeostatic response to the decrease in facilitation. Confocal microscopy was used to examine the structure of the motor terminals and PV expression resulted in smaller motor terminals with fewer synaptic boutons and active zones. This result supports earlier proposals that increased AP activity promotes motor terminal growth through increases in presynaptic [Ca(2+)]i (He, 2018).

GAL4 drivers specific for type Ib and type Is motor neurons in Drosophila

The Drosophila melanogaster larval neuromuscular system is extensively used by researchers to study neuronal cell biology, and Drosophila glutamatergic motor neurons have become a major model system. There are two main types of glutamatergic motor neurons, Ib and Is, with different structural and physiological properties at synaptic level at the neuromuscular junction. To generate genetic tools to identify and manipulate motor neurons of each type, GAL4 driver lines were screened for this purpose. This study describes GAL4 drivers specific for examples of neurons within each Type, Ib or Is. These drivers showed high expression levels and were expressed in only few motor neurons, making them amenable tools for specific studies of both axonal and synapse biology in identified Type I motor neurons (Perez-Moreno, 2018).

Triose-phosphate isomerase deficiency is associated with a dysregulation of synaptic vesicle recycling in Drosophila melanogaster

Numerous neurodegenerative diseases are associated with neuronal dysfunction caused by increased redox stress, often linked to aberrant production of redox-active molecules such as nitric oxide (NO) or oxygen free radicals. One such protein affected by redox-mediated changes is the glycolytic enzyme triose-phosphate isomerase (TPI), which has been shown to undergo 3-nitrotyrosination (a NO-mediated post-translational modification) rendering it inactive. This work uses Drosophila to identify the impacts of altered TPI activity on neuronal physiology, linking aberrant TPI function and redox stress to neuronal defects. Drosophila mutants were used expressing a missense allele of the TPI protein, M81T, resulting in an inactive mutant of the TPI protein (TPI^M81T, wstd¹). Electrophysiological recordings of evoked and spontaneous excitatory junctional currents, alongside high frequency train stimulations and recovery protocols, were applied to the NMJ to investigate synaptic depletion and subsequent recovery. Single synaptic currents were unaltered in the presence of the wstd¹ mutation, but frequencies of spontaneous events were reduced. wstd¹ larvae also showed enhanced vesicle depletion rates at higher frequency stimulation, and subsequent recovery times for evoked synaptic responses were prolonged. A computational model showed that TPI mutant larvae exhibited a significant decline in activity-dependent vesicle recycling, which manifests itself as increased recovery times for the readily-releasable vesicle pool. Confocal images of NMJs showed no morphological or developmental differences between wild-type and wstd(1) but TPI mutants exhibited learning impairments as assessed by olfactory associative learning assays. These data suggests that the wstd¹ phenotype is partially due to altered vesicle dynamics, involving a reduced vesicle pool replenishment, and altered endo/exocytosis processes. This may result in learning and memory impairments and neuronal dysfunction potentially also presenting a contributing factor to other reported neuronal phenotypes (Stone, 2023).

Oxidative stress induces overgrowth of the Drosophila neuromuscular junction

Synaptic terminals are known to expand and contract throughout an animal's life. The physiological constraints and demands that regulate appropriate synaptic growth and connectivity are currently poorly understood. Previous work has identified a Drosophila model of lysosomal storage disease (LSD), spinster (spin), with larval neuromuscular synapse overgrowth. This study identified a reactive oxygen species (ROS) burden in spin that may be attributable to previously identified lipofuscin deposition and lysosomal dysfunction, a cellular hallmark of LSD. Reducing ROS in spin mutants rescues synaptic overgrowth and electrophysiological deficits. Synapse overgrowth was also observed in mutants defective for protection from ROS and animals subjected to excessive ROS. ROS are known to stimulate JNK and fos signaling. Furthermore, JNK and fos in turn are known potent activators of synapse growth and function. Inhibiting JNK and fos activity in spin rescues synapse overgrowth and electrophysiological deficits. Similarly, inhibiting JNK, fos, and jun activity in animals with excessive oxidative stress rescues the overgrowth phenotype. These data suggest that ROS, via activation of the JNK signaling pathway, are a major regulator of synapse overgrowth. In LSD, increased autophagy contributes to lysosomal storage and, presumably, elevated levels of oxidative stress. In support of this suggestion, this study reports that impaired autophagy function reverses synaptic overgrowth in spin. These data describe a previously unexplored link between oxidative stress and synapse overgrowth via the JNK signaling pathway (Milton, 2011).

Reactive oxygen species regulate activity-dependent neuronal plasticity in Drosophila

Reactive oxygen species (ROS) have been extensively studied as damaging agents associated with ageing and neurodegenerative conditions. Their role in the nervous system under non-pathological conditions has remained poorly understood. Working with the Drosophila larval locomotor network, this study showed that in neurons ROS act as obligate signals required for neuronal activity-dependent structural plasticity, of both pre- and postsynaptic terminals. ROS signaling is also necessary for maintaining evoked synaptic transmission at the neuromuscular junction, and for activity-regulated homeostatic adjustment of motor network output, as measured by larval crawling behavior. The highly conserved Parkinson's disease-linked protein DJ-1β was identified as a redox sensor in neurons where it regulates structural plasticity, in part via modulation of the PTEN-PI3 Kinase pathway. This study provides a new conceptual framework of neuronal ROS as second messengers required for neuronal plasticity and for network tuning, whose dysregulation in the ageing brain and under neurodegenerative conditions may contribute to synaptic dysfunction (Oswald, 2018).

This study set out to investigate potential roles for ROS in the nervous system under non-pathological conditions, which are much less well understood. The brain is arguably the most energy demanding organ and mitochondrial oxidative phosphorylation is a major source of ROS. It was therefore asked whether neurons might utilize mitochondrial metabolic ROS as feedback signals to mediate activity-regulated changes. As an experimental model the motor system was used of the fruitfly larva, Drosophila melanogaster allowing access to uniquely identifiable motoneurons in the ventral nerve cord and their specific body wall target muscles. An experimental paradigm was established for studying activity-regulated structural adjustments across an identified motoneuron, quantifying changes at both pre- and postsynaptic terminals. Thermogenetic neuronal over-activation leads to the generation of ROS at presynaptic terminals, and ROS signaling is necessary and sufficient for the activity-regulated structural adjustments. As a cellular ROS sensor the conserved redox sensitive protein DJ-1β, a homologue of vertebrate DJ-1 (PARK7), was identified and the phosphatase and tensin homolog (PTEN) and PI3kinase were identified as downstream effectors of activity-ROS-mediated structural plasticity. ROS signaling is also required for maintaining constancy of evoked transmission at the neuromuscular junction (NMJ) with a separate ROS pathway regulating the amplitude of spontaneous vesicle release events. Behaviourally, ROS signaling is required for the motor network to adjust homeostatically to return to a set crawling speed following prolonged overactivation (Oswald, 2018).

Building on previous work that had shown oxidative stress as inducing NMJ growth (Milton, 2011), this study identified ROS as obligatory signals for activity-regulated structural plasticity. It was further shown that ROS are also sufficient to bring about structural changes at synaptic terminals that largely mimic those induced by neuronal overactivation. A mitochondrially targeted ROS reporter suggests a positive correlation between levels of neuronal activity and ROS generated in mitochondria, potentially as a byproduct of increased ATP metabolism or triggered by mitochondrial calcium influx. Although this study did not specifically investigate the nature of the active ROS in this context, three lines of evidence suggest that H2O2, generated by the dismutation of O2-, is the principal signaling species. First, under conditions of neuronal overactivation (but not control levels of activity) over-expression of the O2- to H2O2 converting enzyme SOD2 potentiated structural plasticity phenotypes. Second, over-expression of the H2O2 scavenger Catalase efficiently counter-acts all activity-induced changes that were quantified, at both postsynaptic dendritic and presynaptic NMJ terminals. Third, over-expression of the H2O2 generator Duox in motoneurons is sufficient to induce NMJ bouton phenotypes that mimic overactivation. In addition to mitochondria, other sources of ROS include several oxidases, notably NADPH oxidases. These have been implicated during nervous system development in the regulation of axon growth and synaptic plasticity. NADPH oxidases can be regulated by NMDA receptor stimulation and activity-associated pathways, including calcium, Protein kinases C and A and calcium/calmodulin-dependent kinase II (CamKII). The precise sources of activity-regulated ROS, potentially for distinct roles in plasticity, will be interesting to investigate (Oswald, 2018).

This study demonstrated that ROS are necessary for activity-dependent structural plasticity of Drosophila motoneurons, at both their postsynaptic dendrites in the CNS and presynaptic NMJs in the periphery. The mechanisms by which ROS intersect with other known plasticity pathways now need to be investigated. Among well documented signaling pathways regulating synaptic plasticity, are Wnts, BMPs, PKA, CREB and the immediate early gene transcription factor AP-1. ROS signaling could be synergistic with other neuronal plasticity pathways, potentially integrating metabolic feedback. Indeed, ROS modulate BMP signaling in cultured sympathetic neurons and Wnt pathways in non-neuronal cells. Biochemically, ROS are well known regulators of kinase pathways via oxidation-mediated inhibition of phosphatases. Redox modifications also regulate the activity of the immediate early genes Jun and Fos, which are required for LTP in vertebrates, and in Drosophila for activity-dependent plasticity of motoneurons, both at the NMJ and central dendrites. It was therefore hypothesized that ROS may provide neuronal activity-regulated modulation of multiple canonical synaptic plasticity pathways (Oswald, 2018).

This study focused on three aspects of synaptic terminal plasticity: dendritic arbor size in the CNS, and bouton and active zone numbers at the NMJ. These were used as phenotypic indicators for activity-regulated changes. By working with identified motoneurons adaptations could be observed across the entire neuron, relating adjustments of postsynaptic dendritic input terminals in the CNS to changes of the presynaptic output terminals at the NMJ in the periphery. For the aCC motoneuron, the degree of neuronal overactivation correlates with changes in synaptic terminal growth: notably reductions of dendritic arbor size centrally and of active zones at the NMJ. Interestingly, presynaptic active zone numbers did not show a linear response profile. Within a certain range low-level activity increases lead to more active zones, associated with potentiation; however, with stronger overactivation active zone number decrease. Reduction of active zones, as was observed at the NMJ, and of Brp levels by increased activation was previously also reported in photoreceptor terminals of the Drosophila adult visual system. At a finer level of resolution it will be interesting to determine how these activity-ROS-mediated structural changes might change active zone cytomatrix composition, which can impact on transmission properties, such as vesicle release probability (Oswald, 2018).

Previous work found that in these motoneurons dendritic length correlates with the number of input synapses and with synaptic drive) Therefore, the negative correlation between the degree of overactivation and the reduction in central dendritic arbors is tentatively interpreted as compensatory. In agreement, it was found that blockade of activity-induced structural adjustment targeted to the motoneurons prevents behavioral adaptation normally seen after prolonged overactivation. Less clear is if and how overactivation-induced structural changes at the NMJ might be adaptive. Unlike many central synapses that facilitate graded analogue computation, the NMJ is a highly specialized synapse with a large safety factor and intricate mechanisms that ensure constancy of evoked transmission in essentially digital format. Rearing larvae at 29°C (which acutely increases motor activity) leads to more active zones at the NMJ and potentiated transmission, yet these larvae crawl at the same default speed as other larvae reared at 25°C (control) or 32°C with reduced numbers of active zones. This suggests that at least with regard to regulating crawling speed, plasticity mechanisms probably operate at the network level, rather than transmission properties of the NMJ. Indeed, recordings of transmission at the NMJ, and those reported by others, show homeostatic maintenance of eEJP amplitude irrespective of changes in bouton and active zone number. Though in this study focus was placed on anatomical changes, these structural adjustments are expected to be linked to, and probably preceded by compensatory changes in neuronal excitability that have been documented (Oswald, 2018).

The observations of activity-regulated adjustments of both dendritic arbor size and NMJ structure give the impression of processes coordinated across the entire neuron. If this was the case, it could be mediated by transcriptional changes, potentially via immediate early genes (AP-1), which are involved in activity and ROS-induced structural changes at the NMJ and motoneuron dendrites (Oswald, 2018).

In neurons the highly conserved protein DJ-1β; is critical for both structural and physiological changes in response to activity-generated ROS. In neurons DJ-1β might act as a redox sensor for activity-generated ROS. In agreement with this idea, DJ-1β has been shown to be oxidized by H2O2 at the conserved cysteine residue C106 (C104 in Drosophila). Oxidation of DJ-1 leads to changes in DJ-1 function, including translocation from the cytoplasm to the mitochondrial matrix, aiding protection against oxidative damage and maintenance of ATP levels. The ability of motoneurons to respond to increased activation is potently sensitive to DJ-1β dosage. It is also blocked by expression of mutant DJ-1βC104A that is non-oxidisable on the conserved Cys104. These observations suggest that DJ-1β is critical to ROS sensing in neurons. They also predict that cell type-specific DJ-1β levels, and associated DJ-1β reducing mechanisms, could contribute to setting cell type-specific sensitivity thresholds to neuronal activity (Oswald, 2018).

The data suggest that DJ-1β could potentially be part of a signaling hub. At the NMJ, this might mediate plasticity across a range, from the addition of active zones associated with potentiation to, following stronger overactivation, the reduction of active zones. This study identified disinhibition of PI3Kinase signaling as one DJ-1β downstream pathway, a well-studied intermediate in metabolic pathways and a known regulator of synaptic terminal growth, including active zone addition. However, with stronger overactivation DJ-1β might engage additional downstream effectors that reduce active zone addition or maintenance, potentially promoting active zone disassembly. While at the presynaptic NMJ PI3Kinase disinhibition explains activity-regulated changes in bouton addition, different DJ-1β effectors likely operate in the somato-dendritic compartment, which responds to overactivation with reduced growth and possibly pruning. Thus, sub-cellular compartmentalization of the activity-ROS-DJ-1β signaling axis could produce distinct plasticity responses in pre- versus post-synaptic terminals (Oswald, 2018).

Previous studies demonstrated a requirement for ROS for LTP and found learning defects in animal models with reduced NADPH oxidase activity, suggesting that synaptic ROS signaling might be a conserved feature of communication in the nervous system. Sharp electrode recordings from muscle DA1 revealed three interesting aspects. First, that changing ROS signaling in the presynaptic motoneuron under normal activity conditions does not obviously impact on NMJ transmission. Second, quenching of presynaptic ROS by expression of Catalase under overactivation conditions led to a significant decrease in eEJP amplitude and concomitantly reduced quantal content. This shows that upon chronic neuronal overactivation ROS signaling is critically required in the presynaptic motoneuron for maintaining eEJP amplitude by increasing vesicle release at the NMJ. This could be achieved by increasing vesicle release probability, which would counteract the reduction in active zone number following a period of neuronal overactivation. In this context it is interesting that components of the presynaptic release machinery, including SNAP25, are thought to be directly modulated by ROS, while others, such as Complexin, might be indirectly affected, for example via ROS-mediated inhibition of phosphatases leading disinhibition of kinase activity. Third, this study found that overactivation of motoneurons leads to reduced mEJP amplitude, also recently reported by others (Yeates, 2017). Curiously, mEJP amplitude, unlike eEJP amplitude, is regulated by DJ-1β, but is not impacted on by artificially increased cytoplasmic levels of the H2O2 scavenger Catalase. How it is that under conditions of neuronal overactivation eEJP and mEJP amplitudes are differentially sensitive to cytoplasmic Catalase versus DJ-1β oxidation is unclear, though it marks these two processes as distinct. One possibility is that cytoplasmic Catalase changes the local redox status, which could directly affect the properties of the presynaptic active zone cytomatrix. In contrast, mEJP amplitude regulation might be indirect and cell non-autonomous, via modulation of glutamate receptors in the postsynaptic target muscle (Oswald, 2018).

Thus, several distinct ROS responsive pathways appear to operate at the NMJ. Structural adjustments in terms of synaptic terminal growth and synapse number are mediated by mechanisms sensitive to DJ-1β oxidation, potentially regulated via local reducing systems, including Catalase. In addition and distinct from these structural changes, at least in part, are the ROS-regulated adjustments in synaptic transmission that show different ROS sensitivities, one maintaining quantal content of evoked transmission while the other reduces mEJP amplitude when neuronal activity goes up. It is conceivable that spatially distinct sources of ROS, for example mitochondria versus membrane localized NADPH oxidases, with different temporal dynamics could potentially mediate such differences in ROS sensitivities at the NMJ (Oswald, 2018).

Experiments exploring the potential behavioral relevance of activity-regulated structural plasticity demonstrated that network drive is regulated by ambient temperature. Acute elevation in ambient temperature produces faster crawling, while acute temperature reduction has the opposite effect. In contrast, with chronic temperature manipulations, larval crawling returns to its default speed (approx. 0.65-0.72 mm/sec). This adaptation to chronic manipulations might overall be energetically more favorable. It also allows larvae to retain a dynamic range of responses to relative changes in ambient temperature (i.e., speeding up or slowing down) (Oswald, 2018).

Where in the locomotor network these adjustments take place remains to be worked out. It is reasonable to assume that proprioceptive sensory neurons, and potentially also central recurrent connections, provide feedback information that facilitates homeostatic adjustment of network output. This study's manipulations of the glutamatergic motoneurons show these cells are clearly important. For example, cell type-specific overactivation of the glutamatergic motoneurons (via dTrpA1) on the one hand, and blockade of activity-induced structural adjustment (by mis-expression of non-oxidizable DJ-1βC104A) on the other demonstrated that ROS-DJ-1β-mediated processes that were showed important for structural adjustment are also required for implementing homeostatic tuning of locomotor network output. The capacity of motoneurons as important elements in shaping motor network output, might be explicable in that these neurons constitute the final integrators on which all pre-motor inputs converge (Oswald, 2018).

In conclusion, this study identified ROS in neurons as novel signals that are critical for activity-induced structural plasticity\. ROS levels regulated by neuronal activity have the potential for operating as metabolic feedback signals. The conserved redox-sensitive protein DJ-1β was further as important to neuronal ROS sensing, and the PTEN/PI3Kinase synaptic growth pathway was identified as a downstream effector pathway for NMJ growth in response to neuronal overactivation. These findings suggest that in the nervous system ROS operate as feedback signals that inform cells about their activity levels. The observation that ROS are important signals for homeostatic processes explains why ROS buffering is comparatively low in neurons. This view also shines a new light on the potential impact of ROS dysregulation with age or under neurodegenerative conditions, potentially interfering with neuronal adaptive adjustments and thereby contributing to network malfunction and synapse loss (Oswald, 2018).

A circuit-dependent ROS feedback loop mediates glutamate excitotoxicity to sculpt the Drosophila motor system

Overproduction of reactive oxygen species (ROS) is known to mediate glutamate excitotoxicity in neurological diseases. However, how ROS burdens can influence neural circuit integrity remains unclear. This study investigated the impact of excitotoxicity induced by depletion of Drosophila Eaat1, an astrocytic glutamate transporter, on locomotor central pattern generator (CPG) activity, neuromuscular junction architecture, and motor function. Glutamate excitotoxicity triggers a circuit-dependent ROS feedback loop to sculpt the motor system. Excitotoxicity initially elevates ROS to inactivate cholinergic interneurons, consequently changing CPG output activity to overexcite motor neurons and muscles. Remarkably, tonic motor neuron stimulation boosts muscular ROS, gradually dampening muscle contractility to feedback-enhance ROS accumulation in the CPG circuit and subsequently exacerbate circuit dysfunction. Ultimately, excess premotor excitation of motor neurons promotes ROS-activated stress signaling to alter neuromuscular junction architecture. Collectively, these results reveal that excitotoxicity-induced ROS can perturb motor system integrity by a circuit-dependent mechanism (Peng, 2019).

Reactive oxygen species (ROS) are generated as the by-product of mitochondrial oxidative phosphorylation. In the central nervous system, under physiological conditions, high energy demand results in higher levels of ROS production relative to those in other body parts. In the past, endogenously generated ROS were recognized as signaling molecules that regulate a range of nervous system processes, including neuronal polarity, growth cone pathfinding, neuronal development, synaptic plasticity, and neural circuit tuning (Li, 2016; Oswald, 2018). By contrast, ROS overproduction and/or overwhelming the antioxidant machinery can generate ROS burdens, termed oxidative stress, in aging and diverse pathological conditions. In turn, excess ROS causes the malfunction and overactivation of ROS-regulated cell signaling pathways. Moreover, the highly oxidative properties of ROS are damaging to nucleotides, proteins, and lipids, eventually leading to neuronal dysfunction or demise. Hence, advancing understanding of the mechanisms underlying ROS-induced neurotoxicity should aid the development of potent therapeutic treatments for neurological disorders (Peng, 2019).

Glutamate acts as the major excitatory neurotransmitter that regulates nearly all activities of the nervous system, with a tight balance between glutamate release and reuptake keeping the micromolar concentration of extracellular glutamate low. In diseases, accumulation of extrasynaptic glutamate results in glutamate-mediated excitotoxicity to the nervous system. Dysfunction of Na+/K+-dependent excitatory amino acid transporters (EAATs) is a key element of glutamate-mediated excitotoxicity. In mammals, there are five EAAT subtypes, that is EAAT1 (GLAST), EAAT2 (GLT1), EAAT3 (EAAC1), EAAT4, and EAAT5. EAAT3, EAAT4 and EAAT5 are expressed in neurons, whereas EAAT1 and EAAT2 are mainly present in astrocytes, where they are enriched in astrocyte terminal processes that form tripartite synapses with neurons and where they take up approximately 90% of released glutamate. Glutamate-mediated excitotoxicity can trigger bulk Ca2+ influx into postsynaptic neurons via NMDA receptors, which causes mitochondrial Ca2+ overload, along with other cellular responses, and which subsequently generates excess amounts of ROS. Notably, it has emerged that dysregulation of neural circuit activity can initiate subsequent disruption of the integrity of other constituents in the same network, resulting in overall circuit dysfunction and even neurodegeneration. However, it still remains unclear whether and how excitotoxicity-induced ROS can influence the integrity of neural circuits (Peng, 2019).

Coordinated animal behaviors are linked to the activity of spinal cord central pattern generators (CPG), which are known to be specialized circuits that integrate inputs from the central brain and sensory neurons, and that subsequently generate rhythmic and patterned outputs to motor neurons. The Drosophila feed-forward locomotor circuit has served as an appropriate model for exploring the pathogenic network mechanisms that underlie neurodegenerative diseases, because it has a relatively simple neural circuitry compared to mammals yet retains conserved functions. This study explored whether glutamate-mediated excitotoxicity impacts locomotor CPG activity, neuromuscular junction (NMJ) architecture, and motor function. Interestingly, it was found that glutamate-mediated excitotoxicity due to depletion of Drosophila Eaat1, the sole Drosophila homolog of human EAAT2, can induce a circuit-dependent ROS feedback loop that impairs the proper activities of the locomotor CPG circuit and muscles, ultimately leading to motor neuron overexcitation, abnormal NMJ growth and strength, and compromised movement. Together, this work reveals a circuit-dependent mechanism for increasing ROS, which mediates glutamate excitotoxicity to sculpt the Drosophila locomotion network (Peng, 2019).

This work utilized a fly model of glutamate excitotoxicity induced by loss of Drosophila eaat1 to explore the impact of glutamate excitotoxicity on the integrity of the motor system. A circuit-dependent feedback mechanism is described for increasing ROS that mediates excitotoxicity to alter premotor circuit activity, NMJ architecture, and motor function. Glutamate excitotoxicity initially alters locomotor CPG activity and hence prolongs CPG output bursts onto motor neurons by ROS-mediated inactivation of the cholinergic interneurons constituting the CPG circuit. Then, tonic premotor stimulation triggers activity-dependent ROS overproduction in both motor neurons and muscles. In muscles, the increased ROS level gradually dampens muscle contractility and consequent sensory input back to the locomotor CPG circuit, with this feedback strengthening ROS accumulation within the CPG circuit to exacerbate circuit dysfunction. Thus, a positive feedback loop between ROS production in the CPG circuit and muscles is established. Finally, in motor neurons, the induced ROS activate JNK stress signaling to promote abnormal NMJ bouton outgrowth and strength. Apart from genetic rescue, pharmacological treatment with the antioxidant AD4 or the K+ channel blocker 4-AP can also significantly alleviate these motor-system deficits (Peng, 2019).

The locomotor CPG circuit for Drosophila larval feed-forward locomotion is positioned in the VNC and is activated by input from the central brain. Furthermore, acute treatment of dissected Drosophila larvae with non-competitive NMDA antagonists has been shown to reduce the initial output burst duration of the locomotor CPG and eventually abolishes all output activity (Cattaert, 2001), suggesting that glutamatergic transmission drives locomotor CPG activity and positively controls its output burst duration during larval movement. Consistent with these latter results, this study has shown that VNC-restricted expression of eaat1-venus using tsh-GAL4 could reverse the prolonged CPG output burst but not the reduced CPG output frequency in eaat1 mutants, indicating that central brain removal of eaat1 reduced burst frequency, whereas VNC removal markedly extended burst duration. The exact neuronal identity and network connections that build up the core components of the Drosophila larval locomotor CPG circuit remain unknown. Intriguingly, the phenotype of prolonged CPG output has also been reported under conditions in which the motor neuron inputs from proprioceptive sensory neurons or period-positive median segmental interneurons (PMSI) are limited. Moreover, RNAi-mediated knockdown of eaat1 extends PMSI-evoked inhibitory postsynaptic currents in motor neurons (MacNamee, 2016). An increase in extrasynaptic glutamate at the axonal synapses of PMSI was noticed when eaat1 is lost, raising an alternative possibility that excess extrasynaptic glutamate may desensitize GluClα to further diminish sensory inhibition feedback. However, this study found that reducing gluRIID but not gluClα in the eaat1 mutant background shortened prolonged CPG output. Hence, upon loss of eaat1, glutamate-mediated excitotoxicity mainly contributes to locomotor CPG circuit dysfunction. In this regard, the CPG outputs to motor neurons may be elongated and/or the potential inhibition from CPG output to PMSI or its upstream interneurons may be abrogated. Further experiments will be needed to unravel the detailed mechanism operating in the CPG circuit upon loss of eaat1 (Peng, 2019).

Targeted relief of the increased ROS in cholinergic interneurons by genetic approaches could significantly alleviate altered CPG activity arising from either eaat1 mutation or short-term exposure to H2O2, indicating that a subset of cholinergic interneurons, which presumably constitutes the locomotor CPG circuit, is vulnerable to and influenced by the ROS increase. Temporal rescue experiments further suggest that the effect of the ROS increase on circuit activity is acute and reversible. In support of the fact that ROS is known to reduce the inactivation of voltage-gated potassium channels in neurons, long-term food-mediated feeding of (or even short-term exposure to) the K+ channel blocker 4-AP led to a restoration of CPG activity in eaat1 mutants. Thus, temporal hypoexcitability of cholinergic interneurons most probably underlies ROS-induced locomotor CPG dysfunction upon eaat1 loss. Interestingly, immediate blockade of glutamatergic transmission shortens the burst duration of the CPG output (Cattaert, 2001). Under this scenario, it is expected that shortened rather than prolonged CPG burst durations should occur upon loss of eaat1. Thus, it is likely that the induced ROS may occur in a restricted way in a certain subset of cholinergic interneurons, resulting in uneven suppression of cholinergic transmission in the locomotor CPG circuit (Peng, 2019).

The GABA neurotransmitter has a crucial role in neuronal inhibition in the central nervous system through its actions on GABA receptors. Notably, regulation of GABAergic transmission by redox signaling is increasingly recognized. ROS, especially those derived from mitochondrial respiration, act to strengthen the neuronal inhibition mediated by GABAA receptors. Thus, it cannot be excluded that, if those ROS-vulnerable cholinergic interneurons also receive GABAergic input, the increased ROS may silence cholinergic transmission of the locomotor CPG circuit by strengthening GABA-mediated inhibition (Peng, 2019).

The pathological roles of ROS in the regulation of skeletal muscles have been studied extensively, and most targets of redox signaling in skeletal muscle participate in muscle contraction. For instance, excess ROS can modulate SR calcium ATPase (SERCA) and ryanodine receptor (RyR) activity, both of which control the Ca2+ homeostasis of sarcoplasmic reticulum. ROS exposure can also oxidize some myofilament proteins, such as myosin heavy chain and troponin C and, in turn, can impair their functions. Consistent with these findings, the ROS increase dampens the muscle contractility of Drosophila larvae. This study found that mitochondrial and cytosolic ROS levels increase upon excess motor neuron stimulation when eaat1 is lost. Moreover, while increasing ROS by dsod1 knockdown reduced muscle contractility and movement velocity, relieving excess ROS in eaat1 mutant muscles improved locomotion. In the motor system, muscles are not only recognized as the end executive tissues for body movement, but also have a crucial role in triggering proprioceptive sensory feedback input to the central circuit. Recent studies in Drosophila have also revealed that proprioceptive sensory feedback plays a vital role in tuning locomotor circuit activity in the homeostatic adjustment of Drosophila larval crawling and in a Drosophila model of amyotrophic lateral sclerosis (ALS). Unexpectedly, this study found that, upon glutamate excitotoxicity, ROS-induced muscle weakness can cause inefficient sensory feedback input to worsen the ROS burden and can negatively impact the functioning of the central locomotor network. Therefore, under pathological conditions, impaired muscle activity can serve as a key mediator for initiating the ROS feedback loop between the CPG circuit and muscles, which may contribute to network dysfunction in excitotoxicity-associated diseases (Peng, 2019).

Excitotoxicity-induced premotor circuit dysfunction elicits activity-dependent synaptic changes ROS are known to activate the JNK/AP-1 signaling pathway that regulates synaptic formation and strength in Drosophila. The mutation in Drosophila spinster (spin), which encodes a late endosome and lysosome protein, causes impaired lysosomal activity and a consequent ROS burden, leading to synaptic bouton outgrowth by activating the JNK signaling pathway. Intriguingly, c-FOS but not c-JUN is important for bouton outgrowth under spin loss. Similarly, it was found that the synaptic bouton phenotypes of eaat1 mutants are dependent on ROS and c-FOS activity. Interestingly, Milton (2011) have shown that 'constitutive' boosting of mitochondria-derived ROS under loss of dsod2 or after paraquat treatment can also promote bouton growth, but in that case it requires both c-FOS and c-JUN activities. By contrast, in eaat1 mutants, the altered CPG pattern possibly elicits a pulsed increase of mitochondrial ROS. Thus, it may be postulated that different resources and temporal generation of ROS may be responsible for engaging different cellular signaling processes. In support of this notion, in addition to mitochondria, NADPH oxidases provide another major source of ROS to control diverse cellular processes. Recently, DJ-1β, a Parkinson's disease-linked protein, has been identified as a redox sensor that mediates the mitochondrial ROS regulating activity-dependent synaptic plasticity at Drosophila NMJ. It will be interesting to further investigate the underlying mechanisms in detail (Peng, 2019).

Potential relevance of ROS-induced motor-circuit dysregulation for neurodegenerative diseases Downregulation of EAAT2 has been demonstrated in patients with Alzheimer's disease or amyotrophic lateral sclerosis (ALS), as well as in ALS rodent models. ALS is a fatal adult-onset disease that predominantly causes NMJ denervation, motor neuron degeneration, and compromised motor function. Spinal removal of mouse EAAT2 is sufficient to elicit motor neuron death. Transgenic expression of EAAT2or treatment with the small compound LDN/OSU-0212320, which mainly increases translation of EAAT2 mRNA, improves the motor performance of an ALS mouse model expressing hSOD1G93A. However, a recent clinical study testing ceftriaxone, an FDA-approved β-lactam antibiotic that can transcriptionally promote EAAT2 expression, in ALS patients concluded that this drug treatment had no therapeutic effect. Therefore, it is questionable whether increasing EAAT2 expression represents a feasible therapeutic strategy for ALS. It has been argued, however, that EAAT2 downregulation largely occurs at the posttranslational and not at the transcriptional level in ALS. There was no evidence for increased EAAT2 in patients treated with ceftriaxone, and ceftriaxone treatment only slightly increases protein levels of EAAT2 in hSOD1G93A mice. In addition, the efficacy of ceftriaxone in hSOD1G93A mice is not consistent among different studies. Hence, more investigations will be needed to strengthen evidence for the pathogenic contribution of EAAT2 dysfunction in ALS (Peng, 2019).

Oxidative stress is known as a hallmark of Alzheimer's disease, Parkinson's disease, and ALS. During aging, neurons are thought to be susceptible to excitotoxicity, and the nervous system and muscles are vulnerable to ROS accumulation because of high oxygen consumption demand. Administration of antioxidants can improve the motor function of hSOD1G93A mice and ALS patients. Nonetheless, how oxidative stress is produced in ALS and how this burden is involved in disease pathogenesis is not well understood. Interestingly, in this study, Drosophila Eaat1 depletion was shown to cause ALS-like characteristics, including motor neuron excitotoxicity, NMJ bouton abnormalities, muscle weakness, and compromised motor performance. In the future, it will be worth exploring whether ROS-induced motor circuit dysfunction might also participate in ALS progression and age-dependent motor system decline (Peng, 2019).

It has previously been shown that reduced excitability of proprioceptive sensory neurons and cholinergic interneurons is causative of locomotor CPG circuit dysfunction and compromised locomotion in Drosophila smn mutants, which are used as a Drosophila model of spinal muscular atrophy (SMA), a motor neuron disease of juveniles. Increasing neuronal excitability by 4-AP treatment reverses these motor system defects. Intriguingly, the current data show that long-term food-mediated feeding of (or even short-term exposure to) the K+ channel blocker 4-AP also rescued altered locomotor CPG activity in eaat1 mutants. Notably, although the precise mechanisms are unknown, 4-AP has been used to treat several motor system-related disorders such as spinal cord injury, Lambert-Eaton syndrome, and hereditary canine spinal muscular atrophy, and it is an FDA-approved therapy for multiple sclerosis. Thus, as supported by the current findings, it seems plausible that neuronal hypoexcitability may be a shared mechanism underlying the motor-system defects displayed in motor-related disorders (Peng, 2019).

Neuron-specific knockdown of Drosophila HADHB induces a shortened lifespan, deficient locomotive ability, abnormal motor neuron terminal morphology and learning disability

Mutations in the HADHB gene induce dysfunctions in the beta-oxidation of fatty acids and result in a Mitochondrial trifunctional protein (MTP) deficiency, which is characterized by clinical heterogeneity, such as cardiomyopathy and recurrent Leigh-like encephalopathy. In contrast, milder forms of HADHB mutations cause the later onset of progressive axonal peripheral neuropathy (approximately 50-80%) and myopathy with or without episodic myoglobinuria. The mechanisms linking neuronal defects in these diseases to the loss of HADHB function currently remain unclear. Drosophila has the CG4581 (dHADHB) gene as a single human HADHB homologue. This study established pan-neuron-specific dHADHB knockdown flies and examined their phenotypes. The knockdown of dHADHB shortened the lifespan of flies, reduced locomotor ability and also limited learning abilities. These phenotypes were accompanied by an abnormal synapse morphology at neuromuscular junctions (NMJ) and reduction in both ATP and ROS levels in central nervous system (CNS). The Drosophila NMJ synapses are glutamatergic that is similar to those in the vertebrate CNS. The present results reveal a critical role for dHADHB in the morphogenesis and function of glutamatergic neurons including peripheral neurons. The dHADHB knockdown flies established herein provide a useful model for investigating the pathological mechanisms underlying neuropathies caused by a HADHB deficiency (Li, 2019).

Non-enzymatic activity of the alpha-Tubulin acetyltransferase alphaTAT limits synaptic bouton growth in neurons

Neuronal axons terminate as synaptic boutons that form stable yet plastic connections with their targets. Synaptic bouton development relies on an underlying network of both long-lived and dynamic microtubules that provide structural stability for the boutons while also allowing for their growth and remodeling. However, a molecular-scale mechanism that explains how neurons appropriately balance these two microtubule populations remains a mystery. It was hypothesized that alpha-tubulin acetyltransferase (alphaTAT), which both stabilizes long-lived microtubules against mechanical stress via acetylation and has been implicated in promoting microtubule dynamics, could play a role in this process. Using the Drosophila neuromuscular junction as a model, this study found that non-enzymatic αTAT activity limits the growth of synaptic boutons by affecting dynamic, but not stable, microtubules. Loss of αTAT results in the formation of ectopic boutons. These ectopic boutons can be similarly suppressed by resupplying enzyme-inactive αTAT or by treatment with a low concentration of the microtubule-targeting agent vinblastine, which acts to suppress microtubule dynamics. Biophysical reconstitution experiments revealed that non-enzymatic alphaTAT1 activity destabilizes dynamic microtubules but does not substantially impact the stability of long-lived microtubules. Further, during microtubule growth, non-enzymatic αTAT activity results in increasingly extended tip structures, consistent with an increased rate of acceleration of catastrophe frequency with microtubule age, perhaps via tip structure remodeling. Through these mechanisms, αTAT enriches for stable microtubules at the expense of dynamic ones. It is proposed that the specific suppression of dynamic microtubules by non-enzymatic αTAT activity regulates the remodeling of microtubule networks during synaptic bouton development (Coombes, 2020).

Drosophila motor neuron boutons remodel through membrane blebbing coupled with muscle contraction

Wired neurons form new presynaptic boutons in response to increased synaptic activity, however the mechanism(s) by which this occurs remains uncertain. Drosophila motor neurons (MNs) have clearly discernible boutons that display robust structural plasticity, being therefore an ideal system in which to study activity-dependent bouton genesis. This study showed that in response to depolarization and in resting conditions, MNs form new boutons by membrane blebbing, a pressure-driven mechanism that occurs in 3-D cell migration, but not previously described to occur in neurons. Accordingly, F-actin is decreased in boutons during outgrowth, and non-muscle myosin-II is dynamically recruited to newly formed boutons. Furthermore, muscle contraction plays a mechanical role, which is hypothesized to promotes bouton addition by increasing MN confinement. Overall, this study identified a mechanism by which established circuits form new boutons allowing their structural expansion and plasticity, using trans-synaptic physical forces as the main driving force (Fernandes, 2023).

Dissection of the mechanisms that regulate activity-dependent bouton formation is critical to understand remodeling strategies required for neuronal growth and wiring. Using live imaging of the Drosophila larval NMJ, a system in which 3-D intercellular and biophysical interactions are preserved, this study showed that bouton formation occurred by membrane blebbing, a mechanism widely used in 3-D cellular migration but, to our knowledge, never reported to be used by neurons to remodel. This study showed that new boutons induced by elevated activity, also called ghost boutons (GB), are bona fide blebs, whose growth does not rely on actin polymerization but is rather pressure driven. Additionally, it was observed that activity-dependent bouton formation was frequently associated with muscle activity and that blocking muscle contraction significantly decreased GB frequency after stimulation. Moreover, the manipulation of synaptic activity and/or muscle contraction resulted in predictable changes in bouton formation in response to stimulation, with a progressive increase in new bouton numbers with higher levels of synaptic activity and/or muscle contraction (Fernandes, 2023).

The results suggest that muscle contraction plays a mechanical role in activity-dependent bouton formation. At the NMJ, the MN is deeply imbedded in the muscle, which by contracting can directly alter neuronal confinement. It is hypothesized that with elevated activity, and in response to a still unclear pre and/or postsynaptic signal to initiate bouton outgrowth, MNs add new boutons by membrane blebbing, and muscle contraction is required to increase neuronal confinement. This confinement results in increased pressure onto the MN membrane, powering bouton outgrowth at places putatively primed for bouton formation. In accordance, it is known that blebs are favored in conditions of high confinement, namely in tissues where cells encounter increased mechanical resistance. Furthermore, cells in compressive 3-D environments naturally go through stiffness gradients, which can polarize the recruitment of molecules required to or that facilitate blebbing in spots of high membrane tension. Likewise, at the NMJ it is possible that regions of higher activity produce more signals for inducing synaptic growth, which is supported by previous studies that showed that synaptic plasticity at the NMJ, including GB formation, requires activity-dependent secretion of postsynaptic signals such as BMP ligands (Maverick and Gbb) and Ca2+-sensitive vesicle regulator Syt-IV, presynaptic Wg and Syt-I mediated NT release and postsynaptic glutamate receptor function. It will be interesting to investigate whether activation of pathways downstream of these factors can converge to determine sites primed for bouton initiation with synaptic activity, by recruiting initiation factors and/or changing cytoskeletal dynamics at these regions (Fernandes, 2023).

Cells migrating in confined environments typically display rounder morphologies and use hydrostatic blebs for movement. Interestingly, it has been shown that neurons from mouse central nervous system (CNS) growing in 3-D matrices, rather than 2-D surfaces, displayed GCs that had an amoeboid-like morphology, characterized by very low adhesive interactions and extensive rounded deformations of the body-wall, challenging the paradigm of the lamellipodia based GC. In the brain, even though there are no muscle compressive forces, synaptic boutons are equally confined and surrounded by other neuronal, astrocytic, or microglial processes. Interestingly, a recent study showed that in the mammalian brain, enlargement of dendritic spines produces mechanical pressure onto boutons thereby enhancing NT release, thus contributing to synaptic strength (a force reported to be comparable to that of muscle contraction). This exciting discovery is in line with the hypothesis that mechanical force directly regulates synaptic function, or as in the case of this study, formation of new boutons. It will be interesting to study how neurons can sense and respond to mechanical signals provided by the cellular environment, and if pathways used for mechanosensing and transduction, likely regulating adhesion and contacts with extracellular partners, are coupled with synaptic transmission machinery during plasticity (Fernandes, 2023).

While the molecular identity of the signal that leads to bouton initiation remains to be discovered, it is well established that blebs nucleate when a small patch of membrane is detached from the actin cortex. This can happen either as a direct consequence of buildup in hydrostatic pressure (that detaches membrane to cortex binding proteins) or by formation of local gaps, resulting from rupture of the cortex in regions of high membrane energy (where accumulation of MyoII helps to weaken the cortex). Our data suggests that, although at the NMJ the two mechanisms may coexist, with elevated activity boutons tend to form mainly because of compressive forces exerted by the muscle. Supporting this, it was found that activity-induced bouton formation was usually fast and correlated with visible muscle contraction, while events with low or no visible muscle contraction were slower and occasional. Additionally, post-stimulation, even though MyoII puncta were observed preceding bouton formation in ~35% of events, boutons were found forming with low MyoII levels and decreasing or inactivation of MyoII did not prevent bouton formation, which suggests that MyoII was not required to propel bouton growth induced by NMJ stimulation (although this result can not directly be extended to a null scenario). Interestingly, MyoII was recruited to ghost boutons in stimulated and unstimulated NMJs, which may explain cases where boutons formed with lessened muscle contractions. The fact that boutons always formed without filamin, which links actin to membrane, further supported that actin polymerization is not required for bouton initiation. Altogether a model is favored in which boutons form in regions of high pressure by detachment of membrane from the actin cortex and that local MyoII activation can facilitate the weaking of the cortex at these sites. Importantly, regardless of the exact signal used to initiate activity-dependent bouton formation, this study showed that growth and stabilization of these boutons requires both actin and MyoII local rearrangements analogous to what is reported for cellular blebs: weakened actomyosin cortex in early-stage boutons and enrichment after expansion (Fernandes, 2023).

This model explains the formation of new synaptic boutons by a physical process-membrane blebbing, which in addition to the more studied transcriptional or biochemical factors, allows fast, on demand expansion of the NMJ. Whether axons are always competent for bleb-dependent bouton expansion or require priming by cytoskeletal regulation remains to be elucidated. Moreover, considering the conservation of presynaptic cytoskeletal components and bouton ultrastructure throughout evolution, it is postulated that this mechanism of synapse remodeling can be present in other organisms, including vertebrates. Overall, the finding that bouton addition at the NMJ occurs as result of a MN-muscle physical interplay highlights the importance of intercellular cooperation during plastic changes. Circuit remodeling as a response to experience requires rapid modulations of bouton number. We speculate that the regulation of confinement by non-neuronal cells (muscle or glia) can be a mechanism widely used by the nervous system to coordinate local activity-dependent structural changes in neurons with its surrounding 3-D cellular microenvironment. Future studies will elucidate whether the understanding of the biochemical and mechanical relations between neurons and their neighboring cells during structural plasticity can help design new strategies to remodel neuronal circuits that have been impaired by neurodevelopmental or neurodegenerative diseases (Fernandes, 2023).

Miles to go (mtgo) encodes FNDC3 proteins that interact with the chaperonin subunit CCT3 and are required for NMJ branching and growth in Drosophila

Analysis of mutants that affect formation and function of the Drosophila larval neuromuscular junction (NMJ) has provided valuable insight into genes required for neuronal branching and synaptic growth. This study reports that NMJ development in Drosophila requires both the Drosophila ortholog of FNDC3 genes; CG42389 (herein referred to as miles to go; mtgo), and CCT3, which encodes a chaperonin complex subunit. Loss of mtgo function causes late pupal lethality with most animals unable to escape the pupal case, while rare escapers exhibit an ataxic gait and reduced lifespan. NMJs in mtgo mutant larvae have dramatically reduced branching and growth and fewer synaptic boutons compared with control animals. Mutant larvae show normal locomotion but display an abnormal self-righting response and chemosensory deficits that suggest additional functions of mtgo within the nervous system. The pharate lethality in mtgo mutants can be rescued by both low-level pan- and neuronal-, but not muscle-specific expression of a mtgo transgene, supporting a neuronal-intrinsic requirement for mtgo in NMJ development. Mtgo encodes three similar proteins whose domain structure is most closely related to the vertebrate intracellular cytosolic membrane-anchored fibronectin type-III domain-containing protein 3 (FNDC3) protein family. Mtgo physically and genetically interacts with Drosophila CCT3, which encodes a subunit of the TRiC/CCT chaperonin complex required for maturation of actin, tubulin and other substrates. Drosophila larvae heterozygous for a mutation in CCT3 that reduces binding between CCT3 and MTGO also show abnormal NMJ development similar to that observed in mtgo null mutants. Hence, the intracellular FNDC3-ortholog MTGO and CCT3 can form a macromolecular complex, and are both required for NMJ development in Drosophila (Syed, 2019)

POU domain motif3 (Pdm3) induces wingless (wg) transcription and is essential for development of larval neuromuscular junctions in Drosophila

Wnt is a conserved family of secreted proteins that play diverse roles in tissue growth and differentiation. Identification of transcription factors that regulate wnt expression is pivotal for understanding tissue-specific signaling pathways regulated by Wnt. This study identified pdm3^m7, a new allele of the pdm3 gene encoding a POU family transcription factor, in a lethality-based genetic screen for modifiers of Wingless (Wg) signaling in Drosophila. Interestingly, pdm3^m7 larvae showed slow locomotion, implying neuromuscular defects. Analysis of larval neuromuscular junctions (NMJs) revealed decreased bouton number with enlarged bouton in pdm3 mutants. pdm3 NMJs also had fewer branches at axon terminals than wild-type NMJs. Consistent with pdm3^m7 being a candidate wg modifier, NMJ phenotypes in pdm3 mutants were similar to those of wg mutants, implying a functional link between these two genes. Indeed, lethality caused by pdm3 overexpression in motor neurons was completely rescued by knockdown of wg, indicating that pdm3 acts upstream to wg. Furthermore, transient expression of pdm3 induced ectopic expression of wg-LacZ reporter and wg effector proteins in wing discs. It is proposed that pdm3 expressed in presynaptic NMJ neurons regulates wg transcription for growth and development of both presynaptic neurons and postsynaptic muscles (Kim, 2020).

Transcription factors play essential roles by inducing genes during the formation of body plans, organ development, tissue specificity, and generation of diverse cell types. Numerous transcription factors are grouped based on similarity in their sequences and domain structures. Pituitary-specific positive transcription factor 1, Octamer transcription factor-1, Uncoordinated-86 domain (POU) transcription factors belong to a subfamily of homeodomain transcription factors, and are highly conserved in all metazoans. POU domain consists of two DNA binding domains, POU homeodomain and POU specific domain, and these two domains are linked by a flexible linker. Based on sequence homology of the POU domain and the linker, POU proteins are grouped into six classes. POU proteins are often expressed in spatiotemporally restricted patterns during development, implying that they may be specialized for differentiation of specific cells or tissues by activating required signal transduction pathways (Kim, 2020).

The class VI Drosophila POU domain motif 3 (Pdm3) protein is reported to function in olfactory receptor neurons (ORNs) by regulating olfactory receptor gene expression and axon targeting, and in ring (R) neurons by regulating the development of ellipsoid body (EB) and axon targeting to EB in the central brain. pdm3 is also important for the axon targeting of a type of tracheal dendrite (td) neurons. In particular, td neurons that normally form synapse in the nerve cord change their target to the central brain by ectopic expression of Pdm3. Besides the neuronal functions of Pdm3, pdm3 also acts as a repressor of abdominal pigmentation in D. melanogaster, and plays a role in female-limited color dimorphism in abdomen of D. montium. Despite these studies, it is still unknown how pdm3 performs these neuronal and non-neuronal functions (Kim, 2020).

pdm3^f00828 and pdm3¹ homozygotes exhibit defects in axon targeting, odor perception, and locomotion. pdm3^f00828 allele has insertion of a piggyback element in an intron near the 3' end of the pdm3 gene, and pdm3¹ has a premature stop codon in the middle of the coding region that results in the deletion of the POU domain. This study identified a new pdm3 allele, pdm3^m7, as a suppressor of lethality induced by Sol narae (Sona) overexpression in a genetic screen. Sona is a fly ADAMTS (A disintegrin and metalloprotease with thrombospondin motif) whose family members are secreted metalloproteases important for cell proliferation, cell survival and development. This study has shown that Sona positively regulates Wingless (Wg) signaling and is essential for fly development, cell survival, and wg processing. wg is a prototype of Wnt family that initiates signal transduction cascade as extracellular signaling proteins, and activation of Wnt signaling leads to transcriptional induction of multiple genes for regulation of cell proliferation, cell survival, cell fate decision, and cell migration. wg is important for the development of all appendages, and the wing imaginal disc has been a great tool to study wg signaling because wg secreted from its dorsal-ventral midline is crucial for growth and development of wings (Kim, 2020).

Wg also plays an essential role in the development of NMJ. During larval development, NMJs continue to form synaptic boutons that are specialized structures with axon terminals of motor neurons surrounded by reticular subsynaptical reticulum (SSR) formed by the plasma membrane of postsynaptic muscle19. Among multiple types of boutons such as type Ib, Is, II, and III, wg is secreted at a high level from the glutamatergic type Ib bouton known as the main localization site of wg protein and wg signaling components, and is absent or at very low levels in other types of boutons (Packard, 2002; Kim, 2020).

Type Ib boutons also have more extensive SSR compared to other bouton types, so are easily detected by the high level of Discs-Large (Dlg) as a postsynaptic marker. Type Ib boutons in NMJs of wg mutants show reduction in bouton number but increase in bouton size. Components in wg signaling such as Arrow (Arr) that positively regulates wg signaling as a coreceptor of wg also shows its mutant phenotype similar to wg, but Shaggy (Sgg)/GSK3β that negatively regulates wg signaling as a kinase shows opposite phenotype to wg. Thus, dynamic regulation of wg signaling is essential for the development of NMJ (Kim, 2020).

Secreted wg also signals to the presynaptic motor neuron to regulate Futsch, one of the microtubule-associated proteins (MAPs). Futsch is a homolog of mammalian MAP1B, and both Futsch and MAP1B are phosphorylated at a conserved site by Sgg/GSK3β. The phosphorylated MAP1B does not bind microtubules, which results in reduced stability of microtubules. Therefore, localization of Futsch at NMJ faithfully reflects the stability of microtubules that is dynamically regulated by wg signaling. Loss of futsch phenotype is similar to the loss of wg phenotype in NMJ (Kim, 2020).

This study reporta that pdm3 is identified as a suppressor of Sona-induced lethality. Based on the involvement of Sona in wg signaling and the neuronal role of Pdm3, the roles of pdm3 in NMJ were specifically studied. Similar to loss of wg, loss of pdm3 in NMJ caused decrease in number but increase in size of boutons. Lethality induced by overexpressed pdm3 was completely rescued by the knockdown of wg in motor neurons but not vice versa. This indicated that pdm3 functions upstream to wg, and prompted a test whether pdm3 can induce wg transcription. Indeed, transient expression of pdm3 in wing discs induced wg transcription and wg effector proteins. Based on these data, it is propose that one of the main functions of pdm3 in NMJ is to induce wg transcription (Kim, 2020).

This study reports that pdm3 regulates growth and development of NMJs. pdm3 mutants showed increase in bouton size and decrease in bouton number, which are similar to the phenotype of wg mutants. Lethality induced by the overexpression of pdm3 was rescued by knockdown of wg in NMJ, indicating that pdm3 functions upstream to wg. Furthermore, overexpression of pdm3 induced wg transcription in wing discs. It is proposed that a major function of pdm3 in motor neurons is to induce wg transcription, and secreted wg from motor neurons regulates growth, development, and maturation of both pre- and post-synaptic regions of NMJ (Kim, 2020).

The mammalian homolog of pdm3 is Brain-5 (Brn-5)/POU class 6 homeobox 1 (POU6F1) mainly expressed in brain and spinal cord. Brn-5 is heavily expressed in embryonic brain but also expressed in adult brain and multiple adult organs such as kidney, lung, testis, and anterior pituitary. In developing brain, Brn-5 is expressed in postmitotic neurons after neuronal progenitor cells exit cell cycle in the early process of terminal neuronal differentiation. Therefore, both pdm3 and Brn-5 function in differentiation of neurons. Interestingly, ectopic expression of Brn-5 inhibits DNA synthesis, which is similar to cell cycle arrest phenotype by wg overexpression. Given the homology between pdm3 and Brn-5 as well as functional similarities, Brn-5 may also induce wnt transcription (Kim, 2020).

Most of pdm3 functions identified so far are related to the maturation of neurons such as olfactory neurons, R neurons and td neurons as well as their postsynaptic partners. Ectopic expression of pdm3 induced lethality without exception, indicating that expression of pdm3 in fly tissues is generally repressed in vivo in order to express wg under the strict spatiotemporal control. An important question is whether pdm3 directly transcribe wg. This study found that wg transcription is induced only after 36 hours of transient overexpression of Pdm3. It is possible that the level of pdm3 needs to be over a threshold to induce wg transcription. Alternatively, pdm3 may need to turn on other components to indirectly induce wg transcription. DNA sequence of Brn-5 binding site has been reported, so analysis on wg and wnt regulatory regions will help understand the mechanism of wnt induction by pdm3 and Brn-5 (Kim, 2020).

This study consistently found more significant NMJ phenotypes in A2 than A3 in both pdm3 and wg mutants. Therefore, pdm3 and wg may play more prominent roles in the A2 than the A3 segment. In fact, the level of pdm3 was higher in the anterior region than the posterior region of ventral ganglion, which suggests that more wg may be present in the NMJs of anterior abdominal segments. Consistent with this idea, the number of type Ib boutons in the A2 segment was 1.8 times more than A3 segment. One difference between pdm3 and wg mutants is the lack of certain phenotypes in the A3 segment of pdm3 NMJs: the size of boutons and the number of axon terminals in A3 were not affected in pdm3 mutant. It is possible that pdm3 turns on both common and segment-specific genes besides wg, and A3 segment-specific components may alleviate the loss of wg phenotype in the A3 segment. Similarly, other proteins induced by pdm3 may also play important roles in NMJ growth, differentiation and maintenance. In fact, multiple signaling pathways including Glass-bottom-boat (Gbb) pathway also play roles in NMJ development. Gbb is secreted from muscles and induces development of both pre- and post-synaptic structures, similar to wg signaling (Kim, 2020).

This study identified a defective hobo element in the pdm3^m7 allele. The hobo element belongs to Ac family found in maize and has short inverted terminal repeats. Laboratory and wild strains of D. melanogaster have average 28 and 22 copies of hobo elements in the genome that are either full-length or defective, respectively. Because other suppressors identified in the genetic screen using Sona overexpression did not have hobo element in the pdm3 gene, the transposition of the hobo element to the pdm3 gene may have occurred subsequent to the generation of a point mutation in the arr gene by EMS. Since both arr and pdm3 are positively involved in wg signaling, this hobo insertion may have helped the original arr^m7 mutation to further decrease the activity of wg signaling under the condition of Sona overexpression (Kim, 2020).

Besides the neuronal roles of Pdm3, all pdm3 mutants show minor but consistent defects in planar cell polarity in a restricted region of the wing as well as adhesion between the dorsal and ventral wing blades. Other phenotypes such as wing drooping and premature death were also observed in all pdm3 mutants, but these may be due to malformation of synaptic structures. pdm3 also plays a role in female-limited color dimorphism in abdomen of D. montium. The authors found in sexually dimorphic females that the first intron of the pdm3 gene has four tandem sets with predicted binding sites for the HOX gene Abdominal-B (Abd-B) and the sex determination gene doublesex (dsx). Interestingly, it has been shown that wg expression is repressed by the combinatory work of Abd-B and Dsx proteins. Taken together, it is possible that transcription of wg and pdm3 is co-repressed by Abd-B and Dsx. Such co-repression of wg and pdm3 transcription may be also required for synaptic growth and differentiation in neurons. Further studies on pdm3 will help understand how this understudied transcription factor is involved in the final differentiation of various cell types (Kim, 2020).

O-GlcNAcase contributes to cognitive function in Drosophila

O-GlcNAcylation is an abundant post-translational modification in neurons. In mice, an increase in O-GlcNAcylation leads to defects in hippocampal synaptic plasticity and learning. O-GlcNAcylation is established by two opposing enzymes O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). To investigate the role of OGA in elementary learning, this study generated catalytically inactive and precise knock-out Oga alleles (Oga(D133N) and Oga(KO), respectively) in Drosophila melanogaster. Adult Oga(D133N) and Oga(KO) flies lacking O-GlcNAcase activity showed locomotor phenotypes. Importantly, both Oga lines exhibited deficits in habituation, an evolutionary conserved form of learning, highlighting that the requirement for O-GlcNAcase activity for cognitive function is preserved across species. Loss of O-GlcNAcase affected number of synaptic boutons at the axon terminals of larval neuromuscular junction. Taken together, this study report behavioral and neurodevelopmental phenotypes associated with Oga alleles and show that Oga contributes to cognition and synaptic morphology in Drosophila (Muha, 2020).

Developmental arrest of Drosophila larvae elicits presynaptic depression and enables prolonged studies of neurodegeneration

Synapses exhibit an astonishing degree of adaptive plasticity in healthy and disease states. This study has investigated whether synapses also adjust to life stages imposed by novel developmental programs for which they were never molded by evolution. Under conditions where Drosophila larvae are terminally arrested, this study has characterized synaptic growth, structure and function at the neuromuscular junction (NMJ). While wild-type larvae transition to pupae after 5 days, arrested third instar (ATI) larvae persist for 35 days, during which NMJs exhibit extensive overgrowth in muscle size, presynaptic release sites, and postsynaptic glutamate receptors. Remarkably, despite this exuberant growth, stable neurotransmission is maintained throughout the ATI lifespan through a potent homeostatic reduction in presynaptic neurotransmitter release. Arrest of the larval stage in stathmin mutants also reveals a degree of progressive instability and neurodegeneration that was not apparent during the typical larval period. Hence, an adaptive form of presynaptic depression stabilizes neurotransmission during an extended developmental period of unconstrained synaptic growth. More generally, the ATI manipulation provides a powerful system for studying neurodegeneration and plasticity across prolonged developmental timescales (Perry, 2020).

Structural and functional synaptic plasticity induced by convergent synapse loss in the Drosophila neuromuscular circuit

Throughout the nervous system, the convergence of two or more presynaptic inputs on a target cell is commonly observed. The question asked in this study is to what extent converging inputs influence each other's structural and functional synaptic plasticity. In complex circuits, isolating individual inputs is difficult because postsynaptic cells can receive thousands of inputs. An ideal model to address this question is the Drosophila larval neuromuscular junction (NMJ) where each postsynaptic muscle cell receives inputs from two glutamatergic types of motor neurons (MNs), known as 1b and 1s MNs. Notably, each muscle is unique and receives input from a different combination of 1b and 1s MNs; this study surveyed multiple muscles for this reason. In this study a cell-specific promoter was identified that allows ablation of 1s MNs post-innervation and structural and functional responses of convergent 1b NMJs were measured using microscopy and electrophysiology. For all muscles examined in both sexes, ablation of 1s MNs resulted in NMJ expansion and increased spontaneous neurotransmitter release at corresponding 1b NMJs. This demonstrates that 1b NMJs can compensate for the loss of convergent 1s MNs. However, only a subset of 1b NMJs showed compensatory evoked neurotransmission, suggesting target-specific plasticity. Silencing 1s MNs led to similar plasticity at 1b NMJs, suggesting that evoked neurotransmission from 1s MNs contributes to 1b synaptic plasticity. Finally, 1s innervation was genetically blocked in male larvae and robust 1b synaptic plasticity was eliminated, raising the possibility that 1s NMJ formation is required to set up a reference for subsequent synaptic perturbations (Wang, 2021).

The nervous system is characterized by complex wiring patterns that include different neurons converging onto the same postsynaptic cell. This wiring paradigm is found in pyramidal neurons that receive input from excitatory and inhibitory contacts, and in esophageal striated muscles that receive enteric and vagal nerve inputs. While dynamic regulation of individual synapses has been examined, interplay between convergent neurons has been predominantly studied by monitoring postsynaptic spine changes. Understanding how convergent neurons respond to such perturbations will shed light on the etiologies of neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS), which display progressive neuronal cell death and devastating functional consequences (Wang, 2021).

Analysis of individual inputs in the central nervous system is complicated by the high density of converging inputs on the same target cell. The Drosophila larval neuromuscular circuit, however, circumvents this with a simple, hard-wired connectivity map. The body plan is segmentally repeated and each hemisegment is bilaterally symmetrical and comprised of 35 motor neurons (MNs) and 30 postsynaptic muscles. Like most vertebrate central synapses, the individual glutamatergic neuromuscular contacts consist of axon terminal swellings, called boutons, and elaborate postsynaptic complexes. Each bouton houses several active zones (AZs) that enable neurotransmission. Larval muscles are innervated by two main MNs, type 1b (big) and 1s (small), which resemble tonic and phasic neurons, respectively, unlike vertebrate skeletal muscles that are monoinnervated. Most 1b MNs innervate a single muscle, whereas 1s MNs innervate functional muscle groups, highlighting important structural and functional distinctions between these classes of neurons (Wang, 2021).

In the larval neuromuscular circuit, 1b and 1s MNs are required for normal locomotion. Each MN type has unique electrophysiological properties: 1b MNs display a higher rate of spontaneous neurotransmitter release but the quantal size is smaller compared to 1s MNs (Newman, 2017; Nguyen, 2016). In traditional neuromuscular junction (NMJ) electrophysiology experiments, excitatory postsynaptic potentials (EPSPs) in muscles represent simultaneous stimulation of both neurons. However, several studies have isolated 1b-derived and 1b+1s-derived EPSPs from different muscles and demonstrated that convergent1b and 1s MNs do not equally contribute to the EPSP amplitude. Several forms of synaptic plasticity have been observed in the larval NMJ including facilitation and depression. As each muscle is co-innervated and individual synaptic activities can be distinguished, this system provides an ideal platform to investigate structural and physiological plasticity changes that enable one MN to compensate for perturbations of a convergent MN. A recent study found that ablating the 1s MN on muscle 1 (m1) elevated evoked neurotransmission of the corresponding 1b NMJ (Aponte-Santiago, 2020). However, each muscle is innervated by a unique 1b-1s MN pair, so it is unclear whether this plasticity is consistent across all muscles (Wang, 2021).

This study examined three separate muscles to test structural and functional plasticity. First, muscle-specific 1b activities were confirmed. Next, 1s MNs were ablated after innervation, and corresponding 1b NMJ compensatory responses were examined. All three 1b NMJs increased their bouton numbers and rate of spontaneous neurotransmitter7 release without a corresponding upregulation of active zones (AZs) and some 1b NMJs also8 partially increased their evoked neurotransmission. An important factor for 1b synaptic plasticity9 is the 1s EPSP as silencing 1s evoked activity caused similar 1b NMJ responses. Additionally,0 robust 1b synaptic plasticity does not occur if the 1s NMJ is never formed, indicating initial 1s innervation is required. These results demonstrate a novel, target-specific synaptic plasticity that2 may be a common feature of convergent neural circuits (Wang, 2021).

This study examined the Drosophila neuromuscular circuit and demonstrate 1b synaptic plasticity upon loss of convergent 1s MNs. The muscles examined in this study, m6, m12, and m4, are co-innervated by unique 1b-1s MN pairs. First, an activity baseline was established in wild type and uncovered that 1b MNs contribute a unique percentage of the total EPSP in a muscle-specific manner. Genetic ablation of 1s MNs (vCE and dCE) after innervation led to expansion of 1b NMJs and elevation of their spontaneous release rates. Furthermore, some 1b MNs elevated evoked neurotransmission while others remained unchanged. A recent study examining m1 found similar 1b functional plasticity when m1-1s was ablated (Aponte-Santiago, 2020), but no structural compensation. This target specificity indicates heterogeneity in synaptic plasticity mechanisms. Silencing 1s MNs yielded partial m4-1b compensation, suggesting 1s evoked neurotransmission is an important factor in eliciting 1b NMJ plasticity. In mutant larvae where the m4-1s NMJs never form, no changes were observed in m4-1b bouton number or spontaneous release rate, and decreased EPSP compensation. These results suggest that initial 1s innervation may set up a reference point for robust 1b synaptic plasticity (Wang, 2021).

A well-established form of plasticity at the larval NMJ is synaptic homeostasis, which can manifest in pre- and/or postsynaptic changes, including neurotransmitter release and neurotransmitter receptor dynamics and abundance, respectively. Presynaptic homeostatic plasticity (PHP) maintains EPSPs at baseline when postsynaptic glutamate receptor function is perturbed, and can be induced in a synapse specific manner. In this study, the loss of 1s MNs could be interpreted as a decrease in glutamate receptor function by the muscle and trigger similar PHP mechanisms to induce compensatory changes at adjacent 1b NMJs. Thus, further analysis of PHP pathways could reveal mechanisms underlying this convergent plasticity (Wang, 2021).

1b NMJs must adapt to accommodate the increased spontaneous and evoked neurotransmission. The results do not reveal robust expansion of AZs or GluRs to support 1b synaptic plasticity, as observed in other studies where NMJ size is altered but total AZs or QCs remain the same (Aponte-Santiago, 2020; Goel, 2019a; Goel, 2019b; Goel, 2020). Therefore, it is proposed that existing AZs may alter their properties. AZs exist in two different states, active or silent, and a subset of AZs specialize in spontaneous release or evoked neurotransmission. Thus, even without an increase in AZs, 1b plasticity mechanisms may modify AZ properties to respond to the loss of 1s inputs. Overall, the data suggest silent AZs may become activated to increase the pools of spontaneous and evoked AZs as ablation of 1s MNs led to enhanced 1b spontaneous release rates, target-specific compensation of EPSPs, and increased QC. Additionally, spontaneous and evoked activities may be independently regulated. Furthermore, the readily-releasable pool (RRP) size is under dynamic control during synaptic plasticity and could modulate m4-1b evoked neurotransmitter release. Detailed examination of AZs will significantly bolster understanding of mechanism underlying 1b NMJ plasticity. Prior studies reported that spontaneous neurotransmitter release regulates synaptic development in both mammals and Drosophila. Thus, the expanded size of all 1b3 NMJs following 1s ablation may be caused by the elevated spontaneous activity. These data also suggest that all 1b MNs can detect and respond to the loss of adjacent 1s inputs. However, the ability to differentially compensate the spontaneous and evoked activity is likely due to independent mechanisms since only some 1b MNs elevate their EPSPs (Wang, 2021).

In complex neural circuits, dissecting contributions of individual inputs to the total postsynaptic activity, also referred to as synaptic weight, remains difficult due to thousands of converging inputs on a single cell. The larval NMJ facilitates the partitioning of synaptic inputs as each muscle is innervated by few MNs. This study combined electrophysiology with calcium imaging and found that 1b synaptic weights differ on m6, m12, and m4. Taken together with the degree of EPSP compensation after ablation of 1s5 MNs, there was a direct correlation with the level of target-specific synaptic weight. Thus, robust 1b MNs that carry more synaptic drive may be endowed with certain synaptic plasticity mechanisms that respond to loss of adjacent inputs. However, regulatory roles for type II and type III MNs that are present on some muscles cannot be rule out (Wang, 2021).

Interestingly, a similar correlation exists in Hebbian plasticity, where stronger synapses are more likely strengthened than weaker ones. This correlation is also reflected in PHP. Two studies examined input-specific PHP on different muscles: on m4, PHP can be only induced at 1b NMJs (Newman, 2017); however, on m6, PHP can be induced on both 1b and 1s NMJs (Genc, 2019). This correlates with the observation that the m4-1b has more synaptic weight than m4-1s, whereas m6-1b and m6-1s have similar synaptic weights. Taken together, homeostatic plasticity varies in target-specific and input-specific manners, suggesting heterogeneous mechanisms (Wang, 2021).

Models of synaptic homeostasis rely on an activity set point to stabilize neurons when confronted with perturbations. Each target neuron must account for all presynaptic inputs to produce a defined output (i.e., the set point). The structural and functional properties of each input thus determine not only its contribution to the postsynaptic activity but also its ability to respond to perturbations in synaptic function. For example, transcription factors not only regulate the temporal expression of ion channels that shape neuronal excitability, but also homeostatic mechanisms. Like many activity-dependent processes, the optimal set point may be established during a narrow time-window of development. This hypothesis was tested in a Drosophila seizure mutant by inhibiting activity during embryonic development and observing suppression of seizures in postembryonic stages. Thus, manipulating activity during an embryonic critical period4 may alter the activity set point (Wang, 2021).

In this study, one intriguing hypothesis is that 1b+1s co-innervation determines the EPSP set point during embryogenesis and is referenced by some 1b NMJs to compensate for the loss of 1s MNs. A model is proposed to describe how 1b NMJs increase their sizes and spontaneous and evoked neurotransmission due to the loss of convergent 1s MNs. The neuromuscular innervation map is formed during late embryonic development, the activity set point is defined in a target-specific manner and how neurons respond to dysfunctional neighbors (Wang, 2021).

Molecular logic of synaptic diversity between Drosophila tonic and phasic motoneurons

Although neuronal subtypes display unique synaptic organization and function, the underlying transcriptional differences that establish these features are poorly understood. To identify molecular pathways that contribute to synaptic diversity, single-neuron Patch-seq RNA profiling was performed on Drosophila tonic and phasic glutamatergic motoneurons. Tonic motoneurons form weaker facilitating synapses onto single muscles, while phasic motoneurons form stronger depressing synapses onto multiple muscles. Super-resolution microscopy and in vivo imaging demonstrated that synaptic active zones in phasic motoneurons are more compact and display enhanced Ca(2+) influx compared with their tonic counterparts. Genetic analysis identified unique synaptic properties that mapped onto gene expression differences for several cellular pathways, including distinct signaling ligands, post-translational modifications, and intracellular Ca(2+) buffers. These findings provide insights into how unique transcriptomes drive functional and morphological differences between neuronal subtypes (Jetti, 2023).

Although pathways driving neuronal development and function are well characterized, how diversity in neuronal subtypes across the brain is established and maintained is poorly understood. With the advent of single-cell transcriptomics, mRNA expression profiles that contribute to neuronal morphology, connectivity, and function are being identified. One feature of diversity is the unique synaptic properties observed across neuronal classes. Synapses represent fundamental building blocks of information processing, exhibiting distinct synaptic vesicle (SV) release probability (Pr), response kinetics, and short-term plasticity. Notable examples of synaptic diversity include Caenorhabditis elegans (C. elegans) AWC and ASH olfactory neurons, invertebrate tonic and phasic motoneurons (MNs), zebrafish ON and OFF bipolar cells, and mammalian hippocampal, cerebellar, and auditory neurons. Functional heterogeneity in synaptic transmission contributes to temporal coding, processing of multisensory information, and circuit computations. Despite these roles, the differentially expressed genes (DEGs) that collectively specify differences in synaptic structure and output are largely unknown (Jetti, 2023).

Neurons displaying tonic or phasic synaptic output are conserved from invertebrates to mammals and often co-innervate postsynaptic targets. Tonic and phasic synapses increase the robustness of information processing by acting as high- and low-pass filters, respectively. Drosophila larvae contain MNs with tonic or phasic output that co-innervate muscles and form glutamatergic neuromuscular junctions (NMJs). Tonic MNs have bigger 'type Ib' boutons and typically innervate single muscles to act as primary drivers of contraction. Phasic MNs form smaller 'type Is' boutons that innervate and coordinate contraction of muscle subgroups. Phasic MN active zones (AZs) display higher Pr and show synaptic depression during stimulation. In contrast, tonic MNs have weaker synaptic output and undergo facilitation. Tonic and phasic MNs also show differences in morphology, inputs, membrane excitability, and synaptic plasticity. The molecular logic that governs these differences is largely unknown, providing a system to characterize DEGs that establish and maintain these distinct properties. This study combined single-neuron Patch-seq RNA profiling with quantal imaging, stimulated emission depletion (STED) nanoscopy, transmission electron microscopy (TEM), optogenetics, electrophysiology, and genetic manipulations to identify and dissect molecular components contributing to the structural and functional diversity of Drosophila tonic and phasic MNs. RNA sequencing (RNA-seq) identified distinct transcriptional profiles for each MN subtype, while genetic analyses indicate multiple DEGs contribute to functional and morphological differences in AZ organization, Pr, and Ca 2+ buffering (Jetti, 2023).

This study describes the nanoscopic, transcriptomic, and post-translational signatures that contribute to distinct features encoding neuropeptide receptors, synaptic cleft proteins, axonal pathfinding and cell adhesion molecules, membrane excitability and ionic balance regulators, and the Arl8 and Unc104 axonal transport factors, were also differentially expressed. Together, these data provide a framework for characterizing molecular mechanisms contributing to unique features of tonic and phasic synaptic diversity. This analysis is consistent with a model in which AZ structural differences and altered Ca 2+ entry and buffering contribute to release differences between Ib and Is MNs. Given that phasic Is synapses have enhanced synaptic strength and higher Pr, their unique stellate AZ architecture and smaller cytomatrix, together with shorter T-bars and larger SVs, could contribute to distinct AZ Ca2+ dynamics and alterations in coupling Ca 2+ influx to SV fusion. Previous studies demonstrated that changes in the coupling distance between SVs and Ca 2+ channels can alter synaptic strength at a number of synapse types. Increased expression of the Cbp53E Ca 2+ buffer in Is MNs lowers resting [Ca 2+ ] and restricts facilitation, which, together with enhanced Ca 2+ influx, provides a synergistic mechanism with the more compact Is AZ nanostructure that could enhance release. Prior studies identified additional differences in SV availability and fusogenicity that also contribute to differences in Ib and Is release properties. Reduced expression of Brp, the decoy SNARE Tomosyn, and the fusion clamp Complexin at Is terminals alters the size of SV pools and their availability for fusion, indicating that Ib synapses have fewer fusogenic SVs available to respond to Ca 2+ entry, in part due to higher expression of these proteins. No changes in Brp, Tomosyn, or Complexin mRNA levels were detected in Isoform-Patch-seq, suggesting that post-translational degradation or altered trafficking underlies differences in their abundance at Is synapses (Jetti, 2023).

Multiple studies indicate that PTMs, such as ubiquitylation, glycosylation, phosphorylation, acetylation, and sumoylation, impact synapse organization and function by modifying protein stability and localization. Isoform-Patch-seq indicated that sialylation and ubiquitination are differentially regulated in Ib and Is MNs. Disruptions of other E3 ubiquitin ligases like Highwire alter synaptic growth at Drosophila NMJs, though they impact both Is and Ib terminals. Identifying targets of the Skp2-CG9003 E3 ligase complex should clarify mechanisms by which cell-type-specific proteolysis contributes to synaptic morphology and function. Sialylation is critical for organization of the cell-surface proteome, regulation of cell adhesion, and control of neuronal excitability. Lack of sialylation impairs surface expression of multiple ion channels and cell adhesion proteins, and further studies should identify the specific targets regulating Ib synaptic organization (Jetti, 2023).

In summary, these data highlight multiple pathways that contribute to synaptic diversity between tonic and phasic MNs, while providing a resource to characterize additional DEGs that specify other structural and functional features of these neuronal subtypes. Neurons with tonic and phasic output represent an evolutionarily conserved design principle, and it will be interesting to determine whether similar DEGs are involved in other species. Recent RNA profiling indicates that Drosophila visual neurons differentially express sialyltransferases and proteolytic regulators, while mammalian cortical GABAergic interneuron subtypes differentially express sialyltransferases that alter the cell-surface proteome. As such, cell-type-specific PTMs may represent a widely used mechanism to generate neuronal diversity beyond their role in Drosophila tonic and phasic MNs (Jetti, 2023).

The conserved alternative splicing factor Caper regulates neuromuscular phenotypes during development and aging

RNA-binding proteins play an important role in the regulation of post-transcriptional gene expression throughout the nervous system. This is underscored by the prevalence of mutations in genes encoding RNA splicing factors and other RNA-binding proteins in a number of neurodegenerative and neurodevelopmental disorders. The highly conserved alternative splicing factor Caper is widely expressed throughout the developing embryo and functions in the development of various sensory neural subtypes in the Drosophila peripheral nervous system. This study found that caper dysfunction leads to aberrant neuromuscular junction morphogenesis, as well as aberrant locomotor behavior during larval and adult stages. Despite its widespread expression, the results indicate that caper function is required to a greater extent within the nervous system, as opposed to muscle, for neuromuscular junction development and for the regulation of adult locomotor behavior. Moreover, Caper was found to interact with the RNA-binding protein Fmrp to regulate adult locomotor behavior. Finally, it was shown that caper dysfunction leads to various phenotypes that have both a sex and age bias, both of which are commonly seen in neurodegenerative disorders in humans (Titus, 2021).

Fragile X Premutation rCGG Repeats Impair Synaptic Growth and Synaptic Transmission at Drosophila larval Neuromuscular Junction

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disease that develops in some premutation (PM) carriers of the FMR1 gene with alleles bearing 55-200 CGG repeats. The discovery of a broad spectrum of clinical and cell developmental abnormalities among PM carriers with or without FXTAS and in model systems suggests that neurodegeneration seen in FXTAS could be the inevitable end-result of pathophysiological processes set during early development. Hence, it is imperative to trace early PM-induced pathological abnormalities. Previous studies have shown that transgenic Drosophila carrying PM-length CGG repeats are sufficient to cause neurodegeneration. This study used the same transgenic model to understand the effect of CGG repeats on the structure and function of the developing nervous system. Presynaptic expression of CGG repeats restricts synaptic growth, reduces the number of synaptic boutons, leads to aberrant presynaptic varicosities, and impairs synaptic transmission at the larval neuromuscular junctions. The postsynaptic analysis shows that both glutamate receptors and subsynaptic reticulum proteins were normal. However, a high percentage of boutons show a reduced density of Bruchpilot protein, a key component of presynaptic active zones required for vesicle release. The electrophysiological analysis shows a significant reduction in quantal content, a measure of total synaptic vesicles released per excitation potential. Together, these findings suggest that synapse perturbation caused by rCGG repeats mediates presynaptically during larval NMJ development. It is also suggested that the stress-activated c-Jun N-terminal kinase protein Basket and CIDE-N protein Drep-2 positively mediate Bruchpilot active zone defects caused by rCGG repeats (Bhat, 2021).

Characterization of a novel stimulus-induced glial calcium wave in Drosophila larval peripheral segmental nerves and its role in PKG-modulated thermoprotection

Insects, as poikilotherms, have adaptations to deal with wide ranges in temperature fluctuation. Allelic variations in the foraging gene that encodes a cGMP dependent protein kinase, were discovered to have effects on behavior in Drosophila by Dr. Marla Sokolowski in 1980. This single gene has many pleiotropic effects and influences feeding behavior, metabolic storage, learning and memory and has been shown to affect stress tolerance. PKG regulation affects motoneuronal thermotolerance in Drosophila larvae as well as adults. While the focus of thermotolerance studies has been on the modulation of neuronal function, other cell types have been overlooked. Because glia are vital to neuronal function and survival, this study determine if glia play a role in thermotolerance as well. In this investigation, a novel calcium wave was discovered at the larval NMJ and set out to characterize the wave's dynamics and the potential mechanism underlying the wave prior to determining what effect, if any, PKG modulation has on the thermotolerance of glia cells. Using pharmacology, it was determined that calcium buffering mechanisms of the mitochondria and endoplasmic reticulum play a role in the propagation of the novel glial calcium wave. By coupling pharmacology with genetic manipulation using RNA interference (RNAi), it was found that PKG modulation in glia alters thermoprotection of function as well as glial calcium wave dynamics (Krill, 2021).

cAMP signals in Drosophila motor neurons are confined to single synaptic boutons

The second messenger cyclic AMP (cAMP) plays an important role in synaptic plasticity. Although there is evidence for local control of synaptic transmission and plasticity, it is less clear whether a similar spatial confinement of cAMP signaling exists. This study suggests a possible biophysical basis for the site-specific regulation of synaptic plasticity by cAMP, a highly diffusible small molecule that transforms the physiology of synapses in a local and specific manner. By exploiting the octopaminergic system of Drosophila, which mediates structural synaptic plasticity via a cAMP-dependent pathway, this study demonstrates the existence of local cAMP signaling compartments of micrometer dimensions within single motor neurons. In addition, evidence is provided that heterogeneous octopamine receptor localization, coupled with local differences in phosphodiesterase activity, underlies the observed differences in cAMP signaling in the axon, cell body, and boutons (Maiellaro, 2016).

The cyclic AMP (cAMP) pathway plays fundamental roles in the nervous system, where it is prominently involved in synaptic plasticity and memory formation. Previous studies in vertebrate and invertebrate models have shown that cAMP can propagate from dendrites to the cell body of neurons, in line with the properties of a small diffusible molecule. However, a local mode of action for cAMP has also been proposed, whereby cAMP signals are localized to the periphery of neurons, namely, dendrites, creating a cAMP microdomain. While the existence of cAMP microdomains in neuronal dendrites is disputed based on the experimental and theoretical data, very little is known about possible cAMP compartmentation in axons and how this may exert local effects at the presynaptic site. In particular, it is unclear how biochemical signals may spread from presynaptic boutons through the axon (Maiellaro, 2016).

To investigate this question in vivo, the neuromuscular junction (NMJ) of Drosophila melanogaster was used, that displays different forms of synaptic plasticity, many of which are dependent on cAMP signaling. Both structural and functional properties of larval neuromuscular synapses are heterogeneous, varying between boutons belonging to the same motor neuron. How such site-specific synaptic differentiation may be achieved at high spatial resolution is currently unknown, though it is tempting to speculate that local cAMP signals play a role. Thus, the developing Drosophila NMJ is a powerful model to investigate the role of cAMP in synaptic plasticity under physiological conditions (Maiellaro, 2016).

This study focused on glutamatergic type Ib motor neurons, which are structurally regulated via G-protein-coupled receptors (GPCRs) for octopamine. Stimulation of these receptors has been shown to induce synaptic bouton outgrowth via a cAMP- and CREB-dependent pathway. Therefore, this study set out to measure spatiotemporal patterns of octopamine-induced cAMP signals in these neurons. To this end, a FRET (Forster resonance energy transfer)-based sensor for cAMP (Epac1-camps) was expressed that has previously been used to image cAMP levels in central Drosophila neurons. The results reveal that cAMP signals are confined to their initiation site, the individual synaptic bouton, and suggest a highly efficient local mechanism for controlling site-specific synaptic plasticity (Maiellaro, 2016).

This study genetically expressed the cAMP sensor, Epac1-camps, at the Drosophila NMJ to monitor and quantify spatiotemporal cAMP dynamics induced by octopamine stimulation. The results reveal an unexpectedly high degree of cAMP compartmentalization in motor neurons, which may serve as a basis for local synaptic plasticity, with cAMP FRET signals being ultimately limited to single synaptic boutons. Three cAMP signaling compartments were identified within the motor neuron: boutons, axon, and cell body. For each cellular compartment, the particular mechanism responsible for the segregation of cAMP increases is described. Specifically, this study found that boutons constitute the most reactive compartment of the motor neuron in terms of cAMP accumulation. The results demonstrate that the production of cAMP is heterogeneous among boutons, with stronger responses to octopamine in large boutons rather than in smaller ones, possibly related to the increased synaptic strength measured at large boutons. Moreover, the activity and the specific localization of PDEs within the synaptic bouton prevent the propagation of cAMP to the cell body and its diffusion from one bouton to the next (Maiellaro, 2016).

In contrast to the boutons, octopamine receptors seem to be absent or inaccessible in the axon, and PDEs were not detected. Accordingly, cAMP FRET signals recorded in the axon differ from those in boutons in terms of amplitude and kinetics. Hence, the axon emerges as the second independent, but not isolated, cAMP signaling compartment. Interestingly, and in contrast to dendrites, cAMP did not propagate along the axon. It remains to be clarified whether this may relate to functional differences between axons and dendrites, between species, or between specific neuron types. Finally, the cell body was determined as the third cAMP signaling compartment within the motor neuron. The cell body has a very low sensitivity toward octopamine, and it was demonstrated that cAMP FRET signals generated in the cell body are not affected by the cell body's physical isolation from the axon. High PDE activity in the cell body contributes to this local suppression of cAMP signaling and may prevent spillover activation of cAMP effectors in the cell body and in the nucleus (Maiellaro, 2016).

Evidence of cAMP microdomains restricted to single boutons provides a biophysical basis for the local control of synaptic plasticity. Spatially constrained cAMP changes help to establish differences in morphology and synaptic content of boutons, suggesting that local cAMP, bouton structure, and synapse formation are intimately linked. The observed confinement of cAMP supports the notion that individual synaptic boutons may represent largely autonomous signaling units, which can receive and integrate signals independently of the other (Maiellaro, 2016).

The concept that cAMP could act as a local messenger was postulated almost 40 years ago. The existence of cAMP microdomains has been demonstrated in other cell types (e.g., cardiomyocytes). However, in neurons, there are contradicting experimental lines of evidence and simulations concerning the existence of cAMP microdomains. The current data clearly show that cAMP can act as a local messenger upon physiological stimulation of a neuron. The detailed spatiotemporal analysis of the dynamics of cAMP reveals that this messenger can be restricted at the micrometer level to induce highly localized physiological responses (Maiellaro, 2016).

Synaptic counts approximate synaptic contact area in Drosophila

The pattern of synaptic connections among neurons defines the circuit structure, which constrains the computations that a circuit can perform. The strength of synaptic connections is costly to measure yet important for accurate circuit modeling. Synaptic surface area has been shown to correlate with synaptic strength, yet in the emerging field of connectomics, most studies rely instead on the counts of synaptic contacts between two neurons. This study quantified the relationship between synaptic count and synaptic area as measured from volume electron microscopy of the larval Drosophila central nervous system. The total synaptic surface area, summed across all synaptic contacts from one presynaptic neuron to a postsynaptic one, can be accurately predicted solely from the number of synaptic contacts, for a variety of neurotransmitters. These findings support the use of synaptic counts for approximating synaptic strength when modeling neural circuits (Barnes, 2021).

Distinct molecular pathways govern presynaptic homeostatic plasticity

Presynaptic homeostatic plasticity (PHP) stabilizes synaptic transmission by counteracting impaired neurotransmitter receptor function through neurotransmitter release potentiation. PHP is thought to be triggered by impaired receptor function and to involve a stereotypic signaling pathway. However, this study demonstrates that different receptor perturbations that similarly reduce synaptic transmission result in different responses at the Drosophila neuromuscular junction. While receptor inhibition by the glutamate receptor (GluR) antagonist γ-D-glutamylglycine (γDGG) is not compensated by PHP, the GluR inhibitors Philanthotoxin-433 (PhTx) and Gyki-53655 (Gyki) induce compensatory PHP. Intriguingly, PHP triggered by PhTx and Gyki involve separable signaling pathways, including inhibition of distinct GluR subtypes, differential modulation of the active-zone scaffold Bruchpilot, and short-term plasticity. Moreover, while PHP upon Gyki treatment does not require genes promoting PhTx-induced PHP, it involves presynaptic protein kinase D. Thus, synapses not only respond differentially to similar activity impairments, but achieve homeostatic compensation via distinct mechanisms, highlighting the diversity of homeostatic signaling (Nair, 2021).

This study demonstrates that neurotransmitter receptor impairment by different GluR antagonists (PhTx and Gyki) induces PHP via distinct mechanisms, including differential inhibition of GluR subtypes, differential modulation of an active zone scaffold, and short-term plasticity during PHP. Importantly, genetic evidence is provided that separable molecular mechanisms promote PHP in response to Gyki and PhTx treatment. On the other hand, the GluR antagonist γDGG did not produce PHP, despite a robust inhibition of postsynaptic receptors. Together, the data suggest that distinct molecular mechanisms mediate PHP in response to GluR inhibition by different antagonists and that GluR inhibition per se is not sufficient for PHP expression (Nair, 2021).

The results contrast with prevalent models of homeostatic synaptic plasticity, which rest on the assumption that synaptic activity changes induce homeostatic signaling. In the case of PHP, the magnitude of the homeostatic increase in presynaptic release scales with the decrease in the amplitude of postsynaptic miniature events. Based on these data, it was proposed that reduced ion flux through the receptors triggers a homeostatic signaling cascade in the postsynaptic cell, which is relayed to the presynaptic compartment where it adjusts release. A prediction that directly follows is that any receptor perturbation with similar effects on the amplitude of synaptic miniature events produces a homeostatic response via similar underlying mechanisms. This study observed that two GluR antagonists, PhTx and Gyki, induced PHP, while one antagonist, γDGG, did not. All three perturbations similarly decreased quantal size (q), indicating a similar reduction of ion flux through the receptors. Although it cannot be excluded that γDGG may have directly blocked PHP expression, the results suggest that ion flux is unlikely the sole signal responsible for PHP induction at the Drosophila NMJ. This agrees with recent observations at the Drosophila and mouse NMJ indicating that Ca2+ flux through the receptor is likely dispensable for PHP induction (Goel, 2017; Wang, 2018). How could pharmacological receptor perturbation induce PHP independent of ion flux through the receptor? One intriguing possibility is that perturbation-specific conformational changes of the receptor may be involved in PHP signaling. Conformational changes of kainate-, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA)-, and N-Methyl-d-aspartate (NMDA)-type GluRs at mammalian synapses are known to signal independent of ion flux in the context of synaptic plasticity, possibly through metabotropic signaling. It is also known that different antagonists stabilize different conformational states of AMPA receptors. Thus, it is conceivable that distinct antagonists may trigger different PHP signaling pathways, depending on the different signaling partners that could be recruited by different conformational states. This hypothesis requires further investigation in relation to molecular signaling underlying PHP induction (Nair, 2021).

Although PhTx and Gyki robustly induced PHP through an increase in RRP size, several differences were observed between PhTx- and Gyki-induced PHP. First, PhTx and Gyki differentially affected GluR subtypes. Whereas PhTx mainly inhibits GluRIIA-containing receptors, Gyki reduced mEPSP amplitude at either GluRIIA or GluRIIB mutant NMJs. Since mEPSPs at the Drosophila NMJs are mediated by GluRIIA- and GluRIIB-containing receptors, these results indicate that Gyki may act on both types of receptor complexes. An intriguing possibility to be tested in the future is that PhTx-dependent receptor inhibition may trigger PHP by affecting a signaling module associated with GluRIIA-containing receptor complexes, whereas Gyki-induced PHP may be triggered by mechanisms beyond GluRIIA-dependent signaling. Second, while Gyki similarly accelerated the decay of miniature excitatory postsynaptic currents (mEPSCs) and EPSCs, the EPSC decay was slower than that of mEPSCs upon PhTx application. The mismatch between the mEPSC and EPSC decay kinetics after PhTx treatment may either result from presynaptic changes, such as the recruitment of synaptic vesicles with a low pr during the EPSC decay phase, or different effects on GluR desensitization, saturation, or diffusion during evoked release. Regardless, as this mismatch was not observed after Gyki treatment, it either reflects a difference in antagonist action or in Gyki- and PhTx-induced PHP. Third, Gyki, but not PhTx, resulted in altered short-term plasticity, indicative of a decrease in pr or increased GluR desensitization and/or saturation. Fourth, while PhTx increased Brp-fluorescence intensity, indicating elevated Brp abundance, Gyki did not. Although it cannot be excluded that Brp modulation at a different time point with regard to Gyki application, this observation suggests differential regulation of this core active zone protein during PhTx- and Gyki-induced PHP. Fifth, this study revealed that Gyki application induced PHP in six mutants that were previously shown to block PHP upon PhTx treatment. Sixth, this study identified a gene (PKD) that is required for Gyki-induced PHP, but not PhTx-induced, PHP. Thus, although the possibility cannot be excluded of partially overlapping molecular mechanisms, these data demonstrate separable molecular pathways between Gyki- and PhTx-induced PHP (Nair, 2021).

Diverging homeostatic signaling has been observed in the context of acute versus chronic receptor perturbations. At the Drosophila NMJ, mutants with intact acute PHP in response to PhTx application displayed impaired chronic PHP upon GluRIICRNAi expression. However, it is difficult to separate whether these differences emerge because of differences in the nature or the timescale of receptor perturbation. Since the curremt experiments focus on acute perturbations, the results support the idea that distinct homeostatic signaling pathways can be triggered depending on the specific way receptors have been perturbed. PhTx, γDGG, and Gyki inhibit AMPA receptors via different modes of antagonism. While PhTx is an activity-dependent pore blocker, γDGG acts as a low-affinity competitive antagonist and Gyki is an allosteric inhibitor. PhTx and γDGG inhibit Drosophila GluRs with characteristics similar to AMPA receptor inhibition. Similarly, the conserved Gyki-interacting residues between rat and Drosophila GluRs highlight the possibility that Gyki could also act similarly on Drosophila and mammalian GluRs. Thus, PhTx, γDGG, and Gyki may inhibit Drosophila GluRs via different modes of antagonism, which may be involved in triggering distinct homeostatic signaling pathways. Differential homeostatic signaling has also been observed in the context of firing rate homeostasis, where molecular responses depend on whether the protein or the conductance of a sodium channel protein is eliminated. Thus, distinct molecular signaling mechanisms that are specific to the nature, rather than the functional effects of a perturbation, may be a general theme in the context of neuronal homeostasis (Nair, 2021).

This study also implicated presynaptic PKD signaling in synaptic homeostasis. Although PKD has been linked to various intracellular processes, such as vesicle sorting, endocytosis, and the regulation of the actin cytoskeleton, its synaptic function is less explored. Recently, PKD was associated with synaptic plasticity (Oueslati Morales, 2020). The current data clearly demonstrate that presynaptic PKD is required for Gyki-induced PHP, but not for PhTx-induced PHP and establish that separable molecular pathways govern Gyki- and PhTx-induced PHP. However, PKD was required for PHP only at low extracellular Ca2+ concentration, similar to a number of genes supporting PhTx-induced PHP. This indicates that PKD signaling may contribute to the robustness of PHP under conditions of low pr. It is worth highlighting that the PHP defect observed upon presynaptic PKD knockdown and in PKD hypomorphs may not be fully penetrant, likely because of the genetic nature of the perturbation, and/or genetic compensation by other members of the Protein Kinase C/Calmodulin-Dependent Kinase family. This genetic compensation is particularly pronounced in PKD null mutants, which precluded the analysis after complete loss of PKD function (Nair, 2021).

Unlike PhTx, Gyki induced PHP in the fully dissected NMJ preparation, which is more amenable to electrophysiology and imaging approaches, and thus allowed probing the dynamics of PHP induction. It is currently unclear why PhTx-induced PHP cannot be observed in the full preparation. One hypothesis is that the muscles/synapses may be stretched in the full preparation, which could lead to a disruption of signaling domains relevant for PhTx-induced PHP. A second possibility could be the potential disruption of neuro-glial signaling that is important for PhTx-induced PHP in the full preparation (Wang, 2020). The fact that Gyki results in PHP after full dissection demonstrates that PHP induction is not limited to the 'semi-intact' preparation per se. It will be interesting to investigate whether different molecular pathways promoting PHP in response to Gyki and PhTx treatment may be differentially affected by the type of the preparation. In this regard, Gkyi induced PHP in Multiplexin (dmp) mutants, which were shown to disrupt PhTx-dependent PHP and neuro-glia signaling (Wang, 2020; Nair, 2021 and regerences therein).

Gyki application to the fully dissected preparation revealed very rapid, low-latency PHP induction kinetics, which maintained EPSP amplitudes constant despite Gyki-induced mEPSP amplitude reduction. However, due to the sampling interval of 35 s, shorter latencies cannot be ruled out. In addition, the exact latency of PHP could not be estimated because of the relatively slow kinetics of mEPSP reduction in these experiments. The reversible GluR block by Gyki also revealed rapid, low-latency PHP reversal after Gyki washout, consistent with previous observations at mouse cerebellar mossy fiber boutons or at mouse NMJs. The results imply continuous, bidirectional PHP signaling that compensates for receptor impairment within ∼35 s, similar to observations at the mouse NMJ. It will be interesting to explore the molecular mechanisms underlying the induction of distinct PHP pathways and whether distinct molecular pathways also rapidly stabilize synaptic efficacy at other synapses (Nair, 2021).

Although the results indicate that PHP signaling depends on specific receptor perturbations, the mechanisms of antagonism of the used antagonists have not been studied for Drosophila GluRs. Moreover, the lack of PHP upon γDGG treatment may result from off-target inhibition of PHP signaling cannot be excluded, nor that Gyki acts independent of GluRs to induce PHP. However, given Gyki's known mechanism of allosteric antagonism of mammalian AMPARs, and its potent reduction of mEPSP amplitudes at the Drosophila NMJ, this is considered unlikely. Furthermore, given the evolutionary conservation of Gyki-interacting residues in Drosophila GluR subunits, it is likely that Gyki acts on all Drosophila GluR subunits. However, the effect of Gyki on individual receptor subunits cannot be tested, because GluRIIC-E subunits are essential for receptor formation. Homozygous Drosophila lines harboring mutations in genes required for PhTx-induced PHP were used to test whether PhTx and Gyki activate different downstream signaling pathways. This analysis did not systematically investigate the effects of potential off-site mutations that may have accumulated over time in these fly lines. However, most strains were kept over a balancer chromosome, and Gyki-induced PHP proceeded normally in six mutants that were previously shown to disrupt PhTx-induced PHP, suggesting that a major contribution of off-target mutations is unlikely (Nair, 2021).

Rapid homeostatic modulation of transsynaptic nanocolumn rings

Robust neural information transfer relies on a delicate molecular nano-architecture of chemical synapses. Neurotransmitter release is controlled by a specific arrangement of proteins within presynaptic active zones. How the specific presynaptic molecular architecture relates to postsynaptic organization and how synaptic nano-architecture is transsynaptically regulated to enable stable synaptic transmission remain enigmatic. Using time-gated stimulated emission-depletion microscopy at the Drosophila neuromuscular junction, it was found that presynaptic nanorings formed by the active-zone scaffold Bruchpilot (Brp) align with postsynaptic glutamate receptor (GluR) rings. Individual rings harbor approximately four transsynaptically aligned Brp-GluR nanocolumns. Similar nanocolumn rings are formed by the presynaptic protein Unc13A and GluRs. Intriguingly, acute GluR impairment triggers transsynaptic nanocolumn formation on the minute timescale during homeostatic plasticity. Distinct phases of structural transsynaptic homeostatic plasticity were revealed, with postsynaptic GluR reorganization preceding presynaptic Brp modulation. Finally, homeostatic control of transsynaptic nano-architecture and neurotransmitter release requires the auxiliary GluR subunit Neto. Thus, transsynaptic nanocolumn rings provide a substrate for rapid homeostatic stabilization of synaptic efficacy (Muttathukunnel, 2022).

This study has identified a stereotypic arrangement of transsynaptically aligned molecular nanocolumns that is regulated in a modular and sequential fashion during homeostatic plasticity at the Drosophila NMJ. Moreover, a GluR subtype-specific nano-organization was revealed and it was discovered that the auxiliary GluR subunit Neto is required for rapid homeostatic modulation of transsynaptic nanocolumn number and neurotransmitter release (Muttathukunnel, 2022).

Previous work demonstrated that a cluster of voltage-gated Ca2+ channels localizes to the Brp ring center at the Drosophila NMJ. Furthermore, Unc13A, a molecule suggested as a molecular correlate of presynaptic release sites, forms ring-like arrays in close proximity to Brp C termini and GluRs. In light of these findings, the results are consistent with a model in which Ca2+ influx at the Brp/AZ center induces neurotransmitter release in the nanocolumn rings. Given that the neurotransmitter content released by a single synaptic vesicle does not activate all GluRs of a given PSD at the Drosophila NMJ and that Drosophila GluRs have a low glutamate affinity, neurotransmitter release may predominantly activate GluRs that are aligned to presynaptic release sites. Some evidence suggests that synaptic transmission predominantly occurs within transsynaptic nanocolumns. Hence, the transsynaptic nanocolumn rings discovered in this study may reflect subsynaptic transmission modules that are activated by a common Ca2+-channel cluster. Future work is needed to relate the molecular nanocolumn topography to synaptic physiology, for example, by assessing how many GluRs are activated by neurotransmitter release from a single synaptic vesicle. In this regard, the slight offset between Unc13A and GluR rings may indicate that a given release site may not only activate a single aligned GluR cluster but also neighboring GluR clusters, consistent with physiology data (Muttathukunnel, 2022).

GluR subunit composition and GluR location with regard to release sites are important factors determining synaptic efficacy. At the Drosophila NMJ, the ratio of slowly and rapidly desensitizing GluRIIA- and GluRIIB-containing receptors is a key regulator of quantal size. This study revealed that transsynaptic nanocolumns harbor a mix of GluRIIA- and GluRIIB-containing receptors, and that ambient receptors, which represent almost half of the GluRs within a PSD, mainly incorporate the GluRIIB subunit. The persistence of transsynaptic nanocolumn rings in GluRIIA and GluRIIB mutants implies that neither of these subunits alone is sufficient for ring formation or transsynaptic alignment. Previous work revealed no defects in spontaneous or AP-evoked synaptic transmission upon GluRIIA overexpression or after GluRIIB loss . Thus, two genetic manipulations that mainly decrease ambient receptor abundance, but not receptors inside the nanocolumn ring, do not induce a corresponding decrease in synaptic transmission. This indicates that synaptic transmission is largely confined to transsynaptic nanocolumn rings and/or that synaptic transmission outside the rings is dominated by rapidly desensitizing GluRIIB-containing receptors. Moreover, the observation of increased mEPSP amplitudes in GluRIIBSP5 mutants suggests that GluRIIB-containing receptors surrounding the nanocolumns have the potential to negatively regulate synaptic transmission by replacing GluRIIA-containing receptors within the nanocolumns (Muttathukunnel, 2022).

A variety of auxiliary subunits control GluR assembly, trafficking, and function. The auxiliary GluR subunit Neto has been implicated in GluR clustering at the Drosophila NMJ (Kim, 2021). This uncovered modular ring arrays of Neto-β that transsynaptically align with Brp C termini, suggesting that this auxiliary GluR subunit is a postsynaptic element of transsynaptic nanocolumn rings. The persistence of transsynaptic nanocolumn rings in hypomorphic neto¹⁰⁹ mutants suggests that neto is not crucial for ring formation or transsynaptic alignment, or that the remaining Neto was sufficient for transsynaptic nanocolumn ring formation. In contrast to neto¹⁰⁹ mutants, in which both Neto-α and Neto-β levels are reduced, loss of Neto-α does not decrease GluR levels or mEPSP amplitude, suggesting that this Neto isoform either does not stabilize GluRs at the Drosophila NMJ or that there is a compensation by Neto-β. While reduced levels of ambient receptors do not impair synaptic transmission in case of GluRIIA overexpression or in GluRIIB^SP5 mutants, the decreased GluR abundance within the rings of neto¹⁰⁹ mutants correlates with a decrease in spontaneous and AP-evoked synaptic transmission, again implying that synaptic transmission predominantly occurs within the rings (Muttathukunnel, 2022).

GluR impairment at the Drosophila NMJ induces a homeostatic increase in release, and there is evidence for the modulation of presynaptic nano-architecture during this form of homeostatic plasticity. A previous study reported increased GluR levels upon sustained pharmacological GluR inhibition for several days. This study demonstrates GluR modulation within 5 min after pharmacological GluR impairment that precedes the modulation of Brp, as well as Neto-β. Although it cannot be excluded that other molecules are modulated prior to GluRs, or that small changes in Brp or Neto-&beta could not be resolved; after PhTX treatment for 5 min, the data imply that GluR modulation precedes Neto-β and presynaptic regulation during homeostatic plasticity. Furthermore, GluR and Brp fluorescence intensity changes detected with confocal microscopy preceded the increase in GluR and Brp cluster numbers at STED resolution. This could either indicate that small nanostructural changes could not be detected with STED microscopy or that the modulation of transsynaptic nano-architecture lags behind the regulation of GluR and Brp levels or distribution. Similar to the data obtained with confocal microscopy, the increase in GluR cluster number preceded Brp cluster regulation upon GluR perturbation, again indicative of a temporal sequence of transsynaptic changes during PHP. Interestingly, while GluR, but not Brp cluster number increased 15 min after PhTX treatment, a larger fraction of transsynaptically aligned Brp clusters was noted. This suggests that transsynaptic nanocolumn formation likely precedes Brp cluster formation. The temporal sequence of GluR and Brp regulation may also explain the existence of GluR clusters within the ring that are not opposed by Brp. Together, these findings are consistent with a model of coordinated, transsynaptic, and modular structural plasticity during PHP that results in the addition of transsynaptic nanocolumns to the ring (Muttathukunnel, 2022).

Apparent changes in GluR fluorescence intensity, GluR cluster number, or homeostatic potentiation of release upon pharmacological GluR perturbation in hypomorphic neto¹⁰⁹ mutants were not observed. This shows that wild-type Neto levels are required for homeostatic control of GluRs and presynaptic release. GluR inhibition also led to a slight but significant increase in Brp fluorescence intensity in neto¹⁰⁹ mutants, which was less pronounced than in wild type. The defect in PHP seen in neto¹⁰⁹ mutants could thus arise from impaired GluR and/or Brp regulation. Although the genetic data establish a causal relationship between the homeostatic regulation of transsynaptic nanocolumns and presynaptic release, future work is required to scrutinize the relationship between transsynaptic nano-architecture and synaptic transmission, and to dissect the molecular mechanisms controlling transsynaptic nano-architecture and its homeostatic regulation. In this regard, it will be exciting to explore which molecules are involved in transsynaptic alignment and ring formation. Synaptic cell-adhesion molecules, such as neurexins and neuroligins, represent obvious candidates (Muttathukunnel, 2022).

Vav independently regulates synaptic growth and plasticity through distinct actin-based processes

Modulation of presynaptic actin dynamics is fundamental to synaptic growth and functional plasticity; yet the underlying molecular and cellular mechanisms remain largely unknown. At Drosophila NMJs, the presynaptic Rac1-SCAR pathway mediates BMP-induced receptor macropinocytosis to inhibit BMP growth signaling. This study shows that the Rho-type GEF Vav acts upstream of Rac1 to inhibit synaptic growth through macropinocytosis. Evidence is presented that Vav-Rac1-SCAR signaling has additional roles in tetanus-induced synaptic plasticity. Presynaptic inactivation of Vav signaling pathway components, but not regulators of macropinocytosis, impairs post-tetanic potentiation (PTP) and enhances synaptic depression depending on external Ca2+ concentration. Interfering with the Vav-Rac1-SCAR pathway also impairs mobilization of reserve pool (RP) vesicles required for tetanus-induced synaptic plasticity. Finally, treatment with an F-actin-stabilizing drug completely restores RP mobilization and plasticity defects in Vav mutants. It is proposed that actin-regulatory Vav-Rac1-SCAR signaling independently regulates structural and functional presynaptic plasticity by driving macropinocytosis and RP mobilization, respectively (Park, 2022).

Loss of Activity-Induced Mitochondrial ATP Production Underlies the Synaptic Defects in a Drosophila Model of ALS

Mutations in the gene encoding vesicle-associated membrane protein B (VAPB) cause a familial form of amyotrophic lateral sclerosis (ALS). Expression of an ALS-related variant of vapb (vapb^P58S) in Drosophila motor neurons results in morphologic changes at the larval neuromuscular junction (NMJ) characterized by the appearance of fewer, but larger, presynaptic boutons. Although diminished microtubule stability is known to underlie these morphologic changes, a mechanism for the loss of presynaptic microtubules has been lacking. By studying flies of both sexes, this study demonstrate the suppression of vapb^P58S) -induced changes in NMJ morphology by either a loss of endoplasmic reticulum (ER) Ca(2+) release channels or the inhibition Ca(2+)/calmodulin (CaM)-activated kinase II (CaMKII). These data suggest that decreased stability of presynaptic microtubules at vapb^P58S NMJs results from hyperactivation of CaMKII because of elevated cytosolic [Ca(2+)]. The Ca(2+) dyshomeostasis is attributed to delayed extrusion of cytosolic Ca(2+) Suggesting that this defect in Ca(2+) extrusion arose from an insufficient response to the bioenergetic demand of neural activity, depolarization-induced mitochondrial ATP production was diminished in vapb^P58S neurons. These findings point to bioenergetic dysfunction as a potential cause for the synaptic defects in vapb^P58S -expressing motor neurons (Karagas, 2022).

Influence of T-Bar on Calcium Concentration Impacting Release Probability

The relation of form and function, namely the impact of the synaptic anatomy on calcium dynamics in the presynaptic bouton, is a major challenge of present (computational) neuroscience at a cellular level. The Drosophila larval neuromuscular junction (NMJ) is a simple model system, which allows studying basic effects in a rather simple way. This synapse harbors several special structures. In particular, in opposite to standard vertebrate synapses, the presynaptic boutons are rather large, and they have several presynaptic zones. In these zones, different types of anatomical structures are present. Some of the zones bear a so-called T-bar, a particular anatomical structure. The geometric form of the T-bar resembles the shape of the letter 'T' or a table with one leg. When an action potential arises, calcium influx is triggered. The probability of vesicle docking and neurotransmitter release is superlinearly proportional to the concentration of calcium close to the vesicular release site. It is tempting to assume that the T-bar causes some sort of calcium accumulation and hence triggers a higher release probability and thus enhances neurotransmitter exocytosis. In order to study this influence in a quantitative manner, a typical T-bar geometry was constructed and the calcium concentration close to active zones (AZs) with and without T-bars was compared. Indeed, a substantial influence was found of the T-bar structure on the presynaptic calcium concentrations close to the AZs, indicating that this anatomical structure increases vesicle release probability. Therefore, this study reveals how the T-bar zone implies a strong relation between form and function. This study answers the question of experimental studies concerning the sense of the anatomical structure of the T-bar (Knodel, 2022).

Synaptic plasticity induced by differential manipulation of tonic and phasic motoneurons in Drosophila

Structural and functional plasticity induced by neuronal competition is a common feature of developing nervous systems. However, the rules governing how postsynaptic cells differentiate between presynaptic inputs are unclear. In this study synaptic interactions was characterized following manipulations of tonic Ib or phasic Is glutamatergic motoneurons that co-innervate postsynaptic muscles of male or female Drosophila melanogaster larvae. After identifying drivers for each neuronal subtype, ablation or genetic manipulations was performed to alter neuronal activity and examined the effects on synaptic innervation and function at neuromuscular junctions (NMJs). Ablation of either Ib or Is resulted in decreased muscle response, with some functional compensation occurring in the Ib input when Is was missing. In contrast, the Is terminal failed to show functional or structural changes following loss of the co-innervating Ib input. Decreasing the activity of the Ib or Is neuron with tetanus toxin light chain resulted in structural changes in muscle innervation. Decreased Ib activity resulted in reduced active zone (AZ) number and decreased postsynaptic subsynaptic reticulum (SSR) volume, with the emergence of filopodial-like protrusions from synaptic boutons of the Ib input. Decreased Is activity did not induce structural changes at its own synapses, but the co-innervating Ib motoneuron increased the number of synaptic boutons and AZs it formed. These findings indicate tonic Ib and phasic Is motoneurons respond independently to changes in activity, with either functional or structural alterations in the Ib neuron occurring following ablation or reduced activity of the co-innervating Is input, respectively (Aponte-Santiago, 2020).

Functional and structural changes in neuronal circuits occur during development and in response to environmental stimuli, learning, and injury. Disruptions of these plasticity pathways contribute to neurodevelopmental diseases and impair rewiring after brain injury, highlighting the importance of the underlying mechanisms. In contrast to mammals, invertebrate nervous systems like that of Drosophila melanogaster are more stereotypical in their organization. Neuroblasts divide and differentiate in a specific order to generate fixed cellular lineages with genetically hardwired synaptic targets. Although Drosophila display stereotypical neuronal connectivity, plasticity can occur throughout development and into adulthood. Structural plasticity is most prominent during metamorphosis, when larval neurons reorganize their processes and synaptic partners form functional adult circuits. Alterations in connectivity also occur in response to changes in environmental stimuli or following acute or chronic manipulations of neuronal activity (Aponte-Santiago, 2020).

Although plasticity occurs broadly across neuronal circuits, the motor system has played an important role in defining mechanisms for activity-dependent structural changes in connectivity. Locomotion is an essential behavior in many animals and requires coordinated output from central pattern generators to orchestrate motoneuron (MN) firing patterns that activate specific muscles. In vertebrates, individual muscle fibers receive transient innervation from many cholinergic motoneurons during early development. As many as 10 distinct motor axons can innervate a single muscle fiber before an activity-dependent competition results in retention of only a single axon. This axonal competition allows a large pool of identical motoneurons to transition from dispersed weak outputs to the muscle field to strong innervation of a smaller subset of muscles (Aponte-Santiago, 2020).

Unlike vertebrate neuromuscular junctions (NMJs), early promiscuity in synaptic partner choice and subsequent synapse elimination does not occur in Drosophila. Instead, the larval motor system is composed of ~36 motoneurons from four subclasses that are genetically programmed by specific transcription factors and guidance molecules to form stereotypical connections to the 30 muscles in each abdominal hemisegment. Although synaptic partner choice is hardwired, activity-dependent plasticity and homeostatic mechanisms have been characterized, making Drosophila an ideal system to study synaptic interactions between motor neurons. Although individual muscles normally restrict innervation to a single neuron from each subclass, it is unclear whether motoneurons interact during innervation of the same muscle target or respond when the output of one motoneuron is altered. Therefore, a system was extablished to differentially manipulate the two primary glutamatergic inputs and characterized the subsequent effects on synaptic morphology and function. Pnly the tonic Ib motoneuron is capable of partially compensating following ablation or silencing of the phasic Is input (Aponte-Santiago, 2020).

To characterize how changes in the presence or activity of tonic Ib versus phasic Is motoneurons alter NMJ development and function in Drosophila, GAL4 drivers specific for each class that innervate M1 were identifed and use to alter the balance of input to the muscle. The data indicate that Ib and Is motoneurons largely form independent inputs that make similar contributions to muscle excitability and contractile force. The Ib subclass was capable of structural and functional changes following manipulations that altered their output or that of the coinnervating Is motoneuron. These changes were observed only during conditions when neuronal activity was decreased or when the Is input was ablated. Functional increases in evoked release without enhanced synapse number were observed in Ib motoneurons following ablation of Is. In contrast, morphologic changes that increased AZ number without enhancing evoked release occurred when Is synaptic output was blocked with tetanus toxin. While Ib motoneurons were capable of several forms of plastic change following reduced input to the muscle, Is motoneurons were insensitive to manipulations of their own activity or that of the Ib input. Unlike the plasticity observed in Ib neurons following reduction in synaptic drive to the muscle, enhancing excitability of either the Ib or Is input was ineffective at triggering changes in either motoneuron class. These data indicate that reductions in activity from either input trigger a structural or functional change primarily from the tonic Ib motoneuron subclass (Aponte-Santiago, 2020).

The stereotypical connectivity found in the abdominal musculature of Drosophila larvae suggest that individual muscles normally allow synaptic innervation from only a single motoneuron of each subclass. However, expanded postsynaptic target choice has been observed following muscle loss induced by laser ablation or genetic mutation, with the affected Ib motoneuron targeting inappropriate nearby muscles without altering the innervation pattern of the correctly targeted Ib neuron. Similarly, ablation of some motoneurons can result in axonal spouting from neighboring connections that target the deinnervated muscle. Misexpression of synaptic cell surface proteins can also alter target choice for some Ib and Is motoneurons. In addition, silencing neuronal activity during development has been demonstrated to induce ectopic NMJs formed primarily by type II neuromodulatory neurons. No axonal sprouting onto M1 from other motoneurons was observed that resulted in altered target choice when MN1-Ib or MNIs was ablated or silenced in these experiments. Given that M1 is the most dorsal muscle of the abdominal musculature, axons from other motoneurons are not present in the direct vicinity, so any signals released from M1 might be insufficient to attract additional innervation. Evidence was found that M1 may attempt to promote synaptic innervation when MN1-Ib was silenced with tetanus toxin. Under these conditions, the MN1-Ib terminal maintained an immature-like state with the presence of filopodial-like extensions. This effect was observed only in Ib motoneurons, highlighting differences in how Is terminals interact with or respond to signals from the muscle. Similar filopodial-like extensions at newly forming NMJ connections have been observed in late embryos and early first instar larvae. These presynaptic filopodial processes contain elevated levels of the Cacophony N-type Ca2+ channel and interact with GluRIIA-rich myopodia, with some progressing to form new synapses during early development. Because of the lack of reinforcement signals caused by the absence of synaptic activity in silenced MN1-Ib motoneurons, it was hypothesized that these processes fail to properly drive AZ assembly and new synapse formation. Indeed, a role for neuronal activity in regulating synaptogenic filopodial stabilization has been characterized in the Drosophila visual system (Aponte-Santiago, 2020).

Many forms of plasticity, including synapse elimination at mammalian NMJs, ocular dominance plasticity, and cerebellar climbing fiber pruning, require Hebbian-like input imbalances to trigger synaptic interactions. As such, it was of interest to see whether changes in the activity of Ib or Is motoneurons that created an imbalance between the output of the two neurons could drive unique changes compared with when one input was missing. In the case of the Ib neuron, this was indeed observed. In the absence of Is input, either because of natural variation in innervation in control animals or following ablation with UAS-RPR, there was no structural response in terms of adding additional release sites. However, the loss of Is triggered a functional increase in evoked release from the Ib neuron. In contrast, when an activity imbalance was created by expressing tetanus toxin in the Is neuron, Ib displayed structural plasticity that increased the number of release sites. Although the underlying molecular pathways that mediate the two distinct responses are unknown, the results suggest that the physical presence of Is likely alters the signaling systems responsible for triggering compensation in Ib motoneurons in response to reduced muscle drive (Aponte-Santiago, 2020).

For every manipulation made beyond increasing excitability of the neurons, a response from the tonic Ib class was detected, while the phasic Is motoneuron displayed less plasticity. Since each muscle is innervated by a single Ib motoneuron, this plasticity may allow more robust and local regulation of muscle function. Although the Is did not show plastic change in response to manipulation of its activity or the coinnervating Ib in these experiments, it cannot be ruled out that Is neurons are capable of such plasticity but display less sensitivity to putative muscle-derived retrograde signals. Given that Is neurons innervate multiple muscles compared with Ib, it is also possible that small plastic changes occurring in Is are not synapse specific and are distributed over a larger population of AZs onto multiple muscles, resulting in little effect at any single postsynaptic target. Similar differences in homeostatic plasticity in Ib versus Is motoneurons have been described following reduced postsynaptic muscle glutamate receptor function, with the Ib motoneuron showing a more robust upregulation of presynaptic release compared with Is. Although homeostatic plasticity has been observed in Is motoneurons in low extracellular Ca2+ (Genc and Davis, 2019), the elevated Pr of Is synapses may occlude further functional increases in release output in higher [Ca2+] normally found in larval hemolymph. Together, these results indicate that tonic Ib motoneurons express distinct plasticity mechanisms that can be triggered by reduced muscle function that are less robust or lacking in the phasic Is subclass. Whether the differential plasticity found in this study is linked directly to the tonic or phasic properties of Ib and Is motoneurons, or is under separate regulatory control, will require further investigation (Aponte-Santiago, 2020).

An important question moving forward is to identify mechanisms that control structural and functional plasticity in Ib motoneurons. Similarly, defining why the Is fails to respond to many of the same manipulations is poorly understood. Whether homeostatic plasticity mechanisms triggered in response to acute or chronic reduction in glutamate receptor function are also activated following the absence or functional silencing of presynaptic inputs as described in this study is unknown. Several molecular pathways contributing to homeostatic plasticity have been described at the NMJ. Beyond Drosophila, studies in crustacean motor systems have shown that long-term alterations in activity can induce cell type-specific changes in tonic or phasic motoneuron structure or release properties. Given that tonic and phasic neurons are abundant in the nervous systems of both invertebrates and vertebrates, it will be interesting to determine whether such properties play a key role in defining their capacity for plastic change. The tools described in this study provide an opportunity to identify the distinct transcriptional profiles of each neuronal subclass in Drosophila to identify candidate mechanisms that mediate the differential plasticity responses of tonic Ib and phasic Is motoneurons (Aponte-Santiago, 2020).

The decoy SNARE Tomosyn sets tonic versus phasic release properties and is required for homeostatic synaptic plasticity

Synaptic vesicle release probability (P(r)) is a key presynaptic determinant of synaptic strength established by cell intrinsic properties and further refined by plasticity. To characterize mechanisms that generate P(r) heterogeneity between distinct neuronal populations, this study examined glutamatergic tonic (Ib) and phasic (Is) motoneurons in Drosophila with stereotyped differences in P(r) and synaptic plasticity. The decoy SNARE Tomosyn is differentially expressed between these motoneuron subclasses and contributes to intrinsic differences in their synaptic output. Tomosyn expression enables tonic release in Ib motoneurons by reducing SNARE complex formation and suppressing P(r) to generate decreased levels of synaptic vesicle fusion and enhanced resistance to synaptic fatigue. In contrast, phasic release dominates when Tomosyn expression is low, enabling high intrinsic P(r) at Is terminals at the expense of sustained release and robust presynaptic potentiation. In addition, loss of Tomosyn disrupts the ability of tonic synapses to undergo presynaptic homeostatic potentiation (PHP) (Sauvola, 2021).

Ca2+-dependent fusion of synaptic vesicles (SVs) is the primary mechanism for neurotransmission and is mediated by the soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) family. Following an action potential, SNARE proteins located on the SV and plasma membrane zipper into an energetically favorable coiled-coil bundle to induce SV fusion. Neurotransmitter release results in a postsynaptic response that varies in size depending on the strength of the synapse, which can be regulated from both pre-and post-synaptic compartments. The postsynaptic cell controls sensitivity to neurotransmitters by governing receptor field composition, while the presynaptic neuron establishes the probability (Pr) of SV fusion. Highly stereotyped differences in Pr exist across neurons, with many neuronal populations broadly classified as tonic or phasic depending on their spiking patterns, Pr and short-term plasticity characteristics. How cell-intrinsic properties establish differences in presynaptic Pr between neuronal classes, and how release strength is further refined via plasticity, remain incompletely understood (Sauvola, 2021).

The Drosophila melanogaster larval neuromuscular junction (NMJ) provides a robust genetic system for characterizing mechanisms mediating synaptic communication and tonic versus phasic release properties. Larval body wall muscles are co-innervated by two glutamatergic motoneuron populations that drive locomotion, including the tonic-like Ib and phasic-like Is subtypes. Tonic Ib terminals display lower initial Pr and sustained release during stimulation, whereas phasic Is terminals show higher intrinsic Pr and rapid depression (Lu, 2016; Newman, 2017). The Drosophila NMJ also undergoes robust presynaptic homeostatic potentiation (PHP) that rapidly increases Pr to compensate for disruptions to postsynaptic glutamate receptor (GluR) function. In addition to intrinsic release differences, the Ib and Is subtypes display distinct capacity for PHP. How tonic and phasic neurons differentially regulate Pr during normal synaptic communication and plasticity is largely unknown (Sauvola, 2021).

The highly conserved SNARE regulatory protein Tomosyn negatively controls SV release and has been proposed to participate in synaptic plasticity. Tomosyn has an N-terminal WD40 repeat domain and a C-terminal SNARE motif with homology to the SV v-SNARE Synaptobrevin 2 (Syb2). Tomosyn inhibits presynaptic release by binding the t-SNAREs Syntaxin1 (Syx1) and SNAP-25 to prevent Syb2 incorporation into the SNARE complex fusion machinery (Sauvola, 2021).

To further examine the role of Tomosyn in synaptic transmission and plasticity, CRISPR was used to generate mutations in the sole Drosophila tomosyn gene. Structure-function analysis revealed the SNARE domain is critical for release inhibition, while the scaffold region promotes enrichment of Tomosyn to SV-rich sites. Despite enhanced evoked release, tomosyn mutants fail to maintain high levels of SV output during sustained stimulation due to rapid depletion of the immediately releasable SV pool. Tomosyn is highly enriched at Ib synapses and generates tonic neurotransmission properties characterized by low Pr and sustained release in this population of motoneurons. Indeed, optogenetic stimulation and optical quantal analysis demonstrate an exclusive role for Tomosyn in regulating intrinsic release strength in tonic motoneurons. PHP expression primarily occurs at tonic synapses and is abolished in tomosyn mutants, suggesting Tomosyn is also essential for acute PHP expression. Together, these data indicate Tomosyn mediates the tonic properties of Ib motoneurons by suppressing Pr to slow the rate of SV usage, while decreasing Tomosyn suppression enables Pr enhancement during PHP. Conversely, the absence of Tomosyn in Is motoneurons facilitates phasic release properties by enabling an intrinsically high Pr that quickly depletes the releasable SV pool, resulting in rapid synaptic depression and reduced capacity for PHP (Sauvola, 2021).

The findings reported in this study indicate the conserved presynaptic release suppressor Tomosyn functions in setting presynaptic output and plasticity differences for a tonic/phasic pair of motoneurons that co-innervate Drosophila larval muscles. CRISPR-generated mutations in Drosophila tomosyn revealed synchronous, asynchronous and spontaneous SV release are all elevated in the absence of the protein. While single evoked responses were enhanced, rapid depression of release was observed during train stimulation, suggesting loss of Tomosyn biases synapses toward a more phasic pattern of SV release. To directly test whether Tomosyn plays a unique role in tonic synapses, Ib and Is motoneurons were separately stimulated using optogenetics to measure their isolated contributions. These experiments revealed a 4-fold increase in output from Ib neurons with no change to Is release. Optical quantal analysis confirmed the Ib specific effect of Tomosyn and demonstrated enhanced evoked responses in tomosyn is due to higher intrinsic Pr across the entire AZ population. Endogenously-tagged Tomosyn was more abundant at Ib synapses than Is, consistent with Tomosyn's role in regulating Ib release. Together, these data indicate the intrinsically high Pr and rapid depression normally found in Is motoneurons is due in part to a lack of Tomosyn inhibition of SV usage at phasic synapses. High-frequency stimulation experiments demonstrate Tomosyn does not regulate the size of the immediately releasable SV pool (IRP) but rather regulates IRP usage to ensure sustained availability of SVs during prolonged stimulation, as the IRP is strongly biased towards early release in tomosyn mutants. A model is proposed where Drosophila synapses are more phasic in release character by default, with tonic release requiring higher levels of Tomosyn to generate a fusion bottleneck that enables extended periods of stable release by slowing the rate of SV usage (Sauvola, 2021).

How Tomosyn normally suppresses SV release has been unclear. The most widely hypothesized mechanism is that Tomosyn competes with Syb2 for binding t-SNAREs. By forming fusion-incompetent SNARE complexes that must be disassembled by NSF, a pool of t-SNAREs is kept in reserve and can be mobilized by alleviating Tomosyn inhibition. Indeed, enhanced SNARE complex formation was found in Drosophila tomosyn mutants, consistent with the model that Tomosyn's SNARE domain acts as a decoy SNARE to inhibit productive SNARE complex assembly. Expression of the Tomosyn scaffold alone failed to rescue the null phenotype, while overexpression of the scaffold had no effect on evoked release. As such, these data indicate that while the scaffold is required for full Tomosyn function, it does not directly inhibit fusion. These observations are consistent with the mechanism proposed in C. elegans, but differ from studies in cultured mammalian cells suggesting the scaffold acts as an independent release suppressor by inhibiting Syt1. Characterization of Drosophila tomosyn/syt1 double mutants demonstrated Tomosyn suppresses release independent of Syt1, arguing the scaffold must serve a function that enhances the inhibitory activity of the SNARE domain independent of Syt1. Indeed, this study found the Tomosyn SNARE motif was mislocalized without the WD40 scaffold, arguing this region indirectly supports Tomosyn's inhibitory activity by ensuring proper localization so the SNARE domain can compete for t-SNARE binding. Similar to studies in C. elegans and mammals, this study found Drosophila Tomosyn co-localized with other SV proteins. Human Tomosyn transgenes also rescued elevated evoked and spontaneous release in tomosyn mutants, indicating functional conservation of its inhibitory properties. Overexpression of either Drosophila or human Tomosyn in a wildtype background also decreased release, demonstrating presynaptic output can be bi-directionally controlled by varying Tomosyn expression levels (Sauvola, 2021).

In addition to intrinsic release differences between tonic and phasic motoneurons, this study found Tomosyn also controls presynaptic homeostatic potentiation (PHP). This form of synaptic plasticity occurs when presynaptic motoneurons upregulate Pr and quantal content to compensate for decreased GluR function and smaller quantal size. Inducing PHP with the allosteric GluR inhibitor Gyki revealed Tomosyn is required for expression of this form of acute PHP at Ib terminals. Removing Tomosyn inhibition at Ib synapses generates a ~ 4 fold enhancement in evoked release, more than sufficient to compensate for a twofold reduction in evoked response size from two equally contributing motoneurons. Indeed, AZ Pr mapping revealed Ib synapses potentiate in the presence of Gyki while Is terminals showed no change, indicating enhanced release from Ib is sufficient to homeostatically compensate for Gyki-induced decreases in quantal size. Although future studies will be required to determine the molecular cascade through which Tomosyn mediates PHP expression, prior work indicates PKA phosphorylation of Tomosyn reduces its SNARE binding properties and decreases its inhibition of SV release. Given Gyki-induced PHP expression requires presynaptic PKD (Nair, 2020), an attractive hypothesis is that PKD phosphorylates Tomosyn and reduces its ability to inhibit SNARE complex formation. Similar to tomosyn mutants, this could promote SV availability by generating a larger pool of free t-SNAREs to support enhanced docking of SVs at AZs. Increased docking would elevate single AZ Pr by increasing the number of fusion-ready SVs upon Ca2+ influx, similar to the effect observed with quantal imaging (Sauvola, 2021).

Despite the importance of Tomosyn in regulating release character between tonic and phasic motoneurons, tomosyn null mutants are viable into adulthood. As such, the entire range of Tomosyn expression can be used by distinct neuronal populations in vivo to set presynaptic output. Tonic Ib terminals shift towards phasic release with no effect on Is output in tomosyn null mutants, resulting in a collapse of presynaptic release diversity between these two neuronal subgroups. Like tomosyn, null mutants in syt7 are viable and show dramatically enhanced evoked release. Tomosyn/syt7 double mutants show even greater increases in release output, arguing multiple non-essential presynaptic proteins can independently fine tune synaptic strength within the presynaptic terminal. Together, these experiments demonstrate Tomosyn is a highly conserved release inhibitor that varies in expression between distinct neuronal subtypes to regulate intrinsic Pr and plasticity, providing a robust mechanism to generate presynaptic diversity across the nervous system (Sauvola, 2021).

Target-wide induction and synapse type-specific robustness of presynaptic homeostasis

Presynaptic homeostatic plasticity (PHP) is an evolutionarily conserved form of adaptive neuromodulation and is observed at both central and peripheral synapses. This work has made several fundamental advances by interrogating the synapse specificity of PHP. This study defines how PHP remains robust to acute versus long-term neurotransmitter receptor perturbation. A general PHP property is described that includes global induction and synapse-specific expression mechanisms. Finally, a novel synapse-specific PHP expression mechanism is detailed that enables the conversion from short- to long-term PHP expression. If these data can be extended to other systems, including the mammalian central nervous system, they suggest that PHP can be broadly induced and expressed to sustain the function of complex neural circuitry (Genc, 2019).

This study has taken advantage of cell-type-specific GAL4 drivers to achieve selective, optogenetic stimulation of single motoneuron inputs to a single muscle fiber. This has enabled exploration of the synapse specificity of PHP. It is demonstrated that the global disruption of postsynaptic glutamate receptors, either pharmacologically or genetically, induces robust PHP at both tonic (Ib) and phasic (Is) presynaptic terminals contacting a single muscle target. The fact that both phasic and tonic synapses participate in presynaptic homeostasis is consistent with previously published work demonstrating that the expression of PHP does not alter the short-term dynamics of presynaptic release when both the phasic and tonic synapses are stimulated simultaneously (Genc, 2019).

It was also demonstrated that PHP is differentially expressed at phasic and tonic synapses when external calcium concentration is decreased. Remarkably, however, the synapse-specific effects of reducing external calcium depend upon how PHP is initiated. Acute induction of PHP is robustly expressed by the phasic synapses but not as robustly at tonic synapses. However, the situation completely reverses during chronic induction of PHP, where PHP is robustly expressed by the tonic synapses but not by the phasic synapses. These data highlight a novel, fundamental difference in the mechanisms responsible for the rapid versus prolonged expression of PHP (Genc, 2019).

It was previously argued that PHP is synapse specific in that it is expressed exclusively by the tonic nerve terminal. These prior experiments included an optical quantal analysis of synaptic transmission in the GluRIIA mutant. This study has confirmed that PHP is expressed solely by the tonic synapse under conditions of diminished release, the GluRIIA mutant background, and low external calcium (conditions that reflect those pursued in the prior optogenetic study, which utilized high concentrations of external magnesium, presumably to stabilize the optical recording conditions). However, by exploring multiple methods of PHP induction at multiple external calcium concentrations, this study has clearly demonstrated that PHP is globally induced and expressed, such that differences in PHP expression emerge only when external calcium is diminished and are dependent on how PHP is induced (acutely versus chronically) (Genc, 2019).

The current understanding of PHP expression is based primarily on the genetic deletion of key molecular components of the presynaptic homeostatic machinery, discovered in forward genetic screens. A presynaptic ENaC channel, composed of subunits transcribed by the PPK11, PPK16, and PPK1 genes, is necessary for both the rapid induction and sustained expression of PHP, driving increased calcium influx through the presynaptic CaV2.1 calcium channel (Younger, 2013). ENaC-channel function is required for both short- and long-term PHP, and this study has now demonstrated that it functions at both phasic and tonic synapses. So, differential activity of the ENaC channel cannot account for synapse-specific PHP expression at low external calcium (Genc, 2019).

It is proposed that the differential expression of acute versus chronic PHP might relate to the EGTA sensitivity of presynaptic release. The chronic induction of PHP is differentially sensitive to EGTA, implying that the chronic expression of PHP includes a population of synaptic vesicles that are weakly coupled to presynaptic calcium channels. This finding could explain the differential synapse-specific expression of PHP at phasic and tonic synapses. During the acute induction of PHP, low-release-probability tonic synapses release neurotransmitters less efficiently at low external calcium, and PHP expression is correspondingly impaired. During the chronic induction, it is proposed that a transformation of the release site allows the participation of weakly coupled vesicles in the release process. It is hypothesized that the different anatomical geometry of release sites (phasic and tonic synapses) causes PHP to selectively fail at phasic synapses. More specifically, phasic synapses might be organized in such a way that weakly coupled vesicles cannot be accessed when external calcium is diminished. Such an effect would only be observed after the chronic induction of PHP combined with diminished external calcium (Genc, 2019).

The trigger that initiates PHP, whether acute or chronic, is the loss or inhibition of postsynaptic neurotransmitter receptors, whether the glutamatergic Drosophila NMJ or the cholinergic mammalian NMJ is considered. On the basis of the data presented in this study, it appears that any synapse at which postsynaptic receptors are perturbed will express PHP. However, it has yet to be determined whether selective disruption of glutamate receptors at one synapse on a given target will induce synapse-selective PHP. The tools to achieve this experiment in a clearly reproducible manner do not yet exist. If PHP is expressed at every synapse that becomes perturbed, then PHP should stabilize the flow of information through complex neural circuitry. The related question of synapse-specific induction will determine whether information transfer can be selectively sustained through the regulation of some, but not all, synapses on a given target. Finally, the data suggest that some synapses might be more or less efficient at acutely and chronically expressing PHP. It is noted that the tonic synapse always expresses PHP in a more variable manner. There is a large range of synapse types with different release probabilities throughout the complex neural circuitry in the mammalian CNS, suggesting that PHP could be more or less robustly expressed at different synapses. As such, the global induction of PHP could differentially affect neural-circuit function in previously unsuspected ways, a possibility that could be important for considering the action of chronically administered neural therapeutics or drugs of addiction (Genc, 2019).

Activity-dependent global downscaling of evoked neurotransmitter release across glutamatergic inputs in Drosophila

Within mammalian brain circuits, activity-dependent synaptic adaptations such as synaptic scaling stabilise neuronal activity in the face of perturbations. Stability afforded through synaptic scaling involves uniform scaling of quantal amplitudes across all synaptic inputs formed on neurons, as well as on the postsynaptic side. It remains unclear whether activity-dependent uniform scaling also operates within peripheral circuits. This study tested for such scaling in a Drosophila larval neuromuscular circuit, where the muscle receives synaptic inputs from different motoneurons. Motoneuron-specific genetic manipulations were employed to increase the activity of only one motoneuron and recordings of postsynaptic currents from inputs formed by the different motoneurons. An adaptation was detected which caused uniform downscaling of evoked neurotransmitter release across all inputs through decreases in release probabilities. This 'presynaptic downscaling' maintained the relative differences in neurotransmitter release across all inputs around a homeostatic set point, caused a compensatory decrease in synaptic drive to the muscle affording robust and stable muscle activity, and was induced within hours. Presynaptic downscaling was associated with an activity-dependent increase in Drosophila vesicular glutamate transporter (DVGLUT) expression. Activity-dependent uniform scaling can therefore manifest also on the presynaptic side to produce robust and stable circuit outputs. Within brain circuits, uniform downscaling on the postsynaptic side is implicated in sleep- and memory-related processes. These results suggest that evaluation of such processes might be broadened to include uniform downscaling on the presynaptic side (Karunanithi, 2020).

Changes in neuronal activity can alter synaptic strength and refine synaptic connections as part of normal developmental and behavioral plasticity. Abnormal levels of activity could disrupt normal activity-dependent synaptic modifications, leading to pathophysiological imbalances in activity within neural circuits. Neurons have however evolved the capacity to functionally adapt and maintain stable activities in the face of perturbations. The most intensely studied adaptation, which stabilizes neuronal activity within mammalian brain circuits, is synaptic scaling. It operates globally by uniformly scaling quantal amplitudes across all synaptic inputs formed on a neuron, through changes in postsynaptic receptor numbers. Synaptic scaling causes compensatory changes in excitatory synaptic drive to the neuron in a direction that enables its firing rate, which has strayed out of set point range, to return back within range. Uniform scaling is also proposed to maintain the relative differences in synaptic weights among inputs, thereby maintaining the ability of neural circuits to appropriately process and interpret divergent inputs with physiological relevance. Considering that maintenance of stable activity is imperative for the robust function of the nervous system as a whole, this study tested whether activity-dependent uniform scaling could also operate within peripheral circuits (Karunanithi, 2020).

Peripheral circuits also exhibit compensatory adaptations and activity-dependent synaptic modifications. One such circuit is the Drosophila larval neuromuscular circuit which drives crawling. This is a converging circuit, where different glutamatergic motoneurons form discrete neuromuscular junctions (NMJs) on each muscle. The highly active 1b and the less active 1s motoneurons form NMJs on virtually all larval muscles. Compensatory adaptations observed at those NMJs are mainly those which preserve strong muscular excitation by homeostatically maintaining synaptic drive to the muscle. They include presynaptic homeostatic potentiation, presynaptic homeostatic depression (PHD), and synaptic homeostasis through structural compensations. Those adaptations are recruited through changes at synapses themselves, rather than through changes in neuronal and muscle activities (firing rates), and are therefore not regarded as activity-dependent. It is unclear whether there are activity-dependent adaptations that manifest at NMJs, involving uniform scaling, to afford robust muscle activity (Karunanithi, 2020).

Activity-dependent synaptic modifications, which appear within the larval neuromuscular circuit following long-term increases in neuronal activity, have been associated with synapse formation and maturation, and with synaptic modifications correlated to information storage. Such work used fly strains where activity was chronically increased in all neurons or motoneurons. With current knowledge regarding activity-dependent compensatory adaptations, it was asked whether such synaptic modifications were fully or partly compensatory to afford robust muscle activity. It was also asked whether compensatory adaptations could be recruited by increasing the activity of only one of the two motoneuron types (Karunanithi, 2020).

Using genetic manipulations, the activity of the 1s motoneuron, innervating muscle 2, was transformed from being lowly active to being highly active. Then whether that transformation recruited compensatory adaptations was assessed by testing for alterations in muscle activity and NMJ properties formed by the genetically manipulated 1s and the unmanipulated 1b motoneurons. A compensatory adaptation was uncovered that was termed 'presynaptic downscaling,' which uniformly downscaled 1b and 1s evoked neurotransmitter release to afford robust and stable muscle activity. Activity-dependent uniform scaling can therefore manifest also within the periphery and on the presynaptic side (Karunanithi, 2020).

Activity-dependent uniform scaling of a determinant of synaptic strength has previously been reported to occur only on the postsynaptic side within central circuits. This work showed that such scaling of a determinant of synaptic strength can also occur on the presynaptic side within peripheral circuits. Presynaptic downscaling describes the uniform decreases in QCs across all inputs, maintaining their relative neurotransmitter release differences around a homeostatic set point. Presynaptic downscaling was induced by increasing the activity of only the 1s motoneuron but was manifest by decreasing release probabilities at both the manipulated 1s and the unmanipulated 1b boutons. Presynaptic downscaling decreased synaptic drive to the muscle: (1) stabilizing muscle firing rates around the downshifted set point and homeostatically maintaining the wide variation in firing rates around that set point; and (2) maintaining robust muscle activity by preventing muscle firing rates from being displaced outside of the physiological range. An activity-dependent increase in 1s DVGLUT expression resulted in presynaptic downscaling, adding support to the idea that regulation of VGLUT expression plays a role in the recruitment of presynaptic compensatory adaptations (Karunanithi, 2020).

The findings regarding muscle activity appear to fit within the framework of biological robustness, a concept in systems biology which accounts for variations in set points to maintain robust function against perturbations. Within that framework, a robust system/component can operate stably at different set points. That framework also accounts for set point shifts, as in this study, where excessive 1s neural drive to the muscle causes the muscle firing rate set point to downshift, without firing rates overshooting the physiological range. Through such shifts, robust muscle activity is afforded to potentially generate a wide range of contractions to drive varied movements under adverse conditions (Karunanithi, 2020).

In synaptic downscaling, which operates centrally on the postsynaptic side, the quantal amplitudes are uniformly downscaled across all inputs, decreasing synaptic drive to the postsynaptic neuron. That decrease in synaptic drive affords stability by lowering firing rates back within the narrow set point range. In presynaptic downscaling, QCs are uniformly downscaled across all glutamatergic inputs through decreases in release probabilities (although uniform downscaling of QCs could also occur through decreases in active release site numbers). The downscaling of QCs decreases synaptic drive, affording robust and stable muscle activity. Activity-dependent uniform scaling appears to therefore operate on both the presynaptic and postsynaptic sides by scaling different determinants of synaptic strength to maintain target cell firing rates within operational range. When the operational range is narrow, as in neurons, uniform downscaling stabilizes firing rates around a set point. But when the range is wide, as in larval muscle, uniform downscaling stabilizes firing rates around a different set point without displacing firing rates outside of the physiological range, maintaining robust outputs. Activity-dependent uniform downscaling appears to therefore operate in different capacities in mammalian neurons and larval muscle (Karunanithi, 2020).

A difference between the induction of synaptic scaling centrally and of presynaptic downscaling peripherally is that the former can be produced cell-autonomously, but not the latter. Synaptic scaling can be produced by directly changing the activity of a postsynaptic neuron. Such scaling can be swift, produced in an hour, as the signals activated in response to changes in activity directly affect the synapses formed on that postsynaptic neuron. Presynaptic downscaling, on the other hand, is produced by increasing the activity of a single input neuron. Induction signals, once initiated in that input neuron, would need to travel anterograde to the target cell, and then retrograde from the target cell to the different synaptic inputs to produce uniform downscaling. Induction signals could also involve signaling between 1b and 1s inputs, possibly like those reported among mammalian glutamatergic inputs. Those signaling pathways suggest that presynaptic downscaling induction is a relatively slow (requiring more than an hour) and nonautonomous process. Some candidate molecules carrying anterograde signals being vesicular glutamate and postsynaptic receptors, and retrograde signals could include Glass bottom boat, Dystrophin, and Semaphorin. Molecules evidenced to mediate signaling among mammalian glutamatergic inputs include glutamate, calcium, protein kinase A, and neurotrophic factors (Karunanithi, 2020).

The experiments argue for presynaptic downscaling resulting from increased 1s DVGLUT expression following increased 1s motoneuron activity. That order of process leading to presynaptic downscaling does not however imply direct causality among those steps. A number of intermediate steps may likely be involved, including retrograde signaling from the muscle (discussed above) to calibrate neurotransmitter release in order to prevent muscle activity from overshooting its physiological range. These possibilities need to be investigated in future work (Karunanithi, 2020).

Uniform scaling is proposed to maintain the relative differences among synapses around a set point to prevent disruption of mechanisms which rely on such differences being preserved. For example, in postsynaptic neurons within brain circuits, synaptic downscaling would enable synaptic inputs which underwent Hebbian potentiation to retain their stronger synaptic weighting relative to other inputs, while concurrently preventing such strengthening from causing runaway excitation. In the periphery, recent work at the larval NMJ shows that 1b synapses primarily drive larval muscle contractions during crawling, whereas 1s synapses drive intersegmental coordination of muscle contractions (Newman, 2017). Neurotransmitter release differences between 1b and 1s synapses underlie the differential execution of these two functions. The current findings therefore suggest that uniform downscaling could preserve the relative neurotransmitter release differences to enable 1b and 1s synapses to continue driving these two functions against perturbations. Uniform downscaling on the presynaptic or postsynaptic sides could therefore afford stability within circuits by maintaining the relative differences among synaptic inputs around a homeostatic set point (Karunanithi, 2020).

Larval NMJs display forms of compensatory adaptations, which preserve strong muscular excitation in the face of perturbations. In those forms, EJP amplitude is homeostatically maintained through compensatory changes in both QC and quantal size. PHD is one such adaptation, where the increases in quantal sizes are offset by compensatory decreases in QCs at both 1b and 1s boutons. PHD is induced by directly overexpressing DVGLUT into both 1b and 1s motoneurons, and as such, has not been shown to be induced by altered activity. By contrast, presynaptic downscaling causes a compensatory decrease in EJP amplitude to afford robust and stable muscle activity. It results following an activity-dependent increase in only 1s DVGLUT expression, causing the uniform downscaling of both 1b and 1s QCs. PHD is therefore a compensatory adaptation that preserves muscular excitation by homeostatically maintaining EJP amplitude, whereas presynaptic downscaling is a compensatory adaptation which stabilizes muscle activity by decreasing EJP amplitude. The two different adaptations are produced by two different patterns of DVGLUT expression, but both involve uniform decreases in QCs across inputs. However, in the case of presynaptic downscaling, the uniform decreases are activity-dependent. In this preparation, compensatory adaptations therefore not only regulate muscle excitation, but also muscle activity (Karunanithi, 2020).

Uniform downscaling has been implicated in processes related to sleep and memory. Work in mammalian brain circuits proposes that uniform downscaling of quantal amplitudes on the postsynaptic side renormalizes synapses during sleep following Hebbian potentiation throughout the day, and prevents runaway excitation following Hebbian potentiation during memory formation. This finding suggests that the scope for evaluating such a process should be broadened to include any uniform downscaling on the presynaptic side, and questions whether such downscaling would involve changes in vesicular neurotransmitter transporter expression (Karunanithi, 2020).

In future work, the molecular mechanisms underlying presynaptic downscaling will need to be elucidated, including identification of retrograde and anterograde signals, and the role of DVGLUT in such signaling. Assessments are also needed of how regulatory molecules, and changes in the activity profiles of the 1b motoneuron and both 1b and 1s motoneurons, contribute to robust and stable circuit function. Finally, detailed analysis of the frequency components within muscle bursts could provide better understanding of the mechanisms shaping muscle activity (Karunanithi, 2020).

Endogenous tagging reveals differential regulation of Ca(2+) channels at single AZs during presynaptic homeostatic potentiation and depression

Neurons communicate through Ca(2+)-dependent neurotransmitter release at presynaptic active zones (AZs). Neurotransmitter release properties play a key role in defining information flow in circuits and are tuned during multiple forms of plasticity. Despite their central role in determining neurotransmitter release properties, little is known about how Ca(2+) channel levels are modulated to calibrate synaptic function. This study used CRISPR to tag the Drosophila CaV2 Ca(2+) channel Cacophony (Cac) and, in males in which all endogenous Cac channels are tagged, investigated the regulation of endogenous Ca(2+) channels during homeostatic plasticity. Heterogeneously distributed Cac was found to be highly predictive of neurotransmitter release probability at individual AZs and differentially regulated during opposing forms of presynaptic homeostatic plasticity. Specifically, AZ Cac levels are increased during chronic and acute presynaptic homeostatic potentiation (PHP), and live imaging during acute expression of PHP reveals proportional Ca(2+) channel accumulation across heterogeneous AZs. In contrast, endogenous Cac levels do not change during presynaptic homeostatic depression (PHD), implying that the reported reduction in Ca(2+) influx during PHD is achieved through functional adaptions to pre-existing Ca(2+) channels. Thus, distinct mechanisms bi-directionally modulate presynaptic Ca(2+) levels to maintain stable synaptic strength in response to diverse challenges, with Ca(2+) channel abundance providing a rapidly tunable substrate for potentiating neurotransmitter release over both acute and chronic timescales (Gratz, 2019).

Diverse synaptic release properties enable complex communication and may broaden the capacity of circuits to communicate reliably and respond to changing inputs. This study has investigated how the regulation of Ca2+ channel accumulation at AZs contributes to the establishment and modulation of AZ-specific release properties to maintain stable communication. Endogenous tagging of Cac allowed tracking of Ca2+ channels live and in fixed tissue without the potential artifacts associated with transgene overexpression. This approach revealed differences in the regulation of endogenous and exogenous Ca2+ channels, underlining the value of developing and validating reagents for following endogenous proteins in vivo (Gratz, 2019).

The abundance of endogenous Cac at individual AZs of single motorneurons is heterogeneous and correlates with single-AZ Pr. This is consistent with previous studies in multiple systems linking endogenous Ca2+ channel levels at individual AZs to presynaptic release probability and efficacy, and a recent investigation of transgenically expressed Cac. This strong correlation suggests Ca2+ channel levels might be regulated to tune Pr during plasticity, so this study investigated the modulation of endogenous Cac levels in several Drosophila models of presynaptic homeostatic plasticity. Previous studies have suggested that the bidirectional regulation of Ca2+ influx at synapses contributes to the modulation of presynaptic release observed during both PHP and PHD. A long-standing question is whether these changes are achieved through the regulation of channel levels, channel function, or through distinct mechanisms. Multiple mechanisms have been proposed to explain the increase in Ca2+ influx observed during the expression of PHP. For example, a presynaptic epithelial sodium channel (ENaC) and glutamate autoreceptor (DKaiR1D) have been implicated in promoting Ca2+ influx during PHP, leading to the model that modulation of presynaptic membrane potential might increase influx through Cac channels. On the other hand, the Eph‐specific RhoGEF Ephexin signals through the small GTPase Cdc42 to promote PHP in a Cac-dependent manner, raising the possibility that it does so through actin-dependent accumulation of new channels. Further, multiple AZ cytomatrix proteins, including Fife, RIM, and RIM-binding protein, are necessary to express PHP and also regulate Ca2+ channel levels during development. However, whether Ca2+ channel abundance is modulated during PHP remained an open question (Gratz, 2019).

This study demonstrates that Cac abundance is indeed enhanced during both the acute and chronic expression of PHP. This increase occurs in conjunction with the accumulation of Brp and enhancement of the RRP, pointing to the coordinated remodeling of the entire neurotransmitter release apparatus during PHP on both timescales. Studies in mammals have found that AZ protein levels are dynamic and subject to homeostatic modification over chronic timescales, indicating that structural reorganization of AZs is a conserved mechanism for modulating release. As ENaC and DKaiR1D-dependent functional modulation occur in tandem with the structural reorganization of AZs, it is interesting to consider why redundant mechanisms may have evolved. One remarkable feature of PHP is the incredible precision with which quantal content is tuned to offset disruptions to postsynaptic neurotransmitter receptor function. It is therefore tempting to hypothesize that PHP achieves this analog scaling of release probability by simultaneously deploying distinct mechanisms to calibrate the structure and function of AZs (Gratz, 2019).

In contrast to the many mechanisms proposed for modulating Ca2+ influx during PHP, far less is known about how Ca2+ influx is regulated during PHD. One attractive idea was a reduction in AZ Ca2+ channel levels based on studies revealing reduced levels of transgenic UAS-Cac-GFP upon vGlut overexpression. However, this study found that endogenous Cac channels do not change in conjunction with vGlut overexpression-induced PHD. Because all Cac channels are tagged in cacsfGFP-N, this observation indicates that a reduction in Cac abundance at AZs is not necessary to achieve PHD. It was determined that the source of the discrepancy is the use of the transgene to report overexpressed versus endogenous Cac levels, demonstrating that exogenous and endogenous channels are regulated differently, at least during this form of PHD. This indicates that a mechanism other than modulation of Cac abundance drives PHD expression. Levels of Brp and RRP size are also unchanged during PHD. Thus, the coordinated reorganization of the AZ appears to be specific to PHP. Interestingly, reversible downregulation of a subset of AZ proteins, but not Cac, was observed at Drosophila photoreceptor synapses following prolonged light exposure. In the future, it will be of interest to determine whether PHP and PHD share any mechanisms to control the bidirectional modulation of Ca2+ influx. PHD signaling operates independently of PHP, and was recently proposed to function as a homeostat responsive to excess glutamate, not synaptic strength, raising the possibility that mechanisms distinct from those that have been elucidated for PHP may regulate presynaptic inhibition during PHD (Gratz, 2019).

Finally, live imaging of CacsfGFP-N during acute PHP enabled the investigation of how baseline heterogeneity in Cac levels and Pr intersects with the homeostatic reorganization of AZs. Monitoring endogenous Cac at the same AZs before and after PhTx treatment, the accumulation was observed of Ca2+ channels across AZs with diverse baseline properties. As with PHP expression over chronic timescales, the findings leave open the possibility of multiple mechanisms acting simultaneously, perhaps to ensure precise tuning and do not rule out additional modulation of channel function or indirect regulation of Ca2+ influx. In fact, a prevailing model posits rapid events that acutely modulate Pr followed by consolidation of the response for long-term homeostasis. Coincident changes in Ca2+ channel function and levels coupled with long-term restructuring of AZs provides an attractive mechanism for this model. This study also found that Cac accumulation is proportional across low- and high-Pr AZs. Therefore, baseline heterogeneity in Cac levels is maintained following the expression of PHP. At mammalian excitatory synapses, proportional scaling of postsynaptic glutamate receptor levels stabilizes activity while maintaining synaptic weights. The findings suggest an analogous phenomenon could be occurring presynaptically at the Drosophila NMJ. Notably, receptor scaling can occur globally or locally. A recent study reported that PHP can be genetically induced and expressed within individual axon branches, demonstrating a similar degree of specificity in the expression of PHP at the Drosophila NMJ. A proportional increase in Cac levels could arise through homeostatic signaling from individual postsynaptic densities responding to similar decreases in quantal size; a strategy that would allow for both the remarkable synapse specificity and precision with which homeostatic modulation of neurotransmitter release operates (Gratz, 2019).

Maintenance of homeostatic plasticity at the Drosophila neuromuscular synapse requires continuous IP3-directed signaling

Synapses and circuits rely on neuroplasticity to adjust output and meet physiological needs. Forms of homeostatic synaptic plasticity impart stability at synapses by countering destabilizing perturbations. The Drosophila melanogaster larval neuromuscular junction (NMJ) is a model synapse with robust expression of homeostatic plasticity. At the NMJ, a homeostatic system detects impaired postsynaptic sensitivity to neurotransmitter and activates a retrograde signal that restores synaptic function by adjusting neurotransmitter release. This process has been separated into temporally distinct phases, induction and maintenance. One prevailing hypothesis is that a shared mechanism governs both phases. This study shows the two phases are separable. Combining genetics, pharmacology, and electrophysiology, a signaling system consisting of PLCbeta, inositol triphosphate (IP3), IP3 receptors, and Ryanodine receptors was shown to be required only for the maintenance of homeostatic plasticity. It was also found that the NMJ is capable of inducing homeostatic signaling even when its sustained maintenance process is absent (James, 2019).

The Drosophila melanogaster NMJ is an ideal model synapse for studying the basic question of how synapses work to counter destabilizing perturbations. At this NMJ, reduced sensitivity to single vesicles of glutamate initiates a retrograde, muscle-to-nerve signaling cascade that induces increased neurotransmitter vesicle release, or quantal content (QC). As a result, the NMJ maintains a normal postsynaptic response level. Mechanistically, this increase in QC depends upon the successful execution of discrete presynaptic events, such as increases in neuronal Ca2+ influx and an increase in the size of the readily releasable pool (RRP) of synaptic vesicles. The field has termed this compensatory signaling process as presynaptic homeostatic potentiation (PHP) (Delvendahl, 2019). Two factors that govern the expression of PHP are the nature of the NMJ synaptic challenge and the amount of time elapsed after presentation of the challenge. Acute pharmacological inhibition of postsynaptic glutamate receptors initiates a rapid induction of PHP that restores synaptic output in minutes. By contrast, genetic lesions and other long-term reductions of NMJ sensitivity to neurotransmitter induce PHP in a way that is sustained throughout life (James, 2019).

Previously work has identified the Plc21C gene as a factor needed for PHP (Brusich, 2015). Plc21C encodes a Drosophila Phospholipase Cβ (PLCβ) homolog known to be neuronally expressed, but recent ribosomal profiling data also indicates possible muscle expression of Plc21C (Chen, 2017). In canonical signaling pathways, once PLCβ is activated by Gαq, it cleaves the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol triphosphate (IP3). DAG can affect synaptic function by activating Protein Kinase C (PKC), while IP3 binds its receptor (IP3R) to trigger release of calcium from intracellular stores. It is not understood which aspects of this signaling machinery are mobilized during PHP. Potential downstream consequences of PLCβ activity at the NMJ include phosphorylation of neuronal proteins, modulation of ion channel activity, and changes in localization of neurotransmission machinery (James, 2019).

This study scrutinized PLCβ-directed signaling further. Tests were performed to see whether PLCβ-directed signaling was required solely for the maintenance of PHP or if it could also be required for induction. In addition to PLCβ, the IP3 Receptor (Drosophila Itpr, herein IP3R) and Ryanodine receptor (Drosophila RyR) were identified as being part of the same signaling process. Neither PLCβ, nor IP3R, nor RyR are required for the rapid induction of PHP. Additionally, it was found that the rapid induction of PHP is still possible in synapses already sustaining PHP. Surprisingly, it was found that NMJs are capable of rapidly inducing PHP -- even when the sustained expression of PHP is already blocked by impairments in PLCβ, IP3R, or RyR signaling. Taken together, these data show that the induction and maintenance of PHP are separable. Even though there is compelling evidence that parts of the induction and maintenance signaling mechanisms overlap (Goel, 2017), it is also true that acute PHP is possible in scenarios where long-term PHP is not (James, 2019).

This study divides the acute induction and chronic maintenance stages of presynaptic homeostatic potentiation. The data support two core findings. The first is that the short-term induction and long-term maintenance of PHP are separable by genetic and pharmacological manipulations. The second is that an IP3-mediated signaling system is specifically required for the maintenance of PHP (see Model depicting PLCβ/IP3R/RyR signaling underling the maintenance of PHP in both muscle and neuron.) (James, 2019).

For several years, one assumption has been that both the acute and chronic forms of PHP are executed in a similar way, and possibly by shared mechanisms. The issue has been clouded by the fact that both PhTox and a GluRIIA deletion mutant -- the primary reagents utilized to induce PHP -- have the same molecular target, that is GluRIIA-containing glutamate receptors. The process of combining these acute and chronic forms of plasticity within a single genotypic background was cumbersome due to a lack of reagents available to conduct temporally separate targeting experiments (James, 2019).

Several groups ascertained insights into temporal requirements by targeting potential homeostatic signaling genes. The main finding has been that the majority of molecules identified are essential to both the acute and chronic forms of PHP. Neurons tightly control neurotransmitter release probability, and the core presynaptic machinery directly responsible for increasing quantal content is shared. These shared components include the CaV2-type voltage-gated calcium channel or factors gating influx through the channel. They also include factors that regulate the size of the readily releasable pool (RRP) of presynaptic vesicles or factors that control the baseline excitability or plasticity of the presynaptic motor neuron, and neurotransmitter fusion events themselves. As a result, both the acute and chronic forms of PHP signaling cause increases in readily releasable pool (RRP) size and CaV2-mediated calcium influx; and in turn, these presynaptic mechanisms underlie the increases in QC which constitute PHP (James, 2019).

This study shows that although the acute and chronic processes might overlap, they are functionally separable. The fact that they are separable is not necessarily surprising. This finding mirrors data for discrete molecules required for long-term PHP maintenance, such as Target of Rapamycin (Tor), the Rho-type guanine exchange factor Ephexin, the transcription factor Gooseberry, C-terminal Src Kinase, innate immune signals molecules IMD, IKKβ, Relish and the kinesin adaptor Arl8. Importantly, this list contains molecules implicated both in neuron and muscle. This study has added PLCβ (Brusich, 2015) and its effectors IP3R and RyR to this list (James, 2019).

Recent studies have augmented the idea of overlapping signaling pathways and added a degree of specificity. Both acute and chronic forms of PHP begin as instructive retrograde signals after perturbations are detected in the muscle. These forms of PHP involve a decrease in phosphorylation of muscle CaMKII levels, and converge upon the same signaling components in the presynaptic neuron. These studies suggest that Tor signaling converges on the same molecular targets as acute forms of PHP (Goel, 2017). However, the precise roles for Tor and CaMKII in either form of PHP are as yet unknown (James, 2019).

These data appear to contradict the idea of PHP pathway convergence (Goel, 2017). Yet, the current findings are not incompatible with this idea. Multiple lines of evidence indicate discrete signaling requirements for acute forms of PHP on both sides of the synapse. A convergence point is undefined. Accounting for the separation of acute and chronic forms of PHP -- as well as their discrete signaling requirements -- long-term maintenance of PHP might integrate multiple signals between the muscle and neuron over time. For future studies, it will be important to clearly define roles of signaling systems underlying PHP and how distinct signaling systems might be linked (James, 2019).

This work presents unexpected findings. The first is that even in the face of a chronic impairment or block of homeostatic potentiation, the NMJ is nevertheless capable of a full rapid induction of PHP. Given that most molecules required for PHP identified to date are needed for both phases, significant functional separation between them was not expected. A priori it was expected that a failure of the chronic maintenance of PHP would make core machinery unavailable for its acute induction. The second unexpected finding is how quickly the chronic maintenance of PHP can be nullified by pharmacology (10 min), resulting in a return to baseline neurotransmitter release probability after only minutes of drug exposure. This study showed that homeostatic potentiation in GluRIIA mutant larvae or GluRIIC knock-down larvae was abrogated by four different reagents previously known to block IP3R or RyR. Those findings are reminiscent of prior work showing that acute blockade of DAG/ENaC channels with the drug benzamil abolishes PHP in both a GluRIIA mutant background, as well as in the presence of PhTox (Younger, 2013). A difference between benzamil application and the pharmacological agents used in the current study is that the drugs employed in this study only abolished PHP in a chronically challenged background (James, 2019)?

It is unclear how signaling systems that drive homeostatic plasticity transition from a state of induction to a state of maintenance. It is also not understood how interdependent short-term and long-term HSP implementation mechanisms are. A more complete understanding of the timing and perdurance of these properties could have important implications for neurological conditions where synapse stability is episodically lost (James, 2019).

The current findings parallel recent data examining active zone protein intensities in the contexts of induction of PHP and maintenance of PHP at the NMJ. There are multiple results informative to this study. First, the expression of any form of PHP (acute or chronic) appears to correlate with an increased intensity of active zone protein levels, such as CaV2/Cacophony, UNC-13, and the Drosophila CAST/ELKS homolog Bruchpilot. Unexpectedly, however, two of these studies also reported that the rapid induction of PHP does not require this protein increase in order to be functionally executed. These are conundrums for future work. How does rapid active zone remodeling happen in minutes on a mechanistic level? In the absence of such remodeling, how is PHP able to be induced rapidly? Moreover, is the observed short-term active zone remodeling the kernel for the longer-term changes to the active zone and release probability - or is some other compensatory system triggered over long periods of developmental time (James, 2019)?

The current findings add a new dimension to those puzzles with the data that IP3 signaling is continuously required to maintain PHP. If active zone remodeling truly is instructive for PHP maintenance, then it will be interesting to test what roles IP3 signaling and intracellular calcium release play in that process. The screen did include a UAS-RNAi line against unc-13 and an upstream GPCR-encoding gene methuselah. Moreover, a previously study of PHP used a UAS-cac[RNAi] line (Brusich, 2015). Chronic PHP maintenance was intact for all of those manipulations. Those findings are not necessarily contradictory to the recent work from other groups. For instance, knockdown of an active zone protein by RNAi is not a null condition. As such, RNAi-mediated knockdown should leave residual wild-type protein around. In theory, that residual protein could be scaled with homeostatic need (James, 2019).

The data strongly suggest that intracellular calcium channel activation and store release fine tune neurotransmitter release that is implemented by PHP. The exact mechanism by which IP3R and RyR function to maintain PHP at the NMJ is unclear. It appears to be a shared process with IP3. If these store-release channels are acting downstream of IP3 activity, then the data suggest that this would be a coordinated activity involving both the muscle and the neuron, with loss of IP3 signaling in the neuron being more detrimental to evoked release (James, 2019).

It remains unclear what signals are acting upstream. PLCβ is canonically activated by Gαq signaling. From prior work, evidence was garnered that a Drosophila Gq protein plays a role in the long-term maintenance of HSP (Brusich, 2015). Logically, there may exist a G-protein-coupled receptor (GPCR) that functions upstream of PLCβ/IP3 signaling. A screen did not positively identify such a GPCR. Several genes encoding GPCRs were examined, including TkR86C, mAChR-A, GABA-B-R1, PK2-R2, methuselah, AdoR, and mGluR. Genes encoding Gβ subunits or putative scaffolding molecules were also examined, including CG7611 (a WD40-repeat-encoding gene), Gβ13F, and Gβ76C, again with no positive screen hits (James, 2019).

The data are consistent with dual pre- and post-synaptic functions of IP3. This could mean dual pre- and post-synaptic roles for calcium store release, through an undetermined combination of RyR and IP3R activities, again either pre- or post-synaptic. Both RyR and IP3R have been shown to be critical for specific aspects of neuroplasticity and neurotransmission. Activities of both RyR and IP3R can activate molecules that drive plasticity, such as Calcineurin and CaMKII. At rodent hippocampal synapses, electrophysiological measures like paired-pulse facilitation and frequency of spontaneous neurotransmitter release are modulated by RyR and/or IP3R function, as is facilitation of evoked neurotransmitter at the rat neocortex. In addition to vesicle fusion apparatus, activity of presynaptic voltage-gated calcium channels is modulated by intracellular calcium. Work from this lab at the NMJ has shown that impairing factors needed for store-operated calcium release can mollify hyperexcitability phenotypes caused by gain-of-function CaV2 amino-acid substitutions (Brusich, 2018; James, 2019 and references therein).

Within the pre-synaptic neuron, IP3R and RyR could activate any number of calcium-dependent molecules to propagate homeostatic signaling. Some candidates were tested in a screen, but none of those tests blocked PHP. One possibility is that the reagents utilized did not sufficiently diminish the function of target molecules enough to impact PHP in this directed screen. Detection of downstream effectors specific to muscle or neuron might also be ham>pered by the fact that attenuation of IP3 signaling in a single tissue is insufficient to abrogate PHP. Another possibility is that presynaptic store calcium efflux via IP3R and RyR may directly potentiate neurotransmitter release, either by potentiating basal calcium levels or synchronously with CaV2-type voltage-gated calcium channels (James, 2019).

Both pre- and post-synaptic voltage-gated calcium channels are critical for the expression of several forms of homeostatic synaptic plasticity. Much evidence supports the hypothesis that store-operated channels and voltage gated calcium channels interact to facilitate PHP. In various neuronal populations, both RyR and IP3R interact with L-type calcium channels physically and functionally to reciprocally impact the opening of the other channel. In presynaptic boutons, RyR calcium release follows action potential firing. Calcium imaging experiments show that both the acute expression and sustained maintenance of PHP requires an increase in presynaptic calcium following an action potential. Because IP3Rs are activated by both free calcium and IP3, elevated IP3 levels in the case of chronically expressed PHP could allow IP3Rs and RyRs to open in a way that is time-locked with CaV2-mediated calcium influx or in a way to facilitate the results of later CaV2-mediated influx (James, 2019).

Dual separable feedback systems govern firing rate homeostasis

Firing rate homeostasis (FRH) stabilizes neural activity. A pervasive and intuitive theory argues that a single variable, calcium, is detected and stabilized through regulatory feedback. A prediction is that ion channel gene mutations with equivalent effects on neuronal excitability should invoke the same homeostatic response. In agreement, this study demonstrates robust FRH following either elimination of Kv4/Shal protein or elimination of the Kv4/Shal conductance. However, the underlying homeostatic signaling mechanisms are distinct. Eliminating Shal protein invokes Kruppel-dependent rebalancing of ion channel gene expression including enhanced slo, Shab, and Shaker. By contrast, expression of these genes remains unchanged in animals harboring a CRISPR-engineered, Shal pore-blocking mutation where compensation is achieved by enhanced IK_DR. These different homeostatic processes have distinct effects on homeostatic synaptic plasticity and animal behavior. It is proposed that FRH includes mechanisms of proteostatic feedback that act in parallel with activity-driven feedback, with implications for the pathophysiology of human channelopathies (Kulik, 2019).

Firing Rate Homeostasis is a form of homeostatic control that stabilizes spike rate and information coding when neurons are confronted by pharmacological, genetic or environmental perturbation. FRH has been widely documented within invertebrate neurons and neural circuits as well as the vertebrate spinal cord, cortical pyramidal neurons and cardiomyocytes. In many of these examples, the genetic deletion of an ion channel is used to induce a homeostatic response. The mechanisms of FRH correct for the loss of the ion channel and precisely restore neuronal firing properties to normal, wild-type levels). To date, little is understood about the underlying molecular mechanisms (Kulik, 2019).

FRH induced by an ion channel gene deletion is truly remarkable. The corrective response is not limited to the de novo expression of an ion channel gene with properties that are identical to the deleted channel, as might be expected for more generalized forms genetic compensation. Instead, the existing repertoire of channels expressed by a neuron can be 'rebalanced' to correct for the deletion of an ion channel. How is it possible to precisely correct for the absence of an essential voltage-gated ion channel? The complexity of the problem seems immense given that many channel types functionally cooperate to achieve the cell-type-specific voltage trajectory of an action potential (Kulik, 2019).

Theoretical work argues that different mixtures of ion channels can achieve similar firing properties in a neuron. These observations have led to a pervasive and intuitively attractive theory that a single physiological variable, calcium, is detected and stabilized through regulatory feedback control of ion channel gene expression. Yet, many questions remain unanswered. There are powerful cell biological constraints on ion channel transcription, translation, trafficking and localization in vivo. How do these constraints impact the expression of FRH? Is calcium the only intracellular variable that is sensed and controlled by homeostatic feedback? There remain few direct tests of this hypothesis. Why are homeostatic signaling systems seemingly unable to counteract disease-relevant ion channel mutations, including those that have been linked to risk for diseases such as epilepsy and autism (Kulik, 2019)?

This study has taken advantage of the molecular and genetic power of Drosophila to explore FRH in a single, genetically identified neuron subtype. Specifically, two different conditions are compared that each eliminate the Shal/Kv4 ion channel conductance and, therefore, are expected to have identical effects on neuronal excitability. Robust FRH is demonstrated following elimination of the Shal protein and, independently, by eliminating the Shal conductance using a pore blocking mutation that is knocked-in to the endogenous Shal locus. Thus, consistent with current theory, FRH can be induced by molecularly distinct perturbations to a single ion channel gene. However, these two different perturbations were found to induce different homeostatic responses, arguing for perturbation-specific effects downstream of a single ion channel gene (Kulik, 2019).

Taken together, these data contribute to a revised understanding of FRH in several ways. First, altered activity cannot be the sole determinant of FRH. Two functionally identical manipulations that eliminate the Shal conductance, each predicted to have identical effects on neuronal excitability, lead to molecularly distinct homeostatic responses. Second, homeostatic signaling systems are sensitive to the type of mutation that affects an ion channel gene. This could have implications for understanding why FRH appears to fail in the context of human disease caused by ion channel mutations, including epilepsy, migraine, autism and ataxia. Finally, the data speak to experimental and theoretical studies arguing that the entire repertoire of ion channels encoded in the genome is accessible to the mechanisms of homeostatic feedback, with a very large combinatorial solution space. The data are consistent with the existence of separable proteostatic and activity-dependent homeostatic signaling systems, potentially acting in concert to achieve cell-type-specific and perturbation-specific FRH (Kulik, 2019).

These data advance mechanistic understanding of FRH in several ways. First, it was demonstrated that FRH can be induced and fully expressed in single, genetically identified neurons. Since changes in the activity of a single motoneuron are unlikely to dramatically alter the behavior of the larvae, these data argue strongly for cell autonomous mechanisms that detect the presence of the ion channel perturbation and induce a corrective, homeostatic response. Second, this study demonstrates that FRH functions to preserve the waveform of individual action potentials. This argues for remarkable precision in the homeostatic response. Third, new evidence is provided that the transcription factor Krüppel is essential for FRH, and selectively controls the homeostatic enhancement of IK_Ca,the voltage-gated sodium current (I_Na) and the voltage-gated calcium current (I_Ca) without altering the baseline ion channel current. Finally, it was demonstrated that different mechanisms of FRH are induced depending upon how the Shal current is eliminated, and these differential expression mechanisms can have perturbation-specific effects on animal behavior (Kulik, 2019).

The existence is proposed of parallel homeostatic mechanisms, responsive to differential disruption of the Shal gene. Different compensatory responses were observed depending upon whether the Shal protein is eliminated or the Shal conductance is eliminated. The following evidence supports the functional equivalence of these manipulations. First, the Shal^W362F mutation completely eliminates somatically recorded fast activating and inactivating potassium current I_KA. Second, a dramatic reduction in I_KA was demonstrated when Shal-RNAi is driven by MN1-GAL4 in a single, identified neuron. Notably, the current-voltage relationship observed for Shal-RNAi is identical to that previously published for the Shal^W362F protein null mutation, being of similar size and voltage trajectory including a + 50 mV shift in voltage activation. This remaining, voltage-shifted, I_KA-like conductance is attributed to the compensatory up-regulation of the Shaker channel on axonal membranes an effect that does not occur in the Shal^W362F mutant. Thus, it seems reasonable to assume that Shal protein elimination and Shal conductance blockade initially create identical effects on neuronal excitability by eliminating Shal function. Subsequently, these perturbations trigger divergent compensatory responses. But, it is acknowledged that direct information is lacking about the immediate effects of the two perturbations (Kulik, 2019).

This study defines FRH as the restoration of neuronal firing rate in the continued presence of a perturbation. This definition is important because it necessitates that the underlying molecular mechanisms of FRH must have a quantitatively accurate ability to adjust ion channel conductances such that firing rate is precisely restored. Mechanistically, a prior example of FRH involves an evolutionarily conserved regulation of sodium channel translation by the translational repressor Pumillio. This work, originally pursued in Drosophila, was extended to mouse central neurons where it was shown that Pumilio-dependent bi-directional changes in the sodium current occur in response to altered synaptic transmission, initiated by application of either AMPA antagonist NBQX or GABA antagonist Gabazine. These data highlight the emerging diversity of molecular mechanisms that can be induced and participate in the execution of FRH (Kulik, 2019).

It is necessary to compare the current results with prior genetic studies of the Shal channel in Drosophila. A prior report, examining the effects of partial Shal knockdown in larval motoneurons, observed a trend toward an increase in the sustained potassium current, but concluded no change. However, the small sample size for potassium current measurements in that study (n = 3 cells) and the incomplete Shal knockdown that was achieved, likely conspired to prevent documentation of the significant increase in IK_DR that was observed. A second prior study examined over-expression of a pore-blocked Shal transgene in cultured Drosophila embryonic neurons, revealing elevated firing rate and a broadened action potential. This was interpreted as evidence against the existence of FRH. However, neuronal precursors were cultured from 5 hr embryos, prior to establishment of neuronal cell fate and prior to the emergence of I_KA currents in vivo, which occurs ~10 hr later in development. It remains unclear whether these cultured neurons are able to achieve a clear cell identity, which may be a prerequisite for the expression of homeostatic plasticity. Another possibility concerns the time-course of FRH, which remains uncertain. Finally, over-expression of the transgene itself might interfere with the mechanisms of FRH, emphasizing the importance of the scarless, CRISPR-mediated gene knock-in approach that was employed (Kulik, 2019).

It is clear from studies in a diversity of systems that FRH can be induced by perturbations that directly alter neuronal activity without genetic or pharmacological disruption of ion channels or neurotransmitter receptors. For example, monocular deprivation induces an immediate depression of neuronal activity in the visual cortex, followed by restoration of normal firing rates. Research on the lobster stomatogastric system ranging from experiments in isolated cell culture to de-centralized ganglia have documented the existence of FRH that is consistent with an activity-dependent mechanism. It is equally clear that FRH can be induced by the deletion of an ion channel gene, including observations in systems as diverse as invertebrate and vertebrate central and peripheral neurons and muscle. But, it has remained unknown whether FRH that is induced by changes in neural activity is governed by the same signaling process that respond to ion channel gene mutations. The current data speak to this gap in knowledge (Kulik, 2019).

Changes in neural activity cannot be solely responsible for FRH. This study compared two different conditions that each completely eliminate the Shal ion channel conductance and, therefore, are expected to have identical effects on neuronal excitability. Robust FRH was demonstrated in both conditions. However, two separate mechanisms account for FRH. Shal-RNAi induces a transcription-dependent homeostatic signaling program. There is enhanced expression of Krüppel and a Krüppel-dependent increase in the expression of the slo channel gene and enhanced IKCA current. By contrast, the Shal^W362F mutant does not induce a change in the expression of Krüppel, slo or any of five additional ion channel genes. Instead, a change was observed in the IK_DR conductance, the origin of which has not yet been identified, but which appears to be independent of a change in ion channel gene transcription (Kulik, 2019).

The existence is proposed of two independent homeostatic signaling systems, induced by separate perturbations to the Shal channel gene. First, it is proposed that Shal-RNAi and the Shal null mutation trigger a homeostatic response that is sensitive to the absence of the Shal protein. In essence, this might represent an ion channel-specific system that achieves channel proteostasis, a system that might normally be invoked in response to errors in ion channel turnover. It is speculated that many, if not all ion channels could have such proteostatic signaling systems in place. In support of this idea, the induction of Kr is specific to loss of Shal, not occurring in eight other ion channel mutant backgrounds, each of which is sufficient to alter neural activity, including eag, para, Shaker, Shab, Shawl, slo, cac and hyperkinetic. Each of these channel mutations is well established to alter neuronal activity. But, Kr responds only to loss of Shal (Kulik, 2019).

Next, it is proposed that eliminating the Shal conductance in the Shal^W362F mutant background induces a separable mechanism of FRH that is independent of ion channel transcription. While the mechanisms of this homeostatic response remain unknown, it is tempting to speculate that this mechanism is activity dependent, consistent with data from other systems. Finally, it remains possible that these homeostatic signaling systems are somehow mechanistically linked. If so, this might provide a means to achieve the precision of FRH. For example, changes in ion channel gene expression might achieve a crude re-targeting of set point firing rates, followed by engagement of activity-dependent processes that fine tune the homeostatic response. Notably, distinct, interlinked negative feedback signaling has been documented in cell biological systems, suggesting a common motif in cell biological regulation (Kulik, 2019).

An interesting prediction of this model is that activity-dependent mechanisms of FRH could be constrained by the action of the channel-specific homeostatic system. For example, loss of Shal induces a Shal-specific gene expression program and activity-dependent homeostatic signaling would be constrained to modulate the Shal-specific response. As such, the homeostatic outcome could be unique for mutations in each different ion channel gene. Given this complexity, it quickly becomes possible to understand experimental observations in non-isogenic animal populations where many different combinations of ion channels are observed to achieve similar firing rates in a given cell. The combined influence of dedicated proteostatic and activity-dependent homeostatic signaling could achieve such complexity, but with an underlying signaling architecture that is different from current theories that focus on a single calcium and activity-dependent feedback processor (Kulik, 2019).

Finally, although the existence is proposed of proteostatic feedback induced by the Shal null mutant and pan-neuronal RNAi, other possibilities certainly exist for activity-independent FRH, inclusive of mechanisms that are sensitive to channel mRNA. For example, the transcriptional compensation that was documented could be considered a more general form of 'genetic compensation'. Yet, the data differ in one important respect, when compared to prior reports of genetic compensation. In most examples of genetic compensation, gene knockouts induce compensatory expression of a closely related gene. For example, it was observed that knockout of β-actin triggers enhanced expression of other actin genes. The compensatory effects that were observed involve re-organization of the expression profiles for many, unrelated ion channel genes. Somehow, these divergent conductances are precisely adjusted to cover for the complete absence of the somato-dendritic A-type potassium conductance. Thus, a more complex form of genetic compensation is favored based upon homeostatic, negative feedback regulation (Kulik, 2019).

How does Kr-dependent control of IK_Ca participate in FRH? IK_Ca is a rapid, transient potassium current. Therefore, it makes intuitive sense that elevated IK_Ca could simply substitute for the loss of the fast, transient I_KA current mediated by Shal. If so, this might be considered an instance of simple genetic compensation. But, if this were the case, then blocking the homeostatic increase in IK_Ca should lead to enhanced firing rates. This is not what was observe. Instead, average firing rates decrease when Kr is eliminated in the background of Shal-RNAi. Thus, the Kr-dependent potentiation of IK_Ca seems to function as a form of positive feedback, accelerating firing rate in order to achieve precise FRH, rather than simply substituting for the loss of Shal. Consistent with this possibility, acute pharmacological inhibition of IK_Ca decreases, rather than increases, average firing rate. However, it should also be emphasized that the role of IK_Ca channels in any neuron are quite complex, with context-specific effects that can either increase or decrease neuronal firing rates. Indeed, it has been argued that BK channels can serve as dynamic range compressors, dampening the activity of hyperexcitable neurons and enhancing the firing of hypoexcitable neurons. This broader interpretation is also consistent with the observed Kr-dependent increase IK_Ca during FRH (Kulik, 2019).

In the stomatogastic nervous system of the crab, single-cell RT-PCR has documented positive correlations between channel mRNA levels, including transcript levels for IK_Ca and Shal. The molecular mechanisms responsible for the observed correlations remain unknown, but it seems possible that these correlations reflect a developmental program of channel co-regulation. Upon homeostatic challenge, the steady-state positive correlations are supplanted by homeostatic compensation, notably enhanced IK_Ca in the presence of 4-AP. The pressing challenge is to define molecular mechanisms that cause the observed correlations and compensatory changes in ion channel expression during homeostatic plasticity. The Kr-dependent control of IK_Ca following loss of Shal is one such mechanism. Clearly, there is additional complexity, as highlighted by the differential response to Shal null and Shal pore blocking mutations and the pumilio-dependent control of sodium channel translation in flies and mice (Kulik, 2019).

Why do ion channel mutations frequently cause disease? If activity-dependent homeostatic signaling is the primary mechanism of FRH, then any ion channel mutation that alters channel function should be detected by changes in neural activity and firing rates restored. One possibility is that FRH is effective for correcting for an initial perturbation, but the persistent engagement of FRH might become deleterious over extended time. Alternatively, each solution could effectively correct firing rates, but have additional maladaptive consequences related to disease pathology. While this remains to be documented in disease, this study has shown that loss of Shal protein throughout the CNS causes deficits in animal behavior that are not observed in animals harboring a pore-blocking channel mutation. Indeed, if one considers that FRH can include altered expression of a BK channel, the potential for maladaptive consequences is high. Altered BK channel function has been repeatedly linked to neurological disease including idiopathic generalized epilepsy, non-kinesigenic dyskinesia and Alzheimer's disease. Thus, there are potentially deleterious ramifications of altering BK channel expression if a homeostatic signaling process is engaged throughout the complex circuitry of the central nervous system. Although the phenotype of maladaptive compensation that was observe is clear, a block in synaptic homeostasis and impaired animal motility, there is much to be learned about the underlying cause. Ultimately, defining the rules that govern FRH could open new doors toward disease therapies that address these maladaptive effects of compensatory signaling (Kulik, 2019).

Myosin VI contributes to synaptic transmission and development at the Drosophila neuromuscular junction

Myosin VI, encoded by jaguar (jar) in Drosophila, is a unique member of the myosin superfamily of actin-based motor proteins. Myosin VI is the only myosin known to move towards the minus or pointed ends of actin filaments. Although Myosin VI has been implicated in numerous cellular processes as both an anchor and a transporter, little is known about the role of Myosin VI in the nervous system. Previous studies recovered jar in a screen for genes that modify neuromuscular junction (NMJ) development and this study reports on the genetic analysis of Myosin VI in synaptic development and function using loss of function jar alleles. The experiments on Drosophila third instar larvae revealed decreased locomotor activity, a decrease in NMJ length, a reduction in synaptic bouton number, and altered synaptic vesicle localization in jar mutants. Furthermore, studies of synaptic transmission revealed alterations in both basal synaptic transmission and short-term plasticity at the jar mutant neuromuscular synapse. Altogether these findings indicate that Myosin VI is important for proper synaptic function and morphology. Myosin VI may be functioning as an anchor to tether vesicles to the bouton periphery and, thereby, participating in the regulation of synaptic vesicle mobilization during synaptic transmission (Kisiel, 2011).

Although Myosin VI function in the vesicle cycle has been implicated in mammalian cells, this report provides the first evidence that Myosin VI is important for maintaining normal peripheral vesicle localization at the bouton. In Drosophila, there are four types of boutons, which are the sites of neurotransmitter release at the NMJ, and they differ in their morphological and chemical properties. Of interest for this study were the largest synaptic boutons found at type I axon terminals, which are present at all NMJs of mature larvae. Visualization of synaptotagmin staining using confocal imaging revealed a mislocalization of synaptic vesicles in jar mutant boutons. An increasing number of jar mutant boutons, corresponding to the severity of Myosin VI loss of function, were found to exhibit diffuse staining over the entire bouton area as opposed to the doughnut-shaped staining pattern present in control boutons. Bouton centre occupancy has previously been observed at Drosophila NMJs of larvae lacking synapsin, a phosphoprotein that reversibly associates with vesicles, using FM1-43 loading under low frequencies. EM analysis confirmed that in synapsin knockouts there was a spread of vesicles into the bouton centre, accompanied by a reduction in the size of the reserve pool. Thus, synapsin is thought to function in maintaining the peripheral distribution of vesicles in Ib boutons. Likewise, the unexpected diffuse synaptotagmin staining of jar mutant boutons suggests Myosin VI participates in restricting vesicles to the bouton periphery. It is possible that Myosin VI is functioning as a regulator of the actin cytoskeleton at the synapse. Mutant studies have revealed that the presynaptic actin cytoskeleton is required for proper synaptic morphogenesis. Myosin VI has already been shown to function in regulating the actin cytoskeleton during the process of spermatid individualization, by acting either as a structural cross-linker or as an anchor at the front edge of the actin cone, and during nuclear divisions in the syncytial blastoderm. However, live imaging of actin dynamics at the synaptic boutons revealed no major defects in the actin cytoskeleton at jar loss of function mutant nerve terminals (Kisiel, 2011).

To assess whether the morphological defects in vesicle localization observed at jar mutant synapses impact synaptic transmission, electrophysiological assays with different stimulation paradigms were used to recruit vesicles from different functional pools. The data add to the knowledge of this protein's physiological role at synapses. Myosin VI mutant mouse hippocampal neurons exhibit defects in the internalization of the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid-type glutamate receptor, responsible for fast glutamatergic transmission, suggesting Myosin VI normally plays a role in AMPAR endocytosis. In addition, basal synaptic transmission is reduced in Myosin VI deficient mouse hippocampal slices compared to wild-type controls. Electrophysiological experiments also indicate that Myosin VI mediates glutamate release induced by brain-derived neurotrophic factor, which is known to modulate synaptic transmission and plasticity in the mammalian central and peripheral nervous system (Kisiel, 2011).

This study is the first to show that Myosin VI's role in synaptic transmission involves mobilization of vesicles from different functional pools, indicating that Myosin VI is important for synaptic plasticity. At the Drosophila NMJ, three pools of vesicles with differential release properties have been identified using FM1-43 staining loaded by various stimulation protocols. The immediately releasable pool (IRP), representing approximately 1% of all vesicles at the NMJ, consists of vesicles docked and primed at active zones for immediate release and experiences rapid depletion within a few stimuli. The readily releasable pool (RRP), making up 14% to 19% of all vesicles at the NMJ, is mobilized by moderate stimulation of ≤3 Hz and maintains exo/endocytosis at these stimulation frequencies. The reserve pool (RP) represents the vast majority of vesicles, 80% to 90%, and is mobilized upon depletion of the RRP. Recruitment from the RP occurs with high frequency stimulation of ≥10 Hz. Spontaneous release was reduced in the most severe jar loss of function mutants. Evoked response at 1 Hz stimulation was also reduced in the jar maternal null mutant. Although less severe jar mutants exhibited a significant decrease in bouton number, they did not experience an accompanying reduction in evoked potential amplitude at low frequency stimulation, suggesting that other homeostatic mechanisms are important for maintaining synaptic strength. The impaired synaptic response in the jar maternal null mutant may be due to a reduction in the probability of RRP vesicle release or in RRP size. If Myosin VI functions to anchor synaptic vesicles, it may act on the RRP to ensure vesicles are localized in manner that makes them readily available for release. Thus, in jar maternal null mutants the reduction in EJP amplitude may occur because a significant number of vesicles were displaced from areas of higher probability release. Alternately, RRP pool size may be reduced at jar mutant synapses (Kisiel, 2011).

Different synaptic vesicle pool properties, such as rate of recruitment of the RP in response to high frequency stimuli, may translate to changes in short-term synaptic plasticity. The increase in EJP amplitude observed at 10 Hz stimulation in 1 mM Ca²⁺ saline may be attributable to enhanced mobilization of the RP for jar³²²/Df(3R)crb87-5 NMJs. Filamentous actin has been implicated in RP mobilization as cytochalasin D, an inhibitor of actin polymerization, has been shown to reduce RP dynamics. This suggests translocation from the RP to the RRP may be mediated by an actin-based myosin motor protein. If Myosin VI functions as a synaptic vesicle tether to regulate recruitment from the RP pool, RP vesicles would be more readily mobilized and transitioned into the RRP upon high frequency stimulation in jar loss of function mutants. Consistent with the idea that RP vesicles were more rapidly incorporated into the RRP, a greater initial depression is observed at jar³²²/Df(3R)crb87-5 mutant synapses during high frequency stimulation in 10 mM Ca²⁺ saline corresponding to the depletion of vesicles at high calcium concentrations. Taken together, the data suggest that Myosin VI mediates synaptic transmission and short-term plasticity by regulating the mobilization of synaptic vesicles from different functional pools. In mammalian cells, Myosin VI has been implicated as a mediator of vesicle endocyctosis and has been shown to transport uncoated vesicles through the actin-rich periphery to the early endosome. The current experiments, however, indicate that endocytosis is not likely affected at jar mutant synapses. Typically, endocytotic mutants are unable to maintain synaptic transmission in response to high frequency stimulation, whereas the Myosin VI loss of function mutants exhibited enhanced EJP amplitude observed at 10 Hz stimulation in 1 mM Ca²⁺ saline. Additional experiments are required to confirm that Myosin VI is functioning as a vesicle tether. Fluorescence recovery after photobleaching analysis can be used to examine the effect of Myosin VI on synaptic vesicle mobility. If Myosin VI is functioning as a vesicle tether, synaptic vesicle mobility is expected to be increased in jar mutants compared to controls (Kisiel, 2011).

In summary, the present work shows that Myosin VI is important for proper synaptic morphology and physiology at the Drosophila NMJ. Myosin VI function in peripheral vesicle localization at the bouton may underlie its contribution to basal synaptic transmission and expression of synaptic plasticity. Future work will address the mechanism by which Myosin VI performs its roles at the synapse, whether as a vesicle tether or by some other involvement in vesicle trafficking (Kisiel, 2011).

Anterograde Transport of Rab4-Associated Vesicles Regulates Synapse Organization in Drosophila

Local endosomal recycling at synapses is essential to maintain neurotransmission. Rab4 GTPase, found on sorting endosomes, is proposed to balance the flow of vesicles among endocytic, recycling, and degradative pathways in the presynaptic compartment. This study reports that Rab4-associated vesicles move bidirectionally in Drosophila axons but with an anterograde bias, resulting in their moderate enrichment at the synaptic region of the larval ventral ganglion. Results from FK506 binding protein (FKBP) and FKBP-Rapamycin binding domain (FRB) conjugation assays in rat embryonic fibroblasts together with genetic analyses in Drosophila indicate that an association with Kinesin-2 (mediated by the tail domain of Kinesin-2α/KIF3A/KLP64D subunit) moves Rab4-associated vesicles toward the synapse. Reduction in the anterograde traffic of Rab4 causes an expansion of the volume of the synapse-bearing region in the ventral ganglion and increases the motility of Drosophila larvae. These results suggest that Rab4-dependent vesicular traffic toward the synapse plays a vital role in maintaining synaptic balance in this neuronal network (Dey, 2017).

The apical compartment of a neuronal cell extends into specific processes forming axons and synapses that are maintained by a distributed endosomal system. For example, vesicle recycling at the synapse, orchestrated by various RabGTPases and associated proteins, renews the readily releasable pool of synaptic vesicles sustaining the neurotransmission. Rab4 is thought to play an important role in balancing the vesicle traffic between the recycling and degradation pathways involved in various cellular processes such as metabolism, cell secretion, and antigen processing. Recruitment of Rab4 on sorting intermediate endosomes allows the transfer of vesicular cargoes from early endosomes to recycling endosomes in axons and at synapses. The activity of Rab4 has also been implicated in the progression of growth cone in Xenopus and the maintenance of dendritic spines in rat hippocampal neurons. Furthermore, endosomal abnormalities found in the cholinergic basal forebrain of patients with Alzheimer's disease correlate with elevated levels of Rab4. All these reports suggest that Rab4-dependent vesicle sorting is essential for the development and maintenance of the nervous system (Dey, 2017).

Vesicular cargoes, including the RabGTPases, originate at the trans-Golgi network, and microtubule-dependent motors transport them to different destinations within a cell. Specific association with motors ensures differential and dynamic subcellular localizations of various RabGTPases according to tissue-specific activities and metabolic demands. For instance, the anterograde trafficking of early, sorting, and late endosomes is dependent on the Kinesin-3 motors KIF13, KIF16B, and KIF1A/IBβ, respectively. Similarly, in Drosophila, Rab5-positive early endosomes associate with Khc-73, which is the ortholog of mammalian KIF13, showing a conserved machinery of Kinesin-Rab interaction. The same vesicles also associate with Dynein for their retrograde movement. It is further indicated that multiple RabGTPases can be grouped together and transported to a destination within a cell where they can engage in different functions. Although the long-range transport of certain presynaptic RabGTPases has been studied in cultured neurons, it is still unclear how the bidirectional movement of Rab-associated vesicles could localize them in distant compartments such as the synapse (Dey, 2017).

This study has shown that Rab4-associated vesicles are transported with an anterograde bias, induced by a specific interaction with the tail domain of Kinesin-2α subunit, which results in its moderate enrichment at the synapses. The rate of pre-synaptic inflow of Rab4 is positively correlated with its activation. Interestingly, this study found that a decreased flow of the Rab4-associated vesicles expanded the region occupied by synapses in the neuropil region of the larval ventral ganglion and enhanced larval motility. Altogether, these results indicate that the flow of Rab4-associated vesicles could maintain synaptic homeostasis in a neuronal network (Dey, 2017).

Heterotrimeric Kinesin-2, implicated in the trafficking of apical endosomes and late endosomal vesicles, was initially thought to bind to its cargoes through the accessory subunit KAP3. However, recent studies have reported that motor subunits could independently interact with soluble proteins through the tail domain. This study shows that the Kinesin-2α tail can also bind to a membrane-associated protein, Rab4. A preliminary investigation in the lab suggests that the interaction in not direct. The RabGTPases are known to recruit a variety of different motors to the membrane, directing intracellular trafficking. Although a previous report indicated that Rab4 could bind to Kinesin-2, this study found that such an interaction leads to the anterograde trafficking of Rab4-associated vesicles in the axon. Rab4 associates with early and recycling endosomes carrying a diverse range of proteins. The FRB-FKBP assay indicated that a particular subset of Rab4-only vesicles associates with Kinesin-2. These vesicles predominantly move toward the synapse in axons. Although Kinesin-2 is also found to bind and transport vesicles containing Acetylcholinesterase (AChE) in the same axon, it was observed that Rab4 does not mark them. Similarly, the post-Golgi vesicles carrying N-cadherin and β-catenin, which are known to associate with Kinesin-2, exclude Rab4. Therefore, the Rab4-associated vesicles transported in the axon are likely to contain a unique set of proteins (Dey, 2017).

An association between RabGTPases and putative transport proteins was identified using coimmunoprecipitation, pull-down, and yeast two-hybrid assays. While these assays provide information on the possible interactions, their nature in a cellular or physiological scenario is unknown. Long-range transport of the Rab27/CRMP2 complex by Kinesin-1 and the Rab3/DENN MADD complex by KIF1A/1Bβ are substantiated by genetic perturbations and imaging. The FKBP-FRB-inducible interaction system used in this study identified Kinesin-2α and KIF13A/B as the key motors for nascent Rab4-associated vesicles. The latter was also shown to associate with Rab5-marked early endosomes. The results excluded the possibility that the Rab4 effectors involved in this interaction could bind to Rab5 and Rab11. In vivo analysis of Rab4-associated vesicular traffic further confirmed that heterotrimeric Kinesin-2 plays a dominant role in the trafficking of nascent Rab4 vesicles toward the synapse (Dey, 2017).

Anterograde axonal movement of Rab4 vesicles is mostly mediated by Kinesin-2, which transports nascent Rab4 vesicles, whereas Kinesin-3 transports sorting endosomes. In a recent study, it was shown that combined activity of Kinesin-1 and Kinesin-2 steers AChE-containing vesicles in the axon (Kulkarni, 2016). A simultaneous association between these two motors was suggested to induce longer runs of AChE-containing vesicles. A similar collaboration between Khc-73 and Kinesin-2 could propel longer runs of Rab4 vesicles. Khc-73 has been implicated in neuronal functions. Khc-73 knockdown in lch5 neurons decreased the motility of Rab4-associated vesicles to a limited extent, which suggests that the motor is only engaged in transporting a relatively small fraction of Rab4-associated vesicles in axons. Khc-73 is a processive motor with an in vitro velocity of 1.54 ± 0.46 &mi;m/s, and this study found that Khc-73 RNAi particularly affected the longer runs. Thus, Khc-73 could support the relatively longer runs (3.05 &mi;m) of Rab4 vesicles in axons. It was previously shown that Rab5-associated vesicles bind to KIF13A/B/Khc-73. Hence, it is inferred that sorting endosomes, positive for both Rab4 and Rab5, can engage both Kinesin-2 and KIF13A/Khc-73 through the cognate adaptors of these two Rabs. However, one needs suitably tagged reagents to establish this conjecture through direct observation in vivo (Dey, 2017).

Local synaptic vesicle recycling mediated by RabGTPases is critical for the availability of fusion-ready synaptic vesicles to sustain neurotransmission. The machinery involved in the translocation of RabGTPases from cell bodies to the synapses is poorly understood. Certain RabGTPases such as Rab3 and Rab27 are known to associate with Kinesin-3 and Kinesin-1, respectively, for their anterograde axonal transport. Rab4 is a protein present on the endosomal sorting intermediates, and it allows for the transfer of cargoes from early endosomes to recycling endosomes. As Rab4 is present in both early and recycling endosomal populations, the logistics of Rab4 transport into the presynaptic terminal has been unclear. The current results show that Rab4-associated vesicles are present throughout the neuron and moderately enriched in the synapse, which is a consequence of a small anterograde bias in their transport (Dey, 2017).

The activity of Rab4 is critical for the physiology of the neuron-like extension of the growth cone. The overexpressed levels and activity of Rab4 found in conjunction with neuropathy, such as Alzheimer's disease, suggest that overactivation of the endosomal system could influence disease progression. In Drosophila, Rab4 is expressed in both neuronal and non-neuronal tissues throughout development with the exception of optic lobe neurons of the pupal brain. These studies project the idea that the expression and activity of Rab4 are crucial for the maintenance of neuronal physiology. This study has shown that the synaptic Rab4 is maintained through axonal transport by heterotrimeric Kinesin-2 and Kinesin-3. As the synaptic localization of Rab4 is dependent on its active form, other factors like PI3K are also implicated in this trafficking. The results further suggest that the function and localization of Rab4 at axon termini are critical for the maintenance of synaptic balance at ventral ganglion. Overactivation of Rab4 in cholinergic neurons reduced the synapse-bearing region of the ventral ganglion, although levels of the marker, Brp, remained unchanged. This observation suggests that local Rab4 activity at the axon termini suppresses synapse formation. Consistent with this conjecture, this study found that the synapse-bearing zone at the neuropil expands when Rab4DN is overexpressed. Although the current data are insufficient to explain the underlying mechanism, they indicate that alteration of the trafficking logistics driven by heterotrimeric Kinesin-2 and Kinesin-3 family motors might play an essential role in the development of the defect. Further analysis of larval behavior and neuronal stability in the ventral ganglion with aging would be useful in uncovering the mechanism (Dey, 2017).

Presynaptic biogenesis requires axonal transport of lysosome-related vesicles

Nervous system function relies on the polarized architecture of neurons, established by directional transport of pre- and postsynaptic cargoes. While delivery of postsynaptic components depends on the secretory pathway, the identity of the membrane compartment(s) supplying presynaptic active zone (AZ) and synaptic vesicle (SV) proteins is unclear. Live imaging in Drosophila larvae and mouse hippocampal neurons provides evidence that presynaptic biogenesis depends on axonal co-transport of SV and AZ proteins in presynaptic lysosome-related vesicles (PLVs). Loss of the lysosomal kinesin adaptor Arl8 results in the accumulation of SV- and AZ-protein-containing vesicles in neuronal cell bodies and a corresponding depletion of SV and AZ components from presynaptic sites, leading to impaired neurotransmission. Conversely, presynaptic function is facilitated upon overexpression of Arl8. These data reveal an unexpected function for a lysosome-related organelle as an important building block for presynaptic biogenesis (Vukoja, 2018).

The KIF1A homolog Unc-104 is important for spontaneous release, postsynaptic density maturation and perisynaptic scaffold organization

The kinesin-3 family member KIF1A has been shown to be important for experience dependent neuroplasticity. In Drosophila, amorphic mutations in the KIF1A homolog unc-104 disrupt the formation of mature boutons. Disease associated KIF1A mutations have been associated with motor and sensory dysfunctions as well as non-syndromic intellectual disability in humans. A hypomorphic mutation in the forkhead-associated domain of Unc-104, unc-104bris, impairs active zone maturation resulting in an increased fraction of post-synaptic glutamate receptor fields that lack the active zone scaffolding protein Bruchpilot. This study shows that the unc-104bris mutation causes defects in synaptic transmission as manifested by reduced amplitude of both evoked and miniature excitatory junctional potentials. Structural defects observed in the postsynaptic compartment of mutant NMJs include reduced glutamate receptor field size, and altered glutamate receptor composition. In addition, there is a marked loss of postsynaptic scaffolding proteins and reduced complexity of the sub-synaptic reticulum, which can be rescued by pre- but not postsynaptic expression of unc-104. These results highlight the importance of kinesin-3 based axonal transport in synaptic transmission and provide novel insights into the role of Unc-104 in synapse maturation (Y. V. Zhang, 2017).

Inhibitory control of synaptic and behavioral plasticity by octopaminergic signaling

Adrenergic receptors and their ligands are important regulators of synaptic plasticity and metaplasticity, but the exact mechanisms underlying their action are still poorly understood. Octopamine, the invertebrate homolog of mammalian adrenaline or noradrenaline, plays important roles in modulating behavior and synaptic functions. Previous work (Koon, 2011) uncovered an octopaminergic positive-feedback mechanism to regulate structural synaptic plasticity during development and in response to starvation. Under this mechanism, activation of Octβ2R autoreceptors by octopamine at octopaminergic neurons initiated a cAMP-dependent cascade that stimulated the development of new synaptic boutons at the Drosophila larval neuromuscular junction (NMJ). However, the regulatory mechanisms that served to brake such positive feedback were not known. This study reports the presence of an alternative octopamine autoreceptor, Octβ1R, with antagonistic functions on synaptic growth. Mutations in octβ1r result in the overgrowth of both glutamatergic and octopaminergic NMJs, suggesting that Octβ1R is a negative regulator of synaptic expansion. As does Octβ2R, Octβ1R functions in a cell-autonomous manner at presynaptic motorneurons. However, unlike Octβ2R, which activates a cAMP pathway, Octβ1R likely inhibits cAMP production through inhibitory Goα. Despite its inhibitory role, Octβ1R is required for acute changes in synaptic structure in response to octopamine and for starvation-induced increase in locomotor speed. These results demonstrate the dual action of octopamine on synaptic growth and behavioral plasticity, and highlight the important role of inhibitory influences for normal responses to physiological stimuli (Koon, 2012).

Adrenaline/noradrenaline and their receptors have emerged as important modulators of synaptic plasticity, metaplasticity, and behavior in the mammalian brain. However, the mechanisms underlying this regulation of synaptic structure are not known (Koon, 2012 and references therein).

In insects, adrenergic signaling is accomplished through octopamine and octopamine receptors and is a powerful modulator of behaviors such as appetitive behavior and aggression. It also regulates synaptic function and synaptic structure (Koon, 2011; Koon, 2012).

A previous study has demonstrated that at the Drosophila larval neuromuscular junction (NMJ) octopamine regulates the expansion of both modulatory and excitatory nerve terminals (Koon, 2011). Larval NMJs are innervated by glutamatergic, octopaminergic, and peptidergic motorneurons. Of these, glutamatergic nerve terminals provide classical excitatory transmission, while octopaminergic nerve endings support global modulation of excitability and synaptic growth. Larval NMJs are continuously expanding to compensate for muscle cell growth and respond to acute changes in activity by extending new synaptic boutons. By binding to the octopamine autoreceptor Octβ2R, octopamine activates a cAMP second messenger pathway that leads to CREB activation and transcription, which in turn promotes the extension of new octopaminergic nerve endings (Koon, 2011). This positive-feedback mechanism was required for an increase in locomotor activity in response to starvation. In addition, this mechanism positively regulated the growth of glutamatergic nerve endings through Octβ2R receptors present in glutamatergic neurons. An important question regards the mechanisms that serve to brake such positive feedback (Koon, 2012).

This study demonstrates the presence of a second octopamine receptor, Octβ1R, which serves as such a brake. Octβ1R is also an autoreceptor in octopaminergic neurons that serves to inhibit synaptic growth. This inhibitory influence is excerpted through the activation of the inhibitory G-protein subunit, Goα, and thus by limiting cAMP production. Like Octβ2R receptors, Octβ1R receptors are also present at excitatory glutamatergic endings. Thus, octopamine release induces a dual excitatory (through Octβ2R) and inhibitory (through Octβ1R) function on the growth of both octopaminergic and glutamatergic endings. The presence of both the excitatory and inhibitory receptors is required for normal structural plasticity at octopaminergic terminals and for normal responses to starvation, as obliterating Octβ1R (this study) or Octβ2R (Koon, 2011) prevents the acute growth of octopaminergic ending in response to octopamine and the increase in locomotor speed in response to starvation. Thus, this study highlights the requirement of both excitatory and inhibitory influences for normal synaptic and behavioral plasticity (Koon, 2012).

The previous study demonstrated that octopamine regulates synaptic and behavioral plasticity through an autoregulatory positive-feedback mechanism involving Octβ2R, which promotes both type I and type II outgrowth (Koon, 2011). This study has now identified an octopamine receptor, Octβ1R, which antagonizes the function of Octβ2R. It is proposed that Octβ1R may serve as a brake for the positive feedback induced by Octβ2R. Octβ1R receptors inhibit the cAMP pathway via the inhibitory G-protein Goα, as loss of Octβ1R or Goα function results in synaptic overgrowth of type I and type II endings in an octopamine cell-autonomous manner, and as octβ1r and goα interact genetically. Notably, defective Octβ1R signaling appears to saturate cAMP levels, occluding the function of Octβ2R. Thus, the loss of Octβ1R function results in insensitivity to octopamine stimulation. In turn, this abolishes starvation-induced behavioral changes that require Octβ2R signaling. While this study centered primarily on Octβ1R function at octopaminergic NMJ terminals, it is important to emphasize that octopamine neurons are also present in the larval brain. Thus, with current tools it is not possible to discern whether the defects are exclusively due to the function of octopaminergic motorneurons, or whether other central octopaminergic neurons contribute to these effects. While the phenotypes on NMJ development are most parsimoniously explained by a local function at NMJ terminals, it is likely that the behavioral effects are more complex, also involving important contribution from brain octopaminergic neurons (Koon, 2012).

At the Drosophila larval NMJ, three type II motorneurons innervate most of the body wall muscles in each segment (Koon, 2011). This layout suggests that octopamine is likely to globally regulate plasticity, by tuning the excitability levels of multiple excitatory synapses on the body wall muscles. Together, the observations in the previous study (Koon, 2011) and this investigation identify the presence of excitatory and inhibitory octopamine receptors that are coexpressed in the same cells. This suggests that global regulation of synapses and behavior by octopamine can be tipped toward excitation or inhibition depending on receptor expression levels, affinity of the receptors for octopamine, and availability of these receptors for binding octopamine on the target cells. This dual mode of controlling excitability likely provides enhanced flexibility, allowing a broader level of control over synaptic functions (Koon, 2012).

An important question is how can Octβ1R and Octβ2R regulate development of innervation and behavior given that they are activated by the same ligand, are localized in the same cells, and their functions are antagonistic. Several alternatives can be proposed. Octβ1R and Octβ2R might have different affinities for octopamine binding. Thus, different levels of octopamine release could differentially activate the receptors. For instance, if Octβ1R receptors have higher affinity for octopamine, and octopamine is normally released at low levels, a stable degree of innervation could be maintained by continuous inhibition of synaptic growth-promoting signals. High levels of octopamine release, as would occur during starvation, would then activate the lower affinity Octβ2R, eliciting synaptic growth. Precedence for this type of regulation has been obtained in honeybees and olive fruit flies, where low concentrations of octopamine are inhibitory while high concentrations are excitatory to cardiac contraction (Koon, 2012).

An alternative possibility is based on the well known internalization of GPCRs upon ligand binding. It is possible that such a mechanism would maintain an appropriate ratio of Octβ1R and Octβ2R at the cell surface, actively keeping or removing octopamine receptor-mediated excitation or inhibition, depending on physiological states. A third alternative is that receptors could be posttranslationally modified upon ligand binding, which might also affect their downstream functions. For example, dimerization of β2-adrenergic receptors can inhibit its adenylate cyclase-activating activity and phosphorylation of β1-adrenergic receptor by PKA reduces its affinity for Gsα and increases its affinity for Giα/oα. Last, Octβ1R and Octβ2R receptors could be spatially separated in neurons, with one receptor being closer and the other distant to sites of octopamine release. In this scenario, the receptors would likely be exposed to different octopamine concentration (Koon, 2012).

Simultaneous expression of excitatory and inhibitory GPCRs in the same neuron has been reported previously. For instance, mammalian dopamine receptors can couple to both stimulatory and inhibitory G-proteins, with the D₁ receptor-like family being coupled to Gsα and the D₂-like family being coupled to Giα/oα (Koon, 2012).

Previous studies have investigated the effect of octopamine on synaptic transmission at the Drosophila first-instar and third-instar larval NMJ. While the studies at the third-instar larval NMJ demonstrated an excitatory effect of octopamine in neurotransmission, the study on the first-instar larval stage substantiated an inhibitory effect. A recent study now provides a potential explanation for such discrepancy between the responses to octopamine at the two larval stages). In particular, it was found that that Octβ1R is expressed at high levels in first instar and at low levels in third instar. In contrast, Octβ2R is expressed at low levels in first instar and at high levels in third instar. The current studies demonstrating an inhibitory role for Octβ1R (this study) and an excitatory role for Octβ2R (Koon, 2011) are in agreement with the idea that octopamine may play an inhibitory role during first instar, but an excitatory role during third instar (Koon, 2012).

Octopamine receptors have been shown to elicit intracellular Ca²⁺ and/or cAMP increase. OAMB, the only α-adrenergic-like receptor in Drosophila, has been implicated to function via Ca²⁺ signaling in the Drosophila oviduct. However, OAMB is expressed in the oviduct epithelium, and not in the oviduct muscle cells. Given that octopamine induces relaxation of oviduct muscles, the presence of an alternative, inhibitory octopamine receptor in oviduct muscles was proposed. The identification of Octβ1R receptor as an inhibitory receptor raises the possibility that this is the inhibitory receptor in the oviduct (Koon, 2012 and references therein).

In apparent contradiction to these findings, a previous study has shown that Octβ1R (also known as OA2) is capable of increasing cAMP. In that study, HEK293 cells transfected with Octβ1R were exposed to different octopamine concentrations, which resulted in an increase in cAMP levels. A potential explanation for the disparate results is that GPCR overexpression might alter its coupling to downstream pathways. For instance, mammalian β2-adrenergic receptors are known to couple to both Gsα and Giα/oα proteins. However, overexpression of β2-adrenergic receptors constitutively couples the receptor to Gsα and not to Giα or Goα. Furthermore, analysis of its binding specificity through immunoprecipitation shows that, when the receptor was overexpressed in transgenic mice, it coprecipitated with Gsα but not with Giα/oα in the absence of agonist. An additional explanation is that human embryonic kidney HEK293 cells are unlikely to express the same transduction pathways as endogenous Drosophila cells. Indeed, a recent study showed that HEK293 cells express virtually no Goα, which could also explain the lack of inhibitory response of overexpressed Octβ1R in this cell line (Koon, 2012).

Goα is expressed in the nervous system of Drosophila and shows a marked increase in levels during the development of axonal tracts. Goα levels are altered in memory mutants including dunce and rutabaga, and Goα is necessary for associative learning. PTX overexpression in mushroom bodies of adult Drosophila severely disrupts memory, suggesting a role of Goα in synaptic plasticity. However, homozygous goα mutants are lethal due to defective development of the heart preventing the use of null mutants in studies of the NMJ or the adult brain. Moreover, overexpression of inhibitory G-proteins is known to sequester available Gβ and Gγ subunits, resulting in unspecific downregulation of other G-protein signaling. Thus, there are significant problems associated with the use of an overexpression approach to study Goα function. Fortunately, the availability of PTX and multiple Goα-RNAi strains allowed downregulation of Goα function in a cell-specific manner to examine synaptic development at the NMJ, which was found to phenocopy defects observed at the NMJ of octβ1r mutants. The presence of genetic interactions between the octβ1r and goα genes further support the notion that the two proteins act in the same signaling pathway to inhibit synaptic growth. These results provide strong evidence for the involvement of Goα in synaptic plasticity at the NMJ (Koon, 2012).

In summary, these studies reveal that octopamine acts both as an inhibitory and excitatory transmitter to regulate synaptic growth and behavior. Thus, the inhibitory function of octopamine in global synaptic growth is as crucial as its excitatory function in maintaining plasticity in a dynamic range (Koon, 2012).

A comparison between the axon terminals of octopaminergic efferent dorsal or ventral unpaired median neurons in either desert locusts (Schistocerca) or fruit flies (Drosophila) across skeletal muscles reveals many similarities. In both species the octopaminergic axon forms beaded fibers where the boutons or varicosities form type II terminals in contrast to the neuromuscular junction (NMJ) or type I terminals. These type II terminals are immunopositive for both tyramine and octopamine and, in contrast to the type I terminals, which possess clear synaptic vesicles, only contain dense core vesicles. These dense core vesicles contain octopamine as shown by immunogold methods. With respect to the cytomatrix and active zone peptides the type II terminals exhibit active zone-like accumulations of the scaffold protein Bruchpilot (BRP) only sparsely in contrast to the many accumulations of BRP identifying active zones of NMJ type I terminals. In the fruit fly larva marked dynamic changes of octopaminergic fibers have been reported after short starvation which not only affects the formation of new branches ('synaptopods') but also affects the type I terminals or NMJs via octopamine-signaling. Starvation experiments of Drosophila-larvae revealed a time-dependency of the formation of additional branches. Whereas after 2 h of starvation a decrease was found in 'synaptopods', the increase is significant after 6 h of starvation. In addition, evidence is provided that the release of octopamine from dendritic and/or axonal type II terminals uses a similar synaptic machinery to glutamate release from type I terminals of excitatory motor neurons. Indeed, blocking this canonical synaptic release machinery via RNAi induced downregulation of BRP in neurons with type II terminals leads to flight performance deficits similar to those observed for octopamine mutants or flies lacking this class of neurons (Stocker, 2018).

A Presynaptic ENaC Channel Drives Homeostatic Plasticity

An electrophysiology-based forward genetic screen has identified two genes, pickpocket11 (ppk11) and pickpocket16 (ppk16), as being necessary for the homeostatic modulation of presynaptic neurotransmitter release at the Drosophila neuromuscular junction (NMJ). Pickpocket genes encode Degenerin/Epithelial Sodium channel subunits (DEG/ENaC). This study demonstrates that ppk11 and ppk16 are necessary in presynaptic motoneurons for both the acute induction and long-term maintenance of synaptic homeostasis. ppk11 and ppk16 are cotranscribed as a single mRNA that is upregulated during homeostatic plasticity. Acute pharmacological inhibition of a PPK11- and PPK16-containing channel abolishes the expression of short- and long-term homeostatic plasticity without altering baseline presynaptic neurotransmitter release, indicating remarkable specificity for homeostatic plasticity rather than NMJ development. Finally, presynaptic calcium imaging experiments support a model in which a PPK11- and PPK16-containing DEG/ENaC channel modulates presynaptic membrane voltage and, thereby, controls calcium channel activity to homeostatically regulate neurotransmitter release (Younger, 2013).

Homeostatic signaling systems are believed to interface with the mechanisms of learning-related plasticity to achieve stable, yet flexible, neural function and animal behavior. Experimental evidence from organisms as diverse as Drosophila, mouse, and humans demonstrates that homeostatic signaling systems stabilize neural function through the modulation of synaptic transmission, ion channel abundance, and neurotransmitter receptor trafficking. In each experiment, the cells respond to an experimental perturbation by modulating ion channel abundance or synaptic transmission to counteract the perturbation and re-establish baseline function. Altered homeostatic signaling is hypothesized to contribute to the cause or progression of neurological disease. For example, impaired or maladaptive homeostatic signaling may participate in the progression of autism-spectrum disorders, posttraumatic epilepsy, and epilepsy (Younger, 2013).

The homeostatic modulation of presynaptic neurotransmitter release has been observed at mammalian central synapses and at neuromuscular synapses in species ranging from Drosophila to mouse and human. The Drosophila neuromuscular junction (NMJ) is a prominent model system for the study of this form of homeostatic plasticity. At the Drosophila NMJ, decreased postsynaptic neurotransmitter receptor sensitivity is precisely counteracted by a homeostatic potentiation of neurotransmitter release, thereby maintaining appropriate muscle excitation. The homeostatic enhancement of presynaptic release is due to increased vesicle release without a change in active zone number (Younger, 2013 and references therein).

This process is referred to as 'synaptic homeostasis,' recognizing that it reflects a subset of homeostatic regulatory mechanisms that have been shown to stabilize neural function through modulation of ion channel gene expression and neurotransmitter receptor abundance (quantal scaling) (Younger, 2013 and references therein).

An electrophysiology-based, forward genetic screen has been pioneered to identify the mechanisms of synaptic homeostasis. To date, a role has been ascribed for several genes in the mechanism of synaptic homeostasis including the Eph receptor, the schizophrenia-associated gene dysbindin, the presynaptic CaV2.1 calcium channel, presynaptic Rab3 GTPase-activating protein (Rab3-GAP), and Rab3-interacting molecule (RIM). An emerging model suggests that, in response to inhibition of postsynaptic glutamate receptor function, a retrograde signal acts upon the presynaptic nerve terminal to enhance the number of synaptic vesicles released per action potential to precisely offset the severity of glutamate receptor inhibition. Two components of the presynaptic release mechanism are necessary for the execution of synaptic homeostasis, increased calcium influx through presynaptic CaV2.1 calcium channels and a RIM-dependent increase in the readily releasable pool of synaptic vesicles. Many questions remain unanswered. In particular, how is a change in presynaptic calcium influx induced and sustained during synaptic homeostasis (Younger, 2013)?

This study reports the identification of two genes, pickpocket16 and pickpocket11, that, when mutated, block homeostatic plasticity. Drosophila pickpocket genes encode Degenerin/Epithelial Sodium channel (DEG/ENaC) subunits. Channels in this superfamily are voltage insensitive and are assembled as either homomeric or heteromeric trimers. Each channel subunit has two transmembrane domains with short cytoplasmic N and C termini and a large extracellular loop implicated in responding to diverse extracellular stimuli (Younger, 2013).

Little is known regarding the function of pH-insensitive DEG/ ENaC channels in the nervous system. DEG/ENaC channels have been implicated as part of the mechanotransduction machinery and in taste perception in both invertebrate and vertebrate systems. In Drosophila, PPK11 has been shown to function as an ENaC channel subunit that is required for the perception of salt taste and fluid clearance in the tracheal system, a function that may be considered analogous to ENaC channel activity in the mammalian lung (Younger, 2013 and references therein).

This study demonstrates that ppk11 and ppk16 are coregulated during homeostatic synaptic plasticity and that homeostatic plasticity is blocked when gene is genetically deleted, when gene expression is disrupted in motoneurons, or when pickpocket channel function is pharmacologically inhibited. Advantage was taken of the fact that presynaptic homeostasis can be blocked pharmacologically to demonstrate that the persistent induction of homeostatic plasticity does not interfere with synapse growth and development. Homeostatic plasticity can be acutely and rapidly erased, leaving behind otherwise normal synaptic transmission. Finally, pharmacological inhibition of this pickpocket channel was demonstrated to abolish the homeostatic modulation of presynaptic calcium influx that was previously shown to be necessary for the homeostatic increase in neurotransmitter release (Younger, 2013).

A model for DEG/ENaC channel function can be based on the well-established regulation of ENaC channel trafficking in the kidney during the homeostatic control of salt balance. Enhanced sodium reabsorption in the principle cells of the cortical collecting duct of the kidney is achieved by increased ENaC channel transcription and trafficking to the apical cell surface, which enhances sodium influx. Sodium is then pumped out of the basolateral. By analogy, a model is proposed for synaptic homeostasis in which the trafficking of DEG/ENaC channels to the neuronal membrane, at or near the NMJ, modulates presynaptic membrane potential to potentiate presynaptic calcium channel activity and thereby achieve precise homeostatic modulation of neurotransmitter release (Younger, 2013).

This study provides evidence that a presynaptic DEG/ENaC channel composed of PPK11 and PPK16 is required for the rapid induction, expression, and continued maintenance of homeostatic synaptic plasticity at the Drosophila NMJ. Remarkably, ppk11 and ppk16 genes are not only required for homeostatic plasticity but are among the first homeostatic plasticity genes shown to be differentially regulated during homeostatic plasticity. Specifically, it was shown that expression of both ppk11 and ppk16 is increased 4-fold in the GluRIIA mutant background. It was also demonstrated that ppk11 and ppk16 are transcribed together in a single transcript and behave genetically as an operon-like, single genetic unit. This molecular organization suggests a model in which ppk11 and ppk16 are cotranscribed to generate DEG/ ENaC channels with an equal stoichiometric ratio of PPK11 and PPK16 subunits. This is consistent with previous models for gene regulation in Drosophila. However, the possibility cannot be ruled out that two independent DEG/ ENaC channels are upregulated, one containing PPK11 and one containing PPK16. The upregulation of ppk11 and ppk16 together with the necessity of DEG/ENaC channel function during the time when synaptic homeostasis is assayed, indicates that these genes are probably part of the homeostat and not merely necessary for the expression of synaptic homeostasis (Younger, 2013).

DEG/ENaC channels are voltage-insensitive cation channels that are primarily permeable to sodium and can carry a sodium leak current. A model for DEG/ENaC channel function during synaptic homeostasis can be based on the well-established regulation of ENaC channel trafficking in the kidney during the homeostatic control of salt balance. Enhanced sodium reabsorption in the principle cells of the cortical collecting duct of the kidney is triggered by aldosterone binding to the mineralocorticoid receptor. This increases ENaC channel transcription and trafficking to the apical cell surface, which enhances sodium influx. Sodium is then pumped out of the basolateral side of the cell, accomplishing sodium reabsorption (Younger, 2013 and references therein).

It is speculated that a retrograde, homeostatic signal from muscle triggers increased trafficking of a PPK11/16-containing DEG/ENaC channel to the neuronal plasma membrane, at or near the NMJ. Since the rapid induction of synaptic homeostasis is protein synthesis independent, the existence is hypothesized of a resting pool of PPK11/16 channels that are inserted in the membrane in response to postsynaptic glutamate receptor inhibition. If postsynaptic glutamate receptor inhibition is sustained, as in the GluRIIA mutant, then increased transcription of ppk11/16 supports a persistent requirement for this channel at the developing NMJ. Once on the plasma membrane, the PPK11/16 channel would induce a sodium leak and cause a moderate depolarization of the nerve terminal. This subthreshold depolarization would lead, indirectly, to an increase in action potential-induced presynaptic calcium influx through the CaV2.1 calcium channel and subsequent neurotransmitter release (Younger, 2013).

There are two major possibilities for how ENaC-dependent depolarization of the nerve terminal could potentiate calcium influx and evoked neurotransmitter release. One possibility, based on work in the ferret prefrontal cortex and Aplysia central synapses, is that presynaptic membrane depolarization causes action potential broadening through potassium channel inactivation, thereby enhancing both calcium influx and release. A second possibility is that subthreshold depolarization of the nerve terminal causes an increase in resting calcium that leads to calcium-dependent calcium channel facilitation. Consistent with this model, it has been shown at several mammalian synapses that subthreshold depolarization of the presynaptic nerve terminal increases resting calcium and neurotransmitter release through low-voltage modulation of presynaptic P/Q-type calcium channels. However, at these mammalian synapses, the change in resting calcium does not lead to an increase in action potential-evoked calcium influx, highlighting a difference between the mechanisms of homeostatic potentiation and the type of presynaptic modulation observed at these other synapses. The mechanism by which elevated basal calcium potentiates release at these mammalian synapses remains under debate. It should be noted that small, subthreshold depolarization of the presynaptic resting potential, as small as 5 mV, are sufficient to cause a 2-fold increase in release at both neuromuscular and mammalian central synapses. This is within a reasonable range for modulation of presynaptic membrane potential by pickpocket channel insertion. Unfortunately, it is not technically feasible to record directly from the presynaptic terminal at the Drosophila NMJ. Finally, it is noted that it remains formally possible that a PPK11/16-containing DEG/ENaC channel passes calcium, based upon the ability of mammalian ASIC channels to flux calcium (Younger, 2013).

This model might provide insight regarding how accurate tuning of presynaptic neurotransmitter release can be achieved. There is a supralinear relationship between calcium influx and release. Therefore, if changing calcium channel number is the mechanism by which synaptic homeostasis is achieved, then there must be very tight and tunable control of calcium channel number within each presynaptic active zone. By contrast, if homeostatic plasticity is achieved by ENaC-dependent modulation of membrane voltage, then variable insertion of ENaC channels could uniformly modulate calcium channel activity, simultaneously across all of the active zones of the presynaptic nerve terminal. Furthermore, if the ENaC channel sodium leak is small, and if presynaptic calcium channels are moderately influenced by small changes in resting membrane potential, then relatively coarse modulation of ENaC channel trafficking could be used to achieve precise, homeostatic control of calcium influx and neurotransmitter release. Again, these are testable hypotheses that will be addressed in the future (Younger, 2013).

Composition and control of a Deg/ENaC channel during presynaptic homeostatic plasticity

The homeostatic control of presynaptic neurotransmitter release stabilizes information transfer at synaptic connections in the nervous system of organisms ranging from insect to human. Presynaptic homeostatic signaling centers upon the regulated membrane insertion of an amiloride-sensitive degenerin/epithelial sodium (Deg/ENaC) channel. Elucidating the subunit composition of this channel is an essential step toward defining the underlying mechanisms of presynaptic homeostatic plasticity (PHP). This study demonstrates that the ppk1 gene (pickpocket) encodes an essential subunit of this Deg/ENaC channel, functioning in motoneurons for the rapid induction and maintenance of PHP. Genetic and biochemical evidence is provided that PPK1 functions together with PPK11 and PPK16 as a presynaptic, hetero-trimeric Deg/ENaC channel. Finally, tight control of Deg/ENaC channel expression and activity is highlighted, showing increased PPK1 protein expression during PHP and evidence for signaling mechanisms that fine tune the level of Deg/ENaC activity during PHP (Orr, 2017a).

Homeostatic signaling systems stabilize neural function through the modulation of presynaptic transmitter release, ion channel abundance and neurotransmitter receptor trafficking. The homeostatic modulation of neurotransmitter release has been observed at mammalian central synapses and at neuromuscular synapses in species ranging from Drosophila to mouse and human. In these systems, decreased neurotransmitter receptor sensitivity is precisely counteracted by a homeostatic potentiation of neurotransmitter release, thereby maintaining appropriate muscle excitation, a process termed presynaptic homeostatic potentiation (PHP) (Orr, 2017a).

Core molecular components of the signaling systems that achieve PHP have begun to emerge through the results of large-scale forward genetic screens at the Drosophila NMJ and additional hypothesis-driven studies. A core finding has been the demonstration that an amiloride-sensitive Deg/ENaC channel functions presynaptically to drive the induction and sustained expression of PHP (Younger, 2013). Presynaptic Deg/ENaC channel activity is not detected at baseline, but is rapidly induced upon postsynaptic neurotransmitter receptor inhibition (Younger, 2013). A current model suggests that ENaC channel insertion causes a sodium leak that depolarizes the presynaptic membrane and subsequently potentiates calcium influx through active-zone localized CaV2.1 calcium channels. This model is supported by a combination of ENaC channel pharmacology and presynaptic calcium imaging (Orr, 2017a).

Deg/ENaC channels encompass a broad family of non-voltage activated sodium and, in some instances, calcium permeable channels that are believed to be heterotrimeric subunit assemblies. This family includes sodium leak channels, channels gated by low pH, chemoreceptors, and mechanoreceptors. Beyond this, a growing list of ligands has been shown to activate these channels including small neuropeptides and opioid peptides. Increasing evidence suggests that channel subunit composition determines the biophysical properties of the channel, as well as the intracellular trafficking and channel localization. Thus, defining the subunit composition of the ENaC channel that drives PHP is an essential step toward defining the feedback control mechanisms that govern the expression of PHP (Orr, 2017a).

In Drosophila, there is the potential for tremendous Deg/ENaC channel heterogeneity. The ppk gene family encodes a diverse family of Deg/ENaC channel subunits and with 31 independent ppk genes encoded in the genome, the combinatorial space for unique ion channel assemblies is tremendous. To date, however, the full subunit composition of Drosophila Deg/ENaC channels with clearly defined biological activities has yet to be achieved and, therefore, very little is known about diversity and biological relevance of this large gene family. By screening a collection of ppk-RNAi transgenes, this study has defined a third essential subunit of the Deg/ENaC channel that is required for presynaptic homeostatic plasticity (PHP) at the Drosophila NMJ. This subunit is encoded by the ppk1 gene. This finding is a surprise, given that PPK1 participates in a very different type of channel in peripheral sensory neurons. In the DA sensory neurons, PPK1 functions with PPK26 as part of a highly-expressed mechanotransduction channel (Gorczyca, 2014; Guo, 2014; Mauthner, 2014). The third subunit of the DA-neuron PPK1-containing mechanotransduction channel has yet to be identified, although it remains possible that the DA-neuron channel is not heterotrimeric. By characterizing a novel PPK1 containing ENaC channel in the context of PHP, this study provides direct evidence that different PPK subunit assemblies can generate channels with very different properties and unique physiological roles in Drosophila neurons (Orr, 2017a).

Deg/ENaC and ASIC ion channels are expressed throughout the mammalian central and peripheral nervous systems, being implicated in learning related plasticity and the response to physiological stress. But, given that ENaC channels have potent activities to control cellular physiology in tissues as diverse as the kidney, lung and colonic epithelium, it seems likely that these studies are only scratching the surface with respect to ENaC and ASIC channel function in the nervous system. Furthermore, even in those systems where ENaC channels are a focus of significant research, there remains much to learn about the regulatory mechanisms that control channel expression, trafficking and surface retention (Orr, 2017a).

The ENaC channel that controls PHP appears to be subject to exquisite post-translational control. First, the channel is either prevented from reaching the plasma membrane or is rendered inactive under baseline conditions. Neither pharmacological inhibition of the channel nor mutations of any of the three subunits influences baseline presynaptic release. Second, ENaC channel activity is induced in seconds to minutes at the isolated NMJ, implying that the channel is either rapidly transferred to the plasma membrane or is activated at that site (Younger, 2013). Third, PHP is a rheostat-like process, adjusting release to the precise level of GluR inhibition. The number of active ENaC channels on the plasma membrane must be precisely controlled to achieve this effect. Fourth, PHP can be sustained for weeks in Drosophila and decades in human. This implies, at least in Drosophila, that the number of active ENaC channels on the plasma membrane is precisely maintained. It will be of great interest to define the regulatory mechanisms that achieve such speed and precision (Orr, 2017a).

By analogy with other systems such as ENaC channel trafficking in the collecting duct of the kidney, and the control of Glut4 surface expression, three types of post-translational control could be employed to regulate ENaC channel levels at the NMJ during PHP. First, there appears to be potent mechanisms to sequester intracellular ENaC channels, effectively preventing them from reaching the plasma membrane. The efficiency of this system is emphasized by the fact that over-expression of a hyper-active ENaC channel in Drosophila motoneurons is without effect. Second, there must be efficient forward trafficking of ENaC channels to the plasma membrane to enable the rapid induction of PHP. Third, regulated recycling of the ENaC channels would be necessary to precisely maintain levels of the channel, given that channel residency is estimated to be as short as 20-30 minutes in other systems. If these regulatory systems exist, the induction of PHP would require dis-inhibition of internal sequestration followed by the engagement of forward trafficking mechanisms. Alternatively, there could be mechanisms that restrict forward trafficking of ENaC channels to the membrane, and these mechanisms would have to be disabled. The trigger for the induction of PHP might immediately act upon these processes. The feedback signaling system that tunes PHP to precise levels would presumably act upon the ENaC channel recycling mechanism, controlling the number of active ENaC channels on the synaptic plasma membrane. Together, these represent testable models for PHP induction and expression BThe discovery that PPK1 is an essential component of a presynaptic ENaC channel expressed in motoneurons is surprising. Prior to this, PPK1 was thought to function primarily in the dendrites of peripheral multi-dendritic sensory neurons. ppk1 is highly expressed in peripheral sensory neurons and the GFP ppk1-promoter fusion line revealed high expression in these cells. It is now apparent that ppk1 is also expressed at lower levels in muscle, trachea and motoneurons based on experiments with the GFP ppk1-promoter fusion line and the results of the genetic rescue and ppk1-RNAi experiments in motoneurons (Orr, 2017a).

In peripheral sensory neurons, PPK1 has been shown to function as part of a mechanosensitive ion channel that is believed to be orthologous to C. elegans DEG/ENaC mechanosensors (Gorczyca, 2014; Guo, 2014). However, PPK1-containing channels that are expressed in Drosophila peripheral sensory neurons can also be gated by low pH, suggesting a parallel with acid-sensitive ion channels (ASIC) that are expressed in mammalian peripheral sensory neurons and implicated in neuropathic pain. There is, as yet, no evidence that PHP at the NMJ is induced by either mechanical or pH-dependent stimuli. PHP can be maximally induced in a relaxed NMJ preparation in the absence of action-potential induced muscle contraction. Similarly, there is no evidence for low pH participating in the induction of PHP. Acid-sensitive currents are generally transient events with rapid inactivation, including those identified by ppk1 in class IV md sensory neurons. This is in contrast to the requirement for a robust, sustained ENaC-mediated effect during the sustained expression of PHP, which can persist for weeks in adult Drosophila. Thus, the demonstration that PPK1 incorporates into DEG/ENaC channels with diverse physiological activities highlights the potential for tremendous DEG/ENaC channel diversity in Drosophila (Orr, 2017a).

There are 31 PPK genes expressed in Drosophila that have been categorized into five sub-classes. While it has been speculated that PPK genes co-assemble within subclasses, this does not seem to be the case given that PPK1 (subclass 5) appears to function with PPK11/16 (subclass 4) in motoneurons. There are other examples that suggest heterologous assembly including evidence that PPK11 (subclass 4) functions with PPK19 (subclass 3) and that PPK23 functions with PPK29 (subclass 1) . As the subunit composition of ENaC channels in Drosophila is gradually resolved, the underlying rules may be discoverednfor channel diversity and function base of PPK subunit composition. Interestingly, there are hints that mammalian ASIC and non-acid sensing ENaC channel subunits can co-assemble, thereby providing the potential for significant channel diversity in mammals as well (Orr, 2017a).

Structural and molecular properties of insect type II motor axon terminals

A comparison between the axon terminals of octopaminergic efferent dorsal or ventral unpaired median neurons in either desert locusts (Schistocerca) or fruit flies (Drosophila) across skeletal muscles reveals many similarities. In both species the octopaminergic axon forms beaded fibers where the boutons or varicosities form type II terminals in contrast to the neuromuscular junction (NMJ) or type I terminals. These type II terminals are immunopositive for both tyramine and octopamine and, in contrast to the type I terminals, which possess clear synaptic vesicles, only contain dense core vesicles. These dense core vesicles contain octopamine as shown by immunogold methods. With respect to the cytomatrix and active zone peptides the type II terminals exhibit active zone-like accumulations of the scaffold protein Bruchpilot (BRP) only sparsely in contrast to the many accumulations of BRP identifying active zones of NMJ type I terminals. In the fruit fly larva marked dynamic changes of octopaminergic fibers have been reported after short starvation which not only affects the formation of new branches ('synaptopods') but also affects the type I terminals or NMJs via octopamine-signaling. Starvation experiments of Drosophila-larvae revealed a time-dependency of the formation of additional branches. Whereas after 2 h of starvation a decrease was found in 'synaptopods', the increase is significant after 6 h of starvation. In addition, evidence is provided that the release of octopamine from dendritic and/or axonal type II terminals uses a similar synaptic machinery to glutamate release from type I terminals of excitatory motor neurons. Indeed, blocking this canonical synaptic release machinery via RNAi induced downregulation of BRP in neurons with type II terminals leads to flight performance deficits similar to those observed for octopamine mutants or flies lacking this class of neurons (Stocker, 2018).

Transsynaptic control of presynaptic Ca(2)(+) influx achieves homeostatic potentiation of neurotransmitter release

Given the complexity of the nervous system and its capacity for change, it is remarkable that robust, reproducible neural function and animal behavior can be achieved. It is now apparent that homeostatic signaling systems have evolved to stabilize neural function. At the neuromuscular junction (NMJ) of organisms ranging from Drosophila to human, inhibition of postsynaptic neurotransmitter receptor function causes a homeostatic increase in presynaptic release that precisely restores postsynaptic excitation. This study addresses what occurs within the presynaptic terminal to achieve homeostatic potentiation of release at the Drosophila NMJ. By imaging presynaptic Ca(2+) transients evoked by single action potentials, a retrograde, transsynaptic modulation of presynaptic Ca(2+) influx was revealed that is sufficient to account for the rapid induction and sustained expression of the homeostatic change in vesicle release. The homeostatic increase in Ca(2+) influx and release is blocked by a point mutation in the presynaptic CaV2.1 channel, demonstrating that the modulation of presynaptic Ca(2+) influx through this channel is causally required for homeostatic potentiation of release. Together with additional analyses, this study establishes that retrograde, transsynaptic modulation of presynaptic Ca(2+) influx through CaV2.1 channels is a key factor underlying the homeostatic regulation of neurotransmitter release (Muller, 2012a).

The homeostatic modulation of presynaptic neurotransmitter release has been observed in organisms ranging from Drosophila to human, at both central and neuromuscular synapses. However, the molecular mechanisms underlying this form of synaptic plasticity are poorly understood. The Drosophila neuromuscular synapse has emerged as a powerful model system to dissect the cellular and molecular basis of this phenomenon. Forward genetic screens at this synapse have begun to identify loss-of-function mutations that prevent this form of neural plasticity. Among the loss-of-function mutations that have been shown to block this process is a mutation in the presynaptic CaV2.1 Ca2+ channel (Frank, 2006). However, these prior genetic data do not inform us regarding whether this calcium channel normally participates in homeostatic plasticity or how it might do so. It remains to be shown that a change in presynaptic Ca2+ influx through the CaV2.1 Ca2+ channel occurs during homeostatic plasticity. It is equally likely that a genetic disruption of the CaV2.1 Ca2+ channel simply occludes this form of plasticity by generally impairing calcium influx or synaptic transmission. Furthermore, if a change in Ca2+ influx occurs during homeostatic plasticity, can it be shown that this change is causally required for the observed homeostatic change in presynaptic release? Finally, if a change in presynaptic Ca2+ influx occurs, can it account for both the rapid induction of homeostatic plasticity as well as the long-term maintenance of homeostatic plasticity, which has been observed to persist for several days (Muller, 2012a)?

To address these outstanding questions, this study probed Ca2+ influx during homeostatic plasticity by imaging presynaptic Ca2+ transients at the Drosophila neuromuscular junction (NMJ). This was done by comparing wild-type controls with animals harboring a mutation in the glutamate receptor subunit IIA (GluRIIA) of the muscle-specific ionotropic GluR at the fly NMJ (GluRII^ASP16). The GluRII^ASP16 mutation causes a reduction in miniature excitatory postsynaptic potential (mEPSP) amplitude, and induces a homeostatic increase in presynaptic release that precisely offsets the postsynaptic perturbation thereby restoring EPSP amplitudes toward wildtype levels. The GluRII^ASP16 mutation is present throughout the life of the animal, and therefore this assay reports the sustained expression of homeostatic plasticity (Muller, 2012a).

If the homeostatic enhancement of release is solely due to a change in presynaptic Ca2+ influx without concomitant changes in the number of releasable vesicles, then repetitive stimulation of homeostatically challenged synapses is expected to result in more pronounced vesicle depletion. Indeed, there is evidence for increased synaptic depression in GluRIIA mutants and following PhTX application. However, in agreement with a recent study, this study observed that homeostatic potentiation was paralleled by an increased number of release-ready vesicles, as assayed by the method of back extrapolation of cumulative excitatory postsynaptic current amplitudes during stimulus trains. The increase in the number of release-ready vesicles detected by this assay could be a direct or an indirect consequence of the homeostatic change in presynaptic Ca2+ influx and might help the potentiated synapse to sustain release during ongoing activity (Muller, 2012a).

Work from several laboratories has provided evidence that chronic manipulation of neural activity in cultured mammalian neurons is associated with a compensatory change in presynaptic neurotransmitter release and a change in presynaptic Ca2+ influx. However, it remains unknown whether these homeostatic changes in release are caused by altered pre- versus postsynaptic activity, and it is unclear whether a change in presynaptic calcium influx is essential for this form of homeostatic plasticity in mammalian central neurons. Ultimately, it will be exciting to determine whether the molecular mechanisms identified in Drosophila, such as those described in this study, will translate to mammalian central synapses (Muller, 2012a).

Extended synaptotagmin localizes to presynaptic ER and promotes neurotransmission and synaptic growth in Drosophila

The endoplasmic reticulum (ER) is an extensive organelle in neurons with important roles at synapses including the regulation of cytosolic Ca2+, neurotransmission, lipid metabolism, and membrane trafficking. Despite intriguing evidence for these crucial functions, how presynaptic ER influences synaptic physiology remains enigmatic. To gain insight into this question, mutations were generated and characterized in the single Extended Synaptotagmin (Esyt) ortholog in Drosophila melanogaster. Esyts are evolutionarily conserved ER proteins with Ca2+-sensing domains that have recently been shown to orchestrate membrane tethering and lipid exchange between the ER and plasma membrane. Esyt was shown to localize to presynaptic ER structures at the neuromuscular junction. It was shown that synaptic growth, structure, and homeostatic plasticity are surprisingly unperturbed at synapses lacking Esyt expression. However, neurotransmission is reduced in Esyt mutants, consistent with a presynaptic role in promoting neurotransmitter release. Finally, neuronal overexpression of Esyt enhances synaptic growth and the sustainment of the vesicle pool during intense activity, suggesting that increased Esyt levels may modulate the membrane trafficking and/or resting calcium pathways that control synapse extension. Thus, this study has identified Esyt as a presynaptic ER protein that can promote neurotransmission and synaptic growth, revealing the first in vivo neuronal functions of this conserved gene family (Kikuma, 2017).

Vps54 regulates Drosophila neuromuscular junction development and interacts genetically with Rab7 to control composition of the postsynaptic density

Vps54 is a subunit of the Golgi-associated retrograde protein (GARP) complex, which is involved in tethering endosome-derived vesicles to the trans-Golgi network (TGN). In the wobbler mouse, a model for human motor neuron (MN) disease, reduction in the levels of Vps54 causes neurodegeneration. However, it is unclear how disruption of the GARP complex leads to MN dysfunction. To better understand the role of Vps54 in MNs, this study has disrupted expression of the Vps54 ortholog in Drosophila and examined the impact on the larval neuromuscular junction (NMJ). Surprisingly, it was shown that both null mutants and MN-specific knockdown of Vps54 leads to NMJ overgrowth. Reduction of Vps54 partially disrupts localization of the t-SNARE, Syntaxin-16, to the TGN but has no visible impact on endosomal pools. MN-specific knockdown of Vps54 in MNs combined with overexpression of the small GTPases Rab5, Rab7, or Rab11 suppresses the Vps54 NMJ phenotype. Conversely, knockdown of Vps54 combined with overexpression of dominant negative Rab7 causes NMJ and behavioral abnormalities including a decrease in postsynaptic Dlg and GluRIIB levels without any effect on GluRIIA. Taken together, these data suggest that Vps54 controls larval MN axon development and postsynaptic density composition through a mechanism that requires Rab7 (Patel, 2020).

GTPase-activating protein TBC1D5 coordinates with retromer to constrain synaptic growth by inhibiting Bone Morphogenetic Protein signaling

Formation and plasticity of neural circuits rely on precise regulation of synaptic growth. At Drosophila neuromuscular junction (NMJ) Bone Morphogenetic Protein (BMP) signaling is critical for many aspects of synapse formation and function. The evolutionarily-conserved retromer complex and its associated GTPase-activating protein TBC1D5 are critical regulators of membrane trafficking and cellular signaling. However, their functions in regulating the formation of NMJ are less understood. This study reports that TBC1D5 is required for inhibition of synaptic growth, and loss of TBC1D5 leads to abnormal presynaptic terminal development, including excessive satellite boutons and branch formation. Ultrastructure analysis reveals that the size of synaptic vesicles and the density of subsynaptic reticulum are increased in TBC1D5 mutant boutons. Disruption of interactions of TBC1D5 with Rab7 and retromer phenocopies the loss of TBC1D5. Unexpectedly, this study found that TBC1D5 is functionally linked to Rab6, in addition to Rab7, to regulate synaptic growth. Mechanistically, this study showed that loss of TBC1D5 leads to upregulated BMP signaling by increasing the protein level of BMP type II receptor Wit at NMJ. Overall, these data establish that TBC1D5 in coordination with retromer constrains synaptic growth by regulating Rab7 activity, which negatively regulates BMP signaling through inhibiting Wit level (Zhou, 2022).

Vesicle clustering in a living synapse depends on a synapsin region that mediates phase separation

Liquid-liquid phase separation is an increasingly recognized mechanism for compartmentalization in cells. Recent in vitro studies suggest that this organizational principle may apply to synaptic vesicle clusters. This study tested this possibility by performing microinjections at the living lamprey giant reticulospinal synapse. Axons are maintained at rest to examine whether reagents introduced into the cytosol enter a putative liquid phase to disrupt critical protein-protein interactions. Compounds that perturb the intrinsically disordered region of synapsin (see Drosophila Synapsin), which is critical for liquid phase organization in vitro, cause dispersion of synaptic vesicles from resting clusters. Reagents that perturb SH3 domain interactions with synapsin are ineffective at rest. These results indicate that synaptic vesicles at a living central synapse are organized as a distinct liquid phase maintained by interactions via the intrinsically disordered region of synapsin (Pechstein, 2020).

Chemical synapses can sustain neurotransmitter release at high rates by mobilizing synaptic vesicles (SVs) from a pool clustered at the release sites. Despite the critical role of SV clusters in neural signaling, the mechanisms underlying their organization remain unclear. Two principal models have been proposed, both of which are centered at synapsins that are essential for the maintenance of the major, distal part of SV clusters. In the first model, called the scaffold model here, the clustering of SVs is suggested to depend on their anchoring via synapsins to the cytoskeleton, because synapsins can bind both SVs and cytoskeletal components like actin, spectrin, and microtubules. Key to the scaffold model is the observation that Ca2+-dependent phosphorylation of synapsin by Ca2+/calmodulin-dependent protein kinase II (CaMKII) causes its dissociation from SVs and actin, thereby enabling vesicle mobilization. A variant of the scaffold model implies that SVs are kept in place by intervesicular filaments, or tethers, that are partly composed of synapsin but also contain unidentified components. In the second model, called the liquid phase model here, SV clustering is suggested to result from phase separation (Milovanovic, 2017). Liquid-liquid phase separation is an increasingly recognized mechanism for membraneless subcellular compartmentalization, in which a distinct liquid phase forms in the cytosol because of weak multimeric interactions between proteins or between RNA and proteins (Banani, 2017; Gomes, 2019). In the case of protein-based liquid phase organization, the interactions typically involve intrinsically disordered regions (IDRs) and/or modular binding domains such as SH2 and SH3 domains. In support of the liquid phase model for SV clustering, in vitro studies have shown that recombinant synapsin, or its isolated IDR (also termed region D), can form droplets in aqueous solution (Milovanovic, 2018). Droplet formation was enhanced by adding synapsin-binding SH3 domains and disrupted by CaMKII phosphorylation. Moreover, small liposomes could be captured in synapsin droplets (Milovanovic, 2018). At present, neither of the two models of SV clustering has been rigorously tested in vivo. Moreover, with regard to liquid-liquid phase separation, in vitro results are not easily transferred to a cellular context (Pechstein, 2020).

This study examined whether the requirements for synapsin droplet formation in vitro pertain to SV clusters at the lamprey giant reticulospinal synapse. Earlier studies in this model have demonstrated that SV clusters in stimulated synapses depend on synapsin. In the present experiments, synapses were maintained at rest to test whether reagents microinjected into the axonal cytosol would enter a putative vesicular liquid phase to disrupt critical protein-protein interactions. This study found that compounds that perturb the synapsin IDR cause dispersion of SVs from resting clusters. However, reagents that perturb SH3 domain interactions with synapsin are ineffective (Pechstein, 2020).

Recent studies with purified components suggest that presynaptic compartments, including the SV cluster and the active zone, may be organized by liquid-liquid phase separation (Milovanovic, 2018, Wu, 2019). However, the precise biological relevance of these data has been unclear. This study used a living giant synapse to examine the conditions underlying organization of the SV cluster. The results fulfill two main criteria predicted by the liquid phase model. First, fusion proteins and antibodies microinjected into the axonal cytosol readily entered, and affected, key interaction sites within resting SV clusters containing thousands of densely packed vesicles. This finding is in line with the property of a liquid phase compartment. The low level of spontaneous vesicle turnover in the lamprey synapse underscores that the SV cluster can be regarded as near a complete resting state. If the cluster had been organized as a rigid scaffold structure, it would have been expected that the reagents penetrated less effectively, thus not giving rise to complete dispersion of clustered vesicles. Second, and most importantly, the intrinsically disordered regions (IDR) defined as critical for synapsin droplet formation in vitro was found to be equally critical for maintaining SV clusters in living synapses. Disturbance of the IDR, by binding of antibodies or the SH3A domain, led to complete dissociation of the distal part of SV clusters. In vitro experiments confirmed the efficacy of the reagents used (Pechstein, 2020).

The SH3A domain potently inhibited droplet formation of the IDR. Its efficacy may suggest that the region within the lamprey IDR that contains the SH3A binding site plays an important role to support IDR-IDR interactions. In mammalian synapsin I, at least two SH3A binding sites have been identifiedr. Thus, it cannot be excluded that occlusion of several SH3A domain binding sites may be required to disrupt liquid droplet formation. Higher than stoichiometric amounts of SH3A domains are more efficient to disperse synapsin IDR phase separation in vitro, suggesting that SH3A acts on other proline-rich regions in synapsin IDR at high concentrations. However, the antibodies stimulated droplet formation in vitro, suggesting that the divalent antibodies may drive aberrant droplet formation with the IDR. In the living synapse, the antibodies may thus have acted in a more complex way by perturbing endogenous IDR-IDR interactions and by forcing formation of antibody-IDR condensates that disrupt recruitment of vesicles to liquid droplets. This interpretation agrees with in vivo observations of electron-dense condensates associated with SVs (Pechstein, 2020).

Although the present results do not formally disprove the scaffold model, several arguments can be raised against it. The original version of the scaffold model implies that SVs are anchored via actin filaments. This possibility appears unlikely for two reasons. First, it would suggest that the C and E regions, which mediate actin binding, would be important, rather than the IDR. Second, actin filaments are mainly localized around SV clusters, with few filaments penetrating their interior. The version of the scaffold model suggesting a role for intervesicular tethers is more difficult to rule out, because their molecular identity is unclear, with only part consisting of synapsin. However, the estimated number of synapsin molecules per vesicle does not correlate well with the number of tethers. Moreover, intervesicular tethers can still be observed after knockout of synapsin, which argues against their primary role in SV clustering (Pechstein, 2020).

It is important to note that the present results only concern the resting synapse, and the situation may be somewhat different during activity. Stimulation causes a fraction of the vesicular synapsin pool to disperse into the axon, at least partly because of its phosphorylation by CaMKII. It is conceivable, and consistent with in vitro data (Milovanovic, 2018), that the mobile synapsin fraction temporarily leaves the liquid phase to reenter it during rest (Pechstein, 2020).

This study found that the antibodies directed to region E of synapsin did not effectively disrupt vesicle clusters at rest. In earlier experiments, these antibodies were shown to deplete the distal vesicle pool when the microinjection was followed by action potential stimulation. This stimulus-dependent effect can be explained by the stimulation-induced dispersal of synapsin discussed earlier. Following its dispersion in the cytosol, synapsin may have been captured by the E region-directed antibodies and thus prevented from reassociating with SVs to maintain a liquid phase. It is possible that the marked stimulus-dependent effect is enhanced by exposure of a normally hidden E region. The E region-directed antibodies produced a minor, non-significant loss of vesicles. The lack of a consistent effect suggests that the E region is not involved in maintaining the liquid phase. It is speculated that the modest effect results from mobility of some vesicles even at rest that may be linked with minute dispersion of synapsin. It is also possible that the E region-directed antibodies exerted a small influence on the adjacent IDR within a liquid phase (Pechstein, 2020).

Another notable observation was the occurrence of numerous scattered SVs and SV aggregates in the axonal cytosol following IDR perturbation, which sheds new light on a long-standing problem. Knockout of synapsins is known to cause a general decrease in the number of SVs, which has led to the proposition that synapsins play a role in SV maintenance, possibly via its ATP-binding C region. In an alternative hypothesis, the loss of SVs was proposed to be secondary to a clustering defect, eventually resulting in SV degradation. The dispersion of SVs following acute IDR perturbation supports a primary role of synapsin in vesicle clustering in central synapses (Pechstein, 2020).

A proximal pool of SVs remained tethered at the active zone after IDR perturbation. The mechanisms underlying the organization of this membrane-proximal pool are presently unclear. It may be organized by scaffolding, with the vesicles tethered to filaments extending from the active zone. Alternatively, these vesicles may compose a distinct liquid phase organized by other proteins than synapsin. The active zone has been proposed to form a distinct liquid phase involving RIM, RIM-BP, and calcium channels, but it is unclear how far into the cytosol this phase may extend (Pechstein, 2020).

Intersectin, amphiphysin, endophilin, and syndapin are all localized in SV clusters at rest. Upon stimulation, they partly redistribute to the periactive zone, where they participate in different aspects of SV endocytosis. Although the endocytic functions of these proteins are well established, their possible roles within the SV cluster remain unclear. No effect on resting SV clusters was observed by disrupting intersectin or amphiphysin SH3 domain interactions with synapsin, which is consistent with results on intersectin- and amphiphysin-deficient mice. In previous studies, no effect on clusters was observed after perturbing endophilin or syndapin SH3 domain interactions. Thus, individual interactions with any one of these four presynaptic SH3 domain-containing proteins are dispensable for the maintenance of resting SV clusters in vivo. However, this study does not rule out the possibility that multivalent interactions simultaneously involving these proteins may promote liquid phase organization of the SV cluster (Pechstein, 2020).

The accumulation of SH3 domain-containing proteins in SV clusters may merely reflect passive buffering, but they may also play active roles at this location. In the case of intersectin, a role in SV mobilization has been proposed, because deletion of intersectins I and II in mice causes enhanced synaptic depression during high-frequency stimulation. Such enhanced depression is consistent with impaired SV mobilization, but it could also be secondary to an endocytosis defect compromising the clearance of release sites. The roles of SH3 domain-containing proteins in SV clusters thus remain an important topic for future studies (Pechstein, 2020).

Postsynaptic cAMP signalling regulates the antagonistic balance of Drosophila glutamate receptor subtypes

The balance among different subtypes of glutamate receptors (GluRs) is crucial for synaptic function and plasticity at excitatory synapses. This study shows that the two subtypes of GluRs (A and B) expressed at Drosophila neuromuscular junction synapses mutually antagonize each other in terms of their relative synaptic levels and affect subsynaptic localization of each other. Upon temperature shift-induced neuromuscular junction plasticity, GluR subtype A increased but subtypeB decreased with a timecourse of hours. Inhibition of the activity of GluR subtype A led to imbalance of GluR subtypes towards more GluRIIA. To gain a better understanding of the signalling pathways underlying the balance of GluR subtypes, an RNA interference screen of candidate genes was performed and postsynaptic-specific knockdown of dunce, which encodes cAMP phosphodiesterase, was found to increase levels of GluR subtype A but decreased subtype B. Furthermore, bidirectional alterations of postsynaptic cAMP signalling resulted in the same antagonistic regulation of the two GluR subtypes. These findings thus identify a direct role of postsynaptic cAMP signalling in control of the plasticity-related balance of GluRs (Zhao, 2020).

A negative correlation between subtype A and B receptors has been reported previously at Drosophila NMJs. However, the mechanism by which the antagonistic balance of different subtypes of GluRs is regulated remains unclear. The present study revealed that bidirectional alterations of cAMP levels in the postsynaptic muscle cells alter the balance of GluR subtypes in a cell-autonomous manner. This study thus provides new insights into the mechanism underlying synaptic plasticity by altering the balance of GluR subtypes (Zhao, 2020).

Most previous conventional microscopy studies have reported substantial colocalization or differential localization of GluRIIA and GluRIIB. This study reports an apparently non-overlapping localization of GluRIIA and GluRIIB at the postsynaptic densities (PSDs) of NMJ synapses (see The subtype B forms a doughnut-shaped ring, with a smaller subtype A ring in the centre). Although clear interpretations are lacking for the distinct localization of GluRIIA and GluRIIB at PSDs, there could be two possibilities: either different classes of receptors might be associated with specific interacting proteins that could mediate, directly or indirectly, the concentric localization of GluR subtypes at PSDs or concentric rings of GluR subtypes A and B in wild-type larvae might associate with their specific biophysical properties. Desensitization is the process by which receptors are inactivated in the prolonged presence of an agonist; it occurs faster in response to a lower concentration of agonist. On the postsynaptic side, GluR subtype A exhibits slower desensitization kinetics than GluR subtype B. It is therefore speculated that the slower desensitization of subtype A receptors might be caused, in part, by a higher concentration of glutamate released on the presynaptic side, because subtype A rings are more closely juxtaposed to presynaptic Cacophony calcium channels than subtype B rings (Zhao, 2020).

This study showed that subtype A rings become enlarged (both the inner and outer ring diameters are increased) when the synaptic levels of GluRIIA are increased, whereas subtype B rings are enlarged in a specific manner, i.e., the inner diameter decreases, but the outer diameter of the ring increases when the level of GluRIIB is increased. A simple explanation for the enlarged GluR rings might therefore be increased synaptic levels of GluRIIA or GluRIIB. Given that GluR-enriched PSDs are confined to specific spatial domains by cell adhesion molecules and the spectrin-actin network, it is possible that an increase in the level of one subtype of GluR might take up the space left by a reduced level of the other (Zhao, 2020).

Synaptic plasticity is the ability of synapses to strengthen or weaken over time, in response to increases or decreases in their activity. It is well established that GluRs are involved in synaptic plasticity at excitatory synapses. However, it is not entirely known how different types or subtypes of GluRs are involved in synaptic plasticity. Drosophila glutamatergic NMJs with two subtypes of GluRs, rather than mammalian NMJs with multiple subtypes of GluRs, are an effective model for studying synaptic plasticity. Hyperexcitable double mutants of eag sh show persistent strengthening of larval NMJs, which represents long-term plasticity. This study found that increased presynaptic release by warm-activated TrpA1 led to increased GluRIIA but normal GluRIIB, which was consistent with increased GluRIIA in eag sh double mutants. In addition, increased GluRIIA but decreased GluRIIB were observed in high temperature-induced synaptic plasticity of long-term strengthening of neurotransmission. Thus, it is speculated that increased presynaptic release might result in long-term plasticity by enhancing postsynaptic responses through increased GluRIIA (Zhao, 2020).

Increased GluRIIA is consistently observed in different models of synaptic plasticity. However, this study observed normal and reduced GluRIIB in TrpA1- and high temperature-induced synaptic plasticity, respectively. The discrepant changes in GluRIIB levels in different models of synaptic plasticity could be caused by different timescales, such as a limited time (8 h) for elevating presynaptic release through activating TRPA1 versus 4 days for raising larvae at 27°C, or the change in the level of GluRIIB might be too low to be detected by overexpressing TRPA1, or both (Zhao, 2020).

Whether the antagonistic balance of GluRs is actively (as a functional requirement) or passively (as a physical competition) regulated depends on specific conditions. It appeared that GluRIIA and GluRIIB competed with each other for the essential subunits when the expression levels of either GluRIIA or GluRIIB were changed, consistent with previous reports. These results support a passive competition between GluRIIA and GluRIIB. However, an actively regulated antagonistic balance of GluRs also occurs. When the essential subunit GluRIIC, GluRIID or GluRIIE was limited, both GluRIIA and GluRIIB decreased. If only passive regulation of GluRIIA and GluRIIB occurs, GluR subtype A and B decreased at similar levels would be expected. However, the ratio of GluRIIA to GluRIIB increased, indicating that the GluRIIA subtype is maintained preferentially when the total GluRs are limited and supporting an active regulation of the balance between GluRIIA and GluRIIB. Given that GluRIIA is mainly responsible for the postsynaptic responses, the relative increase in GluRIIA when an essential subunit of GluRs was knocked down might be a functional compensation for the decrease of synaptic strength (Zhao, 2020).

In addition to the antagonism of GluRIIA and GluRIIB reported in this study, there are a few reports on the regulation of synaptic levels of single GluR subunits. For example, GluRIIA but not GluRIIB receptors are anchored at the PSD by the actin-associated Coracle (Chen, 2005) and are regulated by a signalling pathway involving the Rho-type GEF (Pix) and its effector, Pak kinase (Albin, 2004). Recent studies also showed specific upregulation of GluRIIA but not GluRIIB when the calcium-dependent proteinase calpains were mutated (Zhao, 2020).

A previous study showed that the numbers of terminal varicosities and branches were increased in dnc but not rut mutants. Given that elevated cAMP levels induced an antagonistic balance of GluRs at the postsynaptic side, it was important to test whether the antagonistic balance of GluRs was associated with NMJ overgrowth. The results showed that the number of varicosities remained normal when dnc or rut was knocked down by RNAi in the postsynaptic muscles, suggesting that an alteration in the cAMP pathway at the postsynaptic side did not affect NMJ development (Zhao, 2020).

The importance of the GluR subtype balance in synaptic plasticity has been documented in mammals. The major forms of AMPA receptors in the hippocampus include GluA1/2 and GluA2/3 heteromers, in addition to GluA1 homomers. The relative abundance of GluA1- and GluA2-containing receptors is a well-established determinant of synaptic plasticity in diverse brain circuits; GluA1-containing receptors are recruited to synapses after long-term potentiation, whereas GluA2-containing receptors are required for long-term depression. Together with the mammalian findings, the results support the notion that the GluR subtype balance contributes to synaptic plasticity at excitatory synapses (Zhao, 2020).

It is widely known that cAMP signalling plays an important role in regulating synaptic plasticity by increasing presynaptic neurotransmitter release. However, it is not known whether the cAMP pathway acts postsynaptically in regulating the ratio of GluRs, which plays a crucial role in synaptic plasticity. The present study showed, for the first time, that the cAMP pathway regulates the balance of different GluR subtypes on the postsynaptic side; either increased or reduced cAMP leads to an altered ratio of GluR subtypes at Drosophila NMJ synapses. Thus, an optimal level of cAMP in postsynaptic muscles might be required for the normal ratio of synaptic GluR subtypes (Zhao, 2020).

When cAMP levels are elevated, cAMP binds to the regulatory subunits of PKA and liberates catalytic subunits that then become active. Active PKA in muscles decreases the activity of GluRIIA in Drosophila (Davis, 1998). Thus, an increase in the level of synaptic GluRIIA might compensate for the reduced activity of GluRIIA caused by overexpression of wild-type or constitutively active PKA. Conversely, inhibition of PKA activity in muscles causes a significant increase in the average amplitude of miniature excitatory junctional currents, consistent with the notion that PKA negatively regulates the activity of GluRIIA. Surprisingly, an increase was observed in synaptic GluRIIA when the cAMP level was downregulated in rut mutants or when PKA was knocked down by RNAi. It appears that the negative regulation of GluRIIA activity by PKA is not sufficient to account for the increase of GluRIIA at NMJ synapses (Zhao, 2020).

Analysis of western blots showed that the protein level of GluRIIA increased significantly, regardless of whether postsynaptic cAMP pathway was up- (dnc RNAi and PKAOE) or downregulated (rut RNAi and PKA RNAi), suggesting that the similar antagonistic balance of GluR subtypes induced by both up- and downregulation of cAMP might be caused by an elevated protein level of GluRIIA the cAMP pathway regulates the antagonism between GluRIIA and GluRIIB at two distinct steps, GluRIIA activity and protein level. Exactly how bidirectional changes of cAMP lead to a similar alteration of GluR subtypes remains to be investigated (Zhao, 2020).

Although Dnc and Rut regulate cAMP levels in opposite directions, physiological studies in Drosophila have shown that activity-dependent short-term plasticity is altered in a similar manner at larval NMJs in both dnc and rut mutants. Specifically, synaptic facilitation and post-tetanic potentiation are both weakened, indicating that the bidirectional change of cAMP signalling might result in similar abnormalities in synapse plasticityn. The mechanisms underlying synaptic facilitation and post-tetanic potentiation are exclusively presynaptic. Synaptic facilitation and post-tetanic potentiation both result from increased presynaptic calcium concentrations, leading to an enhanced release of neurotransmitters. A bell-shaped model was proposed to explain this mode of regulation, i.e. mutations in dnc and rut, which regulate cAMP levels in opposite directions, result in a similar plasticity phenotype. It is proposed that the bell-shaped model might also explain a similar increase in GluRIIA at NMJ synapses caused by bidirectional changes in cAMP levels in postsynaptic muscles (Zhao, 2020).

The antagonistic balance of GluRIIA and GluRIIB is induced by the postsynaptic cAMP/PKA pathway. However, whether the antagonism between GluRIIA and GluRIIB requires the cAMP/PKA pathway is unclear. GluRIIA or GluRIIB nulls were recombined with postsynaptic RNAi knockdown of PKA (i.e. inhibition of the cAMP pathway). It is noted that PKA null mutants are lethal at the first larval stage and thus cannot be used for the genetic interaction assay. Compared with simple null mutants of GluRIIA (or GluRIIB), PKA RNAi in the mutant background of GluRIIA (or GluRIIB) did not change the synaptic levels of GluRIIB (or GluRIIA), suggesting that the antagonistic balance of GluRIIA and GluRIIB does not require the cAMP pathway at the postsynaptic side. Thus, an altered cAMP pathway leads to the antagonistic balance of GluRIIA and GluRIIB, but the antagonistic balance of GluRIIA and GluRIIB appears not to be dependent on the cAMP pathway, at least for the antagonism induced by null mutations of GluRIIA or GluRIIB, or the remaining PKA upon RNAi knockdown is sufficient to support the antagonistic balance of GluRIIA and GluRIIB (Zhao, 2020).

It will be of great interest to determine how the cAMP-PKA-GluR signalling pathway acts on the postsynaptic side to contribute to synaptic plasticity and whether this pathway is also effective and conserved in mammalian central synapses (Zhao, 2020).

A Novel Neuron-Specific Regulator of the V-ATPase in Drosophila

The V-ATPase is a highly conserved enzymatic complex that ensures appropriate levels of organelle acidification in virtually all eukaryotic cells. While the general mechanisms of this proton pump have been well studied, little is known about the specific regulations of neuronal V-ATPase. This study characterized CG31030, a previously uncharacterized Drosophila protein predicted from its sequence homology to be part of the V-ATPase family. In contrast to its ortholog ATP6AP1/VhaAC45 which is ubiquitous, it was observed that CG31030 expression is apparently restricted to all neurons, and using CRISPR/Cas9-mediated gene tagging, that it is mainly addressed to synaptic terminals. CG31030 is essential for fly survival and this protein co-immunoprecipitates with identified V-ATPase subunits, and in particular ATP6AP2. Using a genetically-encoded pH probe (VMAT-pHluorin) and electrophysiological recordings at the larval neuromuscular junction, it showed that CG31030 knock-down induces a major defect in synaptic vesicle acidification and a decrease in quantal size, which is the amplitude of the postsynaptic response to the release of a single synaptic vesicle. These defects were associated with severe locomotor impairments. Overall, these data indicate that CG31030, which was renamed VhaAC45-related protein (VhaAC45RP), is a specific regulator of neuronal V-ATPase in Drosophila that is required for proper synaptic vesicle acidification and neurotransmitter release (Dulac, 2021).

AP2 Regulates Thickveins Trafficking to Attenuate NMJ Growth Signaling in Drosophila

Compromised endocytosis in neurons leads to synapse overgrowth and altered organization of synaptic proteins. However, the molecular players and the signaling pathways which regulate the process remain poorly understood. This study shows that α2-adaptin, one of the subunits of the AP2-complex, genetically interacts with Mad, Medea and Dad (components of BMP signaling) to control neuromuscular junction (NMJ) growth in Drosophila. Ultrastructural analysis of α2-adaptin mutants show an accumulation of large vesicles and membranous structures akin to endosomes at the synapse. Mutations in α2-adaptin lead to an accumulation of Tkv receptors at the presynaptic membrane. Interestingly, the level of small GTPase Rab11 was significantly reduced in the α2-adaptin mutant synapses. However, expression of Rab11 does not restore the synaptic defects of α2-adaptin mutations. A model is proposed in which AP2 regulates Tkv internalization and endosomal recycling to control synaptic growth (Choudhury, 2022).

The effects of doxapram (blocker of K2p channels) on resting membrane potential and synaptic transmission at the Drosophila neuromuscular junction

The resting membrane potential of most cells is maintained by potassium K2p channels. The pharmacological profile and distribution of various K2p channel subtypes in organisms are still being investigated. The Drosophila genome contains 11 subtypes; however, their function and expression profiles have not yet been determined. Doxapram is clinically used to enhance respiration in humans and blocks the acid-sensitive K2p TASK subtype in mammals. The resting membrane potential of larval Drosophila muscle and synaptic transmission at the neuromuscular junction are pH sensitive. The present study investigated the effects of doxapram on membrane potential and synaptic transmission using intracellular recordings of larval Drosophila muscles. Doxapram (1 mM and 10 mM) depolarizes the muscle and appears to depolarize motor neurons, causing an increase in the frequency of spontaneous quantal events and evoked excitatory junction potentials. Verapamil (1 and 10 mM) paralleled the action of doxapram. These changes were matched by an extracellular increase in KCl (50 mM) and blocked by Cd(2+). It is assumed that the motor nerve depolarizes to open voltage-gated Ca(2+) channels in presynaptic nerve terminals because of exposure to doxapram. These findings are significant for building models to better understand the function of pharmacological agents that affect K2p channels and how K2p channels contribute to the physiology of tissues. Drosophila offers a genetically amenable model that can alter the tissue-specific expression of K2p channel subtypes to simulate known human diseases related to this family of channels (Vacassenno, 2023).

RNA-binding FMRP and Staufen sequentially regulate the coracle scaffold to control synaptic glutamate receptor and bouton development

Both mRNA-binding Fragile X Mental Retardation Protein (FMRP) and mRNA-binding Staufen regulate synaptic bouton formation and glutamate receptor (GluR) levels at the Drosophila neuromuscular junction (NMJ) glutamatergic synapse. This study tested whether these RNA-binding proteins (RBPs) act jointly in a common mechanism. Both dfmr1 and staufen mutants, and trans-heterozygous double mutants, were shown to display increased synaptic bouton formation and GluRIIA accumulation. With cell-targeted RNAi, a downstream Staufen role within postsynaptic muscle. With immunoprecipitation, this study showed that FMRP binds staufen mRNA to stabilize postsynaptic transcripts. Staufen is known to target actin-binding, GluRIIA anchor Coracle, and this study confirmed that Staufen binds to coracle mRNA. FMRP and Staufen were shown to act sequentially to co-regulate postsynaptic Coracle expression, and show Coracle, in turn, controls GluRIIA levels and synaptic bouton development. Consistently, this study found dfmr1, staufen and coracle mutants elevate neurotransmission strength. FMRP, Staufen and Coracle all suppress pMad activation, providing a trans-synaptic signaling linkage between postsynaptic GluRIIA levels and presynaptic bouton development. This work supports an FMRP-Staufen-Coracle-GluRIIA-pMad pathway regulating structural and functional synapse development (Song, 2022).

This study reveals the mechanism of the established FMRP negative regulation of postsynaptic GluRIIA receptors and presynaptic bouton formation in the Drosophila FXS disease model. Specifically, the mRNA-binding FMRP-positive translational regulator binds to staufen mRNA as predicted, within the postsynaptic cell. Consequently, both dfmr1 and staufen mutants share the elevated GluRIIA level and bouton number phenotypes based on a common postsynaptic pathway function, and genetically interact as trans-heterozygotes to reproduce these phenotypes. Staufen acts as a dsRBP to bind coracle mRNA as predicted; both dfmr1 and staufen mutants exhibit elevated postsynaptic Coracle levels, and genetically interact as trans-heterozygotes to reproduce this phenotype. Coracle acts as a GluRIIA-binding anchoring scaffold within the postsynaptic domain to regulate local receptor accumulation (Chen, 2005). Consequently, dfmr1, staufen and coracle mutants all increase NMJ synaptic functional differentiation to elevate neurotransmission strength. Finally, the elevated postsynaptic GluRIIA levels mediate retrograde BMP receptor trans-synaptic signaling that induces pMad to drive new presynaptic bouton development. dfmr1, staufen and coracle mutants all exhibit elevated presynaptic pMad levels, thereby linking the postsynaptic GluRIIA accumulation and presynaptic supernumerary bouton formation defects shared by all of these mutants (Song, 2022).

The staufen mutant increased synaptic Coracle levels, GluRIIA levels and bouton number are all internally consistent. In a previous study, opposite phenotypes were measured in staufenHL/Df(2R)Pcl7B, which reduces another 14 genes in heterozygous deficiency, including loci involved in neuronal development (e.g. grh, nopo). Importantly, this study similarly found reduced synaptic protein levels and bouton number in staufenHL/Df(2R)Pcl7B, suggesting that heterozygosity of one or more of the neighboring genes impairs synaptic development. However, this study showed that a staufen RNAi that reduces transcript levels by ~90% replicates the staufen mutant NMJ phenotypes of increased GluRIIA levels and synaptic bouton numbers. This was also replicated with a second, independent staufen RNAi line. Moreover, this study showed that the effect is entirely restricted to postsynaptic muscle RNAi, with no effect from presynaptic neuron RNAi, consistent with restricted postsynaptic Staufen function. In addition, postsynaptic staufen rescue of the staufen mutant restored normal synaptic bouton formation, with OE reducing GluRIIA levels in staufen mutants and rescuing GluRIIA levels in dfmr1 mutants. Both staufen mutants and postsynaptic staufen RNAi also share the arrested supernumerary satellite bouton development characterizing dfmr1 null mutants. These many independent lines of evidence confirm the results, and are consistent with the known parallel FMRP role in restricting GluRIIA levels and synaptic bouton formation (Song, 2022).

To regulate Staufen, FMRP binds staufen mRNA and protects targeted staufen transcripts from degradation. FMRP contains at least three distinct RNA-binding domains (RBDs), and Staufen has five RBDs. Staufen reportedly binds a specific RNA hairpin structure formed by long 3' UTRs, but RIP shows that Staufen also binds mRNAs that are not predicted to generate this secondary structure. Although the decreased staufen mRNA levels in both dfmr1 mutants and muscle-targeted dfmr1 RNAi are predicted to be due to the lack of FMRP binding, it is also possible that other unregulated interactors cause the downregulated staufen mRNA expression (Shah et al., 2020). Localized labeling with an anti-Staufen antibody has been reported in the postsynaptic NMJ, which can be confirmed, but it was not possible to reduce labeling in staufen hypomorphic mutants. Therefore Staufen labeling was not shown in the current study. Moreover, western blots have been reported with the same anti-Staufen antibody; however, attempts were unsuccessful. Therefore qPCR was used to measure staufen mRNA levels. Staufen binds to coracle mRNA, but does so in a non-selective manner. This result is consistent with Staufen acting as a very broad spectrum dsRBP, and suggests that Staufen likely acts with a translational regulator partner to generate specificity. FMRP is very well established to partner with other RBPs to mediate the translational regulation of its target transcripts (Song, 2022). ------

The postsynaptic Coracle scaffold acts in a GluRIIA local anchoring mechanism, presumably to link the receptors to the underlying actin cytoskeleton (Chen, 2005). The jointly elevated Coracle and GluRIIA levels in both dfmr1 and staufen mutants are consistent with this scaffold function. Because the dfmr1/+; staufen/+ trans-heterozygotes share this correlated Coracle and GluRIIA upregulation in the postsynaptic domain, a single common signaling pathway is indicated. Coracle also restricts terminal branching development in peripheral sensory neurons. Both coracle mutants and sensory neuron-targeted coracle RNAi also display increased dendritic branch and termini numbers. These phenotypes are similar to the expanded NMJ terminals and increased synaptic bouton development reported in this study. Importantly, both coracle loss of function (mutants and muscle-targeted RNAi) and gain of function (muscle-targeted OE) increase postsynaptic GluRIIA levels and generate supernumerary boutons. Likewise, the knockdown and OE of many other similar scaffolds are known to cause phenocopying defects. Some examples include the muscle chaperone UNC-45, the tight junction scaffold zonula occludens-1 (ZO-1) and synaptic UNC-13. Indeed, both coracle loss and OE similarly cause increased dendritic crossing in Drosophila sensory neurons, similar to the phenocopy of developmental defects reported in this study. Combining the roles of postsynaptic FMRP-Staufen-Coracle in GluRIIA clustering, it was reasoned that this pathway must be a regulatory determinant of synaptic functional development (Song, 2022).

Removing FMRP, Staufen and Coracle strongly enhances functional synaptic differentiation and NMJ neurotransmission strength. This is consistent with expectations from the postsynaptic GluRIIA accumulation in all of these mutants. Elevated GluRIIA levels are well known to be associated with increased evoked functional responses and prolonged channel open times. A GluRIIA pore sequence (MQQ) critically required for the Drosophila channel Ca2+ permeability is conserved in mammalian receptors. This selectivity allows Ca2+-dependent participation in spontaneous (mEJC) and evoked (EJC) neurotransmission. Although enhanced evoked EJC amplitudes are typically accompanied by mEJC alterations, this study found that mEJC amplitude and frequency are unchanged in both the staufen and coracle mutants, and show only minimal changes in the dfmr1 mutants. Classically, both evoked and spontaneous neurotransmission were thought to be mediated by the same vesicles; however, more recent evidence has indicated that spontaneous and evoked neurotransmission have distinct machinery and vesicle pools. Postsynaptic receptors can be segregated into different compartments that are activated by either spontaneous or evoked release. This work supports this growing body of evidence for differential regulation. Importantly, GluRIIA has unique functions, modulating both presynaptic glutamate release and presynaptic bouton development (Song, 2022).

The dfmr1, staufen and coracle mutants all showed upregulated presynaptic pMad correlated with postsynaptic activated GluRIIA accumulation. GluRIIA activation triggers presynaptic pMad signaling via BMP receptors surrounding active zones, which, in turn, stabilizes GluRIIA receptors in the postsynaptic domains. This trans-synaptic signaling mechanism induces new presynaptic bouton development. The targeted postsynaptic RNAi for all three genes confirms this intercellular link. Synaptic BMP signaling involves both the type I serine/threonine kinase receptors and the type II receptor Wit. Although BMP ligand Glass bottom boat (Gbb) signaling via Wit presynaptic receptors is well established at the NMJ to modulate synaptogenesis, the mechanism of presynaptic bouton formation induced by activated GluRIIA signaling does not involve canonical BMP signaling via Gbb. In the dfmr1 mutants, it is suggested that postsynaptic GluRIIA accumulation induces presynaptic bouton development via non-canonical GluRIIA-Wit trans-synaptic retrograde signaling. Similarly, the muscle postsynaptic glypican Dally-like protein (Dlp) negatively regulates NMJ synaptic development by inhibiting this same non-canonical BMP pathway through decreased activated GluRIIA expression. Postsynaptic GluRIIA clustering can thus trigger presynaptic bouton formation, although supernumerary boutons do not always induce reciprocal GluRIIA changes. It is concluded that an FMRP-Staufen-Coracle-GluRIIA-pMad pathway regulates intertwined structural and functional glutamatergic synapse development (Song, 2022).

Gamma-secretase promotes Drosophila postsynaptic development through the cleavage of a Wnt receptor

Developing synapses mature through the recruitment of specific proteins that stabilize presynaptic and postsynaptic structure and function. Wnt ligands signaling via Frizzled (Fz) receptors play many crucial roles in neuronal and synaptic development, but whether and how Wnt and Fz influence synaptic maturation is incompletely understood. This study showed that Fz2 receptor cleavage via the γ-secretase complex is required for postsynaptic development and maturation. In the absence of γ-secretase, Drosophila neuromuscular synapses fail to recruit postsynaptic scaffolding and cytoskeletal proteins, leading to behavioral deficits. Introducing presenilin mutations linked to familial early-onset Alzheimer's disease into flies leads to synaptic maturation phenotypes that are identical to those seen in null alleles. This conserved role for γ-secretase in synaptic maturation and postsynaptic development highlights the importance of Fz2 cleavage and suggests that receptor processing by proteins linked to neurodegeneration may be a shared mechanism with aspects of synaptic development (Restrepo, 2022).

Postsynaptic development and maturation enable the essential transition from nascent, unreliable synapse to robust connection capable of high-fidelity neurotransmission. In Drosophila, the Wg ligand promotes neurodevelopment by activating postsynaptic Fz2 receptors, but the precise downstream events utilized by Fz2 to influence events like synaptic maturation remained controversial and unclear. This study found an unexpected developmental role for postsynaptic γ-secretase in enabling Fz2-mediated postsynaptic development via receptor cleavage. This role is also perturbed by AD patient-derived PSEN1 alleles in Drosophila, suggesting a link between AD and Wnts. These data first solve longstanding mysteries as to the significance of, and factors required for, Fz2 cleavage in postsynaptic maturation. Second, it suggests that proteolytic receptor cleavage may be a shared mechanism between developmental and degenerative processes, raising the possibility of a prior unappreciated neurodevelopmental component to AD (Restrepo, 2022).

In the last 20 years, work at the Drosophila NMJ revealed the importance of Wnts in regulating synaptic function and development through Fz2 and multiple downstream pathways. The roles of each downstream pathway, however, remained unclear. In postsynaptic muscles, Fz2 signals via a Frizzled nuclear import (FNI) pathway resulting in nuclear import of cleaved Fz2 receptor. Three major questions about FNI remained unanswered: (1) what is its physiological significance, (2) how does the Fz2 cleavage site lend to its physiological role, and (3) what protease is necessary for Fz2 cleavage to promotes this physiological role? This work addresses each question, highlighting a role for FNI in postsynaptic development, the function of the cleavage site in synaptic maturation, and identifying γ-secretase as being required for Fz2 cleavage (Restrepo, 2022).

First, it was shown that postsynaptic Fz2 cleavage (and not canonical Wnt signaling) specifically promotes postsynaptic maturation. This does not preclude a postsynaptic Fz2 function in presynaptic growth and active zone establishment. Instead, postsynaptic Fz2 may promote growth via canonical pathways; this will be an important area for future study. Second, identification of γ-secretase in promoting Fz2 cleavage is critical to understand downstream Fz2 pathways and begins to answer longstanding questions in synaptic neurodevelopment and postsynaptic maturation. As the developmental defects in γ-secretase mutants are suppressed by activating the postsynaptic FNI pathway, this indicates that the relevant cleavage target in maturation is Fz2. As maturation defects underlie neurodevelopmental disorders, further understanding the relevant machinery that promotes maturation can inform a grasp of neurodevelopment disease progression (Restrepo, 2022).

Maturation of Drosophila central and NMJ synapses and mammalian dendritic spines all require γ-secretase. γ-secretase promotes axon guidance and early synaptic development, but its role in synaptic maturation is largely unknown. THe data suggest γ-secretase-dependent cleavage and translocation of signaling proteins may be a fundamental feature of postsynaptic development. Indeed, blocking Presenilin::Syt I interaction modestly reduces spine density and mature spines (Zoltowska, 2017). This is consistent with the current data, although the more general blockade of γ-secretase function has a larger effect on spine maturation. The mechanism remains unclear as γ-secretase modulates multiple downstream pathways, including Wnts. More work will be needed to determine if Wnt signaling is conserved in downstream signaling for synaptic maturation across evolution. In vertebrates, Fz5 and Fz8 are homologous to Drosophila Fz2 and contain the same consensus cleavage site. Fz8 is expressed in the nervous system , but its neuronal function and cleavage status remain unknown. Intriguingly, recent work showed that Fz5 can be cleaved in motor neuron-like NSC-34 cells. As Fz5 promotes activity-dependent synaptogenesis and neuronal survival, these data raise a tantalizing prospect that the γ-secretase / Wnt / Fz pathway is conserved in mammalian synaptic development (Restrepo, 2022).

Identifying a role for γ-secretase and its mechanistic basis in neurodevelopment offers insight into the potential function of γ-secretase in disease. PSEN1 mutations are the most widely known genetic cause of Early-Onset Alzheimer Disease (EOAD), but thorough understanding of this genetic link is lacking. The data indicate that neurodevelopmental and neurodegenerative mechanisms may be united by a shared requirement for γ-secretase-dependent receptor processing. In AD, this adds a layer of connection between neurodevelopment and neurodegeneration via synapse-to-nucleus communication. In a model, γ-secretase cleavage enables Fz2-C generation and transition from synapse to nucleus. In AD, the synapse-to-nucleus signals AIDA-1, ATF4, and CRTC1 (that are protein partners of cleaved receptor products) are also altered and blocking the synapse-to-nucleus translocation of Jacob, a protein that couples activity to CREB signaling, suppresses pathogenic Aβ-induced impairments. Neurodevelopmental defects were observed in EOAD mutations indistinguishable from loss-of-function mutants that could be suppressed by restoring nuclear Fz2-C . It will be important for future study to determine the intersection of proteolytic processing, synapse-to-nucleus signaling, and Wnts on AD. The data raise a tantalizing possibility of connecting AD to a previously unappreciated neurodevelopmental process, which can potentially provide earlier hallmarks to assess disease progression and inform therapeutic strategies to ameliorate disease states (Restrepo, 2022).

A prevailing mystery surrounding neurodegenerative diseases involves its onset. Although patients carry gene mutations all their lives, why do these disorders manifest later in life? A further challenge is to identify the earliest changes resulting from neurodegenerative disease gene mutations to better understand if symptoms are causative or correlative. The presence of synaptic maturation defects presents a hypothesis: if neurodegenerative disease risk genes are involved in synaptic maturation, the first reflection of mutations in those genes may be immature synapses that still function but lack the longevity of mature synapses. In advanced age, synapses constructed incorrectly during development may be the first to fail, leading to degeneration. AD models show reductions in postsynaptic proteins that precede neurodegeneration. Further, growing evidence suggests that neurodegenerative diseases have synaptopathic origins and early developmental defects. It is further intriguing that molecules which influence synaptogenesis, like Ephs, are modified by γ-secretase, potentially underlying further connection. Postsynaptic development and maturation may provide a unique angle to consider neurodegeneration, leading to earlier detection. With γ-secretase, maturation defects are suppressed by activating the downstream pathway; the data suggests analogous approaches may have clinical relevance. Whether synaptic NMJ phenotypes indicate a preclinical phenotype in disease models is an open question but examining postsynaptic maturation provides an opportunity to study shared mechanisms of neurodegenerative diseases, inform strategies to combat disease, and better understand neurodevelopment (Restrepo, 2022).

The data indicates that γ-secretase activity is required for Fz2 cleavage. First, γ-secretase and Fz2 localize close enough for interaction. Second, Fz2 and γ-secretase colocalize in the same subcellular compartments and traffic similarly. Third, a structural model predicts that the cleavage site embeds in the membrane, keeping it within the 'lid' distance to be recognized as a cleavage site. Moving the cleavage site so it no longer abuts the membrane prevents the cleavage-dependent functions of Fz2. The most parsimonious interpretation supports Fz2 cleavage by γ-secretase. However, it is important to note that the data do not conclusively imply a direct cleavage event. Fz2 is not a canonical γ-secretase substrate for several reasons. First, γ-secretase typically cleaves single pass transmembrane proteins; Fz2 is a 7-pass transmembrane domain atypical G-protein coupled receptor (GPCR). This is not a requisite, however, as multipass transmembrane proteins like neuregulin-1 can be cleaved by γ-secretase. Second, γ-secretase typically cleaves in a processive fashion: the substrate ectodomain is first shed via an ADAM a disintegrin and metallprotease) enzyme or β-secretase, after which γ-secretase cleavage releases the substrate intracellular domain. There is no evidence that Fz2 undergoes ectodomain shedding and the receptor lacks consensus N-terminal cleavage sites. Third, γ-secretase typically cleaves in the plane of the membrane. The Fz2 cleavage site is cytosolic, although predicted to juxtapose the membrane. Recent years, however, identified noncanonical substrates that serve as exceptions to this rule (Restrepo, 2022).

The data suggest that GPCRs may be atypical γ-secretase cleavage targets. This broadens the potential repertoire of γ-secretase, underscoring its importance in diverse processes. However, alternate interpretations cannot be ruled out, where Fz2 cleavage requires ectodomain shedding or occurs via successive events. In the latter, Fz2 would still be a direct target of γ-secretase that follows a preceding event. Also, γ-secretase may cleave a secondary target that allows Fz2 to be cleaved. In all cases, the activity of γ-secretase is still required for Fz2 cleavage. Future study in a cell-free system will be required for formal proof. Regardless, the complete suppression of the γ-secretase developmental phenotypes by nuclear Fz2-C expression indicates the most relevant cellular substrate in promoting postsynaptic development and maturation via γ-secretase is cleaved Fz2-C (Restrepo, 2022).

Expanded tRNA methyltransferase family member TRMT9B regulates synaptic growth and function

Nervous system function rests on the formation of functional synapses between neurons. This study has identified TRMT9B as a new regulator of synapse formation and function in Drosophila. TRMT9B has been studied for its role as a tumor suppressor and is one of two metazoan homologs of yeast tRNA methyltransferase 9 (Trm9), which methylates tRNA wobble uridines. Whereas Trm9 homolog ALKBH8 is ubiquitously expressed, TRMT9B is enriched in the nervous system. However, in the absence of animal models, TRMT9B's role in the nervous system has remained unstudied. This study generate null alleles of TRMT9B and found it acts postsynaptically to regulate synaptogenesis and promote neurotransmission. Through liquid chromatography-mass spectrometry, it was found that ALKBH8 catalyzes canonical tRNA wobble uridine methylation, raising the question of whether TRMT9B is a methyltransferase. Structural modeling studies suggest TRMT9B retains methyltransferase function and, in vivo, disruption of key methyltransferase residues blocks TRMT9B's ability to rescue synaptic overgrowth, but not neurotransmitter release. These findings reveal distinct roles for TRMT9B in the nervous system and highlight the significance of tRNA methyltransferase family diversification in metazoans (Hogan, 2023).

Precise mapping of one classic and three novel GluRIIA mutants in Drosophila melanogaster

Mutation of the Drosophila melanogaster GluRIIA gene or pharmacological agents targeting it are commonly used to assess homeostatic synaptic function at the larval neuromuscular junction (NMJ). The commonly used mutation, GluRIIA (SP16), is a null allele created by a large and imprecise excision of a P-element which affects GluRIIA and multiple upstream genes. This study mapped the exact bounds of the GluRIIA (SP16) allele, refined a multiplex PCR strategy for positive identification of GluRIIA (SP16) in homozygous or heterozygous backgrounds, and sequenced and characterized three new CRISPR-generated GluRIIA mutants. The three new GluRIIA alleles are apparent nulls that lack GluRIIA immunofluorescence signal at the 3 (rd) instar larval NMJ and are predicted to cause premature truncations at the genetic level. Further, these new mutants have similar electrophysiological outcomes as GluRIIA (SP16), including reduced miniature excitatory postsynaptic potential (mEPSP) amplitude and frequency compared to controls, and they express robust homeostatic compensation as evidenced by normal excitatory postsynaptic potential (EPSP) amplitude and elevated quantal content. These findings and new tools extend the capacity of the D. melanogaster NMJ for assessment of synaptic function (Mallik, 2023).

A Novel Interaction between MFN2/Marf and MARK4/PAR-1 Is Implicated in Synaptic Defects and Mitochondrial Dysfunction

As cellular energy powerhouses, mitochondria undergo constant fission and fusion to maintain functional homeostasis. The conserved dynamin-like GTPase, Mitofusin2 (MFN2)/mitochondrial assembly regulatory factor (Marf), plays a role in mitochondrial fusion, mutations of which are implicated in age-related human diseases, including several neurodegenerative disorders. However, the regulation of MFN2/Marf-mediated mitochondrial fusion, as well as the pathologic mechanism of neurodegeneration, is not clearly understood. This study identified a novel interaction between MFN2/Marf and microtubule affinity-regulating kinase 4 (MARK4)/PAR-1. In the Drosophila larval neuromuscular junction, muscle-specific overexpression of MFN2/Marf decreased the number of synaptic boutons, and the loss of MARK4/PAR-1 alleviated the synaptic defects of MFN2/Marf overexpression. Downregulation of MARK4/PAR-1 rescued the mitochondrial hyperfusion phenotype caused by MFN2/Marf overexpression in the Drosophila muscles as well as in the cultured cells. In addition, knockdown of MARK4/PAR-1 rescued the respiratory dysfunction of mitochondria induced by MFN2/Marf overexpression in mammalian cells. Together, these results indicate that the interaction between MFN2/Marf and MARK4/PAR-1 is fine-tuned to maintain synaptic integrity and mitochondrial homeostasis, and its dysregulation may be implicated in neurologic pathogenesis (Cheon, 2023).

RIM-binding protein links synaptic homeostasis to the stabilization and replenishment of high release probability vesicles

This study defines activities of RIM-binding protein (RBP) that are essential for baseline neurotransmission and presynaptic homeostatic plasticity. At baseline, rbp mutants have a approximately 10-fold decrease in the apparent Ca(2+) sensitivity of release that this study attributes to (1) impaired presynaptic Ca(2+) influx, (2) looser coupling of vesicles to Ca(2+) influx, and (3) limited access to the readily releasable vesicle pool (RRP). During homeostatic plasticity, RBP is necessary for the potentiation of Ca(2+) influx and the expansion of the RRP. Remarkably, rbp mutants also reveal a rate-limiting stage required for the replenishment of high release probability (p) vesicles following vesicle depletion. This rate slows approximately 4-fold at baseline and nearly 7-fold during homeostatic signaling in rbp. These effects are independent of altered Ca(2+) influx and RRP size. It is proposed that RBP stabilizes synaptic efficacy and homeostatic plasticity through coordinated control of presynaptic Ca(2+) influx and the dynamics of a high-p vesicle pool (Muller, 2015).

RIM-binding protein couples synaptic vesicle recruitment to release site

At presynaptic active zones, arrays of large conserved scaffold proteins mediate fast and temporally precise release of synaptic vesicles (SVs). SV release sites could be identified by clusters of Munc13, which allow SVs to dock in defined nanoscale relation to Ca2+ channels. This study shows in Drosophila that RIM-binding protein (RIM-BP) connects release sites physically and functionally to the ELKS family Bruchpilot (BRP)-based scaffold engaged in SV recruitment. The RIM-BP N-terminal domain, while dispensable for SV release site organization, was crucial for proper nanoscale patterning of the BRP scaffold and needed for SV recruitment of SVs under strong stimulation. Structural analysis further showed that the RIM-BP fibronectin domains form a "hinge" in the protein center, while the C-terminal SH3 domain tandem binds RIM, Munc13, and Ca2+ channels release machinery collectively. RIM-BPs' conserved domain architecture seemingly provides a relay to guide SVs from membrane far scaffolds into membrane close release sites (Petzoldt, 2020).

Chemical synapses are the fundamental building blocks of neuronal communication, allowing for a fast and directional exchange of chemical signals between a neurotransmitter releasing presynaptic and receiving postsynaptic target cells. To couple synaptic vesicle (SV) release to electrical stimulation by action potentials, Ca2+ ions entering the cell through voltage-gated Ca2+ channels activate the Ca2+ sensor synaptotagmin that is anchored on SVs to trigger fusion events. The presynaptic site of SV fusion ('active zone' [AZ]) is covered by an electron-dense scaffold ('cytomatrix') formed by a set of large conserved multidomain proteins. How individual AZ scaffold proteins intersect mechanistically with the SV cycle still remains largely enigmatic, though such knowledge was of importance to properly model synapse function in healthy and diseased circuits (Petzoldt, 2020).

On the level of the individual AZs, recent data from both cultivated rodent neurons and Drosophila in vivo neurons suggest that the AZ cytomatrix provides stable SV release sites or 'fusion slots' via the clustering of the critical release factor (m)Unc13. The number of such release sites seems to be determined independently of the mechanisms controlling release probability. How SV release sites might be coupled and integrated with additional processes organized by the AZ scaffold, such as the recruitment of SVs under high demand periods of resupply (e.g., high action potential frequencies), remains a largely open question (Petzoldt, 2020).

The physically extended ELKS family protein (glutamic acid [E], leucine [L], lysine [K],and serine [S]-rich protein) Bruchpilot (BRP) not only operates as the fundamental building block of the Drosophila AZ scaffold but was also shown to promote SV recruitment depending on a binding motif at its extreme, AZ membrane-distal C terminus. Adaptor proteins physically and functionally connecting such extended scaffold proteins, which sample the cytoplasm, and the SV release sites at the AZ membrane might well be relevant in this regard. Rab3-interacting molecule (RIM)-binding proteins (RIM-BPs) are an evolutionarily conserved family of extended AZ scaffold proteins, obviously critical for SV release at Drosophila and mammalian central synapses. RIM-BP family proteins with the C-terminal SH3-II/III domains bind to Ca2+ channels, release factor Unc13A, and RIM, another critical AZ scaffold protein. At Drosophila neuromuscular junction (NMJ) synapses, rim-bp null alleles provoke a most severe functional phenotype, stronger than for rim and rim-bp single mutants at most mammalian synapses. This study exploits the severity of the synaptic Drosophila rim-bp phenotype for a detailed structure-function analysis at Drosophila larval neuromuscular (NMJ) synapses. Elimination of the individual RIM-BP SH3 domains affected transport to AZs severely; however, the low levels of properly locating RIM-BP-ΔSH3 variants were still able to rescue the 'nanoscopic' defects of rim-bp null mutants AZ scaffolds. Crystallographic analysis of the RIM-BP fibronectin III-like (FN-III) domain cluster describes an extended 'hinge' in the protein center, which likely plays conformational roles. Finally, the N-terminal region (NTR) of RIM-BP was required to properly organize the overall nanoscopic architecture of the AZ scaffold. While SV docking and numbers of (m)Unc13 clusters remained at normal levels, the NTR was specifically needed for efficient SV recruitment. Thus, it is suggested that RIM-BP family proteins evolved as adaptors physically and functionally connecting SV release sites with BRP/ELKS-dependent SV recruitment processes (Petzoldt, 2020).

The current view of AZ scaffolds emphasizes their role in providing a 'smart catalytic surface' integrating sub-functionalities that facilitate and control the SV cycle. Providing SV release sites with a proteinaceous 'nano-environment' defining release probability, by precisely defining their spatial distance to voltage-operated Ca2+ channels, is probably one fundamental function using these highly conserved, 'ancient' protein architectures. The SV release sites probably also have to be coupled to processes retrieving SVs from more membrane-distant positions. Concerning protein architectures harvesting SVs, evolutionary solutions might have been somewhat more flexible, obviously adopted to the synapse-type specific needs in physiologically relevant fusion rates. Identifying proteins that might be involved in structurally and functionally coupling recruitment protein architectures with release sites is an emerging topic. This study provides evidence that RIM-BP with its interactions obviously plays a role in both 'nanoscopic locations.' First, through its SH3 domains, it binds RIM, the intercellular C-terminus of the voltage-operated Ca2+ channel and also Unc13A, the release factor whose nanoscale positioning has been recently identified as critical for SV release definition (Reddy-Alla, 2017). Second, this study found that it also binds conserved regions of BRP in the core of the AZ central scaffold. Deleting this interaction surface, though not reducing AZ protein levels, obviously undermines the proper bundling of BRP filaments, at least for a major fraction of AZs. Though it is not easy to prove that individual RIM-BP protein molecules span this distance physically, it is tempting to hypothesize that RIM-BP might literally connect the recruitment of SVs from more membrane distant pools, probably starting at the distal end of the BRP filaments, with their integration into SV release sites (see RIM-BP NTR localizes to the center of the AZ to stabilize the BRP scaffold and promote SV recruitment) (Petzoldt, 2020).

At mammalian synapses, recent multi-loci genetics have demonstrated most severe deficits when eliminating combinations of AZ scaffold proteins between RIM, ELKS, and RIM-BP family members. In this study, quadruple knockouts of RIM1/2, together with RIM-BP1/2 proteins in mice, exhibit a total loss of neurotransmitter release from severe impairments in SV priming and docking, a dramatic loss of AZ scaffold density, with a trans-synaptic effect that impairs the organization of the postsynaptic density. These severe synthetic 'catastrophic' phenotypes, eliminating ultrastructural specializations and release factor targeting, demonstrate a principal functional redundancy between RIM-BPs, RIMs, and ELKS family proteins. Similar results were also retrieved from work on mice in which RIM1αβ and RIM2αβγ, together with ELKS1α and ELKS2α isoforms, have been completely eliminated. Cultured hippocampal synapses of these mutant mice consequently lose Munc13, Bassoon, Piccolo, and RIM-BP, following a mass disassembly of the AZ scaffold (Wang, 2016). Similarly, this study observed that RIM seemingly operates synergistically with RIM-BP in establishing the AZ nanoscopic architecture (Petzoldt, 2020).

Despite the obvious importance of these findings for understanding the collective role of the AZ scaffold, the fact that the domain organization for all specific scaffold proteins has been individually conserved over hundreds of millions of years motivated a search for ways to demonstrate specific functions of these AZ core scaffold proteins. This study has molecularly isolated an additional sub-functionality for the so-far functionally nonconsidered RIM-BP NTR, and provide evidence that its protein architecture might have been conserved for the reason that it operates as an adaptor coupling SV recruitment processes with the membrane-associated SV release sites (Petzoldt, 2020).

Mechanistic analysis of RIM-BPs so far has largely focused on SH3 domains II and III, which bind to Ca2+ channels and RIM in both mammals and Drosophila, and Unc13A in Drosophila. Analysis of RIM-BP at Drosophila NMJ synapses was the first to demonstrate a major role of the protein family in neurotransmission, characterized by a severe reduction in release probability and signs of defective Ca2+ channel clustering. This study has exploited the glutamatergic NMJ synapses for a stringent genetic analysis with multimodal readouts, also including an analysis of transport and nanoscale integration into the AZ scaffold. Deletion of individual SH3 domains majorly affected RIM-BP transport, probably directly reflecting their high affinity binding to JIP-1 homologue Aplip1, whose deletion also interferes with effective axonal transport of BRP/RIM-BP 'packages'. Somewhat surprisingly, however, moderately or even strongly reduced levels of RIM-BPΔSH3-II or RIM-BPΔSH3-III, respectively, still restored RIM-BP functionality when expressed in a null background. It obviously might be argued that only combined elimination of SH3-II together with SH3-III might uncover their function collectively. Indeed, Aplip1 binds both SH3-II and III, and AZ levels were decreased even more strongly in the RIM-BPΔSH3-II/III double-deleted variant. However, at least concerning the role of Ca2+ channel binding, the AZ scaffold can seemingly compensate for the absence of RIM-BP-SH3 mediated interactions, as Ca2+ channels with the highly conserved PXXP RIM-BP binding motif deleted still retained full activity in a genetic rescue assay (instead of resembling the rim-bp null phenotype). It is suggested that redundant interactions stabilize Ca2+ channel: AZ scaffold contacts at NMJ synapses, an idea that might be relevant for other synapses as well. Indeed, it was shown previously that the N terminus of BRP also binds the Ca2+ channel α1 subunit intracellular C terminus. Given the essential character of SH3-III for survival, however, it appears likely that slightly different rules and 'vulnerabilities' apply to other synapse types, for example, in the Drosophila brain. In fact, it was found recently that synapses in the Drosophila brain differ substantially in their BRP content, with, for example, interneuron synapses largely lacking BRP (Fulterer, 2018). This might result in different efficacy of compensation via BRP and be responsible for the essential character of SH3-III. Put differently, it still appears plausible that the C-terminal close SH3 domains might be of truly essential character dependent on synapse type given the results retrieved at mammalian synapses (Petzoldt, 2020).

Finally, the presence of three FN-III domains, despite their invariable character, remained and remains an enigma. FN-III modules are common in extracellular but rarer in intracellular proteins, such as RIM-BP, here found chiefly as the main components of a group of intracellular proteins associated with the contractile apparatus of muscles. The first x-ray structure of the RIM-BPs, which is presented in this study, depicts a typical FN-III-type organization; however, it is oriented in an extended hinge-like arrangement. It is tempting to speculate that the hinge-like architecture might support RIM-BP conformations or conformational change when connecting release sites with recruitment processes (Petzoldt, 2020).

Physiological analysis points toward a discrete function of the RIM-BP N-terminal domain. Analysis of SV recruitment based on the rim-bp null allele is complicated due to the severe release probability deficits dominating the physiological scenario. Nonetheless, a careful analysis, using high Ca2+ concentrations to milden the influence of release probability differences, indeed identified recruitment defects. Remarkably, similar to the NTR-specific deletions, a previous study analyzing the rim-bp null mutant for RIM-BP suggested a rate-limiting function for the replenishment of high release probability SVs following vesicle depletion at NMJ synapses. Moreover, in mouse rim-bp2 knockouts, recruitment/replenishment deficits at auditory hair cells were reported. In rim-bp null mutants, UNC13A levels are strongly reduced, while for the dNTR-2 construct, UNC13A levels and consequently release site number were not reduced (instead rather slightly increased). Thus, the data apparently uncouple the role of RIM-BP for release site organization (via Unc13A clustering) from its role in overall scaffold organization where the NTR plays an important role (Petzoldt, 2020).

The extended BRP filaments in Drosophila probably operate as 'antennae' to harvest SVs from the reserve pool, a process facilitated by the C-terminal amino acids of BRP, made evident by the defects of the brp nude allele lacking only the last 17 amino acids. BRP in its first half is highly homologous to ELKS/CAZ-associated structural proteins (CAST) AZ proteins; however, its C-terminal half is specific for insects and obviously evolved for harvesting SVs. The large vertebrate-specific AZ protein Bassoon was shown to help SV replenishment at the central synapse under conditions of heavy stimulation. Notably, Bassoon binds to the first SH3 domain of RIM-BP, suggestive of convergent evolution where different molecular antennae were used to finally target RIM-BP, the generic 'old' adaptor coupling to the membrane close release sites (Petzoldt, 2020).

In its current form, static STED microscopy as used in this study only samples average epitope distributions, while the relevant proteins likely are dynamically switched in the course of the SV cycle. As an indication of this, the RIM-BP C-terminal SH3 domains were found to bind both the Ca2+ channel C-terminus and Unc13A N terminus. As these binding events can hardly be accomplished by a single Unc13A molecule at a single time point, future analysis will have to address the underlying conformational dynamics likely involved here (Petzoldt, 2020).

Taken together, RIM-BPs might take a generic role of SV replenishment, apart from their obvious role in coclustering of release machinery and Ca2+ channels at the AZ membrane. Indeed, the highly conserved domain architecture might have evolved for exactly this reason, to guide SVs into the proper release environment (Petzoldt, 2020).

A presynaptic glutamate receptor subunit confers robustness to neurotransmission and homeostatic potentiation

Homeostatic signaling systems are thought to interface with other forms of plasticity to ensure flexible yet stable levels of neurotransmission. The role of neurotransmitter receptors in this process, beyond mediating neurotransmission itself, is not known. Through a forward genetic screen, the Drosophila kainate-type ionotropic glutamate receptor subunit DKaiR1D was identified as being required for the retrograde, homeostatic potentiation of synaptic strength. DKaiR1D is necessary in presynaptic motor neurons, localized near active zones, and confers robustness to the calcium sensitivity of baseline synaptic transmission. Acute pharmacological blockade of DKaiR1D disrupts homeostatic plasticity, indicating that this receptor is required for the expression of this process, distinct from developmental roles. Finally, this study demonstrates that calcium permeability through DKaiR1D is necessary for baseline synaptic transmission, but not for homeostatic signaling. It is proposed that DKaiR1D is a glutamate autoreceptor that promotes robustness to synaptic strength and plasticity with active zone specificity (Kiragasi, 2017).

The nervous system is endowed with potent and adaptive homeostatic signaling systems that maintain stable functionality despite the myriad changes that occur during neural development and maturation. The importance of homeostatic regulation in the nervous system is underscored by associations with a variety of neurological diseases, yet the genes and mechanisms involved remain enigmatic. A powerful model of presynaptic homeostatic plasticity has been established at the Drosophila neuromuscular junction (NMJ), a model glutamatergic synapse with molecular machinery that parallels central synapses in mammals. Here, genetic and pharmacological manipulations that reduce postsynaptic (muscle) glutamate receptor function trigger a trans-synaptic, retrograde feedback signal to the neuron that increases presynaptic release to precisely compensate for this perturbation. This process is referred to as 'presynaptic homeostatic potentiation' (PHP), because the expression mechanism requires a presynaptic increase in neurotransmitter release (Kiragasi, 2017).

In recent years, forward and candidate genetic approaches have revealed several new and unanticipated genes necessary for PHP expression. While perturbations to the glutamate receptors in muscle are crucial events in the induction of PHP, whether other ionotropic glutamate receptors (iGluRs) function in PHP or are even expressed at the Drosophila NMJ is unknown. Finally, although evidence has emerged that homeostatic modulation is synapse specific, no roles for neurotransmitter receptors or other factors have been found to enable the presynaptic tuning of release efficacy at individual synapses (Kiragasi, 2017).

This study has identified the kainate-type iGluR subunit DKaiR1D to be necessary for PHP expression at the Drosophila NMJ. DKaiR1D is necessary for the calcium sensitivity of baseline synaptic transmission, as well as for the acute and chronic expression of homeostatic potentiation. Recently, the functional reconstitution of DKaiR1D was achieved in heterologous cells, revealing that these receptors form homomeric calcium-permeable channels with atypical pharmacological properties compared to their vertebrate homologs (Li, 2016). This study has found that DKaiR1D is expressed in the nervous system and not the muscle, is present near presynaptic active zones, and is required specifically in motor neurons to enable the robustness of baseline neurotransmission and homeostatic plasticity. It is proposd that glutamate activates DKaiR1D at presynaptic release sites to translate autocrine activity into the robust stabilization of synaptic strength with active zone specificity (Kiragasi, 2017).

This unexpected role for iGluRs in sensing glutamate at presynaptic terminals indicates an autocrine mechanism that responds to glutamate release to adaptively modulate presynaptic activity at individual active zones (Kiragasi, 2017).

Glutamate receptors have diverse functions in modulating presynaptic excitability and short-term plasticity in addition to their established roles in postsynaptic excitation. Similar to what was observed with DKaiR1D at the Drosophila NMJ, rodent iGluRs also localize to presynaptic active zone, are activated by high concentrations of glutamate, and can modulate release during single action potentials. This suggests conserved autocrine modulatory mechanisms shared between these systems (Kiragasi, 2017).

Rodent autoreceptors are known to modulate presynaptic activity on rapid timescales. In these cases, most of the impact on release is likely to derive from a calcium store-dependent mechanism, or from modulation of the action potential during the repolarization phase, when most of the calcium influx that drives vesicle release occurs. In a similar fashion, activation of presynaptic DKaiR1D during a single action potential could lead to a rapid additional source of presynaptic calcium influx from DKaiR1D itself and/or through modulation of presynaptic membrane potential to drive increased vesicle release. Voltage imaging at the Drosophila NMJ has found the half width of the action potential waveform to be ~5 ms (Ford, 2014), which is sufficient time to be modulated through such a mechanism. Therefore, dynamic changes in voltage or calcium influx at or near active zones could, in principle, drive additional vesicle release during a single action potential. This modulation may be restricted to nearby active zones and compartments relative to the site of glutamate release. Indeed, presynaptic kainate autoreceptors have the capacity to confer short-range, synapse-specific modulation to synaptic transmission (Scott, 2008), while presynaptic ligand-gated ion channels in C. elegans can also rapidly modulate synaptic transmission (Takayanagi-Kiya, 2016). Local activation of DKaiR1D could, therefore, subserve a powerful and flexible means of tuning presynaptic efficacy at or near individual release sites (Kiragasi, 2017).

How does DKaiR1D promote the expression of presynaptic homeostatic plasticity? In contrast to the role of DKaiR1D in baseline release, the data indicate that the DKaiR1D-dependent mechanism that drives PHP is calcium independent. This implies two changes to DKaiR1D functionality that are unique to homeostatic adaptation compared to baseline transmission. First, because presynaptic release is acutely potentiated following application of PhTx, the activity, levels, and/or localization of DKaiR1D receptors must change to acquire a novel influence on neurotransmitter release following PHP induction. The activity of synaptic glutamate receptors can change through associations with additional subunits and auxiliary factors such as Neto. Furthermore, various forms of plasticity are expressed through dynamic changes in the levels and localization of glutamate receptors trafficking between active zones and endosome pools or extra-synaptic membrane. Indeed, when DKaiR1D is overexpressed in motor neurons, it rescues baseline transmission and PHP expression while localizing to heterogeneous puncta of varying distances relative to active zones. Notably, there is evidence that DKaiR1D interacts with other glutamate receptor subunits in vivo (Karuppudurai, 2014), which may contribute to the pharmacological differences observed in this study compared with the in vitro characterization (Li, 2016) and may also be targets of modulation during PHP (Kiragasi, 2017).

Second, calcium signaling through DKaiR1D differentially drives baseline release and homeostatic plasticity. Therefore, mechanisms distinct from calcium permeability of the channel must contribute to PHP expression. One possibility is that DKaiR1D signals through an undefined metabotropic mechanism during PHP, which might contribute to the ability of the calcium impermeable DKaiR1DR transgene, with reduced conductance (Li, 2016), to rescue PHP expression. Alternatively, an attractive possibility is that following PHP induction, glutamate released from nearby active zones may dynamically modulate the presynaptic membrane potential and/or action potential waveform to promote additional synaptic vesicle release. Indeed, small, sub-threshold depolarizations of the presynaptic resting potential, as small as 5 mV, are sufficient to induce a 2-fold increase in presynaptic release. The timescale of this activity could occur within a few milliseconds as discussed above, and studies at the Drosophila NMJ have revealed that glutamate is released from single synaptic vesicles over timescales of milliseconds. Interestingly, epithelial sodium channels (ENaCs) have been proposed to enable PHP expression through changes in the presynaptic membrane potential, and such a mechanism could be shared by DKaiR1D but gated by glutamate release at individual active zones. Thus, DKaiR1D may serve to homeostatically modulate presynaptic release through modulation of presynaptic voltage and, intriguingly, with active zone specificity (Kiragasi, 2017).

This characterization of DKaiR1D has revealed a role for presynaptic glutamate signaling in homeostatic plasticity. In the mammalian CNS, glutamate signaling drives the adaptive regulation of postsynaptic AMPA receptor insertion and removal, known as homeostatic scaling. Further, kainate receptors were recently demonstrated to regulate postsynaptic homeostatic scaling. Together with the present study, these results demonstrate that glutamatergic signaling through kainate receptors orchestrate the potent and adaptive homeostatic control of synaptic strength on both sides of the synapse. Future studies will reveal the integration between synaptic glutamate signaling and other forces that modulate synaptic strength to enable robust, flexible, and stable neurotransmission (Kiragasi, 2017).

The auxiliary glutamate receptor subunit dSol-1 promotes presynaptic neurotransmitter release and homeostatic potentiation

Presynaptic glutamate receptors (GluRs) modulate neurotransmitter release and are physiological targets for regulation during various forms of plasticity. Although much is known about the auxiliary subunits associated with postsynaptic GluRs, far less is understood about presynaptic auxiliary GluR subunits and their functions. At the Drosophila neuromuscular junction, a presynaptic GluR, DKaiR1D, localizes near active zones and operates as an autoreceptor to tune baseline transmission and enhance presynaptic neurotransmitter release in response to diminished postsynaptic GluR functionality, a process referred to as presynaptic homeostatic potentiation (PHP). This study identified an auxiliary subunit that collaborates with DKaiR1D to promote these synaptic functions. This subunit, dSol-1, is the homolog of the Caenorhabditis elegans CUB (Complement C1r/C1s, Uegf, Bmp1) domain protein Sol-1. dSol-1 functions in neurons to facilitate baseline neurotransmission and to enable PHP expression, properties shared with DKaiR1D. Intriguingly, presynaptic overexpression of dSol-1 is sufficient to enhance neurotransmitter release through a DKaiR1D-dependent mechanism. Furthermore, dSol-1 is necessary to rapidly increase the abundance of DKaiR1D receptors near active zones during homeostatic signaling. Together with recent work showing the CUB domain protein Neto2 (see Drosophila Neto) is necessary for the homeostatic modulation of postsynaptic GluRs in mammals, these data demonstrate that dSol-1 is required for the homeostatic regulation of presynaptic GluRs. Thus, it is proposed that CUB domain proteins are fundamental homeostatic modulators of GluRs on both sides of the synapse (Kiragasi, 2020).

Synaptic strength is dynamically tuned during both Hebbian and homeostatic forms of plasticity. One major mechanism that achieves this modulation targets the abundance, localization, and/or functionality of ionotropic glutamate receptors (GluRs). For instance, the expression of long-term potentiation and depression requires bidirectional changes in the abundance of postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors to adjust synaptic strength. Furthermore, some forms of homeostatic plasticity also tune the abundance of N-methyl-D-aspartate (NMDA), AMPA, and kainate receptors at postsynaptic densities to stabilize neuronal activity . Auxiliary subunits associated with GluRs are key factors that control GluR trafficking and dynamics during plasticity, where transmembrane AMPA receptor regulatory protein (TARP), Cornichon, and Neto subunits orchestrate AMPA and kainate receptor function and plasticity. Although much is known about the GluR subtypes and associated auxiliary subunits that regulate GluR trafficking, abundance, and functionality at postsynaptic densities during both Hebbian and homeostatic plasticity, far less is understood about these mechanisms at presynaptic release sites (Kiragasi, 2020).

Presynaptic autoreceptors have emerged as important regulators of neurotransmitter release at glutamatergic synapses. For example, presynaptic kainate receptors are present in hippocampal mossy fibers, where autocrine feedback facilitates neurotransmission during trains of activity. In addition, presynaptic NMDA receptors in the hippocampus mediate presynaptic inhibition in response to excess glutamate release as well as presynaptic facilitation following the induction of long-term potentiation. Furthermore, presynaptic metabotropic receptors play critical roles in various forms of plasticity and can bidirectionally tune presynaptic neurotransmitter release. Finally, ionotropic neurotransmitter receptors at presynaptic terminals can modulate release at neuromuscular junctions (NMJs) in Caenorhabditis elegans and Drosophila. While it is now clear that presynaptic autoreceptors are important bidirectional modulators of neurotransmitter release, how the levels, activity, and localization of these receptors are controlled to establish baseline function, and to what extent they are further modified during plasticity, remains unclear (Kiragasi, 2020).

A kainate-type ionotropic GluR, DKaiR1D, was previously shown to be necessary at the Drosophila NMJ for the expression of presynaptic homeostatic potentiation (PHP). PHP is a fundamental form of synaptic plasticity in which pharmacological and genetic challenges that diminish postsynaptic neurotransmitter receptor functionality trigger a transsynaptic retrograde signal that enhances presynaptic neurotransmitter release to precisely compensate for reduced postsynaptic excitability (19, 20). PHP has been observed at NMJs of Drosophila, rodents, and humans and was recently demonstrated to be rapidly expressed in the mammalian central nervous system. DKaiR1D was identified in a forward genetic screen to be required for the rapid expression of PHP at the fly NMJ. DKaiR1D receptors form homomers that are permeable to both sodium and calcium, localized near presynaptic release sites, and proposed to homeostatically regulate presynaptic voltage following autocrine activation by glutamate. This DKaiR1D-dependent enhancement in neurotransmitter output implies a rapid modulation in the abundance, functionality, and/or localization of these receptors must occur in the course of PHP induction. This regulation could, in principle, be achieved through interactions with auxiliary GluR subunits. However, the precise mechanisms that control DKaiR1D and enable robust and stable neurotransmission at baseline and during plasticity, and whether auxiliary factors are involved, are unknown (Kiragasi, 2020).

A candidate screen of Drosophila GluR modulators and auxiliary subunits was performed to identify potential functions in PHP expression. This effort has discovered an uncharacterized auxiliary GluR subunit that functions in neurons to promote neurotransmitter release and enable homeostatic potentiation. This factor, a homolog of the C. elegans auxiliary GluR subunit Sol-1 (Walker, 2006), contains multiple CUB domains and is structurally similar to the Neto/Sol-2 family of auxiliary GluR subunits. dSol-1 mutants essentially phenocopy DKaiR1D mutants in neurotransmission and PHP. Further experiments demonstrate that dSol-1 functions to homeostatically modulate presynaptic glutamate release by promoting the rapid accumulation of DKaiR1D receptors near active zones. Together, these data indicate that the interactions between CUB domain auxiliary subunits and their associated GluRs are fundamental physiological targets of homeostatic signaling (Kiragasi, 2020).

In the context of baseline neurotransmission, dSol-1 promotes release without measurably changing the abundance or localization of DKaiR1D receptors, indicating a functional role in modulating DKaiR1D activity. However, dSol-1 is necessary during PHP signaling to drive a rapid increase in DKaiR1D receptor abundance at presynaptic terminals. These findings define a CUB domain auxiliary GluR subunit as a central target for the presynaptic modulation of synaptic efficacy and homeostatic plasticity (Kiragasi, 2020).

Several lines of evidence suggest that dSol-1 enhances baseline neurotransmitter release by targeting DKaiR1D receptor functionality. First, dSol-1 promotes baseline neurotransmission in low extracellular Ca2+, a function shared with DKaiR1D. In addition, neurotransmitter release is reduced in dSol-1 mutants and enhanced by neuronal overexpression of dSol-1, indicating a capacity for dSol-1 expression levels to bidirectionally tune release. However, this potentiation in baseline transmission occurs without a significant increase in DKaiR1D receptor abundance, at least when both dSol-1 and DKaiR1D are overexpressed in motor neurons, suggesting a change in DKaiR1D functionality. Interestingly, in C. elegans, Sol-1 regulates GLR1 functionality by modulating channel gating, promoting the open state, and slowing sensitization, without an apparent change in glr1 expression. In heterologous cells, both Sol-1 and dSol-1 promote GLR1 function without altering expression levels. This indicates a potentially conserved function between sol-1 and dSol-1 to confer similar modulations to GluR functionality. In mammals, Neto auxiliary subunits selectively associate with kainate-subtype GluRs, while TARPs such as Stargazin associate with AMPA-type receptors. In Drosophila, Neto is an important auxiliary subunit for the postsynaptic GluRs at the NMJ, which, like DKaiR1D, are generally characterized as non-NMDA, kainate-type GluRs. However, in C. elegans, both Sol-1 and Sol-2/Neto form a complex together with the AMPA receptor subtype GLR1, suggesting some level of promiscuity, at least in invertebrates, between AMPA and kainate GluRs and their auxiliary subunits. One possibility is that at baseline states, a substantial proportion of DKaiR1D receptors do not interact with dSol-1. By increasing levels of dSol-1, more DKaiR1D receptors may become associated with dSol-1, perhaps leading to changes in gating properties that enhance DKaiR1D receptor function. However, the possibility cannot be ruled out that dSol-1 somehow regulates DKaiR1D through a more indirect mechanism. While DKaiR1D receptors can form homomers and traffic to the cell surface when expressed alone in heterologous cells, future in vitro studies will be needed to determine the precise role dSol-1 has in DKaiR1D receptor trafficking and/or functionality (Kiragasi, 2020).

dSol-1 enables PHP expression through a mechanism that is distinct from its role in baseline transmission, although both functions converge on DKaiR1D. The data suggest that PHP signaling leads to a rapid accumulation of DKaiR1D receptors near presynaptic release sites that requires dSol-1. This dSol-1-dependent increase in DKaiR1D levels may be a unique feature of homeostatic signaling in Drosophila, as there is no evidence for worm sol-1 to promote surface levels or changes in GLR1 localization in vivo or in vitro. Although the rapid increase in DKaiR1D receptor levels at synaptic terminals during PHP signaling is surprising, it is not unprecedented. DKaiR1D receptors are present near presynaptic release sites, and many other active zone components rapidly accumulate and/or remodel following PhTx application. An attractive possibility is that DKaiR1D receptors participate in this process of rapid active zone remodeling during PHP signaling. Mechanistically, new protein synthesis of DKaiR1D is unlikely to be involved, as PHP expression and active zone remodeling can occur without new translation. Recently, the lysosomal kinesin adaptor arl-8 and other axonal transport factors were identified to be necessary for the rapid increase in active zone components during PHP signaling. Thus, it is tempting to speculate that DKaiR1D receptors might be cotransported during PHP as part of this pathway. In vertebrates, auxiliary subunits traffic GluRs during synaptic plasticity, so dSol-1 may function similarly in delivering DKaiR1D receptors to the plasma membrane and/or to release sites during PHP signaling. Finally, it is possible that DKaiR1D receptors are constitutively degraded under baseline conditions, and that PHP signaling through dSol-1 inhibits this degradation. The role of protein degradation in PHP signaling has been recently studied in both pre- and postsynaptic compartments at the Drosophila NMJ. Interestingly, inhibition of proteasomal degradation in presynaptic compartments is capable of rapidly enhancing neurotransmission to levels similar to what is observed after overexpression of dSol-1. In both cases, no further increase in neurotransmitter release is observed after PhTx application. While there are apparently distinct roles for dSol-1 in baseline function and homeostatic plasticity, a common point of convergence is DKaiR1D (Kiragasi, 2020).

CUB domains define a structural motif in a large family of extracellular and plasma membrane-associated proteins present in invertebrates to humans. While the specific four extracellular CUB domains that define sol-1/dSol-1 are unique to invertebrates, genes containing multiple CUB domains (between two and eight) are present throughout vertebrate species and function in diverse processes including intercellular signaling, developmental patterning, inflammation, and tumor suppression. CUB domains mediate dimerization and binding to collagen-like regions; this interaction may be relevant to its role in promoting PHP, as the Drosophila collagen member Multiplexin is present in the extracellular matrix and has been proposed to be part of the homeostatic retrograde signaling system. This characterization of dSol-1 contrasts with what is known about another CUB domain auxiliary glutamate receptor in Drosophila, Neto-β. neto-β is highly expressed in the larval muscle, where it is clearly involved in the trafficking and/or stabilization of postsynaptic GluRs at the NMJ. In contrast, dSol-1 is exclusively expressed in the nervous system. Another interesting distinction is that while both dSol-1 and Neto contain multiple extracellular CUB domains and a single transmembrane domain, dSol-1 lacks any intracellular domain while two isoforms of Neto are expressed with one of two alternative intracellular C-terminal cytosolic domains, Neto-α or Neto-β. Neto-β is clearly the major isoform and performs the key functions in controlling postsynaptic GluR levels and composition, while neto-α was recently proposed to function in motor neurons with DKaiR1D and to be necessary for PHP. Interestingly, the C. elegans receptor GLR1 requires the auxiliary subunits Sol-1, Stargazin, and Neto/Sol-2 for functionality in vivo and in vitro. It is therefore possible that Drosophila Neto-α and/or Stargazin-like interact with both dSol-1 and DKaiR1D. In mammals, there is a large body of evidence demonstrating that presynaptic GluRs, including kainate receptors, modulate presynaptic function. While postsynaptic kainate receptors in mammals associate with the CUB domain auxiliary GluR subunit Neto2 to regulate synaptic function and homeostatic plasticity, to what extent Neto2 or other auxiliary subunits function with presynaptic kainate receptors remains enigmatic. This study indicates that CUB domain proteins may be fundamental modulators of GluRs in synaptic function and plasticity on both sides of the synapse (Kiragasi, 2020).

Neto-alpha controls synapse organization and homeostasis at the Drosophila neuromuscular junction

Glutamate receptor auxiliary proteins control receptor distribution and function, ultimately controlling synapse assembly, maturation, and plasticity. At the Drosophila neuromuscular junction (NMJ), a synapse with both pre- and postsynaptic kainate-type glutamate receptors (KARs), this study shows that the auxiliary protein Neto evolved functionally distinct isoforms to modulate synapse development and homeostasis. Using genetics, cell biology, and electrophysiology, this study demonstrates that Neto-α functions on both sides of the NMJ. In muscle, Neto-α limits the size of the postsynaptic receptor field. In motor neurons (MNs), Neto-α controls neurotransmitter release in a KAR (KaiR1D)-dependent manner. In addition, Neto-α is both required and sufficient for the presynaptic increase in neurotransmitter release in response to reduced postsynaptic sensitivity. This KAR-independent function of Neto-α is involved in activity-induced cytomatrix remodeling. It is proposed that Drosophila ensures NMJ functionality by acquiring two Neto isoforms with differential expression patterns and activities (Han, 2020).

Formation of functional synapses during development and their fine-tuning during plasticity and homeostasis relies on ion channels and their accessory proteins, which control where, when, and how the channels function. Auxiliary proteins are diverse transmembrane proteins that associate with channel complexes and mediate their properties, subcellular distribution, surface expression, synaptic recruitment, and associations with various synaptic scaffolds. Channel subunits have expanded and diversified during evolution to impart different channel biophysical properties, but whether auxiliary proteins have evolved to match channel diversity remains unclear (Han, 2020).

Ionotropic glutamate receptors (iGluRs) mediate neurotransmission at most excitatory synapses in the vertebrate CNS and at the neuromuscular junction (NMJ) of insects and crustaceans and include α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), N-methyl-D-aspartic acid receptors (NMDARs), and kainate receptors (KARs). Sequence analysis of the Drosophila genome identified 14 iGluRs genes that resemble vertebrate AMPARs, NMDARs, and KARs. The fly receptors have strikingly different ligand binding profiles; nonetheless, phylogenetic analysis indicates that two of the Drosophila genes code for AMPARs, two code for NMDARs, and 10 code for subunits of the KAR family, which is highly expanded in insects. In flies and vertebrates, AMPARs and KARs have conserved, dedicated auxiliary proteins. For example, AMPARs rely on Stargazin and its relatives to selectively modulate receptors' gating properties, trafficking, and interactions with scaffolds such as PSD-95-like membrane-associated guanylate kinases. Stargazin is also required for the functional reconstitution of invertebrate AMPARs. KARs are modulated by the Neto (Neuropilin and Tolloid-like) family of proteins, including vertebrate Neto1 and Neto2, C. elegans SOL-2/Neto (Wang et al., 2012), and Drosophila Neto. Neto proteins differentially modulate the gating properties of vertebrate KARs. A role for Neto in the biology of KARs in vivo has been more difficult to assess because of the low levels of KARs and Neto proteins. Nevertheless, vertebrate Netos modulate synaptic recruitment of selective KARs by association with synaptic scaffolds such as GRIP and PSD-95, and the PDZ binding domains of vertebrate KAR/Neto complexes are essential for basal synaptic transmission and long-term potentiation (LTP). Post-translational modifications regulate Neto activities in vitro, but the in vivo relevance of many of these observations remains unknown (Han, 2020).

Drosophila NMJ is an excellent genetic system to probe the repertoire of Neto functions. This glutamatergic synapse appears to rely exclusively on KARs, with one presynaptic and five postsynaptic subunits. Previous work has shown that Drosophila Neto is an obligatory auxiliary subunit of the postsynaptic KAR complexes: in the absence of Neto, postsynaptic KARs fail to cluster at synaptic sites and the animals die as paralyzed embryos. Heterologous reconstitution of postsynaptic KARs in Xenopus oocytes revealed that Neto is required for functional receptors. The fly NMJ contains two glutamate receptor (GluR) complexes (types A and B) with different subunit compositions (either GluRIIA or GluRIIB, plus GluRIIC, GluRIID, and GluRIIE) and distinct properties, regulation, and localization patterns. The postsynaptic response to the fusion of single synaptic vesicles (quantal size) is reduced for NMJs with type B receptors only, and the dose of GluRIIA and GluRIIB is a key determinant of quantal size. The fly NMJ is also a powerful model system to study homeostatic plasticity. Manipulations that decrease the responsiveness of postsynaptic GluR (leading to a decrease in quantal size) trigger a robust compensatory increase in presynaptic neurotransmitter release or quantal content (QC). This increase in QC restores evoked muscle responses to normal levels. A presynaptic KAR, KaiRID, has recently been implicated in basal neurotransmission and presynaptic homeostatic potentiation (PHP) at the larval NMJ (Kiragasi, 2017; Li, 2016). The role of KaiRID in modulation of basal neurotransmission resembles GluK2/GluK3 function as autoreceptors (Pinheiro, 2007). The role of KaiRID in PHP must be indirect, because a mutation that renders this receptor Ca2+ impermeable has no effect on the expression of presynaptic homeostasis (Kiragasi, 2017) (Han, 2020).

The fly NMJ reliance on KARs raises the possibility that Drosophila diversified and maximized its use of Neto proteins. Drosophila Neto encodes two isoforms (Neto-α and Neto-β) with distinct intracellular domains generated by alternative splicing. Both cytoplasmic domains are rich in phosphorylation sites and docking motifs, suggesting rich modulation of Neto/KAR distribution and function. Neto-β, the predominant isoform at the larval NMJ, mediates intracellular interactions that recruit PSD components and enables synaptic stabilization of selective receptor subtypes. Neto-α can rescue viability and receptor clustering defects of Neto null. However, the endogenous functions of Neto-α remain unknown (Han, 2020).

This study shows that Neto-α is key to synapse development and homeostasis and fulfills functions distinct from those of Neto-β. Using isoform-specific mutants and tissue-specific manipulations, it was found that loss of Neto-α in the postsynaptic muscle disrupts GluR fields and produces enlarged PSDs. Loss of presynaptic Neto-α disrupts basal neurotransmission and renders these NMJs unable to express PHP. The different functions of Neto-α were mapped to distinct protein domains and Neto-α was shown to be both required and sufficient for PHP, functioning as a bona fide effector for PHP. It is proposed that Drosophila ensured NMJ functionality by acquiring two Neto isoforms with differential expression patterns and activities (Han, 2020).

This study showed that Neto-α is required in both pre- and postsynaptic compartments for the proper organization and function of the Drosophila NMJ. In muscle, Neto-α limits the size of the postsynaptic receptor field; the PSDs are significantly enlarged in muscle where Neto-α has been perturbed. In MNs, Neto-α is required for two distinct activities: (1) modulation of basal neurotransmission in a KaiRID-dependent manner and (2) effector of presynaptic homeostasis response. This is an extremely rare example of a GluR auxiliary protein that modulates receptors on both sides of a particular synapse and plays a distinct role in homeostatic plasticity (Han, 2020).

Vertebrate KARs depend on Neto proteins for their distribution and function (Copits and Swanson, 2012). Because of their reliance on KARs, Drosophila Neto^null mutants have no functional NMJs (no postsynaptic KARs) and consequently die as paralyzed embryos. Previous work has shown that muscle expression of Neto-ΔCTD, or minimal Neto, at least partly rescues the recruitment and function of KARs at synaptic locations. This study reports that neuronal Neto-ΔCTD also rescues the KaiRID-dependent basal neurotransmission. Thus, Neto-ΔCTD, the part of Neto conserved from worms to humans, seems to represent the Neto core required for KAR modulatory activities (Han, 2020).

The intracellular parts of Neto proteins are highly divergent, likely reflecting the microenvironments in which different Neto proteins operate. Similar to mammalian Neto1 and Neto2, Drosophila Neto-α and Neto-β are differentially expressed in the CNS and have different intracellular domains that mediate distinct functions. These large intracellular domains are rich in putative phosphorylation sites and docking motifs and could further modulate the distribution and function of KARs or serve as signaling hubs and protein scaffolds. Post-translational modifications regulate vertebrate Neto activities in vitro, although the in vivo relevance of these changes remains unknown. The current data demonstrate that Neto-α and Neto-β could not substitute for each other. For example, Neto-β, but not Neto-α, controls the recruitment of PAK, a PSD component that stabilizes selective KAR subtypes at the NMJ, and ensures proper postsynaptic differentiation. Conversely, postsynaptic Neto-β alone cannot maintain a compact PSD size; muscle Neto-α is required for this function. Neto-β cannot fulfill presynaptic functions of Neto-α, presumably because is confined to the somato-dendritic compartment and cannot reach the synaptic terminals. Histology and western blot analyses indicate that Neto-α constitutes less than 1/10th of the net Neto at the Drosophila NMJ. These low levels impaired direct visualization of endogenous Neto-α. Several isoform-specific antibodies have been generated, but they could only detect Neto-α when overexpressed. Similar challenges have been encountered in the vertebrate Neto field (Han, 2020).

The two Neto isoforms are limiting in different synaptic compartments. Neto-β limits the recruitment and synaptic stabilization of postsynaptic KARs. In contrast, several lines of evidence indicate that Neto-α is limiting in MNs. First, overexpression of KaiRID cannot increase basal neurotransmission (Kiragasi, 2017); however, neuronal overexpression of Neto-ΔCTD increases basal neurotransmission, indicating that Neto, but not KaiRID, is limiting in the MNs. Second, neuronal overexpression of Neto-α exacerbates the PHP response to PhTx exposure and even rescues this response in KaiRID^null. These findings suggest that KaiRID's function during PHP is to help traffic and stabilize Neto-α, a low-abundance PHP effector. Similarly, studies in mammals reported that KARs trafficking in the CNS do not require Neto proteins; instead, KARs regulate the surface expression and stabilization of Neto1 and Neto2. Nonetheless, the KAR-mediated stabilization of Neto proteins at CNS synapses supports KAR distribution and function. In flies, KaiRID-dependent Neto-α stabilization at synaptic terminals ensures KAR-dependent function, normal basal neurotransmission, and Neto-α-specific activity as an effector of PHP (Han, 2020).

Previous studies showed that presynaptic KARs regulate neurotransmitter release; however, the site and mechanism of action of presynaptic KARs have been difficult to pin down. This study provides strong evidence for Neto activities at presynaptic terminals. First, Neto-α is both required and sufficient for PHP. It has been shown that the PhTx-induced expression of PHP occurs even when the MN axon is severed. In addition, the signaling necessary for PHP expression is restricted to postsynaptic densities and presynaptic boutons. Second, Neto-ΔCTD, but not Neto-β, rescued basal neurotransmission defects in Neto-αnull. Both variants contain the minimal Neto required for KAR modulation, but only Neto-ΔCTD can reach the presynaptic terminal, whereas Neto-β is restricted to the somato-dendritic compartment. This suggests that Neto-ΔCTD (or Neto-α), together with KaiRID, localizes at presynaptic terminals, where KaiRID could function as an autoreceptor. Finally, upon PhTx exposure, Neto-α enabled fast recruitment of Brp at the active zone. Multiple homeostasis paradigms trigger Brp mobilization, followed by remodeling of presynaptic cytomatrix. These localized activities support Neto-α functioning at presynaptic terminals (Han, 2020).

Rapid application of glutamate to outside-out patches from HEK cells transfected with KaiRID indicated that KaiRID forms rapidly desensitizing channels; addition of Neto increases the desensitization rates and open probability for this channel. Neto-α has a large intracellular domain (250 residues) rich in post-translational modification sites and docking motifs, including putative phosphorylation sites for Ca2+/calmodulin-dependent protein kinase II (CaMKII), protein kinase C (PKC), and protein kinase A (PKA). This intracellular domain may engage in finely tuned interactions that allow Neto-α to (1) further modulate the KaiRID properties and distribution in response to cellular signals and (2) function as an effector of presynaptic homeostasis in response to low postsynaptic GluR activity. Mammalian Neto1 and Neto2 are phosphorylated by multiple kinases in vitro (Lomash, 2017); CaMKII- and PKA-dependent phosphorylation of Neto2 restrict GluK1 targeting to synapses in vivo and in vitro. Similarly, Neto-α may function in a kinase-dependent manner to stabilize KaiRID and/or other presynaptic components. Second, Neto-α may recruit Brp or other presynaptic molecules that mediate activity-related changes in glutamate release at the fly NMJ. Besides Brp, several presynaptic components have been implicated in the control of PHP. They include (1) Cacophony (Cac), the α1 subunit of CaV2-type calcium channels and its auxiliary protein α2Δ-3, that control the presynaptic Ca2+ influx; (2) the signaling molecules Eph, Ephexin, and Cdc42 upstream of Cac; and (3) the BMP pathway components, Wit and Mad, required for retrograde BMP signaling. In addition, expression of PHP requires molecules that regulate vesicle release and the RRP size, such as RIM, Rab3-GAP, Dysbindin, and SNAP25 and Snapin. Recent studies demonstrated that trans-synaptic Semaphorin/Plexin interactions control synaptic scaling in cortical neurons in vertebrates and drive PHP at the fly NMJ (Orr, 2017a). Neto-α may interact with one or several such presynaptic molecules and function as an effector of PHP. Future studies on what the Neto-α cytoplasmic domain binds to and how is it modulated by post-translational modifications should provide key insights into the understanding of molecular mechanisms of homeostatic plasticity (Han, 2020).

On the muscle side, Neto-α activities may include (1) engaging scaffolds that limit the PSD size and (2) modulating postsynaptic KAR distribution and function. For example, Neto-α may recruit trans-synaptic complexes such as Ten-a/Ten-m or Nrx/Nlgs that have been implicated in limiting the postsynaptic fields (Banovic, 2010; Mosca, 2012). In particular, DNlg3, like Neto-α, is present in both pre- and postsynaptic compartments and has similar loss-of function phenotypes, including smaller boutons with larger individual PSDs, and reduced EJP amplitudes (Xing, 2014). Neto-α may also indirectly interact with the Drosophila PSD-95 and Dlg and help establish the PSD boundaries. Fly Netos do not have PDZ binding domains, but the postsynaptic Neto/KAR complexes contain GluRIIC, a subunit with a class II PDZ binding domain. It has been reported that mutations that change the NMJ receptors' gating behavior alter their synaptic trafficking and distribution (Petzoldt, 2014). Neto-α could be key to these observations, because it may influence both receptor gating properties and ability to interact with synapse organizers (Han, 2020).

Phylogenetic analyses indicate that Neto-β is the ancestral Neto. In insects, Neto-β is predicted to control NMJ development and function, including recruitment of iGluRs and PSD components, and postsynaptic differentiation. Neto-α appears to be a rapidly evolving isoform present in higher Diptera. This large order of insects is characterized by a rapid expansion of the KAR branch to ten distinct subunits. Insect KARs have unique ligand binding profiles, strikingly different from vertebrate KARs. However, like vertebrate KARs, they all seem to be modulated by Neto proteins. It is speculated that the rapid expansion of KARs forced the diversification of the relevant accessory protein, Neto, and the extension of its repertoire. In flies, the Neto locus acquired an additional exon and consequently an alternative isoform with distinct expression profiles, subcellular distributions, and isoform-specific functions. It will be interesting to investigate how flies differentially regulate the expression and distribution of the two Neto isoforms and control their tissue- and synapse-specific functions. Mammals have five KAR subunits, three of which have multiple splice variants that confer rich regulation. In addition, mammalian Neto proteins have fairly divergent intracellular parts that presumably further integrate cell-specific signals and fine-tune KAR localization and function. In Diptera, KARs have relatively short C tails and thus limited signaling input, whereas Netos have long cytoplasmic domains that could function as scaffolds and signaling hubs. Consequently, most information critical for NMJ assembly and postsynaptic differentiation has been outsourced to the intracellular part of Neto-β. Neto-α-mediated intracellular interactions may also hold key insights into the mechanisms of homeostatic plasticity. This study reveals that Neto functions as a bona fide effector of presynaptic homeostasis (Han, 2020).

Presynaptic spinophilin tunes neurexin signalling to control active zone architecture and function

Assembly and maturation of synapses at the Drosophila neuromuscular junction (NMJ) depend on trans-synaptic neurexin/neuroligin signalling, which is promoted by the scaffolding protein Syd-1 binding to neurexin. This study reports that the scaffold protein spinophilin binds to the C-terminal portion of neurexin and is needed to limit neurexin/neuroligin signalling by acting antagonistic to Syd-1 (RhoGAP100F). Loss of presynaptic spinophilin results in the formation of excess, but atypically small active zones. Neuroligin-1/neurexin-1/Syd-1 levels are increased at spinophilin mutant NMJs, and removal of single copies of the neurexin-1, Syd-1 or neuroligin-1 genes suppresses the spinophilin-active zone phenotype. Evoked transmission is strongly reduced at spinophilin terminals, owing to a severely reduced release probability at individual active zones. It is concluded that presynaptic spinophilin fine-tunes neurexin/neuroligin signalling to control active zone number and functionality, thereby optimizing them for action potential-induced exocytosis (Muhammad, 2015).

Chemical synapses release synaptic vesicles (SVs) at specialized presynaptic membranes, so-called active zones (AZs), which are characterized by electron-dense structures, reflecting the presence of extended molecular protein scaffolds. These AZ scaffolds confer stability and facilitate SV release. Importantly, at individual AZs, scaffold size is found to scale with the propensity to engage in action potential-evoked release. An evolutionarily conserved set of large multi-domain proteins operating as major building blocks for these scaffolds has been identified over the last years: Syd-2/Liprin-α, RIM, RIM-binding-protein (RBP) and ELKS family proteins (of which the the Drosophila homologue is called Bruchpilot (BRP)). However, how presynaptic scaffold assembly and maturation are controlled and coupled spatiotemporally to the postsynaptic assembly of neurotransmitter receptors remains largely unknown, although trans-synaptic signalling via Neurexin-1 (Nrx-1)-Neuroligin-1 (Nlg1) adhesion molecules is a strong candidate for a conserved 'master module' in this context, based on Nrx-Nlg signalling promoting synaptogenesis in vitro, synapses of rodents, Caenorhabditis elegans and Drosophila (Muhammad, 2015).

With respect to scaffolding proteins, Syd-1 was found to promote synapse assembly in C. elegans, Drosophila and rodents. In Drosophila, the Syd-1-PDZ domain binds the Nrx-1 C terminus and couples pre- with postsynaptic maturation at nascent synapses of glutamatergic neuromuscular junctions (NMJs) in Drosophila larvae. Syd-1 cooperates with Nrx-1/Nlg1 to stabilize newly formed AZ scaffolds, allowing them to overcome a 'threshold' for synapse formation. Additional factors tuning scaffold assembly, however, remain to be identified. This study shows that the conserved scaffold protein spinophilin (Spn) is able to fine-tune Nrx-1 function by binding the Nrx-1 C terminus with micromolar affinity via its PDZ domain. In the absence of presynaptic Spn, 'excessive seeding' of new AZs occurred over the entire NMJ due to elevated Nrx-1/Nlg1 signalling. Apart from structural changes, this study shows that Spn plays an important role in neurotransmission since it is essential to establish proper SV release probability, resulting in a changed ratio of spontaneous versus evoked release at Spn NMJ terminals. The trans-synaptic dialogue between Nrx-1 and Nlg1 aids in the initial assembly, specification and maturation of synapses, and is a key component in the modification of neuronal networks. Regulatory factors and processes that fine-tune and coordinate Nrx-1/Nlg1 signalling during synapse assembly process are currently under investigation. These data indicate that Drosophila Spn-like protein acts presynaptically to attenuate Nrx-1/Nlg1 signalling and protects from excessive seeding of new AZ scaffolds at the NMJ. In Spn mutants, excessive AZs suffered from insufficient evoked release, which may be partly explained by their reduced size, and partly by a genuine functional role of Spn (potentially mediated via Nrx-1 binding). In mice, loss of Spn (Neurabin II), one of the two Neurabin protein families present in mammals, was reported to provoke a developmental increase in synapse numbers (Feng, 2000). While Spinophilin was found to be expressed both pre- and post-synaptically (Muly, 2004; Muly, 2004a; Muly, 2004b), its function, so far, has only been analysed in the context of postsynaptic spines (Feng, 2000; Terry-Lorenzo 2005; Allen, 2006; Sarrouilhe, 2005). Given the conserved Spn/Nrx-1 interaction reported in this study, Spn family proteins might execute a generic function in controlling Nrx-1/Nlg1-dependent signalling during synapse assembly (Muhammad, 2015).

This study consistently found that Spn counteracts another multi-domain synaptic regulator, Syd-1, in the control of Nrx-1/Nlg1 signalling. Previous genetic work in C. elegans identified roles of Syd-1 epistatic to Syd-2/Liprin-α in synaptogenesis. Syd-1 also operates epistatic to Syd-2/Liprin-α at Drosophila NMJs. Syd-1 immobilizes Nrx-1, positioning Nlg1 at juxtaposed postsynaptic sites, where it is needed for efficient incorporation of GluR complexes. Intravital imaging suggested an early checkpoint for synapse assembly, involving Syd-1, Nrx-1/Nlg1 signalling and oligomerization of Liprin-α in the formation of an early nucleation lattice, which is followed later by ELKS/BRP-dependent scaffolding events. As Spn promotes the diffusional motility of Nrx-1 over the terminal surface and limits Nrx-1/Nlg1 signalling, and as its phenotype is reversed by loss of a single gene copy of nrx-1, nlg1 or syd-1, Spn displays all the features of a 'negative' element mounting, which effectively sets the threshold for AZ assembly. As suggested by FRAP experiments, Spn might withdraw a population of Nrx-1 from the early assembly process, establishing an assembly threshold that ensures a 'typical' AZ design and associated postsynaptic compartments. As a negative regulatory element, Spn might allow tuning of presynaptic AZ scaffold size and function (Muhammad, 2015).

The C. elegans Spn homologue NAB-1 (NeurABin1) was previously shown to bind Syd-1 in cell culture recruitment assays (Chia, 2012). This study found consistent evidence for Syd-1/Nrx-1/Spn tripartite complexes in salivary gland experiments. Moreover, the PDZ domain containing regions of Spn and Syd-1 interacted in Y2H experiments. It would be interesting to dissect whether the interaction of Spn/Syd-1 plays a role in controlling the access of Nrx-1 to one or both factors. For C. elegans HSN synapses, a previous study showed that loss of NAB-1 results in a deficit of synaptic markers, such as Syd-1 and Syd-2/Liprin-α, while NAB-1 binding to F-actin was also found to be important for synapse assembly. Though at first glance rather contradictory to the results described in this study, differences might result from Chia (2012) studying synapse assembly executed over a short time window, when partner cells meet for the first time. In contrast, this study used a model (Drosophila larval NMJs) where an already functional neuronal terminal adds novel AZs. Despite the efforts of this study, no role of F-actin in the assembly of AZs of late larval Drosophila NMJs was demonstrated. F-actin patches might be particularly important to establish the first synaptic contacts between partner cells. Both the study by Chia et al. and this study, however, point clearly towards important regulatory roles of Spn family members in the presynaptic control of synapse assembly. Further, this study described a novel interaction between the Spn-PDZ domain and the intracellular C-term of Nrx-1 at the atomic level. Interestingly, it was found that all functions of Spn reported in this study, structural as well as functional, were strictly dependent on the ligand-binding integrity of this PDZ domain. It is noteworthy that the Spn-PDZ domain binds other ligands as well, for example, Kalirin-7 and p70^S6K , and further elucidation of its role as a signal 'integrator' in synapse plasticity should be interesting. The fact that Nrx-1 levels were increased at Spn NMJs and, most importantly, that genetic removal of a single nrx-1 gene copy effectively suppressed the Spn AZ phenotype, indicates an important role of the Spn/Nrx-1 interaction in this context. Affinity of Spn-PDZ for the Nrx-1 C-term was somewhat lower than that of the Syd-1-PDZ, both in ITC and Y2H experiments. Nonetheless, overexpression of Spn was successful in reducing the targeting effect of Syd-1 on overexpressed Nrx-1^GFP. It will be interesting to see whether this interaction can be differentially regulated, for example, by (de)phosphorylation. It is worth noting that apart from Syd-1 and Spn, several other proteins containing PDZ domains, including CASK, Mint1/X11, CIPP and Syntenin, were found to bind to the Nrxs C-termini. CASK was previously shown to interact genetically with Nrx-1, controlling endocytic function at Drosophila NMJs. However, when this study tested for an influence of CASK on Nrx-1^GFP motility using FRAP, genetic ablation of CASK had no effect (Muhammad, 2015).

Thus, CASK function seemingly resembles neither Syd-1 nor Spn. Clearly, future work will have to address and integrate the role of other synaptic regulators converging on the Nrx-1 C-term. In particular, CASK (which displays a kinase function that phosphorylates certain motifs within the Nrx-1 C-term) might alternately control Spn- and Syd-1-dependent functions. Presynaptic Nrx-1, through binding to postsynaptic Nlg1 at developing Drosophila NMJ terminals, is important for the proper assembly of new synaptic sites. It is of note, however, that while mammalian Nrxs display robust synaptogenetic activity in cellular in vitro systems, direct genetic evidence for synaptogenetic activity of Nrxs in the mammalian CNS remained rather scarce. Triple knockout mice lacking all α-Nrxs display no gross synaptic defects at the ultrastructural level. Future analysis will have to investigate whether differences here might be explained by specific compensation mechanisms in mammals; for example, by β-Nrxs, or other parallel trans-synaptic communication modules. Genuine functional deficits in neurotransmitter release were also observed after the elimination of presynaptic Spn. Elimination of ligand binding to the PDZ domain rendered the protein completely nonfunctional, without affecting its synaptic targeting. Thus, the Spn functional defects are likely to be mediated via a lack of Nrx-1 binding. Notably, ample evidence connects Nrx-1 function with both the functional and structural maturation of Drosophila presynaptic AZs. This work now promotes the possibility that binding of Spn to Nrx-1 is important for establishing correct release probability, independent of absolute AZ scaffold size. It is noteworthy that Nrx-1 function was previously shown to be important for proper Ca2+ channel function and, as a result, properly evoked SV release. Thus, it will be interesting to investigate whether the specific functional contributions of Spn are mediated via deficits in the AZ organization of voltage-gated Ca2+ channels or Ca2+ sensors, such as synaptotagmin. Taken together, this study found an unexpected function for Spn in addition of AZs at Drosophila glutamatergic terminals, through the integration of signals from both the pre- and postsynaptic compartment. Given that the Spn/Nrx-1 interaction is found to be conserved from Drosophila to rodents, addressing similar roles of presynaptic Spn in mammalian brain physiology and pathophysiology might be informative (Muhammad, 2015).

Neurexin, neuroligin and wishful thinking coordinate synaptic cytoarchitecture and growth at neuromuscular junctions

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Neurexins and Neuroligins have emerged as major players in the organization of excitatory and inhibitory synapses across species. Mutational analyses in Drosophila uncovered specific functions of dnrx and dnlg1 in NMJ synapse organization and growth, where dnrx is expressed pre-synaptically and dnlg1 post-synaptically. Deletion of the extracellular and intracellular regions of dnrx or dnlg1 revealed that both regions are necessary for dnrx and dnlg1 clustering and function at the synapse. Most importantly, the data presented in this study suggest that dnrx and dnlg1 genetically interact with wit as well as the downstream effector of BMP signaling, Mad to allow both the organization and growth of NMJ synapses. The surprising finding that loss of dnrx and dnlg1 leads to decreased wit stability and that dnrx and dnlg1 are required for proper wit localization raises the possibility that these proteins function to coordinate trans-synaptic adhesion and synaptic growth. This is further strengthened from the observations that, in the absence of Wit, the synaptogenic activity from overexpression of dnrx did not manifest into an increased synaptic growth as is seen in the presence of wit. Loss of dnrx and dnlg1 also led to a reduction in the levels of other components of the BMP pathway, namely the Tkv and pMad. Together these findings are the first to demonstrate a functional coordination between trans-synaptic adhesion proteins dnrx and dnlg1 with wit receptor and BMP pathway members to allow precise synaptic organization and growth at NMJ. It would be of immense interest to investigate whether similar mechanisms might be operating in vertebrate systems (Banerjee, 2016).

The trans-synaptic cell adhesion complex formed by heterophilic binding of pre-synaptic Neurexins (Nrxs) and post-synaptic Neuroligins (Nlgs), displayed synaptogenic function in cell culture experiments. In vertebrates and invertebrates alike, Nrxs and Nlgs belong to one of the most extensively studied synaptic adhesion molecules with a specific role in synapse organization and function. In Drosophila, dnrx and dnlg1 mutations cause reductions in bouton number, perturbation in active zone organization and severe reduction in synaptic transmission. These phenotypes are essentially phenocopied in both dnrx and dnlg1 mutants, and double mutants do not cause any significant enhancement in the single mutant phenotypes illustrating that they likely function in the same pathway. From immunohistochemical and biochemical analyses, it is evident that lack of dnrx or dnlg1 causes their diffuse localization and protein instability in each other's mutant backgrounds. These data suggest that trans-synaptic interaction between dnrx and dnlg1 is required for their proper localization and stability, and that these proteins have a broader function in the context of general synaptic machinery involving Nrx-Nlg across phyla. It also raises the possibility that the trans-synaptic molecular complex involving Nrx-Nlg may alter stability of other synaptic proteins and lead to impairments in synaptic function without completely abolishing synaptic structure and neurotransmission. It is therefore not surprising that Nrx and Nlg have been recently reported to be associated with many non-lethal cognitive and neurological disorders, such as schizophrenia, ASD, and learning disability (Banerjee, 2016).

The rescue studies of dnrx and dnlg1 localization using their respective N- and C-terminal domain truncations emphasize a requirement of the full-length protein for proper synaptic clustering. Given that dnrx and dnlg1 likely interact via their extracellular domains, it is somewhat expected that an N-terminal deletion as seen in genotypic combinations of elav-Gal4/UAS-DnrxΔ N;dnrx^-/- and mef2-Gal4/UAS-dnlg1Δ N;dnlg1^-/- would fail to rescue the synaptic clustering of Dnlg1 and Dnrx, respectively. However, the inability of the C-terminal deletions of these proteins as seen in elav-Gal4/UAS-DnrxΔ c;dnrx^-/- and mef2-Gal4/UAS-dnlg1Δ Cdnlg1^-/- to cluster Dnlg1 and Dnrx, respectively, to wild type localization and/or levels suggest that the lack of the cytoplasmic domains of these proteins may render the remainder portion of the protein unstable, thereby leading to its inability to be either recruited or held at the synaptic apparatus (Banerjee, 2016).

Finally, the subcellular localization of Dlg at the SSR and its levels in the dnrx and dnlg1 single and double mutants as well as in the rescue genotypes provides key insights into the stoichiometry of Dnrx-Dnlg1 interactions, and how Dnrx-Dnlg1 might be functioning with other synaptic proteins in their vicinity to organize the synaptic machinery. A significant reduction in Dlg levels in dnrx mutants raises the question of whether Dlg localization/levels are controlled presynaptically? Alternatively, it is possible that Dnrx-Dnlg1 is not mutually exclusive for all their synaptic functions and there might be other Neuroligins that might function with Dnrx. One attractive candidate could be Dnlg2, which is both pre- and postsynaptic. dnrx could also function through the recently identified Neuroligins 3 and 4. Although dnlg1 mutants did not show any significant difference in Dlg levels, a diffuse subcellular localization nevertheless raised the possibility of a structural disorganization of the postsynaptic terminal and defects in SSR morphology, which were confirmed by ultrastructural studies. The rescue analysis of the subcellular localization of Dlg demonstrates that Dlg localization and levels could be restored fully in a presynaptic rescue of dnrx mutants by expression of full length Dnrx, however the localization of Dlg could not be restored by expression of DnrxΔNT. These observations suggest that the extracellular domain of dnrx is essential for the localization and also the levels of Dlg. Whether the extracellular domain influences Dlg via dnlg1 or any of the other three Dnlgs (2, 3 and 4) at the NMJ remains to be elucidated (Banerjee, 2016).

Most studies on Nrx/Nlg across species offer clues as to how these proteins assemble synapses and how they might function in the brain to establish and modify neuronal network properties and cognition, however, very little is known on the signaling pathways that these proteins may potentially function in. It has been previously reported that Neurexin 1 is induced by BMP growth factors in vitro and in vivo and that could possibly allow regulation of synaptic growth and development. In Drosophila, both dnrx and dnlg1 mutants showed reduced synaptic growth similar to wit pathway mutants. Reduced wit levels both from immunolocalization studies in dnrx and dnlg1 mutant backgrounds and biochemical studies from immunoblot and sucrose density gradient sedimentation analyses present compelling evidence towards the requirement of dnrx and dnlg1 for wit stability. Reduction of synaptic dnrx levels in wit mutants argue for interdependence in the localization of these presynaptic proteins at the NMJ synaptic boutons. The findings that synaptic boutons of dnrx and dnlg1 mutants also show reduction in the levels of the co-receptor Tkv suggest that this effect may not be exclusively Wit-specific, and possibly due to the overall integrity of trans-synaptic adhesion complex that ensures Wit and Tkv stability at the NMJ (Banerjee, 2016).

Genetic interaction studies displayed a significant reduction in bouton numbers resulting from trans-heterozygous combinations of wit/+,dnrx/+ and wit/+,dnlg1/+ compared to the single heterozygotes strongly favoring the likelihood of these genes functioning together in the same pathway. Although double mutants of wit,dnrx and wit,dnlg1 are somewhat severe than dnrx and dnlg1 single mutants, they did not reveal any significant differences in their bouton counts compared to wit single mutants. Moreover, genetic interactions between Mad;dnrx and Mad;dnlg1 together with decreased levels of pMad in dnrx and dnlg1 mutants provided further evidence that dnrx and dnlg1 regulate components of the BMP pathway. Interestingly, reduced levels of pMad were observed in dnrx and dnlg1 mutants, in contrast to a recent study that reported higher level of synaptic pMad in dnrx and dnlg1 mutants. The differences in pMad levels encountered in these two studies could be attributed to differences in rearing conditions, nature of the food and genetic backgrounds, as these factors have been invoked to affect synaptic pMad levels. These findings strongly support that dnrx, dnlg1 and wit function cooperatively to coordinate synaptic growth and signaling at the NMJ (Banerjee, 2016).

Loss of either dnrx or dnlg1 does not completely abolish the apposition of pre- and post-synaptic membrane at the NMJ synapses but detachments that occur at multiple sites along the synaptic zone. These observations point to either unique clustering of the Dnlg1/Dnrx molecular complexes or preservation of trans-synaptic adhesion by other adhesion molecules at the NMJ synapses. Indeed, recent studies show nanoscale organization of synaptic adhesion molecules Neurexin 1Β, NLG1 and LRRTM2 to form trans-synaptic adhesive structures (Chamma, 2016). In addition to dnrx and dnlg1 mutants, synaptic ultrastructural analysis showed similarity in presynaptic membrane detachments with a characteristic ruffling morphology in wit mutants as well suggesting that these proteins are required for maintaining trans-synaptic adhesion. Interestingly, double mutants from any combinations of these three genes such as dnlg1,dnrx or wit,dnrx and wit,dnlg1 did not show any severity in the detachment/ruffling of the presynaptic membrane suggesting that there might be a phenotypic threshold that cannot be surpassed as part of an intrinsic synaptic machinery to preserve its very structure and function. It would be interesting to test this possibility if more than two genes are lost simultaneously as in a triple mutant combination. Alternatively, there could be presence of other distinct adhesive complexes that remain intact and function outside the realm of Dnrx, dnlg1 and wit proteins thus preventing a complete disintegration of the synaptic apparatus (Banerjee, 2016).

The same holds true for most of the ultrastructural pre- and postsynaptic differentiation defects observed in the single and double mutants of dnrx, dnlg1 and wit. Barring dnrx,dnlg1 double mutants in which the SSR width showed a significant reduction compared to the individual single mutants, all other phenotypes documented from the ultrastructural analysis showed no difference in severity between single and double mutants. Another common theme that emerged from the ultrastructural analysis was that loss of either pre- or postsynaptic proteins or any combinations thereof, showed a mixture of defects that spanned both sides of the synaptic terminal. For example, presynaptic phenotypes such as increased number of active zones or abnormally long active zones as well as postsynaptic phenotypes such as reduction in width and density of the SSR were observed when presynaptic proteins such as dnrx and wit and postsynaptic dnlg1 were lost individually or in combination. These studies suggest that pre- and postsynaptic differentiation is tightly regulated and not mutually exclusive where loss of presynaptic proteins would result only in presynaptic deficits and vice-versa (Banerjee, 2016).

The postsynaptic SSR phenotypes seen in the single and double mutants of dnrx, dnlg1 and wit might be due to their interaction/association with postsynaptic or perisynaptic protein complexes such as Dlg and Fasciclin II. Alternatively, the postsynaptic differentiation or maturation deficits in these mutants could also be due to a failure of postsynaptic GluRs to be localized properly or their levels maintained sufficiently. It has been shown previously that lack of GluR complexes interferes with the formation of SSR. Deficits in postsynaptic density assembly have been previously reported for dnlg1 mutants, including a misalignment of the postsynaptic GluR fields with the presynaptic transmitter release sites. GluR distribution also showed profound abnormalities in dnrx mutants. These observations suggest that trans-synaptic adhesion and synapse organization and growth is highly coordinated during development, and that multiple molecular complexes may engage in ensuring proper synaptic development. Some of these questions need to be addressed in future investigations (Banerjee, 2016).

In Drosophila, the postsynaptic muscle-derived BMP ligand, Glass bottom boat (Gbb), binds to type II receptor Wit, and type I receptors Tkv, and Saxophone (Sax) at the NMJ. Receptor activation by Gbb leads to the recruitment and phosphorylation of Mad at the NMJ terminals followed by nuclear translocation of pMad with the co-Smad, Medea, and transcriptional initiation of other downstream targets. It is interesting to note that previously published studies revealed that a postsynaptic signaling event occurs during larval development mediated by Type I receptor Tkv and Mad. Based on findings from this study, it is speculated that Dnrx and Dnlg1-mediated trans-synaptic adhesive complex allows recruitment and stabilization of wit and associated components to assemble a larger BMP signaling complex to ensure proper downstream signaling. Loss of dnrx and/or dnlg1 results in loss of adhesion and a decrease in the levels of Wit/Tkv receptors as well as decreased phosphorylation of Mad. Thus a combination of trans-synaptic adhesion and signaling mediated by Dnrx, Dnlg1 and components of the BMP pathway orchestrate the assembly of the NMJ and coordinate proper synaptic growth and architecture (Banerjee, 2016).

The data presented in this study address fundamental questions of how the interplay of pre- and postsynaptic proteins contributes towards the trans-synaptic adhesion, synapse differentiation and growth during organismal development. dnrx and dnlg1 establish trans-synaptic adhesion and functionally associate with the presynaptic signaling receptor wit to engage as a molecular machinery to coordinate synaptic growth, cytoarchitecture and signaling. dnrx and dnlg1 also function in regulating BMP receptor levels (Wit and Tkv) as well as the downstream effector, Mad, at the NMJ. It is thus conceivable that at the molecular level setting up of a Dnrx-Dnlg1 mediated trans-synaptic adhesion is a critical component for molecules such as Wit and Tkv to perform signaling function. It would be of immense interest to investigate how mammalian Neurexins and Neuroligins are engaged with signaling pathways that not only are involved in synapse formation but also their functional modulation, as the respective genetic loci show strong associations with cognitive and neurodevelopmental disorders (Banerjee, 2016).

A neuroprotective role for microRNA miR-1000 mediated by limiting glutamate excitotoxicity

Evidence has begun to emerge for microRNAs as regulators of synaptic signaling, specifically acting to control postsynaptic responsiveness during synaptic transmission. This report provides evidence that Drosophila melanogaster miR-1000 acts presynaptically to regulate glutamate release at the synapse by controlling expression of the vesicular glutamate transporter (VGlut). Genetic deletion of miR-1000 led to elevated apoptosis in the brain as a result of glutamatergic excitotoxicity. The seed-similar miR-137 regulates VGluT2 expression in mouse neurons. These conserved miRNAs share a neuroprotective function in the brains of flies and mice. Drosophila miR-1000 showed activity-dependent expression, which might serve as a mechanism to allow neuronal activity to fine-tune the strength of excitatory synaptic transmission (Verma, 2015).

miRNAs have emerged in recent years as important regulators of homeostatic mechanisms. Changes in miRNA expression and activity have been linked to neurodegenerative disorders. A growing body of evidence suggests that miRNAs are neuroprotective in the aging brain, as well as in the control of synaptic function and plasticity. Mouse miR-134 acts postsynaptically to regulate synapse strength, and miR-181 and miR-223 regulate glutamate receptors, thereby affecting postsynaptic responsiveness to glutamate. miR-1, a muscle-specific miRNA, acts in a retrograde fashion at the neuromuscular junction to regulate the kinetics of synaptic vesicle exocytosis. However, there are few examples of miRNAs acting directly in the presynaptic terminal to control synaptic strength. miR-485, which is found presynaptically, has been shown to control the expression of synaptic vesicle protein SV2A, thereby affecting synapse density and GluR2 receptor levels (Verma, 2015).

This paper reports that Drosophila miR-1000 regulates neurotransmitter release from presynaptic terminals. miR-1000 regulates expression of the VGlut, which loads glutamate into synaptic vesicles. Genetic ablation of miR-1000 leads to glutamate excitotoxicity, resulting in early-onset neuronal death. Presynaptic regulation of miR-1000 is activity dependent and may serve as a mechanism for tuning synaptic transmission. Evidence is presented that this regulatory relationship is conserved in the mammalian CNS, with a seed-similar miRNA, miR-137, conferring neuroprotection through regulation of VGluT2. The consequences of misregulation of glutamatergic signaling can be severe: excitotoxicity due to excessive glutamate release has been implicated in ischemia and traumatic brain injury, as well as in neurodegenerative conditions such as Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis (Verma, 2015).

Although postsynaptic regulation of glutamate receptor activity has been well studied, much less is known about presynaptic regulation of glutamatergic signaling. These findings suggest that miR-1000 acts presynaptically to regulate VGlut expression and thereby control synaptic glutamate release. It is tempting to speculate that this could provide a mechanism for tuning synaptic output and locally modulating synaptic strength. Such a mechanism would be most useful if the miRNA itself could be regulated in an activity-dependent manner. Evidence is provided that miR-1000 expression is regulated by light in vivo, presumably reflecting photoreceptor activity in the eye. This in turn leads to light-regulated regulation of VGlut reporter levels. These findings lend support to the notion of activity-dependent regulation of miR-1000 activity. An in depth exploration of these issues awaits the development of methods to monitor changes in presynaptic miRNA levels in real time (Verma, 2015).

Failure of miR-1000-mediated regulation of VGlut led to excess glutamate release and resulted in excitotoxicity. Consistent with these findings, Gal4-directed overexpression of VGlut has been reported to cause neurodegeneration. Notably, elevated levels of vertebrate VGluTs have been associated with excitotoxicity in animal models of epilepsy and traumatic brain injury. The GAERS rat epilepsy model shows elevated levels of VGluT2 but not of VGluT1. Similarly, in a model of stroke, ischemic injury was found to result in elevated expression of VGluT1 but not of VGluT2. VGluT1 levels are regulated by methamphetamine treatment, likely contributing to excitotoxic consequences of methamphetamine abuse. VGluT1 levels have also been reported to increase in rat brains following antidepressant treatment (Verma, 2015).

In the mouse, miR-223 acts on postsynaptic glutamate receptors and has a neuroprotective role in vivo. These findings provide evidence that miR-1000 has a neuroprotective role mediated through regulation of presynaptic glutamate release and that this regulatory mechanism is conserved for miR-137 and VGluT2 in the mouse. Together, these studies show that miRNA-mediated regulation of glutamatergic activity acts pre- and post-synaptically to modulate synaptic transmission and to protect against excitotoxicity. In this context, it is noteworthy that miR-137 is reported to be enriched at synapses. miR-137 levels were found to be low in a subset of Alzheimer's patients with elevated serine palmitoyltransferase 1 expression leading to increased ceramide production. Single nucleotide polymorphisms affecting miR-137 target sites could lead to low-level constitutive overexpression of its targets, even when the SNP is present in a single copy. A single nucleotide polymorphism affecting miR-137 has also been identified as a risk factor for schizophrenia. It will be of interest to learn whether misregulation of VGluT2 expression contributes to these neurological conditions. The current findings raise the possibility that miRNA mediated regulation makes VGluT and other miRNA targets possible risk factors in neurodegenerative disease (Verma, 2015).

Loss of skywalker reveals synaptic endosomes as sorting stations for synaptic vesicle proteins

Exchange of proteins at sorting endosomes is not only critical to numerous signaling pathways but also to receptor-mediated signaling and to pathogen entry into cells; however, how this process is regulated in synaptic vesicle cycling remains unexplored. This work presents evidence that loss of function of a single neuronally expressed GTPase activating protein (GAP), Skywalker (Sky) facilitates endosomal trafficking of synaptic vesicles at Drosophila neuromuscular junction boutons, chiefly by controlling Rab35 GTPase activity. Analyses of genetic interactions with the ESCRT machinery as well as chimeric ubiquitinated synaptic vesicle proteins indicate that endosomal trafficking facilitates the replacement of dysfunctional synaptic vesicle components. Consequently, sky mutants harbor a larger readily releasable pool of synaptic vesicles and show a dramatic increase in basal neurotransmitter release. Thus, the trafficking of vesicles via endosomes uncovered using sky mutants provides an elegant mechanism by which neurons may regulate synaptic vesicle rejuvenation and neurotransmitter release (Uytterhoeven, 2011).

Synaptic vesicles recycle locally at the synapse, and this study now provides genetic evidence that the synapse holds the capacity to regulate the sorting of synaptic vesicle proteins at 2xFYVE and Rab5 positive endosomes. 2xFYVE-GFP positive endosomes involved in signaling pathways exist at nerve endings. Likewise, membrane invaginations and endosomal-like cisternae form as a result of bulk membrane uptake during intense nerve stimulation, and in various endocytic mutants. However, the current data indicate that endosomes that accumulate in sky mutants are fundamentally different from these 'endocytic cisternae.' First, endocytic cisternae are not enriched for the endosomal markers 2xFYVE and Rab5. Second, endosomal structures in sky mutants do not appear to directly form at the plasma membrane. Third, endocytic cisternae may directly fuse with the membrane, however, based on mEJC and TEM analyses, sky induced endosomes appear to function as an intermediate station. Note that FM 1-43 dye can enter and leave endosomes in sky mutants, suggesting small synaptic vesicles can form at these structures. Together with time-course experiments in sky mutants, endosomes specifically accumulate upon stimulation and dissipate during rest. Therefore, the data suggest that endosomes in sky mutants constitute an intermediate step in the synaptic vesicle cycle. Such a compartment may also be operational in wild-type synapses because a 2xFYVE-GFP endosomal compartment can be largely depleted from synaptic vesicles upon endocytic blockade and because synaptic vesicles harbor proteins that are also commonly found on endosomes (Uytterhoeven, 2011).

Rab GTPases are involved in numerous intracellular trafficking events. In particular in synaptic vesicle traffic, Rab3, and its close isoform Rab27 have been implicated in vesicle tethering and/or priming, while Rab5 has been also implicated in endocytosis. However, an involvement of Rab proteins in other aspects of the synaptic vesicle cycle, including recycling, remains enigmatic. This systematic analysis of CA Rabs now implicates several Rabs in synaptic recycling. First, consistent with previous results, the data suggest that Rab5 mediates transport to synaptic endosomes. Second, Rab7^CA and Rab11^CA appear to inhibit new vesicle formation, likely by controlling post endosome trafficking. Finally, this study found that Rab23^CA and Rab35^CA mediate transport to or retention of synaptic vesicles at sub-boutonic structures. Neither of these Rabs had yet been implicated in synaptic vesicle traffic and hence, these in vivo studies identified different Rab proteins involved in aspects of the synaptic vesicle cycle (Uytterhoeven, 2011).

The expression of Rab5^CA and Rab35^CA results in increased trafficking of vesicles to -or retention of vesicles at- sub-boutonic structures, and unlike expression of Rab23^CA, Rab5^CA and Rab35^CA also result in a facilitation of neurotransmitter release similar to sky mutants. Rab35 has been found to localize to the plasma membrane (Sato, 2008) and this study shows enrichment at NMJ boutons close to the membrane as well. Rab35 has been implicated in endosomal, clathrin-dependent traffic, phagocytic membrane uptake in non-neuronal cells and actin filament assembly during Drosophila bristle development (Allaire, 2010, Sato, 2008, Shim, 2010, Zhang, 2009). Given these roles, Rab35 is ideally posed to also play a role in the endosomal traffic of synaptic vesicles, and the current in vitro and in vivo genetic interaction studies indicate Sky to be a Rab35 GAP in synaptic vesicle trafficking. Although Rab5 mediates endosomal traffic in different cell types, the data indicate that Sky is not a GAP for Rab5 in vivo at the synapse. While this study does not exclude Sky activating the GTPase activity of alternative Rabs in different contexts, the results suggest a Sky-Rab35 partnership that restricts endosomal trafficking of synaptic vesicles (Uytterhoeven, 2011).

In most cell types the ESCRT complex mediates sorting of ubiquitinated proteins into multivesicular bodies; however, such a function has not been characterized for synaptic vesicle components. This study provide evidence that the ESCRT complex mediates synaptic vesicle protein sorting in sky mutants where synaptic vesicles cycle excessively via endosomes. This study found genetic interactions with ESCRT genes. In addition, more efficient clearance of an artificially ubiquitinated synaptic vesicle protein, Ub-nSybHA, was also found in sky mutants, and this effect is ESCRT dependent. Combined, the data indicate endosomal sorting to control the quality of proteins in the synaptic vesicle cycle in sky mutants (Uytterhoeven, 2011).

Increased transmitter release as a result of endosomal sorting e.g. in sky mutants, may appear beneficial in some instances, however in inhibitory neurons for example, such an effect could result in reduced neuronal signaling while in excitatory neurons this feature could over activate postsynaptic cells. Clearly, fine-tuned neurotransmission in neuronal populations may ensure optimal information flow, and misregulation of Sky activity in neuronal populations may cause systemic defects including larval paralysis and death. Further underscoring this notion, in humans, mutations in the Sky orthologue TBC1D24 are causative of focal epilepsy (Uytterhoeven, 2011).

The sorting mechanism uncovered by loss of Sky function or upon expression of Rab35^CA may address the use-dependent decline in protein- or lipid-function at synapses, and the continuous need to rejuvenate the synaptic vesicle protein pool. In addition, it may also constitute a mechanism to remove inappropriately endocytosed membrane components from the vesicle pool. The trafficking pathway inhibited by Sky may also yield the ability of synapses to adapt the functional properties of their synaptic vesicles, thus controlling the nature or abundance of proteins involved in vesicle fusion and neuronal signaling (Uytterhoeven, 2011).

Reduced synaptic vesicle protein degradation at lysosomes curbs TBC1D24/sky-induced neurodegeneration

Synaptic demise and accumulation of dysfunctional proteins are thought of as common features in neurodegeneration. However, the mechanisms by which synaptic proteins turn over remain elusive. This paper studied Drosophila melanogaster lacking active TBC1D24/Skywalker (Sky), a protein that in humans causes severe neurodegeneration, epilepsy, and DOOR (deafness, onychdystrophy, osteodystrophy, and mental retardation) syndrome; endosome-to-lysosome trafficking was identified as a mechanism for degradation of synaptic vesicle-associated proteins. In fly sky mutants, synaptic vesicles traveled excessively to endosomes. Using chimeric fluorescent timers, synaptic vesicle-associated proteins were shown to be younger on average, suggesting that older proteins are more efficiently degraded. Using a genetic screen, it was found that reducing endosomal-to-lysosomal trafficking, controlled by the homotypic fusion and vacuole protein sorting (HOPS) complex, rescued the neurotransmission and neurodegeneration defects in sky mutants. Consistently, synaptic vesicle proteins were older in HOPS complex mutants, and these mutants also showed reduced neurotransmission. These findings define a mechanism in which synaptic transmission is facilitated by efficient protein turnover at lysosomes and identify a potential strategy to suppress defects arising from TBC1D24 mutations in humans (Fernandes, 2014).

Live observation of two parallel membrane degradation pathways at axon terminals

Neurons are highly polarized cells that require continuous turnover of membrane proteins at axon terminals to develop, function, and survive. Yet, it is still unclear whether membrane protein degradation requires transport back to the cell body or whether degradation also occurs locally at the axon terminal, where live observation of sorting and degradation has remained a challenge. This study reports direct observation of two cargo-specific membrane protein degradation mechanisms at axon terminals based on a live-imaging approach in intact Drosophila brains. Different acidification-sensing cargo probes are sorted into distinct classes of degradative 'hub' compartments for synaptic vesicle proteins and plasma membrane proteins at axon terminals. Sorting and degradation of the two cargoes in the separate hubs are molecularly distinct. Local sorting of synaptic vesicle proteins for degradation at the axon terminal is, surprisingly, Rab7 independent, whereas sorting of plasma membrane proteins is Rab7 dependent. The cathepsin-like protease CP1 is specific to synaptic vesicle hubs, and its delivery requires the vesicle SNARE neuronal synaptobrevin. Cargo separation only occurs at the axon terminal, whereas degradative compartments at the cell body are mixed. These data show that at least two local, molecularly distinct pathways sort membrane cargo for degradation specifically at the axon terminal, whereas degradation can occur both at the terminal and en route to the cell body (Jin, 2018).

Neurons must regulate the turnover of membrane proteins in axons, dendrites, and the cell body to ensure normal development and function. Defects in membrane protein degradation are hallmarks of neurodegenerative diseases. Recent progress has identified several mechanisms that are required at axon terminals to prevent dysfunction and degeneration, including the local generation of autophagosomes and endolysosomes. However, it is unclear whether these degradative organelles are principally transported back to the cell body for degradation or whether degradation can occur locally. In addition, the cargo specificity of membrane degradation mechanisms at the axon terminals has remained largely unknown, i.e., it is unclear which membrane proteins are degraded by what mechanisms (Jin, 2018).

Several mechanisms have been directly linked to synapse function or degeneration and have raised questions about cargo specificity and the ultimate locale for degradation. These include (1) local generation of autophagosomes at axon terminals, (2) maturation of autophagosomes and endosomes that depends on the ubiquitous small guanosine triphosphatase (GTPase) Rab7, (3) endosomal sorting that depends on the GTPase Rab35 and RabGAP Skywalker, and (4) endosomal sorting that depends on the neuron-specific synaptic vesicle (SV) proteins neuronal synaptobrevin (n-Syb) and V100. These mechanisms may overlap, and defects in any of them cause neurodegeneration in a variety of neurons. In the case of (macro-) autophagy, the formation of autophagosomes occurs at axon terminals, whereas degradation is thought to occur during and after retrograde transport back to the cell body. As with both the canonical and neuron-specific endolysosomal mechanisms, it remains largely unknown what cargoes are sorted into autophagosomes at axon terminals. The Rab35/Skywalker-dependent endosomal sorting mechanism was recently reported to selectively sort different SV proteins in an activity-dependent manner. Lysosomes have also been shown to localize to dendritic spines in an activity-dependent manner. In both cases, it remains unknown whether degradation occurs locally at synapses and what cargo proteins are affected. Finally, previous work has described a 'neuronal sort-and-degrade' (NSD) mechanism based on the function of the two neuron-specific synaptic genes n-syb (Haberman, 2012) and v100 (Wang, 2014). Similar to the other mechanisms, neither cargo specificity nor the locale of degradation for NSD is known. For all mechanisms, it has remained a challenge to directly observe their local roles in the context of normal development and function in an intact brain (Jin, 2018).

This study reports the direct observation of cargo-specific endolysosomal sorting and degradation at axon terminals in vivo using live imaging in intact Drosophila brains. Axon terminal 'hub' compartments are defined based on their local dynamics, maturation, degradation, continuous mixing through fusion and fission, and budding of retrograde transport vesicles. In addition, two distinct pools of hubs were identified (see Model: Two Parallel Membrane Degradation Mechanisms at Axon Terminals) that function locally in two separate endolysosomal pathways based on different cargo specificities, different molecular sorting, and different maturation mechanisms (Jin, 2018).

Genetically encoded fusions with pHluorin, a pH-sensitive GFP, and mCherry, a particularly pH-resistant red fluorescent protein (RFP), report cargo incorporation into degradative membrane compartments. It is surprising that the only clearly discernible compartments at axon terminals are red live, large, acidified, and spatiotemporally relatively stable endolysosomal compartments. These compartments were named hubs because of their continuous fission, fusion, and budding of smaller retrograde trafficking vesicles. Appearance or disappearance of an entire hub is never reported, but only the formation of hubs by fusion of several smaller vesicles and splitting into multiple smaller compartments that undergo renewed cycles of fusion. These dynamics are reflected in hub composition and maturation: at any point in time, some hubs are marked by early endosomal markers and contain undegraded probes, whereas others are marked by lysosomal markers and contain partially degraded probes (Jin, 2018).

How does this 'hub flux' contribute to the sorting and degradation of dysfunctional membrane proteins? A key insight comes from the characterization of retrograde trafficking vesicles: the axonal vesicles exhibit the same composition and a mix of early and late markers and degraded and undegraded probes. These observations are most straightforwardly explained with random mixing of hubs and random budding of axonal vesicles, irrespective of their maturation stage. In this model, sorting into hubs carries a probabilistic chance of degradation that increases with time (either in hubs or in retrograde trafficking vesicles). Sorting into hubs ensures degradation if membrane proteins cannot be recycled back into the axon terminal; alternatively, degradation and recycling may both be probabilistic. The latter would imply that not only dysfunctional proteins are sorted into hubs. Both mechanisms could ensure a pool of functional synaptic proteins that increases with the amount of endolysosomal flux, as previously observed for the Skywalker/Rab35 mechanism (Jin, 2018).

How SV membrane proteins are specifically sorted into SV hubs is unclear. Because sorting of Syt1-DF into SV hubs is Rab7 independent, SV hub maturation bypasses the requirement for Rab5-to-Rab7 conversion. Neither n-Syb (a vesicle SNARE and membrane fusion factor) nor V100 (part of a proton pump) are required for the continuous fusion and fission of SV hubs. However, the reduced axon terminal numbers of SV hubs are consistent with roles in the sorting of SVs to SV hubs. Different SV retrieval mechanisms, including ultrafast endocytosis, clathrin-mediated endocytosis, and bulk endocytosis, may account for different mechanisms and routes to local degradative hubs. Preassembled plasma membrane cargo complexes may play a role in promoting the different endocytic routes. Colocalization measurements revealed a distinction between two membrane degradation mechanisms with enrichments of SV proteins versus plasma membrane proteins between 3- and 8-fold, but not a 100% separation. Hence, if protein complexes facilitate sorting into a specific endocytic pathway or hub compartments, they may do so in a probabilistic fashion (Jin, 2018).

Continuous flux is a hallmark of endolysosomal compartments and results in low colocalization ratios with dynamically changing molecular markers and difficulties to unambiguously identify a specific compartment at any point in time. Live observation of dynamic hubs as sorting and degradation stations at axon terminals was only made possible by their integrity over time and may provide an inroad to the study of distinct, cargo-specific mechanisms that keep neurons and their synaptic terminals functional (Jin, 2018).

Constitutive turnover of SV hubs was observed prior to synaptogenesis and neuronal activity. In contrast, previous work on SV 'rejuvenation' focused on turnover that increases in response to neuronal activity. Colocalization of axon terminal hubs with the Rab35 GAP Skywalker (Sky) revealed equal overlap with both hub types. This could indicate an intersection of different endolysosomal pathways; alternatively, Sky may only temporarily localize to hubs depending on their maturation stage. The second possibility is favored, because the early endosomal Rab5 and the lysosomal marker Spin exhibit similar colocalization ratios and all known endolysosomal markers depend in some way on the maturation stage Colocalization results implicates Sky in both the canonical and SV pathway. Consistent with this, rab7 affects Sky-dependent rejuvenation and the sky mutant affects the turnover of an n-Syb imaging probe (Jin, 2018).

Autophagy similarly intersects with axon terminal hub compartments based on colocalization with Atg8, albeit this colocalization is significantly higher for the rab7-dependent general PM hubs than for the SV hubs. However, the hubs and axonal trafficking vesicles are distinct from autophagosomes based on their dynamics: Atg8-positive compartments are not part of the 'hub flux,' emerge de novo, and directly enter the axon without prior fission. It is possible that autophagosomes can engulf hub compartments and thus provide an alternative degradative exit to budding of retrograde trafficking vesicles. A Rab26-dependent mechanism was recently proposed for the sorting of SVs to pre-autophagosomal compartments prior to Atg8 recruitment, which may represent a similar hub compartment (Jin, 2018).

This study showed that cathepsin-L-like protease CP1 has specificity for the SV hubs at axon terminals. This finding is consistent with several cystein cathepsins that have been characterized for their tissue-specific expression. In mammalian systems, cathepsin L selectively degrades polyglutamine (polyQ)-containing proteins, but not other types of aggregation-prone proteins lacking polyQ. In HeLa and Huh-7 cells, cathepsin L was reported to degrade autophagosomal membrane markers, but not proteins in the lumen of autophagosomes. In contrast, the major histocompatibility complex (MHC) class-II-associated invariant chain is specifically degraded by cathepsin S, but not cathepsin L, in CD4+ T cells. The current characterization of cargo-specific membrane degradation machinery with a specific protease raises the question to what extent different membrane degradation mechanisms are characterized and may require specific proteases (Jin, 2018).

Activity induces Fmr1-sensitive synaptic capture of anterograde circulating neuropeptide vesicles

Synaptic neuropeptide and neurotrophin stores are maintained by constitutive bidirectional capture of dense-core vesicles (DCVs) as they circulate in and out of the nerve terminal. Activity increases DCV capture to rapidly replenish synaptic neuropeptide stores following release. However, it is not known whether this is due to enhanced bidirectional capture. Experiments at the Drosophila neuromuscular junction, where DCVs contain neuropeptides and a bone morphogenic protein, show that activity-dependent replenishment of synaptic neuropeptides following release is evident after inhibiting the retrograde transport with the dynactin disruptor mycalolide B or photobleaching DCVs entering a synaptic bouton by retrograde transport. In contrast, photobleaching anterograde transport vesicles entering a bouton inhibits neuropeptide replenishment after activity. Furthermore, tracking of individual DCVs moving through boutons shows that activity selectively increases capture of DCVs undergoing anterograde transport. Finally, upregulating fragile X mental retardation 1 protein (Fmr1, also called FMRP) acts independently of futsch/MAP-1B to abolish activity-dependent, but not constitutive, capture. Fmr1 also reduces presynaptic neuropeptide stores without affecting activity-independent delivery and evoked release. Therefore, presynaptic motoneuron neuropeptide storage is increased by a vesicle capture mechanism that is distinguished from constitutive bidirectional capture by activity dependence, anterograde selectivity, and Fmr1 sensitivity. These results show that activity recruits a separate mechanism than used at rest to stimulate additional synaptic capture of DCVs for future release of neuropeptides and neurotrophins (Cavolo, 2016).

Synapses are supplied by anterograde axonal transport from the soma, the site of synthesis of synaptic vesicle proteins and dense-core vesicles (DCVs) that contain neuropeptides and neurotrophins. Delivery to synapses was thought to be based on a one-way anterograde trip until it was discovered that DCVs are subject to sporadic capture while traveling bidirectionally through en passant boutons as part of long-distance vesicle circulation (Wong, 2012). Interestingly, constitutive DCV capture occurs both during fast anterograde and retrograde transport, which are mediated by different motors (i.e., the kinesin 3 family member unc-104/Kif1A and the dynein/dynactin complex, respectively). Balanced capture in both directions is advantageous because DCVs are distributed equally among en passant boutons (Wong, 2012). In principle, bidirectional capture could occur by parallel regulation of anterograde and retrograde motors or by modification of the microtubules that both anterograde and retrograde DCV motors travel on (Cavolo, 2016).

Before the discovery of bidirectional capture of circulating vesicles, activity was shown to replenish the presynaptic neuropeptide pool following release by inducing Ca2+-dependent capture of DCVs being transported through boutons. This result and subsequent experiments (Bulgari, 2014) established that capture, rather than delivery or DCV turnover, limits synaptic neuropeptide stores. Activity-dependent capture was first described with a GFP-tagged neuropeptide in the Drosophila neuromuscular junction (NMJ), but also occurs with neurotrypsin, wnt/wingless, and brain-derived neurotrophic factor. Mechanistically, activity-dependent capture was characterized in terms of the rebound in presynaptic GFP-tagged peptide content following release and correlated with decreased retrograde transport. However, it is now evident that the reduction in retrograde flux could be caused by enhanced bidirectional capture as DCVs travel back and forth through the terminal as part of vesicle circulation (Wong, 2012). Therefore, prior studies support the hypothesis that there is only one synaptic capture mechanism, which is bidirectional and facilitated by activity (Cavolo, 2016).

This study tested the above hypothesis by investigating the directionality of activity-dependent capture. Experiments were performed with multiple approaches, including inhibiting retrograde transport, particle tracking, and simultaneous photobleaching and imaging (SPAIM; Wong, 2012). Furthermore, the effect of fragile X retardation protein (Fmr1, also called FMRP) was examined because it is known to affect bouton size and neuropeptide release. Together, these studies establish that different mechanisms mediate synaptic capture at rest and in response to activity (Cavolo, 2016).

Until recently, it was thought that presynaptic neuropeptide stores were set by controlling synthesis and delivery by fast one-way axonal transport of DCVs. However, studies of the Drosophila NMJ have shown that there is an excess of DCVs delivered to type-I boutons by long-distance vesicle circulation. Therefore, because DCV delivery is not limiting, the presynaptic neuropeptide pool is determined by capture, which was found to be bidirectional (Wong, 2012). However, in addition to constitutive capture, activity induces Ca2+-dependent capture. This is advantageous because tapping into the circulating vesicle pool removes delays associated with synthesis and transport, which can take days in humans, to rapidly replace released peptides. Surprisingly, experiments presented in this study demonstrate that activity-dependent capture is unidirectional and selectively sensitive to a genetic perturbation (i.e., Fmr1 overexpression). Therefore, activity does not simply enhance constitutive bidirectional capture that operates at rest, but instead stimulates an independent synaptic capture mechanism (Cavolo, 2016).

Previously, it was not possible to genetically block activity-dependent capture to determine its contribution to steady-state presynaptic stores. However, this study documented inhibition of activity-dependent capture by Fmr1 overexpression. As this was accompanied by a dramatic decrease in presynaptic DCV number, it is concluded that activity-dependent capture makes a large contribution to steady-state presynaptic peptide stores and hence the capacity for future release. At the Drosophila NMJ, DCVs contain a bone morphogenic protein and neuropeptides. Thus, it is possible that activity-dependent capture affects development and acute synaptic function (Cavolo, 2016).

Capture efficiency measurements revealed that the previously detected decrease in retrograde traffic following activity was an indirect effect of vesicle circulation; activity-induced capture of only anterograde DCVs at each en passant bouton simply leaves fewer DCVs for the retrograde trip back into the axon without changing retrograde capture. Of interest, anterograde selectivity for activity-induced capture rules out mechanisms that would perturb transport in both directions (e.g., microtubule breaks). DCV anterograde transport is mediated by the unc-104/Kif1A motor, which also transports SSV proteins and is required for formation of boutons. Therefore, activity-dependent capture may regulate unc-104/Kif1A to affect synaptic release of both small-molecule transmitters and peptides. However, alternative targets could be involved, including proteins that mediate DCV interaction with this anterograde motor or alter the DCV itself (e.g., its phosphoinositides, which may bind to the unc-104/Kif1A pleckstrin homology domain) (Cavolo, 2016).

Phosphorylation of Complexin by PKA regulates activity-dependent spontaneous neurotransmitter release and structural synaptic plasticity

Synaptic plasticity is a fundamental feature of the nervous system that allows adaptation to changing behavioral environments. Most studies of synaptic plasticity have examined the regulated trafficking of postsynaptic glutamate receptors that generates alterations in synaptic transmission. Whether and how changes in the presynaptic release machinery contribute to neuronal plasticity is less clear. The SNARE complex mediates neurotransmitter release in response to presynaptic Ca(2+) entry. This study shows that the SNARE fusion clamp Complexin undergoes activity-dependent phosphorylation that alters the basic properties of neurotransmission in Drosophila. Retrograde signaling following stimulation activates PKA-dependent phosphorylation of the Complexin C terminus that selectively and transiently enhances spontaneous release. Enhanced spontaneous release is required for activity-dependent synaptic growth. These data indicate that SNARE-dependent fusion mechanisms can be regulated in an activity-dependent manner and highlight the key role of spontaneous neurotransmitter release as a mediator of functional and structural plasticity (Cho, 2015).

These findings indicate that the SNARE-binding protein Cpx is a key PKA target that regulates spontaneous fusion rates and presynaptic plasticity at Drosophila NMJs. Cpx's function can be modified to regulate activity-dependent functional and structural plasticity. In vivo experiments using Cpx phosphomimetic rescues demonstrate that Cpx phosphorylation at residue S126 selectively alters its ability to act as a synaptic vesicle fusion clamp. In addition, S126 is critical for the expression of HFMR, an activity-dependent form of acute functional plasticity that modulates mini frequency at Drosophila synapses. These data indicate a Syt 4-dependent retrograde signaling pathway converges on Cpx to regulate its synaptic function. Additionally, it was found that elevated spontaneous fusion rates correlate with enhanced synaptic growth. This pathway requires Syt 4 retrograde signaling to enhance spontaneous release and to trigger synaptic growth. Moreover, the Cpx S126 PKA phosphorylation site is required for activity-dependent synaptic growth, suggesting acute regulation of minis via Cpx phosphorylation is likely to contribute to structural synaptic plasticity. Together, these data identify a novel mechanism of acute synaptic plasticity that impinges directly on the presynaptic release machinery to regulate spontaneous release rates and synaptic maturation (Cho, 2015).

How does acute phosphorylation of S126 alter Cpx's function? The Cpx C-terminus associates with lipid membranes through a prenylation domain (CAAX motif) and/or the presence of an amphipathic helix. The Drosophila Cpx isoform used in this study (Cpx 7B) lacks a CAAX-motif, but contains a C-terminal amphipathic helix flanked by the S126 phosphorylation site. S126 phosphorylation does not alter synaptic targeting of Cpx or its ability to associate with SNARE complexes in vitro. As such, phosphorylation may instead alter interactions of the amphipathic helix region with lipid membranes and/or alter Cpx interactions with other proteins that modulate synaptic release. Given the well-established role of the Cpx C-terminus in regulating membrane binding and synaptic vesicle tethering of Cpx, phosphorylation at this site would be predicted to alter the subcellular localization of the protein and its accessibility to the SNARE complex. However, no large differences between WT Cpx and CpxS126D were observed in liposome binding. This assay is unlikely to reveal subtle changes in lipid interactions by Cpx, as this study found that C-terminal deletions (CTD) maintained its ability to bind membranes. The ability of the CTD versions of Drosophila Cpx to associate with liposomes is unlike that observed with C. elegans Cpx, and indicate domains outside of Drosophila Cpx's C-terminus contribute to lipid membrane association as well, potentially masking effects from S126 phosphorylation that might occur in vivo. Alternatively, phosphorylation of the Cpx C-terminus could alter its association with other SNARE complex modulators such as Syt 1 (Cho, 2015).

The data indicate that enhanced minis regulate synaptic growth through several previously identified NMJ maturation pathways. The Wit signaling pathway is required for synaptic growth in the background of enhanced minis. The wit gene encodes a presynaptic type II BMP receptor that receives retrograde, transsynaptic BMP signals from postsynaptic muscles. Consistent with these data, other studies have demonstrated that downstream signaling components of the BMP pathway are necessary and sufficient for mini-dependent synaptic growth at the Drosophila NMJ. Additionally, it was found that the postsynaptic Ca++ sensor, Syt 4, is required for enhancing spontaneous release and increasing synaptic growth. The data does not currently distinguish the interdependence of the BMP and Syt 4 retrograde signaling pathways, and other retrograde signaling pathways might contribute to mini-dependent synaptic growth as well. Recently, several retrograde pathways have been identified that regulate functional homeostatic plasticity at the Drosophila NMJ. Future work will be required to fully define the retrograde signaling pathways necessary to mediate mini-dependent synaptic growth (Cho, 2015).

How might elevated spontaneous release through Cpx phosphorylation regulate synaptic growth? It is hypothesized that the switch in synaptic vesicle release mode to a constitutive fusion pathway that occurs over several minutes following stimulation serves as a synaptic tagging mechanism. By continuing to activate postsynaptic glutamate receptors in the absence of incoming action potentials, the elevation in mini frequency would serve to enhance postsynaptic calcium levels by prolonging glutamate receptor stimulation. This would prolong retrograde signaling that initiates downstream cascades to directly alter cytoskeletal architecture required for synaptic bouton budding. Given that elevated rates of spontaneous fusion still occur in cpx and syx3-69 in the absence of Syt 4 and BMP signaling, yet synaptic overgrowth is suppressed in these conditions, it is unlikely that spontaneous fusion itself directly drives synaptic growth. Rather, the transient enhancement in spontaneous release may serve to prolong postsynaptic calcium signals that engage distinct effectors for structural remodeling that fail to be activated in the absence of elevated spontaneous release. Results from mammalian studies indicate spontaneous release can uniquely regulate postsynaptic protein translation and activate distinct populations of NMDA receptors compared to evoked release, so it is possible that spontaneous fusion may engage unique postsynaptic effectors at Drosophila NMJs as well (Cho, 2015).

In the last few decades, intense research efforts have elucidated several molecular mechanisms of classic Hebbian forms of synaptic plasticity that include long-term potentiation (LTP) and long-term depression (LTD), alterations in synaptic function that lasts last minutes to hours. The most widely studied expression mechanism for these forms of synaptic plasticity involve modulation of postsynaptic AMPA-type glutamate receptor (AMPAR) function and membrane trafficking. In contrast, molecular mechanisms of short-term synaptic plasticity remain poorly understood. Several forms of short-term plasticity have been described, such as post-tetanic potentiation (PTP), which involves stimulation-dependent increases in synaptic strength, including changes in mini frequency. Short-term plasticity expression is likely to impinge on the alterations to the presynaptic release machinery downstream of activated effector molecules. For example, Munc 18, a presynaptic protein involved in the priming step of vesicle exocytosis via its ability to associate with members of the SNARE machinery, is dynamically regulated by Ca++-dependent protein kinase C (PKC), and its regulation is required to express PTP at the Calyx of Held. This study demonstrates that the presynaptic vesicle fusion machinery can also be directly modified to alter spontaneous neurotransmission via activity-dependent modification of Cpx function by PKA. Protein kinase CK2 and PKC phosphorylation sites within the C-terminus of mammalian and C. elegans Cpx have been identified. Therefore, activity-dependent regulation of Cpx function via C-terminal phosphorylation may be an evolutionarily conserved mechanism to regulate synaptic plasticity. Moreover, Cpx may lie downstream of multiple effector pathways to modulate various forms of short-term plasticity, including PTP, in a synapse-specific manner. Interestingly, Cpx is expressed both pre- and postsynaptically in mammalian hippocampal neurons and is required to express LTP via regulation of AMPAR delivery to the synapse, suggesting Cpx-mediated synaptic plasticity expression mechanisms may also occur postsynaptically (ACho, 2015 and references therein).

In summary, these results indicate minis serve as an independent and regulated neuronal signaling pathway that contributes to activity-dependent synaptic growth. Previous studies found Cpx’s function as a facilitator and clamp for synaptic vesicle fusion is genetically separable, demonstrating distinct molecular mechanisms regulate evoked and spontaneous release. Evoked and spontaneous release are also separable beyond Cpx regulation, as other studies have demonstrated that minis can utilize distinct components of the SNARE machinery, distinct vesicle pools, and distinct individual synaptic release sites . These findings suggest diverse regulatory mechanisms for spontaneous release that might be selectively modulated at specific synapses (Cho, 2015).

Fife organizes synaptic vesicles and calcium channels for high-probability neurotransmitter release

The strength of synaptic connections varies significantly and is a key determinant of communication within neural circuits. Mechanistic insight into presynaptic factors that establish and modulate neurotransmitter release properties is crucial to understanding synapse strength, circuit function, and neural plasticity. Drosophila Fife , a Piccolo-RIM homolog. has been shown to regulate neurotransmission and motor behavior through an unknown mechanism. This study demonstrates that Fife localizes and interacts with RIM (Rab3 interacting molecule) at the active zone cytomatrix to promote neurotransmitter release. Loss of Fife results in the severe disruption of active zone cytomatrix architecture and molecular organization. Through electron tomographic and electrophysiological studies, a decrease was found in the accumulation of release-ready synaptic vesicles and their release probability caused by impaired coupling to Ca2+ channels. Finally, Fife was found to be essential for the homeostatic modulation of neurotransmission. It is proposed that Fife organizes active zones to create synaptic vesicle release sites within nanometer distance of Ca2+ channel clusters for reliable and modifiable neurotransmitter release (Bruckner 2016).

The strength of synaptic transmission is a critical determinant of information processing in neural circuits. Evoked neurotransmission depends on localized Ca2+ influx triggering neurotransmitter release from synaptic vesicles at specialized domains of presynaptic terminals called active zones. At the active zone membrane, synaptic vesicles are docked and molecularly primed to respond to a rise in Ca2+ concentration by fusing with the membrane to release neurotransmitter. A conserved complex of active zone-associated proteins makes up the active zone cytomatrix. In Drosophila melanogaster, these proteins include the ELKS family protein Bruchpilot, Rab3-interacting molecule (RIM), RIM-binding protein, Unc13, and Fife. Active zone cytomatrix proteins contain many lipid- and protein-binding domains that mediate diverse interactions with key players in synaptic transmission, leading to the model that the active zone cytomatrix spatially organizes presynaptic terminals for the millisecond coupling of neurotransmitter release to action potentials (Bruckner, 2016).

The specific neurotransmitter release properties of an active zone are determined by two key parameters acting in concert: (1) the number of synaptic vesicles docked at the membrane and molecularly primed for Ca2+-triggered release, termed the readily releasable pool, and (2) the release probability of these vesicles. Vesicle release probability is established by multiple parameters, including Ca2+ channel levels, localization and function at active zones, the spatial coupling of Ca2+ channels and release-ready vesicles, and the intrinsic Ca2+ sensitivity of individual vesicles. The observation that the presynaptic parameters of synaptic strength vary significantly even between the synapses of an individual neuron indicates that neurotransmitter release properties are determined locally at active zones and raises the question of how this complex regulation is achieved. Genetic studies in Drosophila, Caenorhabditis elegans, and mice are revealing a key role for the active zone cytomatrix in determining the functional parameters underlying synaptic strength. A mechanistic understanding of how the active zone cytomatrix achieves local control of synaptic release properties will yield fundamental insights into neural circuit function (Bruckner, 2016).

Fife, a Piccolo-RIM-related protein is required for proper neurotransmitter release and motor behavior. This study demonstrates that Fife localizes to the active zone cytomatrix, where it interacts with RIM to promote neurotransmitter release. The active zone cytomatrix is diminished and molecularly disorganized at Fife mutant synapses, and Fife is critical for vesicle docking at the active zone membrane. Not only are the number of release-ready vesicles reduced in the absence of Fife, but their probability of release is also significantly impaired because of disrupted coupling to calcium channels. These results suggest that Fife promotes high-probability neurotransmitter release by organizing the active zone cytomatrix to create vesicle release sites in nanometer proximity to clustered Ca2+ channels. Finally, it was found that in addition to its role in determining baseline synaptic strength, Fife plays an essential role in presynaptic homeostatic plasticity. Together, these findings provide mechanistic insight into how synaptic strength is established and modified to tune communication in neural circuits (Bruckner, 2016).

This study demonstrates that Fife plays a key role in organizing presynaptic terminals to determine synaptic release properties. Fife functions with RIM at the active zone cytomatrix to promote neurotransmitter release. Functional and ultrastructural imaging studies demonstrate that Fife regulates the docking of release-ready synaptic vesicles and, through nanodomain coupling to Ca2+ channels, their high probability of release. It was further found that Fife is required for the homeostatic increase in neurotransmitter release that maintains circuit function when postsynaptic receptors are disrupted. These findings uncover Fife's role as a local determinant of synaptic strength and add to understanding of how precise communication in neural circuits is established and modulated (Bruckner, 2016).

The finding that Fife interacts with RIM provides insight into how Fife functions within the network of cytomatrix proteins. RIM is a central active zone protein that was recently shown to facilitate vesicle priming at mammalian synapses by relieving autoinhibition of the priming factor Munc13. Like Fife, Drosophila RIM promotes Ca2+ channel accumulation at active zones and exhibits EGTA-sensitive neurotransmitter release at the Drosophila NMJ. This suggests that Fife and RIM may promote high-probability neurotransmitter release by acting together to dock and prime synaptic vesicles in close proximity to Ca2+ channels clustered at the cytomatrix. The findings are consistent with previous work in pancreatic β cells, where it was found that Piccolo and RIM2α form a complex that promotes insulin secretion through an unknown mechanism. To date, functional studies at mammalian synapses have focused on investigating interactions between Piccolo and Bassoon, which bind through their common coiled-coil regions. Thus, it will be of interest to investigate the functional relationship between Piccolo and RIM in promoting neurotransmitter release in mouse models. Piccolo also binds CAST1 through coiled-coil domain interactions. Although the Piccolo coiled-coil region is not present in Fife, future studies to determine whether this interaction is preserved through distinct interacting domains will be important, as Fife and Drosophila CAST-related Bruchpilot carry out overlapping functions. Similarly, although neither Fife nor Piccolo contains the conserved SH3-binding domain that mediates the interaction between RIM and RIM-binding protein, the overlap between Fife and RIM-binding protein phenotypes raises the possibility of functional interactions that will also be important to investigate in future experiments (Bruckner, 2016).

Significant alterations to active zone cytomatrix size and structure were observed in Fife mutants, whereas none have been detected in RIM mutants, indicating that Fife carries out this function independently of RIM. Previous ultrastructural analysis in aldehyde-fixed samples revealed occasional free-floating electron-dense structures that resemble active zone cytomatrix material and cluster synaptic vesicles. These unanchored electron-dense structures have also been observed at low frequency in Drosophila RIM-binding protein mutants, which, like Fife mutants, exhibit smaller active zone cytomatrices, and at higher frequency in rodent ribbon synapses lacking Bassoon. These structures were not visible in HPF/FS-prepared electron microscopy samples, likely because protein components of the synapse are not cross-linked upon fixation. That these structures are not observed in control synapses but have been found in multiple active zone cytomatrix mutants argues that the extensive cross-linking of proteins in chemically fixed preparations may enable the visualization of biologically relevant complexes missed with cryofixation. This supports the idea that the two fixation methods may offer different advantages for ultrastructural studies of synapses. In any case, the active zone cytomatrix is significantly reduced in size at Fife active zones in both HPF/FS- and aldehyde-fixed electron micrographs (Bruckner, 2016).

Although diminished or absent cytomatrices have been observed in electron micrographs of RIM-binding protein and Bruchpilot mutants, this phenotype has not been observed in electron micrographs of other active zone cytomatrix mutants, suggesting it represents more than the loss of a single component protein. Rather, the reduced complexity visible in electron micrographs likely reflects a broader underlying molecular disorganization. Further support for this model comes from superresolution imaging, which reveals molecular disorganization at Fife active zones as indicated by the loss of the characteristic ring-shaped localization pattern of Bruchpilot's C terminus. Similar disorganization of Bruchpilot was observed at active zones lacking RIM-binding protein. Superresolution imaging was used to investigate the localization of active zone proteins Cacophony and RIM-binding protein and, although the levels of Cacophony are reduced at Fife active zones, no apparent differences were observed in the patterns of these proteins. Cacophony and RIM-binding protein both localize in smaller puncta than Bruchpilot, so future studies with higher resolution imaging modalities such as stimulated emission depletion microscopy may reveal more subtle abnormalities. Correlations between active zone molecular composition and release probability have been observed at diverse synapses. At the Drosophila NMJ, functional imaging with genetically encoded Ca2+ indicators has demonstrated that active zones display a wide range of release probabilities. Active zones with high release probability contain higher levels of Bruchpilot, which may in turn correlate with higher Ca2+ channel levels. At mouse hippocampal synapses, Bassoon and RIM levels directly correlate with neurotransmitter release probability. Consistently, synaptic probability of release is significantly decreased in Fife mutants (Bruckner, 2016)

Through a combination of morphological and functional studies, this study found that Fife acts to promote the active zone docking of synaptic vesicles and regulates their probability of release. Because the number of readily releasable vesicles appears to scale with active zone cytomatrix size and molecular composition at diverse synapses, a conserved function of the active zone cytomatrix may be to establish release sites for synaptic vesicles. Consistent with this view, the number of release-ready vesicles is also reduced in Drosophila RIM-binding protein-null mutants and isoform-specific bruchpilotΔ170 and bruchpilotΔ190 mutants, which share similar active zone structural abnormalities with Fife. By combining rapid preservation of intact Drosophila larvae by HPF/FS fixation, electron tomography, and extensive segmentation of active zone structures, this study obtained a detailed view of the 3D organization of active zones in near-native state that allowed further dissection of Fife's role in determining the size of the readily releasable vesicle pool. Membrane-docked vesicles are significantly decreased in Fife mutants, whereas more distant vesicles attached to the membrane by long tethers appear unaffected. Correlating physiological and morphological parameters of neurotransmission is an ongoing challenge in the field. It has been proposed that docking and priming are not separable events in the establishment of the readily releasable vesicle pool, but rather the morphological and physiological manifestations of a single process. Although approximately one third of docked vesicles in these preparations lack obvious short connections to the membrane, which are thought to represent priming factors, the possibility cannot be excluded that these filaments are present but obscured, perhaps because the vesicles are more tightly linked to the membrane. As this proportion is unchanged in Fife mutants, it is concluded that Fife acts to promote vesicle docking and may simultaneously facilitate molecular priming-possibly through its interactions with RIM (Bruckner, 2016).

The data indicate that neurotransmitter release at Fife synapses is highly sensitive to EGTA, a slow Ca2+ chelator that has been used to investigate the coupling of Ca2+ influx at voltage-gated Ca2+ channels and Ca2+ sensors on synaptic vesicles. At synapses with high release probability, including inhibitory synapses in the mammalian hippocampus and cerebellum, excitatory synapses of the mature Calyx of held, and the Drosophila NMJ, molecularly primed synaptic vesicles and Ca2+ channel clusters are thought to be positioned within ~100 nm of one another to ensure the tight coupling of Ca2+ influx and Ca2+ sensors that explains observed release properties. The EGTA sensitivity of release at Fife, but not wild-type, NMJs indicates that Fife likely regulates the probability that a docked vesicle is released by positionally coupling release-ready vesicles to Ca2+ channels clustered beneath the active zone cytomatrix. The trend toward fewer docked vesicles associated with the active zone cytomatrix in tomograms of Fife synapses provides morphological support for this model. Building on detailed tomographic studies of the Drosophila NMJ to visualize how Ca2+ channels and vesicles are spatially organized at active zones in different genetic backgrounds will be an important step in advancing understanding of the geometry of release probability and how it is established (Bruckner, 2016).

Finally, this study found that Fife is required for presynaptic homeostasis. In response to decreases in glutamate receptor levels or function, Drosophila motoneurons rapidly increase synaptic vesicle release to maintain postsynaptic excitation. This homeostatic increase in presynaptic neurotransmission is accompanied by an increase in the number of dense projections per active zone and Bruchpilot levels. Cytomatrix proteins RIM, RIM-binding protein, and now Fife have all been shown to function in presynaptic homeostasis, indicating a critical role for the active zone cytomatrix as a substrate for synaptic plasticity. These studies provide insight into the molecular mechanisms through which the active zone cytomatrix determines neurotransmitter release parameters to modulate how information flows in neural circuits (Bruckner, 2016).

An improved catalogue of putative synaptic genes defined exclusively by temporal transcription profiles through an ensemble machine learning approach

Assembly and function of neuronal synapses require the coordinated expression of a yet undetermined set of genes. Previously, an ensemble machine learning model was trained to assign a probability of having synaptic function to every protein-coding gene in Drosophila melanogaster. This approach resulted in the publication of a catalogue of 893 genes which was postulated to be very enriched in genes with a still undocumented synaptic function. Since then, the scientific community has experimentally identified 79 new synaptic genes. This study used these new empirical data to evaluate the original prediction. A series of changes were implemented to the training scheme of this model, and using the new data it was demonstrated that this improves its predictive power. Finally, the new synaptic genes were added to the training set and a new model was trained, obtaining a new, enhanced catalogue of putative synaptic genes. This study presents this new catalogue and announces that a regularly updated version will be available online. This study shows that training an ensemble of machine learning classifiers solely with the whole-body temporal transcription profiles of known synaptic genes resulted in a catalogue with a significant enrichment in undiscovered synaptic genes. Using new empirical data provided by the scientific community, the original approach was validated, improving the model to obtained an arguably more precise prediction. This approach reduces the number of genes to be tested through hypothesis-driven experimentation and will facilitate understanding of neuronal function (Pazos Obregon, 2019).

Drosophila Synaptotagmin 7 negatively regulates synaptic vesicle release and replenishment in a dosage-dependent manner

Synchronous neurotransmitter release is triggered by Ca(2+) binding to the synaptic vesicle protein Synaptotagmin 1, while asynchronous fusion and short-term facilitation is hypothesized to be mediated by plasma membrane-localized Synaptotagmin 7 (SYT7). This study generated mutations in Drosophila to determine if it plays a conserved role as the Ca(2+) sensor for these processes. Electrophysiology and quantal imaging revealed evoked release was elevated 2-fold. Syt7 mutants also had a larger pool of readily-releasable vesicles, faster recovery following stimulation, and intact facilitation. Syt1/Syt7 double mutants displayed more release than Syt1 mutants alone, indicating SYT7 does not mediate the residual asynchronous release remaining in the absence of SYT1. SYT7 localizes to an internal membrane tubular network within the peri-active zone, but does not enrich at active zones. These findings indicate the two Ca(2+) sensor model of SYT1 and SYT7 mediating all phases of neurotransmitter release and facilitation is not applicable at Drosophila synapses (Guan, 2020).

To characterize the location and function of SYT7 in Drosophila the CRISPR-Cas9 system was used to endogenously label the protein and generate null mutations in the Syt7 locus. The findings indicate SYT7 acts as a negative regulator of SV release, active zone probability of release (AZ Pr), the readily releasable pool (RRP) size, and RRP refilling. The elevated P_r across the AZ population in Syt7 mutants provides a robust explanation for why defects in asynchronous release and facilitation are observed in the absence of the protein, as SYT7 levels set the baseline for the amount of evoked release. SYT7's presence on an internal tubular membrane network within the peri-AZ positions the protein to interface with the SV cycle at multiple points to regulate membrane trafficking. In addition, increased SV release in animals lacking both SYT1 and SYT7 indicate the full complement of Ca2+ sensors that mediate the distinct phases of SV release remain unknown (Guan, 2020).

Using a combination of synaptic physiology and imaging approaches, the findings indicate SYT7 acts to reduce SV recruitment and release. Minor defects in asynchronous release and facilitation were identified in Drosophila Syt7 mutants, as observed in mouse and zebrafish models. However, these defects are attrobited to reduced SV availability as a result of increased Pr in Syt7 mutants. Indeed, a key feature of facilitation is its critical dependence on initial Pr. Low Pr synapses increase SV fusogenicity as Ca2+ levels rise during paired-pulses or stimulation trains, resulting in short-term increases in Pr for SVs not recruited during the initial stimuli. In contrast, depression occurs at high Pr synapses due to the rapid depletion of fusion-capable SVs during the initial response. Prior quantal imaging at Drosophila NMJs demonstrated facilitation and depression can occur across different AZs within the same neuron, with high Pr AZs depressing and low Pr AZs facilitating. Given the elevated Pr in Syt7 mutants, the facilitation defects are likely related to differences in initial Pr and depletion of fusion-competent SVs available for release during the 2nd stimuli (Guan, 2020).

A similar loss of SVs due to elevated Pr in Syt7 mutants would reduce fusogenic SVs that are available during the delayed phase of the asynchronous response. Syt1; Syt7 double mutants continue to show asynchronous fusion and facilitation, demonstrating there must be other Ca2+ sensors that mediate these components of SV release. The predominant localization of endogenous SYT7 to an internal tubular membrane compartment at the peri-AZ also places the majority of the protein away from release sites where it would need to reside to directly activate SV fusion. As such, the data indicate SYT7 regulates SV release through a distinct mechanism from SYT1 (Guan, 2020).

It is also concluded that the remaining components of asynchronous fusion and facilitation must be mediated by an entirely different family of Ca2+-binding proteins than Synaptotagmins (or through Ca2+-lipid interactions). Of the seven Syt genes in the Drosophila genome, only 3 SYT proteins are expressed at the motor neuron synapses assayed in this study - SYT1, SYT4 and SYT7. For the remaining SYTs in the genome, SYT-α and SYT-β are expressed in neurosecretory neurons and function in DCV fusion. SYT12 and SYT14 lack Ca2+ binding residues in their C2 domains and are not expressed in motor neurons. In addition, SYT4 is found on exosomes and transferred to postsynaptic cells, where it participates in retrograde signaling. Syt1; Syt4 double mutants display the same SV fusion defects found in Syt1 mutants alone, indicating SYT4 cannot compensate for SYT1 function in SV release. As such, SYT1 and SYT7 are the only remaining SYT isoforms that could contribute to SV trafficking within Drosophila motor neuron terminals (Guan, 2020).

A prior study using a Syt7 exon-intron hairpin RNAi did not result in an increase in evoked release. Although a reduction in ectopic expression of SYT7 in muscles could be seen with Mhc-GAL4 driving the UAS-Syt7 RNAi, the anti-SYT7 antisera does not recognize the endogenous protein in neurons using immunocytochemistry, preventing a determination of presynaptic SYT7 levels following neuronal RNAi. To further examine this issue, western blot analysis was performed with this RNAi and compared those used in the current study. The results confirmed that the RNAi line failed to reduce endogenous GFP-tagged SYT7, although the two commercial RNAi lines used in the current study were highly effective. Based on these data, it is concluded that the previous Syt7 UAS-RNAi line was ineffective in knocking down endogenous SYT7. Given the Syt7^M1 and Syt7^M2 alleles result in early stop codons and lack SYT7 expression by western blot analysis and display elevated levels of fusion, the data indicate SYT7 normally acts to suppress SV release as demonstrated by electrophysiology and optical P_r imaging. SYT7 overexpression reduces SV release even more, further confirming that the levels of SYT7 set the baseline amount of SV fusion at Drosophila NMJ synapses (Guan, 2020).

Although the data indicate SYT7 is not the primary asynchronous or facilitation Ca2+ sensor in Drosophila, this study found it inhibits SV release in a dosage-sensitive manner. The reduction in SV release is not due to changes in the Ca2+ cooperativity of fusion or enhanced presynaptic Ca2+ entry, ruling out the possibility that SYT7 normally acts as a local Ca2+ buffer or an inhibitor of presynaptic voltage-gated Ca2+ channels. The reduction in release is also not due to altered endocytosis, as Syt7 mutants have a normal steady-state rate of SV cycling following depletion of the RRP. Instead, SYT7 regulates SV fusogenicity at a stage between SV endocytosis and fusion. Given the rapid enhanced refilling of the RRP observed in Syt7 mutants, and the suppression of RRP refilling following SYT7 overexpression, the data indicate SYT7 regulates releasable SVs in part through changes in SV mobilization to the RRP. Ca2+ is well known to control the replenishment rate of releasable SVs, with Calmodulin-UNC13 identified as one of several molecular pathways that accelerate RRP refilling in a Ca2+-dependent manner. The findings indicate SYT7 acts in an opposite fashion and slows RRP refilling, providing a Ca2+-dependent counter-balance for SV recruitment into the RRP. Although such an effect has not been described for mammalian SYT7, defects in RRP replenishment have been observed when both SYT1 and SYT7 are deleted or following high frequency stimulation trains (Guan, 2020).

SYT7's role in restricting SV fusion and RRP size also affects spontaneous release. Although Syt7 mutants alone do not show elevated mini frequency, DoubleNull mutants have a 2-fold increase in spontaneous release. Similar increases in spontaneous release were observed at mammalian synapses lacking both SYT7 and SYT1 (or SYT2), with the effect being attributed to a dual role in clamping fusion in the absence of Ca2+ (Luo, 2017; Turecek, 2019). The current results indicate the elevation in spontaneous release at Drosophila synapses is a result of an increase in releasable SVs rather than a clamping function for SYT7. Following overexpression of SYT7, there is a reduction in the number of fusogenic SVs available for both evoked and spontaneous release. The dosage-sensitivity of the various phenotypes indicate SYT7 abundance is a critical node in controlling SV release rate. Indeed, mammalian SYT7 levels are post-transcriptionally modulated by γ-secretase proteolytic activity and APP, linking it to SV trafficking defects in Alzheimer's disease (Guan, 2020).

How does SYT7 negatively regulate recruitment and fusion of SVs? The precise mechanism by which SYT7 reduces release and slows refilling of the RRP is uncertain given it is not enriched at sites of SV fusion. Although the possibility cannot be ruled out that a small fraction of the protein is found at AZs, SYT7 is predominantly localized to an internal membrane compartment at the peri-AZ where SV endocytosis and endosomal sorting occurs. SYT7 membrane tubules are in close proximity and could potentially interact with peri-AZs proteins, endosomes, lysosomes and the plasma membrane. Given its primary biochemical activity is to bind membranes in a Ca2+-dependent manner, SYT7 could mediate cargo or lipid movement across multiple compartments within peri-AZs. In addition, it is possible SYT7 tubules could form part of the poorly defined SV recycling endosome compartment. However, no change was observed in SV density or SV localization around AZs, making it unlikely SYT7 would be essential for endosomal trafficking of SVs. The best characterized regulator of the SV endosome compartment in Drosophila is the RAB35 GAP Skywalker (SKY). Although Sky mutations display some similarities to Syt7, including increased neurotransmitter release and larger RRP size, Syt7 lacks most of the well-described Sky phenotypes such as behavioral paralysis, FM1-43 uptake into discrete punctated compartments, cisternal accumulation within terminals and reduced SV density. In addition, no co-localization was found between SKY-GFP and SYT7RFP within presynaptic terminals (Guan, 2020).

By blocking SV refilling with bafilomycin, the findings indicate the fast recovery of the RRP can occur via enhanced recruitment from the reserve pool and does not require changes in endocytosis rate. The phosphoprotein Synapsin has been found to maintain the reserve SV pool by tethering vesicles to actin filaments at rest. Synapsin interacts with the peri-AZ protein Dap160/Intersectin to form a protein network within the peri-AZ that regulates clustering and release of SVs. Synapsin-mediated phase separation is also implicated in clustering SVs near release sites. SYT7 could similarly maintain a subset of SVs in a non-releasable pool and provide a dual mechanism for modulating SV mobilization. Phosphorylation of Synapsin and Ca2+ activation of SYT7 would allow multiple activity-dependent signals to regulate SV entry into the RRP. As such, SYT7 could play a key role in organizing membrane trafficking and protein interactions within the peri-AZ network by adding a Ca2+-dependent regulator of SV recruitment and fusogenicity (Guan, 2020).

Additional support for a role for SYT7 in regulating SV availability through differential SV sorting comes from recent studies on the SNARE complex binding protein CPX. Analysis of Drosophila Cpx mutants, which have a dramatic increase in minis, revealed a segregation of recycling pathways for SVs undergoing spontaneous versus evoked fusion. Under conditions where intracellular Ca2+ is low and SYT7 is not activated, spontaneously-released SVs do not transit to the reserve pool and rapidly return to the AZ for re-release. In contrast, SVs released during high frequency evoked stimulation when Ca2+ is elevated and SYT7 is engaged, re-enter the RRP at a much slower rate. This mechanism slows re-entry of SVs back into the releasable pool when stimulation rates are high and large numbers of SV proteins are deposited onto the plasma membrane at the same time, allowing time for endosomal sorting that might be required in these conditions. In contrast, SVs released during spontaneous fusion or at low stimulation rates would likely have less need for endosomal re-sorting. Given SYT7 restricts SV transit into the RRP, it provides an activity-regulated Ca2+-triggered switch for redirecting and retaining SVs in a non-fusogenic pool that could facilitate sorting mechanisms (Guan, 2020).

Beyond SV fusion, presynaptic membrane trafficking is required for multiple signaling pathways important for developmental maturation of NMJs. In addition, alterations in neuronal activity or SV endocytosis can result in synaptic undergrowth or overgrowth. No defect was found in synaptic bouton or AZ number, indicating SYT7 does not participate in membrane trafficking pathways that regulate synaptic growth and maturation. However, a decrease in T-bar area and presynaptic Ca2+ influx in Syt7 mutants was found. Although it is unclear how these phenotype arise, they may represent a form of homeostatic plasticity downstream of elevated synaptic transmission. There is also ample evidence that SV distance to Ca2+ channels plays a key role in defining the kinetics of SV release and the size of the RRP, suggesting a change in such coupling in Syt7 mutants might contribute to elevations in P_r and RRP refilling. Further studies will be required to precisely define how SYT7 controls the baseline level of SV release at synapses (Guan, 2020).

Synaptotagmin 7 switches short-term synaptic plasticity from depression to facilitation by suppressing synaptic transmission

Short-term synaptic plasticity is a fast and robust modification in neuronal presynaptic output that can enhance release strength to drive facilitation or diminish it to promote depression. The mechanisms that determine whether neurons display short-term facilitation or depression are still unclear. This study shows that the Ca(2+)-binding protein Synaptotagmin 7 (Syt7) determines the sign of short-term synaptic plasticity by controlling the initial probability of synaptic vesicle (SV) fusion. Electrophysiological analysis of Syt7 null mutants at Drosophila embryonic neuromuscular junctions demonstrate loss of the protein converts the normally observed synaptic facilitation response during repetitive stimulation into synaptic depression. In contrast, overexpression of Syt7 dramatically enhanced the magnitude of short-term facilitation. These changes in short-term plasticity were mirrored by corresponding alterations in the initial evoked response, with SV release probability enhanced in Syt7 mutants and suppressed following Syt7 overexpression. Indeed, Syt7 mutants were able to display facilitation in lower [Ca(2+)] where release was reduced. These data suggest Syt7 does not act by directly sensing residual Ca(2+) and argues for the existence of a distinct Ca(2+) sensor beyond Syt7 that mediates facilitation. Instead, Syt7 normally suppresses synaptic transmission to maintain an output range where facilitation is available to the neuron (Fujii, 2021).

Synaptotagmins (Syts) are a large family of Ca2+ binding proteins, with the Syt1 isoform functioning as the major Ca2+ sensor for synchronous synaptic vesicle (SV) fusion1. Ca2+ also controls presynaptic forms of short-term plasticity, with other Syt isoforms representing promising candidates to mediate these processes. Indeed, Synaptotagmin 7 (Syt7) has been reported to function in facilitation, a form of short-term plasticity that enhances synaptic transmission following consecutive action potentials. Facilitation is believed to be mediated by residual Ca2+ acting to enhance the number of SVs that are released during repetitive action potentials occurring within a short temporal window. Since Syt7 binds Ca2+ with high affinity and slow kinetics, which match requirements for facilitation, the protein has been hypothesized to act as the Ca2+ sensor for this form of presynaptic plasticity. However, the role of Syt7 in facilitation is still unclear (Fujii, 2021).

Drosophila neuromuscular junctions (NMJs) provide an excellent system for testing the role of Syt7 in short-term synaptic plasticity. In particular, embryonic NMJs are highly plastic and allow stable recordings in high Ca2+ concentrations using the myosin heavy chain (Mhc) mutant background to prevent muscle contraction. Together with the lack of compensation that might occur at older synapses, these advantages provide highly reliable measurements of synaptic transmission. Indeed, analysis of Syt1 mutants at embryonic NMJs established this Syt isoform functions as the synchronous Ca2+ sensor for synaptic transmission. Although NMJs in many other species show depression, Drosophila embryonic NMJs are facilitative at physiological Ca2+ concentrations as shown here, similar to many mammalian central synapses such as those in the hippocampus. Using stable recordings from this facilitative and plastic synapse in Drosophila embryos, the absolute value of synaptic currents was quantified in Syt7 mutants and in animals overexpressing Syt7. While loss of Syt7 enhanced presynaptic output, overexpression of Syt7 suppressed release. These changes in the magnitude of presynaptic output were mirrored by changes in short-term presynaptic plasticity. High levels of Syt7 enabled robust facilitative responses while loss of Syt7 switched the normally facilitating synapse into one that displayed short-term depression. This work reveals that Syt7 normally reduces synaptic transmission to scale it to an appropriate range where facilitation is allowed, providing a bi-directional switch for short-term synaptic plasticity (Fujii, 2021).

The current study indicates Syt7 is indispensable for facilitation across the physiological range of Ca2+ concentrations at Drosophila embryonic NMJs as previously shown for mammalian preparations (Jackman, 2016). In the absence of Syt7, the normally facilitating embryonic NMJ now displays depression. Following Syt7 overexpression, facilitation is greatly enhanced. These data indicate the major reason for defective facilitation in Syt7^-/- mutants is due to loss of Syt7's ability to suppress release, which likely causes rapid SV depletion that is non-compatible with short-term synaptic facilitation. Likewise, overexpression of Syt7 reduces SV release and allows for enhanced facilitation. This role for Syt7 contrasts with current models proposed in mammals where Syt7 is hypothesized to bind residual Ca2+ to directly act as a facilitation Ca2+ sensor. Although the current data indicate Syt7 is not the primary Ca2+ sensor for facilitation, the possibility cannot be ruled out that Syt7 has dual roles in both suppressing and facilitating SV fusion as observed for Syt. If Syt7 has a dual role with C2A functioning for clamping and C2B for facilitation, the null mutant would lack both properties. It is possible the lack of clamping is the dominant phenotype, with any facilitative function being masked by SV depletion at higher Ca2+ concentrations. Thus, a Syt7-dependent component of facilitation cannot be ruled out. However, the presence of facilitation in Syt7^-/- mutants at lower [Ca2+] indicates there is a facilitation sensor besides Syt7 that monitors residual Ca2+ to directly activate this form of short-term plasticity. Although a role for residual maternally supplied Syt7 at the embryonic stage in Syt7^-/- mutants cannot be ruled out, there is no evidence from RNA profiling studies that indicate Syt7 is present at earlier stages of embryonic development prior to nervous system formation. Thus, it is unlikely residual Syt7 could sustain normal levels of facilitation as observed in low [Ca2+], consistent with the enhanced synaptic transmission observed across a broad [Ca2+] range in Syt7^-/- mutants. Given facilitation is also present in low [Ca2+] in Syt7^-/- mutants at the 3rd instar stage when any maternal contribution would be depleted, it is concluded that facilitation can occur in the complete absence of Syt7 under conditions where the initial response is reduced (Fujii, 2021).

A key advantage of the Drosophila embryonic NMJ preparation is the ability to unambiguously monitor the absolute baseline values of synaptic strength even in high [Ca2+] using the non-contracting Mhc mutant. In this regard, it is clear that synaptic transmission at Drosophila embryonic NMJs is much stronger in Syt7^-/- mutants at all Ca2+ concentrations tested. Moreover, the stronger transmission is due to higher release probability rather than an increased number of releasable SVs. Thus, the data predict that higher release probability leads to a lower facilitation ratio secondary to vesicle depletion. The precise mechanisms by which Syt7 suppresses SV release to enable facilitation will require further study. Beyond a potential clamping function for Syt7, the protein could alter local Ca2+ buffering or cause increased Ca2+ influx that could contribute to elevated SV release. Syt7 does not localize to SVs and may instead act from the plasma membrane or internal membrane compartments, allowing for several potential mechanisms for Syt7 to suppress release. Ca2+ binding to the C2A and C2B domains of Syt1 have been shown to have distinct functions in SV release, with C2B playing a dominant role in triggering SV fusion and C2A acting to clamp release. It is unclear if the C2A and C2B domains of Syt7 act similarly in Drosophila or have independent functions compared to Syt1. One possibility is that the C2A domain of Syt7 suppresses SV fusion and the C2B domain facilitates release, similar to Syt1. Structure function studies of Syt7 should help elucidate this biology in Drosophila, similar to prior studies of Syt1 function (Fujii, 2021).

As suppression of SV release by Syt7 is dose-dependent (Guan, 2020), increasing levels of Syt7 would elevate the ratio of facilitation. These results suggest the degree of facilitation across distinct neuronal populations may be set by Syt7 levels similar to a potentiometer. The analysis of Syt7^-/- nulls, heterozygotes and overexpression lines support such a model that changes release and short-term plasticity in a graded fashion. Depending on whether a synapse is facilitative or depressive, Syt7 expression could be modulated to gate plasticity to the level that most benefits the local circuit, similar to how Syt1 and Syt2 levels variably control release synchronicity across neuronal populations. Indeed, the squid giant synapse is facilitative only when Ca2+ is lowered from normal saline (artificial sea water), similar to Syt7^-/- mutants, suggesting synapses that normally depress may have reduced levels of Syt7. Indeed, recent evidence suggests that species-specific differences in presynaptic plasticity in rodents is linked to the levels of Syt7. In shrews, the levels of Syt7 are lower in hippocampal CA3 synapses and they show reduced presynaptic plasticity. In contrast, Syt7 levels are much higher in mice, with their CA3 output synapses displaying far greater forms of presynaptic plasticity. Drosophila adults and 3rd instar larvae also have less facilitative NMJs than embryonic NMJs. This difference may contribute to the distinct effects of Syt7 on clamping spontaneous SV release that is observed between embryonic and 3rd instar NMJs. Mammalian studies identified redundant functions for Syt1 and Syt7 in clamping spontaneous fusion at inhibitory synapses. While reductions in Syt7 levels alone did not increase spontaneous SV release, removal of both Syt1 and Syt7 enhanced mini frequency to a far greater level that loss of Syt1 alone. In addition, a Syt7 transgene was able to rescue the elevated miniature frequency in Syt1 mutants. These differences in clamping properties were attributed to an insufficient level of Syt7 expression compared to Syt1. Differences in the Syt1/Syt7 ratio between Drosophila 3rd instar and embryonic NMJs may also contribute to distinct effects on spontaneous SV clamping observed in Syt1 and Syt7 mutants at these distinct developmental stages. In conclusion, controlling expression level of Syt7 provides an attractive mechanism for activity-dependent presynaptic scaling of release probability as a homeostat for both presynaptic output and short-term facilitation, similar to postsynaptic scaling mechanisms previously described for chronic forms of synaptic plasticity (Fujii, 2021).

Rapid regulation of vesicle priming explains synaptic facilitation despite heterogeneous vesicle:Ca(2+) channel distances

Chemical synaptic transmission relies on the Ca(2+)-induced fusion of transmitter-laden vesicles whose coupling distance to Ca(2+)-channels determines synaptic release probability and short-term plasticity, the facilitation or depression of repetitive responses. Using electron- and super-resolution microscopy at the Drosophila neuromuscular junction this study quantitatively mapped vesicle:Ca(2+)-channel coupling distances. These are very heterogeneous, resulting in a broad spectrum of vesicular release probabilities within synapses. Stochastic simulations of transmitter release from vesicles placed according to this distribution revealed strong constraints on short-term plasticity; particularly facilitation was difficult to achieve. It was shown that postulated facilitation mechanisms operating via activity-dependent changes of vesicular release probability (e.g. by a facilitation fusion sensor) generate too little facilitation and too much variance. In contrast, Ca(2+)-dependent mechanisms rapidly increasing the number of releasable vesicles reliably reproduce short-term plasticity and variance of synaptic responses. Activity-dependent inhibition of vesicle un-priming or release site activation are proposed as novel facilitation mechanisms (Kobbersmed, 2020).

This study has described a broad distribution of SV release site:Ca2+ channel coupling distances in the Drosophila NMJ and compared physiological measurements with stochastic simulations of four different release models (single-sensor, dual fusion-sensor, Ca2+-dependent unpriming and site activation model). The two first models (single-sensor and dual fusion-sensor), where residual Ca2+ acts on the energy barrier for fusion and results in an increase in probability of vesicle release (pVr), failed to reproduce facilitation. The two latter models involve a Ca2+-dependent regulation of participating release sites and reproduced release amplitudes, variances and paired-pulse ratios (PPRs). Therefore, the Ca2+-dependent accumulation of releasable SVs is a plausible mechanism for paired-pulse facilitation at the Drosophila NMJ, and possibly in central synapses as well (Kobbersmed, 2020).

Two models can explain paired-pulse facilitation and variance-mean behaviour at the Drosophila NMJ. Both models include a Ca2+-dependent increase in the number of participating (occupied/activated) release sites. In the Ca2+-dependent unpriming model, forward priming happens at a constant rate, but unpriming is inversely Ca2+-dependent, such that increases in residual Ca2+ lead to inhibition of unpriming, thereby increasing release site occupation between stimuli. Ca2+-dependent replenishment has been observed in multiple systems. This has traditionally been implemented in various release models as a Ca2+-dependent forward priming rate. In a previous secretion model in chromaffin cells, a catalytic function of Ca2+ upstream of vesicle fusion was proposed. However, in the context of short term facilitation such models would favour accelerated priming during the AP, which would counteract this facilitation mechanism and might cause asynchronous release, similar to the problem with the dual fusion-sensor model. In the model presented in this study this is prevented by including the Ca2+ dependency on the unpriming rate. Consistent with this idea, recent data in cells and in biochemical experiments showed that the Ca2+-dependent priming protein (M)Unc13 reduces unpriming. Another model that reproduced the electrophysiological data was the site activation model, where sites are activated Ca2+-dependently under docked (but initially unprimed) SVs. In this case, it was necessary to prevent rapid activation-and-fusion during the AP by including an extra, Ca2+-independent transition, which introduces a delay before sites are activated. The two models are conceptually similar in that they either recruit new SVs to (always active) sites, or activate sites underneath dormant SVs. Those two possibilities are almost equivalent when measuring with electrophysiology, but they might be distinguished in the future using flash-and-freeze electron microscopy . Interestingly, Unc13 has recently been shown to form release sites at the Drosophila NMJ. Therefore, the two models also correspond to two alternative interpretations of Unc13 action (to prevent unpriming, or form release sites) (Kobbersmed, 2020).

In this model, all primed vesicles have identical properties, and only deviate in their distance to the Ca2+ channel cluster (positional priming). Alternatively, several vesicle pools with different properties (molecular priming) could be considered, which might involve either vesicles with alternative priming machineries, or vesicles being in different transient states along the same (slow) priming pathway. In principle, if different primed SV states are distributed heterogenously such that more distant vesicles are more primed/releasable, such an arrangement might counteract the effects of a broad distance distribution, although this is speculative. Without such a peripheral distribution, the existence of vesicles in a highly primed/releasable state (such as the 'super-primed' vesicles reported at the Calyx of Held synapse), would result in pronounced STD, and counteract STF, which indeed has been observed (Kobbersmed, 2020).

In this study electrophysiological recordings were performed on muscle 6 of the Drosophila larva which receives input from morphologically distinct NMJs containing big (Ib) and small (Is) synaptic boutons, which have been shown to differ in their physiological properties. This could add another layer of functional heterogeneity in the postsynaptic responses analysed in thus study (the EM and STED analyses shown in this study were focused on Ib inputs). Because the model does not distinguish between Is and Ib inputs, the estimated parameters represent a compound behaviour of all types of synaptic input to this muscle. Future investigations to isolate the contribution of the different input types (e.g. by genetically targeting Is/Ib-specific motoneurons using recently described GAL4 lines) could help distinguish between inputs and possibly further refine the model to identify parameter differences between these input types (Kobbersmed, 2020).

A cartoon summarizes the results for the single-sensor, dual fusion-sensor and unpriming models (see Cartoon illustrations of the single-sensor, the dual fusion-sensor, and the unpriming models during a paired-pulse simulation at 0.75 mM extracellular Ca2+). Facilitation in single and dual fusion-sensor models depend on the increase in release probability from the first AP to the next (compare colored rings representing 25% release probability between row 2 and 4). However, the increase is very small, even for the dual fusion-sensor model, and to nevertheless produce some facilitation, optimisation finds a small Ca2+ influx, which leads to an ineffective use of the broad vesicle distribution (and a too-high estimate of nsites). In the unpriming model a higher fitted Ca2+ influx (QMax) leads to a more effective use of the entire SV distribution, and facilitation results from the combination of incomplete occupancy of release sites before the first AP (row 1), combined with 'overshooting' priming into empty sites between APs (Kobbersmed, 2020).

Molecularly, syt-7 was linked to STF behaviour in mice (Jackman, 2016), and the data does not rule out that syt-7 is essential for STF at the Drosophila NMJ (see Drosophila Synaptotagmin 7 negatively regulates synaptic vesicle release and replenishment in a dosage-dependent manner). However, this study shows clearly that a pVr-based facilitation mechanism (dual fusion-sensor model) cannot account for STF in synapses with heterogeneous distances between release sites and Ca2+ channels. Interestingly, syt-7 was also reported to function in vesicle priming and RRP replenishment. Thus, future work will be necessary to investigate whether the function of syt-7 in STF might take place by Ca2+-dependent inhibition of vesicle unpriming or release site activation (Kobbersmed, 2020).

Similar suggestions that facilitation results from a build-up of primed SVs during stimulus trains were made for the crayfish NMJ and mammalian synapses. This is in line with the results, with facilitation arising from modulation of the number of primed SVs rather than pVr. The models are conceptually simple (e.g. all SVs are equally primed and distinguished only by distance to Ca2+ channels, sometimes referred to as 'positional priming'), and this study has improved conceptually on previous work by using estimated SV release site:Ca2+ channel distributions, stochastic simulations and comparison to variance-mean relationships and a systematic comparison was performed of pVr- and priming-based models. It has not been clear whether increases in primed SVs are also required for paired-pulse facilitation, or only become relevant in the case of 'tonic' synapses that build up release during longer stimulus trains. Paired-pulse facilitation is a more wide-spread phenomenon in synapses than frequency facilitation, and this study shows for the case of Drosophila NMJ that it also seems to require priming-based mechanisms. Thus, Ca2+-dependent increases of the RRP during STP might be a general feature of chemical synapses (Kobbersmed, 2020).

High-probability neurotransmitter release sites represent an energy-efficient design

Nerve terminals contain multiple sites specialized for the release of neurotransmitters. This study tested the hypothesis that high-probability release sites represent an energy-efficient design. Release site probabilities and energy efficiency were examined at the terminals of two glutamatergic motor neurons synapsing on the same muscle fiber in Drosophila larvae. Through electrophysiological and ultrastructural measurements, calculated release site probabilities were found to differ considerably between terminals (0.33 versus 0.11). The energy required to release and recycle glutamate were calculated from the same measurements. The energy required to remove calcium and sodium ions subsequent to nerve excitation was estimated through microfluorimetric and morphological measurements. Energy efficiency was calculated as the number of glutamate molecules released per ATP molecule hydrolyzed, and high-probability release site terminals were found to be more efficient (0.13 versus 0.06). This analytical model indicates that energy efficiency is optimal (~ 0.15) at high release site probabilities (~0.76). As limitations in energy supply constrain neural function, high-probability release sites might ameliorate such constraints by demanding less energy. Energy efficiency can be viewed as one aspect of nerve terminal function, in balance with others, because high-efficiency terminals depress significantly during episodic bursts of activity (Lu, 2016).

This study reports the relationship between the average probability of release from each AZ (P_AZ) and energy efficiency in terminals of two glutamatergic motor neurons (MNs) innervating the same target muscle fiber. P_AZ was three times higher in one of the terminals, and this terminal was also twice as efficient, indicating that a high-probability release site is more energy efficient. Given that the brain's energy demands are high, selection away from low P_AZ synapses because of their low efficiency might be expected to favor the adoption of a uniform high P_AZ design. However, this study found that P_AZ values in situ fell short of the high P_AZ values predicted for optimal energy efficiency. Terminals with the lowest P_AZ and lowest energy efficiency depressed the least at endogenous release rates and performed most of the work during fictive locomotion. The interpretation of these data is that selection away from energy inefficiency has favored high P_AZ but that increased P_AZ is held in check by selection away from an inability to sustain release (Lu, 2016).

Selection away from energy inefficiency is thought to have influenced the size of neurons and properties of their synapses and, in turn, limited neural computational power. This study suggests that selection away from energy inefficiency has selected for high P_AZ synapses, and this study has demonstrated a positive correlation between P_AZ and energy efficiency under physiological conditions. High P_AZ can result from high Ca2+ entry/AZ, sensitivity (S) or cooperativity (nH) of release. At first glance, these parameters seem to have qualitatively different influences on energy efficiency. High S or nH lead to higher P_AZ and, in turn, higher P_AZ leads to higher energy efficiency. More Ca2+ entry/AZ also results in higher P_AZ, but simulation of how changes in Ca2+ entry/AZ affect energy efficiency produced a curve with a distinct optimum. Further analysis revealed that energy efficiency would be optimized if P_AZ were elevated to 0.76 by an increase in Ca2+ entry/AZ. Taken together, these simulation results show that a nerve terminal needs a relatively high P_AZ for optimal energy efficiency but that efficiency diminishes at the highest P_AZ values because the purchase of more Ca2+ yields no more neurotransmitter release in return (Lu, 2016).

Trade-offs between energy efficiency and function have been observed previously. For relay synapses such as the NMJ, the trade-off appears to be between presynaptic energy efficiency and the capacity to sustain neurotransmitter release. Some mammalian central synapses show a capacity for sustained release and are exclusively low P_AZ synapses. This study found that low P_AZ terminals depressed less than high P_AZ terminals, consistent with previous studies showing that low P_AZ synapses are likely to facilitate, whereas high P_AZ synapses are likely to depress. Type-Ib terminals with an endogenous firing rate of about 20 Hz offset the risk of depletion with a low P_AZ, but this measure is attended by low efficiency. The selection potential for low P_AZ becomes apparent when, in combination with large NAZ, it confers a capacity to sustain high levels of release, which may translate to sustaining organismal locomotion without fatigue (Lu, 2016).

While there is a clear rationale for selection against presynaptic design unable to sustain release at a NMJ, the rationale for selection against presynaptic energy inefficiency at a NMJ assumes that the presynaptic terminal consumes (or once consumed) a non-negligible proportion of the NMJ energy budget. Postsynaptic energy consumption can be estimated from EJC measurements that allow calculation of the amount of charge crossing the postsynaptic plasma membrane in response to neurotransmitter released during a single presynaptic AP and the amount of ATP then needed to remove those ions (see the Supplemental Information). Several assumptions that are difficult to defend have to be made to assess postsynaptic energy consumption (Is: 8.16 × 108, and, Ib: 5.18 × 108 ATP molecules). Yet, even if correct only in order of magnitude, it wouls be concluded that the presynaptic terminal consumes ∼1% of the NMJ energy budget. This proportion contrasts starkly with estimates at mammalian central synapses, at which presynaptic energy demands are ∼30% of the total synaptic signaling cost. If presynaptic terminals only consume 1% of the NMJ energy budget, it is suggested that the ability to locomote without fatigue ultimately conferred a greater selection advantage than the energy saved by an efficient terminal and that as a result low P_AZ release sites persisted at the expense of high P_AZ sites (Lu, 2016).

Simulation of a sudden drop-off in distal dense core vesicle concentration in Drosophila type II motoneuron terminals

Recent experimental observations have shown evidence of an unexpected sudden drop-off in the dense core vesicles (DCVs) content at the ends of certain types of axon endings. A mathematical model was developed that is based on the conservation of captured and transiting DCVs in boutons. The model consists of 77 ordinary differential equations and is solved using a standard Matlab solver. It was hypothesized that the drop in DCV content in distal boutons is due to an insufficient supply of anterogradely moving DCVs coming from the soma. This hypothesis was tested by modifying the flux of DCVs entering the terminal, and it was found that the number of most distal boutons left unfilled increases if the DCV flux entering the terminal is decreased. The number of anterogradely moving DCVs in the axon can be increased either by the release of a portion of captured DCVs into the anterograde component or by an increase of the anterograde DCV flux into the terminal. This increase could lead to having enough anterogradely moving DCVs such that they could reach the most distal bouton and then turn around by changing molecular motors that propel them. The model suggests that this could result in an increased concentration of resident DCVs in distal boutons beginning with bouton 2 (the most distal is bouton 1). This is because in distal boutons, DCVs have a larger chance to be captured from the transiting state as they pass the boutons moving anterogradely and then again as they pass the same boutons moving retrogradely (Kuznetsov, 2021).

Drosophila SPG12 ortholog, reticulon-like 1, governs presynaptic ER organization and Ca2+ dynamics

Neuronal endoplasmic reticulum (ER) appears continuous throughout the cell. Its shape and continuity are influenced by ER-shaping proteins, mutations in which can cause distal axon degeneration in Hereditary Spastic Paraplegia (HSP). It was therefore asked how loss of Rtnl1, a Drosophila ortholog of the human HSP gene RTN2 (SPG12), which encodes an ER-shaping protein, affects ER organization and the function of presynaptic terminals. Loss of Rtnl1 depleted ER membrane markers at Drosophila presynaptic motor terminals and appeared to deplete narrow tubular ER while leaving cisternae largely unaffected, thus suggesting little change in resting Ca2+ storage capacity. Nevertheless, these changes were accompanied by major reductions in activity-evoked Ca2+ fluxes in the cytosol, ER lumen, and mitochondria, as well as reduced evoked and spontaneous neurotransmission. Reduced STIM-mediated ER-plasma membrane was found to contact underlie presynaptic Ca2+ defects in Rtnl1 mutants. These results show the importance of ER architecture in presynaptic physiology and function, which are therefore potential factors in the pathology of HSP (Perez-Moreno, 2023).

Synapses, the computational units of nervous systems, are immensely adaptable. The ER is a site of some of the processes that tune synaptic strength. It is a major Ca2+ store of the cell, a site of lipid biosynthesis, and can regulate the physiology of other organelles through contact sites that support exchange of Ca2+ and lipids. Axonal ER comprises an extensive continuous network formed mainly of tubules that are shaped by proteins of the reticulon and REEP families, among others. These contain intramembrane hairpin domains that can curve ER membrane; loss of these proteins can reduce tubule number and curvature of axonal ER and disrupt network continuity (Perez-Moreno, 2023).

The ER network appears to influence axon survival and maintenance; mutations affecting ER-shaping proteins can lead to the axon degeneration disease, Hereditary Spastic Paraplegia (HSP). HSPs show degeneration of the distal regions of longer upper motor axons, consistent with 'dying back' degeneration from axon terminals, preferentially in the longest upper motor axons. The links between HSP and ER-shaping proteins suggest a critical relationship between ER architecture and axonal and presynaptic maintenance. However, a good model for how ER spatial organization affects axonal or presynaptic function and degeneration is lacking. One way in which ER might do this is as an intracellular Ca2+ store. ER Ca2+ stores can either contribute to presynaptic cytosolic Ca2+ responses or sequester excess Ca2+ from the cytosol in response to high frequency stimulation. ER might potentially also influence presynaptic physiology via its interactions with mitochondria. Like ER, mitochondria can also take up Ca2+ when cytosolic Ca2+ is elevated, although this process appears unable to buffer Ca2+ sufficiently to affect synaptic transmission; rather its role appears to be to stimulate ATP synthesis in response to the Ca2+ influx that occurs on synaptic activity. The cytosol, rather than ER, seems to be the main source of Ca2+ for mitochondria in presynaptic terminals (Perez-Moreno, 2023).

As well as being a bulk Ca2+ source or sink, the ER dynamically responds to lumenal Ca2+ levels. STIM (stromal interacting molecule) proteins in the ER membrane, when activated by depletion of ER Ca2+ stores, interact with effectors in the plasma membrane (PM), such as the Orai1 Ca2+ channel, leading to influx of extracellular Ca2+ into the cytosol (store-operated Ca2+ entry, SOCE) and refilling of ER stores. In primary hippocampal neurons, evoked neurotransmitter release correlates with ER Ca2+ content due to a feedback loop dependent on inhibition of voltage-gated Ca2+ channels in the plasma membrane by activated STIM1. Also in primary hippocampal neurons, SOCE, in a STIM2-dependent manner, can transiently increase presynaptic Ca2+ levels and robustly augment spontaneous glutamate vesicular release (Perez-Moreno, 2023).

Since mutations in several ER-shaping proteins can cause axon degeneration, altered ER architecture may elicit degenerative changes in axonal or presynaptic function. One mechanism could be local dysregulation of Ca2+—for example through reduced availability of Ca2+ for synaptic signaling or capacity to remove cytosolic Ca2+, or interference with regulatory effects of the ER STIM Ca2+ sensors. Presynaptic ER comprises a network of interconnected tubules with occasional cisternae While the presence of cisternae might indicate regions specialized in storing Ca2+, the predominance of narrow ER tubules suggests a local regulatory role for this organelle. To test how spatial organization of ER could impair synaptic function, Drosophila was used to study the effects of removing the tubular ER-shaping protein Rtnl1 on presynaptic ER organization, and on the Ca2+ fluxes between compartments during synaptic activity. Rtnl1 is one of two documented reticulons in Drosophila, orthologous to the four RTNs found in mammals (RTN1-4), including the HSP causative gene RTN2. It is distributed continuously in motor axons all the way to presynaptic termini, and its loss leads to partial depletion of axonal ER. Loss of Rtnl1 greatly decreases Excitatory Junction Potential (EJP) amplitude at the larval neuromuscular junction (NMJ), implying major effects on presynaptic physiology, although its effects on presynaptic ER and its role in presynaptic physiology has not been well defined (Perez-Moreno, 2023).

Loss of Rtnl1 depletes tubular ER but not cisternae or ER volume at NMJs, and how this loss significantly decreases ER, mitochondrial, and cytosolic evoked Ca2+ fluxes. This impact of Rtnl1 loss on presynaptic Ca2+ handling is caused by a decrease of STIM-mediated ER-PM contacts. These results suggest a feedback loop, whereby tubular ER and inactivated STIM control evoked Ca2+ entry into the presynaptic compartment and neurotransmitter release (Perez-Moreno, 2023).

While Rtnl1 loss reduces axonal ER levels mainly in longer motor axons (O'Sullivan, 2012; Yalcin., 2017), this study observed that it reduces presynaptic tubular ER independently of axonal length. This phenotype provides a unique model to study the contribution of the presynaptic tubular ER network and ER-shaping HSP proteins to presynaptic Ca2+ handling. These findings display a local role for presynaptic ER tubules, and how synaptic dysfunction might be a consequence of impairing functions of proteins that are mutated in HSP (Perez-Moreno, 2023).

From a body of work mostly in cultured non-neuronal cells, it is known that the ER controls Ca2+ handling. This role appears conserved in presynaptic ER, where both ER-PM contacts and the amount of ER are critical for neurotransmission. Given the relatively larger Ca2+ storage capacity of ER cisternae, are presynaptic ER tubules contributing to Ca2+ dynamics? In this work, the tubular ER-shaping protein Rtnl1 was disrupted and a decrease of presynaptic ER tubules was found. Rtnl1 loss results in a unique model to study the role of ER tubules at the presynaptic region. This model was used to analyze Ca2+ handling, which relates with neurotransmission and the biological basis of neurological disorders associated with tubular ER (Perez-Moreno, 2023).

Loss of either Rtnl1 or REEPs causes a loss of axonal ER preferentially in longer motor neurons of Drosophila larvae (Yalcin, 2017). In contrast, the decrease of presynaptic ER network in Rtnl1 mutants is independent of axon length. The tubular ER network in presynaptic terminals (mouse nucleus accumbens) is more extensive than in axons. It is therefore possible that presynaptic ER is more dependent on the function of tubular ER-shaping proteins than axonal ER. Regarding the continuity of the network, it is possible that neurons can better tolerate loss of ER tubules in presynaptic terminals than in axons, where there are relatively few tubules to begin with. This might explain why loss of Rtnl1 causes ER network discontinuity in axons (Yalcin, 2017), but not at the presynaptic region (Perez-Moreno, 2023).

Increasing the levels of axonal and presynaptic ER (by blocking neuronal autophagy) correlates with elevated Ca2+ release from ER, which in turn increases neurotransmission. In agreement with this, it was shown that lower ER levels correlate with decreased neurotransmission, indicating that the relationship works conversely. Interestingly, a severe decrease was found in the frequency of miniature neurotransmission in Rtnl1 mutants. Since presynaptic ER tubules, but not cisternae, appear to be reduced in Rtnl1 mutants, it is proposed that tubules help regulate Ca2+ handling across presynaptic compartments. Tubule loss abates Ca2+ handling in the ER, cytosol, and mitochondria, in both types of excitatory NMJs in Drosophila larvae. As neurotransmitter release is triggered by cytosolic Ca2+ influx through PM voltage-gated Ca2+ channels, the decrease of postsynaptic Ca2+ responses and presynaptic cytosolic influx corroborate with each other (Perez-Moreno, 2023).

ER-PM contacts have an inhibitory role in cytosolic Ca2+ influx in primary hippocampal neurons via activated STIM1 on emptying of ER stores; other studies in neurons have demonstrated that emptying of Ca2+ from ER stores (thereby activating STIM) promotes spontaneous (miniature), but not evoked vesicle release at excitatory terminals by the action of STIM2. At excitatory terminals it is not expected that STIM playis an inhibitory role to presynaptic Ca2+, as this study observed a decrease in STIM levels and foci in Rtnl1 mutants, as well as a decrease in evoked Ca2+ fluxes, which were rescued by overexpressing STIM. Work in non-neuronal cells indicate that STIM1/STIM2 promotes Ca2+ entry via coupling with Ca2+ channels on the PM, and in vivo data support a similar relationship in motor neurons for the Drosophila STIM1/STIM2 ortholog STIM. Accordingly, a model is proposed for presynaptic compartments, whereby narrow tubular ER modulates Ca2+ entry via STIM. Other components of the network with higher volume, such as cisternae, would work as a Ca2+ store, since both ER volume and resting ER Ca2+ levels are unaffected in Rtnl1 mutants. Evoked Ca2+ uptake by the ER was also decreased by loss of Rtnl1. While most lumenal GFP::HDEL or GCaMP, and hence Ca2+, appears to be in cisternae that would act as Ca2+ reservoirs, evoked ER Ca2+ uptake is reduced upon depletion of the tubular ER network in both phasic and tonic firing boutons. Moreover, this study saw the tubular network influencing the physiological profile of ER Ca2+, with ER Ca2+ release occurring on stimulation of WT Is boutons, and rarely on stimulation of WT Ib boutons. However, with loss of Rtnl1 and depletion of tubular ER, ER in Ib boutons released Ca2+ during neuronal activity in over half of NMJs tested, and thus caused it to act like smaller Is boutons. The results demonstrate a complex relationship between ER structure, and uptake and release of Ca2+ by the ER, suggesting that intact ER cisternae are not sufficient to maintain full levels of synaptic ER Ca2+ release and uptake, but that ER tubules are necessary as well (Perez-Moreno, 2023).

Rtnl1 loss also decreased evoked presynaptic Ca2+ uptake by mitochondria , similar to the ER and cytosolic compartments. As mitochondrial Ca2+ and ATP production are positively correlated, energy generation in both Type Is and Ib boutons might potentially be impaired on depletion of tubular ER. Mitochondria normally receive Ca2+ directly from ER, although synaptic mitochondria can take up Ca2+ from the cytosol. However, the comparative kinetics of the Ca2+ responses of WT cytosol, ER, and mitochondria do not allow distinguishing between mitochondrial Ca2+ uptake from the cytosol or from ER in this situation (Perez-Moreno, 2023).

Beyond the Ca2+ handling defects characterized in this work, it is proposed that Rtnl1 mutants are useful model to explore other biological roles of presynaptic ER, and to understand which presynaptic ER-related processes might be relevant to the mechanisms of HSP. In addition to Ca2+ handling, presynaptic ER depletion might affect other cellular processes such as autophagy, vesicle trafficking, glucose transport, or lipid metabolism. Although this study did not observe any PI(4,5)P2 alterations in Rtnl1 mutants, it is still possible that other ER-dependent lipids might be affected, since at least some ER-PM contacts seem to be reduced (according to the decreased levels observed for STIM1 foci). Failed lipid transfer at ER-PM contacts has been previously related with reduced expansion of the growth cone and with neuronal PM growth in general, but no any defect was observed in morphology of mature motor neurons in Rtnl1 mutants apart from a slight increase in the number of boutons (Perez-Moreno, 2023).

These results reveal at least three aspects of the Rtnl1 mutant phenotype that could be relevant to the disease mechanisms of HSPs caused by mutations in ER-shaping proteins. First, Rtnl1 mutants show a reduced frequency of miniature neurotransmission. This induces bouton fragmentation in young adult Drosophila so as to resemble that of aged Drosophila, whereas elevated minis can delay age-associated fragmentation and prolong motor ability in adult Drosophila]. Second, Rtnl1 mutants show decreased evoked cytosolic Ca2+ influx and neurotransmission at NMJs, which controls muscle contraction. Third, decreased evoked mitochondrial Ca2+ fluxes predict lower ATP generation capacity and potential energy deficits, although these may be mitigated by the reduced levels of synaptic transmission. These results suggest mechanisms whereby altered organization of ER tubules could be a potential mechanism for diseases including HSP by affecting synaptic Ca2+ handling, and thus synapse function (Perez-Moreno, 2023).

Input-specific plasticity and homeostasis at the Drosophila larval neuromuscular junction

Synaptic connections undergo activity-dependent plasticity during development and learning, as well as homeostatic re-adjustment to ensure stability. Little is known about the relationship between these processes, particularly in vivo. This was addressed with novel quantal resolution imaging of transmission during locomotive behavior at glutamatergic synapses of the Drosophila larval neuromuscular junction. Two motor input types, Ib and Is, were found to provide distinct forms of excitatory drive during crawling and differ in key transmission properties. Although both inputs vary in transmission probability, active Is synapses are more reliable. High-frequency firing 'wakes up' silent Ib synapses and depresses Is synapses. Strikingly, homeostatic compensation in presynaptic strength only occurs at Ib synapses. This specialization is associated with distinct regulation of postsynaptic CaMKII. Thus, basal synaptic strength, short-term plasticity, and homeostasis are determined input-specifically, generating a functional diversity that sculpts excitatory transmission and behavioral function (Newman, 2017).

The transfer of information between neurons throughout the nervous system relies on communication across inherently unreliable chemical synapses. Synaptic communication is further complicated by the fact that individual neurons can receive inputs from many functionally diverse neurons. Even synapses formed by one presynaptic neuron and one or more postsynaptic target neurons can vary greatly in neurotransmitter release, postsynaptic sensitivity, and plasticity. Additionally, postsynaptic cells are not passive receivers of information but can produce retrograde signals, including homeostatic signals that modulate synaptic release. The mechanisms that regulate diversity in transmission at individual synapses are not well understood, nor are their relationships to the plasticity and homeostatic mechanisms that adjust synaptic strength. In particular, it is unclear whether retrograde signaling is input or synapse specific and able to maintain input context among diverse convergent synapses (Newman, 2017).

The Drosophila larval neuromuscular junction (NMJ) is a model system for studying glutamatergic transmission, with pre- and postsynaptic molecular machinery similar to that of central excitatory synapses in vertebrates, while also possessing activity-dependent adjustments in synaptic strength, including short- and long-term plasticity, as well as homeostatic plasticity. Two morphologically distinct glutamatergic motor neurons, larger type Ib and smaller type Is, converge onto most of the larval body wall muscles used for locomotion. There is evidence that the transmission properties of these inputs differ (Kurdyak, 1994; Lnenicka, 2000; Lu, 2016), thus providing a powerful system for investigating the role of input and synapse specificity in the regulation of basal synaptic strength, plasticity, and homeostasis. In addition, there is great heterogeneity, in that both the basal release probability (Pr) of evoked release and the frequency of spontaneous release differ greatly between synapses of the same Ib axon (Peled, 2011; Peled, 2014). This study set out to understand how plasticity and homeostasis function in a diverse pool of synapses and to determine whether synaptic homeostasis operates globally or has input specificity (Newman, 2017).

To address these questions requires high-resolution, high- speed analysis of function at many synapses simultaneously. Quantal resolution measurements of excitatory transmission through Ca2+-permeant glutamate receptors (GluRs) has been achieved by imaging chemical or genetically encoded Ca2+ indicators (GECIs) in the postsynaptic cell (Cho, 2015; Guerrero, 2005; Lin, 2016; Melom, 2013; Muhammad, 2015; Peled, 2011; Peled, 2014; Reese, 2015, Reese, 2016; Siegel, 2013). This study has generated a vastly improved postsynaptically targeted GECI based on GCaMP6f. When expressed in Drosophila larval muscle, 'SynapGCaMP6f' enables quantal imaging without voltage clamping. SynapGCaMP6f was combined with an optical platform that immobilizes larvae without anesthetics to measure synaptic transmission simultaneously at hundreds of Ib and Is synapses in the intact, behaving animal (Newman, 2017).

The quantal imaging makes it possible to connect the elementary properties of transmission at single synapses, to the synaptic drive that is generated by convergent synaptic inputs, to the operation of muscle groups in large parts of the animal, and finally to behavior. These observations show that basal synaptic strength, short-term plasticity, and, most strikingly, synaptic homeostasis are input specific, diversifying excitatory transmission and behavioral output (Newman, 2017).

The Ib and Is inputs to the Drosophila larval muscle respectively resemble tonic (low release probability, facilitating) and phasic (high release probability, depressing) inputs seen at neuro-muscular and neuro-neuronal connections in other organisms. Given these differences, the Is input has been proposed to initiate strong, phasic muscle contractions (Schaefer, 2010). However, this study found that the primary excitatory drive and main cause of muscle contraction in larval Drosophila is actually the Ib input. This is despite the fact that Is synapses have a higher basal release probability (Pr) and larger quantal sizes. These functional differences likely reflect the relatively short duration of the Is bursts of activity and the relatively small number of active zones of the Is terminal. Whereas elimination of transmission from Is inputs had little or no effect on muscle contraction during restrained locomotion, just as it had earlier been shown to have negligible effect on general behavior, this study found that the Is input is needed for normal intersegmental coordination of contraction waves. This is consistent with the broad, multi-muscle Is innervation pattern (Newman, 2017).

Ib synaptic activity begins before and ends after Is activity, consistent with their common presynaptic inputs and different intrinsic excitabilities. Recent work has shown that excitatory and inhibitory interneurons regulate the differential recruitment of MNs between hemi- segments and within hemisegments, respectively (Heckscher, 2015; Zwart, 2016). While Ib inputs ramp up their synaptic drive during locomotor bursts, Is inputs abruptly reach a maximum, which they often sustain for the duration of the shorter burst. Given their substantial depression during high-frequency stimulation, the ability of the Is input to sustain a fixed output level during locomotion suggests that it increases the firing frequency during the bursts. The common interneuron drive within the VNC suggests that the progressive increase in synaptic drive during the Ib burst may also be partly due to an increase in frequency during the burst, in addition to the recruitment of silent synapses and increase in Pr of active synapses, which is observed during facilitating high-frequency trains. Thus, a combination of circuit-level and cell-autonomous differences combine to generate the complex behavioral output of the larva from a limited number MNs and muscles (Newman, 2017).

Spontaneous glutamate release has been shown to play a role in synapse development (Choi, 2014). However, this study found that spontaneous release in vivo represents only ~1% of total release, suggesting that evoked release would drown out the influence of spontaneous release. One possible explanation is that in late third-instar larvae, spontaneous release may represent a larger fraction of total synaptic transmission than earlier in development when the developmental influences are exerted. An intriguing alternative explanation derives from the finding that, at both inputs, synapses preferentially participate in either evoked or spontaneous release. This suggests that developmental signals could occur at a subset synapses that are dominated by spontaneous release (Newman, 2017).

Synapses of both inputs were found to differ in key properties, with Is synapses having larger quantal sizes, less total spontaneous release, higher Pr, and short-term depression (as opposed to facilitation in Ib synapses) during high-frequency trains. The difference in short-term plasticity between the inputs is consistent with electrophysiological analysis of responses to separate stimulation of Is or Ib axons (Lnenicka, 2000; Lu, 2016), as well as modeling. These resemble input-specific differences seen in the mammalian brain, such as between parallel and climbing fibers converging on Purkinje cells (Dittman, 2000; Mapelli, 2015) or interneurons in the cortex. The tendency to facilitate or depress is shaped by basal Pr, where high Pr results in greater depletion of immediately releasable vesicles leading to depression. Although overall this agrees with the observation that Is synapses have higher Pr and depress, it was found that despite overlapping distributions of single-synapse Pr, overall release tended to depress in Is and facilitate in Ib, suggesting additional differences in regulation. This greater complexity comes into stark relief when the behavior of hundreds of individual synapses is examined during high-frequency trains of presynaptic firing. This study found that the heterogeneity of short-term plasticity within an input is much greater than was previously appreciated. Local plasticity dynamics were catagorized by how transmission changed during a stimulus train, and it was found that the inputs differ only in two categories, Is inputs having a larger number of active sites that depress and Ib having a larger number of silent sites that are recruited to a releasing state. These observations reveal a previously unknown level of specialization in basal release and plasticity between neighboring synapses of a common input (Newman, 2017).

The Drosophila larval NMJ has proven to be a powerful system for studying synaptic homeostasis. The mechanism of this homeostasis is that reduction in quantal size (because of reduced GluR conductance that results from either mutation of a GluR subunit or partial pharmacological block) triggers a retrograde signal that leads to increased transmitter release, restoring the normal the normal level of excitatory drive (Davis, 2015). The current results reveal a new aspect to homeostatic plasticity by showing that it is specific to input and acts primarily at synapses with certain properties of basal transmission, short-term plasticity and physiological function. Only glutamate release from the Ib input is boosted during homeostatic compensation. This is attributed to the fact that despite the smaller quantal size and unitary Ca2+ influx at Ib synapses, Ib inputs have longer bouts of activity, resulting in much larger aggregate Ca2+ elevation in the Ib postsynapse during locomotion. The larger Ca2+ influx drives a higher activation of CaMKII in the Ib postsynapse. Critically, mutation of the GluR subunit to reduce quantal size at both Ib and Is synapses exclusively reduces activated CaMKII at the Ib postsynapse. Why there is no reduction in activated CaMKII at the Is postsynapse is not clear, though nonlinearity in the relationship between the Ca2+ concentration profile and CaMKII activation (Stratton, 2013) and differences in GluR subunit composition that influence Ca2+ influx may contribute (Newman, 2017).

A second mechanistic difference ensures exclusivity for homeostatic signaling to the Ib input: even when CaMKII activity is inhibited throughout the muscle, only Ib presynapses are boosted in Pr. This could mean that Is presynapses are not responsive to the homeostatic signal. Alternatively, it could mean that Is postsynapses are incapable of generating the homeostatic signal, an idea that would be consistent with the finding that elimination of release from Is axons does not induce homeostatic compensation at Ib synapses. However, this would also require that the retrograde homeostatic signal act very locally so that it could not travel even a few microns from signaling-capable Ib postsynapses to Is boutons that are often located very close by. Another possibility is that basal Pr is high at Is synapses partly due to lower levels of activity in vivo resulting in greater homeostatic enhancement in presynaptic release, which in turn could occlude further increases in synaptic reliability following changes in GluR composition or global postsynaptic CaMKII inhibition (Newman, 2017).

Evidence for signaling compartmentalization can be seen in the structural differences between Ib and Is NMJs, particularly on the postsynaptic side. Ib axons are surrounded by a significantly thicker and more elaborate SSR. There are also substantial differences in the localization of key organizing postsynaptic proteins such as Dlg which is present at higher levels in Ib postsynapses. CaMKII activity has also been shown to affect SSR structure and Dlg localization. Thus, the different activity patterns during native behaviors may also directly regulate postsynaptic properties other than presynaptic release to maintain functional diversity (Newman, 2017).

The presynaptic changes that mediate homeostatic compensation in Pr are only partly known. Homeostasis has been shown to be accompanied by an increase in presynaptic AP-evoked Ca2+ elevation in Ib boutons. Although a number of molecules have been demonstrated to play a role in mediating presynaptic homeostatic compensation (Davis 2015), it remains to be determined how these mechanisms interact with input-specific differences in strength and plasticity (Newman, 2017).

Although homeostasis has been generally thought to be a global mechanism to regulate synaptic strength, evidence has emerged that homeostasis can be regulated with variable degrees of specificity. This study has now demonstrated that homeostatic changes can also propagate retrogradely to the presynaptic neuron in an input-specific and synapse-autonomous manner. Given the divergent innervation of multiple muscles by Is inputs and unique innervation by Ib inputs, a Ib-specific homeostatic mechanism can provide greater functional flexibility (Newman, 2017).

By combining in vivo, physiological measurements of glutamate release with high-resolution quantal analysis at single synapses in the semi-dissected preparation, this study has clarified the relative properties of convergent glutamatergic inputs in the larva during native behaviors. Importantly, this study has demonstrated that the regulation of basal synaptic strength, short-term plasticity, and homeostasis are shaped in a precise input-specific manner. The Ib input has higher levels of in vivo activity, lower Pr synapses, and a propensity for facilitation by recruiting silent synapses during spike trains. Furthermore, the Ib input is the primary determinant for contraction dynamics in the behaving animal. Consistent with its dominant role, when postsynaptic sensitivity to glutamate is altered at both inputs, there is a selective homeostatic adjustment in the amount of neurotransmitter released from Ib input. CaMKII, localized to the postsynaptic density, is ideally placed to detect local changes in postsynaptic activity at both inputs, and postsynaptic inhibition of CaMKII activity is sufficient to enhance release at the Ib input, demonstrating a high degree of signaling compartmentalization within a single muscle cell. Together, these results demonstrate how synaptic activity at the Drosophila larval NMJ is precisely regulated to ensure both functional diversity and stability (Newman, 2017).

The long 3'UTR mRNA of CaMKII is essential for translation-dependent plasticity of spontaneous release in Drosophila melanogaster

A null mutation of the Drosophila calcium/calmodulin-dependent protein kinase II gene (CaMKII) was generated using homologous recombination. Null animals survive to larval and pupal stages due to a large maternal contribution of CaMKII mRNA, which consists of a short 3'-UTR form lacking regulatory elements that guide local translation. The selective loss of the long 3'UTR mRNA in CaMKII null larvae allows testing its role in plasticity. Development and evoked function of the larval neuromuscular junction are surprisingly normal, but the resting rate of miniature excitatory junctional potentials (mEJPs) is significantly lower in CaMKII mutants. Mutants also lack the ability to increase mEJP rate in response to spaced depolarization, a type of activity-dependent plasticity shown to require both transcription and translation. Consistent with this, overexpression of miR-289 in wild-type animals blocks plasticity of spontaneous release. In addition to the defects in regulation of mEJP rate, CaMKII protein is largely lost from synapses in the mutant. All phenotypes are non-sex-specific and rescued by a fosmid containing the entire wild-type CaMKII locus, but only viability and CaMKII localization are rescued by genomic fosmids lacking the long 3'UTR. This suggests that synaptic CaMKII accumulates by two distinct mechanisms: local synthesis requiring the long 3'UTR form of CaMKII mRNA and a process which requires zygotic transcription of CaMKII mRNA. The origin of synaptic CaMKII also dictates its functionality. Locally translated CaMKII has a privileged role in regulation of spontaneous release which cannot be fulfilled by synaptic CaMKII from the other pool (Kuklin, 2017).

CaMKII is both ubiquitous and abundant. In mammals, CaMKII constitutes around 1% of 96 total brain protein and it is also highly expressed in fly heads. Unsurprisingly, CaMKII has been shown to have a plethora of important functions in the nervous system including roles in multiple stages and forms of learning and memory. At the Drosophila neuromuscular junction (NMJ) the roles of CaMKII encompass both development of the synapse and its activity-dependent plasticity. To date, these functions have been revealed using transgenes encoding CaMK II inhibitors or RNAi to decrease kinase activity or activated forms of the kinase to increase activity (Kuklin, 2017).

Early studies at the larval NMJ showed that global inhibition of CaMKII with a heat shock-inducible inhibitor peptide transgene (hs-ala lines) increased branching and bouton number. This same manipulation also increased the amplitude of evoked currents and blocked paired pulse facilitation. Global inhibition of CaMKII was also associatedwith increased presynaptic excitability while expression of constitutively active CaMKII in motor neurons suppressed excitability. Subsequent studies looking at postsynaptic inhibition of CaMKII with both ala peptide and another inhibitor (CaMKIINtide) showed that muscle CaMKII activity could stimulate a retrograde signaling pathway which increased quantal content without changes in mEJP amplitude. The larger quantal content correlated with an increase in morphologically identified release sites and, for high levels of CaMKIINtide expression, an increase in mini frequency (Kuklin, 2017).

More recently, presynaptic expression of either ala peptide or CaMKII RNAi was shown to block activity-dependent bouton sprouting. The abundance of roles is consistent with the presence of the kinase at high levels on both sides of the NMJ, and in multiple cellular compartments (Kuklin, 2017).

The use of genetics to investigate the role of signal transduction molecules in neuronal function has been standard practice for many years in Drosophila. Although Drosophila CaMKII was cloned over 20 years ago, the location of the gene on heterochromatin-rich chromosome complicated standard mutational approaches. To begin genetic analysis of CaMKII, a null mutation was generated in the CaMKII gene by homologous recombination, inserting two stop codons into the N-terminal coding sequence. This study shows that this mutation is comp letely lethal before adulthood in the homozygous state (Kuklin, 2017).

Homozygous mutant animals survive into late larval and pupal stages, due to a large amount of maternally contributed CaMKII mRNA which has a short 3'UTR lacking regulatory information, including binding site for miR-289 which are present in the long form. The fact that null animals survive to pupate, and show essentially normal morphological development of the neuromuscular junction (NMJ), implies that maternally-derived short 3'UTR CaMKII is able to support the majority of basic processes. Indeed transgenic expression of the short 3'UTR form can partially rescue viability indicating that lack of CaMKII protein is the cause of lethality rather than some critical role of the long 3'UTR form of the mRNA. Third instar null animals, however, lack the synaptic enrichment of CaMKII seen in wild-type animals. They also show very specific defects in miniature excitatory junctional potentials (mEJPs) and do not exhibit transcription/translation-dependent plasticity of mEJP frequency. CaMKII derived from newly transcribed mRNA can rescue synaptic localization and viability independent of the 3'UTR, but plasticity of mEJPs requires the long 3'UTR mRNA. Consistent with this, suppression of CaMKII translation in wild-type animals by overexpression of miR-289 also blocks mEJP plasticity. These results argue that synaptic localization of CaMKII can occur via multiple mechanisms, and that locally translated kinase has a special role in plasticity (Kuklin, 2017).

Maternal CaMKII mRNA allows initiation of normal development in null larvae but cannot support metamorphosis. Given the numerous and important functions of CaMKII, it is not unexpected that loss of CaMKII is lethal by adulthood. Surprisingly, however, these mutants appear to be fairly normal with respect to the structure and function of the nervous system in larval stages. This is likely because Drosophila embryos receive large amounts of mRNA from maternally-derived support cells in the ovary. Because the maternal genotype is CaMKII w^+/+, this means that even genetically null oocytes will contain mRNA encoding CaMKII. This mRNA is able to provide normal initial levels of the protein, and accordingly there is no lethality during embryonic development. By late larval stages the amount of CaMKII falls, reflecting either degradation or dilution of maternally encoded kinase. By the time animals reach third instar, functional problems can beseen at the NMJ and there is significant lethality. The severely reduced CaMKII level in pupal stages blocks the ability to complete metamorphosis. Production of new CaMKII mRNA is clearly necessary for continued viability as the animal enters this stage (Kuklin, 2017).

One major difference between the maternal and zygotic mRNAs is that the maternal message has a truncated 3'UTR and lacks sequences known to confer post-transcriptional regulation. Interestingly, viability does not seem to require the long 3'UTR . Animals containing either a rescue fosmid lacking long UTR sequences or expressing a neuronal transgene with a truncated UTR are able to reach adulthood and reproduce. This suggests that producing new protein is sufficient for survival through metamorphosis and that the long UTR form has a specialized role. This underscores the need for future studies to utilize cell-specific and temporally-controlled genetic manipulations of kinase protein and mRNA structure (Kuklin, 2017).

The CaMKII null NMJ phenotype differs from that of animals expressing CaMKII inhibitors. The ability to obtain CaMKII null third instar larvae allowed characterization both the structure of the NMJ and its function in the absence of zygotic transcription. What was immediately obvious was that the phenotypes of the null animals did not resemble the phenotypes reported for animals expressing CaMK II inhibitors or RNAi, manipulations that should affect CaMKII activity levels regardless of the mRNA template. Null animals had no obvious morphological defects or changes in excitability or evoked release, only a decrease in the rate of spontaneous release. In contrast, in animals with strong postsynaptic inhibition of CaMKII an increase in mini rate was reported, likely due to an increase in the number of presynaptic release sites. In the CaMKII null, the number of release sites, as assessed by staining for Brp, is unchanged indicating that the decrease in minis is due to an alteration in release probability (Kuklin, 2017).

These qualitatively distinct phenotypes suggest that inhibition of CaMKII enzymatic activity is not the same as loss of zygotic transcription on a background of maternally-provided kinase. On the face of it, these differences are surprising since both types of manipulation, transgenic inhibition of CaMKII and loss of new transcription of the gene, should produce animals with reduced CaMKII enzyme activity. What might account for these differences? One possibility is that the absolute levels of CaMKII activity might be different. This would imply that different magnitudes of activity loss have qualitatively distinct effects. This possibility seems unlikely, however, given previous findings with the heat shock driven ala lines where different levels of peptide inhibitor were tested: high and low levels of ala expression did not differ qualitatively, only in severity. A second possibility is that the time window in which CaMKII activity is lost is the key difference between the two manipulations. Expression of inhibitors using GAL4 lines that turn on early in development could reduce activity earlier than slow depletion of maternal mRNA does. This would imply that early loss of CaMKII activity has qualitatively different effects than later loss. A third possibility is that RNAi and inhibitor peptides have off-target effects, perhaps on CaMKI, a poorly studied enzyme in the fly. A fourth possibility, and one that is favored, is that inhibition and mutation might be different because they disrupt distinct pools of CaMKII which are specified by both transcriptional and translational mechanisms (Kuklin, 2017).

How is CaMKII from newly transcribed mRNA distinct from that encoded by maternal mRNA? Maternal CaMKII mRNA differs in two ways from zygotic mRNA. First, it differs in structure. Drosophila CaMKII has multiple polyadenylation sites and can have either a short or long 3'UTR. Based on publicly available RNA seq data sets and 3' RACE PCR, mRNA from 0-2 h embryos (which reflects maternal contribution) contains exclusively the short 3'UTR. The RNA seq data also suggest it originates from a distinct transcription start site and differs in its 3'UTR. The second difference is that zygotic mRNA has a different history. Maternal mRNA is synthesized in the nuclei of ovarian nurse cells and never sees the inside of a neuronal nucleus. For CaMK II and other mRNAs the association with mRNA transport machinery occurs in the nucleus. Newly transcribed nuclear mRNAs therefore have preferential access to the machinery that mediates RNA localization. This machinery can be cell type-specific and change over development, meaning that maternal mRNA, even if it has the correct regulatory sequences, may not be competent to localize correctly. Thus while maternal and zygotic CaMKII mRNAs encode the same protein, they do not contain the same regulatory information and may not have the same access to localization or processing factors (Kuklin, 2017).

How do these two differences influence neuronal structure and function? CaMKII null mutants have two obvious defects: a decrease in basal and stimulated mEJP rate and a lack of synaptically localized CaMKII. These two deficits appear to be mechanistically distinct. Rescue of synaptic localization was seen with both the WT gene and a fosmid lacking long UTR sequences. Localization is therefore 3'UTR -independent but appears to require newly transcribed mRNA. Whether this is due to an mRNA-based mechanism (transport of mRNA to synaptic sites and local translation), or whether it is due to preferential transport or diffusion of protein synthesized in the soma from new mRNA templates, will require further investigation (Kuklin, 2017).

In contrast to synaptic CaMKII localization, the presence of the long 3'UTR is absolutely required for establishing a normal basal level of spontaneous release, and for activity-dependent increases in mEJP rate. This plasticity is translation-dependent and is suppressed by miR-289 which has been previously shown to regulate activity-dependent presynaptic synthesis of CaMKII at the NMJr. The partial suppression seen with pre synaptic miR-289 could be due to relative expression levels of the mRNA and miR or to a requirement for other regulators. The postsynaptic suppression of plasticity points to involvement of CaMKII- dependent retrograde signaling in spontaneous release. Taken together, however, these data imply that there is a population of synaptic long 3'UTR CaMKII mRNA that is locally translated and acts to increase the probability of release. Why newly translated CaMKII is required is unknown, but in rodent neurons, synaptically synthesized CaMKII has preferential access to certain binding partners. These results also revealed differences be tween the NMJ and adult olfactory system synapses. In adult projection neurons, the 3'UTR was required for both localization and activity- dependent regulation of CaMKII translation in dendrites. Further investigation of the mechanisms of RNA and protein localization will be required to resolve these differences, but it is likely that there will be multiple mechanisms for regulation of synaptic CaMKII levels (Kuklin, 2017).

Activity-dependent synthesis of CaMKII is clearly a critical feature of the enzyme and is conserved across species and developmental stages. Previous work in the adult fly brain has shown that CaMKII mRNA contains sequences that regulate activity-dependent translation. Importantly, this conserved in mammals where 3'UTR sequences in the CAMK2A gene have been shown to drive localization and activity-dependent translation, though it has been suggested that there may also be a role for 3'UTR sequences. The fly will provide a powerful model system for understanding how and why CaMKII is targeted to multiple subcellular compartments. The discovery that local translation of CaMKII is a key driver of plasticity of mini rate also provides a foothold for obtaining an understanding of this process. Spontaneous release is increasingly being recognized as mechanistically and functionally distinct from evoked release (Kuklin, 2017).

The regulation of spontaneous release, and even the sites at which it occurs, are separate from action potential evoked activity. These miniature events can regulate nuclear gene expression, local translation and participate in developmental processes such as circuit wiring. The dependence of activity-dependent plasticity of spontaneous release on local translation of CaMKII on both sides of the synapse suggests that there are complex mechanisms for fine tuning this important type of synaptic activity (Kuklin, 2017).

Presynaptic DLG regulates synaptic function through the localization of voltage-activated Ca(2+) channels

The DLG-MAGUK subfamily of proteins plays a role on the recycling and clustering of glutamate receptors (GLUR) at the postsynaptic density. discs-large1 (dlg) is the only DLG-MAGUK gene in Drosophila and originates two main products, DLGA and DLGS97 which differ by the presence of an L27 domain. Combining electrophysiology, immunostaining and genetic manipulation at the pre and postsynaptic compartments, this study examined the DLG contribution to the basal synaptic-function at the Drosophila larval neuromuscular junction. The results reveal a specific function of DLGS97 in the regulation of the size of GLUR fields and their subunit composition. Strikingly the absence of any of DLG proteins at the presynaptic terminal disrupts the clustering and localization of the calcium channel DmCa1A subunit (Cacophony), decreases the action potential-evoked release probability and alters short-term plasticity. These results show for the first time a crucial role of DLG proteins in the presynaptic function in vivo (Astorga, 2016).

dlg1 is the only gene of the DLG-MAGUK subfamily in Drosophila. Similar to vertebrate genes, two forms of the gene are expressed as the result of two transcription start sites. DLGA (α form) and DLGS97 (β form) are distinguished by the inclusion of an L27 domain in beta forms located in the amino terminus of the protein. In vertebrates DLG4/PSD95 is predominantly expressed as α form while DLG1/SAP97 is mainly expressed as β form. DLGA is expressed in epithelial tissues, somatic muscle and neurons; in turn, DLGS97 is not expressed in the epithelial tissue. In the larval neuromuscular junction (NMJ), a glutamatergic synapse, both dlg products are expressed pre and postsynaptically. Hypomorphic dlg alleles display underdeveloped subsynaptic reticulum, bigger glutamate receptors fields and an increased size of synaptic boutons, active zones and vesicles. Additionally to these morphological defects, altered synaptic responses such as increased excitatory junction currents (EJC) and increased amplitude of miniature excitatory junction potentials have been observed. The strong morphological defects make difficult to distinguish developmental defects from the role of DLGs in the basal function of the mature synapse. Previously studies have reported form-specific null mutant strains for DLGA (dlgA^40.2) and DLGS97, (dlgS97⁵). These mutants do not show the gross morphological defects observed in hypomorphic mutants, although still show functional synaptic defects, supporting a role of DLG proteins in the mature synaptic function (Astorga, 2016).

Combining genetic, electrophysiology and immunostaining techniques this study dissected the role of DLG proteins at the pre and postsynaptic compartments. The results show the specific requirement of postsynaptic DLGS97 for normal glutamate receptor (GLUR) distribution. In turn, both DLG proteins increase the release probability by promoting voltage-dependent Ca²⁺ channel localization. The results demonstrate for the first time a relevant role to DLG proteins in the presynaptic function contributing to Ca²⁺ mediated short-term plasticity and probability of release (Astorga, 2016).

Flies carrying the severe hypomorph dlg1 mutant allele, dlg^XI-2 and the isoform specific dlgS97 null mutant displayed increased amplitude of the spontaneous excitatory postsynaptic (junctional) potential (mEJP) without changes in frequency. In addition all mutants displayed a decreased quantal content as measured by evoked post-synaptic potentials. The specific defects behind these results were explored. To characterize the synaptic transmission in WT and dlg mutants, post synaptic currents were recorded in HL3.1 solution on muscles 6 or 7 of third instar male larvae of the various genotypes. Recordings of spontaneous excitatory postsynaptic currents (mEJC) were obtained after blocking the voltage activated sodium channels. Thereafter, histogram distributions were constructed with the amplitudes of the miniature events and the quantal size was estimated by the peak value obtained adjusting a Log-Normal distribution in each genotype. It is worth to emphasize that finding a phenotype on dlgA or dlgS97 mutants means that DLGA or DLGS97 proteins by themselves cannot replace DLG function (Astorga, 2016).

The average amplitude of spontaneous postsynaptic potentials were compared and, supporting previous results, it was found that the average amplitude of the mEJC of the mutants dlg^XI-2 (0.99 ± 0.05 nA) and dlgS97 (0.98 ± 0.03 nA) were significantly larger compared to the average amplitudes of the mEJC of Canton-S strain used as WT control (0.81 ± 0.04 nA) and of dlgA (0.78 ± 0.02 nA) specific mutant. The same result was obtained comparing the quantal size. None of the mutants showed a significant change compared to the WT in the frequency of the mEJC. As an additional control, all mutants were recorded over a deficiency covering the dlg gene, finding similar results. These findings are in accordance with the idea that DLGS97 protein and not DLGA is necessary for the quantal size determination (Astorga, 2016).

Bigger quantal size could be of pre or postsynaptic origin as the result of increased neurotransmitter (NT) content in vesicles or increased glutamate receptor field's size respectively. First, to determine the pre or post-synaptic origin of this phenotype, a UAS-dsRNA construct that targets all dlg products, was expressed under the control of the motoneuron promoter OK6-GAL4 or the muscle promoter C57-GAL4. As expected for a post-synaptic defect, the increased quantal size observed in dlgS97 mutants was mimicked only by the decrease of DLG in the muscle. The specific role of DLGS97 in the muscle is supported by the rescue of the dlgS97 mutant phenotype only by the expression of DLGS97 in the muscle and not in the motor neuron. The effect of GAL4 expression was examined in the mutant background in all experiments; neither of the GAL4 lines without the specific UAS constructs changed the phenotype of the mutants. Again, none of the genotypes studied displayed differences with the WT in the frequency of the minis (Astorga, 2016).

Changes in quantal size of postsynaptic origin could be due to higher number of post-synaptic receptors and/or a different composition of the postsynaptic receptors. An increase in the size of glutamate receptors fields has been described in dlg hypomorphic alleles including dlg^XI-2 mutants. Therefore, the size of the glutamate receptor fields was compared among the mutants and with WT, and also the active zones were measured using antibodies for the active zone protein Bruchpilot. Consistently with previous results bigger glutamate receptors fields were found compared to WT only in dlg^XI-2 and dlgS97 mutants but not in dlgA mutants. Surprisingly, an increased number of active zones per bouton was also found in all mutants, a phenotype usually associated with an increase in the frequency of minis that were not observe. In addition, an increased active zone area was found in dlgA and dlg^XI-2 mutants (Astorga, 2016).

As expected for a postsynaptic defect, the bigger size of the glutamate fields in dlgS97 mutants was rescued by the expression of DLGS97 in the muscle but not by its expression in the motor neuron. These results confirm that DLGS97, but not DLGA is responsible for the regulation of the size of the receptors fields in the muscle (Astorga, 2016).

The strict requirement of DLGS97 in the regulation of the size of GLUR fields supports results that have involved other DLGS97 interacting proteins in the regulation of the size of the glutamate receptors fields. METRO, an MPP-like MAGUK protein, has been shown to form a complex with DLGS97 and LIN-7 through the L27 domains present in each of the three proteins. metro mutants display decreased DLGS97 at the synapse and larger GLUR receptors fields than WT, even bigger than dlgS97 mutants. METRO and DLGS97 depend on each other for their stability on the synapse, thus, in dlgS97 mutants, METRO and dLIN-7 are highly reduced at the synapse. The similar post-synaptic phenotype of metro and dlgS97 and the reported interaction between these two proteins suggests the proposal that the increase size of GLUR fields is consequence of the loss of METRO due to the loss of DLGS97 protein (Astorga, 2016).

As stated before, changes in quantal size of postsynaptic origin can also reflect a different composition of the receptors. Drosophila NMJ GLUR receptors are tetramers composed by obligatory subunits and two alternative subunits, GLURIIA and GLURIIB. Receptors composed by one of these two subunits differ in their kinetics; GLURIIB receptors desensitizes faster than GLURIIA receptors. Thus, the kinetic of the spontaneous currents (mEJCs), is associated to the relative abundance of these two types of receptors in the GLUR fields. It has been shown that the abundance of GLURIIB but not of GLURIIA in the synapse is associated with the expression of dlg. To analyze if dlg mutants display a change in the composition of the subunits abundance relative to the control, the kinetics of the mEJCs were studied. Kinetics analyses of the mEJCs revealed that only dlgS97 and the double mutant display a slower kinetic in the off response, which is compatible with a different composition of the glutamate receptors fields regarding the proportion between GLURIIA and B receptors. The value of tau also increased in larvae expressing dsRNA-dlg in the muscle, but not by its expression in the motor neuron. Finally tau-off values recovered the WT value only with the expression of DLGS97 in the muscle. As slower mEJCs were observed, the results suggest an increase in the ratio of GLURIIA/GLURIIB. It is known that receptors containing the GLURIIA subunit display bigger conductance and slower inactivation kinetics than receptors containing the GLURIIB subunit. Thus, synapses with post-synaptic receptors fields containing proportionally less GLURIIB subunits would display bigger and slower mEJCs similar to the phenotype observed in dlgS97 mutants. To confirm this hypothesis, the abundance of GLURIIA and GLURIIB receptors was evaluated by immunofluorescence in the NMJ of WT and dlg mutant larvae. The immunofluorescence that allowed the detection and quantification of GLURIII and GLURIIB fields was performed with paraformaldehyde (PFA) fixative. However, the immunofluorescence to detect GLURIIA receptors only works fixating the tissue with Bouin reagent. Thus, in order to be able to compare between these two fixations, the size of the GLUR fields was normalized by the HRP staining that labels the whole presynaptic bouton. First, as a control, GLURIIA and GLURIII were double stained in the same larvae. The results show that using PFA fixative, GLURIII fields display bigger size only in dlgS97 mutants and not in dlgA mutants. Even more, as predicted from the kinetic data, only dlgS97 and not dlgA mutants display bigger GLURIIA fields while there are not difference in the size of GLURIIB fields between WT and the mutants. Additionally the results show no difference in the number of GLURIIA or GLURIIB clusters between WT and dlg mutants. Immunohistochemical results confirm the prediction from the electrophysiological data revealing that in dlgS97 mutants, GLURIIA subunits are proportionally more abundant in GLUR fields than in control larvae. In conclusion, the results show that dlgS97 mutants display larger quanta and mEJCs with slower kinetic establishing its participation in the regulation of the size of GLUR fields where the increased size is obtained mainly through the recruitment of receptors containing GLURIIA subunits. As a similar result was obtained in another study that observed that the loss of GLURIIB receptors in the NMJ of dlg^XI-2 mutant embryos, these observation suggest that either of the two DLG proteins are necessary for the localization of GLURIIB in the synapse but only DLGS97 is actively limiting the size of the clusters by regulating the number of GLURIIA receptors (Astorga, 2016).

Taking into account previous reports that show the regulation of the synaptic localization of DLG by CAMKII, the regulation of the subunit composition by CAMKII and these results, a mechanism is proposed by which, after a strong activation of CAMKII, the phosphorylation of DLGS97 would detach it from the synapse allowing the increase of the size of the GLUR fields by the recruitment of GLURIIA over GLURIIB. These changes should increase the synaptic response by two different mechanisms (Astorga, 2016).

To determine if DLG proteins modulate the presynaptic release probability, excitatory junction currents (EJC) were recorded in the muscle by stimulating the nerve at 0.5 Hz in low extracellular Ca²⁺ (0.2 mM), both conditions to avoid synaptic depression. For all mutant genotypes the average peak amplitude and quantal content (EJC amplitude/quantal size) of the evoked responses were significantly smaller than WT. In congruence with previous results, the lower amplitude of the current response is accompanied by a decrease in the quantal content. Taking into account the results on the size of the GLUR fields in the mutant's muscles, these results are compatible with a reduction of the neurotransmitter release in dlg mutants. A decreased neurotransmitter release could be associated with a decreased number of release sites in the boutons. However, the number of active zones per bouton is increased in all dlg mutants with bigger active zones in dlg^XI-2 and dlgA mutants (Astorga, 2016).

The decrease in the evoked response could be a consequence of the absence of the specific form of DLG in the postsynaptic side, transmitted by unknown mechanisms or, alternatively, it could be the result of an effect of DLG on the probability of release. In order to explore where this phenotype originates (pre or post-synaptically) DLG levels were downregulated by expressing dsRNA against all forms of dlg. Compatible with a presynaptic defect, the expression of UAS-dsRNA-dlg presynaptically decreases the amplitude of the evoked response while the same construct expressed postsynaptically using C57 promoter did not changed the amplitude of the EJCs. The presynaptic expression of the dsRNA-dlg also mimics the reduction in quantal content of the mutants, displaying a severe reduction in this parameter. On the other hand, the postsynaptic expression of the dsRNA-dlg associates to a moderate but significant decrease on the quantal content, as expected from the effect already reported of the postsynaptic dsRNA-dlg on the quantal size and the lack of effect on the amplitude of the EJCs. The presynaptic effect of DLG is supported further by the rescue experiments. Thus, the amplitude of the evoked response and the quantal content in dlgA^40.2 mutant is completely rescued by the selective expression of DLGA in the presynaptic compartment but not by its expression in the postsynaptic compartment. The pre-synaptic expression of DLGS97 improves the synaptic function increasing the average size of the EJCs such that the difference between the WT and the presynaptic-rescue is not significant, suggesting a complete rescue. However, the average EJC in the presynaptic rescue is not different either from the control mutant animal, which is interpreted as the rescue not being complete and thus the term partial rescue is used. DLGS97 does not, however rescued at al the quantal content. This is explained because although the amplitude of the current increased, the quantal size remains unchanged by the presynaptic expression of DLGS97. In consequence the quantal content does not increase as much as the current. On the other hand, the postsynaptic expression did not increase the amplitude of the evoked current. However, since it does rescue the quantal size the quantal content augmented enough to be different from the mutant control. Notably, DLGA expressed presynaptically in dlgA^40.2mutants not only rescued the EJC amplitude but also the number of active zones per bouton and the size of the active zones. On the other hand DLGS97 expression only partially rescued the increased number of active zones in dlgS97⁵ mutants. These results support a role of DLG proteins in the presynaptic function where DLGA seems to regulate more aspects than DLGS97. Despite the fact that both forms of DLG share most of their protein domains, neither of the two-forms is able to fully rescue the absence of the other, suggesting that both of them participate in a complex. The binding between the SH3 and GUK domains of MAGUK proteins has been described; this interaction (at least in vitro) is able to form intra or intermolecular associations and offers a mechanism by which DLGA and DLGS97 proteins could be associated to recruit proteins to a complex (Astorga, 2016).

Changes in the overall quantal content at these synapses may reveal presynaptic defects. However, genetic background and other independent modification could alter apparent release. To independently scrutinize alteration in the presynaptic release probability two presynaptic properties were examined, the short-term plasticity and the calcium dependency of quantal release (Astorga, 2016).

To explore the EJC phenotype observed in dlg mutants, stimulation paradigms were carried out that allow characterization of aspects of the short term plasticity that are known to depend on presynaptic functionality and give clues about the mechanisms involved in the observed defects. First, the response were studied of the mutants to high frequency stimulation, 150 stimuli at 20 Hz. WT responses at high frequency stimulation show a fast increase in the amplitude of the response that then slows down. The fast initial increase is called facilitation and the second phase with smaller slope is called augmentation. The time constant of the facilitation is believed to reflex the calcium dynamics in the terminal and its slope to be the product of the accumulation of calcium and the consequent calcium dependent increase in the probability of release. The fractional increment in the mutants' responses showed an increased facilitation in all mutants, while an increased augmentation was only significant in dlgA mutants compared to WT. Additionally all mutants showed a trend toward steeper slopes than WT, but only the augmentation slope in dlgA mutants reached statistical significance. These results support that the mutants display a lower probability of release than WT, which could reflect defects in the calcium dynamics or in the response to calcium (Astorga, 2016).

Previous work in dlg mutants did not report defects in short-term plasticity. These works differ from the current one in methodological aspects, mainly that they were carried out in a media with high concentration of magnesium (20 mM) and calcium (1.5 mM). This work was carried out in a media containing low magnesium (4 mM) and calcium (0.2 mM) concentration. It is known that magnesium reduces neurotransmitter release, probably due to partial blockade of VGCC. Additionally, magnesium permeates more than sodium and potassium through GLURs (Astorga, 2016).

To better evaluate the calcium dynamics in the terminal pair pulse (PP) experiments, a well-known paradigm to evaluate presynaptic calcium dynamics, were carried out. In PP, a second depolarization shortly after the first one carried out in low extracellular calcium concentration elicits an increased release of neurotransmitter thought to reflect the increased calcium concentration in the terminal reached after the first stimulus. According to this, and posing as the working hypothesis that DLG affects presynaptic calcium dynamics, a second pulse would be expected to increase the release in a bigger proportion, since the first stimulus did not release much of the ready releasable pool. Conversely, a second stimulus given at high calcium concentration produces a decrease in the release of neurotransmitter, which is considered to originate in the partial depletion of the ready releasable pool at the release sites. Thus, a second pulse at high calcium concentration should elicit a smaller decrease of the release since an inferior entrance of calcium should produce less depletion of the ready releasable pool of vesicles (Astorga, 2016).

Consistently with a decreased calcium entrance, all mutants displayed increased pair pulse facilitation at low calcium concentration and decreased pair pulse depression at high calcium concentration. These results support a defect in the calcium entrance to the terminal as the underlying defect in dlg mutants causing the evoked stimuli defects. To characterize the calcium dependency of the release in the mutants the evoked responses were measured at different calcium concentrations. It can be observed that for all the mutants and at most calcium concentrations, the quantal content of the evoked response is lower than the control. The only exception is seen at 2 mM calcium where the quantal content of the dlgA mutants and the control are not different to each other. However, even at this calcium concentration the quantal content of dlgS97 and the double mutant dlg^XI-2 are significantly lower than the control. To get insight about the release process the responses were fit to a Hill equation. This type of fitting better estimate the maximum responses and the EC50, which is masked in the overall release of different backgrounds. This is observed in the graph with the normalized responses by the maximal quantal predicted. The adjusted curves show that mutants reach the theoretical maximal quantal content at higher calcium concentration than the WT and that the EC50 for the mutants is diminished respect to the WT. To confirm the presynaptic origin of the defect in the calcium dependency, the calcium dependency was carried out in the mutant genotypes expressing DLGA or DLGS97 pre or postsynaptically. The quantal content analysis shows that only the presynaptic expression of DLGA in dlgA mutants completely rescued the calcium dependency, in line with previous results that show the importance of DLGA in the presynaptic compartment. On the other hand the presynaptic expression of DLGS97, although it rescued the calcium dependency, failed to rescue the maximal quantal content. Observing the graph with the normalized responses, DLGA as well as DLGS97 both are able to restore the WT calcium dependency. The inability to rescue the maximal quantal content could be explained by the existence of synaptic compensatory mechanisms that allow to counterweigh the bigger quantal size in dlgS97 mutants, which were shown before not to be rescued by the presynaptic expression of DLGS97 (Astorga, 2016).

Facilitation is thought to depend on the resultant of the calcium entrance, calcium release from intracellular stores and the clearance of cytosolic calcium. So, the defects in facilitation observed in the mutants could be due to a decreased calcium entrance but also they could be due to a defect on the clearance of calcium. In a preliminary experiment, the relative changes were measured of the total intracellular calcium concentration in the bouton using the genetically encoded calcium indicator GCamp6f. GCamp6f expressed in control flies (OK6-GAL4/UAS-GCamp6f) respond with a fast and transitory change in the cytoplasmic calcium of the boutons when they are exposed to a local pulse of potassium. The same experimental approach in dlgS97 mutant larvae reveals that the rise of the calcium response is significantly slower than the control; additionally the recovery of the response is also significantly slower. These preliminary experiments suggest a defect in calcium entrance in the mutants but they also support a defect in the extrusion that hint to additional defects. Further experiments are needed to clarify the calcium kinetics involved since these experiments were measuring the bulk of calcium change and in doing this approximation, the nanodomain changes that are known to be the ones that regulate the neurotransmitter release are being lost (Astorga, 2016).

Since the results described above including the calcium dependency of the release as well as the parameters of the short-term plasticity suggest that the calcium entrance to the terminal is impaired, a view that is supported by the preliminary data measuring the cytosolic calcium, the next experiments focused on the calcium entrance. The main calcium entry to the terminal is the voltage gated calcium channel (VGCC) encoded by the Drosophila gene cacophony. Advantage was taken of a UAS-cacophony1-EGFP transgenic fly (CAC-GFP) to study the distribution of the channel in WT and mutant genotypes. CAC-GFP overexpressed in WT background localizes in the synapse in a strictly plasma membrane-associated manner in big clusters closely associated with release sites. However, CAC-GFP overexpressed in dlgS97 or dlgA mutant background displays a significant decrease in the expression accompanied by a more disperse localization with significantly smaller clusters, suggesting that the Cacophony protein might not be properly delivered or anchored to the plasma membrane in dlg mutants (Astorga, 2016).

It was reasoned that if dlg mutants had a defect on calcium entrance, the over expression of calcium channels should rescue at least partially the phenotype. Advantage was taken of the fact that CAC-GFP construct encodes a functional channel, and recordings were taken from control and dlg mutants overexpressing CAC. As expected and supporting a decreased calcium entrance in the mutants, dlgS97 and dlgA mutants that overexpress CAC-GFP display significantly bigger evoked EJCs compared to dlg mutants, without a change of phenotype in the spontaneous currents. Additionally, the over expression of CAC-GFP partially rescued the pair pulse facilitation and the pair pulse depression as well as the calcium curve (Astorga, 2016).

The disrupted localization of CAC could result from the disturbance of a normal direct association to DLG or it could be affected indirectly. To test an immunoprecipitation assay was carried out using flies that express CAC-EGFP in all neurons. Antibodies against GFP were able to precipitate DLG together with Cacophony-GFP, supporting that Cacophony channel is part of the DLG complex in the boutons (Astorga, 2016).

A possible interaction between DLG and voltage-gated calcium channels (VGCCs is) the VGCC auxiliary subunits. The α₂δ auxiliary subunit (Straightjacket in Drosophila) increases calcium channel activity and plasma membrane expression of CaV2 α₁ subunits and Cacophony. The β auxiliary subunit increases plasma membrane expression of several mammalian VGCC classes. Intriguing β subunits are also MAGUK proteins and they are able to release the VGCC α subunit from the endoplasmic reticulum retention. It may be speculated that DLG through their SH3-GUK domain might be playing the role of the β subunit (Astorga, 2016).

On the other hand, in mammalian cultured neurons it has been proposed that a complex formed by the scaffold proteins LIN-2/CASK, LIN-10/MINT and LIN-7 is involved in the localization of VGCCs at the synapse and that SAP97 forms a complex with CASK. The association of DLGS97 with LIN7 has been reported in the postsynaptic compartment in the Drosophila NMJ. Furthermore, an association between the L27 domain of DLGS97 and the L27 domain of Drosophila CASK has been shown in vitro, however there are no reports of this type of association with DLGA. Another protein involved in the localization of calcium channels in the active zone is RIM. Drosophila rim has been involved in synaptic homeostasis and the modulation of vesicle pools. Surprisingly rim mutants, display low probability of release and altered responses to different calcium concentrations. Recently it was shown that spinophilin mutants display a phenotype with bigger quantal size and GLUR fields size with a higher proportion of GLURIIA subtype of receptors as well as decreased EJCs and decreased pair pulse facilitation. This is a phenotype very similar to the one described here for dlg mutants. The authors in this report did not explore the calcium channels abundance or distribution and the current study did not explore the link of DLG to Neuroligins, Neurexins and Syd. It would be interesting to determine if there is a link between Spinophilin and DLG (Astorga, 2016).

Taken together these results show that dlgS97 is the main isoform responsible for the postsynaptic defects in the dlg^XI-2 mutants; which comprise the increase in the size of the receptors fields and the change in the ratio of GLURIIA/GLURIIB. The results as well support a model in which DLG forms a presynaptic complex that includes Cacophony where the absence of either form of DLG leads to defects in the localization of the voltage dependent calcium channel and to a decrease in the entrance of calcium to the bouton; which in turn affect the probability of release and the short-term plasticity in the mutants. The results described in this study highlight the specificity of the function of DLGS97 and DLGA proteins and describe for the first time an in vivo presynaptic role of DLG proteins (Astorga, 2016).

The Ih channel gene promotes synaptic transmission and coordinated movement in Drosophila melanogaster

Hyperpolarization-activated cyclic nucleotide-gated "HCN" channels, which underlie the hyperpolarization-activated current (Ih), have been proposed to play diverse roles in neurons. The presynaptic HCN channel is thought to both promote and inhibit neurotransmitter release from synapses, depending upon its interactions with other presynaptic ion channels. In larvae of Drosophila melanogaster, inhibition of the presynaptic HCN channel by the drug ZD7288 reduces the enhancement of neurotransmitter release at motor terminals by serotonin but this drug has no effect on basal neurotransmitter release, implying that the channel does not contribute to firing under basal conditions. This study shows that genetic disruption of the sole HCN gene (Ih) reduces the amplitude of the evoked response at the neuromuscular junction (NMJ) of third instar larvae by decreasing the number of released vesicles. The anatomy of the (NMJ) is not notably affected by disruption of the Ih gene. It is proposed that the presynaptic HCN channel is active under basal conditions and promotes neurotransmission at larval motor terminals. Finally, it was demonstrated that Ih partial loss-of-function mutant adult flies have impaired locomotion, and, thus, it is hypothesized that the presynaptic HCN channel at the (NMJ) may contribute to coordinated movement (Hegle, 2017).

A Ca2+ channel differentially regulates Clathrin-mediated and activity-dependent bulk endocytosis

Clathrin-mediated endocytosis (CME) and activity-dependent bulk endocytosis (ADBE) are two predominant forms of synaptic vesicle (SV) endocytosis, elicited by moderate and strong stimuli, respectively. They are tightly coupled with exocytosis for sustained neurotransmission. However, the underlying mechanisms are ill defined. Previous work has shown that the Flower (Fwe) Ca2+ channel present in SVs is incorporated into the periactive zone upon SV fusion, where it triggers CME, thus coupling exocytosis to CME. This study shows that Fwe also promotes ADBE. Intriguingly, the effects of Fwe on CME and ADBE depend on the strength of the stimulus. Upon mild stimulation, Fwe controls CME independently of Ca2+ channeling. However, upon strong stimulation, Fwe triggers a Ca2+ influx that initiates ADBE. Moreover, knockout of rodent fwe in cultured rat hippocampal neurons impairs but does not completely abolish CME, similar to the loss of Drosophila fwe at the neuromuscular junction, suggesting that Fwe plays a regulatory role in regulating CME across species. In addition, the function of Fwe in ADBE is conserved at mammalian central synapses. Hence, Fwe exerts different effects in response to different stimulus strengths to control two major modes of endocytosis (Yao, 2017).

A tight coupling of exocytosis and endocytosis is critical for supporting continuous exocytosis of neurotransmitters. CME and ADBE are well-characterized forms of SV endocytosis triggered by moderate and strong nerve stimuli, respectively. However, how they are coupled with exocytosis under distinct stimulation paradigms remains less explored. A model is proposed based on the present data. When presynaptic terminals are mildly stimulated, SV release leads to neurotransmitter release and the transfer of Fwe channel from SVs to the periactive zone where CME and ADBE occur actively. The data suggest that this channel does not supply Ca²⁺ for CME to proceed. However, intense activity promotes Fwe to elevate presynaptic Ca²⁺ levels near endocytic zones where ADBE is subsequently triggered. Thus, Fwe exerts different activities and properties in response to different stimuli to couple exocytosis to different modes of endocytosis (Yao, 2017).

It has been previously concluded that Fwe-dependent Ca²⁺ influx triggers CME (Yao, 2009). However, the current results suggest alternative explanations. First, the presynaptic Ca²⁺ concentrations elicited by moderate activity conditions, i.e., 1-min 90 mM K⁺/0.5 mM Ca²⁺ or 20-s 10-20 Hz electric stimulation, are not dependent on Fwe. Second, expression of 4% FweE79Q, a condition that abolishes Ca²⁺ influx via Fwe, rescues the CME defects associated with fwe mutants, including decreased FM1-43 dye uptake, a reduced number of SVs, and enlarged SVs. Third, raising the presynaptic Ca²⁺ level has no beneficial impact on the reduced number of SVs observed in fwe mutants. These data are consistent with the observations that a Ca²⁺ influx dependent on VGCCs triggers CME at a mammalian synapse. Hence, Fwe acts in parallel with or downstream to VGCC-mediated Ca²⁺ influx during CME (Yao, 2017).

ADBE is triggered by intracellular Ca²⁺ elevation, which has been assumed to be driven by VGCCs that are located at the active zones. However, the data strongly support a role for Fwe as an important Ca²⁺ channel for ADBE. First, following exocytosis, Fwe is enriched at the periactive zone where ADBE predominates. Second, Fwe selectively supplies Ca²⁺ to the presynaptic compartment during intense activity stimulation, which is highly correlated with the rapid formation of ADBE upon stimulation. Third, 4% FweE79Q expression, which induces very subtle or no Ca²⁺ upon strong stimulation, fails to rescue the ADBE defect associated with loss of fwe. Fourth, treatment with a low concentration of La³⁺ solution that specifically blocks the Ca²⁺ conductance of Fwe significantly abolishes ADBE. Lastly, the role of Fwe-derived Ca²⁺ influx in the initiation of ADBE mimics the effect of Ca²⁺ on ADBE at the rat Calyx of Held. As loss of fwe does not completely eliminate ADBE, the results do not exclude the possibility that VGCC may function in parallel with Fwe to promote ADBE following intense stimulation (Yao, 2017).

Interestingly, Ca²⁺ influx via Fwe does not control SV exocytosis during mild and intense stimulations. How do VGCC and Fwe selectively regulate SV exocytosis and ADBE, respectively? One potential mechanism is that VGCC triggers a high, transient Ca²⁺ influx around the active zone that elicits SV exocytosis. In contrast, Fwe is activated at the periactive zone to create a spatially and temporally distinct Ca²⁺ microdomain. A selective failure to increase the presynaptic Ca²⁺ level during strong stimulation is evident upon loss of fwe. This pinpoints to an activity-dependent gating of the Fwe channel. Consistent with this finding, an increase in the level of Fwe in the plasma membrane does not lead to presynaptic Ca²⁺ elevation at the Calyx of Held when the presynaptic terminals are at rest or subject to mild stimulation. However, previous studies showed that, in shi^ts terminals, blocking CME results in the accumulation of the Fwe channel in the plasma membrane, elevating Ca²⁺ levels. It is possible that Dynamin is also involved in regulating the channel activity of Fwe or that the effects other than Fwe accumulation associated with shi^ts mutants may affect intracellular Ca²⁺ handling. Further investigation of how neuronal activity gates the channel function of Fwe should advance knowledge on the activity-dependent exo-endo coupling (Yao, 2017).

Although a proteomic analysis did not identify ratFwe2 in SVs purified from rat brain, biochemical analyses show that ratFwe2 is indeed associated with the membrane of SVs. The data show that 4% of the total endogenous Fwe channels efficiently promotes CME and ADBE at the Drosophila NMJ. If a single SV needs at least one functional Fwe channel complex during exo-endo coupling, and one functional Fwe complex comprises at least four monomers, similar to VGCCs, transient receptor potential cation channel subfamily V members (TRPV) 5 and 6, and calcium release-activated channel (CRAC)/Orai1, then it is anticipated that each SV contains ~100 Fwe proteins (4 monomers x 25). This suggests that Fwe is highly abundant on the SVs. It is unlikely that many SVs do not have the Fwe, as a 25-fold reduction of the protein is enough to ensure functional integrity during repetitive neurotransmission. Finally, the results for the SypHy and dextran uptake assays at mammalian central synapses indicate the functional conservation of the Fwe channel in promoting different modes of SV retrieval. In summary, the Fwe-mediated exo-endo coupling seems to be of broad importance for sustained synaptic transmission across species. (Yao, 2017).

Neuroligin 4 regulates synaptic growth via the Bone morphogenetic protein (BMP) signaling pathway at the Drosophila neuromuscular junction

The neuroligin (Nlg) family of neural cell adhesion molecules is thought to be required for synapse formation and development, and has been linked to the development of autism spectrum disorders in humans. In Drosophila melanogaster, mutations in the neuroligin 1-3 genes have been reported to induce synapse developmental defects at neuromuscular junctions (NMJs), but the role of neuroligin 4 (dnlg4) in synapse development has not been determined. This study reports that the Drosophila Neuroligin 4 (DNlg4) is different from DNlg1-3 in that it presynaptically regulates NMJ synapse development. Loss of dnlg4 results in reduced growth of NMJs with fewer synaptic boutons. The morphological defects caused by dnlg4 mutant are associated with a corresponding decrease in synaptic transmission efficacy. All of these defects could only be rescued when DNlg4 was expressed in the presynapse of NMJs. To understand the basis of DNlg4 function, genetic interactions were sought, and connections were found with the components of the bone morphogenetic protein (BMP) signaling pathway. Immunostaining and western blot analyses demonstrated that the regulation of NMJ growth by DNlg4 was due to the positive modulation of BMP signaling by DNlg4. Specifically, BMP type I receptor Tkv abundance was reduced in dnlg4 mutants, and immunoprecipitation assays showed that DNlg4 and Tkv physically interacted in vivo. This study demonstrates that DNlg4 presynaptically regulates neuromuscular synaptic growth via the BMP signaling pathway by modulating Tkv (X. Zhang, 2017).

The formation, development, and plasticity of synapses are critical for the construction of neural circuits, and the Drosophila larval neuromuscular junction (NMJ) is an ideal model system to dissect these processes. In the past few decades, several subcellular events and signaling pathways have been reported to be involved in regulating synaptic growth at Drosophila NMJs, such as local actin assembly, endocytosis, ubiquitin-mediated protein degradation, the Wingless pathway, and the bone morphogenetic protein (BMP) pathway. Among these, BMP signaling is thought to be a major retrograde pathway that promotes the synaptic growth of NMJs (X. Zhang, 2017).

At the Drosophila NMJ, the BMP homolog Glass bottom boat (Gbb) is released by muscle cells and binds to the presynaptic type II BMP receptor Wishful thinking (Wit). Wit is a constitutively active serine/threonine kinase and, upon binding to Gbb, forms a complex with the type I BMP receptor Thickvein (Tkv) or saxophone (Sax), which results in their activation by phosphorylation. The activated type I receptor subsequently phosphorylates the downstream R-Smad protein Mothers against decapentaplegic (Mad). Phosphorylated Mad (pMad) then binds to the co-Smad Medea (Med). This complex translocates to the nucleus of motoneurons to activate or repress the transcription of target genes required for NMJ growth. Mutation of any component in the BMP signaling pathway results in a striking deficiency of NMJ growth. In addition, many molecules are reported to affect NMJ growth by negatively regulating BMP signaling at different points in the pathway. This study report that Drosophila Neuroligin 4 (DNlg4), a trans-synaptic adhesion protein, acts as a positive regulator of BMP signaling to regulate NMJ growth (X. Zhang, 2017).

Neuroligins (Nlgs) were initially reported to be the postsynaptic ligands of the presynaptic adhesion proteins neurexins (Nrxs), and loss of function of Nlgs in humans is thought to be associated with several mental disorders, including autism and schizophrenia. Nlgs are an evolutionarily conserved family of proteins encoded by four independent genes in rodents and five independent genes in humans. Nlgs have been reported to induce synapse assembly by co-cultured neurons when expressed in nonneuronal cells, and overexpression of Nlgs in neurons increases synapse density. These in vitro cell culture studies suggest a role of Nlgs in inducing the formation of synaptic contacts. However, an in vivo study showed, despite severe defects in synaptic transmission, that there was no alteration of synapse number in neurons from nlg1-3 triple knock-out mice. Similarly, loss of Nlg1 specifically in the hippocampus or amygdala did not alter the synapse number, suggesting that the role of Nlgs is not to trigger the initial synapse formation. Rather, it is more likely that upon binding to Nrx, Nlg functions in maturation of nascent synapses, including differentiation and stabilization by recruiting scaffolding proteins, postsynaptic receptors, and signaling proteins (X. Zhang, 2017).

In Drosophila, four Nlgs have been identified. Mutations in dnlg1-3 result in defective synapse differentiation that is primarily characterized by abnormal protein levels or the ectopic postsynaptic localization of glutamate receptors in larval NMJs. In addition, loss of dnlg1-3 separately leads to impairment in NMJ synapse development, as indicated by abnormal synaptic bouton number, but the precise underlying mechanism is poorly understood. A recent study showed that flies with a dnlg4 mutation exhibit an autism-related phenotype of behavioral inflexibility, as indicated by impaired reversal learning. DNlg4 also regulates sleep by recruiting the GABA receptor to clock neurons and thus modulating GABA transmission, which suggests a role of DNlg4 in synapse differentiation. However, potential molecular mechanisms underlying the behavioral defects caused by dnlg4 mutation and the role of DNlg4 in synapse development have not been reported (X. Zhang, 2017).

This paper reports the generation of an independent null allele of dnlg4 and characterized the role of DNlg4 in neuromuscular synaptic growth. Loss of DNlg4 led to impaired NMJ synapse growth, as indicated by decreased synaptic bouton numbers and increased bouton size. Presynaptic knockdown of DNlg4 mimicked these phenotypes. These morphological abnormalities in dnlg4 mutants led to corresponding impairment in synaptic transmission efficacy. Unexpectedly, all of these defects were only rescued when DNlg4 was expressed in presynaptic, instead of postsynaptic, areas of NMJs.DNlg4 genetically interacted with components of the BMP pathway and that the presynaptic BMP signaling at NMJs was decreased in dnlg4 mutants. A reduction of the BMP type I receptor Tkv was observed in dnlg4 mutants and DNlg4 physically interacted with Tkv in vivo. Altogether, this study revealed that DNlg4 regulated neuromuscular synaptic growth by positively modulating BMP signaling though Tkv (X. Zhang, 2017).

Sequence analyses showed that there are four nlg genes in the Drosophila genome, and all four DNlgs share significant amino acid sequence homology and protein structures with vertebrate Nlgs. DNlg1 and DNlg2 have a positive effect on synaptic growth of NMJs, as indicated by an obvious reduction in synaptic boutons in the dnlg1 and dnlg2 mutants. Conversely, loss of DNlg3 led to increased numbers of synaptic boutons at NMJs. In the present study, a dnlg4 null mutant was generated by gene targeting, and this mutant exhibited significant defects in NMJ morphology, including fewer synaptic boutons and increased bouton size. However, neuronal overexpression of two copies of the dnlg4 transgene induced a pronounced increase in bouton number. These results demonstrated a positive role of DNlg4 in regulating synapse development. Interestingly, neuronal overexpression of one copy of dnlg4 in the WT background did not induce an increase in bouton number, but it did when expressed in a dnlg4 mutant background. These results suggested a homeostatic adjustment during synapse development in Drosophila, which could somewhat counteract the effect caused by increased DNlg4. As a result, a moderate increase of DNlg4 in WT flies did not lead to increased synaptic growth of NMJs (X. Zhang, 2017).

In Drosophila, loss of DNlgs in the dnlg1-3 mutants also induced synaptic differentiation defects that were characterized by decreased protein levels or impaired distribution of glutamate receptors and other postsynaptic proteins at NMJs. In contrast to other dnlg mutants, statistical alteration in the distribution or protein level of glutamate receptors at NMJs in was not observed dnlg4 mutants. In addition, the distribution and protein level of some presynaptic proteins, such as BRP, CSP, and SYT, were normal in the dnlg4 mutants. However, the ultrastructural analyses of the NMJs showed that there were still some defects in synaptic ultrastructure in the dnlg4 mutants, including the increased bouton area per active zone, longer single PSD, and reduced postsynaptic SSR regions. One striking ultrastructural defect in the dnlg4 mutants was the partial detachment of presynaptic membranes from postsynaptic membranes within the active zone, which was rarely observed in WT flies, suggesting the adhesion function of DNlg4 during synaptogenesis. It was interesting that this defect also appeared in dnlg1 mutants and dnrx mutants, suggesting that the DNlg4 might affect the synaptic architecture by a mechanism similar to that of DNlg1 and DNrx (X. Zhang, 2017).

Functionally, dnlg4 mutants showed a mild impairment in transmitter release at NMJs, as characterized by slightly reduced evoked EJP amplitude and quantal contents. This phenotype was consistent with the morphological impairments and the ultrastructural defects of NMJs, suggesting a probable reduction in the number of total synapses or functional synapses at NMJs. The amplitude of mEJP was not changed in the dnlg4 mutants, which was consistent with the normal protein levels of postsynaptic glutamate receptors. Interestingly, the frequency of mEJPs in the dnlg4 mutants was dramatically increased, indicating that DNlg4 affects spontaneous transmitter release. The detailed mechanism underlying this should be addressed in future work (X. Zhang, 2017).

In mammals, Nlgs are generally considered to function as postsynaptic adhesion molecules and to help form trans-synaptic complexes with presynaptic Nrxs. In Drosophila, DNlg1 and DNlg3 are reported to be located in postsynaptic membranes. However, there are some exceptions to the postsynaptic localization of Nlgs. For example, an Nlg in Caenorhabditis elegans is reported to be present in both presynaptic and postsynaptic regions. DNlg2 is also required both presynaptically and postsynaptically for regulating neuromuscular synaptic growth. These studies support a more complex mechanism of Nlgs in synapse modulation and function. These data add to this complexity by suggesting a presynaptic role of DNlg4 in larval neuromuscular synaptic growth and synaptic functions (X. Zhang, 2017).

First, in the VNC of third-instar larvae, DNlg4 was concentrated in the neuropil region where the synapses aggregated. In NMJs, although endogenous DNlg4 was not detected by anti-DNlg4 antibodies, the exogenous DNlg4 that was expressed in motoneurons was located in type I synaptic boutons of NMJs, suggesting a reasonable presynaptic location of DNlg4 at NMJs. In another assay, DNlg4 was expressed using a DNlg4-Gal4 driver to mimic the endogenous expression pattern of the dnlg4 gene. The DNlg4 promoted by DNlg4-Gal4 was distributed in type I boutons of NMJs and was located in presynaptic areas of boutons, which also supported the presynaptic location of endogenous DNlg4 at NMJs, although a simultaneous postsynaptic localization of DNlg4 at NMJs could not be excluded. Second, knockdown of DNlg4 presynaptically led to morphological defects in NMJs similar to those observed in the dnlg4 mutants, indicated by decreased bouton numbers and increased bouton size. However, knockdown of DNlg4 postsynaptically did not cause the same phenotypes. These morphological defects of NMJs in the dnlg4 mutants could be completely rescued when DNlg4 was expressed in presynaptic neurons, but not when it was expressed in postsynaptic muscles. Third, dnlg4 mutants had a significant increase in spontaneous transmitter release frequency, which was usually interpreted as a presynaptic defect. In addition, all of the functional defects in the dnlg4 mutants, including decreased amplitude of EJPs, reduced quantal contents, and increased mEJP frequency, could be rescued by presynaptic expression of DNlg4. These results indicated that presynaptic DNlg4 was essential for proper proliferation and function of synapses at NMJs. Finally, the number of synaptic boutons at NMJs was significantly increased when two copies of UAS-dnlg4 were overexpressed in presynaptic neurons, whereas this phenomenon was not observed when two copies of UAS-dnlg4 were overexpressed in muscles, suggesting that presynaptic DNlg4 alone was sufficient to promote synaptic growth. Altogether, these data provided convincing evidence that DNlg4 functions as a presynaptic molecule in regulating synaptic growth and transmitter release at NMJs (X. Zhang, 2017).

The BMPs are major retrograde trans-synaptic signals that affect presynaptic growth and neurotransmission both in the CNS and at NMJs. This study presents immunohistochemical and genetic data showing that DNlg4 regulates NMJ growth via the BMP signaling pathway (X. Zhang, 2017).

First, the dnlg4 mutants shared similar phenotypes with the components of the BMP signaling pathway in synapse development, synapse architecture, and synapse functions of NMJs, including reduced synaptic bouton number, increased presynaptic membrane ruffles, and decreased synaptic transmission efficacy. Second, several genetic crosses followed by neuromuscular bouton number analyses showed a definite dosage-sensitive genetic interaction between dnlg4 and the components of the BMP signaling pathway, such as tkv, wit, mad, and dad, suggesting that DNlg4 is required for BMP signaling in regulating NMJ growth. Third, both immunohistochemical and Western blot analyses showed that the pMad, which serves as an indicator of BMP signaling, is decreased in both the synaptic boutons of NMJs and motoneuronal nuclei in the dnlg4 mutants, whereas it is increased in dnlg4-overexpressing flies. Finally, the expression of the BMP signaling target gene trio, is significantly reduced in the dnlg4 mutants, but it was increased in dnlg4-overexpressing flies. Together, these results supported the hypothesis that DNlg4 promotes synaptic growth by positively regulating BMP signaling (X. Zhang, 2017).

The critical question to be addressed, therefore, is the mechanism underlying this regulation. Through Western blot analyses of larval brain homogenates, it is found that the protein level of Tkv (as assessed by ectopically expressed Tkv-GFP) in the dnlg4 mutants is significantly decreased, whereas Wit and Mad, the other two components of the BMP pathway, were not altered. The immunohistochemical assay also showed that the Tkv protein in the dnlg4 mutants is reduced in both the VNC and the synaptic boutons of NMJs. These data indicated the specific positive regulation of Tkv by DNlg4. Previous studies reported that tkv mutants had decreased synaptic bouton number, increased presynaptic membrane ruffles, reduced amplitude of EJP, and unchanged mEJP amplitude. In addition, neuronal overexpression of one copy of transgenic tkv did not induce NMJ overgrowth, but it induced such overgrowth when two copies of transgenic tkv were expressed in neurons. These phenotypes were similar to what was observed in the dnlg4 mutants and dnlg4-overexpressing flies. All of these results strongly demonstrated that DNlg4 regulates BMP signaling by modulating Tkv protein levels (X. Zhang, 2017).

Because the dnlg4 mutants had a level of tkv mRNA comparable with that of the WT controls, DNlg4 did not affect the transcription of tkv. The possibility of Tkv trafficking defects from the cell body to the axon terminal could also be excluded because no retention of Tkv in the soma of motoneurons was observed. Thus, it seems reasonable to speculate that DNlg4 affects the recruitment or stability of Tkvs at the presynapse. In support of this hypothesis, co-localization of Tkv and DNlg4 at presynaptic regions of NMJs was observed by immunostaining. As further confirmation, it was shown that Tkv could be co-immunoprecipitated with an antibody against DNlg4, and a reverse co-immunoprecipitation assay using anti-GFP antibodies also resulted in the precipitation of DNlg4 by Tkv-GFP, suggesting a physical interaction between DNlg4 and Tkv in vivo. Furthermore, using pull-down assay, the acetylcholinesterase-like domain of the N terminus was shown to be essential for DNlg4 interacting with Tkv (X. Zhang, 2017).

If the recruitment of Tkv to the presynaptic membrane entirely depends on DNlg4, the existence of Tkv in the presynaptic membrane would not be detected, and accumulation of Tkv proteins in the presynaptic areas of NMJs would probably be observed in the dnlg4 mutants. However, minor Tkv protein level was detected at the presynaptic membrane, and no accumulation of Tkv was observed at NMJs in the dnlg4 mutants. Thus, a more reasonable hypothesis is that DNlg4 affected the stability of Tkv at the presynaptic membranes. The protein level of Tkv in the presynapses of NMJs was reported to be regulated by several pathways, including direct proteasomal degradation, ubiquitin-mediated degradation, and endocytosis. The simplest model is that the DNlg4 might stabilize Tkv via inhibiting its degradation. Several protein kinases, such as the ribosomal protein S6 kinase-like protein (S6KL) and the serine/threonine kinase Fused, have been reported to interact physically with Tkv in vitro and to facilitate its proteasomal degradation. In particular, S6KL has been demonstrated to degrade Tkv at NMJs. DNlg4 might stabilize Tkv by inhibiting these protein kinases, but the detailed mechanism of such activity still needs to be addressed (X. Zhang, 2017).

In summary, this study demonstrated that DNlg4 positively regulated neuromuscular synaptic growth by modulating BMP signaling through the maintenance of Tkv protein levels in the presynapse of NMJs. To accomplish this function, DNlg4 acted as a presynaptic molecule instead of a postsynaptic molecule. This study further suggested a relationship between Nlgs and BMP signaling and provided a new understanding of the exact role of Nlgs during synapse formation and development (X. Zhang, 2017).

Human APP gene expression alters active zone distribution and spontaneous neurotransmitter release at the Drosophila larval neuromuscular junction

This study provides further insight into the molecular mechanisms that control neurotransmitter release. Experiments were performed on larval neuromuscular junctions of transgenic Drosophila melanogaster lines with different levels of human amyloid precursor protein (APP) production. To express human genes in motor neurons of Drosophila, the UAS-GAL4 system was used. Human APP gene expression increased the number of synaptic boutons per neuromuscular junction. The total number of active zones, detected by Bruchpilot protein puncta distribution, remained unchanged; however, the average number of active zones per bouton decreased. These disturbances were accompanied by a decrease in frequency of miniature excitatory junction potentials without alteration in random nature of spontaneous quantal release. Similar structural and functional changes were observed with co-overexpression of human APP and beta-secretase genes. In Drosophila line with expression of human amyloid-beta42 peptide itself, parameters analyzed did not differ from controls, suggesting the specificity of APP effects. These results confirm the involvement of APP in synaptogenesis and provide evidence to suggest that human APP overexpression specifically disturbs the structural and functional organization of active zone and results in altered Bruchpilot distribution and lowered probability of spontaneous neurotransmitter release (Saburova, 2017).

In vivo single-molecule tracking at the Drosophila presynaptic motor nerve terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. This study describes a technique to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. This study describes how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva (Bademosi, 2018).

Inwardly rectifying potassium (Kir) channels represent a critical ion conductance pathway in the nervous systems of insects

A complete understanding of the physiological pathways critical for proper function of the insect nervous system is still lacking. The recent development of potent and selective small-molecule modulators of insect inward rectifier potassium (Kir) channels, Kir1, Kir2, or Kir3 in Drosophila, has enabled the interrogation of the physiological role and toxicological potential of Kir channels within various insect tissue systems. Therefore, this study aimed to highlight the physiological and functional role of neural Kir channels the central nervous system, muscular system, and neuromuscular system through pharmacological and genetic manipulations. The data provide significant evidence that Drosophila neural systems rely on the inward conductance of K(+) ions for proper function, since pharmacological inhibition and genetic ablation of neural Kir channels yielded dramatic alterations of the CNS spike discharge frequency and broadening and reduced amplitude of the evoked EPSP at the neuromuscular junction. Based on these data, it is concluded that neural Kir channels in insects (1) are critical for proper function of the insect nervous system, (2) represents an unexplored physiological pathway that is likely to shape the understanding of neuronal signaling, maintenance of membrane potentials, and maintenance of the ionic balance of insects, and (3) are capable of inducing acute toxicity to insects through neurological poisoning (Chen, 2018).

The establishment of insecticide resistance within multiple arthropod vectors of human pathogens has been, at least in part, the driving force behind the prolific advancement of the fields of insecticide science and insect molecular physiology. The goal of mitigating the various resistance mechanisms has been a multidisciplinary and transdisciplinary approach that has resulted in a detailed understanding of molecular genetics, transcriptomics, biochemistry, cellular physiology, and neuroendocrinology of non-model insects, such as mosquitoes. In addition to these fields, the reduced efficacy of currently approved classes of insecticides has dramatically increased interest of identifying novel molecular targets for insecticide design and/or development of novel chemical scaffolds targeting previously exploited proteins. A variety of new target sites and chemical scaffolds have been identified and characterized in the past decade that include transient receptor proteins, G-protein coupled receptors, dopaminergic pathways, and K+ ion channels (Chen, 2018).

Inward rectifier potassium (Kir) channels belong to a large 'superfamily' of K+ ion channels that includes the voltage-gated, two-pore, calcium-gated, and cyclic nucleotide-gated channels. Kir channels function as biological diodes due to their unique ability to mediate the inward flow of K+ ions at hyperpolarizing membrane voltages more readily than the outward flow of K+ at depolarizing voltages. On a molecular level, Kir channel are structurally simple ion channels that consists of 4 subunits assembled around a central, water-filled pore, through which K+ ions move down their electrochemical gradient to traverse the plasma membrane. Each subunit consists of a central transmembrane domain, a re-entrant pore-forming loop, and a cytoplasmic domain comprised of amino and carboxyl termini (Chen, 2018).

Recent genetic and pharmacological evidence suggests that Kir channels could represent viable targets for new insecticides. In Drosophila melanogaster, embryonic depletion of Kir1, Kir2, or Kir3 mRNA leads to death or defects in wing development. Reduction of Kir1 and Kir2 mRNA expression in the Malpighian (renal) tubules of Drosophila or inhibition of Kir channels in isolated mosquito Malpighian tubules with barium chloride (BaCl2) dramatically reduces the transepithelial secretion of fluid and K+ (Wu, 2015; Scott, 2004), indicating Kir channels expressed in the Malpighian tubules may be an exploitable insecticide target site. Considering this, high-throughput screens (HTS) of chemical libraries were performed to identify small-molecule modulators of mosquito Kir1 channels, which is the principal conductance pathway in mosquito Malpighian tubules. Structurally distinct small molecules were identified (i.e., VU573, VU590, or VU625) and pharmacological inhibition of Aedes aegypti Kir1 was shown to disrupt the secretion of fluid and K+ in isolated Malpighian tubules, urine production, and K+ homeostasis in intact females. Similarly, a Kir1 inhibitor, termed VU041, was identified in a subsequent HTS campaign and was shown to (1) be highly potent against the Anopheles gambiae Kir1 (ca. 500 nanomolar), (2) exhibit topical toxicity (ca. 1 μg/mosquito) to insecticide-susceptible and carbamate/pyrethroid-resistant strains of mosquitoes, (3) and display high selectivity for mosquito Kir channels over mammalian Kir channel orthologs (Chen, 2018).

Previous work indicates that VU041-mediated toxicity stems from inhibition of the Kir1 channel within the Malpighian tubules to induce tubule failure and an inability to maintain K+ homeostatsis after blood feeding. However, after exposure to lethal doses of VU041, An. gambiae and A. aegypti were found to display both hyperexcitatory and lethargic tendencies that were complexed with uncoordinated movements, which is reminiscent of neurological poisoning. Furthermore, acute toxicity (ca. 1-3 hours) was observed after exposure to VU041, similar to other insecticides that poison the nervous system. Lastly, previous studies have shown that select Kir channel inhibitors were capable of inducing a flightless behavior where mosquitoes were ambulatory, yet were not able to fly, presumably due to failure of the nervous or muscular systems. Although it is possible that the mortality is due to complete systems failure stemming from ubiquitous expression of Kir channels or due to accumulated waste that remains due to impaired Malpighian tubule function, it is also reasonable to predict that VU041 is directly altering the functional capacity of Kir channels expressed in the nervous system to yield toxicity. Unfortunately, there have been no studies to characterize the physiological role of Kir channels in the insect nervous systems, which limits the ability to infer the toxicological potential of these neural proteins. Studies using RT-PCR have shown that the head of A. aegypti is enriched with Kir2B' (vector base accession number: AEL013373) mRNA (Dr. Peter Piermarini, The Ohio State University, personal communication to R. Chen, 2018), suggesting that poisoning of the mosquito central nervous system (CNS) through Kir inhibition is indeed possible. Unfortunately, electrophysiological recordings of mosquito CNS activity have yet to be achieved, which limits the ability to infer the physiological role or toxicological potential of neural Kir channels of mosquitoes. However, electrophysiological recordings from an excised CNS of D. melanogaster is possible and further, the gene encoding Kir2, termed irk2, is highly concentrated in the adult head, CNS, and the thoracic-abdominal ganglia. This suggests that D. melanogaster may represent a suitable substitute for mosquitoes and will enable the characterization of the physiological role Kir channels have in the insect nervous system (Chen, 2018).

Considering (1) the foundational role of Kir channels in mammalian and insect cellular physiology, (2) deletion of irk2 gene in Drosophila is homozygous lethal, (3) the signs of intoxication after exposure to Kir channel modulators being reminiscent of neurological poisoning, and (4) the overexpression of Kir mRNA in mosquito and Drosophila neural tissues, it was hypothesized that Kir channels regulate neuronal signaling and excitability of insect nervous systems and are a critical conductance pathway for proper functioning of the insect nervous system. Therefore, the goals of the present study were to employ electrophysiological methods combined with genetic and pharmacological techniques to determine the physiological importance of Kir channels in insect CNS, neuromuscular junction, and muscular systems that will provide insight into targeting neural Kir channels as a novel insecticide target site. Additionally, data collected in this study begin to bridge the fundamental knowledge gap regarding unexplored physiological pathways in the insect nervous system that will provide a more holistic understanding to neuronal excitability and neurotransmission of insects (Chen, 2018).

Currently, there have been no efforts to characterize the physiological role or toxicological potential of insect neural Kir channels. However, the current findings demonstrate that the recently discovered Kir-directed insecticide, VU0413, is capable of dramatically altering the neural activity of flies and, in a more general sense, that Kir channels constitute a critical K+ ion conductance pathway in the insect nervous system. Despite the nervous system being the target tissue of the extreme majority of deployed insecticides, a complete understanding of the physiological pathways critical for proper function of the insect nervous system is still lacking. This represents a critical gap in knowledge of the complex relationship between the dozens of functionally coupled ion channels, transporters, and enzyme systems that require tight regulation for proper neuronal function. This fundamental gap pertaining to the foundational neural physiology must be filled to develop a holistic understanding of insect nervous system function that will lead to the development of new insecticides (Chen, 2018).

Knowledge of the physiological role and toxicological potential of insect Kir channels is growing rapidly with studies suggesting these channels serve a critical role in Malpighian tubule function of mosquitoes and Drosophila, insect salivary gland function, honey bee dorsal vessel function, and insect antiviral immune pathways. Furthermore, these channels represent a critical K+ conductance pathway in the mammalian nervous system as Kir knockouts in glial cells leads to membrane depolarization, enhanced synaptic potentiation, and reduced spontaneous neural activity. Considering the importance of Kir channels in the function of various insect tissues and the established role of Kir channels in mammalian neuronal tissue, it is hypothesized that Kir channels also serve a critical role in insect neural tissue and aimed to highlight the general influence of Kir channel modulation to the insect nervous system through pharmacological and genetic manipulations of the Kir channel (Chen, 2018).

To begin testing the physiological role of insect neural Kir channels, neurophysiological recordings of the Drosophila CNS were performed using the voltage dependent Kir blocker, BaCl2. BaCl2 is useful pharmacological tool to test the physiological role of Kir channels since, at physiological membrane potentials, Kir channels are up to 1000-fold more sensitive to BaCl2 than other K+ ion channels. This enhanced potency to Kir channels when compared to other K+ ion channels enables selective inhibition of Kir channels at low- to mid-micromolar concentrations of BaCl2. An increase was observed in the spike discharge frequency followed by cessation of firing after exposure of the CNS to mid-micromolar concentrations of BaCl2, providing the first insight that Kir channels constitute a critical conductance pathway in insect CNS. However, the potential for BaCl2 to precipitate out of some saline solutions and the potential of BaCl2 to modulate non-target proteins limits the conclusions that can be drawn from these data. Fortunately, the recent identification of selective and potent small molecules designed to target insect Kir channels has facilitated the characterization of the physiological role of these channels in various insect tissue systems with more certainty than BaCl2 and other divalent cations (Chen, 2018).

This study used the recently discovered insect Kir channel modulator (VU041) and its inactive analog (VU937) to characterize the influence these channels have in insect nervous system function. Exposure of the Drosophila CNS to VU041 dramatically altered the spike discharge frequency in a biphasic manner with low concentrations yielding neuroexcitation and higher concentrations having a depressant effect on CNS activity. A biphasic response is oftentimes observed when multiple pathways are inhibited and it is plausible that VU041 is directly or indirectly altering the functional capacity of other ion channels or transporters, such as delayed rectifier K+ channels or calcium-activated K+ channels. Although off-target effects are possible, they are unlikely since VU937 had no influence to CNS activity, suggesting the observed phenotype is through Kir inhibition. To ensure the observed effect to CNS activity was directly due to Kir2 channel modulation, CNS-specific RNAi-mediated knockdown was performed of the Kir2 encoding gene, irk2. Results from this genetic depletion of irk2 show a dramatic increase in CNS spike discharge frequency that was also substantiated through hyperactive larval behavior. These observed responses to VU041 and irk2 genetic depletion is likely due to the physiological role of only Kir2 since no mRNA reduction was observed in other Kir-encoding genes that are expressed in the CNS or any irk gene within the whole body or carcass. Previous reports have documented compensatory functions of Kir channels that arise after genetic depletion of one Kir channel, which prevents the manifestation of an observable change in phenotype (Wu, 2015). Yet, it does not appear that a compensatory mechanism arose to account for the genetic depletion of irk2 since a direct physiological response was observed and irk1 and irk3 mRNA levels remained unchanged. The influence to expression of other K+ ion transport pathways, such as Na+-K+-2Cl- cotransporter and Na+-K+-ATPase pumps, remains unknown and should be studied prior to drawing absolute conclusions regarding the physiological basis for neural Kir channels. Furthermore, exposure of the neuromuscular junction to VU041 altered the evoked EPSP waveform and muscle excitability. These data indicate that Drosophila, and likely mosquito, central and muscular nervous systems rely on the inward conductance of K+ ions through Kir channels for proper function (Chen, 2018).

The Drosophila genome encodes three Kir channel proteins, termed ir, irk2 and irk3, and all three contain the structural features and biophysical properties that are found in mammalian Kir channel subunits. Although ir and irk3 mRNA has been found to be expressed at low levels in the fly head, the irk2 gene is highly expressed in the adult fly head where it is concentrated in the brain and eye, suggesting that, of the Kir channels, irk2 is the principal inward conductance pathway for K+ ions. The sequence of irk2 is similar to that of ir, and both are highly related to human Kir 2, 3, and 6 proteins, which are constitutively active, GIRK, and ATP-gated Kir channels, respectively. Interestingly, irk2 channels have been shown to be constitutively active in S2 cells, associate with sulphonylurea receptors (SUR) as is seen with KATP channels, and the presence of an Asn223 residue suggests similarity to the GPCR-gated Kirs (Kir3.x; mammalian nomenclature). The variable functional associations have led to the speculation that irk2 may have different mechanisms of gating and regulation based on the cell type the gene is expressed in. Due to this, pharmacological modulators of mammalian GIRK and KATP channels were used to determine the mechanisms of irk2 gating in the Drosophila CNS. The GIRK activator, ML297, is highly selective for mammalian GIRK1/2 subunit combination over other Kir channels and was found to induce neuroexcitation to the Drosophila CNS. The sustained increase in Drosophila CNS activity after ML297 exposure was unexpected since GIRK2 knockouts in mice revealed an epileptic phenotype, suggesting GIRK is responsible for depressing neuronal excitability and thus, an activator of GIRK should reduce CNS spike discharge frequency. It is important to note that ML297 was shown to have moderate activity on the mammalian serotonin (5-Ht2b) receptor, which is expressed in the Drosophila CNS and may be the cause for observed neuroexcitation to the Drosophila CNS. Unfortunately, the severely underdeveloped pharmacological library of GIRK inhibitors prevents further interrogation at this time. To determine if Irk2 is gated by ATP, four structurally distinct activators and inhibitors of mammalian KATP channels were used. No change in CNS spike discharge frequency was observed after exposure to these molecules at concentrations ranging into the upper micromolar range. Since other studies have shown clear effects to various insect systems with mammalian KATP modulators, the lack of response to the Drosophila CNS is likely to be due to the absence of ATP-gated Kir channels and not due to incompatibility of the structural scaffolds with the Drosophila KATP channel. These findings have led to the speculation that (1) irk2 is not likely to be expressed as a KATP channel in the CNS, (2) constitutively active Kir channels are present in the Drosophila CNS, and (3) GIRK-like channels may be present in the CNS yet further studies are required to interrogate this claim (Chen, 2018).

The data presented in this study raise the question as to what the physiological role Kir channels have in nervous system function of insects at the cellular level. In mammals, astrocyte function has received significant interest for their roles in the regulation of synaptic levels of neurotransmitters, in particular glutamate, buffering of extracellular K+, and release of neurotransmitters, all of which have been shown to directly modulate neuronal excitability and transmission. In particular, Kir4.1 channels expressed in astrocytes have been directly linked to K+ influx across neural membranes where cells take up excess extracellular potassium ions, distribute them via gap junctions, and extrude the ions at sites in which extracellular K+ concentrations ([K+]out) is low, which is termed K+ spatial buffering. It is reasonable to predict that the insect nervous system employs this method of K+ transport during neuronal activity since [K+]out is dramatically increased and must be rapidly reversed to prevent membrane depolarization of neurons. Therefore, inhibition of this process through pharmacological blockage of neural Kir channels will lead to depolarization of the nervous system and induce CNS excitation, which was observed in this study at low concentrations of VU041 and after genetic knockdown of Kirs. In mammals, a complete knockout of Kir4.1 yielded a reduction of spontaneous EPSC in pyramidal neurons, similar to what was observed after CNS exposure to concentrations of VU041 greater than 10 µM (Chen, 2018).

It is hypothesized that Kir channels provide a pathway for K+ spatial buffering during neuronal activity of Drosophila and this pathway is critical for proper CNS activity. Excitability and synaptic transmission of insect and mammalian nervous systems are dependent upon [K+]out and alteration of the K+ ion gradient directly affects excitatory neurotransmission. In accordance to this, changes were observed in the CNS spike frequency and complete cessation of evoked EPSP's at the NMJ, which is classically attributed to changes in presynaptic function that may be resultant of altered neurotransmitter release. Similarly, reduced amplitude and broadening of the evoked EPSP waveform at the neuromuscular junction were observed after pharmacological inhibition of Kirs, which may be a result of modification of postsynaptic terminal responsiveness to neurotransmitters. The influence of Kir channel inhibition to pre- and post-synaptic function can be due to changes in either extracellular ion or transmitter levels. This is evidenced by the response of the Drosophila CNS after exposure to BaCl2 and 25 μM VU041. Exposure to these pharmacological agents yielded near maximal spike discharge frequency that culminated in a relatively abrupt termination of this activity. This reduction of CNS spike frequency may be due to depolarization-induced inactivation of Na+ channels due to prolonged exposure to elevated [K+]out, thereby lowering the probability of transmitter release that will reduce neuronal firing. Therefore, it appears as though Kir channels are responsible for regulating the K+ ion gradient that ultimately controls synaptic activity and neurotransmitter release, which is essential for proper neural signaling and activity (Chen, 2018).

Kir channels represent a critical K+ ion conductance pathway within the Drosophila, and likely mosquito, central and neuromuscular nervous systems. Considering this, it is reasonable to suggest that the recently identified Kir-directed mosquitocide, VU041, is capable of inducing toxicity through neurological poisoning in addition to inducing Malpighian tubule failure that leads to toxicity by an inability to perform osmoregulatory actions. These data provides a proof-of-concept that novel chemical scaffolds targeting neural Kir channels in insects represent a novel mechanism of action with insecticide resistance mitigating potential. Based on the data collected in this study, it is hypothesized that the function of Kir channels in the insect nervous system is responsible for reducing [K+]out during neuronal activity by the process known as K+ spatial buffering, similar to that described in mammals. It is important to note that this hypothesis cannot be fully validated until whole-cell electrophysiological recordings are performed to determine the role of Kirs in (1) glutamate and K+ uptake during neural activity, (2) maintenance of neural membrane properties (e.g., Vm, Rm, etc.), and 3) synaptic transmission and plasticity (Chen, 2018).

The Neurexin-NSF interaction regulates short-term synaptic depression

Although Neurexins, which are cell adhesion molecules localized predominately to the presynaptic terminals, are known to regulate synapse formation and synaptic transmission, their role in the regulation of synaptic vesicle release during repetitive nerve stimulation is unknown. This study shows that Drosophila neurexin mutant synapses exhibit rapid short-term synaptic depression upon tetanic nerve stimulation. Moreover, the intracellular region of Neurexin was demonstrated to be essential for synaptic vesicle release upon tetanic nerve stimulation. Using a yeast two-hybrid screen, it was found that the intracellular region of Neurexin interacts with N-ethylmaleimide sensitive factor (NSF), an enzyme that mediates soluble NSF attachment protein receptor (SNARE) complex disassembly and plays an important role in synaptic vesicle release. The binding sites of each molecule were mapped, and it was demonstrated that the Neurexin-NSF interaction is critical for both the distribution of NSF at the presynaptic terminals and SNARE complex disassembly. These results reveal a previously unknown role of Neurexin in the regulation of short-term synaptic depression upon tetanic nerve stimulation and provide new mechanistic insights into the role of Neurexin in synaptic vesicle release (Li, 2015).

Previous studies have shown that the α-NRXs functionally couple Ca2+ channels to the presynaptic machinery to mediate synaptic vesicle exocytosis and that defective synaptic vesicle release can be restored with 1 mm Ca2+ (Li, 2007). In the current experiments, 1.8 mm Ca2+ was used to eliminate the effects of abnormal Ca2+ sensitivity in neurotransmitter release. Under this condition, it was shown that nrx mutant synapses exhibit rapid short term synaptic depression and reduced quantal content of nerve-evoked synaptic currents at the steady state during tetanic stimulation. These observations suggest that NRX regulates activity-dependent synaptic plasticity. Similar observations have been reported in the fast-twitch diaphragm muscle of the α-NRX double knock-out mouse, which indicates that presynaptic efficacy but not presynaptic homeostatic plasticity is normal under basal conditions (Li, 2015).

Deficits in both the presynaptic neurotransmitter release machinery and the postsynaptic neurotransmitter receptors may cause rapid short term synaptic depression. As a presynaptic adhesion molecule, NRX interacts with the postsynaptic adhesion molecule, Neuroligin, and bridges the synaptic cleft that aligns the presynaptic neurotransmitter release machinery with the postsynaptic neurotransmitter receptors. It has been suggested that the activity-dependent regulation of the NRX/Neuroligin interaction mediates learning-related synaptic remodeling and long term facilitation. A recent study reveals that the alternative splicing of presynaptic NRX-3 controls postsynaptic AMPA receptor trafficking and long term plasticity in mice (Aoto, 2013). This study showed that the kinetics of the individual synaptic currents were stable during tetanic stimulation in the nrx mutant synapses. Moreover, NRX was shown to mediate synaptic plasticity through the presynaptic machinery, which was supported by rescue experiments (Li, 2015).

The intracellular sequence of NRX, including the C-terminal PDZ-binding motif, is identical across several vertebrate and invertebrate species. Mammalian NRXs only possesses a short cytoplasmic tail (55 amino acids), including a PDZ-binding motif. To identify the potential binding partners, several laboratories have conducted yeast two-hybrid screening by using the cytosolic tail of mammalian NRX as a bait. Two PDZ-containing proteins, CASK and syntenin, have been identified in the previous screening, and further studies show that the binding between CASK and NRX is abolished by deletion of the last three amino acids of the intracellular C-terminal region of NRX. However, it seems that the previous screenings are not saturated, as the NRX-interacted protein Mints was not identified in these screenings (Li, 2015).

Drosophila NRX contains a long cytoplasmic tail (122 amino acids), including a functional PDZ-binding motif. Thus, it is possible that the long cytoplasmic tail may associate with some partners in a PDZ-independent manner. In this study, normalized Drosophila cDNA libraries were used in the screening, which helps to identify the binding partner with low abundance. In the screening, 23 cDNAs clones that encode the fragments of 18 proteins were recovered. Previous study has demonstrated that the cytoplasmic tail of NRX associates with RFABG and facilitates the retinol transport. This study identified the intracellular region of NRX binds with NSF through the amino acid sequence 1788-1813 but not the PDZ-binding motif of NRX. This NRX/NSF interaction was found to be essential for the NSF recruitment to the presynaptic terminals and plays an important role in synaptic vesicle release. In addition, multiple lines of evidence that NRX associates with NSF at the presynaptic terminals. An alignment analysis of the NSF-binding site of NRX revealed that this sequence is conserved across different species. Moreover, this sequence is present across many proteins that serve different functions, which implies that this sequence may be essential for protein interactions (Li, 2015).

Each NSF molecule contains an N-terminal domain that is responsible for the interaction with α-SNAP and the SNARE complex, a low affinity ATP-binding domain (D1 domain) whose hydrolytic activity is associated with NSF-driven SNARE complex disassembly and a C terminal high affinity ATP-binding domain (D2 domain). This study mapped the NRX-binding sites of NSF to the D2 domain, which mediates the ATP-dependent oligomerization of NSF. An alignment analysis showed that both the D2 domain in NSF and the NSF-binding site in NRX are highly conserved. This result suggests that the NRX/NSF interaction may occur in other species, a possibility that needs to be investigated further (Li, 2015).

It has been documented that the D2 domain of NSF is essential for its hexamerization. This study shows that NRX exhibits a declined NSF binding capacity in high Ca2+ concentration. These observations suggest that NSF can be released from NRX under stimulation. Although NRX and NSF do not bind with Ca2+ directly, it is possible that they undergo Ca2+ signaling-dependent modifications or bind with some Ca2+-binding proteins. In the yeast two-hybrid screening, one potential Ca2+-binding protein (CG33978) has been identified. However, the mechanism of how NRX releases NSF under high Ca2+ concentration need to be further investigated (Li, 2015).

Previous studies have shown that the PDZ-binding motif of NRX associates with several molecules involved in the synaptic vesicle exocytosis machinery, including synaptotagmin and the PDZ domain-containing proteins CASK and Mints. Synaptotagmin functions as a Ca2+ sensor and controls synaptic membrane fusion machinery. Adaptor protein Mints regulates presynaptic vesicle release (Ho, 2006), whereas the other adaptor protein CASK is not essential for the Ca2+-triggered presynaptic release. In contrast, CASK interacts with NRX and protein 4.1 to form a trimeric complex and regulates synapse formation. The process of synaptic vesicle release includes several consecutive steps, docking, priming, and fusion. Depending on the stimulation given to synapses, different synaptic vesicle trafficking steps become rate-limiting for synaptic vesicle release. This causes short term changes in synaptic transmission that determine many higher brain functions such as sound localization, sensory adaptation, or even working memory. Thus, the PDZ-binding motif-linked synaptic vesicle exocytotic machinery might regulate the different steps during synaptic vesicle release. Indeed, nrx mutant synapses that express NRXΔ4 exhibit a rapid current decline over the first several dozen stimulations. In contrast, the expression of NRXΔ4 largely restores the reduced steady-state mean EJCs and quantal content in neurexin mutant synapses. Together with the C-terminally truncated NRX rescue experiments, these data suggest that intracellular regions of NRX other than the PDZ-binding motif also regulate short term synaptic depression. The existing literature extensively documents the roles of NSF in SNARE complex disassembly and short term synaptic depression. This study extended these findings to show that the NRX/NSF interaction facilitates the recruitment of NSF to the presynaptic terminals and promotes the subsequent SNARE complex disassembly (Li, 2015).

Previous studies have established that the NSF hexamer serves as the only active form for SNARE complex disassembly. In this study, the binding assay showed that purified NRX binds to NSF in a concentration-dependent and saturable manner. NRXs appear to serve as scaffold proteins to recruit NSF, and the NRX/NSF interaction may promote SNARE complex disassembly in vivo. Live image studies have shown that NSF mutant (i.e. comt) synapses exhibit defective NSF re-distribution during tetanic nerve stimulation. In this study, immunocytochemical and sedimentation analyses revealed that a lack of the NRX/NSF interaction results in an altered distribution of NSF and an accumulation of 7S SNARE complexes. These results imply that NRX/NSF interaction serves as a potential mechanism to restrict the mobilization of NSF (Li, 2015).

Electrophysiological recordings revealed that synaptic depression was comparable between nrx mutant synapses and wild-type synapses in response to low frequency stimulations. The long intervals between low frequency stimulations may allow the remaining NSF to disassemble the SNARE complexes and generate enough free t-SNARE for the subsequent synaptic vesicle fusion events. Another possibility that needs to be investigated further is that tetanic stimulation may have other effects on the re-distribution of NSF (Li, 2015).

In summary, this study provides evidence that the NRX/NSF interaction recruits NSF to the presynaptic terminals and promotes SNARE complex disassembly. These findings have revealed a previously unknown role of NRX in the regulation of neurotransmitter release and provide a linkage between the presynaptic adhesion molecules and the presynaptic plasticity machinery (Li, 2015).

Characterization of developmental and molecular factors underlying release heterogeneity at Drosophila synapses

Neurons communicate through neurotransmitter release at specialized synaptic regions known as active zones (AZs). Using biosensors to visualize single synaptic vesicle fusion events at Drosophila neuromuscular junctions, this study analyzed the developmental and molecular determinants of release probability (Pr) for a defined connection with ~300 AZs. Pr was heterogeneous but represented a stable feature of each AZ. Pr remained stable during high frequency stimulation and retained heterogeneity in mutants lacking the Ca(2+) sensor Synaptotagmin 1. Pr correlated with both presynaptic Ca(2+) channel abundance and Ca(2+) influx at individual release sites. Pr heterogeneity also correlated with glutamate receptor abundance, with high Pr connections developing receptor subtype segregation. Intravital imaging throughout development revealed that AZs acquire high Pr during a multi-day maturation period, with Pr heterogeneity largely reflecting AZ age. The rate of synapse maturation was activity-dependent, as both increases and decreases in neuronal activity modulated glutamate receptor field size and segregation (Akbergenova, 2018).

This study used quantal imaging, super resolution SIM, and intravital imaging to examine the development of heterogeneity in evoked P_r across the AZ population at Drosophila NMJs. It was first confirmed that release heterogeneity was not caused by summation of fusion events from multiple unresolvable AZs. Indeed, high P_r sites corresponded to single AZs with enhanced levels of BRP. These findings are consistent with previous observations using conventional light microscopy that indicate P_r correlates with BRP levels. By monitoring release over intervals of extensive vesicle fusion during strong stimulation, it was also observed that P_r is a stable feature of each AZ. In addition, loss of the synaptic vesicle Ca2+ sensor Syt1 globally reduced P_r without altering the heterogeneous distribution of P_r across AZs, indicating that AZ-local synaptic vesicle pools with differential Ca2+ sensitivity are not likely to account for P_r heterogeneity. Since voltage gated calcium channel (VGCC) abundance, gating, and organization within the AZ are well established regulators of P_r across synapses, heterogeneity in presynaptic Ca2+ channel abundance was a clear candidate for the generation of P_r heterogeneity at Drosophila NMJs. Indeed, the Cac Ca2+ channel responsible for neurotransmitter release is heterogeneously distributed across the NMJ . Using transgenically labeled Cac lines, it was observed that Cac density at AZs is indeed strongly correlated with Pr. To directly visualize presynaptic Ca2+ influx at single AZs, GCaMP fusions were generated to the core AZ component BRP. Ca2+ influx at single AZs was highly correlated with both Cac density and Pr. The cac^NT27 mutant with decreased conductance also resulted in a global reduction in P_r without disrupting heterogeneity, further confirming that Ca2+ influx regulates P_r across the range of release heterogeneity (Akbergenova, 2018).

Postsynaptically, high P_r AZs were enriched in GluRIIA-containing receptors and displayed a distinct pattern of glutamate receptor clustering. While most synapses showed GluRIIA and GluRIIB spread over the entire PSD, high P_r AZs were apposed by PSDs where GluRIIA concentrated at the center of the AZ, with GluRIIB forming a ring at the PSD periphery. Indeed, anti-glutamate receptor antibody staining of wildtype larvae lacking tagged glutamate receptors had previously identified a GluRIIB ring around the GluRIIA core in some mature third instar NMJ AZs (Marrus , 2004). In addition, activity-dependent segregation of GluRIIA and a GluRIIA gating mutant has been observed at individual AZs in Drosophila (Petzoldt, 2014). The correlation of P_r with GluRIIA accumulation is especially intriguing considering that this subunit has been implicated in homeostatic and activity-dependent plasticity (Davis, 2006; Frank, 2014; Petersen et al., 1997; Sigrist et al., 2003). By following the developmental acquisition of this postsynaptic property as a proxy for P_r from the first through third instar larval stages via intravital imaging in control and mutant backgrounds, it was observed that the earliest formed AZs are the first to acquire this high P_r signature over a time course of ~3 days, and that PSD maturation rate can be modulated by changes in presynaptic activity (Akbergenova, 2018).

Similar to prior observations, this study found that most AZs at the Drosophila NMJ have a low Pr. For the current study, the AZ pool was artificially segregated into low and high release sites, with high releasing sites defined based on having a release rate greater than two standard deviations above the mean. Given that birthdate is a key predictor of glutamate receptor segregation, and by proxy Pr, the AZ pool is expected to actually reflect a continuum of P_r values based on their developmental history. However, using the two standard deviation criteria, 9.9% of AZs fell into the high P_r category, with an average P_r of 0.28. It was also observed that 9.7% of the AZs analyzed displayed only spontaneous release. No fusion events could be detected for either evoked or spontaneous release for another 14.6% of AZs that were defined by a GluRIIA-positive PSD in live imaging. Future investigation will be required to determine whether these cases represent immature AZs with extremely low evoked Pr, or distinct categories reflective of differences in AZ content. The remaining AZs that participated in evoked release had an average P_r of 0.05. Ca2+ channel density and Ca2+ influx at individual AZs was a key determinant of evoked P_r heterogeneity, as P_r and the intensity of Cac channels tagged with either TdTomato or GFP displayed a strong positive correlation. Spontaneous fusion showed a much weaker correlation with both Cac density and Ca2+ influx at individual AZs, consistent with prior studies indicating spontaneous release rates are poorly correlated with external Ca2+ levels at this synapse. With synaptic vesicle fusion showing a steep non-linear dependence upon external Ca2+ with a slope of ~3-4, a robust change in P_r could occur secondary to a relatively modest increase in Ca2+ channel abundance over development. Although the number of VGCCs at a Drosophila NMJ AZ is unknown, estimates of Cac-GFP fluorescence during quantal imaging indicate a ~ 2 fold increase in channel number would be necessary to move a low P_r AZ into the high P_r category. Similar correlations between evoked P_r and Ca2+ channel abundance have been found at mammalian synapses, suggesting this represents a common evolutionarily conserved mechanism for determining release strength at synapses (Akbergenova, 2018).

This study did not test the correlation of P_r with other AZ proteins besides Cac and BRP, but it would not be surprising to see a positive correlation with the abundance of many AZ proteins based on the observation that maturation time is a key determinant for Pr. Indeed, recent studies have begun to correlate P_r with specific AZ proteins at Drosophila NMJs. It was also observed that PSD size was robustly increased by 1.6-fold over a 24 hr period of AZ development during the early larval period in control animals. AZ maturation is likely to promote increased synaptic vesicle docking and availability, consistent with observations that correlate AZ size with either P_r or the readily releasable pool (Akbergenova, 2018).

Several models are considered for how AZs acquire this heterogeneous nature of P_r distribution during a developmental period lasting several days. One possibility is that unique AZs gain high P_r status through a mechanism that would result in preferential accumulation of key AZ components compared to their neighbors. Given that retrograde signaling from the muscle is known to drive synaptic development at Drosophila NMJs, certain AZ populations might have preferential access to specific signaling factors that would alter their P_r state. Another model is that AZs compete for key presynaptic P_r regulators through an activity-dependent process. High P_r AZs might also be more mature than their low P_r neighbors, having a longer timeframe to accumulate AZ components. Given the Drosophila NMJ is constantly forming new AZs at a rapid pace during development, newly formed AZs would be less mature compared to a smaller population of 'older' high P_r AZs (Akbergenova, 2018).

To examine if the release heterogeneity observed at the third instar stage reflects AZ birth order over several days of development, the intravital imaging was extended through a longer time period beginning in the first instar larval stage. GCaMP imaging indicated high P_r sites segregate GluRIIA and GluRIIB differently from low P_r sites, with the IIA isoform preferentially localizing at the center of PSDs apposing high P_r AZs. As such, developmental acquisition of this property was used as an indicator of high P_r sites. Although segregation of glutamate receptors may not perfectly replicate the timing of P_r acquisition during development, it is currently the best tool for estimating P_r during sequential live imaging. Based on the acquisition of GluRIIA/B segregation, the data support the hypothesis that increases in P_r reflect a time-dependent maturation process at the NMJ. The continuous addition of new AZs, which double in number during each day of development, ensures that the overall ratio of high to low P_r sites represents a low percentage as the NMJ grows. Confidence in the age-dependency model of P_r was further established by mapping release after following NMJs intravitally for 24 hr; using this approach, it was found that newly formed AZs are consistently very low Pr. Finally, P_r was mapped in the second instar stage, and it was observed that the heterogeneity at this stage is shifted towards higher releasing sites, with a reduction in the fraction of low-releasing sites (Akbergenova, 2018).

These data indicate AZs are born with low P_r and gain pre- and postsynaptic material over 3 days on an upward trajectory toward higher P_r status. Is AZ age a static determinant of Pr, or can growth rate be regulated to allow faster or slower acquisition of high P_r AZs? To answer this question, whether mutants that alter presynaptic activity influence the rate of PSD growth and GluRIIA/B segregation was assayed. In BRP⁶⁹/def, syt1^null, and nap^TS mutants with decreased presynaptic release, a significant reduction in postsynaptic maturation rate and in the percentage of PSDs with GluRIIA/IIB rings was observed. Conversely, in the shaker, eag double mutant with increased presynaptic excitability, a significantly increased rate of GluRIIB field size and a significant increase in GluRIIA/IIB rings was found compared to controls. To investigate whether differences in synapse growth and maturation rate could be seen between AZs enriched in BRP and Cac versus neighboring AZs that are deficient in these components, development was imaged in the rab3 null mutant. At the first instar stage when AZs are roughly age matched, release sites enriched in presynaptic components had developed large and mature PSDs in stark contrast with non-enriched AZs, whose PSDs appeared immature. These results indicate that PSD maturation can be influenced by presynaptic activity at the resolution of single AZs (Akbergenova, 2018).

Although how AZs are assembled during development is still being established, the current data do not support a model where AZs are fully preassembled during transport and then deposited as a single 'quantal' entity onto the presynaptic membrane. Rather, these data support a model of seeding of AZ material that increases developmentally over time as AZs matures, consistent with previous studies of AZ development in Drosophila. Although no evidence for rapid changes in P_r were detected in the steady-state conditions used in the current study, homeostatic plasticity is known to alter P_r over a rapid time frame (~10 min) at the NMJ. It will be interesting to determine if Cac abundance can change over such a rapid window, or whether the enhanced release is mediated solely through changes in Cac function and Ca2+ influx. Changes in the temporal order of P_r development could also occur secondary to altered transport or capture of AZ material. For example, the large NMJ on muscle fibers 6 and 7 displays a gradient in synaptic transmission, with terminal branch boutons often showing a larger population of higher P_r AZs. If AZ material is not captured by earlier synapses along the arbor, it would be predicted to accumulate in terminal boutons, potentially allowing these AZs greater access to key components, and subsequently increasing their rate of P_r acquisition. Alternatively, the gradient of P_r along the axon could be due to terminal boutons being slightly older than the rest of the arbor (Akbergenova, 2018).

In summary, the current data indicate that heterogeneity in release correlates highly with Ca2+ channel abundance and Ca2+ influx at AZs. Postsynaptically, PSDs apposed to high-releasing AZs display increased GluRIIA abundance and form segregated receptor fields, with GluRIIB forming a ring around a central core of GluRIIA. Release sites accumulate these high P_r markers during a synapse maturation process in which newly formed AZs are consistently low Pr, with AZs gaining signatures of high releasing sites over several days. Finally, mutations that increase or decrease presynaptic activity result in faster or slower rates of PSD maturation, respectively. These data add to the understanding of the molecular and developmental features associated with high versus low P_r AZs (Akbergenova, 2018).

A neuropeptide signaling pathway regulates synaptic growth in Drosophila

Neuropeptide signaling is integral to many aspects of neural communication, particularly modulation of membrane excitability and synaptic transmission. However, neuropeptides have not been clearly implicated in synaptic growth and development. This study demonstrates that cholecystokinin-like receptor (CCK-like receptor at 17D1; CCKLR), and Drosulfakinin (DSK), its predicted ligand, are strong positive growth regulators of the Drosophila melanogaster larval neuromuscular junction (NMJ). Mutations of CCKLR (CCKLR-17D1 but not CCKLR-17D3) or dsk produce severe NMJ undergrowth, whereas overexpression of CCKLR causes overgrowth. Presynaptic expression of CCKLR is necessary and sufficient for regulating NMJ growth. CCKLR and dsk mutants also reduce synaptic function in parallel with decreased NMJ size. Analysis of double mutants revealed that DSK/CCKLR regulation of NMJ growth occurs through the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA)-cAMP response element binding protein (CREB) pathway. These results demonstrate a novel role for neuropeptide signaling in synaptic development. Moreover, because the cAMP-PKA-CREB pathway is required for structural synaptic plasticity in learning and memory, DSK/CCKLR signaling may also contribute to these mechanisms (Chen, 2012).

Proper synaptic growth is essential for normal development of the nervous system and its function in mediating complex behaviors such as learning and memory. The Drosophila larval neuromuscular junction (NMJ) has become a powerful system for studying the molecular mechanisms underlying synaptic development and plasticity, and many of the key synaptic proteins are evolutionarily conserved (Chen, 2012).

Genetic and molecular analysis in Drosophila has uncovered numerous molecules and pathways that regulate NMJ growth, including proteins required for cell adhesion, endocytosis, cytoskeletal organization, and signal transduction via TGF-β, Wingless, JNK, cAMP, and other signaling molecules. For example, previous studies revealed that increased cAMP levels led to a down-regulation of the cell adhesion protein FasII at synapses, and the activation of the cAMP response element binding protein (CREB) transcription factor to achieve long-lasting changes in synaptic structure and function. Despite the identification and characterization of these various positive and negative regulators, understanding of the networks that govern synaptic growth is still incomplete, with many of the key components and mechanisms yet to be uncovered and analyzed (Chen, 2012).

To search for new regulators of synaptic growth, a forward genetic screen was conducted for mutants exhibiting altered NMJ morphology. In this screen, a new mutation was discovered that exhibits strikingly undergrown NMJs, which indicates disruption of a positive regulator of NMJ growth. The affected protein was identified as cholecystokinin (CCK)-like receptor (CCKLR), a putative neuropeptide receptor that belongs to the family of G-protein coupled receptors (GPCRs) sharing a uniform topology with seven transmembrane domains. When activated by their ligands, neuropeptide GPCRs affect levels of second messengers such as cAMP, diacylglycerol, inositol trisphosphate, and intracellular calcium. Through activation of their cognate receptors, secreted neuropeptides mediate communication among various sets of neurons as well as other cell types to regulate several physiological activities, including feeding and growth, molting, cuticle tanning, circadian rhythm, sleep, and learning and memory. In general, neuropeptides act by modulating neuronal activity through both short-term and long-term effects. Short-term effects include modifications of ion channel activity and alterations in release of or response to neurotransmitters. Long-term effects include changes in gene expression through activation of transcription factors and protein synthesis. In contrast with the well-known effects of neuropeptide signaling on neuronal activity and the strength of synaptic transmission, regulation of synaptic growth and development by neuropeptides has not previously been clearly established (Chen, 2012).

This study demonstrates that CCKLR is required presynaptically to promote NMJ growth. Moreover, mutations of drosulfakinin (dsk), which encode the predicted ligand of CCKLR, cause similar NMJ undergrowth and interact genetically with CCKLR mutations, indicating that DSK and CCKLR are components of a common signaling mechanism that regulates NMJ growth. In addition to the morphological phenotypes caused by mutations of CCKLR and dsk, mutant larvae also exhibit deficits in synaptic function. Through phenotypic analysis of double mutant combinations, it was shown that DSK/CCKLR signals through the cAMP-PKA-CREB pathway to regulate NMJ growth. The results suggest a novel role for neuropeptide signaling in regulation of synaptic development. Moreover, because the cAMP-PKA-CREB pathway is required for structural plasticity of synapses in learning and memory, DSK/CCKLR signaling may also contribute to these mechanisms (Chen, 2012).

Neuropeptides, whose effects have been extensively studied at NMJs, are usually described as neuromodulators because they modify the strength of synaptic transmission. For example, proctolin can potentiate the action of glutamate at certain NMJs in insects. However, involvement of neuropeptides in regulating neural development has not been well characterized. Recently, a C-type natriuretic peptide acting through a cGMP signaling cascade was found to be required for sensory axon bifurcation in mice, which suggests that neuropeptides may have a broader role in development than previously appreciated. The current studies demonstrate that DSK and its receptor, CCKLR, are strong positive regulators of NMJ growth in Drosophila (Chen, 2012).

DSK belongs to the family of FMRFamide-related peptides (FaRPs), which is very broadly distributed across invertebrate and vertebrate phyla. Originally identified in clams, FaRPs affect heart rate, blood pressure, gut motility, feeding behavior, and reproduction in invertebrates. They have been shown to enhance synaptic efficacy at NMJs in locust and to modulate presynaptic Ca²⁺ channel activity in crustaceans. In Drosophila, various neuropeptides derived from the FMRFa gene can modulate the strength of muscle contraction when perfused onto standard larval nerve-muscle preparations. To these previously described functions of FaRPs, this study adds a new role as a positive regulator of NMJ development (Chen, 2012).

Transgenic rescue experiments, RNAi expression, and overexpression of WT CCKLR demonstrate that CCKLR functions presynaptically in motor neurons to promote NMJ growth. Downstream components of this pathway were identified on the basis of known biochemistry of GPCRs and phenotypic interactions in double mutant combinations. GPCRs typically function by activating second messenger pathways via G proteins. Because loss-of-function mutations in dgs (which encodes the Gsα subunit in Drosophila) cause NMJ undergrowth, it is hypothesized that CCKLR signals through Gsα. Consistent with this idea, it was found that presynaptic constitutively active dgs overexpression rescues the NMJ undergrowth phenotype of CCKLR mutants. Conversely, dominant dose-dependent interactions were observed between CCKLR-null mutations and mutations of rut, which encodes an AC; or PKA-C1, which encodes a cAMP-dependent protein kinase, resulting in significant reductions in NMJ growth. These data place CCKLR together with the other genes in a common cAMP-dependent signaling pathway that regulates NMJ growth (Chen, 2012).

It is known that the AC encoded by rut is activated by Gsα, and on the basis of the results, it is proposed that Gsα is downstream of CCKLR signaling. However, the NMJ undergrowth in rut¹, which is a presumptive null mutant, is not as severe as that of a CCKLR-null mutant. This is likely due to the fact that the Drosophila genome contains up to seven different AC-encoding genes, all of which are stimulated by Gsα. Presumably, one or more additional AC-encoding genes share some functional overlap with rut in regulation of NMJ growth. This idea is in good agreement with the results of Wolfgang (2004), who found that the NMJ undergrowth phenotype of rut¹ is weaker than that of dgs mutants, which they also interpreted as an indication that multiple ACs are activated by the Gsα encoded by dgs (Chen, 2012).

The primary effector of this pathway is CREB2, a transcriptional regulatory protein that is activated upon phosphorylation by PKA. Consistent with the idea that CCKLR ultimately acts via activation of CREB, loss-of-function mutations of dCreb2 or neuronal overexpression of a dominant-negative dCreb2 transgene cause NMJ undergrowth similar to that of CCKLR-null mutants. Additionally, loss of one copy of dCreb2 in a CCKLR heterozygous background also causes NMJ undergrowth, and overexpression of WT dCreb2 fully rescues the NMJ undergrowth phenotype of CCKLR null, even leading to NMJ overgrowth. Thus, regulation of NMJ growth through the CCKLR signaling pathway is clearly mediated by dCreb2, whose activity is itself necessary and sufficient for regulating NMJ growth. This conclusion differs from another study that suggested that dCreb2 is required for NMJ function but not NMJ growth. One possible explanation for this discrepancy is that a weaker, inducible heat shock-driven transgene was used to express dCreb2 in the earlier work, whereas strong constitutive neuronal drivers were used this study. In any case, the current results demonstrate that in addition to its known role in NMJ function, CREB2 is also a strong positive regulator of NMJ growth and is likely to play a greater role in structural plasticity of synapses in learning and memory in Drosophila than previously suggested. This conclusion is consistent with a recent study indicating that sprouting of type II larval NMJs in response to starvation is stimulated by a cAMP/CREB-dependent pathway via activation of an octopamine GPCR (Chen, 2012).

In addition to being undergrown, NMJs in CCKLR mutant larvae also exhibit a functional deficit. This is perhaps less straightforward than it might seem. Previous analyses of mutations affecting growth of the larval NMJ in Drosophila have shown that there is no simple correlation between the size and complexity of the NMJ and the amplitude of EJPs or amount of neurotransmitter release. These discrepancies arise because of various homeostatic compensatory mechanisms and because some of the affected signaling pathways alter synaptic growth and synaptic function in different ways via distinct downstream targets. For example, a mutation in highwire, which has the most extreme NMJ overgrowth phenotype described, is associated with a decrease in synaptic transmission. Wallenda mutations have been shown to fully suppress the overgrowth phenotype, but have no effect on the deficit in synaptic transmission (Chen, 2012).

In the case of CCKLR mutants, however, there appears to be a very good correspondence between the morphological phenotype and the electrophysiological phenotype: the reduction in the total number of active zones in CCKLR larvae as measured morphologically correlates very well with the reduction in quantal content that was observe. In addition, no difference in CCKLR mutants was detected in calcium sensitivity of transmitter release or in the size or frequency of spontaneous release events. Thus, the synaptic growth phenotype of CCKLR mutant NMJs is sufficient to account for the functional phenotype. However, the possibility cannot be ruled out that DSK/CCKLR signaling also exerts some modulatory effect on NMJ function that is distinct from its effect on NMJ development (Chen, 2012).

DSK is identified as the Drosophila orthologue of CCK, the ligand of CCKLR in vertebrates, on the basis of sequence analysis. The genetic analysis strongly supports the conclusion that DSK is the ligand of CCKLR at the larval NMJ. First, mutations of dsk and expression of dsk RNAi result in NMJ undergrowth phenotypes similar to that of CCKLR mutants. Second, loss of one copy of both dsk and CCKLR in double heterozygotes results in NMJ undergrowth. Third, heterozygosity for dsk does not further enhance the phenotype of a CCKLR-null mutant as expected if DSK regulates NMJ growth through its action on CCKLR. Fourth, overexpression of UAS-dsk does not rescue the undergrowth phenotype of a CCKLR-null mutant, but CCKLR overexpression can rescue NMJ undergrowth of a dsk hypomorphic mutant (Chen, 2012).

The discovery of an entirely novel role for neuropeptide signaling in NMJ growth raises several questions about how this signaling is regulated and the biological significance of this mechanism. Although answers to these questions will require much additional work, an immediate question is whether a paracrine or autocrine mechanism is involved. In the case of octopamine-mediated synaptic sprouting in response to starvation, both autocrine and paracrine signaling are involved in the sprouting of type II and type I NMJs, respectively. In an early immunohistochemical investigation, it was reported that DSK was detected in medial neurosecretory cells in the larval CNS that extended projections anteriorly into the brain and posteriorly to the ventral ganglion. As it was not possible to obtain the original DSK antiserum and raising a new antiserum was not successful, the previous report has not been extended or confirmed. Instead, tissue-specific RNAi experiments were performed to examine the spatial requirement for DSK. Pan-neuronal dsk RNAi expression indicates that DSK expression in neurons is required to promote NMJ growth. In addition, C739-Gal4-driven dsk RNAi also causes NMJ undergrowth, whereas OK-Gal4-driven dsk RNAi in motor neurons does not. The expression pattern of C739-Gal4 overlaps with the DSK-positive cells previously identified by immunohistochemistry, which suggests that DSK produced by those neurosecretory cells is required for normal NMJ growth. Thus, from available data, it seems most likely that DSK is acting in paracrine fashion to regulate NMJ growth. However, further investigation will be necessary to determine the exact source of the DSK that promotes NMJ growth to fully understand how this neuropeptide regulates NMJ development (Chen, 2012).

Studies have demonstrated a role for CREB in long-term synaptic plasticity-structural changes in synaptic morphology that underlie the formation of long-term memories. This study shows that in addition to CREB's role in structural modification of synapses in response to experience after development is complete, it is also a key regulator of growth and morphology during development of the larval NMJ. Moreover, although CREB is the transcriptional effector for many GPCRs, the fact that NMJs in CCKLR mutants are as undergrown as those of CREB mutants suggests that DSK/CCKLR signaling is a major input to CREB during NMJ growth. Many of the genes encoding intermediate components of the pathway such as dnc, rut, and PKA also have effects on NMJ growth and development as well as on synaptic plasticity and learning and memory, further emphasizing an overlap between the mechanisms that regulate synaptic growth during development and those that regulate postdevelopmental structural synaptic plasticity. These results raise the possibility that DSK/CCKLR signaling also plays a role in long-term synaptic plasticity and learning as well as in synaptic development (Chen, 2012).

Heterotrimeric Go protein links Wnt-Frizzled signaling with ankyrins to regulate the neuronal microtubule cytoskeleton

Drosophila neuromuscular junctions (NMJs) represent a powerful model system with which to study glutamatergic synapse formation and remodeling. Several proteins have been implicated in these processes, including components of canonical Wingless (Drosophila Wnt1) signaling and the giant isoforms of the membrane-cytoskeleton linker Ankyrin 2, but possible interconnections and cooperation between these proteins were unknown. This study demonstrates that the heterotrimeric G protein G_o functions as a transducer of Wingless-Frizzled 2 signaling in the synapse. Ankyrin 2 was identified as a target of Go signaling required for NMJ formation. Moreover, the G_o-ankyrin interaction is conserved in the mammalian neurite outgrowth pathway. Without ankyrins, a major switch in the G_o-induced neuronal cytoskeleton program is observed, from microtubule-dependent neurite outgrowth to actin-dependent lamellopodial induction. These findings describe a novel mechanism regulating the microtubule cytoskeleton in the nervous system. This work in Drosophila and mammalian cells suggests that this mechanism might be generally applicable in nervous system development and function (Luchtenborg, 2014).

Ankyrins (Ank) are highly abundant modular proteins that mediate protein-protein interactions, mainly serving as adaptors for linking the cytoskeleton to the plasma membrane. Mammalian genomes encode three Ank genes [AnkR (Ank1), AnkB (Ank2) and AnkG (Ank3)], whereas Drosophila has two [Ank1 (also known as Ank - FlyBase) and Ank2]. Ank2 is expressed exclusively in neurons and exists in several splicing variants. The larger isoforms (Ank2M, Ank2L and Ank2XL) are localized to axons and play important roles in NMJ formation and function. The C-terminal part of Ank2L can bind to microtubules. Despite the well-established role of Ank2 in NMJ formation, its function has been considered somewhat passive and its mode of regulation has not been clarified. This study shows that Gαo binds to Ank2 and that these proteins and the Wg pathway components Wg, Fz2, and Sgg jointly coordinate the formation of the NMJ. The functional Gαo-Ank interaction is conserved from insects to mammals (Luchtenborg, 2014).

Synaptic plasticity underlies learning and memory. Both in invertebrates and vertebrates, activation of Wnt signaling is involved in several aspects of synapse formation and remodeling, and defects in this pathway may be causative of synaptic loss and neurodegeneration. Thus, understanding the molecular mechanisms of synaptic Wnt signaling is of fundamental as well as medical importance. The Drosophila NMJ is a powerful model system with which to study glutamatergic synapses, and the Wnt pathway has been widely identified as one of the key regulators of NMJ formation.

This study provides important mechanistic insights into Wnt signal transduction in the NMJ, identifying the heterotrimeric G_o protein as a crucial downstream transducer of the Wg-Fz2 pathway in the presynapse. It was further demonstrated that Ank2, a known player in the NMJ, is a target of Gαo in this signaling (Luchtenborg, 2014).

This study found that the α subunit of G_o is strongly expressed in the presynaptic cell, and that under- or overactivation of this G protein leads to neurotransmission and behavioral defects. At the level of NMJ morphology, presynaptic downregulation or Ptx-mediated inactivation of Gαo was found to recapitulate the phenotypes obtained by similar silencing of wg and fz2. These data confirm that presynaptic Wg signaling, in addition to the Wg pathway active in the muscle, is crucial for proper NMJ formation, and that G_o is required for this process. Furthermore, neuronal Gαo overexpression can rescue the wg and fz2 loss-of-function phenotypes, demonstrating that, as in other contexts of Wnt/Fz signaling, G_o acts as a transducer of Wg/Fz2 in NMJ formation. In contrast to its evident function and clear localization in the presynapse, Gαo localization on the muscle side of the synapse is much less pronounced or absent. Unlike Gαo, the main Drosophila Gβ subunit is strongly expressed in both the pre- and postsynapse. Thus, a heterotrimeric G protein other than G_o might be involved in the postsynaptic Fz2 transduction, as has been implicated in Fz signaling in some other contexts (Luchtenborg, 2014).

A recent study proposed a role for Gαo downstream of the octopamine receptor Octβ1R. This signaling was proposed to regulate the acute behavioral response to starvation both on type II NMJs (octapaminergic) and on the type I NMJs (glutamatergic) analyzed in this study. In contrast to the current observations, downregulation of Gαo in these NMJs was proposed to increase, rather than decrease, type I bouton numbers. It is suspected that the main reason for the discrepancy lies in the Gal4 lines used. The BG439-Gal4 and C380-Gal4 lines of Koon are poorly characterized and, unlike the well-analyzed pan-neuronal elav-Gal4 and motoneuron-specific OK371-Gal4 and D42-Gal4 driver lines used in the current study, might mediate a more acute expression. In this case, this study reflects the positive role of Gαo in the developmental formation of glutamatergic boutons, as opposed to a role in acute fine-tuning in response to environmental factors as studied by Koon (Luchtenborg, 2014).

Postsynaptic expression of fz2 was found to fully rescue fz2 null NMJs. This study found that presynaptic knockdown of Fz2 (and other components of Wg-Fz2-Gαo signaling) recapitulates fz2 null phenotypes, whereas presynaptic overactivation of this pathway increases bouton numbers; furthermore, presynaptic overexpression of fz2 or Gαo rescues the fz2 nulls, just as postsynaptic overexpression of fz2 does. The current data thus support a crucial role for presynaptic Wg-Fz2-Gαo signaling in NMJ formation. Interestingly, both pre- and postsynaptic re-introduction of Arrow, an Fz2 co-receptor that is normally present both pre- and postsynaptically, as is Fz2 itself, can rescue arrow mutant NMJs. Thus, it appears that the pre- and postsynaptic branches of Fz2 signaling are both involved in NMJ development. A certain degree of redundancy between these branches must exist. Indeed, wild-type levels of Fz2 in the muscle are not sufficient to rescue the bouton defects induced by presynaptic expression of RNAi-fz2, yet overexpression of fz2 in the muscle can restore the bouton integrity of fz2 nulls. One might hypothesize that postsynaptic Fz2 overexpression activates a compensatory pathway - such as that mediated by reduction in laminin A signaling - that leads to restoration in bouton numbers in fz2 mutants. The current data showing that the targeted downregulation of Fz2 in the presynapse is sufficient to recapitulate the fz2 null phenotype underpin the crucial function of presynaptic Fz2 signaling in NMJ formation (Luchtenborg, 2014).

This study found that downregulation of Ank2 produces NMJ defects similar to those of wg, fz2 or Gαo silencing. However, Ank2 mutant phenotypes appear more pronounced, indicating that Wg-Fz2-Gαo signaling might control a subset of Ank2-mediated activities in the NMJ. Ank2 was proposed to play a structural role in NMJ formation, binding to microtubules through its C-terminal region. However, since the C-terminal region was insufficient to rescue Ank2L mutant phenotypes, additional domains are likely to mediate Ank2 function through binding to other proteins. This study demonstrates in the yeast two-hybrid system and in pull-down experiments that the ankyrin repeat region of Ank2 physically binds Gαo, suggesting that the function of Ank2 in NMJ formation might be regulated by Wg-Fz2-Gαo signaling. Indeed, epistasis experiments place Ank2 downstream of Gαo in NMJ formation (Luchtenborg, 2014).

Upon dissociation of the heterotrimeric G_o protein by activated GPCRs such as Fz2, the liberated Gαo subunit can signal to its downstream targets both in the GTP- and GDP-bound state (the latter after hydrolysis of GTP and before re-association with Gβγ). The free signaling Gαo-GDP form is predicted to be relatively long lived, and a number of Gαo target proteins have been identified that interact equally well with both of the nucleotide forms of this G protein. In the context of NMJ formation, this study found that Gαo-GTP and -GDP are efficient in the activation of downstream signaling, and identifies Ank2 as a binding partner of Gαo that interacts with both nucleotide forms. The importance of signaling by Gα-GDP released from a heterotrimeric complex by the action of GPCRs has also been demonstrated in recent studies of mammalian chemotaxis, planar cell polarity and cancer (Luchtenborg, 2014).

Gαo[G203T], which largely resides in the GDP-binding state owing to its reduced affinity for GTP, might be expected to act as a dominant-negative. However, in canonical Wnt signaling, regulation of asymmetric cell division as well as in planar cell polarity (PCP) signaling in the wing, Gαo[G203T] displays no dominant-negative activity but is simply silent, whereas in eye PCP signaling this form acts positively but is weaker than other Gαo forms. Biochemical characterization of the mammalian Gαi2[G203T] mutant revealed that it can still bind Gβγ and GTP, but upon nucleotide exchange Gαi2[G203T] fails to adopt the activated confirmation and can further lose GTP. The current biochemical characterization confirms that Gαo[G203T] still binds GTP. Interestingly, Gαi2[G203T] inhibited only a fraction of Gαi2-mediated signaling, suggesting that the dominant-negative effects of the mutant are effector specific. Thus, it is inferred that a portion of Gαo[G203T] can form a competent Fz2-transducing complex, and a portion of overexpressed Gαo[G203T] resides in a free GDP-loaded form that is also competent to activate downstream targets - Ank2 in the context of NMJ formation (Luchtenborg, 2014).

These experiments place Ank2 downstream of Gαo and also of Sgg (GSK3β). It remains to be investigated whether Ank2 can directly interact with and/or be phosphorylated by Sgg. Meanwhile, it is proposed that the microtubule-binding protein Futsch might be a linker between Sgg and Ank2. Futsch is involved in NMJ formation and is placed downstream of Wg-Sgg signaling, being the target of phosphorylation and negative regulation by Sgg as the alternative target to β-catenin, which is dispensable in Wg NMJ signaling. Abnormal Futsch localization has been observed in Ank2 mutants. In Drosophila wing and mammalian cells in culture, Gαo acts upstream of Sgg/GSK3β. Cumulatively, these data might suggest that the Wg-Fz2-Gαo cascade sends a signal to Futsch through Sgg, parallel to that mediated by Ank2 (Luchtenborg, 2014).

The importance of the Gαo-Ank2 interaction for Drosophila NMJ development is corroborated by findings in mammalian neuronal cells, where it was demonstrated that the ability of Gαo to induce neurite outgrowth is critically dependent on AnkB and AnkG. Knockdown of either or both ankyrin reduces neurite production. Remarkably, upon AnkB/G downregulation, Gαo switches its activity from the induction of microtubule-dependent processes (neurites) to actin-dependent protrusions (lamellopodia). Furthermore, Gαo recruits AnkB to the growing neurite tips. These data demonstrate that the Gαo-ankyrin mechanistic interactions are conserved from insects to mammals and are important for control over the neuronal tubulin cytoskeleton in the context of neurite growth and synapse formation. The novel signaling mechanism that were uncovered might thus be of general applicability in animal nervous system development and function (Luchtenborg, 2014).

Acetylated alpha-tubulin K394 regulates microtubule stability to shape the growth of axon terminals

Microtubules are essential to neuron shape and function. Acetylation of tubulin has the potential to directly tune the behavior and function of microtubules in cells. Although proteomic studies have identified several acetylation sites in α-tubulin, the effects of acetylation at these sites remains largely unknown. This includes the highly conserved residue lysine 394 (K394), which is located at the α-tubulin dimer interface. Using a fly model, this study showed that α-tubulin K394 is acetylated in the nervous system and is an essential residue. Acetylation-blocking mutation in endogenous α-tubulin, K394R, was found to perturb the synaptic morphogenesis of motoneurons and reduces microtubule stability. Intriguingly, the K394R mutation has opposite effects on the growth of two functionally and morphologically distinct motoneurons, revealing neuron-type-specific responses when microtubule stability is altered. Eliminating the deacetylase HDAC6 increases K394 acetylation, and the over-expression of HDAC6 reduces microtubule stability similar to the K394R mutant. Thus, these findings implicate α-tubulin K394 and its acetylation in the regulation of microtubule stability and suggest that HDAC6 regulates K394 acetylation during synaptic morphogenesis (Saunders, 2022).

Drosophila Cbp53E regulates axon growth at the meuromuscular junction

Calcium is a primary second messenger in all cells that functions in processes ranging from cellular proliferation to synaptic transmission. Proper regulation of calcium is achieved through numerous mechanisms involving channels, sensors, and buffers notably containing one or more EF-hand calcium binding domains. The Drosophila genome encodes only a single 6 EF-hand domain containing protein, Cbp53E, which is likely the prototypic member of a small family of related mammalian proteins that act as calcium buffers and calcium sensors. Like the mammalian homologs, Cbp53E is broadly though discretely expressed throughout the nervous system. Despite the importance of calcium in neuronal function and growth, nothing is known about Cbp53E's function in neuronal development. To address this deficiency, novel null alleles of Drosophila Cbp53E were generated, and neuronal development was examined at the well-characterized larval neuromuscular junction. Loss of Cbp53E resulted in increases in axonal branching at both peptidergic and glutamatergic neuronal terminals. This overgrowth could be completely rescued by expression of exogenous Cbp53E. Overexpression of Cbp53E, however, only affected the growth of peptidergic neuronal processes. These findings indicate that Cbp53E plays a significant role in neuronal growth and suggest that it may function in both local synaptic and global cellular mechanisms (Hagel, 2015).

Misregulation of Drosophila Sidestep leads to uncontrolled wiring of the adult neuromuscular system and severe locomotion defects

Holometabolic organisms undergo extensive remodelling of their neuromuscular system during metamorphosis. Relatively, little is known whether or not the embryonic guidance of molecules and axonal growth mechanisms are re-activated for the innervation of a very different set of adult muscles. This study shows that the axonal attractant Sidestep (Side) is re-expressed during Drosophila metamorphosis and is indispensable for neuromuscular wiring. Mutations in side cause severe innervation defects in all legs. Neuromuscular junctions (NMJs) show a reduced density or are completely absent at multi-fibre muscles. Misinnervation strongly impedes, but does not completely abolish motor behaviours, including walking, flying, or grooming. Overexpression of Side in developing muscles induces similar innervation defects; for example, at indirect flight muscles, it causes flightlessness. Since muscle-specific overexpression of Side is unlikely to affect the central circuits, the resulting phenotypes seem to correlate with faulty muscle wiring. It was further shown that mutations in beaten path Ia (beat), a receptor for Side, results in similar weaker adult innervation and locomotion phenotypes, indicating that embryonic guidance pathways seem to be reactivated during metamorphosis (Kinold, 2021).

Distinct regulation of transmitter release at the Drosophila NMJ by different isoforms of nemy

Synaptic transmission is highly plastic and subject to regulation by a wide variety of neuromodulators and neuropeptides. The present study examined the role of isoforms of the cytochrome b561 homologue called no extended memory (nemy) in regulation of synaptic strength and plasticity at the neuromuscular junction (NMJ) of third instar larvae in Drosophila. Specifically, two independent excisions of nemy were generated that differentially affect the expression of nemy isoforms. The nemy⁴⁵ excision, which specifically reduces the expression of the longest splice form of nemy, leads to an increase in stimulus evoked transmitter release and altered synaptic plasticity at the NMJ. Conversely, the nemy^26.2 excision, which appears to reduce the expression of all splice forms except the longest splice isoform, shows a reduction in stimulus evoked transmitter release, and enhanced synaptic plasticity. It was further shown that nemy⁴⁵ mutants have reduced levels of amidated peptides similar to that observed in peptidyl-glycine hydryoxylating mono-oxygenase (PHM) mutants. In contrast, nemy^26.2 mutants show no defects in peptide amidation but rather display a decrease in Tyramine beta hydroxylase activity (TbetaH). Taken together, these results show non-redundant roles for the different nemy isoforms and shed light on the complex regulation of neuromodulators (Knight, 2015).

The guanine exchange factor Gartenzwerg and the small GTPase Arl1 function in the same pathway with Arfaptin during synapse growth

The generation of neuronal morphology requires transport vesicles originating from the Golgi apparatus (GA) to deliver specialized components to the axon and dendrites. Drosophila Arfaptin is a membrane-binding protein localized to the GA that is required for the growth of the presynaptic nerve terminal. This study provides biochemical, cellular and genetic evidence that the small GTPase Arl1 and the guanine-nucleotide exchange factor (GEF) Gartenzwerg are required for Arfaptin function at the Golgi during synapse growth. These data define a new signaling pathway composed of Arfaptin, Arl1, and Garz, required for the generation of normal synapse morphology (Chang, 2015).

Drosophila neuronal injury follows a temporal sequence of cellular events leading to degeneration at the neuromuscular junction

There is a critical need to improve understanding of the molecular and cellular mechanisms that drive neurodegeneration. At the molecular level, neurodegeneration involves the activation of complex signaling pathways that drive the active destruction of neurons and their intracellular components. This study used an in vivo motor neuron injury assay to acutely induce neurodegeneration in order to follow the temporal order of events that occur following injury in Drosophila. Sites of injury can be rapidly identified based on structural defects to the neuronal cytoskeleton that result in disrupted axonal transport. Additionally, the neuromuscular junction accumulates ubiquitinated proteins prior to the neurodegenerative events, occurring at 24 hours post injury. These data provide insights into the early molecular events that occur during axonal and neuromuscular degeneration in a genetically tractable model organism. Importantly, the mechanisms that mediate neurodegeneration in flies are conserved in humans. Thus, these studies will facilitate the identification of biomedically relevant targets for future treatments (Lincoln, 2015).

Protein phosphatase 2A restrains DLK signaling to promote proper Drosophila synaptic development and mammalian cortical neuron survival

Protein phosphatase 2A (PP2A) is a major cellular phosphatase with many protein substrates. In post-mitotic neurons, the microtubule associated protein Tau is a particularly well-studied PP2A substrate as hyperphosphorylation of Tau is a hallmark of Alzheimer's disease. This study finds that activation of a single pathway can explain important aspects of the PP2A loss-of-function phenotype in neurons. PP2A inhibits activation of the neuronal stress kinase DLK and its Drosophila ortholog Wallenda. In the fly, PP2A inhibition activates a DLK/Wallenda-regulated transcriptional program that induces synaptic terminal overgrowth at the neuromuscular junction. In cultured mammalian neurons, PP2A inhibition activates a DLK-dependent apoptotic program that induces cell death. Contrary to expectations, in the absence of Tau PP2A inhibition still activates DLK and induces neuronal cell death, demonstrating that hyperphosphorylated Tau is not required for cell death in this model. Moreover, hyperphosphorylation of Tau following PP2A inhibition does not require DLK. Hence, loss of PP2A function in cortical neurons triggers two independent neuropathologies: 1) Tau hyperphosphorylation and 2) DLK activation and subsequent neuronal cell death. These findings demonstrate that inhibition of the DLK pathway is an essential function of PP2A required for normal Drosophila synaptic terminal development and mammalian cortical neuron survival (Hayne, 2022).

Diminished MTORC1-dependent JNK activation underlies the neurodevelopmental defects associated with lysosomal dysfunction

This study evaluated the mechanisms underlying the neurodevelopmental deficits in Drosophila and mouse models of lysosomal storage diseases (LSDs). Lysosomes promote the growth of neuromuscular junctions (NMJs) via Rag GTPases and mechanistic target of rapamycin complex 1 (MTORC1). However, rather than employing S6K/4E-BP1, MTORC1 stimulates NMJ growth via JNK, a determinant of axonal growth in Drosophila and mammals. This role of lysosomal function in regulating JNK phosphorylation is conserved in mammals. Despite requiring the amino-acid-responsive kinase MTORC1, NMJ development is insensitive to dietary protein. This paradox is attributed to anaplastic lymphoma kinase (ALK), which restricts neuronal amino acid uptake, and the administration of an ALK inhibitor couples NMJ development to dietary protein. These findings provide an explanation for the neurodevelopmental deficits in LSDs and suggest an actionable target for treatment (Wong, 2015).

Mucolipidosis type IV (MLIV) and Batten disease are untreatable lysosomal storage diseases (LSDs) that cause childhood neurodegeneration. MLIV arises from loss-of-function mutations in the gene encoding TRPML1, an endolysosomal cation channel belonging to the TRP superfamily. The absence of TRPML1 leads to defective lysosomal storage and autophagy, mitochondrial damage, and macromolecular aggregation, which together initiate the protracted neurodegeneration observed in MLIV). Batten disease arises from the absence of a lysosomal protein, CLN3), and results in psychomotor retardation. Both diseases cause early alterations in neuronal function. For instance, brain imaging studies revealed that MLIV and Batten patients display diminished axonal development in the cortex and corpus callosum, the causes of which remain unknown (Wong, 2015).

To better understand the etiology of MLIV in a genetically tractable model, flies were generated lacking the TRPML1 ortholog. The trpml-deficient (trpml¹) flies have led to insight into the mechanisms of neurodegeneration and lysosomal storage (Wong, 2015).

This study reports that trpml¹ larvae exhibit diminished synaptic growth at the NMJ, a well-studied model synapse. Lysosomal function supports Rag GTPases and MTORC1 activation, and this is essential for JNK phosphorylation and synapse development (Wong, 2015).

Drosophila larvae and mice lacking CLN3 also exhibit diminished Rag/ MTORC1 and JNK activation, suggesting that alterations in neuronal signaling are similar in different LSDs and are evolution- arily conserved. Interestingly, the NMJ defects in the two fly LSD models were suppressed by the administration of a high-protein diet and a drug that is currently in clinical trials to treat certain forms of cancer. These findings inform a pharmacotherapeutic strategy that may suppress the neurodevelopmental defects observed in LSD patients (Wong, 2015).

This study shows that lysosomal dysfunction in Drosophila MNs results in diminished bouton numbers at the larval NMJ. Evidence is presented that lysosomal dysfunction results in decreased activation of the amino-acid-responsive cascade involving Rag/MTORC1, which are critical for normal NMJ development (Wong, 2015).

Despite the requirement for MTORC1 in NMJ synapse development, previous studies and the current findings show that bouton numbers are independent of S6K and 4E-BP1. Rather, MTORC1 promotes NMJ growth via a MAP kinase cascade culminating in JNK activation. Therefore, decreasing lysosomal function or Rag/MTORC1 activation in hiw^ND8 suppressed the associated synaptic overgrowth. However, the 'small-bouton' phenotype of hiw^ND8 was independent of MTORC1. Thus, MTORC1 is required for JNK-dependent regulation of bouton numbers, whereas bouton morphology is independent of MTORC1. Furthermore, although both rheb expression and hiw loss result in Wallenda-dependent elevation in bouton numbers, the supernumerary boutons in each case show distinct morphological features. Additional studies are needed for deciphering the complex interplay between MTORC1-JNK in regulating the NMJ morphology (Wong, 2015).

Biochemical analyses revealed that both JNK phosphorylation and its transcriptional output correlated with the activity of MTORC1, which are consistent with prior observations that cln³ overexpression promotes JNK activation and that tsc1/tsc2 deletion in flies result in increased JNK-dependent transcription. These findings point to the remarkable versatility of MTORC1 in controlling both protein trans lation and gene transcription (Wong, 2015).

Using an in vitro kinase assay, this study demonstrates that Wallenda (Wnd) is a target of MTORC1. Because axonal injury activates both MTORC1 and DLK/JNK, these findings imply a functional connection between these two pathways. Interestingly, the data also suggest that MTORC1 contains additional kinases besides MTOR that can phosphorylate Wnd. One possibility is that ULK1/Atg1, which associates with MTORC1, could be the kinase that phosphorylates Wnd. Consistent with this notion, overexpression of Atg1 in the Drosophila neurons has been shown to promote JNK signaling and NMJ synapse overgrowth via Wnd) (Wong, 2015).

This study also found that developmental JNK activation in axonal tracts of the CC and pJNK levels in cortical neurons were compromised in a mouse model of Batten disease. Thus, the signaling deficits identified in Drosophila are also conserved in mammals. The activity of DLK (the mouse homolog of Wnd) and JNK signaling are critical for axonal development in the mouse CNS. Therefore, decreased neuronal JNK activation during development might underlie the thinning of the axonal tracts observed in many LSDs (Wong, 2015).

Although the findings of this study demonstrate a role for an amino-acid- responsive cascade in the synaptic defects associated with lysosomal dysfunction, simply elevating the dietary protein content was not sufficient to rescue these defects. These findings were reminiscent of an elegant study that showed that the growth of Drosophila neuroblasts is uncoupled from dietary amino acids owing to the function of ALK, which suppresses the uptake of amino acids into the neuroblasts (Cheng, 2011). Indeed, simultaneous administration of an ALK inhibitor and a high-protein diet partially rescued the synaptic growth defects associated with the lysosomal dysfunction, and improved the rescue of pupal lethality associated with trpml¹. Although these studies do not causally link the defects in synapse development with pupal lethality, they do raise the intriguing possibility that multiple phenotypes associated with LSDs could be targeted using ALK inhibitors along with a protein-rich diet (Wong, 2015).

Although LSDs result in lysosomal dysfunction throughout the body, neurons are exceptionally sensitive to these alterations. The cause for this sensitivity remains incompletely understood. Given the findings of this study that mature neurons do not efficiently take up amino acids from the extracellular medium, lysosomal degradation of proteins serves as a major source of free amino acids in these cells. Therefore, disruption of lysosomal degradation leads to severe shortage of free amino acids in neurons, regardless of the quantity of dietary proteins, thus explaining the exquisite sensitivity of neurons to lysosomal dysfunction (Wong, 2015).

σ2-adaptin facilitates basal synaptic transmission and is required for regenerating endo-exo cycling pool under high frequency nerve stimulation in Drosophila

The functional requirement of AP2 complex (see AP-2α) in synaptic membrane retrieval by clathrin mediated endocytosis (CME) is not fully understood. This study isolated and functionally characterized a mutation that dramatically altered synaptic development. Based on the aberrant neuromuscular junction synapse, this mutation was named angur (a Hindi dialect meaning grapes). Loss-of-function alleles of angur show more than two-fold overgrowth in bouton numbers and dramatic decrease in bouton size. angur mutation was mapped to σ2-adaptin, the smallest subunit of the adapter complex 2. Reducing neuronal level of any of the subunits of AP2 complex or disrupting AP2 complex assembly in neurons phenocopied σ2-adaptin mutation. Genetic perturbation of σ2-adaptin in neurons leads to a reversible temperature sensitive paralysis at 38 degrees °C. Electrophysiological analysis of the mutants revealed reduced evoked junction potentials and quantal content. Interestingly, high frequency nerve stimulation caused prolonged synaptic fatigue at the NMJs. The synaptic level of subunits of AP2 complex and clathrin but not other endocytic proteins were reduced in the mutants. Moreover, the BMP/TGFβ signalling was altered in these mutants and was restored by normalizing σ2-adaptin in neurons. Thus, these data suggest that - 1) while σ2-adaptin facilitates SV recycling for basal synaptic transmission, its activity is also required for regenerating SV during high frequency nerve stimulation; and 2) σ2-adaptin regulates NMJ morphology by attenuating TGFβ signalling (Choudhury, 2016).

Synaptic transmission requires fusion of synaptic vesicles (SVs) at the active zones followed by their efficient retrieval and recycling through endocytic mechanisms. Retrieval and sorting of membrane lipids and vesicular proteins at the synapse are mediated by a well-orchestrated and coordinated action of several adapter and endocytic proteins. Clathrin-mediated endocytosis (CME) is the primary pathway operative at the synapses for membrane retrieval. Genetic analysis of the components of the CME pathway in Caenorhabditis elegans and Drosophila has revealed that this pathway is required for SV re-formation, and in many cases, blocking CME at synapses results in temperature-sensitive paralysis. Additionally, CME plays a crucial role in regulating synaptic morphology. At Drosophila NMJs, blocking CME results in enhanced bone morphogenetic protein (BMP) signaling and affects synaptic growth (Choudhury, 2016).

The heterotetrameric adapter protein 2 (AP2) complex is a major effector of the CME pathway. AP2 serves as a major hub for a large number of molecular interactions and links plasma membrane, cargo/signaling molecules, clathrin, and accessory proteins in the CME pathway and hence can directly influence synaptic signaling. The AP2 complex is pseudo-asymmetric and contains four subunits-one each of large α and β₂ subunits, one medium μ₂ subunit, and a small σ₂ subunit. Depletion of clathrin or its major adapter, AP2, in either Drosophila or mammalian central synapses results in accumulation of endosome-like vacuoles and reduction of SVs, suggesting that CME may not be essential for membrane retrieval. Similarly, genetic perturbation of μ₂-adaptin or α-adaptin shows only mild defects in vesicle biogenesis at C. elegans synapses, but simultaneous loss of both adaptins leads to severely compromised SV biogenesis and accumulation of large vacuoles at nerve terminals. While Drosophila loss-of-function mutations in α-adaptin are embryonic lethal, hypomorphic mutants exhibit reduced FM1-43 uptake, suggesting a compromised endocytosis in these mutants. Whether reduced endocytosis reflects a defect in membrane retrieval or a defect in SV biogenesis remains unclear. Moreover, the consequences of AP2 reduction on synaptic morphology and physiology remain unknown (Choudhury, 2016).

This study presents an analysis of Drosophila σ₂-adaptin in the context of regulating NMJ morphological plasticity and physiology. A mutation was identified that dramatically altered NMJ morphology. Next, this mutation was mapped to σ₂-adaptin by deficiency mapping. This study shows that AP2-dependent vesicle endocytosis regulates both synaptic growth and transmitter release. The AP2 complex is a heterotetramer, and these studies in Drosophila show that the four subunits are obligate partners of each other and are required for a functional AP2 complex. This finding is in contrast to the hemicomplex model in C. elegans, in which α/σ₂ and β₂/μ₂ can sustain the function, if any one of the subunits is mutated. Loss of AP2 disrupts stable microtubule loops of the presynaptic cytoskeleton and exacerbates growth signaling through the phosphorylated Mothers Against Decapentaplegic (pMAD) pathway, suggesting that normal AP2 constrains the TGFβ signaling module. Reducing σ₂-adaptin level results in synaptic fatigue at the larval NMJ synapses during high-frequency stimulation and causes temperature-sensitive paralysis in adults. Based on these results, it is suggestd that AP2 is essential for attenuating synaptic growth signaling mediated by the TGFβ pathway in addition to its requirement in regenerating SVs under high-frequency nerve firing (Choudhury, 2016).

AP2/CME has been proposed to play an essential role in SV endocytosis. Moreover, mutation in the proteins affecting CME also results in altered NMJ development in Drosophila, suggesting regulation of synaptic signaling by CME. This study analyzed the role of AP2, one of the major adapters for clathrin at synapses, in the context of its physiological relevance and synaptic signaling. The results demonstrate that AP2 facilitates basal synaptic transmission and SV endocytosis but also is essential during high-frequency nerve firing to regenerate SVs. Moreover, evidence is provided that AP2 regulates morphological plasticity at the Drosophila NMJ by stabilizing the microtubule loops and attenuating the BMP signaling pathway (Choudhury, 2016).

The biogenesis of rhabdomeres is regulated by endocytosis and intracellular trafficking. Components of the endocytic machinery, when perturbed, lead to defects in rhabdomere formation. One such example is the disruption of the rhabdomere base when shi^ts flies are grown at restrictive temperature for 4 hr. Rh1-Gal4-driven shi^ts1 flies raised at 19° led to an enlargement of photoreceptor cell bodies with the inter-rhabdomeric space being reduced. Similarly, when α-adaptin was knocked down using GMR-Gal4, rhabdomere biogenesis was disrupted, with smaller rhabdomeres. In accordance with these observations, the TEM analysis of angur mutant eye clones showed no detectable rhabdomeres. Consistent with the defect in rhabdomere formation, no membrane depolarization was observed in angur mutant eye clones. The visual cascade in Drosophila begins with rhodopsin being converted to meta-rhodopsin by light, followed by a subsequent phosphorylation by GPRK1. The phosphorylated meta-rhodopsin binds to arrestins, where its activity is quenched. The rhodopsin-arrestin complex is endocytosed, and rhodopsin is degraded in the lysosomes. Thus, endocytosis regulates rhodopsin turnover in the photoreceptor cells, and blocking of the endocytic pathway components by mutations in Arr1, Arr2, or AP2 leads to retinal degeneration and photoreceptor cell death. This further establishes a strong link between endocytosis and retinal degeneration. Thus, it is likely that defective endocytosis in angur mutant eye clones affects meta-rhodopsin turnover, resulting into defective rhabdomeres and disorganized ommatidia. Moreover, endocytosis of several signaling receptors also has been shown to regulate morphogenesis during Drosophila eye development. The Notch signaling pathway has been studied extensively, and it has been shown that the Notch extracellular domain is transendocytosed during this process into the Delta-expressing cells. The localization of Notch and Delta is disrupted when endocytosis is acutely blocked, and this prevents internalization of Notch in the Delta-expressing cells. It is thus speculated that the endocytic defect in angur mutant clones affects turnover and internalization of signaling molecules that may lead to defective rhabdomere development (Choudhury, 2016).

The functional analysis of the σ₂-adaptin mutants as well as of other subunits of AP2 reveals its requirement in maintaining basal synaptic transmission and regeneration of synaptic vesicles under high-frequency nerve firing at Drosophila NMJ synapses. The data are consistent with previous reports showing the requirement for Drosophila α-adaptin in SV recycling at NMJ synapses. However, in contrast to its role at mammalian central synapses, this study found that loss of AP2 at Drosophila NMJ synapses affects basal synaptic transmission and SV endocytosis, albeit not very strongly. Moreover, the kinetics of SV re-formation in σ₂-adaptin mutants was only mildly affected. Consistent with the recent observation that loss of the AP2 complex at mammalian central synapses or its subunits in C. elegans affects synaptic vesicle biogenesis, compromised synaptic transmission and SV re-formation was observed after high-frequency stimulation, suggesting that at Drosophila NMJ synapses, AP2 function is required not only for SV trafficking but also for SV regeneration under high-frequency nerve stimulation. A direct estimation of vesicle pool size in σ₂-adaptin mutants further supports its requirement in the regulation of SV pool size and is consistent with a recent report. Because loss of AP2 in Drosophila does not completely abrogate SV re-formation, it remains possible that other known synaptic clathrin adapters such as AP180, Eps15, and Epsin might partially compensate for loss of the AP2 complex. This is corroborated by the observation that even a 96% reduction in synaptic α-adaptin level caused only an ~50% reduction in clathrin, suggesting that other clathrin adapters might contribute to retention and/or stability of synaptic clathrin (Choudhury, 2016).

The striking physiological defect in σ₂-adaptin mutants or neuronally reduced α-adaptin was their inability to recover from synaptic depression even after 4 min of cessation of high-frequency stimulation. Consistent with these observations, the endo-exo cycling pool did not recover to its initial value even after 4 min of rest following high-frequency nerve stimulation. This suggests that in addition to its requirement in synaptic vesicle endocytosis, AP2 complex functions at a relatively slower step downstream of membrane retrieval, possibly during membrane sorting from endosomes to regenerate fusion-competent synaptic vesicles (Gu, 2013; Kononenko, 2014; Choudhury, 2016 and references therein).

Several studies in cell culture support a model that indicates that the AP2 complex is an obligate heterotetramer for subunit stability and function. However, studies of AP2 function in C. elegans suggest that α/σ₂ subunits and β₂/μ₂ subunits may constitute two hemicomplexes that can carry out the minimal function of the AP2 complex independent of one another. Does AP2 in Drosophila form hemicomplexes and contribute to vesicle trafficking? The results do not support this model for Drosophila. First, unlike C. elegans, in which individual mutants for subunits of the AP2 complex are viable, the Drosophila mutant for α-adaptin is early larval lethal. Consistent with these findings, neuronal knockdown of α-adaptin or β₂-adaptin resulted in third instar lethality. Second, if the hemicomplexes were functional in Drosophila, one would observe significant levels of synaptic β₂-adaptin in α-adaptin knockdown or σ₂-adaptin mutants. However, it was observed that removing any of the subunits of the Drosophila AP2 complex in neurons resulted into an unstable AP2 complex and degradation of α- and β₂-adaptins. Third, inhibiting AP2 complex assembly by reducing synaptic PI(4,5)P2 levels also resulted into an unstable AP2 complex. These observations strongly suggest that in contrast to C. elegans, all the subunits of AP2 in Drosophila are obligate partners for a stable AP2 complex for clathrin-dependent SV trafficking (Choudhury, 2016).

Mutations in genes implicated in regulating CME, BMP signaling, or actin cytoskeleton dynamics all show abnormal NMJ development in Drosophila, characterized by either supernumerary boutons or an increased number but smaller boutons. The NMJ phenotype due to loss of σ₂-adaptin is consistent with that of other known endocytic mutants implicated in CME. The contrasting synaptic overgrowth observed in σ₂-adaptin mutants suggests a role for AP2 in mediating presynaptic growth signaling. Such synaptic overgrowth also was observed when any of the subunits of AP2 were downregulated or its assembly was interfered with by downregulating neuronal PI(4,5)P2. This suggests a pathway in which PI(4,5)P2 and the AP2 complex interact obligatorily to regulate synapse growth. The NMJ phenotype in angur mutants is strikingly more severe from that of the other synaptic mutants and points toward multiple growth signaling pathways that are possibly affected by an AP2-dependent endocytic deficit (Choudhury, 2016).

The morphology of the synapse is a consequence of the neuronal cytoskeleton network that shapes the growing synapse. Futsch, a protein with MAP1B homology, regulates the synaptic microtubule cytoskeleton, thereby controlling synaptic growth at the Drosophila NMJ. The hypothesis that the microtubule organization could be altered was strengthened by the dramatic decrease in the number of Futsch-positive loops in the σ₂-adaptin mutants. Futsch-positive loops have long been known to be associated with stable synaptic boutons, while the absence or disruption of these loops is indicative of boutons undergoing division or sprouting. The dramatic increase in the number of boutons in these mutants with a corresponding decrease in the number of loops correlates well with the fact that these boutons might be undergoing division. The phenotype associated with futsch mutants is fewer and larger boutons with impaired microtubule organization, which is an expected phenotype when bouton division is impaired. The σ₂-adaptin mutants, however, show a larger number but smaller-sized boutons, indicating that bouton division is enhanced. One of the signaling events that has been shown to dictate this process is BMP signaling. Further, BMP signaling is also required during developmental synaptic growth. Consistent with this, elevated pMAD levels were found in σ₂-adaptin mutants, indicating that BMP signaling is upregulated in these mutants. It has also been demonstrated that BMP signaling plays a role in maintenance of the presynaptic microtubule network. Drosophila spichthyin and spartin mutants have upregulated BMP signaling with significantly increased microtubule loops. It has been proposed that Futsch acts downstream of BMP signaling to regulate synaptic growth. The σ₂-adaptin mutants also show upregulated BMP signaling, but in contrast to spichthyin and spartin mutants, σ₂-adaptin mutants have fewer Futsch loops, which also appeared fragmented. This suggests that deregulated BMP signaling, whether upregulation or downregulation, impairs microtubule stability. dap160 mutants also show supernumerary boutons, with fragmented Futsch staining suggesting that the microtubule dynamics are misregulated in these mutants. Interestingly, Nwk levels are drastically altered in dap160 mutants, suggesting that Nwk levels are important for maintaining microtubule stability. Because σ₂-adaptin mutants have normal Nwk levels, the observed synaptic overgrowth is independent of Nwk and suggests regulation of Futsch through Nwk-independent pathways (Choudhury, 2016).

Tomosyn-dependent regulation of synaptic transmission is required for a late phase of associative odor memory

Synaptic vesicle secretion requires the assembly of fusogenic SNARE complexes. Consequently proteins that regulate SNARE complex formation can significantly impact synaptic strength. The SNARE binding protein tomosyn has been shown to potently inhibit exocytosis by sequestering SNARE proteins in nonfusogenic complexes. The tomosyn-SNARE interaction is regulated by protein kinase A (PKA), an enzyme implicated in learning and memory, suggesting tomosyn could be an important effector in PKA-dependent synaptic plasticity. This hypothesis was tested in Drosophila, in which the role of the PKA pathway in associative learning has been well established. It was first determined that panneuronal tomosyn knockdown by RNAi enhanced synaptic strength at the Drosophila larval neuromuscular junction, by increasing the evoked response duration. Next memory performance was assayed 3 min (early memory) and 3 h (late memory) after aversive olfactory learning. Whereas early memory was unaffected by tomosyn knockdown, late memory was reduced by 50%. Late memory is a composite of stable and labile components. Further analysis determined that tomosyn was specifically required for the anesthesia-sensitive, labile component, previously shown to require cAMP signaling via PKA in mushroom bodies. Together these data indicate that Tomosyn has a conserved role in the regulation of synaptic transmission and provide behavioral evidence that Tomosyn is involved in a specific component of late associative memory (Chen, 2011).

Synaptic transmission is dependent on the formation of SNARE complexes between the vesicle SNARE synaptobrevin and the plasma membrane SNAREs syntaxin and SNAP-25. SNARE complex assembly produces fusion-competent (primed) vesicles, docked at the plasma membrane. Originally identified as a syntaxin-binding protein, tomosyn has emerged as a negative regulator of secretion by directly competing with synaptobrevin to form nonfusogenic tomosyn SNARE complexes (Fujita, 1998; Hatsuzawa, 2003; Pobbati, 2004). The N terminus of tomosyn also promotes SNARE complex oligomerization, sequestering SNARE monomers required for priming, and impedes the function of the calcium-sensor synaptotagmin. By these means tomosyn is involved in regulating SNARE complex assembly and controlling the size of the readily releasable pool of synaptic vesicles. Evidence suggests that the interaction between tomosyn and the SNARE machinery can be modulated by cAMP-dependent protein kinase A (PKA) phosphorylation of tomosyn, a second messenger cascade previously implicated in several forms of behavioral and synaptic plasticity. This study characterized the synaptic function of Drosophila tomosyn and probed the functional relevance of tomosyn in the regulation of behavioral plasticity (Chen, 2011).

The results demonstrate that tomosyn suppresses synaptic function and is necessary in mushroom body intrinsic neurons specifically for a long-term cAMP-dependent component of associative olfactory learning in Drosophila. The prominent biophysical change observed at the fly NMJ after tomosyn RNAi is a prolonged EJC resulting in an increased total charge transfer, similar to that observed at C. elegans tomosyn mutant synapses. Although the underlying cause of the altered EJC duration in either C. elegans or Drosophila has yet to be determined, one potential explanation is the occurrence of late fusion events possibly resulting from ectopically primed vesicles distal to release sites. This hypothesis is supported by ultrastructural data from C. elegans tomosyn mutants, in which a twofold increase in the number of morphologically docked vesicles was observed, the additional vesicles positioned further from the presynaptic density (Gracheva, 2006). Analyses of priming defective unc-13 and syntaxin C. elegans mutants, in which docked vesicles were found to be greatly reduced, suggest that vesicle docking is a morphological correlate of priming. On the basis of these data, it has been proposed that distally primed vesicles in C. elegans tomosyn mutants exhibit delayed release relative to those close to the presynaptic density, the presumed site of calcium entry, leading to a broadened evoked response (Chen, 2011).

The observed increase in synaptic release at the fly NMJ after tomosyn RNAi adds to a growing body of evidence that tomosyn suppresses synaptic strength. For example, the twofold increase in docked vesicles observed at C. elegans tomosyn mutant synapses correlates with a doubling of the readily releasable pool assessed by applying hyperosmotic saline and corresponds to the enhanced evoked EJC charge integral. The increase in quantal content and reduction in PPD at the fly NMJ after tomosyn RNAi is also consistent with an enhanced primed vesicle pool. Similar conclusions were reached for changes in paired-pulse facilitation at central synapses of mouse tomosyn mutants (Chen, 2011).

The observation that tomosyn knockdown at the fly NMJ results in the addition of mEJCs with slower decay kinetics could also be a manifestation of ectopic vesicle priming. Alternatively, this change in fly mEJCs could reflect a change in fusion pore dynamics in the absence of tomosyn, a possibility given previously observed genetic and/or physical interactions between tomosyn and several key components of the exocytic machinery, including the SNARE proteins synaptotagmin, Munc-13, and Munc-18. Because the presynaptic density is responsible for localizing elements of the vesicle fusion machinery such as UNC-13 and calcium channels, spatial misregulation of vesicle docking in the absence of tomosyn may be related to these changes in fusion properties. However, definitive evidence for ectopically primed vesicles at the fly NMJ after tomosyn RNAi is not yet available, and therefore the cause of the altered evoked response kinetics remains speculative (Chen, 2011).

Evidence indicates that vertebrate tomosyn is a PKA target. Although it cannot yet be definitively establish that tomosyn function is down-regulated by PKA phosphorylation at fly synapses pending the availability of a tomosyn null mutant, the fact that tomosyn RNAi phenocopies the NMJ response to cAMP activation and that these two treatments show nonadditivity supports this notion (Chen, 2011).

On the basis of this analysis of NMJ function, it is predicted that, as in mouse tomosyn mutants, synapses within the fly CNS will experience similar increases in synaptic strength after tomosyn RNAi or cAMP activation. This prediction is supported by the specific ASM aversive odor learning deficit observed in the fly olfactory CNS after tomosyn knockdown in mushroom body Kenyon cells, the site of cAMP-mediated associative memory formation (Chen, 2011).

Drosophila aversive odor learning has long been used to investigate the molecular and cellular mechanisms underlying associative memory formation. In the Drosophila mushroom bodies, neuronal signals representing odor cues and electric shock converge onto type I adenylyl cyclase encoded by rutabaga (rut-AC I) to initiate cAMP signals necessary and sufficient to form engrams underlying associative odor memory. These instructive cAMP signals localize to the Kenyon cells in which presynaptic changes are thought to represent a particular odor memory. Stabilization of aversive odor memory over time requires signals from dorsal-paired medial (DPM) neurons, the putative release sites of amnesiac peptide — the fly homolog to mammalian pituitary adenylate cyclase-activating polypeptide (PACAP) — onto mushroom body Kenyon cells. Amnesiac neuropeptides are known to stimulate cAMP production, whereas the major fly PKA is required from 30 min to 3 h after acquisition to sustain late odor memory. The two distinct components of late odor memory, ASM and ARM, differ in both temporal dynamics and molecular mechanisms. Whereas ARM requires the active zone protein Bruchpilot, ASM is not only tomosyn-dependent, according to the current observations, but also requires synapsin, a conserved PKA phosphorylation target associated with synaptic vesicles. Adult synapsin mutant flies exhibit impaired ASM in aversive odor learning. Furthermore, PKA-dependent phosphorylation of synapsin within Kenyon cells is necessary to support larval appetitive odor learning. Thus, both tomosyn and synapsin are required for the cAMP-dependent ASM phase of associate learning (Chen, 2011).

How might synapsin and tomosyn function together in ASM? Mechanistically, PKA phosphorylation of synapsin is implicated in the mobilization and supply of synaptic vesicles from the reserve pool to the active zone, whereas PKA phosphorylation of tomosyn promotes SNARE complex assembly (Baba, 2005). This suggests that enhanced vesicle delivery and increased priming capacity through PKA regulation of synapsin and tomosyn, respectively, may act in concert to maintain synaptic strength in support of ASM. Because knockdown of either protein disrupts ASM, it seems that both vesicle mobilization and enhanced priming capacity are required for this phase of synaptic plasticity (Chen, 2011).

Several lines of evidence suggest that PKA-dependent modulation of tomosyn function provides a possible molecular mechanism for the transduction of cAMP signaling into synaptic plasticity within the olfactory system underlying ASM. First, at the level of the NMJ, it was shown that tomosyn regulates synaptic strength and that this regulation occludes cAMP-dependent synaptic enhancement. Second, within the olfactory neuronal network, it was demonstrated that tomosyn function is likely required within Kenyon cells to support ASM: the cells that receive instructive signals to initiate cAMP-dependent synaptic changes underlying appropriate behavioral plasticity. Third, synaptic output from Kenyon cells is necessary to support both early and late odor memories. Fourth, DPM signaling onto Kenyon cells is required for late aversive odor memory, and DPM neurons stain positive for the amnesiac peptide, which is known to stimulate cAMP production. Fifth, enhanced synaptic transmission in cultured neurons induced by the vertebrate amnesiac homolog PACAP requires PKA-dependent tomosyn phosphorylation (Baba, 2005). Sixth, postacquisitional PKA activity is necessary for late aversive odor memory and is likely mediated by an A kinase-anchoring protein (AKAP)-bound pool of PKA holoenzymes within Kenyon cells. Finally, biochemical evidence demonstrating that PKA-dependent phosphorylation of tomosyn reduces its affinity for the SNARE machinery suggests a potential mechanistic link between cAMP signaling within Kenyon cells and the up-regulation of synaptic strength (Chen, 2011).

On the basis of this evidence, it is postulated that loss of tomosyn inhibitory function leads to a generalized up-regulation of synaptic strength at Drosophila synapses. In Kenyon cells enhanced synaptic transmission resulting from loss of tomosyn likely occludes cAMP-dependent plasticity. This speculation is supported by the observed occlusion of forskolin-dependent synaptic enhancement at the NMJ after tomosyn RNAi. Similarly, PACAP-induced synaptic plasticity in cultured neurons is occluded when tomosyn is no longer phosphorylatable by PKA (Baba, 2005). It is further speculated that within Kenyon cells the phosphorylation of Drosophila tomosyn is due to postacquisitional, Amnesiac-induced PKA activity. This hypothesis would fit with the observed requirement of AKAP-bound PKA for late but not early ASM. It is thus tempting to speculate that tomosyn phosphorylation is dependent on localized signaling via AKAPs at specific subdomains of Kenyon cells that occur after early ASM is established (Chen, 2011).

Hsc70-4 deforms membranes to promote synaptic protein turnover by endosomal microautophagy

Synapses are often far from their cell bodies and must largely independently cope with dysfunctional proteins resulting from synaptic activity and stress. To identify membrane-associated machines that can engulf synaptic targets destined for degradation, a large-scale in vitro liposome-based screen was performed followed by functional studies. A presynaptically enriched chaperone Hsc70-4 was identified that bends membranes based on its ability to oligomerize. This activity promotes endosomal microautophagy and the turnover of specific synaptic proteins. Loss of microautophagy slows down neurotransmission while gain of microautophagy increases neurotransmission. Interestingly, Sgt, a cochaperone of Hsc70-4, is able to switch the activity of Hsc70-4 from synaptic endosomal microautophagy toward chaperone activity. Hence, Hsc70-4 controls rejuvenation of the synaptic protein pool in a dual way: either by refolding proteins together with Sgt, or by targeting them for degradation by facilitating endosomal microautophagy based on its membrane deforming activity (Uytterhoeven, 2016).

Rab3-GEF controls active zone development at the Drosophila neuromuscular junction

Synaptic signaling involves the release of neurotransmitter from presynaptic active zones (AZs). Proteins that regulate vesicle exocytosis cluster at AZs, composing the cytomatrix at the active zone (CAZ). At the Drosophila neuromuscular junction (NMJ), the small GTPase Rab3 controls the distribution of CAZ proteins across release sites, thereby regulating the efficacy of individual AZs. This study identifies Rab3-GEF as a second protein that acts in conjunction with Rab3 to control AZ protein composition. At rab3-GEF mutant NMJs, Bruchpilot (Brp) and Ca(2+) channels are enriched at a subset of AZs, leaving the remaining sites devoid of key CAZ components in a manner that is indistinguishable from rab3 mutant NMJs. As the Drosophila homologue of mammalian DENN/MADD and Caenorhabditis elegans AEX-3, Rab3-GEF is a guanine nucleotide exchange factor (GEF) for Rab3 that stimulates GDP to GTP exchange. Mechanistic studies reveal that although Rab3 and Rab3-GEF act within the same mechanism to control AZ development, Rab3-GEF is involved in multiple roles. It was shown that Rab3-GEF is required for transport of Rab3. However, the synaptic phenotype in the rab3-GEF mutant cannot be fully explained by defective transport and loss of GEF activity. A transgenically expressed GTP-locked variant of Rab3 accumulates at the NMJ at wild-type levels and fully rescues the rab3 mutant but is unable to rescue the rab3-GEF mutant. These results suggest that although Rab3-GEF acts upstream of Rab3 to control Rab3 localization and likely GTP-binding, it also acts downstream to regulate CAZ development, potentially as a Rab3 effector at the synapse (Bae, 2016).

A pre-synaptic regulatory system acts trans-synaptically via Mon1 to regulate Glutamate receptor levels in Drosophila

Mon1 is an evolutionarily conserved protein involved in the conversion of Rab5 positive early endosomes to late endosomes through the recruitment of Rab7. This study has identified a role for Drosophila Mon1 in regulating glutamate receptor levels at the larval neuromuscular junction. Mutants were generated in Dmon1 through P-element excision. These mutants are short-lived with strong motor defects. At the synapse, the mutants show altered bouton morphology with several small supernumerary or satellite boutons surrounding a mature bouton; a significant increase in expression of GluRIIA and reduced expression of Bruchpilot. Neuronal knockdown of Dmon1 is sufficient to increase GluRIIA levels suggesting its involvement in a pre-synaptic mechanism that regulates post-synaptic receptor levels. Ultrastructural analysis of mutant synapses reveals significantly smaller synaptic vesicles. Overexpression of vglut suppresses the defects in synaptic morphology and also downregulates GluRIIA levels in Dmon1 mutants suggesting that homeostatic mechanisms are not affected in these mutants. It is proposed that DMon1 is part of a pre-synaptically regulated trans-synaptic mechanism that regulates GluRIIA levels at the larval neuromuscular junction (Deivasigamani, 2015).

Neurotransmitter release at the synapse is modulated by factors that control synaptic growth, synaptic vesicle recycling, and receptor turnover at postsynaptic sites. Endolysosomal trafficking modulates the function of these factors and therefore plays an important role in regulating synaptic development and function. Intracellular trafficking is regulated by Rabs, which are small GTPases. These proteins control specific steps in the trafficking process. A clear understanding of the role of Rabs at the synapse is still nascent. Drosophila has 31 Rabs, and most of these are expressed in the nervous system. Rab5 and Rab7, present on early and late endosomes, respectively, are critical regulators of endolysosomal trafficking and loss of this regulation affects neuronal viability underscored by the fact that mutations in Rab7 are associated with neurodegeneration. Rab5 along with Rab3 is present on synaptic vesicles, and both play a role in regulating neurotransmitter release. In Drosophila, Rab3 is involved in the assembly of active zones by controlling the level of both Bruchpilot-a core active zone protein-and the calcium channels surrounding the active zone. In hippocampal and cortex neurons, Rab5 facilitates LTD through removal of AMPA receptors from the synapse. In Drosophila, Rab5 regulates neurotransmission; it also functions to maintain synaptic vesicle size by preventing homotypic fusion. Compared to Rab5 or Rab3, less is known about the roles of Rab7 at the synapse. In spinal cord motor neurons, Rab7 mediates sorting and retrograde transport of neurotrophin-carrying vesicles. In Drosophila, tbc1D17-a known GAP for Rab7-affects GluRIIA levels; the effect of this on neurotransmission has not been evaluated. Excessive trafficking via the endolysosomal pathway also affects neurotransmission. This has been observed in mutants for tbc1D24-a GAP for Rab35. A high rate of turnover of synaptic vesicle proteins in these mutants is seen to increase neurotransmitter release (Deivasigamani, 2015 and references therein).

This study has examined the synaptic role of DMon1-a key regulator of endosomal maturation. Multiple synaptic phenotypes are found associated with Dmon1 loss of function, and one of these is altered synaptic morphology. Boutons in Dmon1 mutants are larger with more satellite or supernumerary boutons-a phenotype strongly associated with endocytic mutants. Formation of satellite boutons is thought to occur due to loss of bouton maturation, with the initial step of bouton budding being controlled postsynaptically and the maturation step being regulated presynaptically. Supporting this, a recent study shows that miniature neurotransmission is required for bouton maturation. The presence of excess satellite boutons in Dmon1 mutants suggests that the number of 'miniature' events is likely to be affected in these mutants. The fact that this phenotype can be rescued upon expression of vGlut supports this possibility. However, this does not fit with the observed decrease in size and intensity of Brp positive puncta in these mutants. Active zones with low or nonfunctional Brp are known to be more strongly associated with increased spontaneous neurotransmission. Considering the involvement of postsynaptic signaling in initiating satellite bouton formation, it is thought that altered neurotransmission possibly together with impaired postsynaptic or retrograde signaling, contributes to the altered synaptic morphology in Dmon1 mutants. This may also explain why no satellite boutons are observed in neuronal RNAi animals (Deivasigamani, 2015).

A striking phenotype associated with loss of Dmon1 is the increase in GluRIIA levels. This phenotype seems presynaptic in origin since neuronal loss of Dmon1 is sufficient to increase GluRIIA levels. Is the increase in GluRIIA due to trafficking defects in the neuron? This seems unlikely for the following reasons: First, it has been shown that although neuronal overexpression of wild-type and dominant negative Rab5 alters evoked response in a reciprocal manner, there is no change in synaptic morphology, glutamate receptor localization and density, or change in synaptic vesicle size. The role of Rab7 at the synapse is less clear. In a recent study, loss of tbc1D15-17, which functions as a GAP for Rab7, was shown to increase GluRIIA levels at the synapse. Selective knockdown of the gene in muscles, and not neurons, was seen to increase GluRIIA levels, indicating that the function of the gene is primarily postsynaptic. These data are not consistent with the current results from neuronal knockdown of Dmon1, suggesting that the presynaptic role of Dmon1 in regulating GluRIIA levels is likely to be independent of Rab5 and Rab7 and therefore novel (Deivasigamani, 2015).

The current experiments to evaluate the postsynaptic role of Dmon1 have been less clear. Although a modest increase in GluRIIA levels are seen upon knockdown in muscles, the increase is not always significant when compared to controls. However, the fact that muscle expression of Dmon1 can rescue the GluRIIA phenotype in the mutant suggests that it is likely to be one of the players in regulating GluRIIA postsynaptically. Further, it is to be noted, that while overexpression of vGlut leads to down-regulation of the receptor at the synapse, the receptors do not seem to get trapped in the muscle, suggesting that multiple pathways are likely to be involved in regulating receptor turnover in the muscle, and the DMon1-Rab7-mediated pathway may be just one of them (Deivasigamani, 2015).

How might neuronal Dmon1 regulate receptor expression? One possibility is that the increase in receptor levels is a postsynaptic homeostatic response to defects in neurotransmission, given that Dmon1^Δ181 mutants have smaller synaptic vesicles. However, in dvglut mutants, presence of smaller synaptic vesicles does not lead to any change in GluRIIA levels, given that receptors at the synapse are generally expressed at saturating levels. Therefore, it seems unlikely that the increase in GluRIIA is part of a homeostatic response, although one cannot rule this out completely. The other possibility is that DMon1 is part of a transsynaptic signaling mechanism that regulates GluRIIA levels in a post-transcriptional manner. The observation that presynaptically expressed DMon1 localizes to postsynaptic regions and the results from neuronal RNAi and rescue experiments support this possibility. The involvement of transsynaptic signaling in regulating synaptic growth and function has been demonstrated in the case of signaling molecules such as Ephrins, Wingless, and Syt4. In Drosophila, both Wingless and Syt4 are released by the presynaptic terminal via exosomes to mediate their effects in the postsynaptic compartment. It was hypothesized that DMon1 released from the boutons either directly regulates GluRIIA levels or facilitates the release of an unknown factor required to maintain receptor levels. The function of DMon1 in the muscle is likely to be more consistent with its role in cellular trafficking and may mediate one of the pathways regulating GluRIIA turnover. These possibilities will need to be tested to gain a mechanistic understanding of receptor regulation by Dmon1 (Deivasigamani, 2015).

Drosophila homolog of human KIF22 at the autism-linked 16p11.2 loci influences synaptic connectivity at larval neuromuscular junctions

Copy number variations at multiple chromosomal loci, including 16p11.2, have recently been implicated in the pathogenesis of autism spectrum disorder (ASD), a neurodevelopmental disease that affects ~1%-3% of children worldwide. The aim of this study was to investigate the roles of human genes at the 16p11.2 loci in synaptic development using Drosophila larval neuromuscular junctions (NMJ), a well-established model synapse with stereotypic innervation patterns. A preliminary genetic screen based on RNA interference was conducted in combination with the GAL4-UAS system, followed by mutational analyses. The result indicated that disruption of klp68D, a gene closely related to human KIF22, caused ectopic innervations of axon branches forming type III boutons in muscle 13, along with less frequent re-routing of other axon branches. In addition, mutations in klp68D, of which gene product forms Kinesin-2 complex with KLP68D, led to similar targeting errors of type III axons. Mutant phenotypes were at least partially reproduced by knockdown of each gene via RNA interference. Taken together, these data suggest the roles of Kinesin-2 proteins, including KLP68D and KLP64D, in ensuring proper synaptic wiring (Park, 2016).

Recent clinical studies on ASD have revealed gross alterations in the structure of nervous system. For instance, total brain size and the rate of neuronal proliferation in the prefrontal cortex are significantly increased in ASD patients. Such structural changes may reflect altered neuronal connectivity between specific brain regions. Moreover, the proposed candidate genes responsible for ASD include various synaptic proteins that play important roles in neurite outgrowth, axonal guidance, axonal targeting and synaptogenesis, suggesting structural abnormalities at a synaptic level responsible for expression of ASD phenotypes. Based on these findings, it was hypothesized that genetic perturbation at the 16p11.2 loci would lead to aberrant synaptic connectivity, thus underlying functional disturbances that lead to ASD. Results demonstrate significant axon targeting errors caused by defects in KLP68D, a Drosophila Kinesin-2 protein closely related to human KIF22 at the autism-linked 16p11.2 loci (Park, 2016).

Hetero-trimeric Kinesin-2 complex in Drosophila, consisting of KLP68D, KLP64D and DmKAP, has been implicated in microtubule organization and axonal transport of synaptic proteins such as choline acetyltransferase. However, experimental evidence is missing to support the idea that Kinesin-2 complex may participate in delivering molecules important for axon targeting. Potential cargos of KLP68D and KLP64D motors have been estimated to include Unc-51/ATG1, Fasciclin II, EB1, Armadillo, Bazooka, and DE-cadherin, most of whom have been well characterized for their roles in synaptogenesis. It will be important to investigate whether disruptions of any of these potential cargos lead to aberrant axon targeting phenotypes observed in Klp68D and Klp64D mutants (Park, 2016).

It should be noted that Drosophila Nod ("no distributive disjunction"), mostly involved in chromosomal segregation has been recognized as a homolog for human KIF22. However, similar levels of sequence homology to KIF22 were found in both Nod and KLP68D. In fact, a blast analysis results in higher sequence identity between KIF22 and KLP68D than Nod (41% vs. 33%). The specificity of motor protein cargos is often predicted to depend on the amino acid composition of motor proteins outside their core motor domain. Therefore, relatively lower level of homology between human KIF22 and Drosophila KLP68D may correspond to their distinct molecular functions. In contrast to KLP68D, the role of KIF22 in the mammalian nervous system has not been extensively investigated, but only limited to chromosomal segregation and genomic stability. Whether Drosophila KLP68D can be functionally replaced by human KIF22 in transgenic animals awaits further investigations (Park, 2016).

Na+ /H+ -exchange via the Drosophila vesicular glutamate transporter (DVGLUT) mediates activity-induced acid efflux from presynaptic terminals

Neuronal activity can result in transient acidification of presynaptic terminals and such shifts in cytosolic pH (pHcyto) likely influence mechanisms underlying forms of synaptic plasticity with a presynaptic locus. As neuronal activity drives acid loading in presynaptic terminals it was hypothesized that the same activity might drive acid efflux mechanisms to maintain pHcyto homeostasis. To better understand the integration of neuronal activity and pHcyto regulation this study investigated the acid extrusion mechanisms at Drosophila glutamatergic motorneuron terminals. Expression of a fluorescent genetically-encoded pH-indicator (GEpHI), named 'pHerry', in the presynaptic cytosol revealed acid efflux following nerve activity to be greater than that predicted from measurements of the intrinsic rate of acid efflux. Analysis of activity-induced acid transients in terminals deficient in either endocytosis or exocytosis revealed an acid efflux mechanism reliant upon synaptic vesicle exocytosis. Pharmacological and genetic dissection in situ and in a heterologous expression system indicate that this acid efflux is mediated by conventional plasmamembrane acid transporters, and also by previously unrecognized intrinsic H+ /Na+ exchange via the Drosophila vesicular glutamate transporter (DVGLUT). DVGLUT functions not only as a vesicular glutamate transporter but also serves as an acid extruding protein when deposited on the plasma membrane (Rossano, 2016).

Neuronal activity generates large cytosolic pH (pH_cyto) transients in presynaptic terminals and such transients are likely to influence mechanisms underlying neurotransmitter release. Minute changes in pH_cyto can influence presynaptic processes such as voltage-gated Ca²⁺ channel gating, endocytosis, synaptic vesicle (SV) filling, cytosolic Ca²⁺ buffering, and both Ca²⁺/calmodulin-dependent kinase II and cyclic-AMP-based forms of synaptic plasticity. pH_cyto in quiescent neurons is set by the equilibrium between standing acid influx and efflux. Neuronal activity, however, results in enhanced acid influx, primarily due to electroneutral H⁺/Ca²⁺ exchange across the plasma membrane Ca²⁺-ATPase (PMCA) as it clears cytosolic Ca²⁺. pH homeostasis would be well served if nerve activity triggered acid efflux to offset acid influx (Rossano, 2016).

Nerve activity may enhance acid efflux in many ways. Standing efflux mechanisms might be directly enhanced by decreasing pH_cyto or through intracellular signalling linked to Ca²⁺ entry. For example, anion exchangers (AEs) are known to be modulated by Ca²⁺/calmodulin. Also, various mammalian Na⁺/H⁺ exchangers (NHEs) are modified by Ca²⁺-dependent kinases and protein kinase A and C pathways. Finally, but perhaps most significantly, activity-induced acid efflux could be mediated by translocation of acid extruders, such as the vesicular H⁺ ATPase (vATPase), from intracellular compartments to the plasmamembrane (PM), as described for cholinergic mouse motorneuron (MN) terminals. While mammalian NHE5, NHE6 and NHE9 are present on intracellular membranes in neurons, there are no reports of NHE translocation to the PM during nerve activity (Rossano, 2016).

Whether activity-induced acid efflux occurs at glutamatergic nerve terminals is unknown, but Drosophila glutamatergic MN terminals represent a tractable system for investigation. vATPases are present in these terminals and may extrude acid when deposited on the PM. Drosophila NHE and AE expression patterns have only been grossly characterized. Vesicular glutamate transporters (VGLUTs) have not been previously implicated in pH_cyto homeostasis but a recent report of VGLUT1 cation/H⁺ exchange activity in mammalian SVs (Preobraschenski, 2014) led to the speculation that VGLUTs contribute to acid efflux (Rossano, 2016).

VGLUTs transport glutamate into vesicles using the electrical portion (ΔΨ_H+) rather than the chemical portion (ΔpH) of the electrochemical proton gradient (Δμ_H+), and most models of VGLUT bioenergetics suggest VGLUTs are coupled to cation/H⁺ exchangers (Goh, 2011; Preobraschenski, 2014) or a Cl⁻ shunt. While these mechanisms have never been validated beyond in vitro preparations, H⁺/cation exchange by VGLUT may provide a novel acid efflux mechanism when VGLUTs are deposited on the PM during SV exocytosis and exposed to the strong PM electrochemical Na⁺ gradient (Δμ_Na+) (Rossano, 2016).

In this study, enhanced activity-induced acid transients under exocytotic blockade suggest that Drosophila MN terminals usually extrude acid through the translocation of vesicular acid extruders to the PM. While translocation of the vATPase accounts for some acid efflux, pharmacological and genetic tools revealed a portion of the efflux is mediated directly by the Drosophila vesicular glutamate transporter (DVGLUT). This is the first report of pH_cyto modulation in situ by a VGLUT in any cell. Expression of DVGLUT in Xenopus oocytes revealed intrinsic Na⁺/H⁺ exchange, which marks the first description of ion transport by DVGLUT. Modulation of activity-induced pH_cyto transients by DVGLUT though Na⁺/H⁺ exchange at the PM demonstrates novel integration of pH_cyto, vesicular recycling, glutamate loading and [Ca²⁺]_cyto in presynaptic terminals (Rossano, 2016).

This study examined the extent to which SV exocytosis shapes activity-induced pH_cyto transients in glutamatergic MN terminals through translocation of acid transporters to the PM. Cytosolic expression of the pseudo-ratiometric fluorescent GEpHI pHerry permitted measurement of acid dynamics within individual MN terminals. While intrinsic acid extrusion from MN terminals accelerates when pH_cyto falls, acid extrusion following action potentials trains is much faster than predicted by activation of acid extruders by low pH_cyto. Activity-induced acid extrusion partially offsets Ca²⁺-dependent acidification during nerve activity and continues for seconds after nerve activity. Complementary genetic and pharmacological approaches revealed this activity-induced acid efflux to be mediated by NHEs, the vATPase and Na⁺/H⁺ exchange through DVGLUT (Rossano, 2016).

Previous work has shown depolarization produces significant acid loading in soma, axons and presynaptic terminals of many neuronal preparations. While less common, there are several reports of depolarization-induced acid efflux from neurons. This report expands upon previous studies by characterizing mechanisms which shape rapid pH_cyto transients in the context of established models of vesicular trafficking and [Ca²⁺]_cyto dynamics. The latter is particularly important as [Ca²⁺]_cyto is tightly regulated in presynaptic terminals and [Ca²⁺]_cyto levels drive rapid acid influx and efflux mechanisms through direct Ca²⁺/H⁺ exchange via the PMCA and Ca²⁺-dependent trafficking of vesicular H⁺ transporters, respectively (Rossano, 2016).

Careful analysis of the relationship between pH_cyto and [Ca²⁺]_cyto was necessary to determine if alterations in activity-induced pH_cyto transients were directly due to changes in the location and activity of acid transporters or secondary to changes in [Ca²⁺]_cyto levels. To this end the relationships between [Ca²⁺]_cyto, J_H+ and acid extrusion (τ_rec) as well as resting pH_cyto and [Ca²⁺]_e were quantified. Many manipulations produced changes in resting pH_cyto which cannot be explained by alterations in Ca²⁺ handling as resting pH_cyto is independent of Ca²⁺ in Drosophila MN terminals. Similarly, manipulations which altered τ_rec probably altered acid efflux directly as τ_rec is independent of Ca²⁺ loading during stimulation. Interpreting manipulations which only altered J_H+ during stimulation proved the most challenging as J_H+ is proportional to Ca²⁺ loading during stimulation and thus changes in J_H+ may represent changes in acid influx due to changes in Ca²⁺ handling or direct changes in acid efflux. MN terminals with altered expression of DVGLUT only differed from their controls with respect to J_H+. As there were no differences in bulk [Ca²⁺]_cyto at rest or during stimulation between genotypes with different DVGLUT expression levels, differences in J_H+ between genotypes are probably not due to changes in Ca²⁺ handling, with the caveat that potential alterations in the Ca²⁺ and pH near-membrane micro-environments are not addressed in this analysis (Rossano, 2016).

The data in this study indicate that activity-induced acid efflux is mediated by multiple PM and vesicular acid extruders, namely NHEs, the vATPase and DVGLUT. The contribution of NHEs is unsurprising as NHE gene products are highly expressed in Drosophila larvae (Giannakou, 2001) and spontaneous vesicular fusion at the larval NMJ is sensitive to the NHE inhibitor amiloride (Caldwell, 2013). These results are corroborated in this study as application of the amiloride derivative EIPA decreased resting pH_cyto and delayed acid extrusion in MN terminals. The contributions of the vATPase and DVGLUT to pH_cyto transients are probably attributable to selective trafficking of these transporters to the PM during exocytosis as they are primarily vesicular proteins, although BafA1-sensitive vATPases are constitutively present on the PM of many cells as well. Furthermore, genetic manipulations to impair vesicular endocytosis and exocytosis revealed that stopping vesicular fusion impaired acid extrusion while locking vesicular membrane at the PM enhanced acid clearance following both NH₄⁺ withdrawal and activity-induced acid loading. The conclusion that both the vATPase and DVGLUT are functional components of the recycling pool of vesicular proteins which shape activity-induced pH_cyto transients is further supported by the observation that application of both the glutamate transporter inhibitor EB and BafA1, an established vATPase inhibitor, decrease acid clearance following activity-induced acid loading. These results agree with a previous report that trafficking of the vATPase to the PM can alkalinize the cytosol of mouse cholinergic MN terminals (Zhang, 2010; Rossano, 2016 and references therein).

The notion that the DVGLUT can function at the PM as an acid extruder requires careful consideration as exact transport mechanisms of VGLUTs are unclear and no previous studies have been conducted to elucidate the transport mechanisms of DVGLUT, although it is reasonable to assume its transport modalities are generally similar to those of mammalian VGLUT1 (Preobraschenski, 2014). Here, a combined pharmacological and genetic approach provided the most compelling evidence for acid efflux mediated by DVGLUT. This conclusion is further supported by the observation that EB can inhibit DVGLUT-mediated Na⁺/H⁺ exchange in oocytes. Experiments in which EB has been used to inhibit VGLUTs have been historically interpreted by assuming a primary effect on glutamate transport, not H⁺ dynamics. If intrinsic Na⁺/H⁺ exchange is a shared property of mammalian VGLUTs it is possible that prior work with EB has erroneously ascribed changes in neurotransmitter loading to direct inhibition of glutamate transport rather than secondary effects of inhibited cation/H⁺ exchange (Rossano, 2016).

The bioenergetics of vesicular glutamate transporters are undoubtedly vital to modulation of neurotransmitter release. Studies of mammalian VGLUTs in heterologous expression systems suggest that VGLUTs are primarily driven by ΔΨ_H+ of Δμ_H+ across the vesicular membrane, which is established by the vATPase. Maintenance of ΔΨ_H+ requires dissipation of ΔpH via H⁺ exchange with another cation to enable continuous proton pumping and glutamate transport (Goh, 2011). It is unclear if H⁺/cation exchange is due to co-transport with another ion transporter, as has been described in insect midgut where co-expression of vATPase and Na⁺-coupled nutrient amino acid transporters form a functional NHE (Harvey, 2009), or is an intrinsic property VGLUTs, as has been described in mammalian VGLUT1 (Preobraschenski, 2014). The data presented in this study provide the first direct evidence that intrinsic EB-sensitive Na⁺/H⁺ exchange is a property of DVGLUT. Taken with the observation that the in situ effects of EB are only additive with those of BafA1 in the presence of significant DVGLUT expression it is very likely that Na⁺/H⁺ exchange via DVGLUT contributes to activity-enhanced acid efflux across the PM of MN terminals. The electroneutrality of ion exchange by DVGLUT requires further investigation as changes in V_m upon removal of Na⁺ from oocytes expressing DVGLUT is probably attributable to decrease in the PM Na⁺ gradient rather than electrogenic Na⁺/H⁺ exchange by DVGLUT (Rossano, 2016).

The following model of pH_cyto regulation by DVGLUT reconciles the observation that DVGLUT is a mediator of activity-induced acid extrusion with the available data from mammalian VGLUT ion transport mechanisms. In quiescent nerve terminals NHEs mediate a standing acid efflux and DVGLUT is primarily on the vesicular membrane where it loads glutamate into the vesicular lumen via ΔΨ_H+ generated by the vATPase. The growing ΔpH across the vesicular membrane is dissipated by DVGLUT through cation/H⁺ exchange. Under these conditions K⁺/H⁺ exchange is most likely as K⁺ is much more abundant than Na⁺ in the cytosol and previous work has demonstrated functional K⁺/H⁺ exchange across organellar membranes in rat synaptosomes (Goh, 2011) and amphibian vestibular hair cells. Upon exocytosis to the PM the directionality of DVGLUT reverses and acid efflux is mediated by Na⁺/H⁺ exchange driven by the strong Δμ_Na+ at the PM. Upon cessation of neuronal activity, DVGLUT contributes to an early phase of accelerated acid efflux until it is retrieved from the PM by endocytosis. In recently endocytosed vesicles the elevated Cl⁻ concentration of the vesicular lumen drives glutamate into the vesicle by a Cl⁻ shunt mechanism (Schenck, 2009; Preobraschenski, 2014). As the vesicular ΔΨ_H+ generated by the vATPase is re-established the cycle completes. The model presented above describes a mechanism by which acid efflux can scale to effectively clear the overall net acid load through enhanced trafficking of acid-extruding proteins, including DVGLUT, to the PM, thus maintaining tight control of pH_cyto in the face of large PMCA-medicated acid loads during nerve activity (Rossano, 2016).

Kinesin Khc-73/KIF13B modulates retrograde BMP signaling by influencing endosomal dynamics at the Drosophila neuromuscular junction

Retrograde signaling is essential for neuronal growth, function and survival; however, little is known about how signaling endosomes might be directed from synaptic terminals onto retrograde axonal pathways. This study identified Khc-73, a plus-end directed microtubule motor protein, as a regulator of sorting of endosomes in Drosophila larval motor neurons. The number of synaptic boutons and the amount of neurotransmitter release at the Khc-73 mutant larval neuromuscular junction (NMJ) are normal, but a significant decrease in the number of presynaptic release sites was found. This defect in Khc-73 mutant larvae can be genetically enhanced by a partial genetic loss of Bone Morphogenic Protein (BMP) signaling or suppressed by activation of BMP signaling in motoneurons. Overexpression of the type II TGFβ receptor Wit enhanced presynaptic pMad levels. In Khc-73 mutants, this enhancement was significantly suppressed. Similarly, muscle overexpression of the ligand Gbb enhanced pMAD levels in presynaptic boutons. Consistently, activation of BMP signaling that normally enhances the accumulation of phosphorylated form of BMP transcription factor Mad in the nuclei, can be suppressed by genetic removal of Khc-73. Using a number of assays including live imaging in larval motor neurons, loss of Khc-73 was shown to curb the ability of retrograde-bound endosomes to leave the synaptic area and join the retrograde axonal pathway. These findings identify Khc-73 as a regulator of endosomal traffic at the synapse and modulator of retrograde BMP signaling in motoneurons (Liao, 2018).

Khc-73 function plays a supporting role in retrograde BMP signaling under basal conditions. However under conditions of enhanced BMP signaling, this endosomal coordination by Khc-73 becomes critical to transmit the retrograde signal from the synapse to the neuronal cell body (Liao, 2018).

Efficient retrograde signaling from synaptic terminals back to the neuronal soma is critical for appropriate neuronal function and survival. Nevertheless, little is known about the molecular steps that facilitate the routing of synaptic endosomes destined for retrograde axonal pathways. This study describes several lines of evidence for a potential role for Khc-73 in this process. Khc-73 mutant larvae develop grossly normal synaptic structure and function at the Drosophila larval neuromuscular junction (NMJ), but this study finds a reduction in the number of presynaptic release sites. Through genetic interaction experiments, this defect was shown to most likely be the result of abnormal BMP signaling in motoneurons: transheterozygous combinations of Khc-73 and Medea or wit mutants show a significant loss of presynaptic release sites compared to control. Khc-73 becomes even more critical, when higher demand is put on the motoneuron by activating BMP signaling: loss of Khc-73 largely blocks the retrograde enhancement in synaptic release in response to activation of BMP pathway in motor neurons. Consistently it has been shown previously that transgenic knock down of Khc-73 in motoneurons blocks the ability of the NMJ to undergo retrograde synaptic homeostatic compensation (Tsurudome, 2010). The current findings show that when BMP signaling is activated, loss of Khc-73 reduces the accumulation of pMad in motoneuron nuclei, suggesting a role for Khc-73 in the regulation of retrograde signaling. Immunohistochemical assessment and live imaging analysis of Khc-73 mutant larvae provide evidence for involvement of Khc-73 in at least two steps in endosomal dynamics in motoneurons. On the one hand, Khc-73 is required for normal dynamics of internalized endosomes through late endosomal and multivesicular stages, and on the other Khc-73 plays a role in facilitating the routing of endosomes onto the retrograde pathway. These defects have two main consequences: first, an accumulation of BMP receptors was found at the NMJ (possibly in multivesicular bodies) without increased local signaling, suggesting that these receptor containing endosomes might be trapped in a state between late endosomal and lysosomal stage. Second, a dampening was seen of the ability of retrograde bound Rab7:GFP tagged endosomes to join the retrograde pathway, illustrating a defect in retrograde movement of vesicles and possibly providing an underlying explanation for the reduction in pMAD when retrograde BMP signaling is activated in Khc-73 mutants. These results together present Khc-73, a plus-end microtubule motor, in the unexpected role of regulation of endosomal traffic from synapse to the soma in motoneurons with a role for ensuring the efficiency of retrograde BMP signaling (Liao, 2018).

The findings point to a model in which Khc-73 facilitates the routing of retrograde bound vesicles onto the retrograde axonal pathway. This model predicts coordination between endosomes, dynein motors and kinesin Khc-73. The coordinated involvement of dynein and kinesin motor proteins in the transport and sorting of endosomes has been previously proposed and examples supporting this model are mounting. Previously published data for Khc-73 and KIF13B have provided evidence that interaction between early endosomes, dynein motors and microtubules are possible. Khc-73/KIF13B is capable of binding to the GTPase Rab5 (found on early endosomes), thus allowing Khc-73 to localize directly to Rab5 endosomes. As a kinesin motor protein, Khc-73 could then transport these endosomes to the retrograde pathway by moving along the microtubule network in the synapse (Liao, 2018).

Compelling evidence for a dynein interaction with Khc-73 has been previously demonstrated during mitotic spindle formation. The Khc-73/KIF13B stalk domain is phosphorylated by Par1b and this creates a 14-3-3 adapter protein binding motif. It has been proposed that physical interaction between Khc-73 stalk domain and the dynein interacting protein NudE via 14-3-3 ε/ζ might underlie the interaction between Khc-73 and dynein that is necessary for appropriate mitotic spindle formation. Interestingly, transgenic knock down of NudE in Drosophila larval motoneurons leads to a reduction in the number of presynaptic release sites, a phenotype reminiscent of Khc-73 loss of function. Thus, Khc-73 contains domains and protein-protein interactions that are capable of coordinating endosomes, microtubules and dynein. It is proposed that Khc-73 is necessary for the normal endosomal sorting and exit of endosomes from the NMJ to support efficient retrograde BMP signaling (Liao, 2018).

Tao negatively regulates BMP signaling during neuromuscular junction development in Drosophila

The coordinated growth and development of synapses is critical for all aspects of neural circuit function and mutations that disrupt these processes can result in various neurological defects. Several anterograde and retrograde signaling pathways, including the canonical Bone Morphogenic Protein (BMP) pathway, regulate synaptic development in vertebrates and invertebrates. At the Drosophila larval neuromuscular junction (NMJ), the retrograde BMP pathway is part of the machinery that controls NMJ expansion concurrent with larval growth. This study sought to determine whether the conserved Hippo pathway, critical for proportional growth in other tissues, also functions in NMJ development. Neuronal loss of the serine-threonine protein kinase Tao, a regulator of the Hippo signaling pathway, results in supernumerary boutons, each of which contain a normal number of active zones. Tao is also required for proper synaptic function, as reduction of Tao results in NMJs with decreased evoked excitatory junctional potentials. Surprisingly, Tao function in NMJ growth is independent of the Hippo pathway. Instead, the experiments suggest that Tao negatively regulates BMP signaling as reduction of Tao leads to an increase in pMad levels in motor neuron nuclei and an increase in BMP target gene expression. Taken together, these results support a role for Tao as a novel inhibitor of BMP signaling in motor neurons during synaptic development and function (Politano, 2019).

AP2 Regulates Thickveins Trafficking to Attenuate NMJ Growth Signaling in Drosophila

Compromised endocytosis in neurons leads to synapse overgrowth and altered organization of synaptic proteins. However, the molecular players and the signaling pathways which regulate the process remain poorly understood. This study shows that σ2-adaptin, one of the subunits of the AP2-complex, genetically interacts with Mad, Medea and Dad (components of BMP signaling) to control neuromuscular junction (NMJ) growth in Drosophila Ultrastructural analysis of σ2-adaptin mutants show an accumulation of large vesicles and membranous structures akin to endosomes at the synapse. Mutations in σ2-adaptin lead to an accumulation of Tkv receptors at the presynaptic membrane. Interestingly, the level of small GTPase Rab11 was significantly reduced in the σ2-adaptin mutant synapses. However, expression of Rab11 does not restore the synaptic defects of σ2-adaptin mutations. A model is proposed in which AP2 regulates Tkv internalization and endosomal recycling to control synaptic growth (Choudhury, 2023).

The Strip-Hippo pathway regulates synaptic terminal formation by modulating actin organization at the Drosophila neuromuscular synapses

Synapse formation requires the precise coordination of axon elongation, cytoskeletal stability, and diverse modes of cell signaling. The underlying mechanisms of this interplay, however, remain unclear. This study demonstrates that Strip, a component of the striatin-interacting phosphatase and kinase (STRIPAK) complex that regulates these processes, is required to ensure the proper development of synaptic boutons at the Drosophila neuromuscular junction. In doing so, Strip negatively regulates the activity of the Hippo (Hpo) pathway, an evolutionarily conserved regulator of organ size whose role in synapse formation is currently unappreciated. Strip functions genetically with Enabled, an actin assembly/elongation factor and the presumptive downstream target of Hpo signaling, to modulate local actin organization at synaptic termini. This regulation occurs independently of the transcriptional co-activator Yorkie, the canonical downstream target of the Hpo pathway. This study identifies a previously unanticipated role of the Strip-Hippo pathway in synaptic development, linking cell signaling to actin organization (Sakuma, 2016).

Since the Hippo (Hpo) pathway was discovered as the key regulator to ensure the appropriate final tissue size by coordinating cell proliferation and cell death, large-scale genetics studies have identified numerous regulators of the Hpo pathway. While most pathway components identified thus far are positive regulators of Hpo, some negative regulators were recently reported. One such negative regulator is the STRIPAK (STRiatin-Interacting Phosphatase And Kinase) complex, which is evolutionarily conserved and regulates various cellular processes including cell cycle control and cell polarity. The core component of the STRIPAK complex is the striatin family of proteins: striatins serve as B‴ subunits (one of the subfamily of regulatory B subunits) of the protein phosphatase 2A (PP2A) complex. Beyond this, the A and C subunits of PP2A, Mob3, Mst3, Mst4, Ysk1, Ccm3, Strip1, and Strip2 form the core mammalian STRIPAK complex. It has been previously reported that Strip, the Drosophila homolog of mammalian Strip1 and 2, is involved in early endosome formation, which is essential for axon elongation. Building on these findings, it was hypothesized that the Strip-Hpo pathway may also be involved in neuronal synaptic development (Sakuma, 2016).

The Drosophila larval neuromuscular junction (NMJ) is an ideal model for studying synaptic development because of its identifiable, stereotyped morphology, accessibility, broad complement of available reagents, and suitability for a wide range of experimental approaches. Furthermore, the Drosophila NMJ, like vertebrate central synapses, is glutamatergic, suggesting that the molecular mechanisms that regulate synaptic development in Drosophila NMJ might be applicable to vertebrates. Motor neuron axons are genetically hardwired to target specific muscles by the end of the embryonic stage. There, axonal growth cones subsequently differentiate into presynaptic termini, called boutons, each of which contains multiple active zones). During the larval stage, muscle size increases nearly 100-fold and boutons are continuously and proportionately added to maintain constant innervation strength. Various molecules can negatively or positively regulate the growth of synaptic termini. Amongst the many factors, elements of the actin cytoskeleton are key effectors of morphological change, functioning downstream of several cell surface receptors and signaling pathways. Of the two types of actin filaments (branched and linear), the activity of Arp2/3 complex, responsible for nucleation of branched F-actin, the first step of actin polymerization, should be strictly regulated. Arp2/3 hyperactivation results in synaptic terminal overgrowth characterized by excess small boutons emanating from the main branch that are termed satellite boutons (Sakuma, 2016).

This study shows that Strip negatively regulates the synapse terminal development through tuning the activity of the core Hpo kinase cassette. Loss or reduction of strip function in motor neurons increased the number of satellite boutons, which could be suppressed by reducing the genetic dosage of hpo. Similarly, activation of the core Hpo kinase cassette also increased satellite boutons. In this context, the presumptive downstream target of the core Hpo kinase cassette is Enabled (Ena), a regulator of F-actin assembly and elongation that was reported to antagonize the activity of Arp2/3. The canonical downstream transcriptional co-activator, Yorkie (Yki), appears dispensable for Hpo-mediated synaptic terminal development. It is proposed that the evolutionarily conserved Strip-Hpo pathway regulates local actin organization by modulating Ena activity during synaptic development (Sakuma, 2016).

This study has identified Strip and components of the Hpo pathway as regulators of synaptic morphology. In addition to the intensely investigated function of Hpo in growth control in mitotic cells, a few postmitotic roles of the Hpo pathway have recently been uncovered, such as dendrite tiling in Drosophila sensory neurons and cell fate specification of photoreceptor cells in Drosophila retina. This study now finds an additional postmitotic role for Hpo in synaptic terminal development. The results indicate that Strip and the core Hpo kinase cassette regulate satellite bouton formation by regulating the activity of Ena, an actin regulator that is involved in the initiation, extension, and maintenance of linear actin filaments at the barbed end. Although it cannot be excluded that there might be other targets of Yki in motor neurons than diap1 or bantam whose transcriptional activations were not observed in this study, Yki, a well-known downstream target of the core Hpo kinase cassette, was dispensable for proper synaptic morphology. Ena phosphorylation causes its inactivation; therefore, it was reasoned that Strip can act as a positive regulator of Ena by inactivating the Hpo pathway. A model is proposed for the regulation of satellite bouton formation by Strip and Hpo pathway components (see The Strip-Hpo pathway regulates satellite bouton formation with Ena, a regulator of F-actin organization). As the presynaptic localization of endogenous Strip was punctate and non-uniform, it is expected that Strip localization could be critical for regulating the phosphorylation status of Hpo, Wts, and Ena, which locally alters actin organization and eventually specifies the position of satellite bouton formation that could be a marker for new bouton outgrowth. When Strip is present, the core Hpo kinase cassette is inactivated, which in turn locally increases the expression of the active (unphosphorylated) form of Ena. However, the core Hpo kinase cassette can be activated in the absence of Strip, which subsequently phosphorylates and inactivates Ena. Ena prevents Arp2/3-induced branching, suggesting that Ena inactivation activates Arp2/3 and results in satellite bouton formation, similar to Rac activation. It is reported that Arp2/3 is involved in bouton formation and axon terminal branching downstream of WAVE/SCAR complex in NMJ. Indeed, the cureent findings support this hypothesis. F-actin organization was altered by strip knockdown in motor neurons. When the GFP-moe reporter was expressed in motor neuron, the GFP fused to the C-terminal actin-binding domain of Moesin, which is widely used as an F-actin reporter. The intensity of actin puncta became higher and puncta were unevenly distributed when strip was knocked down. This data suggests that Strip function is important for the proper organization of F-actin (Sakuma, 2016).

There are many indications that Strip and other STRIPAK components (Mst3, Mst4, and Ccm3) regulate the actin network. For example, Strip1, Strip2, Mst3, and Mst4 regulate the actomyosin contractions which regulate cell migration in cancer cells. In addition to regulating the actin network, STRIPAK has been suggested to function in microtubule organization. Mutants of Drosophila Mob4, a member of the core STRIPAK complex and homolog of mammalian Mob3, show abnormal microtubule morphology at NMJs and muscles. Furthermore, it has been reported that Strip forms a complex with Glued, the homolog of mammalian p150^Glued, a component of the dynactin complex required for dynein motor-mediated retrograde transport along microtubules. Strip also affects microtubule stability. As previously mentioned, microtubules are also key effectors of synaptic development downstream of several receptors and signaling pathways. Taken together, the STRIPAK complex can act as a regulatory hub for multiple cellular signals including Hpo pathway-mediated actin organization, endocytic pathway-dependent BMP signals, and microtubule stability for proper synaptic development (Sakuma, 2016).

The Hpo pathway has been reported to act as a sensor of the local cellular microenvironment, such as mechanical cues, apico-basal polarity and actin architecture to balance cell proliferation and cell death. Although synaptic morphogenesis is a postmitotic process, it is very plastic and depends on a dynamically changing extracellular environment, as exemplified by the nearly 100-fold expansion of muscle size during larval development. Thus, this study demonstrates an intriguing function for the Strip-Hippo pathway in the homeostatic control of neuronal synaptic morphology and function (Sakuma, 2016).

The innate immune receptor PGRP-LC controls presynaptic homeostatic plasticity

It is now appreciated that the brain is immunologically active. Highly conserved innate immune signaling responds to pathogen invasion and injury and promotes structural refinement of neural circuitry. However, it remains generally unknown whether innate immune signaling has a function during the day-to-day regulation of neural function in the absence of pathogens and irrespective of cellular damage or developmental change. This study shows that an innate immune receptor, a member of the peptidoglycan pattern recognition receptor family (PGRP-LC), is required for the induction and sustained expression of homeostatic synaptic plasticity. This receptor functions presynaptically, controlling the homeostatic modulation of the readily releasable pool of synaptic vesicles following inhibition of postsynaptic glutamate receptor function. Thus, PGRP-LC is a candidate receptor for retrograde, trans-synaptic signaling, a novel activity for innate immune signaling and the first known function of a PGRP-type receptor in the nervous system of any organism (Harris, 2015).

The homeostatic modulation of presynaptic neurotransmitter release has been observed at mammalian central synapses and at neuromuscular synapses in species ranging from Drosophila to mouse and human. The homeostatic enhancement of presynaptic release following inhibition of postsynaptic glutamate receptors is achieved by an increase in presynaptic calcium influx through presynaptic CaV2.1 calcium channels and the simultaneous expansion of the readily releasable pool (RRP) of synaptic vesicles. To date, the retrograde, trans-synaptic signaling system that initiates these presynaptic changes following disruption of postsynaptic glutamate receptors remains largely unknown (Harris, 2015).

A large-scale forward genetic screen for homeostatic plasticity genes, using synaptic electrophysiology at the neuromuscular junction (NMJ) as the primary assay, identified mutations in the PGRP-LC locus that block presynaptic homeostasis. In Drosophila, PGRP-LC is the primary receptor that initiates an innate immune response through the immune deficiency (IMD) pathway. In mammals, there are four PGRPs including a 'long' isoform (PGRP-L or PGlyRP2) that appears to have both secreted and membrane-associated activity. In mice, the PGlyRP2 protein has two functions in the innate immune response: a well-documented extracellular enzymatic (amidase) activity and a pro-inflammatory signaling function that is independent of its extracellular enzymatic activity. As yet, there are no known functions for PGRPs in the nervous system of any organism (Harris, 2015).

Innate immune signaling has been found to participate in neural development and disease including a role for the C1q component of the complement cascade, Toll-like receptor signaling, and tumor necrosis factor signaling. In these examples, however, the innate immune response is induced within microglia or astrocytes. Far less clear is the role of innate immune signaling within neurons, either centrally or peripherally, although there are clear examples of downstream signaling components such as Rel and NFκB having important functions during learning related neural plasticity. This study places the innate immune receptor, PGRP-LC at the presynaptic terminal of Drosophila motoneurons and demonstrate that this receptor is essential for robust presynaptic homeostatic plasticity. It is speculated, based on the current data, that PGRP-LC could function as a receptor for the long-sought retrograde signal that mediates homeostatic signaling from muscle to nerve (Harris, 2015).

A distinguishing feature of presynaptic homeostatic plasticity is that it can be both rapidly induced and sustained for prolonged periods. It is possible that innate immune signaling activity is adjusted to fit the requirements of presynaptic homeostasis in motoneurons. This would be consistent with the established diversity of innate immune signaling functions during development such as dorso-ventral patterning. Alternatively, the induction of innate immune signaling might serve a unique function during presynaptic homeostasis. Many homeostatic signaling systems incorporate feed-forward signaling elements. By analogy, PGRP-LCx could function as a feed-forward signaling receptor that acts more like a 'switch' to enable presynaptic homeostasis in the nerve terminal. The accuracy of the homeostatic response would be determined by other signaling elements. In favor of this idea, the induction of innate immune signaling is rapid, occurring in seconds to minutes, and can be maintained for the duration of an inducing stimulus, consistent with recent evidence that presynaptic homeostasis is rapidly and continually induced at synapses in the presence of a persistent postsynaptic perturbation. Finally, it remains formally possible that PGRP-dependent signaling is a permissive signal that allows expression presynaptic homeostasis (Harris, 2015).

Control of synaptic connectivity by a network of Drosophila IgSF cell surface proteins

A network of interacting Drosophila cell surface proteins has been defined in which a 21-member IgSF subfamily, the Dprs, binds to a nine-member subfamily, the DIPs. The structural basis of the Dpr-DIP interaction code appears to be dictated by shape complementarity within the Dpr-DIP binding interface. Each of the six dpr and DIP genes examined here is expressed by a unique subset of larval and pupal neurons. In the neuromuscular system, interactions between Dpr11 and DIP-γ affect presynaptic terminal development, trophic factor responses, and neurotransmission. In the visual system, dpr11 is selectively expressed by R7 photoreceptors that use Rh4 opsin (yR7s). Their primary synaptic targets, Dm8 amacrine neurons, express DIP-γ. In dpr11 or DIP-γ mutants, yR7 terminals extend beyond their normal termination zones in layer M6 of the medulla. DIP-γ is also required for Dm8 survival or differentiation. These findings suggest that Dpr-DIP interactions are important determinants of synaptic connectivity (Carrillo, 2015).

This study has defined a network of interacting Drosophila IgSF CSPs in which 21 Dpr proteins bind to 9 DIPs. The structure of the Dpr-DIP complex resembles that of neural and immune cell adhesion complexes. Each of the six dpr and DIP genes examined in this study is expressed by a different subset of neurons in the larval VNC and pupal OL. In the larval neuromuscular system, Dpr11 and its binding partner DIP-γ regulate presynaptic terminal development and neurotransmission. In the pupal OL, they are required for normal formation of synapses between a Dpr11-expressing sensory neuron and a DIP-γ expressing interneuron (Carrillo, 2015).

The crystal structure shows that Dprs and DIPs belong to a group of IgSF CSPs that interact via their N-terminal Ig domains. These include immune cell receptors such as CD2, CD58, JAML, CAR, B7-1, and CTLA-4, and Nectin/Nectin-like (Necl) proteins. The nine Nectin/Necls interact with each other, forming a small network. Although DIPs resemble Nectins/Necls, their closest vertebrate counterpart is the five-member IgLON subfamily, which is also expressed in neurons. Dprs have no clear mammalian orthologs. DIPs and Dprs are distinguished from IgLONs and Nectins in that their interactions are across subfamilies, not within a subfamily. The closest structural homolog of the Dpr-DIP complex is the SYG-1-SYG-2 complex, known to be involved in synapse specification (Carrillo, 2015).

The Dpr-DIP complex has an interface involving no charge pairs, suggesting that binding specificity is encoded through shape complementarity. The Dpr-DIP interaction code may be created by substitution of larger or smaller residues within the binding interface in order to create more or less complementary surfaces between individual interacting Dpr-DIP pairs. This differs substantially from the electrostatic complementarity model, in which receptor-ligand specificity is created primarily through hydrogen bonding interactions and salt bridges. Interestingly, for Dscam homophilic interactions, where each of the many thousands of possible variants binds primarily to itself, both electrostatic and shape complementarity play crucial roles. Each Dscam variant has to find a single binding solution, which is a task that can be solved in many ways. By contrast, the complex cross-reactivity observed for Dpr-DIP interactions may impose restrictions on encoding of specificity that mandate the selection of shape complementarity as the primary mechanism (Carrillo, 2015).

The larval neuromuscular system is a genetic model system for glutamatergic synapses in mammals. In mutants lacking either Dpr11 or DIP-γ, NMJs contain many small clustered boutons called satellites. The satellite bouton phenotypes are rescued by either pre- or postsynaptic expression of the proteins. mEPSP amplitude and frequency are increased to similar extents in dpr11 and DIP-γ mutants. These data, together with the fact that the two loci genetically interact, indicate that the two proteins have linked functions, and suggest that the phenotypes are due to loss of Dpr11-DIP-γ adhesion complexes (Carrillo, 2015).

BMPs are trophic factors for mammalian neurons, and retrograde BMP signaling controls NMJ arbor growth in Drosophila. Satellites are observed in mutants in which BMP signaling is upregulated. Consistent with this, presynaptic pMad staining, which reports on the magnitude of the BMP signal, is increased in dpr11 mutants, and dpr11 and DIP-γ interact with genes encoding BMP signaling components (Carrillo, 2015).

Each dpr and DIP examined is expressed in a unique subset of neurons that project to specific layers in the OL neuropils. Identifying these neurons can define relationships between dpr/DIP expression and synaptic connectivity, because detailed synaptic maps for units of the first two areas of the OL, the La and Me, have been created using electron microscopic reconstruction (Carrillo, 2015).

Axons of UV-sensitive R7 photoreceptors synapse in layer M6 of the Me onto Dm8, Tm5a, Tm5b, and other targets. dpr11 is selectively expressed by yR7s, which express Rh4 opsin and are in ~70% of ommatidia. Dpr11 is the first cell surface protein to be associated with a subclass of R7s. DIP-γ is expressed by Dm8s, which arborize in M6 and receive more R7 synapses than any other neurons (Carrillo, 2015).

To examine whether formation of synapses between yR7s and Dm8s involves interactions between Dpr11 and DIP-γ, a marker for existing active zones, Brp-shortmCherry, was expressed in yR7s. In control animals, yR7 terminals are bulb-shaped and regularly arranged in M6. In dpr11 and DIP-γ mutants, the main bodies of yR7 terminals have altered shapes, and active zone and membrane markers are found in extensions projecting into deeper Me layers. These data suggest that synapses between yR7 and its M6 targets do not form normally in the absence of Dpr11 or DIP-γ. Because most M6-projecting DIP-γ-positive cells seen in the FLP-out analysis are Dm8s, and because Dpr11's other partner, DIP-β, does not label M6, it is infered that the loss of Dpr11 or DIP-γ is likely to primarily affect yR7-Dm8 synapses in M6 (Carrillo, 2015).

In DIP-γ mutants, there are large gaps in M6 labeling by DIP-γ or Dm8 reporters. The number of OrtC2b+, DIP-γ+ cells is reduced by >3-fold, suggesting that most DIP-γ-expressing Dm8s die. Alternatively, they might turn off expression of the OrtC2b-GAL4 driver, although this seems less likely. This effect on cell fate suggests that DIP-γ is required for reception of a neurotrophic signal. Since dpr11 mutants have no DIP-γMiMIC M6 gaps, implying that they have normal numbers of OrtC2b+, DIP-γ+ cells, this signal might be communicated through Dprs 15, 16, and/or 17, the other Dprs that bind to DIP-γ. Other OL neurons are also dependent on trophic factors for survival. R cell growth cones secrete the Jelly Belly (Jeb) ligand, which binds to its receptor Alk on L3 neurons, and L3s die in the absence of Jeb or Alk. The functions of DIP-γ in mediating normal development of yR7-Dm8 connectivity, as assayed by displacement of the active zone marker in yR7s, may be distinct from its roles in Dm8 survival, because about half of the overshoots in DIP-γ mutants appear to grow through a Dm8 arbor labeled by the DIP-γ reporter (Carrillo, 2015).

dpr11 is expressed by subsets of direction-selective T4 and T5 neurons that arborize in the Lop layers activated by front-to-back and back-to-front motion, and DIP-γ is expressed by three LPTCs, which receive synaptic input from T4s and T5s. These data suggest that Dpr11 and DIP-γ expression patterns might have evolved to facilitate assembly of synaptic circuits for specific sensory responses: near-UV vision for yR7-Dm8 connections and movement along the anterior-posterior axis for T4/T5 subset-LPTC connections. In a conceptually similar manner, a specific type of vertebrate amacrine neuron, VG3-AC, forms synapses on W3B retinal ganglion cells, which are specialized for detecting object motion. Both VG3-ACs and W3B-RGCs selectively express the IgSF protein Sidekick2 (Sdk2), and Sdk2-mediated homophilic adhesion is required for their connectivity (Carrillo, 2015).

An accompanying paper on gene expression in La neurons (Tan, 2015) presents ten instances in which a La neuron expressing a Dpr is synaptically connected to a Me neuron expressing a DIP to which that Dpr binds in vitro. In nine of these, as well as in the two cases described in this study (yR7 -> Dm8 and T4/T5 -> LPTC), the Dpr is in the presynaptic neuron and the DIP in the postsynaptic neuron. Each dpr and DIP gene examined in the two papers is expressed in a different subset of OL neurons, each of which projects to a distinct set of neuropil layers, and neurons can express multiple Dprs or DIPs or a combination of the two (Tan, 2015). This means that there are hundreds of different synaptic matches in the OL that could potentially be programmed by the Dpr-ome network. Dprs and DIPs are also expressed by subsets of neurons in other areas of the larval and pupal brain. These expression patterns, together with the phenotypic data presented here for one Dpr-DIP binding pair, suggest that Dpr-DIP interactions are likely to be important determinants of synaptic connectivity during brain development (Carrillo, 2015).

An auxiliary subunit of the presynaptic calcium channel, α2δ-3, is required for rapid transsynaptic homeostatic signaling

The homeostatic modulation of neurotransmitter release, termed presynaptic homeostatic potentiation (PHP), is a fundamental type of neuromodulation, conserved from Drosophila to humans, that stabilizes information transfer at synaptic connections throughout the nervous system. This study demonstrates that α2δ-3 (straitjacket), an auxiliary subunit of the presynaptic calcium channel, Cacophony, is required for PHP. The α2δ gene family has been linked to chronic pain, epilepsy, autism, and the action of two psychiatric drugs: gabapentin and pregabalin. Loss of α2δ-3 blocks both the rapid induction and sustained expression of PHP due to a failure to potentiate presynaptic calcium influx and the RIM-dependent readily releasable vesicle pool. These deficits are independent of α2δ-3-mediated regulation of baseline calcium influx and presynaptic action potential waveform. α2δ proteins reside at the extracellular face of presynaptic release sites throughout the nervous system, a site ideal for mediating rapid, transsynaptic homeostatic signaling in health and disease (Wang, T., 2016).

Presynaptic homeostatic potentiation (PHP) can be initiated by disruption of postsynaptic neurotransmitter receptors and is expressed as a change in presynaptic vesicle release. As such, PHP requires retrograde, transsynaptic signaling. The homeostatic potentiation of presynaptic release is mediated by increased presynaptic calcium influx without a change in the presynaptic action potential waveform. A remarkable property of presynaptic homeostatic plasticity is that it can be induced in a time frame of seconds to minutes and can be stably maintained throughout the life of an organism -- months in Drosophila and decades in humans. Equally remarkable, presynaptic homeostasis can precisely offset the magnitude of postsynaptic perturbations that vary widely in severity. This implies the existence of profoundly stable and remarkably precise homeostatic modifications to the presynaptic release apparatus. Transsynaptic signaling systems that are capable of achieving the rapid, accurate, and persistent control of presynaptic vesicle release are generally unknown (Wang, T., 2016).

In a large-scale forward genetic screen for homeostatic plasticity genes, mutations were identified in the α2δ-3 auxiliary subunit of the Ca_V2.1 calcium channel. α2δ genes encode a family of proteins that are post-translationally processed into a large glycosylated extracellular α2 domain that is linked through disulfide bonding to a short, membrane-associated δ domain. Existing loss-of-function data are consistent with the primary function of α2δ being the trafficking and synaptic stabilization of pore-forming α1 calcium channel subunits, with which they associate in the ER. There is also evidence that α2δ subunits control calcium channel kinetics in a channel-type- and cell-type-specific manner. However, the function of the α2δ gene family extends beyond calcium channel trafficking and membrane stabilization, including activities related to synapse formation and stability. As such, the large, glycosylated extracellular domain in α2δ may have additional, potent signaling activities at the active zone (Wang, T., 2016).

Importantly, the α2δ gene family is associated with a wide range of neurological diseases, including autism spectrum disorders (ASDs), neuropathic pain, and epilepsy. The α2δ-1 and α2δ-2 proteins are the primary targets of gabapentin and pregabalin, two major drugs used to treat neuropathic pain and epilepsy. This study demonstrates that α2δ-3 is essential for PHP. Thus, while α2δ-3 is an extracellular component of the extended presynaptic calcium channel complex (Davies, 2010), it nonetheless has a profound ability to modulate the intracellular neurotransmitter release mechanism. It is proposed that α2δ-3 relays signaling information from the synaptic cleft to the cytoplasmic face of the presynaptic active zone during PHP, an activity that could reasonably be related to the function of α2δ-3 during neurological disease (Wang, T., 2016).

This study demonstrates that α2δ-3 is essential for the rapid induction and sustained expression of presynaptic homeostatic potentiation (PHP). α2δ-3 encodes a glycosylated extracellular protein known to interact with matrix proteins that reside within the synaptic cleft. As such, it is proposed that α2δ-3 mediates homeostatic, retrograde signaling by connecting signaling within synaptic cleft to effector proteins within the presynaptic terminal, such as RIM. Since α2δ-3 associates with the pore-forming α1 subunit of calcium channels, it is ideally positioned to relay signaling to the site of high-release probability vesicle fusion adjacent to the presynaptic calcium channels (Wang, T., 2016).

It was previously demonstrated that PHP requires not only potentiation of presynaptic calcium influx but also a parallel homeostatic expansion of the readily releasable pool (RRP). Several lines of evidence argue against the possibility that the homeostatic potentiation of presynaptic calcium influx fully accounts for the observed potentiation of the RRP. First, it is well established in mammalian systems and the Drosophila NMJ (Müller, 2015) that the calcium-dependence of the RRP is sub-linear. Therefore, the relatively small change in presynaptic calcium influx that occurs during PHP (12%-25%) would not be sufficient to account for the observed doubling of the RRP, an effect that has been quantified across a wide range of extracellular calcium (0.3-15 mM [Ca²⁺]_e) (Müller, 2015). Second, the homeostatic increase of presynaptic calcium influx and the homeostatic expansion of RRP are genetically separable processes (Harris, 2015). Since loss of α2δ-3 completely blocks the homeostatic expansion of the RRP, it appears that α2δ-3 has an additional activity that is directed at the homeostatic modulation of the RRP (Wang, T., 2016).

Collectively, these data argue that α2δ-3 functions with Rab3 interacting molecule (rim), either directly or indirectly, to achieve a homeostatic potentiation of the RRP. First, the loss of function phenotype of α2δ-3 is strikingly similar to that observed in rim mutants. Both mutations cause a deficit in presynaptic release that is associated with diminished baseline presynaptic calcium influx, diminished size of the baseline RRP, and enhanced sensitivity to application of EGTA-AM. Second, this study demonstrates a strong trans-heterozygous interaction between rim/+ and α2δ-3/+, suggesting that both genes function to control the same presynaptic processes during PHP. Since the rim mutation selectively disrupts the homeostatic modulation of the RRP, this genetic interaction could reflect a failure to homeostatically modulate the RRP (Wang, T., 2016).

Both RIM and α2δ-3 bind the pore-forming α1 subunit of the Ca_V2.1 calcium channel. As such, signaling could be relayed from α2δ-3 to RIM through molecular interactions within the extended Ca_V2.1 calcium channel complex. However, not all evidence is consistent with this possibility. For example, RNAi-mediated depletion of Ca_V2.1 channels, sufficient to decrease release by ∼80%, does not prevent presynaptic homeostasis. Thus, loss of α2δ-3 blocks PHP, whereas loss of the Ca_V2.1 α1 subunit does not. In addition, the double-heterozygous mutant of rim/+ and α2δ-3/+ blocks PHP but does not disrupt baseline vesicle release, arguing that this genetic interaction is not due to a decrease in the number or organization of presynaptic α1 calcium channel subunits. Thus, it is speculated that α2δ-3 conveys signaling through a co-receptor on the plasma membrane to participate in the homeostatic modulation of the RRP. There are very few extracellular proteins known to establish baseline levels of primed, fusion-competent synaptic vesicles. Since α2δ proteins should reside at chemical synapses throughout the nervous system, this signaling could reasonably be related to the neurological and psychiatric diseases associated with α2δ genes (Wang, T., 2016).

Presynaptic CamKII regulates activity-dependent axon terminal growth

Spaced synaptic depolarization induces rapid axon terminal growth and the formation of new synaptic boutons at the Drosophila larval neuromuscular junction (NMJ). This study identified a novel presynaptic function for the Calcium/Calmodulin-dependent Kinase II (CamKII) protein in the control of activity-dependent synaptic growth. Consistent with this function, both total and phosphorylated CamKII (p-CamKII) are were found to be enriched in axon terminals. Interestingly, p-CamKII appears to be enriched at the presynaptic axon terminal membrane. Moreover, levels of total CamKII protein within presynaptic boutons globally increase within one hour following stimulation. These effects correlate with the activity-dependent formation of new presynaptic boutons. The increase in presynaptic CamKII levels is inhibited by treatment with cyclohexamide suggesting a protein-synthesis dependent mechanism. Previous work has found that acute spaced stimulation rapidly downregulates levels of neuronal microRNAs (miRNAs) that are required for the control of activity-dependent axon terminal growth at this synapse. The rapid activity-dependent accumulation of CamKII protein within axon terminals is inhibited by overexpression of activity-regulated miR-289 in motor neurons. Experiments in vitro using a CamKII translational reporter show that miR-289 can directly repress the translation of CamKII via a sequence motif found within the CamKII 3' untranslated region (UTR). Collectively, these studies support the idea that presynaptic CamKII acts downstream of synaptic stimulation and the miRNA pathway to control rapid activity-dependent changes in synapse structure (Nesler, 2016).

Acute spaced synaptic depolarization rapidly induces the formation of new synaptic boutons at the larval NMJ. These immature presynaptic outgrowths, also known as "ghost boutons", are characterized by the presence of synaptic vesicles but by a lack of active zones and postsynaptic specializations. IA wild-type third instar larval NMJ will typically have about 2 ghost boutons. Using an established synaptic growth protocol, a robust increase in the number of ghost boutons was observed following 5 x K+ spaced stimulation. It has been shown that activity-dependent ghost bouton formation involves both new gene transcription and protein synthesis. Furthermore, new presynaptic expansions can form within 30 min of stimulation even after the axon innervating the NMJ has been severed. These findings suggest that a local mechanism (i.e. local signaling and/or translation) is required for the budding and outgrowth of new axon terminals. As expected, application of the translational inhibitor cyclohexamide during the recovery phase prevented the formation of new ghost boutons (Nesler, 2016).

It has been shown previously that the outgrowth of new synaptic boutons in response to spaced depolarization requires the function of activity-regulated neuronal miRNAs including miR-8, miR-289, and miR-958 (Nesler, 2013). This implies that mRNAs encoding for synaptic proteins might be targets for regulation by these miRNAs. Focused was placed on CamKII for three reasons. (1) CamKII has been shown to have a conserved role in the control of long-term synaptic plasticity and its expression at synapses requires components of the miRNA pathway. Furthermore, the fly CamKII mRNA contains two predicted binding sites for activity-regulated miR-289. (2) CamKII and PKA both phosphorylate and actives synapsin. At the fly NMJ, a synapsin-dependent mechanism is required for a transient increase in neurotransmitter release in response to tetanic stimulation. Synapsin also redistributes to sites of activity-dependent axon terminal growth and regulates outgrowth via a PKA-dependent pathway. (3) Presynaptic CamKII has been shown to function in axon pathfinding in cultured Xenopus neurons. It seemed likely that activity-dependent ghost bouton formation and axon guidance might share similar molecular machinery (Nesler, 2016).

It was postulated that presynaptic CamKII was required to control activity-dependent axon terminal growth at the larval NMJ. To address this question, CamKII expression was disrupted in motor neurons using two transgenic RNAi constructs. Depletion of presynaptic CamKII with both transgenes prevented the formation of new ghost boutons in response to spaced stimulation. Thus, presynaptic CamKII is necessary to control the formation of new synaptic boutons (Nesler, 2016).

To further confirm that presynaptic CamKII function was required for activity-dependent growth, a transgenic line was used that inducibly expressed an inhibitory peptide (UAS-CamKII^Ala). As in mammals, the activation of Drosophila CamKII by exposure to calcium leads to the autophosphorylation of a conserved threonine residue within the autoinhibitory domain (T287 in Drosophila). Activation of CamKII then confers an independence to calcium levels that persists until threonine-287 is dephosphorylated. The synthetic Ala peptide mimics the autoinhibitory domain and its transgenic expression is sufficient to substantially inhibit endogenous CamKII activity. Expression of the Ala inhibitory peptide in larval motor neurons disrupted the formation of new ghost boutons following spaced synaptic depolarization. These observations are consistent with results from CamKII RNAi (Nesler, 2016).

Together, these data suggest that presynaptic CamKII function is required to control new ghost bouton formation in response to acute synaptic activity. Similarly, presynaptic CamKII has been implicated in controlling both bouton number and morphology during development of the larval NMJ. Reducing neuronal CamKII levels by RNAi has recently been shown to significantly reduce the number of type 1b boutons at the larval NMJ suggesting that presynaptic CamKII is required to control normal synapse development (Gillespie, 2013). In contrast, presynaptic expression of the Ala inhibitory peptide has no effect on the total number of type 1 synaptic boutons. Given that the Ala peptide does not completely inhibit CamKII autophosphorylation, it is suggested that the activation of CamKII in response to acute spaced synaptic depolarization is likely to be more sensitive to disruption then during NMJ development (Nesler, 2016).

It was next asked if presynaptic CamKII could induce activity-dependent axon terminal growth at the NMJ. The overexpression of genes that are necessary for the control of ghost bouton formation generally does not cause an increase in the overall number of new synaptic boutons following 5 x high K+ spaced. Instead, overexpression often leads to an increased sensitization of the synapse to subsequent stimuli (for example, significant growth is observed after 3 x instead of 5 x high K+). The overexpression of a wild-type CamKII transgene in motor neurons caused an increase of 71% in ghost bouton numbers in 3 x K+ spaced stimulation larvae compared to 0 x K+ controls. While this is trending towards an increase, it did not reach statistical significance, even though expression levels were substantially higher than endogenous CamKII. Thus, increased CamKII is not sufficient to stimulate activity-dependent axon terminal growth (Nesler, 2016).

To further investigate the role of presynaptic CamKII in activity-dependent axon terminal growth, the effect of transgenic neuronal overexpression of either an overactive form of CamKII (CamKII^T287D) or a form that is incapable of remaining active in the absence of elevated calcium (CamKII^T287A). Much like C380-Gal4/+ controls, presynaptic expression of either transgene had no significant effect on the number of ghost boutons in 3 x high K+ stimulation. Again, levels of CamKII protein in axon terminals in both transgenic lines were elevated relative to controls. Collectively, these data suggest that constitutive activation of CamKII is not sufficient to sensitize the NMJ to stimulation (Nesler, 2016).

The results suggest that the temporal and/or spatial regulation of CamKII expression or activation is likely required to control activity-dependent growth. In support, the Drosophila CamKII protein has been shown to phosphorylate and regulate the activity of the Ether-a-go-go (Eag) potassium channel in motor neuron axon terminals. In turn, CamKII is bound and locally activated by phosphorylated Eag. This local activation can persist even after calcium levels have been reduced. CamKII autophosphorylation and Eag localization to synapses requires the activity of the membrane-associated Calcium/Calmodulin-associated Serine Kinase, CASK. The presynaptic coexpression of CASK with CamKII^T287D reverses (to wild-type levels) the increase in type 1b boutons observed when CamKII^T287D is overexpressed alone. Thus, a mechanism exists at the larval NMJ that allows for the persistence of local CamKII activation in the absence of additional stimul (Nesler, 2016).

After establishing that CamKII has a novel presynaptic function in activity-dependent ghost bouton formation, the distribution of CamKII protein at the larval NMJ was examined closely. It has previously been reported that CamKII strongly colocalizes with postsynaptic Discs large (DLG), the Drosophila ortholog of mammalian PSD-95, around the borders of type 1 synaptic boutons. In support, an anti-CamKII antibody coimmunoprecipitates DLG from larval body wall extracts. Interestingly, while DLG is pre-dominantly postsynaptic at the developing NMJ it is also initially expressed in the presynaptic cell and at least partially overlaps with presynaptic membrane markers in axon terminals. It has been demonstrated that while fly CamKII colocalizes with DLG within dendrites of adult olfactory projection neurons (PNs), it also localizes to presynaptic boutons within those same neurons. Consistent with the latter observations (using a different CamKII antibody), it has been shown that CamKII is substantially enriched in presynaptic terminals of type 1b boutons. To resolve these inconsistent results, both antibodies against CamKII were used to more closely analyze the localization of CamKII at the third instar larval NMJ. First, double labeling of wild-type NMJs with a monoclonal CamKII antibody and anti-horseradish peroxidase (HRP), a marker for Drosophila neurons, confirmed that CamKII was enriched in presynaptic boutons in a pattern very similar to that of HRP. A closer examination of confocal optical sections revealed that almost all CamKII localized to the presynaptic terminal and was not significantly enriched either (1) at sites surrounding presynaptic boutons, or (2) in the axons innervating synaptic arbors (Nesler, 2016).

Within boutons, CamKII appeared to be predominantly cytoplasmic but was sometimes localized to discrete puncta that were reminiscent of antibody staining for active zones. Prolonged depolarization of hippocampal neurons with K+ leads to mobilization of CamKII from the cytoplasm to sites near active zones. Moreover, using a fluorescent reporter for CamKII activity, high frequency stimulation causes the very rapid (on the order of minutes) activation of presynaptic CamKII and promotes its translocation from the cytoplasm to sites near active zones. To address this possibility, larval NMJs were double labeled with antibodies targeting both CamKII and DVGLUT, the Drosophila vesicular glutamate transporter, in order to visualize active zones. As predicted, it was found that some presynaptic CamKII colocalized with DVGLUT in type 1b and 1s boutons. Thus, in some type 1 synaptic boutons, CamKII protein is enriched in or near active zones (Nesler, 2016).

To confirm that CamKII was enriched in presynaptic boutons, wild-type NMJs were double labelled with a polyclonal CamKII antibody and anti-DLG. CamKII did partially colocalize with DLG at the border of type 1 synaptic boutons. However, in this study, CamKII was primarily localized to the presynaptic side of the synapse. Collectively, this study provides strong evidence that CamKII is expressed on both the pre- and postsynaptic side of the synapse but that it is clearly enriched within presynaptic boutons at the larval NMJ. This localization is analogous to CamKII distribution in mammalian axons (Nesler, 2016).

After demonstrating that total CamKII was enriched in presynaptic axon terminals, it was next asked if any of this protein was active by assessing phosphorylation of threonine-287 using a phospho-specific polyclonal antibody. It was found that pT287 CamKII staining intensity was strong and fairly uniform in presynaptic boutons and weakly stained axons innervating synaptic arbor. Closer examination of confocal optical sections revealed that almost all p-CamKII colocalized with HRP in the presynaptic terminal and only sparsely stained the body wall muscle (Fig. 3A′). Presynaptic CamKII RNAi almost completely disrupted p-CamKII in axon terminals leaving some residual staining in the presynaptic bouton and surrounding muscle suggesting that the antibody is specific. To further demonstrate this presynaptic localization, it was found that p-CamKII staining clearly does not overlap with postsynaptic DLG but does colocalize strongly with immunostaining using the monoclonal total CamKII antibody (Nesler, 2016).

Collectively, three different antibodies were used to show that CamKII enriched in presynaptic axon terminals. Next, it was asked as to how this enrichment was occurring. In Drosophila and mammalian neurons, the CamKII mRNA is transported to dendritic compartments and locally translated in response to synaptic stimulation. This spatial and temporal regulation requires sequence motifs found within the 5' and 3' UTRs of the CamKII transcript. In contrast, the localization of CamKII to axon terminals of Drosophila PNs does not strictly require the CamKII 3'UTR suggesting that enrichment in presynaptic boutons occurs through a mechanism that does not strictly require local translation. In mammalian neurons, CamKII is enriched in axon terminals where it can associate with synaptic vesicles and synapsin I. Recently, it has been shown that mammalian CamKII and the synapsin proteins are both conveyed to distal axons at rates consistent with slow axonal transport, with a small fraction of synapsin cotransported with vesicles via fast transport (Nesler, 2016).

Because activity-dependent growth at the larval NMJ requires the miRNA pathway and new protein synthesis, it was asked if the localization of CamKII protein to axon terminals might require the CamKII 3'UTR. As expected, when expression was specifically driven in larval motor neurons, a transgenic CamKII:EYFP fusion protein regulated by the CamKII 3'UTR localized strongly to presynaptic boutons at the larval NMJ. However, very similar results were observed using the same CamKII:EYFP fusion protein regulated by a heterologous 3'UTR. Taken together, these data suggest that localization of CamKII protein to presynaptic boutons at the NMJ does not require mRNA transport and local translation. Thus, it is concluded that most of the Drosophila CamKII protein found in motoneuron axon terminals is likely there due to the transport of cytosolic CamKII from the cell body to synapses via a mechanism involving axonal transport. (Nesler, 2016).

It was of interest to determining how CamKII might be regulating activity-dependent axon terminal growth, and it was speculated that either the levels or distribution of CamKII protein might be altered in response to spaced depolarization. It first asked if high K+ stimulation resulted in an increase in CamKII protein within motoneuron axon terminals. Larval preparations were stimulated, and changes in the levels of CamKII protein in presynaptic boutons was examined by immunohistochemistry and quantitative confocal microscopy. Following spaced stimulation, CamKII staining within boutons rapidly increased (in ~ 1 h) by an average of 26%. This increase in immunofluorescence was global and did not appear to be localized to particular regions of the NMJ (i.e., near obvious presynaptic outgrowths). CamKII has been reported to very rapidly translocate to regions near active zones in response to high frequency stimulation. However, when compared to DVGLUT levels in unstimulated and stimulated larvae, no significant increase in CamKII immunofluorescence was observed indicating that translocation does not occur or does not persist in the current assay. To determine if this increase in CamKII enrichment required new protein synthesis, larval preparations were incubated with the translational inhibitor cyclohexamide during the recovery phase. Surprisingly, this treatment completely blocked the activity-dependent affects on presynaptic CamKII enrichment within axon terminals. Thus, spaced high K+ stimulation results in a rapid increase in CamKII levels in presynaptic boutons via some mechanism that requires activity-dependent protein synthesis (Nesler, 2016).

Next, it was asked if the levels or distribution of p-CamKII changed in response to spaced stimulation. Larval preparations were stimulated exactly as described above and analyzed by confocal microscopy. Interestingly, p-CamKII staining was enriched at the presynaptic membrane of many axon terminals following spaced depolarization (Nesler, 2016).

Given the requirement for new protein synthesis, it was speculated that the additional CamKII protein in axon terminals could be derived from a pool of CamKII mRNA that is rapidly transcribed and translated in the soma in response to spaced depolarization. This newly translated CamKII would then be actively transported out to axon terminals via standard mechanisms. If this were true, it would be expected that elevated CamKII levels could be detected in the larval ventral ganglion. To examine this process more closely, global total CamKII expression levels within the larval ventral ganglion were assayed by Western blot analysis. It was found that two distinct isoforms of CamKII are expressed in explanted larval ventral ganglia, Surprisingly, no increase was observed in CamKII protein levels in the ventral ganglion (Nesler, 2016).

What is the source of this new presynaptic CamKII protein? Three possible explanations are proposed. First, new CamKII protein might be transcribed and translated in the motor neuron cell body. However, this new protein would be rapidly transported away to axon terminals in response to spaced depolarization. Second, some CamKII protein is found in the axons innervating the NMJ (seen using the p-CamKII antibody). It is possible that activity stimulates the rapid transport of an existing pool of CamKII protein from distal axons into axon terminals. This process would be sensitive to translational inhibitors. Finally, a pool of CamKII mRNA might be actively transported into axon terminals and then locally translated in response to spaced depolarization. This would account for the both the dependence on translation and for increased CamKII enrichment in presynaptic boutons (Nesler, 2016).

Thus far, this study has shown that activity-dependent ghost bouton formation correlates with a protein synthesis-dependent increase in CamKII levels within presynaptic boutons at the larval NMJ. The activity-dependent translation of CamKII in olfactory neuron dendrites in the adult Drosophila brain requires components of the miRNA pathway. Within the CamKII 3'UTR, there are two putative binding sites for activity-regulated miR-289. These two binding sites were of particular interest. It was previously shown that levels of mature miR-289 are rapidly downregulated in the larval brain in response to 5 x high K+ spaced training (Nesler, 2013). Moreover, presynaptic overexpression of miR-289 significantly inhibits activity-dependent ghost bouton formation at the larval NMJ (Nesler, 2013). Based on these data, it was speculated that CamKII might be a target for regulation by miR-289 (Nesler, 2016).

To determine if CamKII is a target for repression by miR-289 in vivo, a transgenic construct containing the primary miR-289 transcript was overexpressed in motor neurons and CamKII enrichment was examined by anti-CamKII immunostaining and quantitative confocal microscopy. Relative to controls, the presynaptic overexpression of miR-289 completely abolished the observed activity-dependent increase in CamKII immunofluorescence. When analyzing global CamKII levels within axon terminals during NMJ development, presynaptic miR-289 expression led to a slight decrease in CamKII immunofluorescence. This trend is similar to results observed following treatment with cyclohexamide during the recover period. The lack of full repression by miR-289 is not surprising given that one miRNA alone is often not sufficient to completely repress target gene expression (Nesler, 2016).

To directly test the ability of miR-289 to repress translation of CamKII, a reporter was developed where the coding sequence for firefly luciferase (FLuc) was fused to the regulatory CamKII 3'UTR (FLuc-CamKII 3'UTR). When this wild-type reporter was coexpressed with miR-289 in Drosophila S2 cells, expression of FLuc was significantly reduced. In contrast, when this reporter was coexpressed with miR-279a, a miRNA not predicted to bind to the CamKII 3'UTR, no repression was observed. To confirm that repression of the FLuc-CamKII reporter by miR-289 was via a specific interaction, the second of two predicted miR-289 binding sites was mutagenized. Binding site 2 (BS2) was a stronger candidate for regulation because it is flanked by AU-rich elements (AREs) and miR-289 has been shown to promote ARE-mediated mRNA instability through these sequences. Moreover, it is well established that the stabilization and destabilization of neuronal mRNAs via interactions between AREs and ARE-binding factors plays a significant role in the establishment and maintenance of long-term synaptic plasticity in both vertebrates and invertebrates. Altering three nucleotides within BS2 in the required seed region binding site was sufficient to significantly disrupt repression of the reporter by miR-289. The minimal predicted BS2 sequence was cloned into an unrelated 3'UTR and it was asked if miR-289 could repress translation. Coexpression of the FLuc-SV-mBS2 reporter with miR-289 led to significant repression. Taken together, these results indicate that the BS2 sequence is both necessary and sufficient for miR-289 regulation via the CamKII 3′UTR (Nesler, 2016).

The most important conclusion of this study is that presynaptic CamKII is required to control activity-dependent axon terminal growth at the Drosophila larval NMJ. First, it was shown that CamKII is necessary to control ghost bouton formation in response to spaced synaptic depolarization. Next, it was demonstrated that spaced stimulation correlates with a rapid protein synthesis dependent increase in CamKII immunofluorescence in presynaptic boutons. This increase is suppressed by presynaptic overexpression of activity-regulated miR-289. Previous work has shown that overexpression of miR-289 in larval motor neurons can suppress activity-dependent axon terminal growth (Nesler, 2013). This study demonstrated that miR-289 can repress the translation of a FLuc-CamKII 3'UTR reporter via a specific interaction with a binding site within the CamKII 3'UTR ( Fig. 6C-E). Collectively, this experimental evidence suggests that CamKII functions downstream of the miRNA pathway to control activity-dependent changes in synapse structure. (Nesler, 2016).

Thus, CamKII protein is expressed in the right place to regulate rapid events that are occurring within presynaptic boutons. Several questions remain regarding CamKII function in the control of activity-dependent axon terminal growth. First, it is unclear what the significance might be of a rapid increase of total CamKII in presynaptic terminals. Why is the pool of CamKII protein that is already present not sufficient to control these processes? Similar questions have been asked regarding activity-dependent processes occurring within dendrites. It is postulated that the CamKII mRNA might be locally translated in axon terminals. It has been proposed that local mRNA translation might be (1) required for efficient targeting of some synaptic proteins to specific sites, or (2) local translation may in and of itself be required to control activity-dependent processes at the synapse. Second, the impact of spaced depolarization on CamKII function needs to be assessed and downstream targets of CamKII phosphorylation involved in these processes need to be identified. One very strong candidate is synapsin which, at the Drosophila NMJ, has been shown to rapidly redistribute to sites of new ghost bouton outgrowth in response to spaced stimulation. Finally, the idea that CamKII might work through a Eag/CASK-dependent mechanism to control activity-dependent axon terminal growth needs to be examined (Nesler, 2016).

The equilibrium between antagonistic signaling pathways determines the number of synapses in Drosophila

Using the Drosophila larval neuromuscular junction, this study shows a PI3K-dependent pathway for synaptogenesis which is functionally connected with other previously known elements including the Wit receptor, its ligand Gbb, and the MAPkinases cascade. Based on epistasis assays, the functional hierarchy within the pathway was determined. Wit seems to trigger signaling through PI3K, and Ras85D also contributes to the initiation of synaptogenesis. However, contrary to other signaling pathways, PI3K does not require Ras85D binding in the context of synaptogenesis. In addition to the MAPK cascade, Bsk/JNK undergoes regulation by Puc and Ras85D which results in a narrow range of activity of this kinase to determine normalcy of synapse number. The transcriptional readout of the synaptogenesis pathway involves the Fos/Jun complex and the repressor Cic. In addition, an antagonistic pathway was identified that uses the transcription factors Mad and Medea and the microRNA bantam to down-regulate key elements of the pro-synaptogenesis pathway. Like its counterpart, the anti-synaptogenesis signaling uses small GTPases and MAPKs including Ras64B, Ras-like-a, p38a and Licorne. Bantam downregulates the pro-synaptogenesis factors PI3K, Hiw, Ras85D and Bsk, but not AKT. AKT, however, can suppress Mad which, in conjunction with the reported suppression of Mad by Hiw, closes the mutual regulation between both pathways. Thus, the number of synapses seems to result from the balanced output from these two pathways (Jordan-Alvarez, 2017).

The epistasis assays have determined the in vivo functional links between PI3K and other previously known pro-synaptogenesis factors. Epistasis assays are based on the combined expression of two or more UAS constructs. Several double combinations in this study have produced a phenotype in spite of the apparent ineffectiveness of the single constructs. This type of results underscores the necessity to use epistasis assays in order to reveal functional interactions in vivo, hence, biologically relevant. In addition to the pro-synaptogenesis signaling, the study has revealed an anti-synaptogenesis pathway that composes a signaling equilibrium to determine the actual number of synapses. The magnitude of the synapse number changes elicited by the factors tested here are mostly within the range of 20%-50%. Are these values significant to cause behavioral changes? Reductions in the order of 30% of excitatory or inhibitory synapses in adult Drosophila local olfactory interneurons transform perception of certain odorants from attraction to repulsion and vice versa. In schizophrenia patients, a 16% loss of inhibitory synapses in the brain cortex has been reported. In Rhesus monkeys, the pyramidal neurons in layer III of area 46 in dorsolateral prefrontal cortex show a 33% spine loss, and a significant reduction in learning task performance during normal aging. Thus, it seems that behavior is rather sensitive to small changes in synapse number irrespective of the total brain mass (Jordan-Alvarez, 2017).

The signaling interactions analyzed here were chosen because they were reported in other cellular systems and species previously. Some of these interactions have been confirmed (e.g., Gbb/Wit), while others have proven ineffective in the context of synaptogenesis (e.g., Ras85D/PI3K binding). Likely, the two signaling pathways, pro- and anti-synaptogenesis, are not the only ones relevant for synapse formation. For example, in spite of the null condition of the gbb and wit mutant alleles used here, the resulting synaptic phenotypes are far less extreme than expected if these two factors would be the only source of signaling for synaptogenesis. Although it could be argued that the incomplete absence of synapses in the mutant phenotypes could result from maternal perdurance, Wit is not part of the oocyte endowment while Gbb is. Three alternative possibilities may be considered, additional ligands for Wit, additional receptors for Gbb, and a combination of the previous two. Beyond the identity of these putative additional ligands and receptors, the stoichiometry between ligands and receptors may certainly be relevant. Actually, Gbb levels are titrated by Crimpy. An equivalent quantitative regulation could operate on Wit. The reported data on Wit illustrate already the diversity of the functional repertoire of this receptor. Wit can form heteromeric complexes with Thick veins (Tkv) or Saxophone (Sax) receptors to receive Dpp/BMP4 or Gbb/BMP7 as ligands. However, the same study also showed that Wit could dimerize with another receptor, Baboon, upon binding of Myoglianin to activate a different and antagonistic signaling pathway, TGFβ/activin-like (Jordan-Alvarez, 2017).

The Gbb/Wit/PI3K signaling analyzed in this study is likely not the only pro-synaptogenesis pathway in flies and vertebrates. The ligand Wingless (Wg), member of the Wnt family, and the receptors Frizzled have been widely documented as relevant in neuromuscular junction development, albeit data on synapse number are scant. Interestingly, however, the downstream intermediaries can be as diverse as those mentioned above for Wit. Although generally depicted as linear pathways, a more realistic image would be a network of cross-interacting signaling events whose in situ regulation and cellular compartmentalization remains fully unexplored (Jordan-Alvarez, 2017).

The quantitative regulation of receptors is most relevant to understand their biological effects. In that context, is worth noting that Tkv levels are distinctly regulated from those of Wit and Sax through ubiquitination in the context of neurite growth. On the other hand, although the receptor Wit is considered a RSTK type, the functional link with PI3K is a feature usually associated to the RTK type instead. The link of Wit with a kinase has a precedent with LIMK1 that binds to, and is functionally downstream from, Wit in the context of synapse stabilization. Thus, Wit should be considered a wide spectrum receptor in terms of its ligands, co-receptor partners and, consequently, signaling pathways elicited. Actually, the Wit amino acid sequence shows both, Tyr and Ser/Thr motifs justifying its initial classification as a 'dual' type of receptor. In this report this study did not determine if Wit heterodimerizes with other receptors, as canonical RSTKs do, or if it forms homodimers, as canonical RTKs do. However, the lack of synaptogenesis effects by the putative co-receptors, Tkv and Sax, and the phenotypic similarity with the manipulation of the standard RTK signaling effector Cic, leaves open the possibility that Wit could play RTK-like functions, at least in the context of synaptogenesis (Jordan-Alvarez, 2017).

Consistent with the proposal of a dual mechanism for Wit, its activation seems to be a requirement to elicit two independent signaling steps, PI3K and Ras85D, that could reflect RTK and RSTK mechanisms, respectively. Both steps are independent because the mutated form of PI3K unable to bind Ras85D, PI3KΔRBD, is as effective as the normal PI3K to elicit synaptogenesis. PI3K and Ras85D signaling, however, seem to converge on Bsk revealing a novel feature of this crossroad point. The activity level of Bsk is known to be critical in many signaling processes. The peculiarities of Bsk/JNK activity include its coordinated regulation by p38a and Slpr in the context of stress heat response without interference on the developmental context. Another modulator, Puc, was described as a negative feed-back loop in the context of oxidative stress. The Puc mediated loop is operative also for synaptogenesis, while that of p38a/Slpr is relevant for p38a only, as shown here. Further, Ras85D represents an additional regulator in the neural scenario. The triple regulation of Bsk/JNK by Ras85D, Puc and the MAPKs seems to stablish a narrow range of activity thresholds within which normal number of synapses is determined (Jordan-Alvarez, 2017).

The concept of signaling thresholds is also unveiled in this study by the identification of another signaling pathway that opposes synapse formation. The pro- and anti-synaptogenesis pathways have similar constituents, including small GTPases, MAPKs and transcriptional effectors, Mad/Smad, which are canonical for RSTK receptors. The RSTK type II receptor Put, which can mediate diverse signaling pathways according to the co-receptor bound can be discarded in either the pro- or the anti-synaptogenesis pathways. Thus, the main receptor for the anti-synaptogenesis pathway remains to be identified (Jordan-Alvarez, 2017).

Concerning small GTPases, the pro-synaptogenesis pathway uses Ras85D while its counterpart uses the poorly studied Ras64B. The anti-synaptogenesis pathway includes an additional member of this family of enzymes, Rala. This small GTPase plays a role in the exocyst-mediated growth of the muscle membrane specialization that surrounds the synaptic bouton as a consequence of synapse activity. That is, Rala can influence synapse physiology acting from the postsynaptic side. The experimental expression of a constitutively active form of Rala in the neuron does not seem to affect the overall synaptic terminal branching. However, the null ral mutant shows reduced synapse branching and its vertebrate homolog is expressed in the central nervous system. This study found that Rala under-expression in neurons yields an elevated number of synapses. Thus, it is likely that this small GTPase acts as a break to synaptogenesis, hence its inclusion in the antagonistic pathway (Jordan-Alvarez, 2017).

Synaptogenesis and neuritogenesis are distinct processes since each one can be differentially affected by the same mutant (e.g.: Hiw). Both features, however, share some signals (e.g., Wnd, Hep). This signaling overlap is akin to the case of axon specification versus spine formation for constituents of the apico/basal polarity complex Par3-6/aPKC. These and other examples illustrated in this study underscore the need to discriminate between synapses and boutons. This study is focused on the cell autonomous signaling that takes place in the neuron. Non-cell autonomous signals (e.g., originated in the glia or hemolymph circulating) have not been considered. The active role of glia in axon pruning and bouton number has been the subject of other studies. Considering the reported role of Hiw through the midline glia in the remodeling of the giant fiber interneuron it is not unlikely that the glia-to-neuron signaling may share components with the neuron autonomous signaling addressed here (Jordan-Alvarez, 2017).

The summary scheme (see Summary diagram of antagonistic signaling pathways for synaptogenesis and their interactions) describes the scenario where two signaling pathways mutually regulate each other. Epistasis assays are the only experimental approach for in vivo studies of more than one signaling component, albeit this type of assay is only feasible in Drosophila Thus, it is plausible that vertebrate synaptogenesis will be regulated by a similar antagonistic signaling (Jordan-Alvarez, 2017).

The regulatory equilibrium as a mechanism to determine a biological parameter is the most relevant feature in this scenario for several reasons. First, because this type of mechanism can respond very fast to changes in the physiological status of the cell, and, second because it provides remarkable precision to the trait to be regulated, synapse number in this case. Although bi-stable regulatory mechanisms are known in other contexts, the case of synapse number may seem unexpected because the highly dynamic nature of synapse number has been recognized only recently. Consequently, a molecular signaling mechanism endowed with proper precision and time resolution must sustain this dynamic process. The balanced equilibrium uncovered in this study, although most likely still incomplete in terms of its components, offers such a mechanism (Jordan-Alvarez, 2017).

Vonhoff, F. and Keshishian, H. (2017). In Vivo Calcium Signaling during Synaptic Refinement at the Drosophila Neuromuscular Junction. J Neurosci 37(22): 5511-5526. PubMed ID: 28476946

In Vivo Calcium Signaling during Synaptic Refinement at the Drosophila Neuromuscular Junction

Neural activity plays a key role in pruning aberrant synapses in various neural systems, including the mammalian cortex, where low frequency (0.01 Hz) calcium oscillations refine topographic maps. However, the activity-dependent molecular mechanisms remain incompletely understood. Activity-dependent pruning also occurs at embryonic Drosophila neuromuscular junctions (NMJs), where low frequency Ca2+ oscillations are required for synaptic refinement and the response to the muscle-derived chemorepellant Sema2a. This study examined embryonic growth-cone filopodia in vivo to directly observe their exploration and to analyze the episodic Ca2+ oscillations involved in refinement. Motoneuron filopodia repeatedly contacted off-target muscle fibers over several hours during late embryogenesis, with episodic Ca2+ signals present in both motile filopodia as well as in later-stabilized synaptic boutons. The Ca2+ transients matured over several hours into regular low frequency (0.03Hz) oscillations. In vivo imaging of intact embryos of both sexes revealed that the formation of ectopic filopodia is increased in Sema2a heterozygotes. Genetic evidence is provided suggesting a complex presynaptic Ca2+-dependent signaling network underlying refinement that involves the phosphatases Calcineurin and PP1, as well the serine/threonine kinases CaMKII and PKA. Significantly, this network influenced the neuron's response to the muscle's Sema2a chemorepellant, critical for the removal of off-target contacts (Vonhoff, 2017).

As Ca2+ signals were detected at both ectopic and native motoneuron terminals during the critical period for synaptic refinement, the effect of knocking-down Ca2+ channels on synaptic connectivity and development was tested. In separate experiments, RNAi constructs were expressed pan-neurally during embryonic and larval development to target each of the known genes that encode α subunits of voltage-gated Ca2+ channels (VGCCs): the Ca(v)1 channel gene Dmca1D, the Ca(v)2.1 gene cacophony (also known as Dmca1A), and Ca(v)3 gene Dmca1G. Compared with control larvae, animals expressing RNAi-knockdowns of either cacophony or Dmca1G had an elevated frequency of ectopic contacts. By contrast, RNAi-knockdown of Dmca1D did not have a miswiring phenotype, although Dmca1D-RNAi has been shown to reduce Ca2+ currents in Drosophila larval motoneurons. An elevated ectopic frequency was also observed in larvae expressing the RNAi construct for the auxiliary Ca2+ channel β subunit that is required for channel function (Vonhoff, 2017).

Skywalker-TBC1D24 has a lipid-binding pocket mutated in epilepsy and required for synaptic function

Mutations in TBC1D24 cause severe epilepsy and DOORS syndrome, but the molecular mechanisms underlying these pathologies are unresolved. This study solved the crystal structure of the TBC domain of the Drosophila ortholog Skywalker, revealing an unanticipated cationic pocket conserved among TBC1D24 homologs. Cocrystallization and biochemistry showed that this pocket binds phosphoinositides phosphorylated at the 4 and 5 positions. The most prevalent patient mutations affect the phosphoinositide-binding pocket and inhibit lipid binding. Using in vivo photobleaching of Skywalker-GFP mutants, including pathogenic mutants, it was shown that membrane binding via this pocket restricts Skywalker diffusion in presynaptic terminals. Additionally, the pathogenic mutations cause severe neurological defects in flies, including impaired synaptic-vesicle trafficking and seizures, and these defects are reversed by genetically increasing synaptic PI(4,5)P2 concentrations through synaptojanin mutations. Hence, this study has discovered that a TBC domain affected by clinical mutations directly binds phosphoinositides through a cationic pocket and that phosphoinositide binding is critical for presynaptic function (Fischer, 2016).

Drosophila Syncrip modulates the expression of mRNAs encoding key synaptic proteins required for morphology at the neuromuscular junction

Localized mRNA translation is thought to play a key role in synaptic plasticity, but the identity of the transcripts and the molecular mechanism underlying their function are still poorly understood. This study shows that Syncrip, a regulator of localized translation in the Drosophila oocyte and a component of mammalian neuronal mRNA granules, is also expressed in the Drosophila larval neuromuscular junction, where it regulates synaptic growth. RNA-immunoprecipitation followed by high-throughput sequencing and qRT-PCR were used to show that Syncrip associates with a number of mRNAs encoding proteins with key synaptic functions, including msp-300, syd-1 (RhoGAP100F), neurexin-1, futsch, highwire, discs large, and alpha-spectrin. The protein levels of MSP-300, Discs large, and a number of others are significantly affected in syncrip null mutants. Furthermore, syncrip mutants show a reduction in MSP-300 protein levels and defects in muscle nuclear distribution characteristic of msp-300 mutants. These results highlight a number of potential new players in localized translation during synaptic plasticity in the neuromuscular junction. It is proposed that Syncrip acts as a modulator of synaptic plasticity by regulating the translation of these key mRNAs encoding synaptic scaffolding proteins and other important components involved in synaptic growth and function (McDermott, 2014).

Localized translation is a widespread and evolutionarily ancient strategy used to temporally and spatially restrict specific proteins to their site of function and has been extensively studied during early development and in polarized cells in a variety of model systems. It is thought to be of particular importance in the regulation of neuronal development and in the plastic changes at neuronal synapses that underlie memory and learning, allowing rapid local changes in gene expression to occur independently of new transcriptional programs. The Drosophila neuromuscular junction (NMJ) is an excellent model system for studying the general molecular principles of the regulation of synaptic development and plasticity. Genetic or activity-based manipulations of synaptic translation at the NMJ has previously been shown to affect the morphological and electrophysiological plasticity of NMJ synapses. However, neither the mRNA targets nor the molecular mechanism by which such translational regulation occurs are fully understood (McDermott, 2014).

Previous work identified CG17838, the fly homolog of the mammalian RNA binding protein SYNCRIP/hnRNPQ, which was named Syncrip (Syp). Mammalian SYNCRIP/hnRNPQ is a component of neuronal RNA transport granules that contain CamKIIα, Arc, and IP3R1 mRNAs and is thought to regulate translation via an interaction with the noncoding RNA BC200/BC1, itself a translational repressor. Moreover, SYNCRIP/hnRNPQ competes with poly(A) binding proteins to inhibit translation in vitro and regulates dendritic morphology via association with, and localization of, mRNAs encoding components of the Cdc-42/N-WASP/Arp2/3 actin nucleation-promoting complex. Drosophila Syp has a domain structure similar to its mammalian homolog, containing RRM RNA binding domains and nuclear localization signal(s), as well as encoding a number of protein isoforms. It was previously shown that Syp binds specifically to the gurken (grk) mRNA localization signal together with a number of factors previously shown to be required for grk mRNA localization and translational regulation. Furthermore, syp loss-of-function alleles lead to patterning defects indicating that syp is required for grk and oskar (osk) mRNA localization and translational regulation in the Drosophila oocyte (McDermott, 2014).

This study shows that Syp is detected in the Drosophila third instar larval muscle nuclei and also postsynaptically at the NMJ. Syp is required for proper synaptic morphology at the NMJ, as syp loss-of-function mutants show a synaptic overgrowth phenotype, while overexpression of Syp in the muscle can suppress NMJ growth. Syp protein associates with a number of mRNAs encoding proteins with key roles in synaptic growth and function including, msp-300, syd-1, neurexin-1 (nrx-1), futsch, highwire (hiw), discs large 1 (dlg1), and α-spectrin (α-spec). The protein levels of a number of these mRNA targets, including msp-300 and dlg1, are significantly affected in syp null mutants. Furthermore, in addition to regulating MSP-300 protein levels, Syp is required for correct MSP-300 protein localization, and syp null mutants have defects in myonuclear distribution and morphology that resemble those observed in msp-300 mutants. It is proposed that Syp coordinates the protein levels from a number of transcripts with key roles in synaptic growth and is a mediator of synaptic morphology and growth at the Drosophila NMJ (McDermott, 2014).

The results demonstrate that Syp is required for the appropriate branching of the motoneurons and the number of synapses they make at the muscle. These observations are potentially explained by the finding that Syp is also required for the correct level of expression of msp-300, dlg1 and other mRNA targets. Given that it was previously shown that Syp regulates mRNA localization and localized translation in the Drosophila oocyte, and studies by others have shown that mammalian SYNCRIP/hnRNPQ inhibits translation initiation by competitively binding poly(A) sequences, these functions of Syp as occurring at the level of translational regulation of the mRNAs to which Syp binds. The data are also consistent with other work in mammals showing that SYNCRIP/hnRNPQ is a component of neuronal RNA transport granules that can regulate dendritic morphology via the localized expression of mRNAs encoding components of the Cdc-42/N-WASP/Arp2/3 actin nucleation-promoting complex (McDermott, 2014 and references therein).

Translation at the Drosophila NMJ is thought to provide a mechanism for the rapid assembly of synaptic components and synaptic growth during larval development, in response to rapid increases in the surface area of body wall muscles or in response to changes in larval locomotion. The phenotypes observed in this study resemble, and are comparable to, those seen when subsynaptic translation is altered genetically or by increased locomotor activity. In syp null mutants, NMJ synaptic terminals are overgrown, containing more branches and synaptic boutons. Similarly, bouton numbers are increased by knocking down Syp in the muscle using RNAi. In contrast, overexpression of Syp in the muscle has the opposite phenotype, resulting in an inhibition of synaptic growth and branching. Furthermore, expressing RNAi against syp in motoneurons alone does not result in a change in NMJ morphology, indicating that Syp acts postsynaptically in muscle, but not presynaptically at the NMJ to regulate morphology. Interestingly, pan-neuronal syp knockdown or overexpression using Elav-GAL4 also results in NMJ growth defects, revealing that some of the defects observed in the syp null mutant may be attributed to Syp function in neuronal cell types other than the motoneurons, such as glial cells, which are known to influence NMJ morphology. Finally, while Syp is not required in the motoneuron to regulate synapse growth and is not detected in the motoneuron, the possibility cannot be excluded that Syp is present at low levels in the presynapse and regulates processes independent of synapse morphology. A further detailed characterization of the cell types and developmental stages in which Syp is expressed and functions is required to better understand the complex phenotypes that were observe (McDermott, 2014).

RNA binding proteins have emerged as critical regulators of both neuronal morphology and synaptic transmision, suggesting that protein production modulates synapse efficacy. Consistent with this, it has been shown in a parallel study that Syp is required for proper synaptic transmission and vesicle release and regulates the presynapse through expression of retrograde Bone Morphogenesis Protein (BMP) signals in the postsynapse. In this role, Syp may coordinate postsynaptic translation with presynaptic neurotransmitter release. These observations provide a good explanation for how Syp influences the presynapse despite being only detectable in the postsynapse. This study has shown that Syp associates with a large number of mRNAs within third instar larvae, many of which encode proteins with key roles in synaptic growth and function. Syp mRNA targets include msp-300, syd-1, nrx-1, futsch, hiw, dlg1, and α-spec. Syp negatively regulates Syd-1, Hiw, and DLG protein levels in the larval body wall but positively regulates MSP-300 and Nrx-1 protein levels. Dysregulation of these multiple mRNA targets likely accounts for the phenotypes that were observed. Postsynaptically expressed targets with key synaptic roles that could explain the synaptic phenotypes that were observed in syp alleles include MSP-300, α-Spec, and DLG. For example, mutants in dlg1 and mutants where postsynaptic DLG is destabilized or delocalized have NMJ morphology phenotypes similar to those observed upon overexpression of Syp in the muscle. Presynaptically expressed targets include syd-1, nrx-1, and hiw. However, this study has shown that syp knockdown in presynaptic motoneurons does not result in any defects in NMJ morphology. The RIP-Seq experiments were carried out using whole larvae and will, therefore, identify Syp targets in a range of different tissues and cells, the regulation of which may or may not contribute to the phenotype that were observed in syp mutants. It is, therefore, possible that Syp associates with these presynaptic targets in other neuronal cell types such as the DA neurons of the larval peripheral nervous system. It is also possible that Nrx-1 or Hiw are expressed and required postsynaptically in the muscle, but this has not been definitively determined. syp alleles may provide useful tools to examine where key synaptic genes are expressed and how they are regulated (McDermott, 2014).

The identity of localized mRNAs and the mechanism of localized translation at the NMJ are major outstanding questions in the field. To date, studies have shown that GluRIIA mRNA aggregates are distributed throughout the muscle. The Syp targets identified in this study, such as msp-300, hiw, nrx-1, α-spec, and dlg1, are now excellent candidates for localized expression at the NMJ. Ultimately, conclusive demonstration of localized translation will involve the visualization of new protein synthesis of targets during activity-dependent synaptic plasticity. Biochemical experiments will also be required to establish the precise mode of binding of Syp to its downstream mRNA targets, the basis for interaction specificity, and the molecular mechanism by which Syp differentially regulates the protein levels of its mRNA targets at the Drosophila NMJ. Despite the fact that mammalian SYNCRIP is known to associate with poly(A) tails, this study and other published work have revealed that Syp can associate with specific transcripts. How Syp associates with specific mRNAs is unknown, and future studies are needed to uncover whether the interaction of Syp with specific transcripts is dictated by direct binding of the three Syp RRM RNA binding domains or by binding to other specific mRNA binding proteins. It is also possible that specific mRNA stem–loops, similar to the gurken localization signal, are required for Syp to bind to its mRNA targets (McDermott, 2014).

This study shows that msp-300 is the most significant mRNA target of Syp. MSP-300 is the Drosophila ortholog of human Nesprin proteins. These proteins have been genetically implicated in various human myopathies. For example, Nesprin/Syne-1 or Nesprin/Syne-2 is associated with Emery-Dreifuss muscular dystrophy (EDMD) as well as severe cardiomyopathies. Moreover, Syp itself is increasingly linked with factors and targets that can cause human neurodegenerative disorders. Recent work has revealed that SYNCRIP/hnRNPQ and Fragile X mental retardation protein (FMRP) are present in the same mRNP granule, and loss of expression of FMRP or the ability of FMRP to interact with mRNA and polysomes can cause cases of Fragile X syndrome. Separate studies have also shown that SYNCRIP interacts with wild-type survival of motor neuron (SMN) protein but not the truncated or mutant forms found to cause spinal muscular atrophy, and Syp genetically interacts with Smn mutations in vivo. Understanding Syp function in the regulation of such diverse and complex targets may, therefore, provide new avenues for understanding the molecular basis of complex disease phenotypes and potentially lead to future therapeutic approaches (McDermott, 2014).

The EHD protein Past1 controls postsynaptic membrane elaboration and synaptic function

Membranes form elaborate structures that are highly tailored to their specialized cellular functions, yet the mechanisms by which these structures are shaped remain poorly understood. This study shows that the conserved membrane-remodeling C-terminal Eps15 Homology Domain (EHD) protein Past1 is required for the normal assembly of the subsynaptic muscle membrane reticulum (SSR) at the Drosophila melanogaster larval neuromuscular junction (NMJ). past1 mutants exhibit altered NMJ morphology, decreased synaptic transmission, reduced glutamate receptor levels, and a deficit in synaptic homeostasis. The membrane-remodeling proteins Amphiphysin and Syndapin colocalize with Past1 in distinct SSR subdomains, and collapse into Amphiphysin-dependent membrane nodules in the SSR of past1 mutants. These results suggest a mechanism by which the coordinated actions of multiple lipid-binding proteins lead to the elaboration of increasing layers of the SSR, and uncover new roles for an EHD protein at synapses (Koles, 2015).

Dozens of lipid-binding proteins dynamically remodel membranes, generating diverse cell shapes, sculpting organelles, and promoting traffic between subcellular compartments. Although the activities of many of these membrane-remodeling proteins have been studied individually, what is lacking is an understanding of how membrane-remodeling factors work together to generate specialized membranes in vivo (Koles, 2015).

C-terminal Eps15 Homology Domain (EHD)-family proteins encode large membrane-binding ATPases with structural similarity to dynamin and function at a variety of steps of membrane transport (Naslavsky, 2011). These proteins contain an ATPase domain, a helical lipid-binding domain, and a carboxy-terminal EH domain that interacts with Asn-Pro-Phe (NPF)-containing binding partners (Naslavsky, 2011). Although their mechanism of action is not fully understood, it is postulated that C-terminal EHD proteins bind and oligomerize in an ATP-dependent manner on membrane compartments, where they are involved in the trafficking of cargo. The mouse and human genomes each contain four highly similar EHD proteins (EHD1-4), which have both unique and overlapping functions. EHD proteins interact with several members of the Bin/Amphiphysin/Rvs167 (BAR) and Fes/Cip4 homology-BAR (F-BAR) protein families, which themselves can remodel membranes via their crescent-shaped dimeric BAR domains. In mammals, EHD proteins associate with the NPF motifs of the F-BAR proteins Syndapin I and II, and these interactions are critical for recycling of cargo from endosomes to the plasma membrane in cultured cells. In Caenorhabditis elegans, the sole EHD protein Rme-1 colocalizes and functions with the BAR protein Amphiphysin and the F-BAR protein Syndapin, also via their NPF motifs. Further, EHD1 has been suggested to drive the scission of endosomal recycling tubules generated by the membrane-deforming activities of Syndapin 2 and another NPF-containing protein, MICAL-L1. However, the combined membrane-remodeling activities that might arise in vivo from the shared functions of C-terminal EHD and NPF-containing proteins remain unclear (Koles, 2015).

The Drosophila neuromuscular junction (NMJ) is a powerful system in which to study membrane remodeling. On the postsynaptic side of the NMJ, a highly convoluted array of muscle membrane infoldings called the subsynaptic reticulum (SSR) incorporates neurotransmitter receptors, ion channels, and cell adhesion molecules. Assembly of the SSR during larval growth involves activity-dependent targeted exocytosis mediated by the small GTPase Ral and its effector, the exocyst complex, as well as the t-SNARE (target soluble N-ethylmaleimide-sensitive factor attachment protein receptor) receptor gtaxin/Syx18 and scaffolding proteins such as Discs Large (Dlg). Many proteins with predicted membrane-remodeling activities, including Drosophila homologues of Syndapin (Synd) and Amphiphysin (Amph), localize extensively to SSR membranes, making them prime candidates to facilitate SSR elaboration. Amph regulates the postsynaptic turnover of the trans-synaptic cell adhesion molecule FasII, but its role in organizing the SSR is unknown (Koles, 2015).

The Drosophila melanogaster genome encodes a single C-terminal EHD protein called Putative achaete/scute target (Past1). Past1 mutants exhibit defects in endocytic recycling in larval nephrocytes, sterility and aberrant development of the germline, and short lifespan (Olswang-Kutz, 2009), but the functions of Past1 at the NMJ have not been explored. Mammalian EHD1 localizes to the mouse NMJ, but its function there has been difficult to ascertain, perhaps due to redundancy with other EHD proteins. This study takes advantage of the fact that Past1 encodes the only Drosophila C-terminal EHD protein and define its role at the NMJ (Koles, 2015).

Putting together the current observations at the NMJ and in S2 cells with previous results from other groups, a new working model is proposed for how Past1 functions in synaptic membrane elaboration. The first key observation is that Past1 is required for normal elaboration of the SSR and that this function depends on its ATP-binding and thus membrane-remodeling activity. Next, in wild-type SSR, Amph was found to localize to a domain proximal to the bouton, whereas Past1 and Synd localize to a more extended tubulovesicular domain. By contrast, in the absence of Past1, the SSR rearranges into highly organized subdomains, with a core of Synd surrounded by a shell of Amph (likely corresponding to membrane sheets. Amph was found to be required for the formation of the sheets (perhaps by regulating the tight curvature at the tips of these membrane structures) and for consolidation of Synd into nodules. Further, FRAP data indicate that the nodules result in significantly increased membrane flow within the SSR relative to wild-type SSR, suggesting reduced complexity. Finally, S2 cell data indicate that Past1 may activate the membrane-binding/remodeling activity of Synd (Koles, 2015).

These results suggest a novel mechanism for SSR elaboration at the wild-type NMJ involving sequential steps of membrane remodeling. In this model, Amph localizes and generates membrane tubules proximal to the bouton, and Past1 and Synd work together to further elaborate the tubules distal to the bouton. Successive rounds of these events could lead to the growth and expansion of layers of reticulum. In past1 mutants, this process is severely compromised, resulting in nodules containing a core of inactive Synd packed by Amph-dependent membrane sheets (Koles, 2015).

One issue that remains to be resolved is whether direct physical interactions among Past1, Amph, and Synd (within the SSR subdomains to which they colocalize) contribute to Past1-dependent membrane remodeling at the NMJ, as they do in other systems. S2 cell data suggest that Past1 and Synd functionally interact in vivo. However, no Past1-Amph or Past1-Synd complexes were detected using coprecipitation experiments in extracts from Drosophila larvae or S2 cells or with purified proteins, suggesting that either they do not directly interact or their interactions are not preserved in solution under the conditions tested. Genetic experiments at the NMJ using mutations that disrupt putative Past1-Synd and Past1-Amph interactions are unlikely to be informative because synd and amph single mutants exhibit no dramatic phenotype in SSR organization, perhaps due to redundancy with other membrane-remodeling proteins. In fact, in addition to Amph and Synd, it was found that the BAR proteins Cip4 and dRich are localized to nodules in past1 mutants, suggesting that multiple membrane-remodeling proteins are available to function in the Past1-dependent pathway. In the future, it will be important to build into a working model the additional roles of these and other SSR-localized membrane-remodeling proteins, as well as the timing of exocyst-dependent membrane addition (Koles, 2015 and references therein).

The results demonstrate that postsynaptic Past1 plays critical roles in the structure and function of the Drosophila NMJ. Past1 mutant NMJs exhibit aberrant morphology and excess ghost boutons. These ghost boutons are unlikely to be due to defective clearance of excess neuronal membrane, since large amounts of neuronal debris were not observed. They are also unlikely to be related to excess ghost boutons seen in Wingless (Wg) signaling pathway mutants, since past1 mutants do not phenocopy many other aspects of reduced Wg signaling, including increased GluR levels, disrupted presynaptic function, and reduction in bouton number. The likeliest interpretation is that Past1 functions directly in SSR membrane elaboration, consistent with EM observations, and ghost boutons may arise when membrane nodules become too severe to allow SSR assembly around boutons that form toward the end of larval development (Koles, 2015).

Another prominent synaptic phenotype that found in past1 mutants is a strong and specific reduction in localization of GluRIIA to postsynaptic specializations, resulting in decreased mEPSP amplitude. This decrease in GluRIIA could potentially arise by many mechanisms, including altered transcriptional or translational regulation or GluR traffic to or from the synapse. Indeed, expression of a dominant-negative EHD1 suppresses AMPA glutamate receptor recycling in hippocampal dendritic spines. Although there has been little evidence that Drosophila GluRs are regulated by membrane traffic, the data implicating the membrane-remodeling protein Past1 indicate that this may be the case. Finally, unlike the great majority of perturbations that reduce GluRIIA levels, past1 mutants surprisingly fail to compensate for this loss by homeostatic up-regulation of presynaptic release, suggesting that Past1 could be involved in relaying an as-yet-unidentified retrograde signal for synaptic homeostasis. Further work exploring mechanisms of GluRIIA regulation and retrograde signaling will be required to understand the role of Past1 in these events (Koles, 2015).

The present data cannot distinguish whether the function of Past1 in GluR traffic or homeostasis is directly related to its role in SSR elaboration, and it is possible that membrane compartments independent of the SSR are required for these functions and are disrupted in the mutant. The finding that GluRIIA levels are still reduced in amph; past1 double mutants although SSR nodules are suppressed supports the conclusion that GluR localization defects are independent of aberrant SSR morphogenesis. Of note, many mutants with severely defective SSR and/or reduced GluR levels exhibit normal homeostasis (e.g., GluRIIA, which also has reduced SSR, Dlg, Gtaxin, and Pak1, suggesting that homeostasis is a specific function of Past1 rather than a general SSR- or GluR-related defect (Koles, 2015).

Past1 represents the sole EHD homologue in Drosophila, whereas mammals express four EHD proteins with distinct functions. Of importance, many of the roles for EHD proteins at the NMJ and in muscle are likely to be conserved. Past1 localizes to the NMJ, the muscle cortex, and myotendinous junctions. However, unlike EHD1, Past1 does not significantly localize to t-tubules. The activities identified for Past1 at the Drosophila NMJ may inform mechanisms by which EHD2 participates in sarcolemmal repair at the muscle cortex, EHD3 functions in cardiac muscle physiology , and EHD1 and EHD4 act at the mouse NMJ (Mate, 2012). The current findings set the stage for uncovering how neuromuscular synapses are formed and elaborated and illustrate how cooperation between lipid-remodeling proteins can create highly complex membrane structures (Koles, 2015).

Mucin-type core 1 glycans regulate the localization of neuromuscular junctions and establishment of muscle cell architecture in Drosophila

T antigen (Galβ1-3GalNAcα1-Ser/Thr), a core 1 mucin-type O-glycan structure, is synthesized by Drosophila core 1 β1,3-galactosyltrasferase 1 (dC1GalT1) and is expressed in various tissues. dC1GalT1 synthesizes T antigen expressed in hemocytes, lymph glands, and the central nervous system (CNS) and dC1GalT1 mutant larvae display decreased numbers of circulating hemocytes and excessive differentiation of hematopoietic stem cells in lymph glands. dC1GalT1 mutant larvae have also been shown to have morphological defects in the CNS. However, the functions of T antigen in other tissues remain largely unknown. This study found that glycans contributed to the localization of neuromuscular junction (NMJ) boutons. In dC1GalT1 mutant larvae, NMJs were ectopically formed in the cleft between muscles 6 and 7 and connected with these two muscles. dC1GalT1 synthesized T antigen, which was expressed at NMJs. In addition, the function of mucin-type O-glycans in muscle cells was determined. In dC1GalT1 mutant muscles, myofibers and basement membranes were disorganized. Moreover, ultrastructural defects in NMJs and accumulation of large endosome-like structures within both NMJ boutons and muscle cells were observed in dC1GalT1 mutants. Taken together, these results demonstrated that mucin-type O-glycans synthesized by dC1GalT1 were involved in the localization of NMJ boutons, synaptogenesis of NMJs, establishment of muscle cell architecture, and endocytosis (Itoh, 2016).

Tenectin recruits integrin to stabilize bouton architecture and regulate vesicle release at the Drosophila neuromuscular junction

Assembly, maintenance and function of synaptic junctions depend on extracellular matrix (ECM) proteins and their receptors. This study reports that Tenectin (Tnc), a Mucin-type protein with RGD motifs, is an ECM component required for the structural and functional integrity of synaptic specializations at the neuromuscular junction (NMJ) in Drosophila. Using genetics, biochemistry, electrophysiology, histology and electron microscopy, this study shows that Tnc is secreted from motor neurons and striated muscles and accumulates in the synaptic cleft. Tnc selectively recruits alphaPS2/betaPS integrin at synaptic terminals, but only the cis Tnc/integrin complexes appear to be biologically active. These complexes have distinct pre- and post-synaptic functions, mediated at least in part through the local engagement of the spectrin-based membrane skeleton: the presynaptic complexes control neurotransmitter release, while postsynaptic complexes ensure the size and architectural integrity of synaptic boutons. This study reveals an unprecedented role for integrin in the synaptic recruitment of spectrin-based membrane skeleton (Wang, 2018).

The extracellular matrix (ECM) and its receptors impact every aspect of neuronal development, from axon guidance and migration to formation of dendritic spines and neuromuscular junction synaptic junctions and function. The heavily glycosylated ECM proteins provide anchorage and structural support for cells, regulate the availability of extracellular signals, and mediate intercellular communications. Transmembrane ECM receptors include integrins, syndecans and the dystrophin-associated glycoprotein complex. Integrins in particular are differentially expressed and have an extensive repertoire, controlling multiple processes during neural development. In adults, integrins regulate synaptic stability and plasticity. However, integrin roles in synapse development have been obscured by their essential functions throughout development. How integrins are selectively recruited at synaptic junctions and how they engage in specific functions during synapse development and homeostasis remain unclear (Wang, 2018).

One way to confer specificity to ECM/integrin activities is to deploy specialized ECM ligands for the synaptic recruitment and stabilization of selective heterodimeric integrin complexes. For example, at the vertebrate NMJ, three laminins containing the β2 subunit (laminin 221, 421 and 521, that are heterotrimers of α2/4/5, β2 and γ1 subunits) are deposited into the synaptic cleft and basal lamina by skeletal muscle fibers and promote synaptic differentiation. However, only laminin 421 interacts directly with presynaptic integrins containing the α3 subunit and anchors a complex containing the presynaptic Cavα and cytoskeletal and active zone-associated proteins. Studies with peptides containing the RGD sequence, recognized by many integrin subtypes, have implicated integrin in the morphological changes and reassembly after induction of long-term potentiation (LTP). Several integrin subunits (α3, α5, α8, β1 and β2) with distinct roles in the consolidation of LTP have been identified, but the relevant ligands remain unknown (Wang, 2018).

Drosophila neuromuscular junction (NMJ) is a powerful genetic system to examine the synaptic functions of ECM components and their receptors. In flies, a basal membrane surrounds the synaptic terminals only in late embryos; during development, the boutons ‘sink’ into the striated muscle, away from the basal membrane. The synaptic cleft relies on ECM to withstand the mechanical tensions produced by the muscle contractions. The ECM proteins, including laminins, tenascins/teneurins (Ten-a and -m) and Mind-the-gap (Mtg), interact with complexes of five integrin subunits (αPS1, αPS2, αPS3, βPS, and βν). The αPS1, αPS2 and βPS subunits localize to pre- and post-synaptic compartments and have been implicated in NMJ growth. The αPS3 and βν are primarily presynaptic and control activity-dependent plasticity. The only known integrin ligand at the fly NMJ is Laminin A, which is secreted from the muscle and signals through presynaptic αPS3/βν and Focal adhesion kinase 56 (Fak56) to negatively regulate the activity-dependent NMJ growth. Teneurins have RGD motifs, but their receptor specificities remain unknown. Mtg secreted from the motor neurons influences postsynaptic βPS accumulation, but that may be indirectly due to an essential role for Mtg in the organization of the synaptic cleft and the formation of the postsynaptic fields. The large size of these proteins and the complexity of ECM-integrin interactions made it difficult to recognize relevant ligand-receptor units and genetically dissect their roles in synapse development (Wang, 2018).

This study reports the functional analysis of Tenectin (Tnc), an integrin ligand secreted from both motor neurons and muscles; Tnc accumulates at synaptic terminals and functions in cis to differentially engage presynaptic and postsynaptic integrin. tnc, which encodes a developmentally regulated RGD-containing integrin ligand, in a screen for ECM candidates that interact genetically with neto, a gene essential for NMJ assembly and function. This study found that Tnc selectively recruits the αPS2/βPS integrin at synaptic locations, without affecting integrin anchoring at muscle attachment sites. Dissection of Tnc functions revealed pre- and postsynaptic biologically active cis Tnc/integrin complexes that function to regulate neurotransmitter release and postsynaptic architecture. Finally, the remarkable features of this selective integrin ligand were explored to uncover a novel synaptic function for integrin, in engaging the spectrin-based membrane skeleton (Wang, 2018).

The ECM proteins and their receptors have been implicated in NMJ development, but their specific roles have been difficult to assess because of their early development functions and the complexity of membrane interactions they engage. This study has shown that Tnc is a selective integrin ligand that enables distinct pre- and post-synaptic integrin activities mediated at least in part through the local engagement of the spectrin-based cortical skeleton. First, Tnc depletion altered NMJ development and function and correlated with selective disruption of αPS2/βPS integrin and spectrin accumulation at synaptic terminals. Second, manipulation of Tnc and integrin in neurons demonstrated that presynaptic Tnc/integrin modulate neurotransmitter release; spectrin mutations showed similar disruptions of the presynaptic neurotransmitter release. Third, postsynaptic Tnc influenced the development of postsynaptic structures (bouton size and SSR complexity), similar to integrin and spectrin. Fourth, presynaptic Tnc/integrin limited the accumulation and function of postsynaptic Tnc/integrin complexes. Fifth, secreted Tnc bound integrin complexes at cell membranes, but only the cis complexes were biologically active; trans Tnc/integrin complexes can form but cannot function at synaptic terminals and instead exhibited dominant-negative activities. These observations support the model that Tnc is a tightly regulated component of the synaptic ECM that functions in cis to recruit αPS2/βPS integrin and the spectrin-based membrane skeleton at synaptic terminals and together modulate the NMJ development and function (Wang, 2018).

Tnc appears to fulfill unique, complementary functions with the other known synaptic ECM proteins at the Drosophila NMJ. Unlike Mtg, which organizes the active zone matrix and the postsynaptic domains, Tnc does not influence the recruitment of iGluRs and other PSD components. LanA ensures a proper adhesion between the motor neuron terminal and muscle and also acts retrogradely to suppress the crawling activity-dependent NMJ growth. The latter function requires the presynaptic βν integrin subunit and phosphorylation of Fak56 via a pathway that appears to be completely independent of Tnc. Several more classes of trans-synaptic adhesion molecules have been implicated in either the formation of normal size synapses, for example Neurexin/Neuroligin, or in bridging the pre- and post-synaptic microtubule-based cytoskeleton, such as Teneurins. However, genetic manipulation of Tnc did not perturb synapse assembly or microtubule organization, indicating that Tnc functions independently from these adhesion molecules. Instead, Tnc appears to promote expression and stabilization of αPS2/βPS complexes, which in turn engage the spectrin-based membrane skeleton (SBMS) at synaptic terminals. On the presynaptic side these complexes modulate neurotransmitter release. On the postsynaptic side, the Tnc-mediated integrin and spectrin recruitment modulates bouton morphology. A similar role for integrin and spectrin in maintaining tissue architecture has been reported during oogenesis; egg chambers with follicle cells mutant for either integrin or spectrin produce rounder eggs (Wang, 2018).

The data are consistent with a local function for the Tnc/βPS-recruited SBMS at synaptic terminals; this is distinct from the role of spectrin in endomembrane trafficking and synapse organization. Embryos mutant for spectrins have reduced neurotransmitter release, a phenotype shared by larvae lacking presynaptic Tnc or βPS integrin. However, Tnc perturbations did not induce synapse retraction and axonal transport defects as seen in larvae with paneuronal α- or β- spectrin knockdown. Spectrins interact with ankyrins and form a lattice-like structure lining neuronal membranes in axonal and interbouton regions. This study found that Tnc manipulations did not affect the distribution of Ankyrin two isoforms (Ank2-L and Ank2-XL) in axons or at the NMJ; also loss of ankyrins generally induces boutons swelling, whereas Tnc perturbations shrink the boutons and erode bouton-interbouton boundaries. Like tnc, loss of spectrins in the striated muscle shows severe defects in SSR structure. Lack of spectrins also disrupts synapse assembly and the recruitment of glutamate receptors. In contrast, manipulations of tnc had no effect on PSD size and composition. Instead, tnc perturbations in the muscle led to boutons with altered size and individualization and resembled the morphological defects seen in spectrin tetramerization mutants, spectrin^R22S. spectrin^R22S mutants have more subtle defects than tnc, probably because spectrin is properly recruited at NMJs but fails to crosslink and form a cortical network. Spectrins are also recruited to synaptic locations by Teneurins, a pair of transmembrane molecule that form trans-synaptic bridges and influence NMJ organization and function. Drosophila Ten-m has an RGD motif; this study found that βPS levels were decreased by 35% at ten-mMB mutant NMJs. Thus, Ten-m may also contribute to the recruitment of integrin and SBMS at the NMJ, a function likely obscured by the predominant role both play in cytoskeleton organization (Wang, 2018).

Previous work has shown that α-Spectrin is severely disrupted at NMJs with suboptimal levels of Neto, such as neto¹⁰⁹- a hypomorph with 50% lethality. These mutants also had sparse SSR, reduced neurotransmitter release, as well as reduced levels of synaptic βPS. In this genetic background, lowering the dose of tnc should further decrease the capacity to accumulate integrin and spectrin at synaptic terminals and enhance the lethality. This may explain the increased synthetic lethality detected in the genetic screen (Wang, 2018).

In flies or vertebrates, the ECM proteins that comprise the synaptic cleft at the NMJ are not fully present when motor neurons first arrive at target muscles. Shortly thereafter, the neurons, muscles and glia begin to synthesize, secrete and deposit ECM proteins. At the vertebrate NMJ, deposition of the ECM proteins forms a synaptic basal lamina that surrounds each skeletal myofiber and creates a ~ 50 nm synaptic cleft. In flies, basal membrane contacts the motor terminal in late embryos, but is some distance away from the synaptic boutons during larval stages. Nonetheless, the NMJ must withstand the mechanical tensions produced by muscle contractions. The current data suggest that Tnc is an ideal candidate to perform the space filling, pressure inducing functions required to engage integrin and establish a dynamic ECM-cell membrane network at synaptic terminals. First, Tnc is a large mucin with extended PTS domains that become highly O-glycosylated, bind water and form gel-like complexes that can extend and induce effects similar to hydrostatic pressure. In fact, Tnc fills the lumen of several epithelial tubes and forms a dense matrix that acts in a dose-dependent manner to drive diameter growth. Second, the RGD and RGD-like motifs of Tnc have been directly implicated in αPS2/βPS-dependent spreading of S2 cells (Fraichard, 2010). Third, secreted Tnc appears to act close to the source, presumably because of its size and multiple interactions. In addition to the RGD motifs, Tnc also contains five complete and one partial vWFC domains, that mediate protein interactions and oligomerization in several ECM proteins including mucins, collagens, and thrombospondins. The vWFC domains are also found in growth factor binding proteins and signaling modulators such as Crossveinless-2 and Kielin/Chordin suggesting that Tnc could also influence the availability of extracellular signals. Importantly, Tnc expression is hormonally regulated during development by ecdysone (Fraichard, 2010). Tnc does not influence integrin responsiveness to axon guidance cues during late embryogenesis; unlike integrins, the tnc mutant embryos have normal longitudinal axon tracks. Instead, Tnc synthesis and secretion coincide with the NMJ expansion and formation of new bouton structures during larval stages. Recent studies have reported several mucin-type O-glycosyltransferases that modulate integrin signaling and intercellular adhesion in neuronal and non-neuronal tissues, including the Drosophila NMJ. Tnc is likely a substrate for these enzymes that may further regulate Tnc activities (Wang, 2018).

In flies as in vertebrates, integrins play essential roles in almost all aspects of synaptic development. Early in development, integrins have been implicated in axonal outgrowth, pathfinding and growth cone target selection. In adult flies, loss of αPS3 integrin activity is associated with the impairment of short-term olfactory memory. In vertebrates, integrin mediates structural changes involving actin polymerization and spine enlargement to accommodate new AMPAR during LTP, and ‘lock in’ these morphological changes conferring longevity for LTP. Thus far, integrin functions at synapses have been derived from compound phenotypes elicited by use of integrin mutants, RGD peptides, or enzymes that modify multiple ECM molecules. Such studies have been complicated by multiple targets for modifying enzymes and RGD peptides and by the essential functions of integrin in cell adhesion and tissue development (Wang, 2018).

In contrast, manipulations of Tnc, which affects the selective recruitment of αPS2/βPS integrin at synaptic terminals, have uncovered novel functions for integrin and clarified previous proposals. This study demonstrated that βPS integrin is dispensable for the recruitment of iGluRs at synaptic sites and for PSD maintenance. An unprecedented role was reveaked for integrin in connecting the ECM of the synaptic cleft with spectrin, in particular to the spectrin-based membrane skeleton. These Tnc/integrin/spectrin complexes are crucial for the integrity and function of synaptic structures. These studies uncover the ECM component Tnc as a novel modulator for NMJ development and function; these studies also illustrate how manipulation of a selective integrin ligand could be utilized to reveal novel integrin functions and parse the many roles of integrins at synaptic junctions (Wang, 2018).

The HSPG glypican regulates experience-dependent synaptic and behavioral plasticity by modulating the non-canonical BMP pathway

Under food deprivation conditions, Drosophila larvae exhibit increases in locomotor speed and synaptic bouton numbers at neuromuscular junctions (NMJs). Octopamine, the invertebrate counterpart of noradrenaline, plays critical roles in this process; however, the underlying mechanisms remain unclear. This study shows that a glypican (Dlp) negatively regulates type I synaptic bouton formation, postsynaptic expression of GluRIIA, and larval locomotor speed. Starvation-induced octopaminergic signaling decreases Dlp expression, leading to increases in synapse formation and locomotion. Dlp is expressed by postsynaptic muscle cells and suppresses the non-canonical BMP pathway, which is composed of the presynaptic BMP receptor Wit and postsynaptic GluRIIA-containing ionotropic glutamate receptor. During starvation, decreases in Dlp increase non-canonical BMP signaling, leading to increases in GluRIIA expression, type I bouton number, and locomotor speed. These results demonstrate that octopamine controls starvation-induced neural plasticity by regulating Dlp and provides insights into how proteoglycans can influence behavioral and synaptic plasticity (Kamimura, 2019).

Food deprivation induces behavioral and synaptic plasticity in Drosophila larvae through octopamine signaling. This study demonstrates that octopamine signaling dynamically regulates Dlp expression at the NMJ during starvation. Dlp was found to inhibits postsynaptic GluRIIA expression, and starvation and octopamine function were found to reduce postsynaptic Dlp expression. The inhibition of Dlp results in increased GluRIIA expression, which this study proposes is maintained for several hours during starvation by the activation of the non-canonical BMP pathway. Increased GluRIIA expression causes increased type I bouton growth at the NMJ and increased starvation-dependent larval locomotion. It has previously been shown that during the non-starvation or normal development of NMJs, postsynaptic GluRIIA expression and type I bouton growth are regulated by distinct biochemical pathways (Sulkowski, 2016). GluRIIA/GluRIIB postsynaptic composition is regulated by non-canonical BMP signaling, while type I bouton growth is regulated independently by canonical BMP signaling. However, this study finds that during starvation-dependent growth, GluRIIA expression and type I bouton growth become interrelated. Starvation increases GluRIIA expression in a Dlp-dependent manner, and type I bouton growth requires GluRIIA expression. It is not certain why this change in regulation occurs, but it is noted that independent regulatory mechanisms allow more flexibility for NMJ formation during normal development, while starvation-dependent growth may simply require a quick and transient increase in synaptic activity that may not need to be as flexible. This study found that Dlp regulates starvation-dependent changes at least in part through the regulation of non-canonical BMP signaling. However, non-canonical BMP signaling is likely only one component of how Dlp regulates NMJ plasticity and larval locomotion. This study finds that GluRIIA amounts increase upon the suppression of Dlp even in wit null mutants, indicating that BMP signaling functions to maintain increases in GluRIIA, but not necessarily to induce it. Thus, Dlp must inhibit GluRIIA expression through a pathway that is independent of non-canonical BMP signaling. Furthermore, although starvation-dependent increases in type I boutons depend on Wit and GluRIIA, there is also a component to this increase that is Wit and GluRIIA independent. This suggests that Dlp may also regulate type I boutons through a GluRIIA-independent mechanism (Kamimura, 2019).

Octopamine is required to maintain the basal expression levels of postsynaptic Dlp, and thus it positively regulates Dlp expression under normal rearing conditions. However, after food deprivation, octopamine signaling turns into a negative regulator of Dlp expression during the early phase of starvation. In the late phase of starvation, octopamine signaling reverts back to being a positive regulator. Koon (2012) and Koon (2011) revealed that type II bouton formation was not only positively regulated by Octβ2R autoreceptors but also negatively regulated by Octβ1R autoreceptors. To explain the acute growth of type II bouton growth after starvation, they postulated that Octβ1R exhibited a higher affinity for octopamine than did Octβ2R. Under normal rearing conditions, high-affinity Octβ1R may be dominantly activated by low concentrations of octopamine. Under this condition, low-affinity Octβ2R may be weakly activated. Thus, basal levels of type II bouton growth are determined by the balanced activities of these antagonistic octopamine receptors. After food deprivation, high concentrations of octopamine released from type II endings may strongly activate positive Octβ2R receptors, leading to the acute growth of type II boutons. A similar mechanism may be involved in the regulation of Dlp expression. One possibility is that high-affinity octopamine receptors (HOctRs) and low-affinity octopamine receptors (LOctRs) at type I synaptic regions positively and negatively, respectively, regulate the postsynaptic localization of Dlp. If this is the case, then the basal postsynaptic levels of Dlp may be affected by the dominant activation of positive HOctRs. In the early phase of starvation, during which high levels of octopamine are present, postsynaptic Dlp levels may be decreased by the activation of negative LOctRs. In the late phase of starvation, octopamine levels may be decreased by the exhaustion of octopaminergic neurons, leading to the dominant activation of HOctRs again. This may result in increases in postsynaptic Dlp expression. Thus, the flexible roles of octopamine signaling may be explained by differences in the octopamine receptors mainly activated in each phase. Future studies are needed to identify the octopamine receptors that regulate Dlp expression (Kamimura, 2019).

It remains unclear how octopamine signaling at type I synaptic regions regulates postsynaptic Dlp levels. Broadie and collaborators revealed that ECM metalloproteinases (Mmp1 and Mmp2) and the tissue inhibitor of metalloproteinases (Timp) play critical roles in synapse formation and activity-dependent plasticity in the Drosophila NMJ (Dear, 2016; Dear, 2017; Shilts, 2017). Mmp2 from the presynaptic and postsynaptic compartments has been shown to negatively regulate synaptic Dlp levels at the NMJ, possibly by the proteolytic cleavage of Dlp core proteins (Wang, 2014). However, Mmp1 is known to associate with Dlp via heparan sulfate (HS) chains and positively regulate Dlp expression levels at type I endings (Dear, 2016). Octopamine signaling after food deprivation may activate the proteolytic activity of Mmp2 at type I synaptic regions, leading to a decrease in postsynaptic Dlp levels. Mmp 1 may be activated in the later phase of starvation, leading to an increase in postsynaptic Dlp levels. A previous study revealed that Timp regulated the activities of these MMPs at the NMJ to restrict trans-synaptic Gbb signaling (Shilts, 2017). Thus, complex cooperation among Mmps, Timp, and Dlp may be involved in the experience-dependent plasticity of the NMJ. The local translation of Dlp may also be involved, particularly in the later phase (Kamimura, 2019).

This study has revealed that Dlp suppressed the GluRIIA-mediated non-canonical BMP signaling pathway, which forms a positive feedback loop between presynaptic pMad accumulation and postsynaptic type A iGluR expression (Sulkowski, 2016). Thus, decreases in Dlp expression after food deprivation may acutely promote this signaling pathway, leading to rapid increases in type A iGluR and pMad accumulation at the NMJ. However, Dlp expression was increased during the late phase of starvation, which may have suppressed this BMP pathway. A positive feedback loop may lead to a runaway condition with no brakes. In this sense, Dlp appears to critically regulate the levels of this GluRIIA-mediated BMP signaling as a brake during starvation. How, then, does Dlp regulate non-canonical BMP signaling? Since Dlp exclusively localizes at postsynaptic regions, it may directly associate with GluRIIA and destabilize type A iGluRs at synapses. After food deprivation, the postsynaptic expression levels of Dlp decrease, and thus type A iGluRs may be stabilized, leading to the promotion of presynaptic local Wit signaling and pMad accumulation. It is also conceivable that Dlp inhibits the trans-synaptic association between presynaptic BMP receptor complexes and postsynaptic type A iGluRs proposed by Sulkowski (2016). Many of the functions of HSPGs are mediated by HS moieties, which bind with various signaling molecules in a sulfation pattern-dependent manner. The development and function of the NMJ are known to be regulated in a complex manner by HS co-polymerase (encoded by ttv), N-deacetylase/N-sulfotransferase (encoded by sfl), HS 6-O-sulfotransferase, and extracellular sulfatase (Sulf1) (Kamimura, 2019).

These findings suggest that various signaling pathways at the NMJ are intricately regulated by structural changes in HS chains. The structure of HS chains on Dlp may be altered under specific conditions such as starvation, and Dlp may selectively regulate a particular signaling pathway among multiple target pathways in an HS sulfation pattern-dependent manner. LAR is known to associate with Dlp and Sdc through HS chains at the developing NMJ, in which Dlp showed higher affinity for LAR than Sdc, possibly because of differences in HS structures. Presynaptic LAR is activated by postsynaptic Sdc, leading to type I bouton growth, whereas Dlp competitively inhibits this signaling. Therefore, the decrease observed in Dlp expression after food deprivation may disinhibit LAR signaling and promote type I bouton growth. In addition, Dlp has been shown to regulate Wg signaling through the HS moieties during NMJ development. Wg is secreted from presynaptic terminal and glia and participates in synaptic plasticity. Therefore, it is also possible that Wg and Dlp regulate the starvation-induced synaptic and behavioral plasticities. Furthermore, a recent study revealed that neurexin synaptic organizing proteins are expressed as HSPGs. HS modifications in presynaptic neurexins are critical for high-affinity interactions with the postsynaptic ligands neuroligins and the leucine-rich repeat transmembrane neuronal proteins (LRRTMs), and are essential for synapse development at the mouse hippocampus. Drosophila Neurexin (Dnrx) is also an HSPG and positively regulates larval locomotion speed and type I bouton growth at NMJs in an HS-dependent manner. Furthermore, it has been suggested that Dnrx, Neuroligin (Dnlg1), and Wit form signaling complexes at the NMJ. Therefore, presynaptic Dnrx and postsynaptic Dlp may competitively regulate type I bouton formation and larval locomotor activity as antagonistic HSPGs, in which the HS chains of Dlp may inhibit the Dnrx-Dnlg1 interaction in a sulfation pattern-dependent manner. Further studies are needed to examine whether these pathways and HS modifications regulate starvation-induced synaptic and behavioral plasticities (Kamimura, 2019).

Only three type II octopaminergic neurons bilaterally innervate most of the body wall muscles in each abdominal segment, suggesting that type II octopaminergic neurons globally regulate the properties of type I terminals, coordinating the activities of the body wall musculature. This is similar to the mammalian brain noradrenergic system. Noradrenaline-producing neurons mainly cluster in a small nucleus called the locus coeruleus, which is located in the pons with global projections throughout the brain. The locus coeruleus noradrenergic system is activated by many stressors. For example, noradrenaline is released in many brain regions during emotional arousal and enhances long-term memory, an extreme example of which is post-traumatic stress disorder (PTSD). It has been shown that that noradrenaline facilitated the synaptic delivery of GluR1-containing AMPA receptors in the hippocampus, which lowered the threshold for long-term potentiation (LTP), thereby enhancing learning and memory. This is similar to the starvation-induced synaptic localization of type A iGluRs in the Drosophila NMJ, which is regulated by Dlp in an octopamine-dependent manner. Thus, further studies are needed to investigate whether the emotional enhancement of memory is regulated by glypican in the hippocampus. A previous study reported that astrocyte-secreted glypican 4 induced the release of the AMPA receptor clustering factor neuronal pentraxin 1 from presynaptic terminals through the signaling of RPTPδ, a LAR family receptor protein tyrosine phosphatase. This induced the formation of active synapses by recruiting AMPA receptors to postsynaptic sites. In addition, noradrenaline has been shown to activate astrocyte networks in the cortex and enhance their responsiveness to local changes in neuronal activity. Therefore, noradrenaline may stimulate the secretion of glypican 4 from astrocytes in a neuronal activity-dependent manner, thereby enhancing active synapse formation. In this case, the direction of glypican 4 activities appears to be opposite that of Dlp. However, glypicans often display opposing activities in a context-dependent manner. Thus, the interplay among Dlp, type A iGluR, LAR, and octopamine at the Drosophila NMJ may become a simple model to study the molecular mechanisms underlying emotion-enhanced memory formation and thus PTSD (Kamimura, 2019).

In the adult mammalian brain, the synapses of a subset of neurons are enwrapped with a specialized chondroitin sulfate proteoglycan-rich ECM called the perineuronal net (PNN). PNNs are mainly composed of tenascin-R, hyaluronan, and lectican family proteoglycans such as aggrecan, versican, and neurocan. The ECM enwrapping type I synaptic boutons at the Drosophila larval NMJ is an HSPG-based ECM containing Dlp, syndecan, perlecan, laminin, and the secreted lectin Mind-the-Gap. The orthologs of hyaluronan synthase and lectican family proteoglycans are not found in Drosophila, and therefore an HSPG-based ECM may be used at the synapses of Drosophila. In spite of their different compositions, both synaptic ECMs play critical roles in experience-induced neural plasticity, as shown by the present study and previous studies on mammalian ocular dominance plasticity. This study has revealed that Dlp regulated the localization of type A iGluRs during starvation. PNNs also contribute to the synaptic localization of AMPA receptors. Detailed comparative studies are needed to reveal functional and structural similarities between the PNNs and the ECM of the Drosophila NMJ (Kamimura, 2019).

Abnormalities in the expression of glypicans have been linked to various neurological disorders, such as autism spectrum disorders (ASDs), schizophrenia, attention-deficit/hyperactivity disorder (ADHD), and neuroticism. Dysfunctions in the noradrenergic system have also been reported in ASDs, schizophrenia, and ADHD. Thus, studies on the octopamine-mediated control of Dlp expression may contribute to a deeper understanding of the molecular mechanisms responsible for the pathogeneses of these disorders (Kamimura, 2019).

The matricellular protein Drosophila CCN is required for synaptic transmission and female fertility

Within the extracellular matrix, matricellular proteins (MCPs) are dynamically expressed non-structural proteins that interact with cell surface receptors, growth factors, and proteases, as well as with structural matrix proteins. The CCN (Cellular Communication Network Factors) family of MCPs serve regulatory roles to regulate cell function and are defined by their conserved multi-modular organization. This study characterize the expression and neuronal requirement for the Drosophila CCN family member. Drosophila CCN (dCCN) is expressed in the nervous system throughout development including in subsets of monoamine-expressing neurons. dCCN-expressing abdominal ganglion neurons innervate the ovaries and uterus and the loss of dCCN results in reduced female fertility. In addition, dCCN accumulates at the synaptic cleft and is required for neurotransmission at the larval neuromuscular junction. Analyzing the function of the single Drosophila CCN family member will enhance the ability to understand how the microenvironment impacts neurotransmitter release in distinct cellular contexts and in response to activity (Garrett, 2023).

Shank modulates postsynaptic wnt signaling to regulate synaptic development

Prosap/Shank scaffolding proteins regulate the formation, organization, and plasticity of excitatory synapses. Mutations in SHANK family genes are implicated in autism spectrum disorder and other neuropsychiatric conditions. However, the molecular mechanisms underlying Shank function are not fully understood, and no study to date has examined the consequences of complete loss of all Shank proteins in vivo. This study characterized the single Drosophila Prosap/Shank family homolog. Shank is enriched at the postsynaptic membrane of glutamatergic neuromuscular junctions and controls multiple parameters of synapse biology in a dose-dependent manner. Both loss and overexpression of Shank result in defects in synaptic bouton number and maturation. It was found that Shank regulates a noncanonical Wnt signaling pathway in the postsynaptic cell by modulating the internalization of the Wnt receptor Fz2. This study identifies Shank as a key component of synaptic Wnt signaling, defining a novel mechanism for how Shank contributes to synapse maturation during neuronal development (Harris, 2016).

The postsynaptic density (PSD) of excitatory synapses contains a complex and dynamic arrangement of proteins, allowing the cell to respond to neurotransmitter and participate in bidirectional signaling to regulate synaptic function. Prosap/Shank family proteins are multidomain proteins that form an organizational scaffold at the PSD. Human genetic studies have implicated SHANK family genes as causative for autism spectrum disorder (ASD) (Uchino and Waga, 2013; Guilmatre, 2014), with haploinsufficiency of SHANK3 considered one of the most prevalent causes (Betancur and Buxbaum, 2013). Investigations of Shank in animal models have identified several functions for the protein at synapses, including regulation of glutamate receptor trafficking, the actin cytoskeleton, and synapse formation, transmission, and plasticity (Grabrucker, 2011; Jiang and Ehlers, 2013). However, phenotypes associated with loss of Shank are variable, and it has been challenging to fully remove Shank protein function in vivo as a result of redundancy between three Shank family genes and the existence of multiple isoforms of each Shank. There is a single homolog of Shank in Drosophila (Liebl and Featherstone, 2008), presenting the opportunity to characterize the function of Shank at synapses in vivo in null mutant animals (Harris, 2016).

Wnt pathways play important roles in synaptic development, function, and plasticity. Like Shank and several other synaptic genes, deletions and duplications of canonical Wnt signaling components have been identified in individuals with ASD. A postsynaptic noncanonical Wnt pathway has been characterized at the Drosophila glutamatergic neuromuscular junction (NMJ), linking release of Wnt by the presynaptic neuron to plastic responses in the postsynaptic cell. In this Frizzled-2 (Fz2) nuclear import (FNI) pathway, Wnt1/Wg is secreted by the neuron and binds its receptor Fz2 in the postsynaptic membrane. Surface Fz2 is then internalized and cleaved, and a C-terminal fragment of Fz2 (Fz2-C) is imported into the nucleus in which it interacts with ribonucleoprotein particles containing synaptic transcripts. Mutations in this pathway result in defects of synaptic development at the NMJ (Harris, 2016 and references therein).

In this study, a null allele of Drosophila Shank was created, allowing investigation of the consequences of removing all Shank protein in vivo. Loss of Shank is shown to impair synaptic bouton number and maturity and results in defects in the organization of the subsynaptic reticulum (SSR), a complex system of infoldings of the postsynaptic membrane at the NMJ. It was also demonstrated that overexpression of Shank has morphological consequences similar to loss of Shank and that Shank dosage is critical to synaptic development. Finally, the results indicate that Shank regulates the internalization of Fz2 to affect the FNI signaling pathway, revealing a novel connection between the scaffolding protein Shank and synaptic Wnt signaling (Harris, 2016).

By generating Drosophila mutants completely lacking any Shank protein, this study identified a novel function of this synaptic scaffolding protein in synapse development. Aberrant expression of Shank results in defects affecting synapse number, maturity, and ultrastructure, and a subset of these defects is attributable to a downregulation of a noncanonical Wnt signaling pathway in the postsynaptic cell (Harris, 2016).

The defects observed in Shank mutants are mostly consistent with defects described from in vivo and in vitro rodent models of Shank. Synaptic phenotypes reported from Shank mutants vary, likely reflecting incomplete knockdown of Shank splice variants, and heterogeneity in the requirement for Shank between the different brain regions and developmental stages analyzed (for review, see Jiang and Ehlers, 2013). Nevertheless, taken collectively, analyses of Shank1-Shank3 mutant mice indicate that Shank genes regulate multiple parameters of the structure and function of glutamatergic synapses, including the morphology of dendritic spines and the organization of proteins in the PSD (Harris, 2016 and references therein).

By removing all Shank protein in Drosophila, this study identified essential functions for Shank at a model glutamatergic synapse. Shank mutants exhibit prominent abnormalities in synaptic structure, including a decrease in the total number of synaptic boutons, which results in an overall decrease in the number of AZs. In addition, a subset of synaptic boutons fails to assemble a postsynaptic apparatus. Finally, even in mature boutons, the SSR has fewer membranous folds and makes less frequent contact with the presynaptic membrane, indicating a defect in postsynaptic development. The SSR houses and concentrates important synaptic components near the synaptic cleft, including scaffolding proteins, adhesion molecules, and glutamate receptors. Thus, defects in SSR development can affect the assembly and regulation of synaptic signaling platforms. These findings indicate that Shank is a key regulator of synaptic growth and maturation (Harris, 2016).

The findings also indicate that gene dosage of Shank is critical for normal synapse development at Drosophila glutamatergic NMJs. The morphological phenotypes that were observed scale with the level of Shank expression, with mild phenotypes seen with both 50% loss and moderate overexpression of Shank, and severe phenotypes seen with both full loss and strong overexpression of Shank. The observation of synapse loss in heterozygotes of the Shank null allele is significant, because haploinsufficiency of SHANK3 is well established as a monogenic cause of ASD (Harris, 2016).

Consistent with the observation that excess Shank is detrimental, duplications of the SHANK3 genomic region (22q13) are known to cause a spectrum of neuropsychiatric disorders. Large duplications spanning SHANK3 and multiple neighboring genes have been reported in individuals with attention deficit-hyperactivity disorder (ADHD), schizophrenia, and ASD. Smaller duplications, spanning SHANK3 and only one or two adjacent genes, have been reported in individuals with ADHD, epilepsy, and bipolar disorder. Furthermore, duplication of the Shank3 locus in mice results in manic-like behavior, seizures, and defects in neuronal excitatory/inhibitory balance. Thus, the requirement for proper Shank dosage for normal synaptic function may be a conserved feature (Harris, 2016).

One unexpected finding from this study was the identification of a previously unappreciated aspect of Shank as a regulator of Wnt signaling. Shank regulates the internalization of the transmembrane Fz2 receptor, thus affecting transduction of Wnt signaling from the plasma membrane to the nucleus. Downregulation of this pathway is implicated in impaired postsynaptic organization, including supernumerary GBs and SSR defects. The physical proximity of Shank and Fz2 at the postsynaptic membrane suggests that Shank directly or indirectly modulates the internalization of Fz2. Shank is a scaffolding protein with many binding partners that could contribute to such an interaction. One intriguing possibility is the PDZ-containing protein Grip. Shank2 and Shank3 have been reported to bind Grip1. Furthermore, Drosophila Grip transports Fz2 to the nucleus on microtubules to facilitate the FNI pathway (Ataman, 2006). Thus, an interaction between Shank, Fz2, and Grip to regulate synaptic signaling is an attractive model (Harris, 2016).

Although loss of Shank is associated with impaired internalization of the Fz2 receptor, how excess Shank leads to FNI impairment remains an open question. One possibility is that an increase in the concentration of the Shank scaffold at the synapse physically impedes the transport of Fz2 or other components of the pathway or saturates binding partners that are essential for Fz2 trafficking. Both overexpression and loss of function of Shank ultimately lead to a failure to accumulate the cleaved Fz2 C terminus within the nucleus, in which it is required to interact with RNA binding proteins that facilitate transport of synaptic transcripts to postsynaptic compartments. Although Shank and Wnt both play important synaptic roles, this study is the first demonstration of a functional interaction between Shank and Wnt signaling at the synapse (Harris, 2016).

Intriguingly, no obvious defects were found in glutamate receptor levels or distribution in the absence of Shank. This was surprising given the role for Shank in regulating the FNI pathway, and downregulation of FNI was shown previously to lead to increased GluR field size. Several studies have reported changes in the levels of AMPA or NMDA receptor subunits in Shank mutant mice, although others have also observed no changes. Levels of metabotropic glutamate receptors are also affected in some Shank mutant models. Moreover, transfected Shank3 can recruit functional glutamate receptors in cultured cerebellar neurons. It is possible that Drosophila Shank mutants have defects in GluRs that are too subtle to detect with current methodology. Another possibility is that Shank is involved in signaling mechanisms that are secondary to FNI and that lead to compensatory changes in GluRs at individual synapses. Indeed, the results are consistent with Shank having additional functions at the synapse in addition to its role in FNI, particularly affecting synaptic bouton number. In conclusion, this study has fpind that the sole Drosophila Shank homolog functions to regulate synaptic development in a dose-dependent manner, providing a new model system to further investigate how loss of this scaffolding protein may underlie neurodevelopmental disease (Harris, 2016).

The influence of postsynaptic structure on missing quanta at the Drosophila neuromuscular junction

Synaptic transmission requires both pre- and post-synaptic elements for neural communication. The postsynaptic structure contributes to the ability of synaptic currents to induce voltage changes in postsynaptic cells. At the Drosophila neuromuscular junction (NMJ), the postsynaptic structure, known as the subsynaptic reticulum (SSR), consists of elaborate membrane folds that link the synaptic contacts to the muscle, but its role in synaptic physiology is poorly understood. This study investigated the role of the SSR with simultaneous intra- and extra-cellular recordings that allow identification of the origin of spontaneously occurring synaptic events. Data from Type 1b and 1s synaptic boutons, which have naturally occurring variations of the SSR, were compared with genetic mutants that up or down-regulate SSR complexity. Some synaptic currents do not result in postsynaptic voltage changes, events that were called 'missing quanta'. The frequency of missing quanta is positively correlated with SSR complexity in both natural and genetically-induced variants. Rise-time and amplitude data suggest that passive membrane properties contribute to the observed differences in synaptic effectiveness. It is concluded that electrotonic decay within the postsynaptic structure contributes to the phenomenon of missing quanta. Further studies directed at understanding the role of the SSR in synaptic transmission and the potential for regulating 'missing quanta' will yield important information about synaptic transmission at the Drosophila NMJ (Nguyen, 2016).

Hebbian plasticity guides maturation of glutamate receptor fields in vivo

Synaptic plasticity shapes the development of functional neural circuits and provides a basis for cellular models of learning and memory. Hebbian plasticity describes an activity-dependent change in synaptic strength that is input-specific and depends on correlated pre- and postsynaptic activity. Although it is recognized that synaptic activity and synapse development are intimately linked, a mechanistic understanding of the coupling is far from complete. Using Channelrhodopsin-2 to evoke activity in vivo, this study investigated synaptic plasticity at the glutamatergic Drosophila neuromuscular junction. Remarkably, correlated pre- and postsynaptic stimulation increased postsynaptic sensitivity by promoting synapse-specific recruitment of GluR-IIA-type glutamate receptor subunits into postsynaptic receptor fields. Conversely, GluR-IIA was rapidly removed from synapses whose activity failed to evoke substantial postsynaptic depolarization. Uniting these results with developmental GluR-IIA dynamics provides a comprehensive physiological concept of how Hebbian plasticity guides synaptic maturation and sparse transmitter release controls the stabilization of the molecular composition of individual synapses (Ljaschenko, 2013).

Repeated light-triggered neurotransmitter release from presynaptic active zones provoked synaptic depression via a decrease in quantal content. Interestingly, muscle depolarization itself also led to a drop in quantal content despite bypassing synapses (Post animals). The latter observation is highly reminiscent of homeostatic communication whereby a retrograde pathway of inverted polarity operates to increase quantal content in response to reduced muscle excitability. Future studies can now test whether molecular components involved in the homeostatic upregulation of quantal content also contribute to its downregulation following postsynaptic ChR2 stimulation. (Ljaschenko, 2013).

Pairing pre- and postsynaptic depolarizations repetitively (pre and post) triggered a synapse-specific increase in postsynaptic GluR-IIA-type GluRs. Hence, correlated activity initiated a Hebbian form of synaptic plasticity at the Drosophila NMJ. A comparison of the cluster size distributions following brief and long pulses suggests two phases of plasticity. The first phase of activity-induced plasticity (15 ms pulses) promotes an evenly distributed increase in the number of clusters. Therefore, despite an increase in total number, the average size of GluR-IIA clusters is not significantly altered. The next phase (2 s pulses) then leads to an increase mainly in the number of large clusters, and hence the average GluR-IIA cluster size increases. Neurotransmitter pr varies across active zones at the NMJ. Because the size of GluR clusters is largest opposite high-pr active zones, it is to be expected that functional recordings of synaptic currents preferentially sample large receptor fields. For this reason, GluR-IIA incorporation likely remained below the detection threshold in electrophysiological recordings following brief light pulses (Ljaschenko, 2013).

In view of the unchanged total number of receptor fields (anti-GluR-IID staining) and active zones, paired stimulation did not appear to give rise to the formation of new synapses. Instead, GluR-IIA was likely incorporated into receptor fields with previously undetectable IIA levels (Ljaschenko, 2013).

In vivo imaging suggests that positive feedback initially promotes GluR-IIA incorporation during synapse growth and that GluR-IIA entry is specifically restrained with further maturation, whereas the rate of GluR-IIB recruitment remains constant. The physiological signals that guide these synapse-specific molecular dynamics are unknown. It is argued that the Hebbian mechanism identified in the present study represents the signal that promotes GluR-IIA entry during synapse development. This is consistent with the observed increase in small clusters following short pulses. Furthermore, in this framework, paired pre- and postsynaptic stimulation would be able to override the inhibition of GluR-IIA incorporation at relatively mature receptor fields and thereby restore the 'juvenile behavior' of the PSDs (Ljaschenko, 2013).

At the developing Drosophila NMJ, receptor field growth is accompanied by BRP-dependent, active zone maturation. Correspondingly, large receptor fields are found opposite high-pr active zones that are rich in BRP. Therefore, small, growing receptor fields opposite immature, low-pr active zones will tend to be exposed to glutamate only when pr is elevated, e.g., during trains of action potentials. Because a large number of other synapses will also be active at these time points, transmitter release will coincide with strong postsynaptic depolarization, leading to Hebbian GluR-IIA incorporation (Ljaschenko, 2013).

A comprehensive model conversely demands a signal to remove GluR-IIA from mature receptor fields in order to describe their diminished rate of IIA incorporation in vivo and to limit receptor-field growth. It was reasoned that such a physiological cue could be provided by sparse (i.e., unsynchronized) transmitter release that preferentially occurs at high-pr, mature synapses and does not trigger substantial muscle depolarization. This hypothesis is experimentally supported by GluR-IIA removal from synapses when muscle depolarization is prevented during neurotransmission (Ljaschenko, 2013).

This study introduces a physiological model in which GluR-IIA is increased at simultaneously active synapses via Hebbian plasticity and is decreased at solitarily active synapses. Such solitary activity may be provided by spontaneous transmitter release (i.e., minis). The physiological function of minis has been controversially discussed. The results suggest that they contribute to 'taming the beast'; in other words, restraining the extent of Hebbian plasticity. The model can account for developmental, synapse-specific receptor subunit dynamics, and explains why GluR-IIA levels are higher opposite low-pr Ib motorneurons than opposite high-pr Is motorneurons. This conceptual framework can account for an increase in GluR-IIA following chronic activity elevation and is consistent with low synaptic IIA levels in the presence of ambient extracellular glutamate, although, intriguingly, sustained glutamate exposure also affects GluR-IIB (Ljaschenko, 2013).

Trains of action potentials are likely the physiological equivalent of paired pre- and postsynaptic depolarization, which simply triggers the Hebbian change more efficiently than solely presynaptic ChR2 stimulation. Notably, rapid GluR-IIA exit can be acutely provoked. This observation is compatible with fast GluR dynamics in mammals, which can operate on a timescale of minutes and well below. Hence, rapid receptor trafficking also occurs in Drosophila, though this probably remains concealed when receptor exit is not explicitly provoked during time-lapse imaging of synapse development in vivo (Ljaschenko, 2013).

Perhaps most conspicuously, activity-dependent bidirectional GluR-IIA mobility is reminiscent of subunit-specific AMPA receptor trafficking at mammalian central synapses, which mediates manifold forms of synaptic plasticity. Local activity has been shown to drive synapse-specific accumulation of GluR1 AMPA receptors. Whereas high-frequency stimulation triggers LTP and synaptic GluR1 incorporation, low-frequency stimulation triggers LTD and GluR1 removal. Collectively, these considerations support the notion that fundamental mechanisms of synaptic plasticity have been strongly conserved during evolution (Ljaschenko, 2013).

Dbo/Henji modulates synaptic dPAK to gate glutamate receptor abundance and postsynaptic response

In response to environmental and physiological changes, the synapse manifests plasticity while simultaneously maintains homeostasis. This study analyzed mutant synapses of henji, also known as diablo (dbo), at the Drosophila neuromuscular junction (NMJ). In henji mutants, NMJ growth is defective with appearance of satellite boutons. Transmission electron microscopy analysis indicates that the synaptic membrane region is expanded. The postsynaptic density (PSD) houses glutamate receptors GluRIIA and GluRIIB, which have distinct transmission properties. In henji mutants, GluRIIA abundance is upregulated but of GluRIIB is not. Electrophysiological results also support a GluR compositional shift towards a higher IIA/IIB ratio at henji NMJs. Strikingly, dPAK, a positive regulator for GluRIIA synaptic localization, accumulates at the henji PSD. Reducing the dpak gene dosage suppresses satellite boutons and GluRIIA accumulation at henji NMJs. In addition, dPAK associated with Henji through the Kelch repeats which is the domain essential for Henji localization and function at postsynapses. It is proposed that Henji acts at postsynapses to restrict both presynaptic bouton growth and postsynaptic GluRIIA abundance by modulating dPAK (Wang, X., 2016).

Coordinated action and communication between pre- and postsynapses are essential in maintaining synaptic strength and plasticity. Presynaptic strength or release probability of synaptic vesicles involves layers of regulation including vesicle docking, fusion, and recycling, as well as endocytosis and exocytosis. Also, how postsynapses interpret the signal strength from presynapses depends largely on the abundance of neurotransmitter receptors at the synaptic membrane. During long-term potentiation, lateral diffusion of extrasynaptic AMPA receptor to synaptic sites is accelerated and the exocytosis of AMPAR is enhanced near the postsynaptic density (PSD), causing an accumulation of synaptic receptors. In contrast, under the long-term depression condition, synaptic AMPAR is reduced by hastened endocytosis. While molecular mechanisms are proposed to play roles in regulating and fine-tuning postsynaptic glutamate receptor (GluR) abundance in plasticity models, the developmental regulation of GluR abundance at the synaptic surface still needs to be elucidated. Synapses at the Drosophila neuromuscular junction (NMJ) use glutamate as the neurotransmitter, and have properties reminiscent of mammalian central excitatory synapses. Homologous to vertebrate AMPAR and kainate receptors, Drosophila GluR subunits assemble as tetramers to gate ion influx. Each functional receptor contains essential subunits (GluRIIC, GluRIID and GluRIIE) and either GluRIIA or GluRIIB; therefore, synaptic GluRs can be classified according to their subunit compositions as either A- or B-type receptors. These two types of receptors exhibit distinct developmental and functional properties. Newly-formed PSDs tend to accumulate more GluRIIA channels, while the IIA/IIB ratio becomes more balanced when PSDs mature. In addition, GluRIIB channels have much faster desensitization kinetics, which results in smaller quantal size than GluRIIA channels. Therefore, the synaptic composition of these two types of GluRs greatly influences the postsynaptic interpretation of neuronal activities. The Drosophila homolog of p21-activated kinase (dPAK) regulates GluRIIA abundance at the PSD; GluRIIA receptor clusters at the postsynaptic membrane are strongly reduced in dpak mutants. However, overexpression of dPAK in postsynapses is not sufficient to increase GluRIIA cluster size, suggesting that dPAK activity in regulating GluRIIA abundance is tightly controlled (Wang, X., 2016).

Ubiquitination and deubiquitination play critical roles in regulating synaptic functions. In loss-of-function mutants for highwire, a gene encoding a conserved E3 ubiquitin ligase, NMJs overgrow, producing supernumerary synaptic boutons. This phenotype is duplicated by overexpression of the deubiquitinating enzyme Fat facets (Faf) in presynapses. These studies underline the importance of balanced ubiquitination in synapse formation and function. Cullin-RING ubiquitin ligases (CRLs) are large protein complexes that confer substrate ubiquitination. Importantly, CRLs promote ubiquitination through substrate receptors that provide specific recognition of substrates for ubiquitination. The BTB-Kelch proteins are suggested to be the substrate receptors for Cul3-scaffolded CRLs. This study identified a BTB-Kelch-containing protein, Henji, also known as Dbo, which regulates NMJ growth and synaptic activity by restricting the clustering of GluRIIA. Synaptic size of henji mutants was significantly expanded, as viewed under transmission electron microscopy (TEM). Immunostaining for dPAK and GluRIIA also suggests larger areas of PSDs in the absence of Henji, and the intensity of each fluorescent punctum becomes stronger, indicating abnormal accumulation of these PSD proteins. By genetically reducing one gene dosage of dpak in henji mutants, GluRIIA accumulation and abnormal bouton morphology was suppressed. In contrast, reducing the gluriia gene dosage in henji mutants restored bouton morphology but failed to suppress dPAK accumulation. Thus, Henji regulates bouton morphology and GluRIIA clustering levels likely through a control of dPAK. Interestingly, while overexpression of dPAK, either constitutively active or dominantly negative, had no effects on GluRIIA clustering, overexpression of these dPAK forms in henji mutants modulated GluRIIA levels, indicating that Henji limits the action of dPAK to regulate GluRIIA synaptic abundance. Henji localized to the subsynaptic reticulum (SSR) surrounding synaptic sites, consistent with the idea that Henji functions as a gatekeeper for synaptic GluRIIA abundance (Wang, X., 2016).

This study shows that Henji functions at the postsynapse to regulate synaptic development and function at the NMJ. The PSD area is expanded and GluRIIA clusters abnormally accumulate at the PSD. Genetic evidences are provided to support that the elevation of GluRIIA synaptic abundance is at least partially caused by a corresponding accumulation of dPAK in henji mutants. Henji is sufficient to downregulate dPAK and GluRIIA levels and the Kelch repeats of Henji play the most critical role in this process. Henji tightly gates dPAK in regulating GluRIIA abundance, as dPAK enhances GluRIIA cluster abundance only when Henji is absent. Therefore, this study has identified a specific negative regulation of dPAK at the postsynaptic sites that contributes to the PSD formation and GluR cluster formation at the NMJ (Wang, X., 2016).

PAK proteins transduce various signaling activities to impinge on cytoskeleton dynamics. Through kinase activity-dependent and -independent mechanisms, PAK regulates not only actin- and microtubule-based cytoskeletal rearrangement but also the activity of motors acting on these cytoskeletal tracks. In mammalian systems, PAKs participate in many synaptic events including dendrite morphogenesis, neurotransmitter receptor trafficking, synaptic strength modulation, and activity-dependent plasticity. Pathologically, PAK dysregulation also contributes to serious neurodegenerative diseases, Huntington's disease and X-linked mental retardation (Wang, X., 2016).

At Drosophila NMJs, dPAK has divergent functions; loss of dpak causes a dramatic reduction in both Dlg and GluRIIA synaptic abundance, but the underlying molecular mechanisms have not been revealed. The current data show that Henji functions to restrict GluRIIA clustering but has no effect on Dlg levels, suggesting that Henji regulates one aspect of dPAK activities, probably via the SH2/SH3 adaptor protein Dock. Alternatively, Henji may function to limit dPAK protein levels locally near the postsynaptic region, rendering its influence on GluRIIA clustering, while dPAK that regulates Dlg may localize outside of the Henji-enriched region. Supporting this idea, Henji is specifically enriched around the SSR region instead of dispersed throughout the muscle cytosol. Moreover, ectopic Myc-dPAK localized at the postsynapse only when henji was mutated, indicating that Henji regulates dPAK postsynaptic localization (Wang, X., 2016).

The interaction with Rac, Cdc42, or both triggers autophosphorylation and subsequent conformational changes of PAK, resulting in kinase activation. The myristoylated dPAK that has been shown to be active in growth cones failed to enhance GluRIIA abundance at the NMJ. This result shows that dPAK is necessary to regulate GluRIIA synaptic abundance, but is itself tightly regulated at the synaptic protein level or the kinase activity. Indeed, evidence is provided to show specific negative regulation of dPAK by Henji; overexpression of dPAK CA that could not enhance GluRIIA abundance in WT larvae further increased the already enhanced GluRIIA levels in the henji mutant. Similar to the CA form, the DN form also showed no effect on GluRIIA when simply overexpressed in the WT background, but exhibited strong suppression of GluRIIA in the henji mutant background. Thus, regardless of the possible conformational differences between the CA and DN forms, Henji appears to confer a constitutive negative regulation of dPAK at postsynapses, suggesting a tight control that could be at subcellular localization. In contrast to CA and DN forms, activation of dPAK requires binding to Rac1 and Cdc42, and subsequent protein phosphorylation. This additional layer of regulation may serve as a limiting factor rendering dPAK WT from recruiting GluRIIA to PSDs regardless in WT or henji mutant background (Wang, X., 2016).

The structural feature suggests that Henji could function as a conventional substrate receptor of the Cul3-based E3 ligase complex. At Drosophila wing discs, Dbo functions as a Cul3-based E3 ligase to promote Dishevelled (Dsh) downregulation. Similar to the henji alleles, it was confirmed that the dbo [Δ25.1] allele and dbo RNAi were competent to induce dPAK and GluRIIA accumulation at the postsynapse. An immunoprecipitation experiment detected Henji and dPAK in the same complex, and dPAK also forms a complex with the C-terminal substrate-binding Kelch-repeats region. However, no notable or consistent increase was detected in Henji-dependent dPAK poly-ubiquitination in both S2 cells and larval extracts. Also, the Cul3-binding BTB domain of Henji seems dispensable in the suppression of dPAK levels in henji mutants. Importantly, Cul3 knockdown in muscle cells failed to cause any accumulation of GluRIIA and dPAK at the NMJ. Sensitive genetic interaction between henji and Cul3 failed to induced dPAK and GluRIIA accumulation. Dbo functions together with another BTB-Kelch protein Kelch (Kel) to downregulate Dsh. However, Kel negatively regulates GluRIIA levels without affecting dPAK localization at the postsynaptic site. This data argues that Kel functions in a distinct pathway to Henji in postsynaptic regulation of GluRIIA. Taken together, no direct evidence was found to support that dPAK is downregulated by Henji through ubiquitination-dependent degradation. Alternately, Henji could bind dPAK near the postsynaptic region and this interaction may block the recruitment or localization of dPAK onto postsynaptic sites. Under this model, dPAK is less restricted and has a higher propensity to localize at postsynaptic sites in the absence of Henji, resulting in synaptic accumulation of dPAK and GluRIIA expansions (Wang, X., 2016).

As many synaptic events require rapid responses, local regulation of protein levels becomes crucial in synapses. To achieve accurate modulation, certain synaptic proteins should be selectively controlled under different developmental or environmental contexts. Indeed, emerging evidence shows that various aspects of synapse formation and function are under the control of the ubiquitin proteasome system (UPS), including synapse formation, morphogenesis, synaptic pruning and elimination, neurotransmission, and activity-dependent plasticity. In particular, the membrane abundance of postsynaptic GluR that modulates synaptic function can be regulated by components of the UPS. When Apc2, the gene encoding Drosophila APC/C E3 ligase, is mutated, GluRIIA shows excess accumulation but the molecular mechanism was not elucidated. Similarly, loss of the substrate adaptor BTB-Kelch protein KEL-8 in C. elegans also results in the stabilization of GLR-1-ubiquitin conjugates. However, no evidence shows direct ubiquitination and degradation of GLR-1 by KEL-8. Also, absence of the LIN-23-APC/C complex in C. elegans affects GLR-1 abundance at postsynaptic sites without altering the level of ubiquitinated GLR-1. Therefore, GLR-1 receptor endocytosis and recycling or ubiquitination and degradation of GLR-1-associated scaffold proteins are proposed to be the underlying mechanism for E3 ligase regulation. In mammals, endocytosis of AMPAR can be influenced by poly-ubiquitination and degradation of the prominent postsynaptic scaffold protein PSD-95 (Wang, X., 2016).

This study describes a novel regulation by the BTB-Kelch protein Henji on synaptic GluRIIA levels. By limiting GluRIIA synaptic levels, Henji modulates the postsynaptic output in response to presynaptic glutamate release. In the absence of Henji, quantal size is elevated, coinciding with an increase in the postsynaptic GluRIIA/GluRIIB ratio. In a previous study, increases in the GluRIIA/GluRIIB ratio by overexpressing a GluRIIA transgene in the muscle or by reducing the gene copy of gluriib promote NMJ growth, but co-expression of both GluRIIA and GluRIIB did not alter the bouton number. Combined with the current findings, those data provide a link between an increased GluRIIA-mediated postsynaptic response and bouton addition at NMJs. However, satellite boutons were not detected following GluRIIA overexpression. One possibility is that satellite boutons are considered as immature boutons and their appearance may indicate the tendency for NMJ expansion, as in the case of excess BMP signaling. Failure to become mature boutons may be caused by the lack of cooperation with other factors such as components of the presynaptic endocytic pathway, actin cytoskeleton rearrangement or neuronal activity. No significant alterations in endocytosis and the BMP pathway in the henji mutant. Nevertheless, it cannot be ruled out that Henji may modulate other presynaptic events that are defective in henji mutants to interfere with bouton maturation (Wang, X., 2016).

Filamin, a synaptic organizer in Drosophila, determines glutamate receptor composition and membrane growth

Filamin is a scaffolding protein that functions in many cells as an actin-crosslinker. FLN90, an isoform of the Drosophila ortholog Filamin/cheerio that lacks the actin-binding domain, is shown in this study to govern the growth of postsynaptic membrane folds and the composition of glutamate receptor clusters at the larval neuromuscular junction. Genetic and biochemical analyses reveal that FLN90 is present surrounding synaptic boutons. FLN90 is required in the muscle for localization of the kinase dPak and, downstream of dPak, for localization of the GTPase Ral and the exocyst complex to this region. Consequently, Filamin is needed for growth of the subsynaptic reticulum. In addition, in the absence of filamin, type-A glutamate receptor subunits are lacking at the postsynapse, while type-B subunits cluster correctly. Receptor composition is dependent on dPak, but independent of the Ral pathway. Thus two major aspects of synapse formation, morphological plasticity and subtype-specific receptor clustering, require postsynaptic Filamin (Lee, 2016).

Proper postsynaptic function depends on appropriate localization of receptors and signaling molecules. Scaffolds such as PSD-95/SAP90 and members of the Shank family are critical to achieving this organization. While usually without intrinsic enzymatic activity, scaffolds recruit, assemble, and stabilize receptors and protein networks through multiple protein-protein interactions: they can bind to receptors, postsynaptic signaling complexes, and the cytoskeleton at the postsynaptic density. Mutations in these proteins are associated with neuropsychiatric disorders. While understanding synapse assembly has begun, much remains to be investigated (Lee, 2016).

The Drosophila larval neuromuscular junction (NMJ) is a well-studied and genetically accessible glutamatergic synapse. Transmission is mediated by AMPA-type receptors, and several postsynaptic proteins important for its development and function have related proteins at mammalian synapses, including the PDZ-containing protein Discs-Large (DLG) and the kinase Pak. In addition, the postsynaptic membrane forms deep invaginations and folds called the subsynaptic reticulum (SSR), which are hypothesized to create subsynaptic compartments comparable to dendritic spines. Recently, it was found that the SSR is a plastic structure whose growth is regulated by synaptic activity. This phenomenon may be akin to the use-dependent morphological changes, such as growth of dendritic spines, that occur postsynaptically in mammalian brain. The addition of membrane and growth of the SSR requires the exocyst complex to be recruited to the synapse by the small GTPase Ral; the SSR fails to form in ral mutant larvae. Moreover, the localization of Ral to a region surrounding synaptic boutons is likely to direct the selective addition of membrane to this domain. Ral thus provided a tractable entry point for better understanding postsynaptic assembly. The mechanism for localizing the Ral pathway, however, was unknown. The present study determined that Ral localization is dependent on cheerio, a gene encoding filamin, which is critical for proper development of the postsynapse (Lee, 2016).

Filamin is a family of highly conserved protein scaffolds with a long rod-like structure of Ig-like repeats. With over 90 identified binding partners, some of which are present also at the synapse, mammalian filamin A (FLNA) is the most abundant and commonly studied filamin. Filamin can bind actin and has received the most attention in the context of actin cytoskeletal organization. Drosophila filamin, encoded by the gene cheerio (cher), shares its domain organization and 46% identity in amino acid sequence with human FLNA. Drosophila filamin has a well-studied role in ring canal formation during oocyte development, where it recruits and organizes actin filaments. This study now shows that filamin has an essential postsynaptic role at the fly NMJ. An isoform of this scaffold protein that lacks the actin-binding domain acts via dPak to localize GluRIIA receptors and Ral; filamin thereby orchestrates both receptor composition and membrane growth at the synapse (Lee, 2016).

Filamin is a highly conserved protein whose loss of function is associated with neurodevelopmental disorders. In humans, mutations in the X-linked FLNA cause periventricular heterotopia, a disorder of cortical malformation with a wide range of clinical manifestations such as epilepsy and neuropsychiatric disturbances. Studies in rodent models have shown that abnormal filamin expression causes dendritic arborization defects in a TSC mouse and that filamin influences neuronal proliferation. Filamin is present in acetylcholine receptor clusters at the mammalian NMJ, but its function there is unknown. In lysates of the mammalian brain, filamin associates with known synaptic proteins such as Shank3, Neuroligin 3, and Kv4.2. A recent report indicated that filamin degradation promotes a transition from immature filopodia to mature dendritic spines, a phenomenon that is likely to be related to the actin-bundling properties of the long isoform of filamin. Data in the present study have uncovered a novel pathway that does not require the actin-binding domain of filamin. In this pathway, postsynaptically localized filamin, via Pak, directs two distinct effector modules governing synapse development and plasticity: (1) the Ral-exocyst pathway for activity-dependent membrane addition and (2) the composition of glutamate receptor clusters. These pathways determine key structural and physiological properties of the postsynapse (Lee, 2016).

Although loss of filamin had diverse effects on synapse assembly, they were selective. Muscle-specific knockdown or the cher^Q1415sd allele disrupted type-A but not type-B GluR localization at the postsynaptic density. Likewise, the phenotypes for muscle filamin were confined to the postsynaptic side: the presynaptic active zone protein Brp and overall architecture of the nerve endings were not altered by muscle-specific knockdown. The specificity of its effect on particular synaptic proteins, and the absence of the actin-binding region in FLN90, suggests that filamin's major mode of action here is not overall cytoskeletal organization, but rather to serve as a scaffold for particular protein-protein interactions (Lee, 2016).

Analysis of the distribution of the SSR marker Syndapin and direct examination of the subsynaptic membrane by electron microscopy revealed that formation of the SSR required filamin. Genetic analysis uncovered a sequential pathway for SSR formation from filamin to the Pak/Pix/Rac signaling complex, to Ral, to the exocyst complex and consequent membrane addition. The SSR is formed during the second half of larval life and may be an adaptation for the low input resistance of third-instar muscles. Like dendritic spines, the infoldings of the SSR create biochemically isolated compartments in the vicinity of postsynaptic receptors and may shape physiological responses, although first-order properties of the synapse, such as mini- or EPSP amplitude are little altered in mutants that lack an SSR. The formation of the SSR requires transcriptional changes driven by Wnt signaling and nuclear import, proteins that induce membrane curvature (such as Syndapin, Amphiphysin, and Past1), and Ral-driven, exocyst-dependent membrane addition. The activation of Ral by Ca2+ influx during synaptic transmission allows the SSR to grow in an activity-dependent fashion. The localization of Ral to the region surrounding the bouton appears crucial to determining the site of membrane addition because Ral localization precedes SSR formation and exocyst recruitment and because exocyst recruitment occurred selectively surrounding boutons even when Ca2+-influx occurred globally in response to calcimycin. This study has now shown that Ral localization, and consequently exocyst recruitment, membrane growth, and the presence of the SSR marker Syndapin, are all dependent on a local action of filamin at the synapse. FLN90, the filamin short isoform, localized to sites of synaptic contact and indeed surrounded the boutons just as does Ral. When this postsynaptic filamin was removed by muscle-specific filamin knockdown or the cher^Q1415sd allele, the downstream elements of the pathway, Pak, Rac, Ral, the exocyst, and Syndapin, were no longer synaptically targeted. The mislocalization is not a secondary effect of loss of the SSR but likely a direct consequence of filamin loss, as Pak and Ral can localize subsynaptically even in the absence of the SSR. Unlike the likely mode of action of nuclear signaling by Wnt, the delocalization of Ral was not a consequence of altered protein production; its expression levels did not change. Thus filamin may be viewed as orchestrating the formation of the SSR and directing it to the region surrounding synaptic boutons (Lee, 2016).

The second major feature of the filamin phenotype was the large reduction in the levels of the GluRIIA receptor subunit from the postsynaptic membranes. GluRIIA and GluRIIB differ in their electrophysiological properties and subsynaptic distribution. Because type B GluRs, which contain the IIB subunit, desensitize more rapidly than type A, the relative abundance of type A and type B GluRs is a key determinant of postsynaptic responses and changes with synapse maturation. The selective decrease in GluRIIA at filamin-null NMJs is likely a consequence of dPak mislocalization: filamin-null NMJs lack synaptic dPak, and dPak null NMJs lack synaptic GluRIIA. Moreover, the first-order electrophysiological properties at NMJs lacking filamin resembled those reported at NMJs missing dPak. In the current study, though, only the change in mEPSP frequency was statistically significant. At filamin-null NMJs, the decrease in GluRIIA is accompanied by an increase in GluRIIB, suggestive of a partial compensation that could account for the relatively normal synaptic transmission. Because the IIA and IIB subunits differ in desensitization kinetics and regulation by second messengers, functional consequences of filamin loss may become more apparent with more extensive physiological characterizations at longer time scales (Lee, 2016).

While both SSR growth and receptor composition required the kinase activity of dPak, receptor composition was independent of Ral and thus represents a distinct branch of the pathway downstream of dPak. As with Ral, the loss of GluRIIA from the synapse was due to delocalization and not a change in expression of the protein, consistent with unaltered GluRIIA transcripts in dPak null animals. Thus filamin, via dPak, alters proteins with functional significance for the synapse as well as its structural maturation (Lee, 2016).

Mammalian filamin, via its many Ig-like repeats, has known scaffold functions in submembrane cellular compartments and filamin is therefore likely also to serve as a scaffold at the fly NMJ. These epistasis data indicate that filamin recruits Ral through recruitment of a signaling complex already known to function at the fly NMJ: dPak and its partners dPix and Rac. Mammalian filamin is reported to directly interact with Ral during filopodia formation, however the details of their interaction at the fly NMJ are less clear. Because Ral localization requires filamin to recruit dPak and dPix and specifically requires the kinase activity of dPak, it is possible that either Ral or filamin need to be phosphorylated by dPak to bind one another. Mammalian filamin interacts with some components of the Pak signaling complex and is a substrate of Pak. This study has now shown that Drosophila filamin and PAK interact when coexpressed in HEK cells, and thus a direct scaffolding role for FLN90 in the recruitment of Pak and the organization of the postsynapse is likely (Lee, 2016).

The overlapping but different distributions of filamin and its downstream targets indicate that its scaffolding functions must undergo regulation by additional factors. The proteins discussed in this study take on either of two patterns at the synapse. Some, like Ral, Syndapin, and filamin itself, are what can be termed subsynaptic and, like the SSR, envelope the entire synaptic bouton. Others, like dPak and its partners and the GluRIIA proteins, are concentrated in much smaller regions, immediately opposite presynaptic active zones, where the postsynaptic density (PSD) is located. It is hypothesized that filamin interacts with additional proteins, including potentially transsynaptic adhesion proteins, that localize filamin to the subsynaptic region and also govern to which of the downstream effectors it will bind. Indeed, it appears paradoxical that dPak, though predominantly at the postsynaptic density is nonetheless required for Ral localization throughout the subsynaptic region. If dPak is needed to phosphorylate either filamin or Ral to permit Ral localization, the phosphorylations outside the PSD may be due to low levels of the dPak complex in that region; synaptic dPak was previously shown to be a relatively mobile component of the PSD (Lee, 2016).

Filamin was the first nonmuscle actin-crosslinking protein to be discovered. With an actin-binding domain at the N terminus, the long isoform of filamin and its capacity to integrate cellular signals with cytoskeletal dynamics have subsequently been the focus of the majority of the filamin literature. At the NMJ, however, this was not the case. Several lines of evidence indicated that the short FLN90 isoform of filamin, which lacks the actin-binding domain, plays an essential role in postsynaptic assembly. First, the short FLN90 isoform was the predominant and perhaps the only isoform of filamin found expressed in the muscles. Second, both endogenous and overexpressed FLN90 localized subsynaptically. Third, loss of the short isoform disrupted localization of postsynaptic components while lack of just the long isoform had little or no effect. Lastly, exogenous expression of just the short isoform in filamin null background sufficiently rescued the defect in SSR growth. The modest postsynaptic phenotypes of the cher1allele, which predominantly disrupts the long isoform, may be due to small effects of the allele on expression of the short isoform or may be an indirect consequence of the presence of the long isoform in the nerve terminals (Lee, 2016).

The existence of the short isoform has been reported in both flies and mammals and may be produced either by transcriptional regulation or calpain-mediated cleavage. The short isoform can be a transcriptional co-activator, but its functional significance and mechanisms of action have been largely elusive. The short isoform has little or no affinity for actin, but most of the known sites for other protein-protein interactions are shared by both isoforms. Thus the structure of FLN90, with nine predicted Ig repeats and likely protein-protein interactions, is consistent with a scaffolding function to localize key synaptic molecules independent of interactions with the actin cytoskeleton (Lee, 2016).

This study has introduced filamin as a major contributor to synapse development and organization. The severity of the phenotypes indicates filamin has a crucial role that is not redundant with other scaffolding proteins. The effects of filamin encompass several much-studied aspects of the Drosophila NMJ: the clustering and subunit subtype of glutamate receptors and the plastic assembly of specialized postsynaptic membrane structures. The pathways that govern these two phenomena diverge downstream of Pak kinase activity and are dependent on filamin for the proper localization of key signaling modules in the pathways. By likely acting as a scaffold protein, the short isoform of filamin may function as a link between cell surface proteins, as yet unidentified, and postsynaptic proteins with essential localizations to and functions at the synapse. Because many of the components of these pathways at the fly NMJ are also present at mammalian synapses and can interact with mammalian filamin, a parallel set of functions in CNS dendrites merits investigation (Lee, 2016).

TOR is required for the retrograde regulation of synaptic homeostasis at the Drosophila neuromuscular junction

Homeostatic mechanisms operate to stabilize synaptic function; however, little is known about how they are regulated. Exploiting Drosophila genetics, a critical role was uncovered for the target of rapamycin (TOR) in the regulation of synaptic homeostasis at the Drosophila larval neuromuscular junction. Loss of postsynaptic TOR disrupts a retrograde compensatory enhancement in neurotransmitter release that is normally triggered by a reduction in postsynaptic glutamate receptor activity. Moreover, postsynaptic overexpression of TOR or a phosphomimetic form of S6 ribosomal protein kinase, a common target of TOR, can trigger a strong retrograde increase in neurotransmitter release. Interestingly, heterozygosity for eIF4E, a critical component of the cap-binding protein complex, blocks the retrograde signal in all these cases. These findings suggest that cap-dependent translation under the control of TOR plays a critical role in establishing the activity dependent homeostatic response at the NMJ (Penney, 2012).

A growing consensus by neurobiologists suggests that a balance exists between forces that promote and those that hinder synaptic growth and function, ensuring proper synaptic connectivity and functional stability in the nervous system. A robust retrograde signaling mechanism at the Drosophila NMJ carries out the task of adjusting synaptic strength in response to a reduction in postsynaptic receptor function in GluRIIA mutants. Genetic analysis suggests that postsynaptic activity of TOR plays a key role in the ability of this retrograde signaling to carry out its function. The current findings are consistent with a model in which TOR, through activation of S6K and inhibition of 4E-BP, ensures the efficiency of cap-dependent translation in muscles and allows for the retrograde compensation to take place (Penney, 2012).

Interestingly, a moderate to strong reduction in TOR activity in the Tor^E161K/Tor^ΔP mutant combination does not influence normal synaptic growth and has only a mild effect on baseline synaptic transmission. However, the findings indicate that once synaptic activity is compromised, i.e., in GluRIIA mutants, TOR becomes critical for the retrograde induction of homeostatic signaling. Furthermore, the findings suggest that TOR activity is required throughout larval development, as its inhibition by rapamycin for 12 hr during late stages of larval development is sufficient to block the retrograde signal. In addition, it was found that TOR can induce a retrograde increase in neurotransmitter release in wild-type animals, indicating that TOR can also act as an instructive force to regulate synaptic strength. These results together lead one to envision that under metabolic stress, during dietary restriction or as a result of aging perhaps, TOR could function as a modulator of neuronal function. As such, the identification of TOR as a key player in establishing retrograde signaling across synapses offers new insights into how defects in this aspect of translational regulation may underlie the destabilization of synaptic activity in neural circuits leading to abnormal neural function and behavior associated with diseases such as tuberous sclerosis complex (TSC), autism, mental retardation, and schizophrenia, where regulation of TOR activity may be altered. In fact, in animal models for TSC, hyperactivity in circuits and the susceptibility to epileptic activity can be diminished in response to rapamycin treatment. Based on the current results, it is conceivable that in TSC animals, when TOR is upregulated, synaptic activity in circuits is enhanced due to the retrograde action of TOR on neurotransmitter release, in a manner independent of growth related phenotype associated with TOR gain of function. Therefore, the results reveal a role for TOR in the retrograde regulation of neurotransmitter release in neurons, an avenue to explore aimed at potential therapeutic approaches (Penney, 2012).

Based on genetic interaction experiments and biochemical assessment, it is concluded that TOR normally acts downstream of synaptic activity. It was observed that postsynaptic phosphorylation of S6K, a bona fide TOR target, is increased in GluRIIA mutants, suggesting that TOR signaling may be upregulated in these mutants. Consistently, the genetic experiments show that removal of one gene copy of either Tor or S6k is sufficient to block the homeostatic response in GluRIIA mutants. Furthermore, when TOR is overexpressed in GluRIIA mutants no additional increase in quantal content is observed. This lack of an additive effect suggests that a common molecular pathway may be utilized by GluRIIA mutants and larvae overexpressing TOR. This is further supported by the observations that the enhancement in neurotransmission in response to TOR (or S6K) overexpression and that triggered in GluRIIA loss of function are both highly dependent on wild-type availability of eIF4E. These results together support the idea that TOR functions downstream of synaptic activity at the NMJ. Further experiments are needed to understand how changes in synaptic activity may regulate the activity of TOR (Penney, 2012).

The findings are consistent with a growing body of evidence that implicates the involvement of TOR/S6K in the regulation of synaptic plasticity in mammals. The results indicate that TOR/S6K may be exerting their function through a retrograde mechanism to enhance neurotransmission. As such, the findings reveal a novel mode of action for TOR, through which it can modulate circuit activity in higher organisms. Further experiments are required to verify if this mode of action is conserved in higher organisms (Penney, 2012).

One potential way in which general translational mechanisms can lead to specific changes in synaptic function is through localized translation. In both vertebrates and invertebrates, local postsynaptic translation is required for normal synaptic plasticity and is itself modulated by synaptic function. This is perhaps best demonstrated in cultured hippocampal neurons, where local protein synthesis at postsynaptic sites is regulated by postsynaptic activity. It appears that this modulation is, at least in part, mediated by the action of synaptic activity on the function of elongation factor, eEF2. Interestingly, blocking NMDA mediated miniatures leads to phosphorylation and inhibition of eEF2 (Sutton, 2007). The current findings are consistent with a role for miniature synaptic activity in the regulation of postsynaptic translation: in GluRIIA mutants, a reduction in miniature amplitude (and perhaps in postsynaptic calcium influx) leads to an upregulation of TOR activity as evident in the increase in S6K phosphorylation. However, at this point it cannot be conclusively show that the effect of postsynaptic TOR is localized. Further experiments are needed to verify whether these changes occur at specific postsynaptic loci at the NMJ (Penney, 2012).

GluRIIA mutants is critically dependent on the efficiency of the cap-binding protein complex but is less sensitive to the availability of the ternary complex, which mediates the binding of Met-tRNA_i^Met to the 40S ribosomal subunit (Sonenberg, 2009). Similarly, while very strong inhibition of translation at the level of elongation using cycloheximide can block the retrograde compensation, revealing that retrograde compensation relies on de novo protein synthesis, moderate genetic interference with translation elongation does not interfere with retrograde signaling. These results together highlight the critical role of the cap-dependent protein complex in the retrograde regulation of synaptic strength (Penney, 2012).

The major task of the cap-binding complex is binding to the 5′UTR of the mRNA and unwinding it, so that the ribosome can interact with the mRNA and initiate translation (Ma, 2009). The results suggest that this stage of translation is the most critical for the induction of retrograde compensation. As the results suggest, once the 5′UTR is unwound, changes in the availability of the ternary complex and translation elongation are less critical for the induction of retrograde signaling. On the other hand, TOR would have a two-fold function in this scenario: one through its inhibitory action on 4E-BP, promoting eIF4Es ability to bind the 5′ cap structure, and another through its activation of S6K, which would ultimately increase the helicase ability of eIF4A to unwind mRNA 5′UTR secondary structure. This notion was supported by the results of an in vivo reporter assay showing a significant increase in translation of a reporter that bore a complex 5′UTR in response to TOR overexpression. Based on the current findings, it is speculated that perhaps genes with highly structured 5′UTRs are among the mRNAs triggered when postsynaptic activity is reduced in GluRIIA mutants or when TOR is overexpressed in muscles. The next challenge is to identify and characterize these genes, a discovery that will likely lead to a better understanding of how homeostatic mechanisms are regulated at the synapse (Penney, 2012).

The maintenance of synaptic homeostasis at the Drosophila neuromuscular junction is reversible and sensitive to high temperature

Homeostasis is a vital mode of biological self-regulation. The hallmarks of homeostasis for any biological system are a baseline set point of physiological activity, detection of unacceptable deviations from the set point, and effective corrective measures to counteract deviations. Homeostatic synaptic plasticity (HSP) is a form of neuroplasticity in which neurons and circuits resist environmental perturbations and stabilize levels of activity. One assumption is that if a perturbation triggers homeostatic corrective changes in neuronal properties, those corrective measures should be reversed upon removal of the perturbation. This study tests the reversibility and limits of HSP at the well-studied Drosophila melanogaster neuromuscular junction (NMJ). At the Drosophila NMJ, impairment of glutamate receptors causes a decrease in quantal size, which is offset by a corrective, homeostatic increase in the number of vesicles released per evoked presynaptic stimulus, or quantal content. This process has been termed presynaptic homeostatic potentiation (PHP). Taking advantage of the GAL4/GAL80^TS/UAS expression system, PHP was triggered by expressing a dominant-negative glutamate receptor subunit at the NMJ. PHP was then reversed by halting expression of the dominant-negative receptor. The data show that PHP is fully reversible over a time course of 48-72 h after the dominant-negative glutamate receptor stops being genetically expressed. As an extension of these experiments, it was found that when glutamate receptors are impaired, neither PHP nor NMJ growth is reliably sustained at high culturing temperatures (30-32°C). These data suggest that a limitation of homeostatic signaling at high temperatures could stem from the synapse facing a combination of challenges simultaneously (Yeates, 2017).

Homeostasis is a strong form of biological regulation. It permits individual cells or entire systems of cells to maintain core physiologic properties that are compatible with life. In the nervous system, decades of study have shown that while synapses and circuits are inherently plastic, they also possess robust homeostatic regulatory systems to maintain physiologic stability. Homeostatic plasticity in the nervous system is a non-Hebbian strategy to counteract challenges to neuronal function that may threaten to disrupt essential neuronal and circuit activities (Turrigiano, 2017). Depending on the synaptic preparation examined and the environmental challenge presented to the synapse, homeostatic responses may be executed via compensatory adjustments to presynaptic neurotransmitter release (Cull-Candy, 1980; Petersen, 1997; Murthy, 2001; Thiagarajan, 2005; Frank, 2006; Davis, 2015), postsynaptic neurotransmitter receptor composition (O'Brien, 1998; Turrigiano, 1998; Rongo , 1999; Turrigiano, 2008), neuronal excitability (Marder, 2002; Marder, 2006; Marder and Bucher, 2007; Bergquist, 2010; Parrish, 2014), or even developmentally, via changes in synaptic contact formation and maintenance (Davis and Goodman, 1998; Burrone, 2002; Wefelmeyer, 2016; Yeates, 2017 and references therein).

Bidirectionality has been documented in several homeostatic systems, perhaps most prominently in the case of synaptic scaling of neurotransmitter receptors. For vertebrate neuronal culture preparations -- such as cortical neurons or spinal neurons—global silencing of network firing can induce increases in excitatory properties, such as increased AMPA-type glutamate receptor accumulation; by contrast, global enhancement of activity can induce the opposite type of response (O'Brien, 1998; Turrigiano, 1998; Wierenga, 2005; Turrigiano, 2008). Bidirectionality is also a key feature underlying homeostatic alterations of neurotransmitter release at peripheral synapses such as the neuromuscular junction (NMJ). At the NMJs of Drosophila melanogaster and mammals, impairing neurotransmitter receptor function postsynaptically results in diminished sensitivity to single vesicles of transmitter. Electrophysiologically, this manifests as decreased quantal size. NMJs respond to this challenge by enhancing neurotransmitter vesicle release (Cull-Candy, 1980; Plomp, 1992, 1995; Petersen, 1997; Davis, 1998; Frank, 2009). By contrast, perturbations that enhance quantal size (for example, overexpression of a vesicular neurotransmitter transporter in Drosophila) can result in decreased quantal content (Daniels, 2004; Gavino, 2015; Yeates, 2017 and references therein).

Synapses and circuits possess myriad solutions to assume appropriate functional outputs in the face of perturbations (Marder, 2002; Marder, 2006). Therefore, a corollary to bidirectional regulation is that homeostatic forms of regulation should also be reversible. There are experimental difficulties of presenting and removing a synaptic challenge in the context of a living synapse, so homeostatic reversibility has not been rigorously studied in an in vivo system or over extended periods of developmental time. Understanding how homeostatic regulatory systems are reversibly turned on and off could have profound implications for elucidating fundamental properties of circuit regulation (Yeates, 2017).

This study exploits the Drosophila NMJ as a living synapse to test homeostatic reversibility. At the Drosophila NMJ, a canonical way to challenge synapse function is through glutamate receptor impairment (Frank, 2014), either genetically (Petersen, 1997) or pharmacologically (Frank, 2006). Impairments of muscle glutamate receptor function decrease quantal size. Decreased quantal size spurs muscle-to-nerve signaling that ultimately results in a homeostatic increase in presynaptic vesicle release, a process that has been termed presynaptic homeostatic potentiation (PHP). The most widely used experimental homeostatic challenges to Drosophila NMJ function are not easily reversed. These challenges include genetic deletion of the glutamate receptor subunit GluRIIA (Petersen, 1997) and the mostly irreversible pharmacological inhibition of glutamate receptors with Philanthotoxin-433 (PhTox; Frank, 2006; Yeates, 2017 and references therein).

For this study, a way was engineered to challenge NMJ function in vivo for significant periods of time, verify the effectiveness of the challenge at a defined developmental time point, remove the challenge, and then assess the homeostatic capacity of the NMJ at a later developmental time point. By using the temporal and regional gene expression targeting (TARGET) GAL4/GAL80TS/UAS expression system (McGuire, 2003) to temporally control the expression of a dominant-negative GluRIIA receptor subunit (DiAntonio, 1999), this study found that homeostatic potentiation of neurotransmitter release is fully reversible. In the course of conducting these studies, a high temperature limitation of homeostatic potentiation at the NMJ was uncovered (Yeates, 2017).

Thus study presents evidence that PHP at the Drosophila neuromuscular synapse is a reversible process. In doing so, prior findings were confirmed showing that there is a tight inverse relationship between quantal amplitude and QC at the NMJ. Those findings were complemented with the results of temperature shift experiments. PHP is measurable at an early stage of larval development and can be erased over a matter of days. Interestingly, at high temperatures, PHP induced by impairing glutamate receptor function either fails or falls short of full compensation. This failure appears to correlate with impaired NMJ growth in the same animals. Why is reversibility slow after dominant-negative GluRIIAM/R removal (Yeates, 2017)?

There was a robust expression of PHP for NMJs of MHC-Gal4 >> UAS-GluRIIAM/R larvae with the TubP-Gal80TS transgene raised at 29°C for 48 h after egg-laying. Once the expression of the dominant-negative UAS-GluRIIAM/R transgene was halted, this expression of PHP was erased over a slow 48- to 72-h period (Yeates, 2017).

If PHP is a readily reversible homeostatic process, why is there a days-long time lag to reverse it? The data likely reflect a constraint of the dominant-negative GluRIIAM/R experimental perturbation, rather than the NMJ's capacity to respond quickly to the changed environment. In a prior study, researchers expressed functional, tagged GluRIIA transgenic subunits at the NMJ and performed fluorescence recovery after photobleaching (FRAP) experiments. Those experiments demonstrated that receptor turnover rates at the Drosophila NMJ are extremely slow: it appears that once postsynaptic densities (PSDs) reach a critical size, GluRIIA subunits are stably incorporated. For the current study, this likely means that the temperature downshifts in the reversibility experiments represented an opportunity for the NMJ to incorporate endogenous WT GluRIIA into a significant number of new PSDs while it continued to grow. Given sufficient growth, the endogenously expressed GluRIIA would gradually overcome the previously incorporated dominant-negative GluRIIAM/R subunits. This would restore electrophysiological parameters to normal levels, which is consistent with the data. Reversibility of rapid and sustained forms of homeostatic plasticity (Yeates, 2017).

The majority of recent studies about synaptic homeostasis at the Drosophila NMJ have emphasized that presynaptic adjustments to neurotransmitter release properties must occur within minutes of drug-induced (PhTox) postsynaptic receptor inhibition to induce a rapid and offsetting response to PhTox challenge. Some important parameters uncovered through these studies include regulation of presynaptic Ca2+ influx; regulation of the size of the readily releasable pool (RRP) of presynaptic vesicles; control of presynaptic SNARE-mediated fusion events; control of neuronal excitability; and recently, implication of endoplasmic reticulum calcium-sensing activities; presynaptic glutamate receptor activity ; and finally, identification of a retrograde, trans-synaptic signaling system governed by the Semaphorin 2b ligand and the Plexin B receptor (Yeates, 2017).

For almost all of the cases in which a mutation or an experimental condition blocks the short-term induction of homeostatic signaling, the same perturbation has also been shown to block its long-term maintenance. Interestingly, however, the converse is not true. Additional studies have shown that the long-term consolidation (or expression) of homeostatic signaling at the NMJ can be genetically uncoupled from its induction. Select molecules seem to be dedicated to a long-term maintenance program that involves protein translation and signaling processes in both the neuron and the muscle. Recent data suggest that such long-term processes may take 6 h or more to take full effect (Yeates, 2017).

As more molecular details about HSP are elucidated, it will be interesting to test whether the rapid induction and sustained consolidation of PHP can be reversed by similar or separate mechanisms, and what the time courses of those reversal mechanisms are. At the mouse NMJ, reversibility was recently demonstrated pharmacologically. d-Tubocurarine was applied at a subblocking concentration to impair postsynaptic acetylcholine receptors. Within seconds of drug application, QC increased—and then within seconds of drug washout, it decreased again (Wang, X., 2016). Follow-up experiments suggested that those rapid, dynamic changes in PHP dynamics at the mouse NMJ were mediated by a calcium-dependent increase in the size of the RRP of presynaptic vesicles (Wang, X., 2016). Because there seem to be several similarities between the mouse NMJ and the Drosophila NMJ, it is possible that PHP at the insect NMJ can also be rapidly reversed (Yeates, 2017).

It is instructive to consider mammalian synaptic preparations and study how homeostatic forms of synaptic plasticity are turned on and off. Groundbreaking work on cultured excitatory vertebrate synapses showed that in response to activity deprivation (or promotion), synapses employ scaling mechanisms by adding (or subtracting) AMPA-type glutamate receptors to counteract the perturbation. Bidirectional scaling suggested that reversible mechanisms likely dictate homeostatic scaling processes. Complementary studies testing scaling reversibility have borne out this prediction. Additionally, evidence for reversible forms of homeostatic scaling have been found in rodent sensory systems, such as auditory synapses after hearing deprivation (and restoration to reverse) and in the visual cortex after light deprivation. Collectively, the vertebrate and invertebrate studies support the notion that reversible fine-tuning is an efficient strategy used to stabilize activities in metazoan nervous systems. One advantage offered by the Drosophila system is a genetic toolkit to uncover possible reversibility factors. Partial or failed homeostatic signaling at high temperatures (Yeates, 2017).

Are there environmental limitations for homeostatic potentiation at the Drosophila NMJ? Clearly there are. The data suggest that a combination of high temperature (30°C-32°C) plus impaired glutamate receptor function can severely limit the NMJ's ability to compensate for reduced neurotransmitter sensitivity. High temperature alone does not seem to be a severe enough restriction to impair PHP, because Gal4 driver controls at 30°C have somewhat reduced quantal size, but a fully (or nearly fully) offsetting increase in QC. Likewise, reduced glutamate receptor function alone does not appear to be a sufficient barrier to impair PHP. For example, quantal size is severely diminished when the dominant negative UAS-GluRII^AM/R transgene is expressed at 25°C, but PHP is nevertheless intact (Yeates, 2017).

It is not clear what the molecular or anatomic basis of this limit on PHP is. It is known that it is not an issue of NMJ excitation at high temperatures, because evoked neurotransmission for WT (or driver control) NMJs remains remarkably robust over a range of temperatures, including 30°C. Nor does it seem to be an elimination of PHP in general, because PHP was still present in the case of GluRIIA^SP16 animals raised at 30°C, revealing a distinction between the null GluRIIA condition and the dominant-negative GluRIIA condition. The limitation seems to be on homeostatic signaling that supports PHP at high temperatures in the face of the dominant-negative transgene expression (Yeates, 2017).

Temperature effects on neurophysiology are well documented. Recent work in crustaceans demonstrates that robust and reliable circuits such as the neurons driving the rhythmicity stomatogastric nervous system can fail under extreme temperature challenges. For the Drosophila NMJ, prior studies of larval development documented a significant enhancement of synaptic arborization when larvae were raised at higher temperatures. Additional studies have shown that NMJ growth plasticity can be additionally affected by mutations that affect neuronal excitability. Given the backdrop of these data, it is not unreasonable to hypothesize that the tolerable limits of synaptic activity challenge could be different at different temperatures (Yeates, 2017).

For the current experiments, 29°C-32°C represents a potential failure zone for homeostatic potentiation at the Drosophila NMJ. It must be noted that the data suggest that the coping capacity of the NMJ is dependent on genotype. WT NMJs cope at all temperatures. By contrast, for dominant-negative GluRIIA-expressing NMJs, 29°C is a point at which PHP becomes partial, and 30°C is a point at which it fails. For GluRII^ASP16 subunit deletion NMJs, there is robust, but partial PHP at 30°C, not unlike the compensation seen for the dominant-negatives at 29°C. GluRII^ASP16 NMJs fail to execute PHP only when temperature is pushed to an extreme range, such as 32°C. Why do these differences persist? It is not clear. The answer could relate to the well-documented temperature-induced alterations in NMJ growth, or alternatively, a limited availability of synaptic factors that are needed to cope with a double challenge of high temperature and particular impairment of glutamate receptor function. Future molecular and physiologic work will be needed to unravel those possibilities in the contexts of different genetic backgrounds and culturing conditions (Yeates, 2017).

The Drosophila postsynaptic DEG/ENaC channel ppk29 contributes to excitatory neurotransmission

The protein family of Degenerin/Epithelial Sodium Channels (DEG/ENaC) is comprised of diverse animal-specific, non-voltage-gated ion channels that play important roles in regulating cationic gradients across epithelial barriers. However, the specific neurophysiological functions of most DEG/ENaC-encoding genes remain poorly understood. This study demonstrates that ppk29 contributes specifically to the postsynaptic modulation of excitatory synaptic transmission at the larval neuromuscular junction (NMJ). Electrophysiological data indicate that the function of ppk29 in muscle is necessary for normal postsynaptic responsivity to neurotransmitter release, and for normal coordinated larval movement. The ppk29 mutation does not affect gross synaptic morphology and ultrastructure, which indicates the observed phenotypes are likely due to defects in glutamate receptor function. Together, these data indicate that DEG/ENaC ion channels play a fundamental role in the postsynaptic regulation of excitatory neurotransmission (Hill, 2017).

The identities and functions of genes that regulate neuronal synaptic functions in health and disease remains a major goal of neuroscience research. Although the principle molecular mechanisms that regulate synapse formation and function are relatively well understood, mechanisms for synaptic plasticity, especially at the physiological timescale, are still mostly unknown. This study describes a novel role for pickpocket (ppk) 29, a member of the degenerin/epithelial sodium channel (DEG/ENaC) family of non-voltage-gated sodium channels, in the postsynaptic modulation of baseline excitatory neurotransmission at the Drosophila larval neuromuscular junction (NMJ) (Hill, 2017).

Members of the DEG/ENaC family are exclusively found in animal genomes. They function as trimeric cation channels, which are expressed in both neuronal and non-neuronal tissues. DEG/ENaC channels can be gated by diverse extracellular stimuli, including extracellular ligands and mechanical forces (Ben-Shahar, 2011; Eastwood, 2012; Hill, 2017 and references therein).

Several studies in invertebrate and mammalian species suggest that some DEG/ENaC family members also directly contribute to synaptic functions (Younger, 2013; Urbano, 2014; Ievglevskyi, 2016; Miller-Fleming, 2016), which may explain their reported contributions to long-term potentiation, learning and memory (Wemmie, 2002), and addiction (Kreple, 2014). In addition, mutations in DEG/ENaC-encoding genes have been implicated in neuropathologies such as multiple sclerosis and epilepsy (Wemmie, 2013). However, whether the observed neuronal and behavioral phenotypes of mutations in DEG/ENaC-encoding genes are due to presynaptic or postsynaptic processes is not well understood (Hill, 2017).

In contrast to mammalian genomes, which typically harbor eight to nine independent DEG/ENaC-encoding genes, the genome of the fruit fly Drosophila melanogaster encodes >30 independent family members, named ppk genes. Analyses of mutations in several ppk genes indicates that Drosophila DEG/ENaC channels contribute to diverse sensory functions such as salt taste, water sensing, and the detection of mating pheromone. In addition, some ppk genes have been implicated in the maintenance of synaptic homeostasis (Younger, 2013). Nevertheless, the specific molecular mechanisms by which these channels exert their synaptic functions remain elusive (Hill, 2017).

This study reports that ppk29, which has been reported previously as a neuronally enriched Drosophila DEG/ENaC subunit implicated in pheromone-sensing functions (Thistle, 2012; Mast, 2014; Vijayan, 2014; Yuan, 2014), is also required for normal neurotransmission at the larval NMJ, a model glutamatergic synapse, via postsynaptic processes, possibly via modulation of postsynaptic glutamate receptors (Hill, 2017).

Despite their emerging importance in neurological and cognitive pathologies, the precise neurophysiological functions of DEG/ENaC channels remain elusive. This study demonstrates that a Drosophila DEG/ENaC-encoding gene, ppk29, is required for normal synaptic functions. However, in contrast to the ppk11/ppk16 complex (Younger, 2013), ppk29 action is restricted to the postsynaptic site and is associated with baseline spontaneous neurotransmission but not PhTX-dependent synaptic homeostasis. Therefore, it is proposed that individual DEG/ENaC-like channels may play independent roles in regulating synaptic functions, which may explain some of the contradicting reports about their functions in the mammalian synapse (Hill, 2017).

Previous studies have shown that some DEG/ENaC-encoding genes are expressed in human skeletal muscles, with their function remaining unknown. In Caenorhabditis elegans, the DEG/ENaC-encoding gene unc-105 is also expressed in muscles, where it is important for proper muscle organization, growth, and contraction. The current data indicate that, in addition to the contributions of DEG/ENaC proteins to muscle development and physiology, they also contribute to excitatory neurotransmission via postsynaptic processes in muscle. Nonetheless, because the fly NMJ is glutamatergic, the findings presented in this study could also provide important insights about postsynaptic functions of DEG/ENaC signaling in glutamatergic central synapses of vertebrates (Hill, 2017).

The postsynaptic impact of ppk29 on excitatory signaling may be mediated directly by PPK29 or indirectly via interaction with other proteins. Since currently available tools did not allow localization of PPK29 to specific subcellular compartments, it is too early to conclude whether the observed phenotypes in the ppk29 mutants are the consequence of a direct synaptic function or possibly of an indirect function via its action in other subcellular domains. It is also noted the that PPK29 may play more than one role in muscles and, therefore, that the electrophysiological and behavioral data may not be mediated through the same mechanism (Hill, 2017).

Nevertheless, one intriguing way that PPK29 might directly contribute to synaptic transmission is by acting as a direct postsynaptic receptor for glutamate or other molecules that are coreleased during spontaneous excitatory neurotransmission. For example, in mouse brain slices, extracellular protons increase with the stimulation of glutamatergic inputs, which can activate acid-sensitive ion channels, which are also members of the DEG/ENaC family (Du, 2014). Therefore, although it is not known whether PPK29 is an acid-activated channel, the corelease of protons with glutamate during spontaneous neurotransmission at the Drosophila NMJ may directly activate PPK29 channels (Hill, 2017).

The ppk29 gene may also affect neurotransmission indirectly through the modulation of expression or function of other synaptic proteins. Postsynaptic glutamate receptors are promising candidates to mediate an indirect impact of ppk29 on synaptic physiology since these ionotropic receptors are the main mediators of excitatory neurotransmission at the larval NMJ. This study found that ppk29 mutant animals exhibit decreased spontaneous amplitude and current flow, suggesting altered function of the postsynaptic ionotropic GluRs, which may be mediated by direct interaction between PPK29 and the GluR complex, or by indirect interaction via other postsynaptic signaling mechanisms. In line with this hypothesis, direct physical interactions between other DEG/ENaC proteins and potassium channels and sodium/chloride cotransporters have been reported in mammalian systems. It is further hypothesized that the observed differences in GluR expression levels are due to compensatory transcriptional changes. It is not known whether the ppk29 mutation independently impacts both GluRIIA and GluRIIB. Yet, previous studies have shown that genetic manipulation of expression levels of either GluRIIA or GluRIIB affect expression levels of the other (Marrus, 2004); therefore, the ppk29 mutation may directly or indirectly impact one or both subunit types (Hill, 2017).

To date, studies of spontaneous neurotransmitter release at the Drosophila larval NMJ have suggested that its main function is to regulate the development and maintenance of excitatory synaptic transmission by regulating presynaptic morphology and the postsynaptic clustering of glutamate receptors. However, spontaneous neurotransmitter release at central synapses has also been shown to impact local protein synthesis in dendrites as well as dendritic summation of EPSPs at much shorter timescales. This study has identified an important function for DEG/ENaC channels at the physiological timescale, which has an impact on both neurophysiological and behavioral phenotypes. Although it is not known how the molecular action of PPK29 might affect synapses and behavior, it is argued that these findings about the contribution of DEG/ENaC-encoding genes to spontaneous excitatory neurotransmission at the Drosophila larval NMJ may serve as an excellent model for understanding the function of spontaneous baseline excitatory neurotransmission in regulating synaptic organization. Better understanding of these processes at the physiological timescale is essential for understanding behavioral and neural plasticity in health and disease (Hill, 2017).

Synaptic excitation is regulated by the postsynaptic dSK channel at the Drosophila larval NMJ

In the mammalian CNS, the postsynaptic small-conductance Ca2+-dependent K+ (SK) channel has been shown to reduce postsynaptic depolarization and limit Ca2+ influx through NMDA receptors. To examine further the role of the postsynaptic SK channel in synaptic transmission, its action was studied at the Drosophila larval NMJ. Repetitive synaptic stimulation produced an increase in postsynaptic membrane conductance leading to depression of EPSP amplitude and hyperpolarization of the resting membrane potential (RMP). This reduction in synaptic excitation was due to the postsynaptic Drosophila SK (dSK) channel; synaptic depression, increased membrane conductance and RMP hyperpolarization were reduced in dSK mutants or after expressing a Ca2+ buffer in the muscle. Ca2+ entering at the postsynaptic membrane was sufficient to activate dSK channels based upon studies in which the muscle membrane was voltage clamped to prevent opening voltage-dependent Ca2+ channels. Increasing external Ca2+ produced an increase in resting membrane conductance and RMP that was not seen in dSK mutants or after adding the glutamate-receptor blocker philanthotoxin. Thus, it appeared that dSK channels were also activated by spontaneous transmitter release and played a role in setting membrane conductance and RMP. In mammals, dephosphorylation by protein phosphatase 2A (PP2A) increased the Ca2+ sensitivity of the SK channel; PP2A appeared to increase the sensitivity of the dSK channel since PP2A inhibitors reduced activation of the dSK channel by evoked synaptic activity or increased external Ca2+. It is proposed that spontaneous and evoked transmitter release activate the postsynaptic dSK channel to limit synaptic excitation and stabilize synapses (Gertner, 2014).

Stimulation of the neuromuscular synapses at 20 Hz resulted in an increase in resting G_in (input conductance), and a decrease in synaptic excitation due to membrane hyperpolarization and a decrease in EPSP amplitude. Simulations predicted that the increase in resting G_in during stimulation accounted for about one-third of the depression of EPSP amplitude due to shunting of the synaptic current. Synaptic depression is generally held to be due to a reduction in transmitter release; thus the remaining decrease in EPSP amplitude may have resulted from a decline in transmitter release. Nonetheless, evidence is provided for a change in postsynaptic conductance contributing to synaptic depression. The increase in G_in and RMP was consistent with activation of a g_KCa; this was further supported by the reduced increase in G_in and RMP seen after expressing th Ca²⁺ buffer parvalbumin in the muscle (Gertner, 2014).

Larval muscle fibers contain two types of Ca²⁺-dependent K⁺ channels producing g_CF and g_CS. g_CF was found to result from the slowpoke channel, a BK-type channel showing voltage- and Ca²⁺-dependent activation and voltage-dependent inactivation, and the dSK channel was proposed to produce g_CS (Abou Tayoun, 2011). Western blots showed a single dSK isoform in larval brain and muscle; a Western blot of adult fly brains showed two closely spaced bands, which could have represented two dSK isoforms or a single isoform along with its posttranslationally altered form (Abou Tayoun, 2011). SK channels are not voltage-gated, and calmodulin acts as their Ca²⁺ sensor, resulting in a relatively high Ca²⁺ sensitivity. Repetitive synaptic stimulation activated the postsynaptic dSK channels, since the increase in G_in and RMP was eliminated in dSK⁻ or 24B/dSK^DN larvae, but not in slo¹ larva. As expected, there was reduced synaptic depression in dSK mutants and PV-expressing larvae; however, it was surprising that synaptic depression was completely eliminated. This suggests that there was also less depression of transmitter release in these larvae (Gertner, 2014).

It is assumed that the dSK channel is activated during evoked transmitter release in vivo, since the 20-Hz stimulation applied in this study likely falls within the physiological range of activity. The Ib synapses on muscle fiber 6 fire on average 20–30 Hz, and the Is terminals fire less than 10 Hz in dissected larvae; however, these firing frequencies may be greater in vivo since the waves of contraction are slow in dissected larvae, probably due to lack of appropriate sensory input. Also, since HL3 with low Ca²⁺ was used for the stimulation procedure, the postsynaptic Ca²⁺ influx was less than would occur under physiological conditions (Gertner, 2014).

Ca²⁺ entering at the postsynaptic membrane was sufficient to activate dSK channels, since evoked transmitter release produced an increase in G_in even when the muscle was voltage clamped at −60 mV. Under these conditions, it is expected that Ca²⁺ entry is limited to glutamate receptors, since voltage-dependent Ca²⁺ channels in larval muscle open at membrane potentials more positive than −30 mV. However, it cannot be ruled that there was a voltage drop across the subsynaptic reticulum (SSR), such that voltage-dependent Ca²⁺ channels near the glutamate receptors remained unclamped. In this case, both the glutamate receptors and nearby Ca²⁺ channels could have been the source of Ca²⁺ that activated the dSK channel. In fact, dSK channels in larval muscle can be activated by Ca²⁺ entering through voltage-dependent Ca²⁺ channels since depolarization of the muscle in the absence of synaptic activity produced an increase in g_CS. Additional evidence that the glutamate receptor can act as the Ca²⁺ source comes from the activation of the dSK channel by spontaneous transmitter release; here, it seems unlikely that the SSR resistance would be great enough for quantal currents to open voltage-dependent Ca²⁺ channels. It appears that dSK channels exist at the postsynaptic membrane near the glutamate receptors, and this is consistent with findings in mammals, where the SK channel has been shown to be closely coupled to postsynaptic nicotinic acetylcholine receptors and NMDA receptors (Gertner, 2014).

The dSK channels were activated at rest and were responsible for the previously reported dependence of the RMP on external Ca²⁺. This was based upon the finding that the increase in G_in and RMP typically seen when increasing external Ca²⁺ from 0 to 1.5 mM was not seen in dSK⁻ larvae. In fact, it appeared that activation of the dSK channel is responsible for much of the normal variability in the RMP, since resting G_in and the RMP were positively correlated in wild-type larvae, but not in dSK⁻ larvae. Spontaneous transmitter release apparently activates dSK channels to set the resting G_in and RMP, since blocking the glutamate receptors prevented the rise in G_in and RMP produced by increasing external Ca²⁺. This is a novel function for spontaneous transmitter release, but not entirely unexpected, since spontaneous transmitter release produced postsynaptic Ca²⁺ transients similar in amplitude to evoked release (Gertner, 2014).

The spontaneous Ca²⁺ transients must have produced an increase in resting G_in that outlasted the Ca²⁺ signal, since the frequency of spontaneous events in this muscle is usually about 1 Hz, and the spontaneous Ca²⁺ transients had a decay time constant of ∼50 ms. The increase in G_in produced by repetitive synaptic stimulation also appeared to outlast the increase in [Ca²⁺]_i, since it persisted for about 10 min after stimulation. Previous studies have found that after 5 s of 10-Hz stimulation, postsynaptic [Ca²⁺]_i decayed with a time constant of about 100 ms, and this decay was largely due to the plasma membrane Ca²⁺ ATPase, which appeared to be highly enriched in the SSR. The SSR may limit Ca²⁺ diffusion to enhance the amplitude of postsynaptic Ca²⁺ transients; however, it seems likely that it also reduces their duration by providing efficient postsynaptic Ca²⁺ extrusion. Thus it seems improbable that postsynaptic [Ca²⁺]_i remained elevated for 10 min after the stimulation train (Gertner, 2014).

There was an increase in leakage conductance (g_L) in dSK⁻ larvae. This is consistent with previous studies showing covariation of ion conductances. In particular, reduced activation of Ca²⁺-dependent K⁺ currents in Drosophila cultured neurons resulted in a compensatory upregulation of the transient K⁺ current, apparently due to increased expression of transient K⁺ current channels. Although the channels responsible for g_L have not been characterized in larval muscle, they may include ORK1 which was identified as a K⁺ leak channel in Drosophila and appears to be expressed in adult muscle; it is a member of the two-pore domain K⁺ channels identified in mammals. The nonselective cation channel NALCN produces the Na⁺ leak conductance in mammalian neurons and its ortholog na is expressed in Drosophila neurons, raising the possibility that it could also be responsible for the Na⁺ leak conductance in larval muscle (Gertner, 2014).

In mammals, CK2 and PP2A are constitutively bound to the SK channel and regulate the phosphorylation state of calmodulin, which determines the Ca²⁺ sensitivity and deactivation rate of the SK channel. Dephosphorylation of calmodulin by PP2A can reduce EC₅₀ to 0.3 μM Ca²⁺ and phosphorylation of the SK channel by CK2 can increase EC₅₀ to 2.0 μM. An increase in [Ca²⁺]_i should shift the balance toward dephosphorylation and produce an increase in Ca²⁺ sensitivity, since CK2 cannot phosphorylate calmodulin when the channel is open. The current results showed that the dSK channel is also regulated by PP2A, since inhibiting PP2A reduced the increase in G_in and RMP produced by repetitive stimulation or increasing external Ca²⁺. During repetitive stimulation, the increase in postsynaptic [Ca²⁺]_i may have increased the Ca²⁺ sensitivity of the dSK channel. Similarly, it may be that the Ca²⁺ transients produced by spontaneous transmitter release produced an increase in Ca²⁺ sensitivity so that the dSK channel is partially activated by resting [Ca²⁺]_i. If the Ca²⁺-dependent increase in Ca²⁺ sensitivity persisted, this could explain the lasting increase in G_in seen after the end of 20-Hz stimulation and after spontaneous Ca²⁺ transients (Gertner, 2014).

The current results demonstrate that Ca²⁺ entering at the postsynaptic membrane during transmitter release provides negative feedback on synaptic excitation. This could rapidly stabilize the synapse and dampen the effects of changes in impulse activity, transmitter release or postsynaptic sensitivity. The effect of g_SK on synaptic excitation was modeled using a G_syn of 500 nS. The resting g_SK in 1 mM (HL3) and 1.5 mM external Ca²⁺ (HL3.1) would make the EPSP peak about 3 mV and 14 mV more negative, respectively, mainly due to hyperpolarization of the RMP. An increase in g_SK from 0 to 1 μS (approximately the value seen after 20 Hz stimulation) made the EPSP peak 22 mV more negative due to both RMP hyperpolarization and a decrease in EPSP amplitude. Activation of the dSK channel by evoked transmitter release is consistent with this negative-feedback mechanism, but what is the function of dSK channel activation by spontaneous transmitter release? minEPSP frequency is elevated after repetitive synaptic activity, and this could produce a persistent reduction in synaptic excitation. Also, since greater minEPSP frequency and amplitude would predict a larger EPSP, spontaneous transmitter release could set g_SK to oppose strong synaptic excitation (Gertner, 2014).

This action of the postsynaptic dSK channel is similar to that found at mammalian central nervous system synapses, where the SK channel counteracts synaptic changes: experiments blocking SK channels with apamin, or overexpressing them, showed that activation of SK channels increased the threshold for the induction of LTP and impaired learning. At these synapses, it was proposed that SK channels act specifically to restrict Ca²⁺ entry through NMDA receptors limiting LTP and learning and memory. The current findings show that dSK channels play a more general role in regulating synaptic excitation (Gertner, 2014).

The dSK channel could act in concert with forms of homeostatic synaptic plasticity; these have involved compensatory changes in transmitter release at the NMJ, and most operate over long time scales. A failure of the dSK channel to maintain appropriate synaptic excitation could result in compensatory changes in transmitter release; this is supported by the apparent decrease in transmitter release seen when dSK channel activity was reduced in dSK mutants or by expressing PV in the muscle. This is consistent with previous findings that expressing additional K⁺ channels in Drosophila larval muscle resulted in a homeostatic increase in transmitter release (Gertner, 2014).

At mammalian central synapses, the SK channel reduced the amplitude of the EPSC; single EPSPs and EPSCs were larger after adding the SK-channel blocker apamin. Thus the SK channel was rapidly activated during synaptic transmission so that the resultant EPSC was a composite of the inward current through the postsynaptic receptor and outward current through the SK channel. This study did not directly examined whether the dSK channel influences the amplitude of the EPSC; this is made difficult since apamin does not block dSK channels. Evidence is available that dSK currents reduced the duration of the EPSC, but further experiments are required to characterize fully the contribution of the dSK current to the EPSC (Gertner, 2014).

The SMAD2/3 interactome reveals that TGFβ controls m⁶A mRNA methylation in pluripotency

The TGFβ pathway has essential roles in embryonic development, organ homeostasis, tissue repair and disease. These diverse effects are mediated through the intracellular effectors SMAD2 and SMAD3 (hereafter SMAD2/3), whose canonical function is to control the activity of target genes by interacting with transcriptional regulators. Therefore, a complete description of the factors that interact with SMAD2/3 in a given cell type would have broad implications for many areas of cell biology. This study describes the interactome of SMAD2/3 in human pluripotent stem cells. This analysis reveals that SMAD2/3 is involved in multiple molecular processes in addition to its role in transcription. In particular, a functional interaction was identified with the METTL3-METTL14-WTAP complex, which mediates the conversion of adenosine to N6-methyladenosine (m6A) on RNA4. SMAD2/3 promotes binding of the m6A methyltransferase complex to a subset of transcripts involved in early cell fate decisions. This mechanism destabilizes specific SMAD2/3 transcriptional targets, including the pluripotency factor gene NANOG, priming them for rapid downregulation upon differentiation to enable timely exit from pluripotency. Collectively, these findings reveal the mechanism by which extracellular signalling can induce rapid cellular responses through regulation of the epitranscriptome. These aspects of TGFβ signalling could have far-reaching implications in many other cell types and in diseases such as cancer (Bertero, 2018)

Regulation of neuromuscular junction organization by Rab2 and its effector ICA69 in Drosophila

Mechanisms underlying synaptic differentiation, which involves neuronal membrane and cytoskeletal remodeling, are not completely understood. This study performed a targeted RNAi-mediated screen of Drosophila BAR-domain proteins and identified islet cell autoantigen 69 kDa (dICA69) as one of the key regulators of morphological differentiation of larval neuromuscular junction (NMJ). Drosophila ICA69 colocalizes with α-Spectrin at the NMJ. The conserved N-BAR domain of dICA69 deforms liposomes in vitro. Full length and ICAC but not the N-BAR domain of dICA69 which induces filopodia in cultured cells. Consistent with its cytoskeleton regulatory role, dICA69 mutants show reduced α-Spectrin immunoreactivity at the larval NMJ. Manipulating levels of dICA69 or its interactor dPICK1 alters synaptic level of ionotropic glutamate receptors (iGluRs). Moreover, reducing dPICK1 or dRab2 levels phenocopies dICA69 mutation. Interestingly, dRab2 regulates not only synaptic iGluR but also dICA69 levels. Thus, these data suggest that: a) dICA69 regulates NMJ organization through a pathway that involves dPICK1 and dRab2, and b) dRab2 genetically functions upstream of dICA69 and regulates NMJ organization and targeting/retention of iGluRs by regulating dICA69 levels (Mallik 2017).

This study demonstrates that ICA69 regulates NMJ structural organization and synaptic levels of glutamate receptor clusters. The findings suggest a model in which Rab2 functions genetically upstream of ICA69 to regulate its synaptic level, which in turn regulates the Spectrin cytoskeleton and iGluRs at the NMJ (Mallik, 2017).

The requirement of ICA69 for Drosophila NMJ organization is strongly supported by its enrichment in the postsynaptic Spectrin-rich scaffold. Consistent with this idea, ICA69 mutants or animals with downregulated ICA69 levels show reduced arborization and bouton numbers at the NMJ. Several studies have shown that cytoskeletal regulation is a key process for NMJ development. Multiple lines of evidence suggest that ICA69 promotes NMJ growth by regulating the cytoskeletal network surrounding the SSR. First, ICA69 is highly enriched at the NMJ in the same microdomain as Spectrin. Second, ICA69 induces filopodia in cultured cells and relocalizes positive regulators of actin polymerization at the filopodia. Third, mutation in ICA69 significantly reduces α-Spectrin levels. The Actin-Spectrin scaffold at the postsynapse has been implicated in regulation of NMJ organization in postembryonic development in Drosophila. This study reveals a crucial requirement of ICA69 in regulating synaptic α-Spectrin levels and indicates that ICA69 is required for the assembly of Actin-Spectrin scaffolds surrounding the SSR. Whether localization and/or stability of postsynaptic Spectrin-Actin scaffold depends on direct interaction between scaffold components and ICA69 or on some unknown signaling mechanism needs to be further investigated (Mallik, 2017).

For the proper establishment of NMJ connections, neurons as well as muscles require trafficking of various synaptic proteins. Rab GTPases and their regulators are considered to be some of the most important signaling molecules for intracellular trafficking. Interestingly, nearly half of all the Drosophila Rab proteins function specifically in neurons and a few of them localize to the NMJs. ICA69 physically associates with Rab2 and has been suggested as one of its effectors in regulating dense core vesicle maturation in Caenorhabditis elegans. This study found that Rab2 endogenous regulatory sequence-driven Rab2EYFP is detectable in the larval muscles as punctate structures, suggesting its involvement in NMJ organization. This idea is supported by four compelling pieces of evidence. First, ubiquitous or muscle-specific knockdown of Rab2 phenocopies ICA69 mutants. Second, knockdown of Rab2 significantly reduces synaptic α-Spectrin levels. Third, Rab2 directly regulates synaptic ICA69 levels. Fourth, co-expressing an ICA69 transgene and Rab2 RNAi rescues the morphological defects of Rab2 RNAi. Based on these observations, it is suggested that Drosophila Rab2 functions genetically upstream of ICA69. Like Rab2, PICK1 depletion reduced synaptic ICA69 levels and phenocopied the NMJ morphological defects observed in ICA69 mutants or after Rab2 depletion. Moreover, simultaneous knockdown of ICA69 and PICK1 or of ICA69 and Rab2 did not show an additive effect on the NMJ structural defects. These observations support the notion that ICA69, PICK1 and Rab2 might function in the same genetic pathway to regulate NMJ structural organization (Mallik, 2017).

In mammalian neurons, ICA69 is, surprisingly, not enriched at the synapses and negatively regulates AMPA receptor trafficking. Hence, it is expected that ICA69 mutants would have normal, if not more, iGluR clusters at the NMJ. Contrary to this expectation, reducing the ICA69 level resulted in reduced GluRIIA as well as GluRIIB glutamate receptor clusters. A recent study has shown that ICA69 and PICK1 stability is interdependent in Drosophila brain. Thus, it is likely that iGluR clusters at the NMJ are regulated by levels of ICA69 and PICK1 in muscles (Mallik, 2017).

How does ICA69 reduce iGluR levels both in knockdown and overexpression scenarios? It is suggested that the endogenous stoichiometry of ICA69 and PICK1 is crucial for normal synaptic targeting of iGluRs at the Drosophila NMJ. Reducing ICA69 destabilizes the ICA69-PICK1 heteromeric complex thereby reducing PICK1 availability for synaptic targeting of iGluRs. Overexpression of ICA69 forms more of the ICA69-PICK1 inhibitory complexes, which reduces synaptic targeting of iGluRs. Hence, the idea that the endogenous level of ICA69 is crucial for maintaining normal glutamate receptor clusters at the synapses is supported (Mallik, 2017).

The data suggest that ~40% simultaneous reduction of GluRIIA/IIB/III at Drosophila NMJ synapses has no major consequence on larval synaptic physiology. Three possibilities are suggested to explain this. First, the relative levels of GluRIIA and GluRIIB subunits are crucial for determining the efficacy of synaptic transmission at the Drosophila NMJ synapse . The decrease for each of the GluRIIA, -IIB and -III subunits in the ICA69 mutant is almost identical; ~40% compared with controls. This hints towards a homeostatic compensatory mechanism whereby ~60% of the receptor subunits are sufficient to form enough functional receptor complexes, which can maintain normal synaptic strength. Second, the amount of GluRIII is reflective of the sum of GluRIIA and -IIB complexes together, and GluRIII is essential for the localization of GluRIIA and IIB subunits. A 40% decrease in GluRIII staining correlates well with an identical decrease in GluRIIA and -IIB staining. It is plausible that there is essentially negligible change in functional glutamate receptor assembly at ICA69 mutant synapses. Third, ICA69 possibly plays a role in trafficking glutamate receptors to the postsynaptic density and is not rate limiting in the formation of functional glutamate receptor complexes. Thus, ICA69 mutants exhibit normal synaptic physiology without embracing other compensatory mechanisms such as reduced quantal size or increased quantal content (Mallik, 2017).

How might the iGluR levels relate to the NMJ growth? A tight correlation exists between the amount of synaptic glutamate receptors and the NMJ morphology. Downregulation of iGluRs in muscles has been shown to reduce the number of boutons. Similarly, hypomorphic mutants of GluRIII or GluRIIA have reduced bouton numbers. Consistent with this, overexpression of GluRIIA induces arborization and bouton number. Moreover, mutants with altered synaptic iGluR levels also show altered bouton numbers. For instance, neto and filamin (cheerio) mutants show reduced iGluR levels and bouton numbers. One of the possible mechanisms by which glutamate receptors can alter the NMJ morphology is through regulation of synaptic phospho-MAD levels. As the iGluRs (for instance, GluRIID) have also been shown to localize in central neuropil, it remains a possibility that the endogenous pattern of central electrical activity could also play crucial roles in sculpting the NMJ during development (Mallik, 2017).

Development of a tissue-specific ribosome profiling approach in Drosophila enables genome-wide evaluation of translational adaptations

Recent advances in next-generation sequencing approaches have revolutionized understanding of transcriptional expression in diverse systems. However, measurements of transcription do not necessarily reflect gene translation, the process of ultimate importance in understanding cellular function. To circumvent this limitation, biochemical tagging of ribosome subunits to isolate ribosome-associated mRNA has been developed. However, this approach, called TRAP, lacks quantitative resolution compared to a superior technology, ribosome profiling. This study reports the development of an optimized ribosome profiling approach in Drosophila. First, successful ribosome profiling was demonstrate from a specific tissue, larval muscle, with enhanced resolution compared to conventional TRAP approaches. Next the ability of this technology to define genome-wide translational regulation was validated. This technology was leveraged to test the relative contributions of transcriptional and translational mechanisms in the postsynaptic muscle that orchestrate the retrograde control of presynaptic function at the neuromuscular junction. Surprisingly, no evidence was found that significant changes in the transcription or translation of specific genes are necessary to enable retrograde homeostatic signaling, implying that post-translational mechanisms ultimately gate instructive retrograde communication. Finally, it was shown that a global increase in translation induces adaptive responses in both transcription and translation of protein chaperones and degradation factors to promote cellular proteostasis. Together, this development and validation of tissue-specific ribosome profiling enables sensitive and specific analysis of translation in Drosophila (Chen, 2017).

Acute fasting regulates retrograde synaptic enhancement through a 4E-BP-dependent mechanism

While beneficial effects of fasting on organismal function and health are well appreciated, little is known about the molecular details of how fasting influences synaptic function and plasticity. Genetic and electrophysiological experiments demonstrate that acute fasting blocks retrograde synaptic enhancement that is normally triggered as a result of reduction in postsynaptic receptor function at the Drosophila larval neuromuscular junction (NMJ). This negative regulation critically depends on transcriptional enhancement of eukaryotic initiation factor 4E binding protein (4E-BP) under the control of the transcription factor Forkhead box O (Foxo). Furthermore, these findings indicate that postsynaptic 4E-BP exerts a constitutive negative input, which is counteracted by a positive regulatory input from the Target of Rapamycin (TOR). This combinatorial retrograde signaling plays a key role in regulating synaptic strength. These results provide a mechanistic insight into how cellular stress and nutritional scarcity could acutely influence synaptic homeostasis and functional stability in neural circuits (Kauwe, 2016).

Many forms of dietary restriction can reduce cellular stress, improve organismal health, and in many instances extend lifespan in a number of model organisms. A major cellular function that is highly sensitive to nutrient intake from yeast to mammals is cap-dependent translation under the regulation of the target of rapamycin (TOR). TOR promotes cap-dependent translation primarily through phosphorylation of 4E-BPs (eukaryotic initiation factor 4E binding proteins) and p70 S6Ks (S6 ribosomal protein kinases). Phosphorylation of 4E-BP suppresses its ability to bind and inhibit the interaction between eIF4E (eukaryotic initiation factor 4E) and the initiation factor eIF4G, a critical step for translation initiation. In addition to the regulation by TOR, 4E-BP undergoes upregulation in response to dietary restriction and starvation. Together these two responses result in a strong inhibition of protein synthesis and act as a metabolic brake. Multiple lines of evidence suggest that fasting-induced increase in ketone bodies influences neuronal excitability and aspects of neurotransmitter release; however, little is known about how different forms of dietary restriction, by influencing protein translation, can exert an effect on the regulation of synaptic function and plasticity (Kauwe, 2016).

At the Drosophila larval neuromuscular junction (NMJ), the genetic removal of GluRIIA, one of five glutamate receptor subunits, reduces the postsynaptic response to unitary release of neurotransmitter. As a result of this reduced response to neurotransmitter, a retrograde signal is triggered in the postsynaptic muscle that ultimately leads to a compensatory enhancement in presynaptic release from the motor neuron, a process that is conserved at the vertebrate NMJs. The maintenance of this homeostatic synaptic compensation or retrograde synaptic enhancement is highly sensitive to postsynaptic cap-dependent translation in Drosophila; mutations in either Target of Rapamycin (TOR) or eIF4E can dominantly suppress the synaptic compensation in GluRIIA mutants (Penney, 2012). Interestingly, postsynaptic overexpression of TOR or S6K, in an otherwise wild-type muscle, is also sufficient to trigger a retrograde enhancement in presynaptic neurotransmitter release, suggesting that normal synaptic strength may be affected by a postsynaptic signal from the muscle (Penney, 2012; Kauwe, 2016 and references therein).

Previous findings have demonstrated that postsynaptic translation plays a critical role in the regulation of retrograde synaptic enhancement at the NMJ. Therefore, in light of the effect of dietary restriction on TOR-dependent translation, this study set out to investigate the consequence of nutrient restriction on retrograde synaptic compensation in GluRIIA mutants. Electrophysiological analysis indicates that acute fasting, but not amino acid restriction, blocks this retrograde synaptic compensation. This block is not merely due to reduced TOR activity, but rather a result of transcriptional upregulation of postsynaptic 4E-BP under the control of the transcription factor Foxo. These results indicate that the retrograde regulation of synaptic strength at the NMJ depends on the balance between 4E-BP and TOR (Kauwe, 2016).

A few hours of fasting can have a strong impact on retrograde synaptic enhancement at the Drosophila larval NMJ. Removal of food source acutely activates 4E-BP transcription in postsynaptic muscles in a Foxo-dependent manner, thereby leading to the inhibition of retrograde synaptic enhancement at the NMJ. The results indicate that Foxo and 4E-BP act cell autonomously in postsynaptic muscles to exert a retrograde negative regulation on presynaptic neurotransmitter release. Future studies are needed to test whether fasting-induced alterations in insulin signaling underlie the transcriptional upregulation of 4E-BP via its effect on Foxo in postsynaptic muscles. While 4E-BP-mediated suppression of synaptic enhancement as a result of fasting could be considered undesirable during development, it can be beneficial under conditions of abnormally high synaptic activity. As such, 4E-BP-mediated inhibition of retrograde synaptic enhancement and the subsequent dampening of circuit activity might provide an explanation for the beneficial effects of fasting in reducing seizures in some cases. Similarly, in cases where dysregulation of TOR activity is thought to underlie abnormal circuit activity, such as in TSC models, intermittent fasting could potentially dampen the increase in synaptic release through a 4E-BP-dependent inhibition, thereby stabilizing neuronal circuits (Kauwe, 2016).

In addition to its role as a molecular responder to stress, 4E-BP exerts a constitutive negative regulation on presynaptic neurotransmitter release at the NMJ. Electrophysiological analysis of loss-of-function mutant larvae indicates that 4E-BP functions in postsynaptic muscles to constitutively provide a retrograde negative influence on synaptic strength. In light of these findings, a two-pronged scheme is proposed for the retrograde regulation of synaptic strength at the NMJ. On the one hand, a positive input from TOR is mediated through S6K/eIF4A and eIF4E to enhance postsynaptic translation. Synaptic compensation in GluRIIA mutant larva appears to rely mostly on this axis as evidenced by strong sensitivity to S6K heterozygosity and no change in the proportion of phosphorylated 4E-BP versus non-phosphorylated 4E-BP levels. Opposing this positive input, 4E-BP inhibits translation by sequestering eIF4E and adjusting the degree of retrograde compensation. Indeed, loss of 4E-BP leads to a strong increase in quantal content that is highly sensitive to eIF4E heterozygosity but not sensitive to S6K heterozygosity. The balance between these two forces reveals itself also when 4E-BP loss-of-function mutants are rescued by a non-phosphorylatable 4E-BP transgene. In this combination TOR can no longer inhibit 4E-BP, and this study finds that the presynaptic neurotransmitter release is lower than wild-type, similarly to what is observed in TOR hypomorphic mutants (Penney, 2012). A working model is proposed in which the negative force of 4E-BP is under constant check via phosphorylation by TOR, and the positive input from TOR/S6K is constitutively countered by 4E-BP’s ability to sequester eIF4E, a dynamic duel that ensures a tight regulation of synaptic strength (Kauwe, 2016).

Disparate postsynaptic induction mechanisms ultimately converge to drive the retrograde enhancement of presynaptic efficacy

Retrograde signaling systems are fundamental modes of communication synapses utilize to dynamically and adaptively modulate activity. However, the inductive mechanisms that gate retrograde communication in the postsynaptic compartment remain enigmatic. This study investigated retrograde signaling at the Drosophila neuromuscular junction, where three seemingly disparate perturbations to the postsynaptic cell trigger a similar enhancement in presynaptic neurotransmitter release. This study shows that the same presynaptic genetic machinery and enhancements in active zone structure are utilized by each inductive pathway. However, all three induction mechanisms differ in temporal, translational, and CamKII activity requirements to initiate retrograde signaling in the postsynaptic cell. Intriguingly, pharmacological blockade of postsynaptic glutamate receptors, and not calcium influx through these receptors, is necessary and sufficient to induce rapid retrograde homeostatic signaling through CamKII. Thus, three distinct induction mechanisms converge on the same retrograde signaling system to drive the homeostatic strengthening of presynaptic neurotransmitter release (Goel, 2017).

The Drosophila neuromuscular junction (NMJ) is an established system to study retrograde synaptic signaling. At this model glutamatergic synapse, genetic or pharmacological perturbations to postsynaptic receptor functionality trigger retrograde signaling that instructs the neuron to precisely increase presynaptic neurotransmitter release, maintaining stable levels of synaptic strength. This process is termed presynaptic homeostatic potentiation (PHP) and can be induced through two distinct disruptions to postsynaptic glutamate receptor functionality. First, acute pharmacological blockade of receptors by application of philanthotoxin-433 (PhTx) reduces miniature excitatory postsynaptic potential (mEPSP) amplitude, initiating rapid expression of PHP (increase in quantal content) within 10 min. Second, genetic loss of the postsynaptic glutamate receptor subunit GluRIIA leads to a similar reduction in mEPSP amplitudes over chronic timescales (days) and a similar expression of PHP. Although these perturbations each disrupt receptors and lead to adaptive increases in presynaptic neurotransmitter release, PhTx- and GluRIIA-mediated PHP signaling exhibit important differences. First, some genes have been identified that are only necessary for GluRIIA-dependent PHP expression, whereas PHP is robustly expressed following acute PhTx application in larvae with mutations in these genes (Frank, 2009, Kauwe, 2016, Penney, 2016, Spring, 2016, Tsurudome, 2010). In addition, PhTx-induced PHP expression is translation-independent (Frank, 2006), whereas GluRIIA-induced PHP is blocked by inhibitions to postsynaptic translation through loss of the translational regulator target of rapamycin (Tor) (Kauwe, 2016, Penney, 2012). Although several genes and mechanisms necessary for the expression of PHP in the presynaptic neuron have been identified, far less is known about the mechanistic differences in postsynaptic transduction between PhTx- and GluRIIA-induced PHP signaling (Goel, 2017).

Recently, a novel manipulation to the postsynaptic muscle that does not affect glutamate receptors was demonstrated to induce retrograde PHP signaling at the Drosophila NMJ. This was accomplished by postsynaptic overexpression of the non-specific translational regulator Tor (Tor-OE) (Penney, 2012), which leads to a chronic and global increase in muscle protein synthesis (Chen, 2017). Although Tor-OE does not functionally affect glutamate receptors, somehow the increased muscle protein synthesis is converted into an instructive retrograde signal that appears to induce an enhancement in presynaptic glutamate release of a magnitude comparable with that observed in PhTx- and GluRIIA-mediated PHP (Penney, 2012). Although PhTx application, loss of GluRIIA, and Tor-OE each induce a similar enhancement in presynaptic release, to what extent they utilize separate or shared postsynaptic induction pathways, retrograde signaling systems, and modulations to presynaptic function is not known (Goel, 2017).

This study has characterized PHP signaling and expression when induced through PhTx application, loss of GluRIIA, and Tor-OE. This analysis has revealed that a common retrograde signaling system drives similar homeostatic adaptations in the presynaptic terminal but that separate inductive pathways differentially respond to glutamate receptor perturbation, Ca2+/calmodulin-dependent protein kinase II (CamKII) activity, and protein synthesis (Goel, 2017).

There appears to be a core set of genes necessary for both acute and chronic PHP expression, including ones involved in the homeostatic modulation of synaptic vesicle trafficking, presynaptic excitability, calcium channel activity, and active zone remodeling. However, other genes appear to be dispensable for this core program and may rather be involved in secondary functions, such as maintaining PHP expression over chronic timescales or supporting other aspects of homeostatic adaptation. Interestingly, the existence of multiple retrograde signaling pathways may be one reason for the failure of forward genetic approaches to identify any individual genes required in the muscle for the core process of PHP induction, suggesting some level of redundancy. This convergence of diverse induction mechanisms in the postsynaptic cell enables multiple pathways to detect and respond to homeostatic challenges by feeding into a unitary retrograde signaling system that potentiates presynaptic neurotransmitter release to stabilize synaptic strength (Goel, 2017).

CamKII activity plays a crucial role in gating diverse forms of synaptic plasticity. At the Drosophila NMJ, transgenic manipulations that affect postsynaptic CamKII activity have been reported to modulate the expression of PHP in GluRIIA mutants (Haghighi, 2003, Newman, 2017). The current results indicate that pCamKII levels are reduced to similar levels in GluRIIA mutants or following acute pharmacological receptor blockade, consistent with CamKII activity being capable of modulation in seconds at postsynaptic compartments. An attractive model would be that a reduction in calcium influx, either over 10 min or during chronic timescales, triggers diminished pCamKII levels and activates PHP signaling. However, this study found that pharmacological blockade of receptors is necessary and sufficient to reduce pCamKII levels at postsynaptic densities, independent of extracellular calcium, and that incubation in calcium-free saline alone is not sufficient to acutely induce PHP expression. Although there are several indications that reduced calcium influx in the postsynaptic muscle over chronic timescales likely contributes to PHP signaling, perhaps necessitating translation-dependent pathways, a calcium-independent system drives the acute expression of PHP following PhTx application, implying a distinct mechanism (Goel, 2017).

Two possibilities are considered to explain how PhTx application to glutamate receptors is transduced into PHP retrograde signaling without requiring calcium signaling through extracellular sources. First, PhTx binding to receptors may induce a conformational perturbation, distinct from ion influx through the receptor, to initiate PHP signaling. Such a mechanism could operate through a metabotropic mechanism, which would be unanticipated but not unprecedented. For example, at mammalian central synapses, the induction of N-methyl-D-aspartate (NMDA) receptor-dependent long term depression (LTD) does not require calcium influx through NMDA, but, rather, pharmacological perturbation to the receptor is sufficient. A metabotropic pathway has been proposed. Further, mammalian kainate receptors, to which the Drosophila glutamate receptors are homologous, are also capable of signaling through metabotropic mechanisms. Thus, pharmacological perturbation to GluRIIA-containing receptors could, in principle, initiate PHP signaling through an undefined metabotropic mechanism. However, at present, there is no evidence for such a mechanism in Drosophila (Goel, 2017).

Alternatively, pharmacological disruption of glutamate receptors may lead to local signaling at the NMJ through interactions with scaffolds such as Discs large (DLG)/PSD-95 and Dalcium/calmodulin dependent serine protein kinase (CASK). These scaffolds are known to be in complexes with CamKII and capable of modulating CamKII activity and phosphorylation at the subsynaptic reticulum (SSR). Intriguingly, defects in the elaboration of the SSR have recently been reported to disrupt retrograde homeostatic plasticity (Koles, 2015). CamKII signaling during PHP appears to be restricted to postsynaptic densities of type 1b boutons (Newman, 2017), suggesting that compartmentalized signaling at the SSR orchestrates local PHP signal transduction. In contrast, Tor-OE is capable of initiating PHP signaling independent of pCamKII reduction, where it promotes translation throughout the cell. This implies that protein synthesis modulates retrograde signaling downstream of or in parallel to CamKII signal transduction but ultimately feeds back into local post-translational signaling pathways. Future experiments probing the interactions between glutamate receptors, postsynaptic scaffolds, translation, and CamKII activity will clarify the signaling at this compartmentalized synapse (Goel, 2017).

The finding that PHP can be acutely induced by pharmacological perturbation of glutamate receptors and not through reductions in calcium influx over rapid timescales may help to explain perplexing observations regarding the phenomenology of PhTx-mediated PHP. For example, it was noted that PHP can be induced and expressed by a 10-min incubation of PhTx with only mEPSP events occurring. Although a reduction in calcium during these mEPSP events was discussed as a possible induction mechanism, estimates are that, at most, six mEPSP events occur per active zone during this induction time, a very low level and frequency of activity to reliably and robustly produce PHP expression. Indeed, a recent study demonstrated that mEPSP events account for a very small fraction (<1%) of the total postsynaptic calcium signal at individual NMJs (Newman, 2017), making a reduction in calcium even more implausible to explain acute PHP induction. Hence, pharmacological perturbation of postsynaptic glutamate receptors, rather than a reduction in calcium through these receptors, is an attractive mechanism to explain the characteristics of the acute induction of PHP by PhTx and raises interesting questions for future studies about how pharmacological receptor perturbation is transduced into PHP induction (Goel, 2017).

Why might a single retrograde signaling system exist to homeostatically stabilize synaptic strength at the Drosophila NMJ? In central neurons, diverse forms of synaptic plasticity, including Hebbian and homeostatic, dynamically operate over multiple timescales to bi-directionally adjust synaptic strength. Further, translation-dependent and independent processes also contribute to retrograde homeostatic signaling in the hippocampus following AMPA receptor blockade. In contrast, the NMJ is built for stable excitation and is acutely sensitive to reductions in receptor function. However, when neurotransmitter sensitivity in muscle is enhanced by increased receptor expression, no retrograde signaling system exists to homeostatically downregulate presynaptic efficacy. Thus, the muscle is endowed with multiple signaling systems to respond to perturbations but appears limited to signal retrograde increases in neurotransmitter release. Hence, a single retrograde signaling system might provide an efficient means to ensure non-additive potentiation in synaptic strength and prevent hyper-excitation when conflicting signals and multiple inductive mechanisms are simultaneously activated (Goel, 2017).

A postsynaptic PI3K-cII dependent signaling controller for presynaptic homeostatic plasticity

Presynaptic homeostatic plasticity stabilizes information transfer at synaptic connections in organisms ranging from insect to human. By analogy with principles of engineering and control theory, the molecular implementation of PHP is thought to require postsynaptic signaling modules that encode homeostatic sensors, a set point, and a controller that regulates transsynaptic negative feedback. The molecular basis for these postsynaptic, homeostatic signaling elements remains unknown. In this study, an electrophysiology-based screen of the Drosophila kinome and phosphatome defines a postsynaptic signaling platform that includes a required function for PI3K-cII, PI3K-cIII and the small GTPase Rab11 during the rapid and sustained expression of PHP. Evidence is presented that PI3K-cII localizes to Golgi-derived, clathrin-positive vesicles and is necessary to generate an endosomal pool of PI(3)P that recruits Rab11 to recycling endosomal membranes. A morphologically distinct subdivision of this platform concentrates postsynaptically where it is proposed to functions as a homeostatic controller for retrograde, trans-synaptic signaling (Hauswirth, 2018).

Homeostatic signaling systems stabilize the functional properties of individual neurons and neural circuits through life. Despite widespread documentation of neuronal homeostatic signaling, many fundamental questions remain unanswered. For example, given the potent action of homeostatic signaling systems, how can neural circuitry be modified during neural development, learning, and memory? Although seemingly contradictory, the homeostatic signaling systems that stabilize neural function throughout life may actually enable learning-related plasticity by creating a stable, predictable background upon which learning-related plasticity is layered. Therefore, defining the underlying molecular mechanisms of homeostatic plasticity may not only be informative about the mechanisms of neurological disease, these advances may be informative regarding how complex neural circuitry is able to accomplish an incredible diversity of behaviorally relevant tasks and, yet, retain the capacity for life-long, learning-related plasticity (Hauswirth, 2018).

Neuronal homeostatic plasticity encompasses a range of compensatory signaling that can be sub-categorized based upon the cellular processes that are controlled, including ion channel gene expression, neuronal firing rate, postsynaptic neurotransmitter receptor abundance and presynaptic vesicle release. Presynaptic homeostatic potentiation (PHP) is an evolutionarily conserved form of neuronal homeostatic control that is expressed at the insect, rodent and human neuromuscular junctions (NMJ) and has been documented at mammalian central synapses. PHP is initiated by the pharmacological inhibition of postsynaptic neurotransmitter receptors. The homeostatic enhancement of presynaptic vesicle release can be detected in a time frame of seconds to minutes, at both the insect and mouse NMJ. This implies the existence of postsynaptic signaling systems that can rapidly detect the disruption of neurotransmitter receptor function and convert this into retrograde, trans-synaptic signals that accurately adjust presynaptic neurotransmitter release. Notably, the rapid induction of PHP is transcription and translation independent, and does not include a change in nerve terminal growth or active zone number (Hauswirth, 2018).

There has been considerable progress identifying presynaptic effector molecules responsible for the expression of PHP. There has also been progress identifying postsynaptic signaling molecules that control synaptic growth at the Drosophila NMJ as well as the long-term, translation-dependent maintenance of PHP. However, to date, nothing is known about the postsynaptic signaling systems that initiate and control the rapid induction and expression of PHP (Hauswirth, 2018).

This paper reports the completion of an unbiased, forward genetic screen of the Drosophila kinome and phosphatome, and the identification of a postsynaptic signaling system for the rapid expression of PHP that is based on the activity of postsynaptic Phosphoinoside-3-Kinase (PI3K) signaling. There are three classes of PI3-Kinases, all of which phosphorylate the 3 position of phosphatidylinositol (PtdsIns). Class I PI3K catalyzes the conversion of PI(4,5)P₂ to PI(3,4,5)P₃ (PIP3) at the plasma membrane, enabling Akt-dependent control of cell growth and proliferation, and participating in the mechanisms of long-term potentiation. Class II and III PI3Ks (PI3K-cII and PI3K-cIII, respectively) both catalyze the conversion of PI to PI(3)P, which is a major constituent of endosomal membranes. PI(3)P itself may be a signaling molecule with switch like properties, functioning in the endosomal system as a signaling integrator. The majority of PI(3)P is synthesized by PI3K-cIII, which is involved in diverse cellular processes. By contrast, the cellular functions of PI3K-cII remain less well defined. PI3K-cII has been linked to the release of catecholamines, immune mediators, insulin, surface expression and recycling of integrins, and GLUT4 translocation to the plasma membrane, a mediator of metabolic homeostasis in muscle cells. This study demonstrates that Class II and Class III PI3K-dependent signaling are necessary for the rapid expression of PHP, controlling signaling from Rab11-dependent, recycling endosomes. By doing so, this study defines a postsynaptic signaling platform for the rapid expression of PHP and defines a novel action of PI3K-cII during neuronal homeostatic plasticity. This is the first established postsynaptic function for PI3K-cII at a synapse in any organism (Hauswirth, 2018).

Recently, it has become clear that the endosomal system has a profound influence on intracellular signaling and neural development. There is evidence that early and recycling endosomes can serve as sites of signaling intersection and may serve as signaling integrators and processors. Furthermore, protein sorting within recycling endosomes, and novel routes of protein delivery to the plasma membrane, may specify the concentration of key signaling molecules at the cell surface. The essential role of endosomal protein trafficking is underscored by links to synapse development and neurodegeneration. Yet, connections to homeostatic plasticity remain to be established. Based upon the data presented in this study and building upon prior work on endosomal signaling in other systems, it is speculated that postsynatpic PI3K-cII and Rab11-dependent recycling endosomes serve as as a postsynaptic 'homeostatic controller' that is essential for the specificity of retrograde, transsynaptic signaling (Hauswirth, 2018).

The Drosophila kinome and phosphatome were screened for genes that control the rapid expression of PHP. This screen identified three components of a conserved, postsynaptic lipid signaling pathway that is essential for the robust expression of PHP including: (1) class II PI3K, (2) class III PI3K (Vps34) and a gene encoding the Drosophila orthologue of PI4K (not examined in detail in this study). Pi3K68D was shown to be is essential, postsynaptically for PHP. Pi3K68D resides on a Clathrin-positive membrane compartment that is positioned directly adjacent to Golgi membranes, throughout muscle and concentrated at the postsynaptic side of the synapse. Pi3K68D is necessary for the maintenance of postsynaptic PI(3)P levels and the recruitment of Rab11 to intracellular membranes, likely PI(3)P-positive recycling endosomes. Postsynaptic Rab11 and Vps34 knockdown block PHP in an unusual, calcium-dependent manner that phenocopies Pi3K68D. Thus, this study has identified a postsynaptic signaling platform, centered upon the formation of PI(3)P and Rab11-positive recycling endosomes, that is essential for PHP (Hauswirth, 2018).

First it is considered whether postsynaptic Pi3K68D, Vps34 and Rab11 might alter PHP through modulation of postsynaptic glutamate receptor abundance. There is no consistent change in mEPSP amplitude in Pi3K68D mutants or following muscle-specific knockdown of Rab11 or Vps34 that could account for altered PHP. Therefore, functionally, there is no evidence for a change in glutamate receptor abundance at the postsynaptic membrane that could drive the phenotypic effects that were observe. Anatomically, data is presented examining GluR staining levels. In the Pi3K68D mutants, no change was found in GluRIIA levels. GluRIIA subunit containing receptors are the primary mediator of PhTx-dependent PHP. This study also reports a very modest (16%), though statistically significant, increase in GluRIIB levels. Based on these combined data, it seems unlikely that a change in GluR trafficking is a causal event leading to altered expression of PHP. It is noted that previous work showed limited GluRIIA receptor mobility within the PSD at the Drosophila NMJ. Thus, it is speculated that the function of Pi3K68D, Vps34 and Rab11 during PHP is not directly linked to postsynaptic GluR trafficking (Hauswirth, 2018).

Any model to explain the role of PI3K, Vps34 and Rab11-dependent endosomal signaling during homeostatic plasticity must account for the phenotypic observation that PHP is only blocked at low extracellular concentrations. More specifically, in animals deficient for Pi3K68D, Rab11 or Vps34, PHP is fully expressed at elevated calcium, following PhTX application or in the GluRIIA mutant. However, PHP completely fails when extracellular calcium is acutely decreased (following induction) below 0.7 mM [Ca2+]e. Clearly, the PHP induction mechanisms remain fully intact. Instead, the presynaptic expression of PHP has been rendered calcium-dependent. It is important to note that PHP can be fully induced in the absence of extracellular calcium, so the concentration of calcium itself is not the defect. In addition, this study documents trans-heterozygous interactions of Pi3K68D with presynaptic rim and dmp, arguing for the loss of trans-synaptic signaling and a specific function of Pi3K68D in the mechanisms of PHP. In very general terms, it is concluded that PI3K and Rab11-dependent endosomal signaling platform is necessary to enable the normal expression of PHP. Ultimately, some form of retrograde signaling must be defective due to either: 1) the absence of a retrograde signal that should have normally participated in PHP or 2) the presence of an aberrant or inappropriate signal that dominantly obstructs normal PHP expression. Both of these ideas in greater depth (Hauswirth, 2018).

First, the possibility is considered that the absence of postsynaptic PI3K and Rab11 signaling could alter the molecular composition or development of the presynaptic terminal due to the persistent absence of a retrograde signal that controls generalized synapse development or growth. Several observations demonstrate that impaired PHP is not a secondary consequence of a general defect in synapse development. Three independent postsynaptic manipulations are reported (postsynaptic expression of kinase dead Pi3K68D, postsynaptic knockdown of Rab11, and postsynaptic knockdown of Vps34) that have no effect on presynaptic release at any [Ca2+]e, yet block PHP at low [Ca2+]e. In addition, no obvious defect was found in anatomical synapse development (Hauswirth, 2018).

Next, the possibility is considered that postsynaptic PI3K and Rab11 signaling eliminate a retrograde signal that is specific for PHP. It was recently demonstrated that Semaphorin2b (Sema2b) and PlexinB (PlexB) define a retrograde signal at the Drosophila NMJ that is necessary for PHP. However, both Sema2b and PlexB are essential for the rapid induction of PHP, inclusive of experiments at low and elevated extracellular calcium. Further, acute application of recombinant Sema2b is sufficient to fully induce PHP. Since the induction of PHP remains fully intact in the Pi3K68D mutant, and since PHP is rendered calcium sensitive, it suggests that altered Sema2b secretion is not the cause of impaired PHP in the Pi3K68D mutant. Nevertheless, this possibility will be directly tested in the future (Hauswirth, 2018).

Next, the possibility is considered that the loss of PI3K and Rab11 signaling causes aberrant or inappropriate retrograde signaling, thereby impairing the expression of PHP. This is a plausible scenario because the induction of presynaptic homeostatic plasticity suffers from a common problem inherent to many intra-cellular signaling systems: two incompatible outcomes (1. presynaptic homeostatic potentiation and 2. presynaptic homeostatic depression -- PHD) are produced from a common input, and it remains unclear how signaling specificity is achieved. The topic of signaling specificity has been studied in several systems. One system, budding yeast, is a good example. Different pheromone concentrations can induce several distinct behaviors in budding yeast despite having a common input (pheromone concentration) and underlying signaling systems. Signaling specificity degrades in the background of mutations that affect Map Kinase scaffolding proteins. In a similar fashion, presynaptic homeostatic plasticity is induced by a change in mEPSP amplitude. A decrease in mEPSP amplitude causes the induction of PHP, whereas an increase in mEPSP amplitude causes the induction of presynaptic homeostatic depression (PHD). If a common sensor is employed to detect deviations in average mEPSP amplitude, how is this converted into the specific induction of either PHP or PHD? It has been shown that PHD and PHP can be sequentially induced. But, it remains unknown what would happen if the mechanisms of PHP and PHD were simultaneously induced. Under normal conditions this would never occur because mEPSP amplitudes cannot be simultaneously increased and decreased. But, if signaling specificity were degraded in animals lacking postsynaptic PI3K or Rab11, then the expression of PHP and PHD might coincide and create a mechanistic clash within the presynaptic terminal (Hauswirth, 2018).

Signaling and recycling endosomes are, in many respects, ideally suited to achieve signaling specificity during homeostatic plasticity. Signaling specificity can be achieved by mechanisms including sub-cellular compartmentalization of pathways, physically separating signaling elements with protein scaffolds, or through mechanisms of cross-pathway inhibition. Well-established mechanisms of protein sorting within recycling endosomes could physically compartmentalize signaling underlying PHP versus PHD. Alternatively, recycling endosomes can serve as a focal point for signal digitization, integration, and, perhaps, cross-pathway inhibition. Thus, it is proposed that the loss of postsynaptic PI3K and Rab11 compromises the function of the postsynaptic endosomal platform that this study has identified, thereby degrading homeostatic signaling specificity. As such, this platform could be considered a 'homeostatic controller' that converts homeostatic error signaling into specific, homeostatic, retrograde signaling for either PHP or PHD (Hauswirth, 2018).

Other models are considered, but are not favored. It remains formally possible that the calcium-sensitivity of PHP expression could be explained by a partially functioning PHP signaling system. This seems unlikely given that the same phenotype is observed in four independent genetic manipulations including a null mutation in Pi3K68D, postsynaptic expression of kinase dead Pi3K68D, postsynaptic knockdown of Rab11, and postsynaptic knockdown of Vps34. Furthermore, prior experiments examining hypomorphic and trans-heterozygous genetic interactions among essential PHP genes suggest that PHP is either diminished across the entire calcium spectrum or fully functional. So, there is no evidence that partial disruption of PHP could account for calcium-sensitive expression of PHP. Finally, the experiments argue against the possibility that compensatory changes in Vps34 expression partially rescue the Pi3K68D mutant phenotype (Hauswirth, 2018).

Another common signaling module that emerged from the genetic screen is considered. Both CamKII and CamKK were identified as potential hits. The identification of CamKII is supported by prior work showing the expression of dominant negative CamKII transgenes disrupt the long-term maintenance of PHP in the GluRIIA mutant background (Haghighi, 2003). It has been assumed that postsynaptic calcium is used to detect the PhTX or GluRIIA-dependent perturbation. But, the logic remains unclear. PHP is induced by diminished GluR function and, therefore, diminished postsynaptic calcium influx. This should diminish activation of CamKII and yet, loss of CamKII blocks PHP. An interesting alternative model is that calcium and calmodulin-dependent kinase activity facilitate the function of the postsynaptic endosomal membrane system. Both calcium and calmodulin are necessary for endosomal membrane fusion. In this manner, the action of CamKK and CamKII would be entirely consistent with the identification of Class II/III PI3K and Rab11 as homeostatic plasticity genes (Hauswirth, 2018).

This study has uncovered novel postsynaptic mechanisms that drive homeostatic plasticity. Eventually, continued progress in this direction may make it possible to not only reveal how stable neural function is achieved throughout life, but to uncover new rules that are essential for the processing of information throughout the nervous system. In particular, PHP has a very large dynamic range, whether one considers data from Drosophila or human NMJ or mammalian central synapses. The homeostatic control of presynaptic release can achieve a 7-fold change in synaptic gain, and yet retains the ability to offset even small changes in postsynaptic neurotransmitter receptor function. Thus, it is expected that the regulatory systems that achieve PHP will be complex and have a profound impact on brain function. This study has defined a postsynaptic signaling system responsible for the rapid expression of PHP and a novel, albeit speculative, model is proposed for the postsynaptic control of PHP, taking into account the need for signaling specificity. The identification of this pathway paves the way for future advances in understanding how homeostatic signaling is designed and implemented at a cellular and molecular level (Hauswirth, 2018).

Akt regulates glutamate receptor trafficking and postsynaptic membrane elaboration at the Drosophila neuromuscular junction

The Akt family of serine-threonine kinases integrates a myriad of signals governing cell proliferation, apoptosis, glucose metabolism, and cytoskeletal organization. Akt affects neuronal morphology and function, influencing dendrite growth and the expression of ion channels. Akt is also an integral element of PI3Kinase-target of rapamycin (TOR)-Rheb signaling, a pathway that affects synapse assembly in both vertebrates and Drosophila. Recent findings demonstrated that disruption of this pathway in Drosophila is responsible for a number of neurodevelopmental deficits that may also affect phenotypes associated with tuberous sclerosis complex, a disorder resulting from mutations compromising the TSC1/TSC2 complex, an inhibitor of TOR. Therefore, this study examined the role of Akt in the assembly and physiological function of the Drosophila neuromuscular junction (NMJ), a glutamatergic synapse that displays developmental and activity-dependent plasticity. The single Drosophila Akt family member, Akt1 selectively altered the postsynaptic targeting of one glutamate receptor subunit, GluRIIA, and was required for the expansion of a specialized postsynaptic membrane compartment, the subsynaptic reticulum (SSR). Several lines of evidence indicated that Akt1 influences SSR assembly by regulation of Gtaxin (Syntaxin18), a Drosophila t-SNARE protein in a manner independent of the mislocalization of GluRIIA. These findings show that Akt1 governs two critical elements of synapse development, neurotransmitter receptor localization, and postsynaptic membrane elaboratio (Lee, 2013).

This study explored Akt function in synapse development and function using a well-characterized model system, the Drosophila neuromuscular junction. There is a single Akt homolog in Drosophila, Akt1, facilitating the genetic and cellular studies of Akt function in synapse assembly (see Model for Akt1's regulatory role at the NMJ). Akt1 was specifically required for the correct assembly of A-type glutamate receptors. Reductions of Akt1 function either by mutation or RNA interference resulted in a loss of GluRIIA at the synapse paired with accumulation into intracellular structures. Reduction of Akt1 influenced the levels and localization of proteins shown to affect GluRIIA, Dorsal, and Cactus. Therefore, Akt1 could affect GluRIIA at least in part via control of these proteins. Akt1 was also required for the normal expansion of a specialized postsynaptic membrane compartment, the SSR. Evidence is provided that Akt1 mediates its effects on SSR via control of the t-SNARE Gtaxin. RNA interference of Gtaxin did not affect GluRIIA localization, showing that the control of SSR expansion and glutamate receptor composition mediated by Akt1 occurs via different molecular mechanisms (Lee, 2013).

The analysis of Akt1 reported in this study examined physiological, morphological, and cellular phenotypes, using both traditional Akt1 mutant alleles and cell-type directed knockdown achieved with either of two different UAS-Akt1^RNAi lines. The results from these different genetic tools were consistent and showed that Akt1 function is critical for both GluRIIA localization and SSR expansion. In particular, combinations of Akt1 alleles resulted in the redistribution of GluRIIA into intracellular bands, a phenotype found to be even more pronounced in muscle-directed RNAi of Akt1. This remarkable phenotype was also observed in larvae expressing both Akt1^RNAi and a UAS-transgene-derived GluRIIA-RFP in the muscle, the latter detected by either endogenous fluorescence or anti-RFP antibody. It was of note that fluorescent signal from the GluRIIA-RFP was reduced at the synapse but receptor mislocalization to intracellular compartments was detected only with anti-RFP antibody. Akt1-dependent events were clearly required for the proper formation of the folded RFP domain of the recombinant GluRIIA protein while the polypeptide, detected with the anti-RFP antibody was present and redirected to an alternative cellular location, as was observed for the endogenous GluRIIA. These data implicate Akt1 in processes of folding, stabilization, or assembly of GluRIIA (Lee, 2013).

A number of experiments were conducted to evaluate if Akt1 was required for the localization of specific postsynaptic proteins, or rather served a more generalized role in directing a variety of proteins to this membrane specialization. The correct localization of GluRIIB, GluRIIC, Basigin, Discs large, andSyndapin in animals with Akt1 knockdown in the muscle demonstrated that Akt1 has specific targeting functions for GluRIIA and is not a general factor for delivery of all postsynaptic proteins. Levels of these postsynaptic proteins were reduced in Akt1^RNAi bearing animals, not surprisingly given the substantial size reduction in the SSR (Lee, 2013).

At the Drosophila NMJ, two types of glutamate receptors have been defined by their distinct compositions and physiological properties. The shifting between A- and B-type receptors provides a mechanism for modulating postsynaptic responses to variable presynaptic inputs during development. There is considerable evidence that modulation of GluRIIA and B representation at the NMJ is governed by different signaling systems. Coracle, a homolog of protein 4.1 in Drosophila, has been shown to specifically influence the targeting of GluRIIA but not IIB. A physical interaction between Coracle and GluRIIA was essential for actin-dependent trafficking of GluRIIA-containing vesicles to the plasma membrane. Conversely, DLG has been shown to be required for GluRIIB but not GluRIIA localization at the NMJ. The current finding supports the conclusion that A and B receptor subunits are differentially regulated and show that Akt1 serves a role in A but not B subunit control (Lee, 2013).

There is evidence that the assembly and localization of GluRIIA into the postsynaptic density at the NMJ is accomplished following delivery to the plasma membrane. This conclusion is based upon the observation that fluorescence photobleaching of the entire muscle delays accumulation of new GluRIIA to synaptic sites more so than local bleaching at the NMJ (Rasse, 2005). The effects of Akt1 on GluRIIA localization could therefore be mediated by either regulated delivery of GluRIIA-containing vesicles to the plasma membrane, or by affecting the localization to the postsynaptic density following insertion into the plasma membrane. The accumulation of GluRIIA into an intracellular membrane compartments argues for a trafficking-based mechanism. This model is further supported by the results from the developmental timing experiments, where Akt1 function was removed during different stages in synapse assembly. Loss of Akt1 in a 2 day window early in development produced the phenotypes observed with continuous loss of Akt1, whereas a 2 day loss in third instar did not. If Akt1 simply served to retain GluRIIA at the synapse, there should have been time for new synthesis to repopulate the NMJ. Therefore, a model is favored where Akt1 affects developmental processes required for the selective delivery of GluRIIA from the endoplasmic reticulum into functional receptor units that arrive at the plasma membrane. It is notable that in mammalian systems, Akt is critical for the insulin-stimulated exocytosis of glucose transporter containing vesicles to the plasma membrane. Perhaps Akt1 governs similar exocytic processes at synapses. Akt1 signaling has also shown to be essential for AMPA receptor trafficking in hippocampal neurons, further supporting a role for Akt1 in trafficking of synaptic proteins (Lee, 2013).

A striking phenotype of animals with reduced Akt1 function in muscles was a severe reduction in the SSR and disruption of intracellular membrane organization. These phenotypes were similar to those found in a Gtaxin mutant and suggested the possibility that Akt1 and Gtaxin are involved in the same cellular process. A number of observations reported in this study indicate Akt1 activity is mediated at least in part by control of Gtaxin. First, Gtaxin levels at the SSR are greatly reduced in animals with reduced Akt1 function in the muscle cells. Second, muscle-directed overexpression of a constitutively active form of Akt1 (Akt1^CA) produced ectopic membranous structures; a phenotype also observed with Gtaxin overexpression and elevated levels of Gtaxin. Third, inhibition of Gtaxin blocks the effects of the constitutively active Akt1 in the muscle cell. Gtaxin does contain a consensus site for Akt1 phosphorylation and could therefore be a direct target of Akt1 kinase activity in regulating SNARE complex assembly (Lee, 2013).

The regulatory roles of Akt1 in glutamate receptor composition and postsynaptic membrane expansion could be accomplished through separate or identical downstream effectors. The fact that Gtaxin mutants did not disrupt GluRIIA distribution suggests different downstream effectors regulated by Akt1. The regulation of GluRIIA localization by Akt1 does not involve Gtaxin but could be mediated via Dorsal and Cactus. Dorsal and Cactus influence glutamate receptor delivery and are known effectors of Akt activity in mammalian cells. The levels of both Dorsal and Cactus were reduced in animals with knockdown of Akt1 in the muscle. Notably, in some animals expressing Akt1^RNAi in the muscle, Dorsal showed an altered intracellular distribution that overlapped with the mislocalized GluRIIA. However, because Dorsal and Cactus mutants are not reported to mislocalize GluRIIA into intracellular bands, Akt1 is likely to have additional downstream targets that influence GluRIIA localization and delivery to the postsynaptic specialization (Lee, 2013).

Physiological measures of synaptic transmission showed that Akt1 function is required for normal synapse function. Akt1 transheterozygous mutants (Akt1¹/Akt1⁰⁴²²⁶) showed reduced EJP amplitudes and altered decay kinetics of the EJP. These same phenotypes were observed in animals with muscle-specific inhibition of Akt1 function, with the severity correlating to the degree of Akt1 inhibition. These changes in EJP kinetics were not accompanied by alterations of nonvoltage-dependent membrane capacitance or resistance, suggesting that voltage-gated channels contributing to EJP rise and decay times may be affected by Akt1. These findings contrast published work with Akt1 mutant animals describing changes in long-term depression but not in EJP properties. However, it is noted that the physiological studies reported in this paper were conducted at a higher Ca²⁺ concentration, which could account for these different measures of EJP properties in Akt1 mutants. It is important to point out that the physiological changes documented in this study observed in both Akt1 mutant larvae as well as animals with RNA interference of Akt1 in the muscle cell. The physiological changes observed in Akt1 compromised animals are logical consequences of observed changes in NMJ composition. Loss of GluRIIA-containing receptors and an overall decrease in functional GluRs at the synapse could decrease the EJP amplitude. The altered EJP decay pattern in animals with reduced Akt1 is consistent with the involvement of Gtaxin, as has been documented in this study. Gtaxin mutants showed similar changes in EJP decay, indicating that this feature of Akt1 mediated physiological change is associated with the consequences of compromising the function of this t-SNARE (Lee, 2013).

There is a precedent for Akt-mediated regulation of neurotransmitter receptor localization to the cell surface. The NMDA receptor subunit NR2C is developmentally regulated in cerebellar granule cells and Akt-mediated phosphorylation is critical for cell surface expression of NR2C-containing receptors. Akt has also proven to be important in the elaboration of dendritic complexity in Drosophila sensory neurons, suggesting that this kinase is of general importance in the control of nervous system receptive fields. Selective control of Akt or its downstream targets could provide a powerful method of influencing synaptic transmission and the receptive properties of neurons (Lee, 2013).

Regulation of SH3PX1 by dNedd4-long at the Drosophila Neuromuscular Junction

Drosophila Nedd4 (dNedd4) is a HECT E3 ubiquitin ligase present in two major isoforms: short (dNedd4S) and long (dNedd4Lo), with the latter containing two unique regions (N-terminus and Middle). While dNedd4S promotes neuromuscular synaptogenesis (NMS), dNedd4Lo inhibits it and impairs larval locomotion. To explain how dNedd4Lo inhibits NMS, mass spectrometry was performed to find its binding partners and identified SH3PX1, which binds dNedd4Lo unique Middle region. SH3PX1 contains SH3, PX and BAR domains and is present at neuromuscular junctions, where it regulates active zone ultrastructure and presynaptic neurotransmitter release. This study demonstrates direct binding of SH3PX1 to the dNedd4Lo Middle region (which contains a Pro rich sequence) in vitro and in cells, via the SH3PX1-SH3 domain. In Drosophila S2 cells, dNedd4Lo overexpression reduces SH3PX1 levels at the cell periphery. In vivo overexpression of dNedd4Lo post-synaptically, but not pre-synaptically, reduces SH3PX1 levels at the subsynaptic reticulum and impairs neurotransmitter release. Unexpectedly, larvae that overexpress dNedd4Lo post-synaptically and are heterozygous for a null mutation in SH3PX1 display increased neurotransmission compared to dNedd4Lo or SH3PX1 mutant larvae alone, suggesting a compensatory effect from the remaining SH3PX1 allele. These results suggest a postsynaptic - specific regulation of SH3PX1 by dNedd4Lo (Wasserman, 2018).

Calcium-activated Calpain specifically cleaves Glutamate Receptor IIA but not IIB at the Drosophila neuromuscular junction

Calpains are calcium-dependent, cytosolic proteinases active at neutral pH. They do not degrade but cleave substrates at limited sites. Calpains are implicated in various pathologies, such as ischemia, injuries, muscular dystrophy, and neurodegeneration. Despite so, the physiological function of calpains remains to be clearly defined. Using the neuromuscular junction of Drosophila of both sexes as a model, RNAi screening was performed, and calpains were found to negatively regulated protein levels of the glutamate receptor GluRIIA but not GluRIIB. Calpains enrich at the postsynaptic area, and the calcium-dependent activation of calpains induced cleavage of GluRIIA at Q788 of its C terminus. Further genetic and biochemical experiments revealed that different calpains genetically and physically interact to form a protein complex. The protein complex was required for the proteinase activation to downregulate GluRIIA. These data provide a novel insight into the mechanisms by which different calpains act together as a complex to specifically control GluRIIA levels and consequently synaptic function (Metwally, 2019).

Calpains are a family of calcium-activated cytoplasmic cysteine proteases, which are ubiquitously expressed in various mammalian tissues and are functionally active at neutral pH. Members of the calpain family act in pathological processes associated with calcium overload, such as ischemia and Alzheimer's disease. For example, calpains cleave multiple synaptic proteins, including both inotropic and metabotropic glutamate receptors in excitotoxic conditions, as well as in synaptic plasticity (Chan, 1999; Xu, 2007; Doshi, 2009; Baudry, 2016). However, little is known of the precise physiological function of calpain-dependent cleavage of target proteins at synapses during normal development (Metwally, 2019).

Calpains exist in organisms ranging from bacteria to humans. To date, 15 different calpains have been identified in mammals and four in Drosophila. Unlike most proteases, calpains do not destroy but cleave their substrates at limited sites to modulate their function. Calpain 1 (μ-calpain) and calpain 2 (m-calpain) are the most ubiquitous and well-studied calpain family members. They are activated in vitro by micromolar and millimolar concentrations of calcium, respectively. Conventional calpains 1 and 2 are heterodimers composed of a large catalytic subunit with four domains (dI-dIV) and a small regulatory subunit with two domains (dV and dVI). Heterodimerization of the large and small subunits occurs through a unique interaction between their C-terminal domains. There are two calcium-binding sites within the crystal structure of the enzymatically active domain II of μ-calpain. These sites, together with domains IV and VI, interact with calcium and are required for full enzymatic activity. Calpastatin is an endogenous calpain inhibitor that binds and inhibits calpains via its calpain-inhibitor domains when the proteases are activated by calcium (Hanna, 2008). Unlike mammalian calpains, Drosophila calpains comprise a large subunit only and calpastatin has not been identified in Drosophila (Metwally, 2019).

The efficiency of neurotransmission is determined by the level of neurotransmitter receptors at the postsynaptic densities (PSDs). However, the molecular mechanism by which the level of neurotransmitter receptors at synapses is regulated is not well understood. At Drosophila neuromuscular junction (NMJ) synapses, there are two subtypes of glutamate receptors (GluR), GluRIIA and GluRIIB, which are developmentally, biophysically, and pharmacologically distinct. For example, the postsynaptic sensitivity to glutamate is much higher at synapses enriched for GluRIIA than at synapses enriched for GluRIIB. Previous studies show that GluRIIA and GluRIIB receptors are differentially regulated via independent pathways, although the molecular mechanisms that control different subtypes of GluRs remain elusive. To uncover new pathways that control GluR expression at NMJ synapses, a genetic screen was carried out identified a critical role was identified for calpains in specifically regulating the abundance of GluRIIA, but not GluRIIB, at the Drosophila NMJs. Valpains enrich at the postsynaptic area and cleave GluRIIA, but not GluRIIB, at its C-terminal in a calcium-dependent manner. As calcium is a highly versatile intracellular signal that regulates many different cellular functions, the finding of calpain proteinases activated by calcium under physiological conditions at NMJ synapses offers novel insights into the myriad of calcium-mediated processes at other cellular contexts (Metwally, 2019).

Neurotransmission efficiency is determined by the level of neurotransmitter receptors at the PSDs. Therefore, the regulation of glutamate receptors at synapses has been under intensive studies. However, the molecular mechanism responsible for regulating the expression of neurotransmitter receptors at synapses is still poorly understood. Using genetic screening, together with optogenetic manipulation and calcium treatment, this study has revealed that GluRIIA protein levels were specifically and negatively regulated by calcium-dependent calpains during development (Metwally, 2019).

Extensive studies have demonstrated robust homeostatic regulation at the Drosophila NMJ. Genetic or pharmacological manipulations that impair the protein level or activity of postsynaptic GluRs cause a compensatory increase in presynaptic action potential-evoked neurotransmitter release; the increase in evoked release precisely compensates for the decrease in GluR sensitivity and, thereby, maintains a normal level of transmission. This study has shown that calpains negatively regulated mEJP amplitude and frequency at NMJs, consistent with previous studies demonstrating that quantal size and mEJP frequency are increased when GluRIIA is overexpressed. The increase in mEJP amplitude and frequency in calpain mutants can be explained by the elevated levels of GluRIIA and Brp protein at synapses. GluRIIA mutant exhibits a large decrease in mEJP amplitude with no change in EJP amplitude, probably due to homeostatic regulation, whereas overexpression of GluRIIA leads to an increase in mEJP amplitude with an increase of EJP amplitude, indicating that there is no compensatory downregulation of quantal content. In the latter case, different effects on EJP amplitude were observed in a more quantitative manner by using a different gene dosage of GluRIIA. Specifically, increased EJP amplitude is observed at synapse overexpressing a low level of GluRIIA, whereas overexpression of GluRIIA at a high level leads to normal EJP amplitude. This latter case is consistent with the finding that a high threefold increase of GluRIIA level in calpains mutant did not lead to upregulation of EJP amplitude. It is possible that the increase in mEJP amplitude is compensated by a downregulation of quantal content to maintain normal EJP amplitude in calpain mutants. The finding of normal EJP amplitudes regardless of upregulation or downregulation of GluRIIA abundance upon calpain manipulations further support homeostatic regulation at Drosophila NMJ (Metwally, 2019).

Previous studies showed that GluRIIA and GluRIIB exhibit competing effects and interdependence as a result of the selective absence of GluRIIA or GluRIIB type at NMJ. If the primary effect of calpains was specifically on GluRIIA, it would not be expected to see an opposite change in GluRIIB level. However, this is not what was observed. Instead, it was observed that GluRIIB was normally localized and its levels were unchanged when GluRIIA was upregulated, resulting from calpain mutation or knockdown, suggesting a distinct regulation of subunit composition by calpains at the NMJ (Metwally, 2019).

At the Drosophila NMJ, the postsynaptic calcium has been speculated to regulate the sensitivity and synaptic localization of GluRIIA. However, how calcium regulates GluRIIA at NMJ is yet to be investigated. The findings in the present study showed a critical role of calcium in regulating postsynaptic GluRIIA levels via calpain. In Drosophila muscles and S2 cells, calcium-dependent activation of calpain resulted in the C terminus cleavage of GluRIIA but not GluRIIB. It is noted that the percentage of cleaved GluRIIA by calpains is low by Western analysis. This low cleavage efficiency is consistent with previous findings. However, immunostaining showed apparent changes in GluRIIA level at NMJ upon manipulating calpain expressions. Multiple possibilities could explain the discrepancy between low cleavage efficiency by Western analysis and apparent changes in GluRIIA level by immunostaining. First, synaptic GluRIIA is part of the total GluRIIA and calpain may preferentially cleave a subset of synaptic GluRIIA, which is physically accessible and sensitive to calpain cleavage due to specific post-translational modifications. The enzymatic activity of calpains could also be differentially regulated at different cellular locations. Alternatively, calpain may indirectly regulate GluRIIA level by other mechanism, such as translation regulation (Metwally, 2019).

There are a few reports that calpain is involved in the regulation of protein synthesis. For example, ischemia-induced calpain activation targets translation initiation factor 4G1 for cleavage, leading to reduced translation. On the other hand, calpain stimulates protein synthesis by truncating B56a, the regulatory subunit of PP2A which inhibits the Akt/mTOR/S6 signaling during mGluR-dependent LTD. Although multiple independent lines of evidence support that Drosophila calpains specifically target GluRIIA for cleavage, there is still a possibility that calpain negatively regulates GluRIIA level through other unknown mechanisms, including translational regulation (Metwally, 2019).

Where does GluRIIA get cleaved by calpains within the cell? As calpains are enriched in the postsynaptic area where calcium transients are produced by spontaneous and evoked transmitter release, it is possible that calpain cleaves GluRIIA at the postsynaptic area. Alternatively, calpain might cleave GluRIIA at the endoplasmic reticulum when it is synthesized. As an obvious decrease in GluRIIA intensity was observed upon calcium treatment for a short time of 30 min, the first scenario is favored, in which calpain cleaves GluRIIA mostly at the postsynaptic site (Metwally, 2019).

Calpain interacts with and cleaves dozens of target proteins. The present studyfound that calpains specifically target GluRIIA but not other synaptic proteins at the Drosophila NMJ terminals. A physical interaction between calpain and GluRIIA in a calcium-dependent manner. This is the first demonstration of calcium-facilitated interaction of calpain with its target protein in cultured cells. It will be of interest to test whether other substrates of calpains identified so far also interact with calpains in a calcium-dependent manner (Metwally, 2019).

Calpain activation is triggered by abnormally high calcium levels associated with pathologies, such as ischemic insults and neurodegeneration, but the in vivo role of calpain under physiological conditions is largely unknown. This genetic and optogenetic results indicate that calcium-induced activation of calpains is likely to occur under certain physiological conditions at NMJ synapses during development. As calcium signaling is involved in a variety of molecular and cellular processes in development and disease, the current findings offer a novel insight into the role of calcium signaling pathways. How and where calpains are activated by calcium within a cell remain to be elucidated (Metwally, 2019).

Multiple independent lines of evidence showed that different calpains, at least calpains A, B, and D, form a complex in which each is essential for the normal proteinase activity to control GluRIIA level. Drosophila calpain B was originally demonstrated to work as monomers based on in vitro studies of Escherichia coli-produced recombinant protein (Park, 2008), rather than as heterodimers, such as mammalian calpains 1 and 2. However, the current results showed that different calpains acted together in vivo to downregulate GluRIIA. Specifically, this study showed that different calpains colocalized and interacted physically in vivo and in cultured cells. Drosophila calpains do not form homodimers, at least for calpain A (this study) and calpain B (Park, 2008), but instead formed multimers consisting of different calpains, although the domain mediating calpain-calpain interaction remains to be defined. The finding of a calpain complex is supported by a previous study demonstrating that different calpains in Drosophila function synergistically downstream of calcium transients to trigger dendritic pruning of sensory neurons during metamorphosis (Kanamori, 2013). Similarly, mammalian calpains 8 and 9, which are specifically expressed in the gastrointestinal tract, interact physically to form a protein complex called 'gastric calpain' involving in gastric mucosal defense. Thus, in addition to the conventional calpain heterodimers, such as calpains 1 and 2, consisting of a large and a small subunit, heterodimers or heteromultimers of different calpains without a small subunit are emerging as a new mode of calpain activity regulation (Metwally, 2019).

Ttm50 facilitates calpain activation by anchoring it to calcium stores and increasing its sensitivity to calcium

Calcium-dependent proteolytic calpains are implicated in a variety of physiological processes, as well as pathologies associated with calcium overload. However, the mechanism by which calpain is activated remains elusive since intracellular calcium levels under physiological conditions do not reach the high concentration range required to trigger calpain activation. From a candidate screening using the abundance of the calpain target glutamate receptor GluRIIA at the Drosophila neuromuscular junction as a readout, this study uncovered that calpain activity was inhibited upon knockdown of Ttm50, a subunit of the Tim23 complex known to be involved in the import of proteins across the mitochondrial inner membrane. Unexpectedly, Ttm50 and calpain are co-localized at calcium stores Golgi and endoplasmic reticulum (ER), and Ttm50 interacts with calpain via its C-terminal domain. This interaction is required for calpain localization at Golgi/ER, and increases calcium sensitivity of calpain by roughly an order of magnitude. These findings reveal the regulation of calpain activation by Ttm50, and shed new light on calpain-associated pathologies (Metwally, 2020).

Cul3 and insomniac are required for rapid ubiquitination of postsynaptic targets and retrograde homeostatic signaling

At the Drosophila neuromuscular junction, inhibition of postsynaptic glutamate receptors activates retrograde signaling that precisely increases presynaptic neurotransmitter release to restore baseline synaptic strength. However, the nature of the underlying postsynaptic induction process remains enigmatic. In this study a forward genetic screen is described to discover factors in the postsynaptic compartment necessary to generate retrograde homeostatic signaling. This approach identified insomniac (inc), a putative adaptor for the Cullin-3 (Cul3) ubiquitin ligase complex, which together with Cul3 is essential for normal sleep regulation. Interestingly, it was found that Inc and Cul3 rapidly accumulate at postsynaptic compartments following acute receptor inhibition and are required for a local increase in mono-ubiquitination. Finally, it was shown that Peflin, a Ca(2+)-regulated Cul3 co-adaptor, is necessary for homeostatic communication, suggesting a relationship between Ca(2+) signaling and control of Cul3/Inc activity in the postsynaptic compartment. This study suggests that Cul3/Inc-dependent mono-ubiquitination, compartmentalized at postsynaptic densities, gates retrograde signaling and provides an intriguing molecular link between the control of sleep and homeostatic plasticity at synapses (Kikuma, 2019).

By screening >300 genes with putative functions at synapses, this study has identified inc as a key postsynaptic regulator of retrograde homeostatic signaling at the Drosophila NMJ. The data suggest that Inc and Cul3 are recruited to the postsynaptic compartment within minutes of glutamate receptor perturbation, where they promote local mono-ubiquitination. Inc/Cul3 appear to function downstream of or in parallel to CaMKII and upstream of retrograde signaling during PHP. Pef was identified as a putative co-adaptor that may work with Inc/Cul3 to link Ca2+ signaling in the postsynaptic compartment with membrane trafficking and retrograde communication. Altogether, these findings implicate a post translational signaling system involving mono-ubiquitination in the induction of retrograde homeostatic signaling at postsynaptic compartments (Kikuma, 2019).

Although forward genetic screens have been very successful in identifying genes required in the presynaptic neuron for the expression of PHP, these screens have provided less insight into the postsynaptic mechanisms that induce retrograde homeostatic signaling. It seems clear that many genes acting presynaptically are individually required for PHP, with loss of any one completely blocking PHP expression. Indeed, ~25 genes that function in neurons have thus far been implicated in PHP expression. In contrast, forward genetic screens have largely failed to uncover new genes functioning in the postsynaptic muscle during PHP, implying some level of redundancy. The specific postsynaptic induction mechanisms driving retrograde PHP signaling have therefore remained unclear, and are further complicated by cap-dependent translation and metabolic pathways that contribute to sustaining PHP expression over chronic, but not acute, time scales. Therefore, it is perhaps not surprising that despite screening hundreds of mutants, this study found only a single gene, insomniac, to be required for PHP induction. Inc is expressed in the nervous system and can traffic to the presynaptic terminals of motor neuron. In the context of PHP signaling, however, inc was found to be required in the postsynaptic compartment, where it functions downstream of or in parallel to CaMKII. One attractive possibility is that a reduction in CaMKII-dependent phosphorylation of postsynaptic targets enables subsequent ubiquitination by Cul3-Inc complexes, and that this modification ultimately drives retrograde signaling during PHP. Indeed, reciprocal influences of phosphorylation and ubiquitination on shared targets are a common regulatory feature in a variety of signaling systems. The dynamic interplay of phosphorylation and ubiquitination in the postsynaptic compartment may enable a sensitive and tunable mechanism for controlling the timing and calibrating the amplitude of retrograde signaling at the NMJ (Kikuma, 2019).

The substrates targeted by Inc and Cul3 during PHP induction are not known, but the identification of mono-ubiquitination in the postsynaptic compartment during PHP signaling and the putative Cul3 co-adaptor Peflin provides a foundation from which to assess possible candidates and pathways. In mammals, Pef forms a complex with another Ca2+ binding protein, ALG2, to confer Ca2+ regulation to membrane trafficking pathways. Moreover, Pef/ALG2 were recently found to serve as target-specific co-adaptors for Cul3-KLHL12. In particular, SEC31 and other components involved in ER-mediated membrane trafficking pathways were shown to be targeted for mono-ubiquitination, which in turn modulate Collagen secretion. One attractive possibility, therefore, is that Cul3/Inc could respond to changes in Ca2+ in the postsynaptic compartment through regulation by Pef during PHP signaling to control membrane trafficking pathways. Importantly, the subsynaptic reticulum (SSR) is a complex and membraneous network at the Drosophila NMJ, where electrical, Ca2+-dependent, and membrane trafficking pathways in the postsynaptic compartment are integrated (Teodoro, 2013; Nguyen, 2016). Indeed, Multiplexin, a fly homolog of Collagen XV/XVIII and a proposed retrograde signal, is secreted into the synaptic cleft and is required for trans-synaptic retrograde signaling during PHP (Wang, 2014). In addition, another proposed retrograde signal and secreted protein, Semaphorin 2B, was recently shown to function postsynaptically in retrograde PHP signaling (Orr, 2017b). However, inc does not appear to be the closest Drosophila ortholog to KLHL12, and it is therefore possible that Pef and Cul3/Inc regulate postsynaptic PHP signaling through a more indirect mechanism (Kikuma, 2019).

While the precise relationships between CaMKII, Inc, Cul3, and Pef are currently unclear, the activity of membrane trafficking pathways could ultimately be targeted for modulation by Ca2+- and Cul3/Inc-dependent signaling during PHP induction. First, a role for postsynaptic membrane trafficking and elaboration during PHP signaling has already been suggested. In addition, extracellular Ca2+ does not appear to be involved in rapid PhTx-dependent PHP induction. It is therefore tempting to speculate that Ca2+ release from the postsynaptic SSR during rapid PHP signaling may influence Cul3/Inc activity through Pef-dependent regulation, as transient changes in ER-derived Ca2+-signaling controls Pef-dependent recruitment of Cul3 (McGourty, 2016). Alternatively, postsynaptic scaffolds and/or glutamate receptors themselves may be targeted by Cul3/Inc at the Drosophila NMJ, given that these proteins are involved in ubiquitin-mediated signaling and remodeling at dendritic spines. Consistent with this idea, there is evidence that signaling complexes composed of neurotransmitter receptors, CaMKII, and membrane-associated guanylate kinases are intimately associated at postsynaptic densities in Drosophila, as they are in the mammalian central nervous system. There has been speculation that these complexes are targets for modulation during PHP signaling. Although these models are not mutually exclusive, further studies will be required to determine the specific substrates and signal transduction mechanisms through which Cul3/Inc and Pef initiate and sustain retrograde homeostatic communication in postsynaptic compartments (Kikuma, 2019).

While it is well established that the ubiquitin proteasome system can sculpt and remodel synaptic architecture, the importance of mono-ubiquitination at synapses is less studied. Ubiquitin-dependent pathways play key roles in synaptic structure, function, and degeneration, and also contribute to activity-dependent dendritic growth. However, the fact that some proteins persist for long periods at synapses suggests that modification of these proteins by ubiquitin likely include non-degredative and reversible mechanisms. Indeed, a recent study revealed a remarkable heterogeneity in the stability of synaptic proteins, with some short lived and rapidly turned over, while others persisting for long time scales, with half lives of months or longer. At the Drosophila NMJ, rapid ubiquitin-dependent proteasomal degradation at presynaptic terminals is necessary for the expression of PHP through modulation of the synaptic vesicle pool (Wentzel, 2018). In contrast, postsynaptic proteasomal degradation does not appear to be involved in rapid PHP signaling, suggesting that ubiquitin-dependent pathways in the postsynaptic compartment contribute to PHP signaling by non-degradative mechanisms. The current data demonstrate that Cul3, Inc, and Pef function in muscle to enable retrograde PHP signaling, and suggest that Cul3/Inc rapidly trigger mono-ubiquitination at postsynaptic densities following glutamate receptor perturbation. Interestingly, synaptic proteins can be ubiquitinated in <15 s following depolarization-induced Ca2+ influx at synapses (Chen, 2003) and changes in intracellular Ca2+ can activate Pef and Cul3 signaling with similar rapidity. Therefore, both poly- and mono-ubiquination may function in combination with other rapid and reversible processes, including phosphorylation at postsynaptic compartments to enable robust and diverse signaling outcomes during the induction of homeostatic plasticity (Kikuma, 2019).

A prominent hypothesis postulates that a major function of sleep is to homeostatically regulate synaptic strength following experience-dependent changes that accrue during wakefulness. Several studies have revealed changes in neuronal firing rates and synapses during sleep/wake behavior, yet few molecular mechanisms that directly associate the electrophysiological process of homeostatic synaptic plasticity and sleep have been identified. The finding that inc is required for the homeostatic control of synaptic strength provides an intriguing link to earlier studies, which implicate inc in the regulation of sleep. It remains to be determined to what extent the role of inc in controlling PHP signaling at the NMJ is related to the impact of inc on sleep and, if so, whether Inc targets the same substrates to regulate these processes. Interestingly, virtually all neuropsychiatric disorders are associated with sleep dysfunction, including those associated with homeostatic plasticity and Fragile X Syndrome, and sleep behavior is also disrupted by mutations in the Drosophila homolog of FMRP, dfmr1. Further investigation of this intriguing network of genes involved in the homeostatic control of sleep and synaptic plasticity may help solve the biological mystery that is sleep and also shed light on the etiology of neuropsychiatric diseases (Kikuma, 2019).

Retrograde signaling by Syt 4 induces presynaptic release and synapse-specific growth

The molecular pathways involved in retrograde signal transduction at synapses and the function of retrograde communication are poorly understood. This study demonstrates that postsynaptic calcium 2+ ion (Ca2+) influx through glutamate receptors and subsequent postsynaptic vesicle fusion trigger a robust induction of presynaptic miniature release after high-frequency stimulation at Drosophila neuromuscular junctions. An isoform of the synaptotagmin family, synaptotagmin 4 (Syt 4), serves as a postsynaptic Ca2+ sensor to release retrograde signals that stimulate enhanced presynaptic function through activation of the cyclic adenosine monophosphate (cAMP)-cAMP-dependent protein kinase pathway. Postsynaptic Ca2+ influx also stimulates local synaptic differentiation and growth through Syt 4-mediated retrograde signals in a synapse-specific manner (Yoshihara, 2005).

Neuronal development requires coordinated signaling to orchestrate pre- and postsynaptic maturation of synaptic connections. Synapse-specific enhancement of synaptic strength as occurs during long-term potentiation, as well as compensatory homeostatic synaptic changes, have been suggested to require retrograde signals for their induction. Although retrograde signaling has been implicated widely in synaptic plasticity, the molecular mechanisms that transduce postsynaptic Ca²⁺ signals during enhanced synaptic activity to alterations in presynaptic function are poorly characterized. Because postsynaptic Ca²⁺ is essential for synapse-specific potentiation, it is important to characterize how Ca²⁺ can regulate retrograde communication at synapses (Yoshihara, 2005).

To dissect the mechanisms underlying activity-dependent synaptic plasticity, test were performed to see whether newly formed Drosophila glutamatergic neuromuscular junctions (NMJs), which have ∼30 active zones, show physiological changes after 100-Hz stimulation (5-1552+ chelator EGTA from the patch pipette caused a modest suppression of HFMR, whereas the fast Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) induced strong suppression by 2.5 min of perfusion. Longer perfusion with BAPTA for 5 min before stimulation abolished HFMR, indicating HFMR is induced after postsynaptic Ca²⁺ influx (Yoshihara, 2005).

Ca²⁺-induced vesicle fusion in presynaptic terminals provides a temporally controlled and spatially restricted signal essential for synaptic communication. Postsynaptic vesicles within dendrites have been visualized by transmission electron microscopy, and dendritic release of several neuromodulators has been reported. To test whether postsynaptic vesicle fusion might underlie the Ca²⁺-dependent release of retrograde signals, postsynaptic vesicle recycling was blocked by using the dominant negative shibire^ts1 mutation, which disrupts endocytosis at elevated temperatures. shibire^ts1 was expressed specifically in postsynaptic muscles by driving a UAS-shibire^ts1 transgene with muscle-specific myosin heavy chain (Mhc)-Gal4, keeping presynaptic activity intact. At the permissive temperature (23°C), high-frequency stimulation induced normal HFMR. However, raising the temperature to 31°C suppressed HFMR in the presence of postsynaptic shibire^ts1, whereas wild-type animals displayed normal HFMR at 31°C. Basic synaptic properties in Mhc-Gal4, UAS-shibire^ts1 animals were not affected at either the permissive or the restrictive temperature. The suppression of HFMR is not due to irreversible damage induced by postsynaptic UAS-shibire^ts1 expression, because a second high-frequency stimulation after recovery to the permissive temperature induced normal HFMR (Yoshihara, 2005).

The synaptic vesicle protein synaptotagmin 1 (Syt 1) is the major Ca²⁺ sensor for vesicle fusion at presynaptic terminals but is not localized postsynaptically. It has recently been shown that another isoform of the synaptotagmin family, synaptotagmin 4 (Syt 4), is present in the postsynaptic compartment (Adolfsen, 2004), suggesting Syt 4 might function as a postsynaptic Ca²⁺ sensor. Syt 4 immunoreactivity is observed in a punctate pattern surrounding presynaptic terminals, suggesting Syt 4 is present on postsynaptic vesicles. Postsynaptic vesicle recycling was blocked by using the UAS-shibire^ts1 transgene driven with Mhc-Gal4. Without a temperature shift, Syt 4-containing vesicles showed their normal postsynaptic distribution surrounding presynaptic terminals. When the temperature was shifted to 37^oC for 10 min in the presence of high-K⁺ saline containing 1.5 mM Ca²⁺ to drive synaptic activity, Syt 4-containing vesicles translocated to the plasma membrane. After recovery at 18^oC for 20 min, postsynaptic vesicles returned to their normal position. Removing extracellular Ca²⁺ during the high-K⁺ stimulation resulted in vesicles that did not translocate to the postsynaptic membrane (Yoshihara, 2005).

To further test whether the Syt 4 vesicle population undergoes fusion with the postsynaptic membrane as opposed to mediating fusion between intracellular compartments, transgenic animals were constructed expressing a pH-sensitive green fluorescent protein (GFP) variant (ecliptic pHluorin) fused at the intravesicular N terminus of Syt 4. Ecliptic pHluorin increases its fluorescence 20-fold when exposed to the extracellular space from the acidic lumen of intracellular vesicles during fusion. Expression of Syt 4-pHluorin in postsynaptic muscles resulted in intense fluorescence at specific subdomains in the postsynaptic membrane, defining regions where Syt 4 vesicles undergo exocytosis. The fluorescence was not diffusely present over the postsynaptic membrane but directed to restricted compartments. Mhc-Gal4, UAS-Syt 4-pHluorin larvae were costained with antibodies against the postsynaptic density protein, DPAK, and nc82, a monoclonal antibody against a presynaptic active zone protein. Syt 4-pHluorin colocalized with DPAK and localized adjacent to nc82, demonstrating that Syt 4-pHluorin translocates from postsynaptic vesicles to the plasma membrane at postsynaptic densities opposite presynaptic active zones (Yoshihara, 2005).

To examine the function of Syt 4-dependent postsynaptic vesicle fusion, the phenotypes of a syt 4 null mutant (syt 4^BA1) and a syt 4 deficiency (rn¹⁶) were tested. Mutants lacking Syt 4 hatch from the egg case 21 hours after egg laying at 25^oC, similar to wild type, and grow to fully mature larvae that pupate and eclose with a normal time course. To determine whether postsynaptic vesicle fusion triggered by Ca²⁺ influx is required for HFMR, the effects of high-frequency stimulation in syt 4 mutants were analyzed. In contrast to controls, the increase of miniature release was eliminated in syt 4 mutants. Postsynaptic expression of a UAS-syt 4 transgene completely restored HFMR in the null mutant, demonstrating that postsynaptic Syt 4 is required for triggering enhanced presynaptic function. Presynaptic expression of a UAS-syt 4 transgene did not restore HFMR. In addition, postsynaptic expression of a mutant Syt 4 with neutralized Ca²⁺-binding sites in both C2A and C2B domains did not rescue HFMR, indicating that retrograde signaling by Syt 4 requires Ca²⁺ binding (Yoshihara, 2005).

The large increase in miniature frequency observed during HFMR is similar to the enhancement of presynaptic release after activation of cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) described in Aplysia and Drosophila. Bath application of forskolin, an activator of adenylyl cyclase, results in a robust enhancement of miniature frequency at Drosophila NMJs similar to that observed during HFMR, suggesting retrograde signals may function to increase presynaptic cAMP. To test the role of the cAMP-PKA pathway in HFMR, DC0 mutants were assayed for the presence of HFMR. DC0 encodes the major catalytic subunit of PKA in Drosophila and has been implicated in olfactory learning. Similar to the lack of forskolin-induced miniature induction, DC0 null mutants lacked HFMR. Bath application of forskolin in syt 4 mutants resulted in enhanced miniature frequency, suggesting activation of the cAMP pathway can bypass the requirement for Syt 4 in synaptic potentiation (Yoshihara, 2005).

To further explore the role of retrograde signaling at Drosophila synapses, the role of activity was tested in synapse differentiation and growth. During Drosophila embryonic development, presynaptic terminals undergo a stereotypical structural change from a flat path-finding growth cone into varicose synaptic terminals through dynamic reconstruction. Such developmental changes in synaptic structure may share common molecular mechanisms with morphological changes induced during activity-dependent plasticity. Synaptic transmission was eliminated by using a deletion mutation that removes the postsynaptic glutamate receptors, DGluRIIA and DGluRIIB (referred to as GluRs). Postsynaptic currents normally induced by nerve stimulation were completely absent in the mutants (gluR). Miniatures were also eliminated, even at elevated extracellular Ca²⁺ concentrations of 4 mM. In the absence of GluRs, the presynaptic morphology of motor terminals is abnormal, even though GluRs are only expressed in postsynaptic muscles. GluR-deficient terminals maintain a flattened growth cone-like structure and fail to constrict into normal synaptic varicosities. Synaptic development was also assayed in a null mutant of the presynaptic plasma membrane t-SNARE [SNAP (soluble N-ethylmaleimide-sensitive factor attachment protein) receptor], syntaxin (syx), which eliminates neurotransmitter release, providing an inactive synapse similar to that in the gluR mutant. syx null mutants also have abnormal growth cone-like presynaptic terminals with less varicose structure (Yoshihara, 2005).

Because activity is required for synapse development, whether Syt 4-dependent vesicle fusion may be required, similar to its role in acute retrograde signaling during HFMR, was tested. Physiological analysis revealed that the amplitude of evoked currents in mutants lacking Syt 4 was moderately reduced compared with wild type, suggesting weaker synaptic function or development. Similar to the morphological phenotype of the gluR mutant, syt 4 null mutant embryos showed defective presynaptic differentiation. Nerve terminals lacking Syt 4 displayed reduced varicose structure, whereas wild-type terminals have already formed individual varicosities at this stage of development. Postsynaptic expression with a UAS-syt 4 transgene rescued the physiological and morphological phenotypes. Syt 4 Ca²⁺-binding deficient mutant transgenes did not rescue either the morphological immaturity or the reduced amplitude of evoked currents, even though Syt 4 immunoreactivity at the postsynaptic compartment was restored by muscle-specific expression of the mutant syt 4 transgene, similar to the wild-type syt 4 transgene and endogenous Syt 4 immunoreactivity (Yoshihara, 2005).

Mammalian syt 4 was originally identified as an immediate-early gene that is transcriptionally up-regulated by nerve activity in certain brain regions. Therefore, this study analyzed gain-of-function phenotypes caused by postsynaptic Syt 4 overexpression specifically in muscle cells to increase the probability of postsynaptic vesicle fusion. Syt 4 overexpression induced overgrowth of presynaptic terminals in mature third instar larvae, in contrast to overexpression of Syt 1, which does not traffic to Syt 4-containing postsynaptic vesicles. In addition to synaptic overgrowth, Syt 4 overexpression occasionally induced the formation of abnormally large varicosities. Postsynaptic overexpression of the Syt 4 Ca²⁺-binding mutant did not induce synaptic overgrowth, indicating that retrograde signaling by Syt 4 also requires Ca²⁺ binding to promote synaptic growth (Yoshihara, 2005).

To determine whether the cAMP-PKA pathway is important in activity-dependent synaptic growth, the effects of PKA on synaptic morphology were assayed. Expression of constitutively active PKA presynaptically using a motor neuron-specific Gal4 driver induced not only synaptic overgrowth but also larger individual varicosities in mature third instar larvae, similar to those induced by postsynaptic overexpression of Syt 4. These observations are consistent with the presynaptic overgrowth observed in the learning mutant, dunce, which disrupts the enzyme that degrades cAMP, and with studies in Aplysia implicating PKA in synaptic varicosity formation. The loss-of-function phenotype of PKA mutants (DC0^B3) were characterized at the embryonic NMJ to compare with gluR and syt 4 mutants. Presynaptic terminals in the DC0 mutant were morphologically aberrant, with abnormal growth cone-like features and less varicose structure. Postsynaptic expression of a constitutively active PKA transgene in the DC0 or syt 4 mutant backgrounds rescued the immature morphology, suggesting activation of PKA is downstream of Syt 4-dependent release of retrograde signals (Yoshihara, 2005).

Similar to the role of Syt 1-dependent synaptic vesicle fusion in triggering synaptic transmission at individual synapses, Syt 4-dependent vesicle fusion might trigger synapse-specific plasticity and growth. To test synapse specificity, advantage was taken of the specific properties of the Drosophila NMJ at muscle fibers 6 and 7, where two motorneurons innervate both muscle fibers 6 and 7 during development. Syt 4 was expressed specifically in embryonic muscle fiber 6 but not muscle fiber 7 by using the H94-Gal4 driver. If Syt 4-dependent retrograde signals induce general growth of the motorneuron, one would expect to see a proliferation of synapses on both muscle fibers. Alternatively, if Syt 4 promoted local synaptic growth, one would expect specific activation of synapse proliferation only on target muscle 6, releasing the Syt 4-dependent signal. UAS-syt 4 driven by H94-Gal4 increased innervation on muscle fiber 6 compared with that on muscle fiber 7 in third instar larvae. Control experiments with Syt 4 Ca²⁺-binding deficient mutant transgenes, or a transgene encoding Syt 1, did not result in proliferation. Thus, synaptic growth can be preferentially directed to specific postsynaptic targets where Syt 4-dependent retrograde signals predominate, allowing differential strengthening of active synapses via local rewiring (Yoshihara, 2005).

On the basis of the results described in this study, a local feedback model is proposed for activity-dependent synaptic plasticity and growth at Drosophila NMJs. Synapse-specific Ca²⁺ influx triggers postsynaptic vesicle fusion through Syt 4. Fusion of Syt 4-containing vesicles with the postsynaptic membrane releases locally acting retrograde signals that activate the presynaptic terminal, likely through the cAMP pathway. Active PKA then triggers cytoskeletal changes by unknown effectors to induce presynaptic growth and differentiation. Moreover, PKA is well known to facilitate neurotransmitter release directly, triggering a local synaptic enhancement of presynaptic release as shown in HFMR. Therefore, postsynaptic vesicular fusion might initiate a positive feedback loop, providing a localized activated synaptic state that can be maintained beyond the initial trigger (Yoshihara, 2005).

As a general mechanism for memory storage, Hebb postulated that potentiated synapses maintain an activated state until structural changes occur to consolidate alterations in synaptic strength. The current results demonstrate that acute plasticity and synapse-specific growth require Syt 4-dependent retrograde signaling at Drosophila NMJs. The feedback mechanism described in this study could be a molecular basis for both input-specific postsynaptic tagging and an output-specific presynaptic mark or tag for long-lasting potentiation. The regenerative nature of a positive feedback signal allows individual synapses to be tagged in a discrete all-or-none manner until synaptic rewiring is completed. The synaptic tag is maintained as a large increase in miniature frequency at Drosophila NMJs, suggesting a previously unknown role for miniature release in neuronal function. The spatial resolution for input and output specificity would result from the accuracy insured by Ca²⁺-dependent vesicle fusion and subsequent diffusion, similar to the precision of presynaptic neurotransmitter release (Yoshihara, 2005).

Postsynaptic Syntaxin 4 negatively regulates the efficiency of neurotransmitter release

Signaling from the postsynaptic compartment regulates multiple aspects of synaptic development and function. Syntaxin 4 (Syx4) is a plasma membrane t-SNARE that promotes the growth and plasticity of Drosophila neuromuscular junctions (NMJs) by regulating the localization of key synaptic proteins in the postsynaptic compartment. This study describes electrophysiological analyses and reports that loss of Syx4 leads to enhanced neurotransmitter release, despite a decrease in the number of active zones. A requirement is described for postsynaptic Syx4 in regulating several presynaptic parameters, including Ca(2+) cooperativity and the abundance of the presynaptic calcium channel Cacophony (Cac) at active zones. These findings indicate Syx4 negatively regulates presynaptic neurotransmitter release through a retrograde signaling mechanism from the postsynaptic compartment (Harris, 2018).

Syntaxin 4 regulates multiple aspects of synaptic biology. This study reports that loss of Syx4 leads to synaptic enhancement, a surprising finding give that Syx4 mutant synapses have significantly fewer active zones than the control animals. An increase in evoked release and a reduction in paired-pulse facilitation were observed in Syx4 mutants. Two mechanisms were identified that are likely to contribute to the increase in neurotransmission: an increase in the levels of the presynaptic Ca2+ channel Cac at individual active zones, and a decrease in Ca2+ cooperativity. These two potentiation mechanisms could be linked – for example, an increase in Cac channels, leading to changes in Ca2+ influx and the spatial arrangement of the channels, could contribute to changes in the sensitivity of the exocytotic machinery to Ca2+. However, the possibility cannot be ruled out that they are distinct phenomena. As all of these phenotypes are rescued by postsynaptic, but not presynaptic, expression of Syx4, the data indicate a retrograde signaling mechanism by which Syx4 regulates active zones (Harris, 2018).

Cac clustering at active zones is regulated by components of the active zone cytomatrix, including Brp. Of these, Brp has the largest effect, with an approximate 50% reduction in Cac levels at active zones of brp null mutants. Although Syx4 mutants have no obvious defects in the size or intensity of Brp clusters, the distribution other cytomatrix proteins has not yet been examined. One mechanism for Cac regulation downstream of Syx4 signaling could be through changes in the levels of other active zone cytomatrix components (Harris, 2018).

One possible explanation for the potentiation observed in Syx4 mutants is that it is the result of homeostatic compensation. Many studies have described homeostatic mechanisms of potentiation and depression at the fly NMJ. In presynaptic homeostatic potentiation (PHP), perturbations that inhibit the function of postsynaptic glutamate receptors by acute pharmacological blockade or genetic loss, though most studies have not reported Cac levels. Presynaptic homeostatic depression (PHD) is a distinct phenomenon in which overexpression of the vesicular glutamate transporter, resulting in more glutamate packaged per synaptic vesicle, is offset by compensatory decreases in neurotransmitter release. PHD has been shown to involve a decrease in presynaptic Ca2+ influx and a decrease in Cac levels at active zones. Thus, the synapse employs multiple mechanisms during homeostatic plasticity, including regulation of Cac channels and Ca2+ influx (Harris, 2018).

An important distinction is that during homeostatic compensation the compensatory changes typically restore muscle depolarization precisely, whereas in Syx4 mutants a significant enhancement of neurotransmission is seen, well beyond control levels. Nevertheless, it may be interesting to investigate whether any of the known homeostatic pathways are required for potentiation in Syx4 mutants. It is also possible that Syx4 itself is engaged in homeostatic mechanisms. For example, if Syx4 is involved in downregulating Cac channels, it could potentially participate in a previously described PHD mechanism, which would therefore be impaired in Syx4 mutant animals. Syx4 could also interact with other retrograde pathways that affect presynaptic release probability, such as signaling through the importin Imp13, which functions postsynaptically to regulate release probability and presynaptic intracellular Ca2+ (Harris, 2018).

One intriguing observation from the current study is that paired-pulse facilitation is enhanced, compared to controls, when Syx4 is expressed postsynaptically in Syx4 mutants. This is surprising since simple overexpression of Syx4 in a wildtype animal does not affect facilitation. While it is not yet understood how this enhanced facilitation arises, one possibility could be differential expression of Syx4 isoforms. All of the overexpression and rescue experiments described in this study were conducted using a full-length Syx4 cDNA (Syx4A). However, there is a second isoform of Syx4 (Syx4B), encoding a shorter N-terminus, which is redundant with the A isoform with respect to all other phenotypes characterized for Syx4 to date. However no evidence was found that Syt4 or Nlg participate with Syx4 to regulate the Syx4 presynaptic enhancement phenotypes. Thus, it is likely that Syx4, as a postsynaptic t-SNARE, mediates the release of additional retrograde signals, and that multiple overlapping Syx4-dependent pathways are involved in establishing normal synaptic morphology, plasticity, and function (Harris, 2018).

This study has described electrophysiological analysis of animals lacking the postsynaptic t-SNARE Syx4. Syx4 mutants exhibit synaptic enhancement accompanied by presynaptic changes at active zones, including an increase in presynaptic Ca2+ channels at active zones and a decrease in Ca2+ cooperativity. All of these features are rescued by restoring postsynaptic Syx4. It is concluded that retrograde pathways regulated by Syx4 inhibit active zone potentiation, and that Syx4 modulates multiple postsynaptic signaling pathways with overlapping function (Harris, 2018).

Molecular interface of neuronal innate immunity, synaptic vesicle stabilization, and presynaptic homeostatic plasticity

This study define a homeostatic function for innate IMD immune signaling within neurons. A genetic analysis of the innate immune signaling genes IMD, IKKbeta, Tak1, and Relish demonstrates that each is essential for presynaptic homeostatic plasticity (PHP). Subsequent analyses define how the rapid induction of PHP (occurring in seconds) can be coordinated with the life-long maintenance of PHP, a time course that is conserved from invertebrates to mammals. This study defines a novel bifurcation of presynaptic innate immune signaling. Tak1 (Map3K) acts locally and is selective for rapid PHP induction. IMD, IKKbeta, and Relish are essential for long-term PHP maintenance. This study then define how Tak1 controls vesicle release. Tak1 stabilizes the docked vesicle state, which is essential for the homeostatic expansion of the readily releasable vesicle pool. This represents a mechanism for the control of vesicle release, and an interface of innate immune signaling with the vesicle fusion apparatus and homeostatic plasticity (Harris, 2018).

This study has tested the core signaling components of an innate immune signaling pathway (IMD signaling) for a role during presynaptic homeostatic plasticity. The data support a model in which IMD signaling bifurcates downstream of the presynaptic innate immune receptor PGRP-LC to achieve immediate, local modulation of the presynaptic release apparatus via Tak1, and prolonged maintenance of the homeostatic response via the transcription factor Relish. This model allows the innate immune signaling system to rapidly alter presynaptic release (seconds to minutes) and simultaneously initiate a Rel-dependent consolidation of PHP. It is noteworthy that the consolidation of PHP can persist for months in insects and decades in humans. It has become clear that the molecular mechanisms responsible for the rapid induction and sustained expression are genetically separable. These findings provide an explanation for how the rapid induction and sustained expression of PHP are mechanistically coordinated (Harris, 2018).

The canonical function of innate immunity is to recognize invading pathogens or non-normal molecular patterns and induce a rapid, inflammatory reaction that is sustained for as long as the invasion persists. During an innate immune response, Tak1-dependent signaling turns on more rapidly, and turns off more rapidly, than Rel-mediated transcription. As such, Tak1 signaling can be considered as a feedforward activator of the cellular immune response. Based on the data, it is proposed that innate immune signaling in the nervous system is transfigured to detect non-normal neurophysiology, although the molecular event that reports altered neural (synaptic) function and is subsequently detected by pre-synaptic PGRP-LC remains a mystery. In the current study, Tak1 is placed at the presynaptic release site where homeostatic plasticity is rapidly induced. Thus, just as in canonical innate immunity, Tak1 is ideally situated to act as a feedforward potentiometer that controls vesicle fusion, thereby achieving a rapid compensatory change in presynaptic release following postsynaptic glutamate receptor inhibition. Subsequent activation of Rel- mediated transcription provides a sustained response that can be maintained for the duration of the perturbation (Harris, 2018).

Evidence is provided that Tak1 has a potent, constitutive function to stabilize the docked vesicle pool, defined both electrophysiologically and at the EM level. This can explain the dramatic defect in spontaneous miniature release frequency, which occurs without a change in the total number of presynaptic release sites. Fewer docked vesicles can explain the defect in EPSC amplitude despite normal action-potential-induced calcium influx. It was also demonstrated that vesicles can be mobilized to the release site during a stimulus train, temporarily achieving WT release rates before declining back to a reduced baseline. This effect reflects a stimulus-dependent re-population of the docked vesicle pool, as demonstrated by EM analysis directly following a train of APs. The stimulus, and presumably calcium-dependent, re-population of the docked vesicle pool explains why there is pronounced short-term synaptic facilitation in the Tak1 mutants compared to short-term depression in WT. Essentially, the number of docked vesicles is increased in a stimulus-dependent manner, something that does not occur in WT, based on EM analysis. Finally, analysis of recovery from vesicle depletion pinpoints the time at which Tak1 activity becomes essential. The recovery of synaptic transmission is completely normal in Tak1 for the first 500 ms following a stimulus train. It is only after this time point that recovery becomes defective (Harris, 2018).

As a working model, it is proposed that Tak1 functions to inhibit the rate of vesicle undocking and, thereby, persistently stabilizes the docked/primed vesicle pool. Immediately following a stimulus train, calcium- dependent mechanisms actively mobilize vesicles to the active zone and drive calcium-dependent vesicle docking and priming. Thus, calcium-dependent potentiation of the forward rate of docking/priming transiently overcomes the enhanced un-docking rate caused by loss of Tak1 and the number of docked vesicles is restored to WT levels. As intra-terminal calcium levels drop following the stimulus train, the rates of docking and un-docking re-equilibrate in the Tak1 mutant, reaching a steady state with many fewer docked vesicles compared to WT (Harris, 2018).

This model can also explain the failure of PHP in a Tak1 mutant. The rapid induction of PHP occurs following inhibition of postsynaptic glutamate receptors and is expressed in response to a single AP. This requires an expansion of the resting pool of docked/primed vesicles and this cannot be achieved in the absence of Tak1. Accordingly, enhanced Tak1 activity, following activation of the innate immune receptor PGRP, would inhibit the rate of vesicle un-docking and potentiate the pool of docked/primed vesicles, necessary for the rapid induction of PHP. Thus, Tak1 functions as a molecular potentiometer controlling the docked/primed vesicle pool during PHP. A question for future experimentation concerns how baseline Tak1 activity is established to achieve highly reproducible, baseline synaptic transmission in WT (Harris, 2018).

Most of the knowledge regarding the organization of innate immune signaling pathways is based on assays that detect the nuclear translocation of Rel/NF-κB, or quantify Rel/NF-κB-mediated transcription. Thus, the logic and spatial organization of innate immune signaling in neurons has yet to be clearly defined. The data argue for several novel features of signaling organization in a neuron, beyond the local synaptic action of Tak1. In canonical innate immune signaling, receptor activation catalyzes the assembly of an intracellular complex that includes Imd, Tak1, and IKKβ. Based on the evidence in Drosophila motoneurons, and consistent with published models of IMD signaling, it is proposed that activation of the PGRP receptor drives assembly of the IMD signaling complex that includes a poly-ubiquitin chain as well as the three proteins IMD, Tak1, and IKKβ. Then, it is suggested that IKKβ is necessary for this signaling complex to either assemble correctly or signal efficiently. Thus, IKKβ is necessary for both the short- and long-term forms of PHP, just like the PGRP receptor. Finally, once this complex is assembled and activated, two independent signal transduction cascades are initiated. One requires Tak1 and acts locally within the presynaptic terminal to achieve the rapid induction of PHP. The second pathway requires the function of IMD and signals via Rel-mediated transcription, necessary for the long-term maintenance of PHP. It is noted, however, that other models for IKKβ activation should also be considered. Finally, the data raise an important question regarding how signaling is conveyed from synaptic IKKβ to nuclear Rel, a topic of future studies (Harris, 2018).

Postsynaptic glutamate receptors regulate local BMP signaling at the Drosophila neuromuscular junction

Effective communication between pre- and post-synaptic compartments is required for proper synapse development and function. At the Drosophila neuromuscular junction (NMJ), a retrograde BMP signal functions to promote synapse growth, stability and homeostasis and coordinates the growth of synaptic structures. Retrograde BMP signaling triggers accumulation of the pathway effector pMad in motoneuron nuclei and at synaptic termini. Nuclear pMad, in conjunction with transcription factors, modulates the expression of target genes and instructs synaptic growth; a role for synaptic pMad remains to be determined. This study reports that pMad signals are selectively lost at NMJ synapses with reduced postsynaptic sensitivities. Despite this loss of synaptic pMad, nuclear pMad persisted in motoneuron nuclei, and expression of BMP target genes was unaffected, indicating a specific impairment in pMad production/maintenance at synaptic termini. During development, synaptic pMad accumulation followed the arrival and clustering of ionotropic glutamate receptors (iGluRs) at NMJ synapses. Synaptic pMad was lost at NMJ synapses developing at suboptimal levels of iGluRs and Neto, an auxiliary subunit required for functional iGluRs. Genetic manipulations of non-essential iGluR subunits revealed that synaptic pMad signals specifically correlate with the postsynaptic type-A glutamate receptors. Altering type-A receptor activities via protein kinase A (PKA) revealed that synaptic pMad depends on the activity and not the net levels of postsynaptic type-A receptors. Thus, synaptic pMad functions as a local sensor for NMJ synapse activity and has the potential to coordinate synaptic activity with a BMP retrograde signal required for synapse growth and homeostasis (Sulkowski, 2013).

Previous work has described Neto as the first nonchannel subunit required for the clustering of iGluRs and formation of functional synapses at the Drosophila NMJ. Neto and iGluR complexes associate in the striated muscle and depend on each other for targeting and clustering at postsynaptic specializations. This study shows that Neto/iGluR synaptic complexes induce accumulation of pMad at synaptic termini in an activity-dependent manner. The effect of Neto/iGluR clusters on BMP signaling is selective, and limited to synaptic pMad; nuclear accumulation of pMad appears largely independent of postsynaptic glutamate receptors. This study demonstrates that synaptic pMad mirrors the activity of postsynaptic type-A receptors. As such, synaptic pMad may function as an acute sensor for postsynaptic sensitivity. Local fluctuations in synaptic pMad may provide a versatile means to relay changes in synapse activity to presynaptic neurons and coordinate synapse activity status with synapse growth and homeostasis (Sulkowski, 2013).

Drosophila NMJs maintain their evoked potentials remarkably constant during development, from late embryo to the third instar larval stages. This coordination between motoneuron and muscle properties requires active trans-synaptic signaling, including a retrograde BMP signal, which promotes synaptic growth and confers synaptic homeostasis. Nuclear pMad accumulates in motoneurons during late embryogenesis. However, embryos mutant for BMP pathway components hatch into the larval stages, indicating that BMP signaling is not required for the initial assembly of NMJ synapses and instead modulates NMJ growth and development. This study demonstrates that synaptic accumulation of pMad follows GluRIIA arrival at nascent NMJs and depends on optimal levels of synaptic Neto and iGluRs. As type-A receptors have been associated with nascent synapses, and type-B receptors mark mature NMJs, accumulation of synaptic pMad appears to correlate with a growing phase at NMJ synapses. Furthermore, synaptic pMad correlates with the activity and not the net levels of postsynaptic type-A receptors. In fact, expression of a GluRIIA variant with a mutation in the putative ion conduction pore triggered reduction of synaptic pMad levels. Thus, synaptic pMad functions as a molecular sensor for synapse activity and may constitute an important element in synapse plasticity (Sulkowski, 2013).

The synaptic pMad pool has been localized primarily to the presynaptic compartment. However, a contribution for postsynaptic pMad to the pool of synaptic pMad is also possible. Postsynaptic pMad accumulates in response to glia-secreted Mav, which regulates gbb expression and indirectly modulates the Gbb-mediated retrograde signaling (Fuentes-Medel, 2012). RNAi experiments revealed that knockdown of mad in muscle induces a decrease in synaptic pMad, albeit much reduced in amplitude compared with knockdown of mad in motoneurons (Fuentes-Medel, 2012). Also, knockdown of wit in motoneurons, but not in muscle, and knockdown of put in muscle, but not in motoneurons, triggers reduction of synaptic pMad (Fuentes-Medel, 2012). Intriguingly, the synaptic pMad is practically abolished in GluRIIA and neto¹⁰⁹ mutants and cannot be further reduced by additional decrease in Mad levels. Whereas loss of postsynaptic pMad could be due to a Mav-dependent feedback mechanism that controls Gbb secretion from the muscle, the absence of presynaptic pMad demonstrates a role for GluRIIA and Neto in modulation of BMP retrograde signaling (Sulkowski, 2013).

As BMP signals are generally short lived, synaptic pMad probably reflects accumulation of active BMP/receptor complexes at synaptic termini. Recent evidence suggests that BMP receptors traffic along the motoneuron axons, with Gbb/receptors complexes moving preferentially in a retrograde direction. By contrast, Mad does not appear to traffic. Thus, Mad is likely to be phosphorylated and maintained locally by a pool of active Gbb/BMP receptor complexes that remain at synaptic termini for the time postsynaptic type-A receptors are active (Sulkowski, 2013).

The activity of type-A glutamate receptors may control synaptic pMad accumulation (1) indirectly via activity-dependent changes that are relayed to both pre- and postsynaptic cells, or (2) directly by influencing the production and signaling of varied Gbb ligand forms or by localizing Gbb activities. For example, inhibition of postsynaptic receptor activity induces trans-synaptic modulation of presynaptic Ca²⁺ influx. Such Ca²⁺ influx changes may trigger events that induce a local change in synaptic pMad accumulation. One possibility is that changes in Ca²⁺ influx may recruit Importin-β11 at presynaptic termini, which in turn mediate synaptic pMad accumulation (Sulkowski, 2013).

At the Drosophila NMJ, Gbb is secreted in the synaptic cleft from both pre- and postsynaptic compartments. The secretion of Gbb is regulated at multiple levels, transcriptionally and post-translationally. Furthermore, the Gbb prodomain could be processed at several cleavage sites to generate Gbb ligands with varying activities. The longer, more active Gbb ligand retains a portion of the prodomain that could influence the formation of Gbb/BMP receptor complexes. Synaptic pMad may result from signaling by selective forms of Gbb. Or type-A receptors could modulate secretion and processing of Gbb in an activity-dependent manner. Understanding the function of different pools and active forms of Gbb within the synaptic cleft will help explain the multiple roles for Gbb at Drosophila NMJs (Sulkowski, 2013).

Alternatively, active postsynaptic type-A receptor complexes may directly engage and stabilize presynaptic Gbb/BMP receptor signaling complexes via trans-synaptic interactions. CUB domains can directly bind BMPs; thus Neto may utilize its extracellular CUB domains to engage Gbb and/or presynaptic BMP receptors. As synaptic pMad mirrors active type-A receptors, such trans-synaptic complexes will depend on Neto in complexes with active type-A receptors. No capture has yet been shown of a direct interaction between Gbb and Neto CUB domains in co-immunoprecipitation experiments. Nonetheless, a trans-synaptic complex that depends on the activity of type-A receptors could offer a versatile means for relaying synapse activity status to the presynaptic neuron via fast assembly and disassembly (Sulkowski, 2013).

Irrespective of the strategy that correlates synaptic pMad pool with the active type-A receptor/Neto complexes, further mechanisms must act to maintain the Gbb/BMP receptor complexes at synapses and protect them from endocytosis and retrograde transport. Such mechanisms must be specific, as general modulators of BMP receptors endocytosis impact both synaptic and nuclear pMad. A candidate for differential control of BMP/receptor complexes is Importin-β11. Loss of synaptic pMad in importin-β11 is rescued by neuronal expression of activated BMP receptors, by blocking retrograde transport, but not by neuronal expression of Mad. As Mad does not appear to traffic, presynaptic Importin-β11 must act upstream of the BMP receptors, perhaps to stabilize active Gbb/BMP receptor complexes at the neuron membrane. By contrast, local pMad cannot be restored at Neto-deprived NMJs by overactivation of presynaptic BMP receptors or by blocking retrograde transport. As neto and gbb interact genetically, it is tempting to speculate that postsynaptic Neto/type-A complexes localize Gbb activities and stabilize Gbb/BMP receptor complexes from the extracellular side. Additional extracellular factors, for example heparan proteoglycans, or intracellular modulators, such as Nemo kinase, may control the distribution of sticky Gbb molecules within the synaptic cleft and their binding to BMP receptors, or may stabilize Gbb/BMP receptor complexes at synaptic termini (Sulkowski, 2013).

Synaptic pMad may act locally and/or in coordination with the transcriptional control of BMP target genes to ensure proper growth and development of the synaptic structures. A presynaptic pool of pMad maintained by Importin-β11 neuronal activities ensures normal NMJ structure and function. Like importin-β11, GluRIIA and Neto-deprived synapses show a significantly reduced number of boutons. Intriguingly, the absence of GluRIIA induces up to 20% reduction in bouton numbers, whereas knockdown of GluRIIB does not appear to affect NMJ growth. Although the amplitude of the growth phenotypes observed in normal culturing conditions (25°C) was modest, this phenomenon may explain the requirement for GluRIIA reported for activity-dependent NMJ development (at 29°C). Furthermore, knockdown of Neto or any iGluR essential subunit affect synaptic pMad and NMJ growth in a dose-dependent manner. Not significant changes were found in nuclear pMad or expression of BMP target genes in GluRIIA or Neto-deprived animals, but the restoration of synaptic pMad by presynaptic constitutively active BMP receptors rescues the morphology and physiology of importin-β11 mutant NMJs. The smaller NMJs observed in the absence of local pMad may reflect a direct contribution of synaptic pMad to retrograde BMP signaling, a pathway that provides an instructive signal for NMJ growth. Thus, BMP signaling may integrate synapse activity status with the control of synapse growth (Sulkowski, 2013).

Synaptic pMad may also contribute to synapse stability. Mutants in BMP signaling pathway have an increased number of 'synaptic footprints': regions of the NMJ where the terminal nerve once resided and has retracted. It has been proposed that Gbb binding to its receptors activates the Williams Syndrome-associated Kinase LIMK1 to stabilize the NMJ. Synaptic pMad may further contribute to the stabilization of synapse contacts by engaging in interactions that anchor the Gbb/BMP receptor complexes at synaptic termini. During neural tube closure, local pSmad1/5/8 mediates stabilization of BMP signaling complexes at tight junction via binding to apical polarity complexes. Flies may utilize a similar anchor mechanism that relies on pMad-mediated interactions for stabilizing BMP signaling complexes and other components at synaptic junctions. Local active BMP signaling complexes are thought to function in this manner in the maintenance of stemness and in epithelial-to-mesenchymal transition (Sulkowski, 2013).

Separate from its role in synapse growth and stability, BMP signaling is required presynaptically to maintain the competence of motoneurons to express homeostatic plasticity. The requirements for BMP signaling components for the rapid induction of presynaptic response may include a role for synaptic pMad in relaying acute perturbations of postsynaptic receptor function to the presynaptic compartment. At the very least, attenuation of local pMad signals, when postsynaptic type-A receptors are lost or inactive, may release local Gbb/BMP receptor complexes and allow them to traffic to neuron soma and increase the BMP transcriptional response, promoting expression of presynaptic components and neurotransmitter release. In addition, synaptic pMad-dependent complexes may influence the composition and/or activity of postsynaptic glutamate receptors. Although future experiments will be needed to address the nature and function of local pMad-containing complexes, the current findings clearly demonstrate that synaptic pMad constitutes an exquisite monitor of synapse activity status, which has the potential to relay information about synapse activity to both pre- and postsynaptic compartments and contribute to synaptic plasticity. As BMP signaling plays a crucial role in synaptic growth and homeostasis at the Drosophila NMJ, the use of synaptic pMad as a sensor for synapse activity may enable the BMP signaling pathway to monitor synapse activity then function to adjust synaptic growth and stability during development and homeostasis (Sulkowski, 2013).

MAN1 restricts BMP signaling during synaptic growth in Drosophila

Bone morphogenic protein (BMP) signaling is crucial for coordinated synaptic growth and plasticity. This study shows that the nuclear LEM-domain protein MAN1 is a negative regulator of synaptic growth at Drosophila larval and adult neuromuscular junctions (NMJs). Loss of MAN1 is associated with synaptic structural defects, including floating T-bars, membrane attachment defects, and accumulation of vesicles between perisynaptic membranes and membranes of the subsynaptic reticulum. In addition, MAN1 mutants accumulate more heterogeneously sized vesicles and multivesicular bodies in larval and adult synapses, the latter indicating that MAN1 may function in synaptic vesicle recycling and endosome-to-lysosome trafficking. Synaptic overgrowth in MAN1 is sensitive to BMP signaling levels, and loss of key BMP components attenuate BMP-induced synaptic overgrowth. Based on these observations, it is proposed that MAN1 negatively regulates accumulation and distribution of BMP signaling components to ensure proper synaptic growth and integrity at larval and adult NMJs (Laugks, 2016).

Jelly belly trans-synaptic signaling to anaplastic lymphoma kinase regulates neurotransmission strength and synapse architecture

In Drosophila, the secreted signaling molecule Jelly Belly (Jeb) activates anaplastic lymphoma kinase (Alk), a receptor tyrosine kinase, in multiple developmental and adult contexts. Jeb and Alk are highly enriched at Drosophila synapses within the CNS neuropil and neuromuscular junction (NMJ), and a conserved intercellular signaling function was been postulated. At the embryonic and larval NMJ, Jeb is localized in the motor neuron presynaptic terminal whereas Alk is concentrated in the muscle postsynaptic domain surrounding boutons, consistent with anterograde trans-synaptic signaling. This study shows that neurotransmission is regulated by Jeb secretion by functional inhibition of Jeb-Alk signaling. Jeb is a novel negative regulator of neuromuscular transmission. Reduction or inhibition of Alk function results in enhanced synaptic transmission. Activation of Alk conversely inhibits synaptic transmission. Restoration of wild-type postsynaptic Alk expression in Alk partial loss-of-function mutants rescues NMJ transmission phenotypes and confirms that postsynaptic Alk regulates NMJ transmission. The effects of impaired Alk signaling on neurotransmission are observed in the absence of associated changes in NMJ structure. Complete removal of Jeb in motor neurons, however, disrupts both presynaptic bouton architecture and postsynaptic differentiation. Nonphysiologic activation of Alk signaling also negatively regulates NMJ growth. Activation of Jeb-Alk signaling triggers the Ras-MAP kinase cascade in both pre- and postsynaptic compartments. These novel roles for Jeb-Alk signaling in the modulation of synaptic function and structure have potential implications for recently reported Alk functions in human addiction, retention of spatial memory, cognitive dysfunction in neurofibromatosis, and pathogenesis of amyotrophic lateral sclerosis (Rohrbough, 2013).

The results support an anterograde signaling model in which presynaptically secreted Jeb activates postsynaptic Alk. The data to support this hypothesis derives from multiple tests. First, immunolabeling shows Jeb is concentrated within presynaptic boutons, while Alk is present in the surrounding postsynaptic subsynaptic reticulum (SSR) (Rohrbough, 2011). Second, targeted postsynaptic Alk expression in Alk LOF mutants is sufficient to rescue synaptic transmission defects, a strong demonstration that Alk is required in the postsynaptic muscle to regulate neurotransmission. Third, post-synaptic inhibtion of Alk by tissue specific RNAi results in 2- fold increased accumulation of perisynaptic Jeb. Fourth, the MARCM clonal approach demonstrates Jeb may be required within presynaptic motor neurons to regulate postsynaptic molecular assembly. Fifth, elevated presynaptic Jeb expression activates postsynaptic Ras/MAPK/ERK activation, while inhibition of postsynaptic Alk reduces Ras/MAPK/ERK activitation (Rohrbough, 2013).

In structurally normal NMJs, strong effects on neurotransmission were found as a consequence of perturbations in Jeb-Alk signaling. The clearest, most consistent results derive from techniques that activate or inhibit Jeb-Alk signaling postsynaptically. Postsynaptic hyperactivation of Alk weakens NMJ synaptic transmission. This functional phenotype parallels the negative regulation of synaptic growth by postsynaptic Alk activation. Consistent with the inhibitory effect of Alk activation on neurotransmission, enhanced neurotransmission was observed as a consequence of muscle specific reductions in Alk levels by transgenic RNAi. Additional confirmation for Alk-dependent inhibition of neurotransmission is provided by analysis of a hypomorphic temperature sensitive allele of Alk. Partial loss of Alk function results in strongly increased NMJ neurotransmission. The implication is that Alk activity limits or negatively regulates synaptic strength. It was also shown that muscle-specific Alk expression in the strongest alk^ts/alk^f01491 partial loss of function genotype rescues reduced neurotransmission to near wild-type levels, a conclusive demonstration that postsynaptic Alk function negatively regulates the strength of NMJ neurotransmission. This function is novel: Jeb-Alk transynaptic signaling is the only known negative regulator of synaptic transmission (Rohrbough, 2013).

Presynaptic manipulation of Jeb yields less strong though still consistent results. Transmission is uneffected by increased pan-neuronal Jeb expression, though this activates Ras/MAPK/ERK both centrally and presynaptically at the NMJ and, to a lesser degree, within the postsynaptic muscle. Motor neuron electrical activity activates neuronal Ras/MAPK/ERK signaling, and this presynaptic Ras/MAPK/ERK activation is positively linked to both structural and functional NMJ synaptic remodeling. Motor neuron specific over expression of Jeb does produce a modest but statistically significant reduction in neuromuscular transmission. Ectopic expression of Jeb in muscle results in substantial inhibiton of neuromuscular transmission. One hypothesis that may account for the diffence between panneuronal and motor neuron or muscle specific manipulation of Jeb-Alk signalling is that the effects of manipulating pan-neuronal Jeb represent a composite of central and peripheral effects on the motor neuron. In first instar larvae it was found that both jeb and alk mutants display impaired central output to motor neurons most consistent with a central synaptic defect (Rohrbough, 2011). The integrated physiologic function subserved by Jeb-Alk signaling in the NMJ, which has yet to be determined, will provide the essential context for interpretting these results (Rohrbough, 2013).

The novel inhibitory role of Jeb-Alk signaling in NMJ transmission implies that it is part of a transynaptic regulatory network that integrates neuronal activity and responses with other homeostatic mechanisms. This study provides indirect evidence that Jeb secretion is regulated. The physiologic regulation of Jeb secretion is a critical missing component of understanding how Jeb-Alk signaling fits into the regulation of synaptic plasticity. Jeb-Alk signaling regulates postembryonic NMJ synaptic growth and patterning Jeb-Alk signaling is not required for embryonic NMJ synaptogenesis or differentiation, although jeb and alk null mutants display impaired locomotion and reduced NMJ transmission (Rohrbough and Broadie 2011). At later developmental stages, removing Jeb in motor neurons strongly disrupts late larval NMJ synaptic terminal architecture and bouton morphology. Postsynaptic Dlg scaffolding and GluR clustering are strongly perturbed in association with jeb mutant terminals. The mosaic analysis supports a cell-autonomous, anterograde signaling function for Jeb. One mechanistic hypothesis is that Jeb-Alk nerve-to-muscle signaling regulates NMJ morphogenesis by recruiting or regulating cell adhesion molecules (CAMs). In the developing adult visual system, anterograde Jeb-Alk signaling induces the expression of postsynaptic adhesion molecules Dumbfounded/Kirre, Roughest/IrreC and Flamingo to shape the optic neuropil target environment. At the larval NMJ, adhesion molecules such as fasciclins and integrins regulate activity-dependent synaptic growth and structural remodeling. The current results imply that Jeb-Alk signaling either directly regulates Dlg localization or indirectly drives Dlg-dependent postsynaptic differentiation. Dlg has demonstrated roles in NMJ morphogenesis and GluR expression and field regulation, and directly binds and regulates fasciclin II and βPS integrin. Future work will test the hypothesis that Jeb-Alk signaling organizes or regulates adhesion receptors and postsynaptic scaffolding to control bouton differentiation and shape functional synaptic architecture (Rohrbough, 2013).

In other systems, Jeb-Alk signaling has been studied primarily at the level of behavior. In C. elegans, the Jeb homolog Hen-1 was identified in a forward genetic behavioral screen for impaired ability to integrate conflicting sensory input (Ishihara, 2002). The Hen-1 phenotype is non-developmental and can be rescued only by adult Hen-1 expression. There is no uniquely identified mammalian Jeb/Hen-1 homolog, but ALK is expressed in the mammalian nervous system during development and at maturity. Alk is expressed in the mouse hippocampus and Alk loss of function enhances behavioral performance in tests dependent on hippocampal function. Similarly, Drosophila learning has shown a dependence on the Ras/MAPK/ERK cascade, which is activated by Jeb-Alk signaling and is probably inhibited by Drosophila neurofibromin (dNf1). Genetic or pharmacologic inhibtion of Jeb-Alk signaling enhances associative learning while increased Jeb-Alk signaling or loss of dNf1 impairs learning. Inhibition of Alk rescues dNf1 mutant learning deficits. These studies suggest that the Jeb-Alk trans-synaptic pathway acts in concert with other, negative regulators of Ras/MAPK/ERK signaling to balance developmental and learning-related synaptic structural and functional changes. Strikingly, a whole-genome association study recently identified human ALK as one of a small number of genes associated with sporadic amyotrophic lateral sclerosis (ALS), a devistating neurodegerative disease of central motor units. If Alk has a conserved inhibitory role in synaptic physiological regulation, hypofunctional human Alk variants may result in augmented motor unit activity and contribute to excitotoxicity and progressive motor unit degeneration in ALS. Pharmacologic activation of Alk has already been hypothesized to have therapeutic benefit in treating ALS. Further insight from future studies should be gained into the mechanism by which the Jeb-Alk signaling pathway regulates synaptic adaptivity in both normal and pathological states (Rohrbough, 2013).

Modeling spinal muscular atrophy in Drosophila links Smn to FGF signaling

Spinal muscular atrophy (SMA), a devastating neurodegenerative disorder characterized by motor neuron loss and muscle atrophy, has been linked to mutations in the Survival Motor Neuron (SMN) gene. Based on an SMA model developed in Drosophila, which displays features that are analogous to the human pathology and vertebrate SMA models, the fibroblast growth factor (FGF) signaling pathway was functionally linked to the Drosophila homologue of SMN, Smn through interaction of the FGF receptor breathless with Smn. This study functionally characterize this relationship and demonstrates that Smn activity regulates the expression of FGF signaling components and thus FGF signaling. Furthermore, it was shown that alterations in FGF signaling activity are able to modify the neuromuscular junction defects caused by loss of Smn function and that muscle-specific activation of FGF is sufficient to rescue Smn-associated abnormalities (Sen, 2011).

Given the variability of the SMA phenotype and the proven relationship between the severity of the disease and small changes in wild-type SMN activity, there is a significant possibility that any modifiers of SMN activity, either direct or indirect, will have therapeutic value. To systematically explore the genome for genes that are capable of modulating SMN function in vivo, advantage was taken of the existence of an SMA model offered by Drosophila to search for Smn genetic interactors. The model that was developed is based on the lethality and an associated neuromuscular junction phenotype linked to loss of Smn function, a phenotype remarkably similar to the NMJ phenotype reported for human patients. Though the role of SMN in biogenesis of snRNPs has been well documented, its regulators and downstream effectors have not been systematically delineated, nor has the link between mutations in SMN and the specific loss of motor neurons seen in SMA patients been uncovered. It may be the case that the specificity of this phenotype is reflective of either specialized SMN functions at the NMJ or a particular sensitivity of motor neurons to the loss of SMN activity. Among the genes the genetic strategy revealed as Smn loss of function modifiers was breathless, encoding an FGF receptor, thus establishing a link between Smn and the FGF pathway (Sen, 2011).

Importantly, in addition to this link, it was also found that FGF signaling is independently involved in NMJ morphogenesis, a function demonstrated in vertebrates but not previously attributed to this pathway in Drosophila despite extensive characterization of its essential role in branching morphogenesis of the tracheal system, migration of multiple cell types, as well as the proper patterning of the mesoderm. The morphological effects that were observed, caused by the modulation of several pathway elements, plainly reveal an involvement of FGF signaling at the NMJ, a role confirmed by the electrophysiological analyses. The down-regulation of FGF signals in muscle results in a reduction of bouton numbers and is associated with increased mEJP amplitudes. The opposite effect is observed when FGF signaling is increased in muscles, suggesting that FGF signaling inversely regulates quantal size. Thus, FGF perturbation in muscle alters both presynaptic growth and specific aspects of synaptic transmission. These observations imply the existence of functional trans-synaptic homeostatic mechanisms, which have been previously shown to compensate for similar changes by increasing presynaptic bouton numbers and transmitter release. However, in this specific instance, only synaptic growth (bouton number) but not transmitter release (quantal content) is affected, the precise mechanisms for which remain unclear. Moreover, the fact that mEJP amplitudes are affected suggests that postsynaptic receptivity to glutamate release from the presynapse is altered. Similar quantal size phenotypes have been observed in several instances previously. For instance, postsynaptic PKA and NF-kappaB are known to regulate quantal size through changes in DGluRs. Directly altering the expression of various GluR subunits also predictably influences quantal size. The genetic interaction this study has demonstrated between FGF and Smn can be described as an epistatic relationship in which the FGF pathway functions downstream of Smn and is consistent with the observation that neuromuscular defects associated with loss of Smn function in muscle can be rescued by muscle-specific activation of FGF signaling. Intriguingly, the relationship described in this study between Smn and FGF is valid beyond the NMJ, as loss of Smn function genetic mosaics in the wing disc clearly result in the down-regulation of FGF signaling. Although the precise molecular mechanism underlying this relationship is still elusive, Smn activity affects transcript and protein levels of the FGF receptor, as well as the expression of additional elements of the FGF pathway. Whether this defines a cascade of interrelated events or whether each of these changes reflects an independent Smn-related regulatory event remains to be determined. Given the fact that Smn mutants in Drosophila display altered postsynaptic currents and severely compromised postsynaptic receptor clustering in muscles, it is conceivable that FGF signaling represents a link between Smn activity and postsynaptic glutamate receptor levels (Sen, 2011).

It should be noted that a link between SMN and the FGF pathway has been suggested by a series of studies in vertebrates where a molecular interaction between an FGF-2 isoform and the SMN protein has been described.These studies raise the possibility that FGF-2 may negatively interfere with SMN complex function through SMN itself. Such observations would, on first appearance, suggest that the epistatic relationship between SMN and FGF signaling in vertebrate cells may be the reverse of what was observed in Drosophila. In point of fact however, the differences in the experimental parameters and approaches between these studies do not allow meaningful comparisons (Sen, 2011).

An important question raised by the above phenotypic analyses is whether the abnormalities associated with FGF and/or Smn perturbations reflect developmental or maintenance issues. It may be the case that the larval system in Drosophila is not ideally suited to differentiate between these alternatives as larval tissue is destined to undergo programmed cell death (histolysis) during metamorphosis. One advantage that flies do offer, however, is the ability to dissociate the development of the adult neuromuscular system from its maintenance as the entirety of its development occurs during the pupal stage, before emergence of the adult. Thus, the Drosophila pupa/adult may provide a platform to address these issues, as Drosophila displays Smn-dependent adult phenotypes. In light of the relationship that was established between Smn and FGF signaling and the known involvement of FGF signaling in the development of both the larval and adult musculature, it will be particularly interesting to examine the effects of modulating FGF activity on the aforementioned processes. Such studies may be of particular relevance to SMA where it is quite difficult to discern the developmental consequences of SMN loss in humans, as neurodegenerative symptoms displayed by patients may obscure basic problems resulting from altered developmental programs such as neuronal pathfinding, initial NMJ formation, etc (Sen, 2011).

In vertebrates, synaptic development and maintenance use at least three distinct signaling mechanisms: the TGF-β, wingless, and FGF pathways. In Drosophila, it is noteworthy that the first two have been demonstrated to function in a similar fashion at the NMJ. Remarkably, the genetic screens involving Smn have identified elements of all three of these pathways as modifiers of Smn-related phenotypes. These connections are considered particularly significant as they raise the possibility that Smn may serve as a node, integrating signaling events crucial for NMJ function, potentially leaving this structure particularly vulnerable to the loss of Smn. Though further correspondence between the Drosophila model and the human condition remains to be determined, the Smn-FGF relationship observed in Drosophila raises the possibility that pharmacological manipulation of FGF signals might mitigate SMN motor neuron-related abnormalities (Sen, 2011).

The Drosophila beta-amyloid precursor protein homolog promotes synapse differentiation at the neuromuscular junction

Although abnormal processing of beta-amyloid precursor protein (APP) has been implicated in the pathogenic cascade leading to Alzheimer's disease, the normal function of this protein is poorly understood. To gain insight into APP function, a molecular-genetic approach was taken to manipulate the structure and levels of the Drosophila APP homolog APPL. Wild-type and mutant forms of APPL were expressed in motoneurons to determine the effect of APPL at the neuromuscular junction (NMJ). APPL was transported to motor axons and that its overexpression caused a dramatic increase in synaptic bouton number and changes in synapse structure. In an Appl null mutant, a decrease in the number of boutons was found. Examination of NMJs in larvae overexpressing APPL revealed that the extra boutons had normal synaptic components and thus were likely to form functional synaptic contacts. Deletion analysis demonstrated that APPL sequences responsible for synaptic alteration reside in the cytoplasmic domain, at the internalization sequence GYENPTY and a putative G(o)-protein binding site. To determine the likely mechanisms underlying APPL-dependent synapse formation, hyperexcitable mutants, which also alter synaptic growth at the NMJ, were examined. These mutants with elevated neuronal activity changed the distribution of APPL at synapses and partially suppressed APPL-dependent synapse formation. A model is proposed by which APPL, in conjunction with activity-dependent mechanisms, regulates synaptic structure and number (Torroja, 1999).

The Drosophila β-amyloid precursor protein (APP) homolog APPL is a pan-neural protein belonging to the conserved APP family. This family includes APP and APLP1/2 in mammals, apl-1 in nematodes, and APP747 in Xenopus. APP is synthesized as a transmembrane glycoprotein composed of extracellular, transmembrane, and cytoplasmic domains. APPs undergo proteolytic cleavage, releasing the ectodomain. Homology among APP members is paralleled by their ability to functionally substitute for each other: transgenes encoding Drosophila APPL or a human neural APP both rescue behavioral defects of Appl null flies (Torroja, 1999).

Expression studies reveal a relationship between stages of APP synthesis and neurite outgrowth and synaptogenesis. In mammals, transmembrane APP is associated with elongating axons, whereas secreted APP is correlated with synaptogenesis. In Drosophila, APPL is enriched in growing axons and areas of synapse formation (Torroja, 1999).

The effects of APP on neuronal development and function have been extensively studied because of their implication in Alzheimer's disease. APP exhibits neurite outgrowth-promoting activities in vitro through interactions with the extracellular matrix and changes in intracellular calcium. Experiments with neuronal cultures suggest that secreted APP modulates excitability via Ca2+-dependent K+ channels and NMDA receptor activity (Torroja, 1999).

In vivo evidence supports a role for APP in synapse formation, maintenance, and plasticity. APP administration increases synaptic density and memory retention, and exposure of hippocampal slices to secreted APP enhances long-term potentiation and modifies the induction of long-term depression. APP knock-out mice show impaired learning and memory. However, experiments with APP overexpression are contradictory, demonstrating enhancement of synaptic density or Alzheimer's-like pathology and lack of synaptotrophic effects. Much of this controversial evidence may arise from the inability, in these systems, to analyze APP function at the level of single synapses (Torroja, 1999).

To investigate the role of APP at single, identified synapses, the Drosophila neuromuscular junction (NMJ) was used. APPL overexpression in motoneurons results in a dramatic increase in the number of synaptic boutons. Conversely, NMJs of Appl^null larvae exhibit a significant decrease in synaptic bouton number. The synapse-promoting function required a conserved internalization sequence and a putative Go binding site at the cytoplasmic domain. This function was partially suppressed by mutations with increased excitability, which by themselves regulate synapse growth and APPL expression at the NMJ (Torroja, 1999).

An overexpression approach was used to determine whether APPL plays a role in the regulation of synapse formation. Overexpression of APPL in motoneurons resulted in morphologically distinct NMJs, characterized by an increase in both parent and satellite boutons. To define the APPL domains essential for these effects, an analysis was performed with Appl deletion proteins that were expressed in motoneurons. This demonstrated that a cytoplasmic internalization sequence is essential for satellite bouton formation, whereas a cytoplasmic Go-protein binding sequence and extracellular domains are essential in promoting excess parent bouton formation. Furthermore, it was observed that, in eag Sh mutants (elevated neuronal activity), APPL overexpression-associated effects are partially suppressed, and the endogenous pattern of APPL expression at the NMJ is altered. These results indicate that APPL localization and trafficking at the synaptic bouton membrane can be regulated in an activity-dependent manner and may influence synaptic growth (Torroja, 1999).

The normal appearance of NMJs in larvae lacking APPL in the loss-of-function mutation demonstrates that APPL is not required for synaptogenesis and/or synapse maintenance. However, bouton numbers are reduced, indicating a possible regulatory role. This notion is bolstered by the striking increase in the number of parent and satellite boutons observed when APPL is elevated. Similar to APPL, human neuronal APP also elicited an increase in satellite and parent bouton number. This provides further support for the notion that APP695 (an alternatively spliced form of App) and APPL are structurally and functionally conserved (Torroja, 1999).

An increase in bouton number has been observed in several Drosophila mutants that affect synaptic activity, such as eag Sh and dunce. In eag Sh, the increase in bouton number appears to derive from longer and more branched neuronal processes containing normal synaptic boutons. In contrast, APPL overexpression results in satellite boutons that protrude from a larger parent bouton, although parent bouton number is also increased (Torroja, 1999).

During the postembryonic stage, there is tremendous growth of the NMJ, including elongation and formation of branches, as well as increases in bouton number, number of active zones, and bouton area. These studies suggest that NMJ expansion involves sprouting and elongation of a process, followed by the differentiation of new terminal boutons. Satellite boutons resulting from APPL overexpression may be caused by increased sprouting and abnormal bouton differentiation (Torroja, 1999).

The distribution of different APPL forms in larval brains suggests that the transmembrane and soluble forms play specific roles in Drosophila neurons (Torroja, 1996). Indeed, it was found that the secretion-deficient APPL form is as effective as wild-type APPL in promoting satellite bouton formation, whereas constitutively secreted APPL has no effect when expressed in one dose and has an effect similar to the Appl null mutant with two doses of the transgene. The observation that two doses of APPLs result in a reduction in the percentage of satellites may indicate an inhibitory role on the satellite bouton-promoting activities (Torroja, 1999).

Deletion of the APPL cytoplasmic domain abolished both the satellite bouton and parent bouton-promoting activity of APPL. However, these activities can be dissected by smaller deletions of the cytoplasmic and extracellular domains. Deletion of the internalization sequence prevented the formation of satellites but did not prevent the increase in parent boutons. In contrast, deletion of the putative Go-protein binding site prevented the increase in the number of parents, but the satellite bouton-promoting activity remained intact. Similar results were found when portions of the extracellular domain (E1 and E2), which may serve to bind a ligand to regulate Go function (Okamoto et al., 1995;Brouillet et al., 1999), were deleted (Torroja, 1999).

In vertebrates, the internalization signal is crucial for regulating APP processing and trafficking. It interacts with several proteins, including X11 and Fe65, which may participate in the regulation of APP metabolism. Thus, APPL-dependent satellite bouton formation is likely to involve APPL internalization. Alternatively, or in addition, APPL may signal by interacting with cytoplasmic proteins, leading to the formation of satellite boutons. The low levels of APPL detected in wild-type NMJs suggests that APPL turnover may be high at the plasma membrane, similar to APP turnover in vertebrates (Torroja, 1999).

Internalization of synaptic proteins plays an important role in the regulation of synaptic growth. For example, the Aplysia cell adhesion molecule ApCaM and its Drosophila homolog Fasciclin II are internalized at synapses in an activity-dependent manner. This promotes synapse growth, presumably by decreasing adhesion between presynaptic and postsynaptic membranes (Torroja, 1999).

Studies in vertebrates show that APP695 behaves as a Go-linked receptor; its cytoplasmic region binds to Go-protein. However, whether APP stimulates or downregulates Go is unclear. The observation that deletion of the putative APPL Go-binding site abolishes the ability of APPL to increase parent bouton number indicates that Go activity may be involved. This is consistent with observations implicating Go in growth cone motility and synapse plasticity and with high levels of Go-protein in developing insect neurites (Torroja, 1999).

Processing and trafficking of APP are known to be affected by neuronal activity. For example, in cortical neurons, the axonal pool of APP holoprotein at the surface was found to be increased after Ca2+ entry. In eag Sh larvae, endogenous APPL signal was consistently increased, especially at the distal-most boutons. Furthermore, when APPL was overexpressed in the hyperexcitable mutant, there was a reduction in satellite bouton number and an increase in parent boutons. This phenotype is similar to the phenotype associated with overexpression of APPL lacking the internalization signal. Thus, deletion of the internalization signal, or high activity, are both likely to increase surface APPL (presumably through a decrease in internalization) and have similar effects on synaptic growth (Torroja, 1999).

It is suggested that APPL is involved in synaptic plasticity, because APPL is nonessential for formation and maintenance of synapses but can promote synapse formation and appears to be affected by neuronal activity. NMJ phenotypes resulting from the expression of APPL proteins lacking specific domains suggest that APPL trafficking and APPL-dependent signal transduction are two processes that regulate APPL-induced synaptic growth. The evidence indicates that (1) APP can be rapidly internalized; (2) processing and trafficking of APPL and APP is affected by activity; and (3) plasma membrane APP, and possibly APPL, behave as Go-protein linked receptors (Torroja, 1999).

It is proposed that NMJ expansion occurs by two consecutive steps: the formation of sprouts and the consolidation of some of these sprouts into differentiated boutons. Bouton differentiation entails the proper arrangement of presynaptic and postsynaptic components, as well as the enlargement of the sprout to accommodate all the elements required for synaptic transmission. Both sprouting and differentiation are modulated by APPL. It is suggested that plasma membrane APPL induces sprouting and that this response is independent of APPL signal transduction. In contrast, bouton differentiation is regulated by APPL signal transduction, which may involve Go. Internalization of APPL stops both activities. It is proposed that a satellite bouton is formed when APPL-induced sprouting is initiated, followed by rapid internalization of APPL, thus reducing APPL-dependent signal transduction and, therefore, bouton differentiation. As a result, some degree of differentiation, such as the formation of active zones and transport of vesicles, does occur, but other aspects, such as bouton enlargement, do not (Torroja, 1999).

Several predictions from this model are in line with these findings. For instance, decreasing APPL internalization (ΔCI) would reduce formation of satellites. A persistent increase in APPL activation, as a result of decreased internalization, is predicted to promote the differentiation of sprouts, thus effectively increasing the number of parent boutons. In contrast, overexpression of APPL variants that are unable to undergo ligand-dependent receptor activation (ΔE1, ΔE2, ΔCg) would reduce bouton differentiation, preventing the increase of parents but not of satellites. Full-length APPL (APPL+, APPLsd) would stimulate both sprouting and differentiation and could be rapidly internalized as in wild type, thus promoting both parent and satellite bouton formation (Torroja, 1999).

Rapid APPL internalization appears to be key to the normally low level of plasma membrane APPL. Increases in plasma membrane-associated APPL result from increased neuronal activity, a factor shown to increase bouton number (Budnik, 1990). Strikingly, overexpression of APPLsd (which has a cleavage site deleted) in eag Sh results in reduction of satellites, with a concomitant increase in the number of parent boutons. This further supports the idea that neuronal activity can drive APPL-mediated bouton differentiation (Torroja, 1999).

Based on these observations, it is concluded that APPL has functional significance for the regulation of synapse formation. Moreover, this study has shown that this process involves the APPL domains that are likely to affect APPL signal transduction, suggesting a novel mechanism for the regulation of the size of synaptic arbors (Torroja, 1999).

Drosophila Neuroligin3 regulates neuromuscular junction development and synaptic differentiation

Neuroligins (Nlgs) are a family of cell adhesion molecules thought to be important for synapse maturation and function. Studies in mammals have shown that different Nlgs have different roles in synaptic maturation and function. The functions of Drosophila Neuroligin1 (DNlg1), DNlg2, and DNlg4 have also been examined. This study reports the role of DNlg3 in synaptic development and function by using Drosophila neuromuscular junctions (NMJs) as a model system. DNlg3 was found to be expressed in both CNS and NMJs where it was largely restricted to the postsynaptic site. By generating and examining dnlg3 mutants, the mutants mutants were found to exhibit an increased bouton number and reduced bouton size compared to the wild-type. Consistent with alterations in bouton properties, pre- and postsynaptic differentiations were also affected including abnormal synaptic vesicle endocytosis, increased PSD length and reduced GluRIIA recruitment. Additionally, synaptic transmission was reduced. Altogether, this study shows that DNlg3 is required for NMJ development, synaptic differentiation and function (Xing, 2014).

Anterograde Activin signaling regulates postsynaptic membrane potential and GluRIIA/B abundance at the Drosophila neuromuscular junction

Members of the TGF-beta superfamily play numerous roles in nervous system development and function. In Drosophila, retrograde BMP signaling at the neuromuscular junction (NMJ) is required presynaptically for proper synapse growth and neurotransmitter release. This study analyzed whether the Activin branch of the TGF-beta superfamily also contributes to NMJ development and function. Elimination of the Activin/TGF-beta type I receptor babo, or its downstream signal transducer smox, does not affect presynaptic NMJ growth or evoked excitatory junctional potentials (EJPs), but instead results in a number of postsynaptic defects including depolarized membrane potential, small size and frequency of miniature excitatory junction potentials (mEJPs), and decreased synaptic densities of the glutamate receptors GluRIIA and B. The majority of the defective smox synaptic phenotypes were rescued by muscle-specific expression of a smox transgene. Furthermore, a mutation in actβ, an Activin-like ligand that is strongly expressed in motor neurons, phenocopies babo and smox loss-of-function alleles. These results demonstrate that anterograde Activin/TGF-beta signaling at the Drosophila NMJ is crucial for achieving normal abundance and localization of several important postsynaptic signaling molecules and for regulating postsynaptic membrane physiology. Together with the well-established presynaptic role of the retrograde BMP signaling via Glass bottom boat and Wishful thinking, these findings indicate that the two branches of the TGF-beta superfamily are differentially deployed on each side of the Drosophila NMJ synapse to regulate distinct aspects of its development and function (Kim, 2014).

Numerous reports have now implicated the Activin/TGF-β and BMP branches of the TGF-β superfamily in regulating neuronal development, synaptic plasticity and cognitive behavior. Accordingly, members from both subfamilies are widely expressed in the nervous system and are co-expressed in multiple regions of vertebrate and invertebrate brains. It is therefore quite likely that ligands of both subfamilies co-exist within the extracellular space and in some cases, act on the same neurons. Lending support to this idea, pyramidal neurons in the CA3 region of the rat hippocampus are known to accumulate both phosphorylated Smad2 and Smad1/5/8, transcriptional transducers of the canonical Activin/TGF-β and BMP-type signaling, respectively. The activation of these two closely-related signaling pathways in common sets of neurons, or different cells of a common neuronal circuit raises the intriguing question of whether the two pathways play different or redundant roles during neuronal development and function (Kim, 2014).

This study utilized the Drosophila neuromuscular junction to address this issue since ligands of both Activin/TGF-β and BMP families are expressed in both muscle and motor neurons. The data, together with previous studies on the role of BMP signaling at the NMJ, clearly demonstrate that the two pathways influence NMJ synaptogenesis in different ways. The Activin/TGF-β pathway is necessary for achieving the proper densities of GluRIIA, GluRIIB and Dlg in postsynaptic muscle membrane, while the BMP pathway has a smaller effect on the distribution of these postsynaptic proteins. In addition, the Activin/TGF-β pathway was dispensable for maintaining overall synaptic growth and homeostasis, both of which are strongly affected by mutations in the BMP pathway. In addition, tissue-specific rescue experiments indicate that the postsynaptic reception of Activin/TGF-β signaling is important in regulating synaptic GluR abundance, whereas BMP signal reception is known to act in the presynaptic motor neurons to promote synaptic growth. These observations suggest that each pathway influences NMJ synapse development and function by acting mainly in either the pre- or postsynaptic cell (Kim, 2014).

Interestingly, the BMP and Activin/TGF-β pathways have also been recently found to control different aspects of the Drosophila innate immune response (Clark, 2011). In this case BMP signaling suppresses the expression of multiple antimicrobial peptide genes following wounding, whereas the Activin/TGF-β pathway limits melanization after bacterial infection in adult flies. Therefore, it appears that the division of labor between these subpathways is not limited to just the nervous system, rather it may be the norm when these related signaling pathways act in concert to regulate a common biological process (Kim, 2014).

The fact that the pathways actually differ in how they affect a complex biological process is not surprising given that the different R-Smads are likely to have different selectivity in gene activation. Within motor neurons, BMP signaling promotes microtubule formation in axons and directly regulates expression of trio, a Rac GEF, that acts as a major regulator of actin cytoskeleton in many types of cells. Thus, it is likely that BMP signaling modulates synaptic growth, in part, by changing the structure and dynamics of the actin and microtubule cytoskeleton within motor neurons. BMP signaling also regulates the transcription of twit, a gene encoding a L-6 neurotoxin-like molecule that controls the frequency of presynaptic spontaneous vesicle release (Kim, 2012; Kim, 2014 and references therein).

Targets of Drosophila Activin/TGF-β signaling in any tissue are less well characterized. Within the central brain, glial-derived Myo signals through Smox to control expression of the Ecdysone B1 receptors in remodeling mushroom body neurons. However, it is not clear if EcR-B1 is a direct or indirect target of smox transcriptional regulation. It is also unclear if Ecdysone signaling plays a role in regulating synaptogenesis at the NMJ, although it may play a role during metamorphic remodeling of the NMJ as it does for the mushroom body neurons. The only other known targets of Smox are InR, Pi3K and Akt, all of which are Insulin signaling components and are reduced in the Drosophila prothoracic gland in the absence of Activin/TGF-β signaling. Once again the effect may be indirect, but this finding is interesting since Insulin signaling components have been shown to control synaptic clustering of GluRs (Kim, 2014).

The clustering of GluRs and Dlg at the NMJ have been shown to be regulated by both transcriptional and post-transcriptional mechanisms. For example, a recent genetic screen identified longitudinals lacking (lola), a BTN-Zn finger transcription factor, as an essential regulator of GluR and dPak expression in muscles. In contrast, the current studies on Activin/TGF-β signaling suggest, at least for GluRIIA, that this pathway functions at the post-transcriptional level since this study found that overexpression of glurIIA-gfp using an exogenous promotor and transcriptional activator does not lead to an enrichment of GluRIIA^GFP at synaptic sites of Activin/TGF-β pathway mutants. This phenotype is reminiscent of that found for certain mutants in the NF-κB signaling system. Loss of Dorsal (an NF-κB homolog), Cactus (an IκB related factor), or Pelle (an IRAK kinase) leads to a substantial reduction of GluRIIA and a slight reduction of Dlg postsynaptic localization at the NMJ and a concomitant reduction in mEJP size. In addition, as was found for loss of Activin/TGF-β signaling, exogenously-expressed GluRIIA-myc did not reach the synaptic surface in NF-κB signaling mutants consistent with a possible role of Activin/TGF-β signaling in regulating NF-κB signaling. However, even if future studies show that the relationship is true, the Activin/TGF-β pathway likely regulates additional factors since its loss also affects GluRIIB levels and muscle resting potential, neither of which is altered in NF-κB pathway mutants. Interestingly, the regulation of GluRIIB levels by Activin/TGF-β signaling does appear to be at the level of transcription, indicating that this signaling pathway likely affects GluR clustering at the NMJ via both transcriptional and post-transcriptional mechanisms (Kim, 2014).

Analysis of Activin/TGF-β signaling at the NMJ, coupled with previous studies on BMP signaling and the novel ligand Maverick, indicates that TGF-β ligands are produced in, and act upon, all three cell types that contribute to NMJ function, specifically the motor neuron, wrapping glia, and muscle (see Model of controlling NMJ development and function by Activin/TGF-β and BMP pathways). This leads to the important issue of how directionality of TGF-β signaling at the NMJ is regulated. One possibility is that ligands are sequestered, either inside the secreting cells or on their surfaces, so that they have limited access to receptors on the opposing pre or postsynaptic membrane. For example, Gbb is produced both in muscle and motor neurons, leading to the issue of how directional signaling from muscle to motor neurons is achieved. On the postsynaptic muscle, Gbb release is potentiated by dRich (Rho GTPase activating protein at 92B), a Cdc42 selective Gap while in the presynaptic neuron Crimpy, a Drosophila homolog of the vertebrate Crim1 gene, has been shown to bind to a precursor form of Gbb. The Gbb/Crimpy complex is thought to either interfere with secretion or activation of motor neuron-derived Gbb thus ensuring that only muscle-derived Gbb activates the retrograde BMP signal at the NMJ. Since there are a large number of characterized TGF-β superfamily binding proteins, Drosophila homologs of some of these factors such as the BMP binding proteins Cv-2, Sog, Tsg and Dally, or the Activin-binding protein Follistatin, may sequester and regulate levels of active ligands within the NMJ. Sequestering mechanisms may also provide direction control by facilitating autocrine as opposed to juxtacrine signaling. If ligand-binding proteins are associated with the membrane surface of the ligand-producing cell, they may facilitate delivery of the ligand to receptors on the producing cell, thus enhancing autocrine signaling. It is interesting in this regard that in the developing Drosophila retina, Actβ appears to signal in an autocrine fashion to control photoreceptor connectivity in the brain (Kim, 2014).

Activin-type ligands are secreted from glia, motor neuron and muscle. The Activin-type ligands induce Babo-mediated phosphorylation of Smox that facilitates association with Med. In the muscle, the phospho-Smox/Med complexes activate the transcription of glurIIB and an unknown factor controlling post-transcriptional process or stability of glurIIA mRNA. In the motor neuron, the phospo-Smox/Med complex controls spontaneous release of synaptic vesicles via unknown mechanism(s). On the other hand, glia-secreted Mav stimulates Mad phosphorylation in the muscle resulting in increased gbb transcription. Gbb protein is released from the muscle and binds Tkv/Sax and Wit complex on the motor neuron leading to an accumulation of phospho-Mad in the nuclei by an unknown mechanism. The resultant phospho-Mad/Med complex activates the transcription of trio whose product promotes synaptic bouton formation (Kim, 2014).

Another important mechanism to control signal direction is likely to be tissue-specific receptor expression. For example, Wit is highly enriched in motor neurons compared to muscle, and this may help ensure that Gbb released from the postsynaptic muscle signals to the presynaptic motor neuron. Type I receptor diversity may be even more important in controlling directionality since at least 2 isoforms of Tkv and three isoforms of Babo have been identified. In the case of Babo, Activin-like ligands have a clear preference for signaling through different receptor isoforms, and these isoforms show differential tissue expression (Kim, 2014).

An additional factor to be considered in understanding TGF-β superfamily signal integration within different NMJ cell types is the possibility of canonical versus non-canonical and/or cross-pathway signaling. For example, in mushroom body neurons Babo can signal in a non-Smad dependent manner through Rho1, Rac and LIM kinase1 (LIMK1) to regulate axon growth and target recognition. Whether this mechanism, or another non-canonical pathway is operative at the NMJ is unclear. Cross-pathway signaling has also recently been identified in Drosophila. In this example, loss of Smox protein in the wing disc has been shown to up-regulate Mad activity in a Babo-dependent manner. Double mutants of babo and smox suppress the cross-pathway signal. As is described in this study, smox protein null mutations lead to significantly more severe GluR and mEJP defects than strong babo mutations alone, and this phenotype is suppressed in double mutants. Thus, as in wing discs, loss of Smox protein likely leads to ectopic Mad activity in muscles that further decrease GluR expression and/or localization at the NMJ. Consistent with this view, this study found that loss of Mad actually increases GluRIIB localization, suggesting that Mad acts negatively to regulate GluRIIB in muscle. One possible model to explain the Smox/Mad data is that normally the Babo/Smox signal inhibits Mad signaling which is itself a repressive signal for GluR accumulation. Thus, in babo mutants, total GluR levels decrease due to the loss of smox and therefore an increase in the repressive Mad signal. In the smox protein null mutant even more repressive Mad signal is generated by Babo further hyperactivating Mad activity leading to even lower levels of GluR accumulation. In medea mutants the activity of both pathways is reduced thereby returning the level of GluR levels close to normal. Additional experiments employing various single and double mutants, together with tissue-specific expression of various ligands, receptor isoforms and ligand-binding proteins will be needed to fully elucidate how vectorial TGF-β signaling is accomplished at the NMJ. Likewise, the identifcation of directly responding target genes and how they are influenced by both Smox and Mad signals is needed to fully appreciate how these two TGF-β signaling branches regulates NMJ functional activity (Kim, 2014).

Excess glutamate release triggers subunit-specific homeostatic receptor scaling

Ionotropic glutamate receptors (GluRs) are targets for modulation in Hebbian and homeostatic synaptic plasticity and are remodeled by development, experience, and disease. This study has probed the impact of synaptic glutamate levels on the two postsynaptic GluR subtypes at the Drosophila neuromuscular junction, GluRIIA and GluRIIB. It was demonstrated that GluRIIA and GluRIIB compete to establish postsynaptic receptive fields, and that proper GluR abundance and composition can be orchestrated in the absence of any synaptic glutamate release. However, excess glutamate adaptively tunes postsynaptic GluR abundance, echoing GluR scaling observed in mammalian systems. Furthermore, when GluRIIA vs. GluRIIB competition is eliminated, GluRIIB becomes insensitive to glutamate modulation. In contrast, GluRIIA is now homeostatically regulated by excess glutamate to maintain stable miniature activity, where Ca(2+) permeability through GluRIIA receptors is required. Thus, excess glutamate, GluR competition, and Ca(2+) signaling collaborate to selectively target GluR subtypes for homeostatic regulation at postsynaptic compartments (Han, 2023).

By generating null mutations in GluRIIA and GluRIIB subunits, this study has shown a competition exists between GluR subtypes that establishes stable postsynaptic fields. While synaptically released glutamate is not required to organize this process, excess glutamate triggers an adaptive downscaling of both GluR subtypes in the postsynaptic compartment. However, when this GluR subtype competition is eliminated, a clear and distinctive relationship is revealed between excess glutamate and GluR plasticity: GluRIIB receptors become completely insensitive to excess glutamate, with stable GluRIIB levels maintained. In contrast, GluRIIA receptors constitute the 'plastic' receptor subtype, homeostatically tuned to excess synaptic glutamate release to maintain stable miniature activity. Further, Ca2+ influx through GluRIIA receptors is a key transducer of this signaling system, rendering GluRIIA non-plastic when Ca2+ permeability is lost. Together, these results highlight the interplay between GluR subtype competition, synaptic glutamate, and Ca2+ signaling at postsynaptic compartments and reveal the existence of homeostatic receptor scaling at the Drosophila NMJ (Han, 2023).

Several layers of regulation operate at postsynaptic compartments to establish GluR fields at the fly NMJ. First, the relative level of GluR transcription and translation between subtypes can ultimately set GluRs at synapses. This is demonstrated by overexpression of either GluRIIA or GluRIIB subunits, which can saturate the entire GluR field at postsynaptic compartments and lead to the concomitant loss of the other GluR subtype. Second, post-translational processes, mediated by such factors as enzymatic cleavage, phosphorylation, and degradation modulate GluR activity and abundance. For example, Ca2+-dependent protein cleavage by calpain, phosphorylation control by p21-activated kinase, and proteosomal degradation by the E3 ubiquitin ligase adapter Diablo have all been shown to modulate GluRs at the fly NMJ, which parallel findings in vertebrates. Third, while glutamate released from synaptic vesicles is not necessary to establish or maintain GluR fields in Drosophila or in mammals there is evidence that ambient glutamate modulation from non-vesicular glutamate release and glial transporters might regulate GluR clustering and receptor field size, while excess vesicular glutamate release triggers adaptive reductions in GluRs. There is also evidence that correlated or diminished activity selectively regulates GluRIIA abundance (Han, 2023).

In C. elegans, parallel processes regulate GluR trafficking and plasticity. It will be of interest to determine how these many layers of control intersect and are coordinated to establish GluR fields during development and remodeling in plasticity. There appears to be a hierarchy of regulatory steps controlling GluR plasticity in response to excess glutamate. While excess glutamate downregulates both GluRIIA and GluRIIB abundance when both receptor subtypes are present, this plasticity is adaptive but not homeostatic-miniature amplitude is still enhanced. However, when GluRIIA vs. GluRIIB competition is eliminated, a complete distinction in GluR behavior is revealed: GluRIIB is not responsive to excess glutamate, while GluRIIA receptors are sensitively tuned to glutamate to now enable the homeostatic control of miniature activity. Ca2+ influx through GluRIIA is crucial to this plasticity, where loss of this secondary messenger converts GluRIIA to behave like static GluRIIB receptors. It is possible that excess glutamate drives Ca2+-related signaling through GluRIIA in postsynaptic compartments that ultimately acts on both GluRIIA and GluRIIB when both receptor subtypes are present, at least at MN-Ib NMJs, and this signaling may be lost in GluRIIB-only NMJs. Although the downstream effectors that respond to excess glutamate and Ca2+ to modulate postsynaptic GluRs are not known, an attractive candidate is the auxiliary KAR subunit Neto, which regulates GluR abundance at the fly NMJ46 and functionally modulates AMPARs in worms (Han, 2023).

Interestingly, KARs in mammals are also under homeostatic control, where the auxiliary subunit Neto controls key properties of these receptors. What purpose might two GluR subtypes, differing in their current amplitudes, biophysics, and plasticity, subserve? One idea is that GluRIIB receptors provide a basal signal at postsynaptic compartments to maintain synaptic dialogue, while GluRIIA receptors are the potent subtype that sets synaptic strength, drives muscle contraction, and is targeted for plasticity. These differential functions may be reflected in their distinct subsynaptic localizations, with GluRIIA enriched opposite active zone centers where glutamate is released, while GluRIIB is enriched in the outside periphery of these areas. Another possibility, not mutually exclusive, is that GluRIIBs serve as 'back-up' receptors to maintain NMJ transmission and locomotion when GluRIIAs are blocked, a phenomenon that occurs naturally at larval NMJs due to toxins injected by parasitoid wasps and other organisms. Indeed, presynaptic homeostatic potentiation, a conserved form of retrograde plasticity modeled at the fly NMJ, is induced when GluRIIA is lost or pharmacologically inhibited to maintain stable NMJ excitation (Han, 2023).

Hence, stable GluRIIB receptors provide robustness to buffer NMJ function from perturbations while also allowing flexibility for GluRIIA receptors to dynamically change with plasticity (Han, 2023).

Although the Drosophila NMJ has long been used as a model to study presynaptic forms of adaptive plasticity such as homeostatic potentiation and depression,recent work has found parallel modes of adaptive plasticity that target postsynaptic GluR abundance at this model glutamatergic synapse. In addition to excess glutamate targeting GluRs, at least three additional examples of adaptive GluR plasticity have been observed at the fly NMJ. First, in synaptic undergrowth mutants, where presynaptic innervation is reduced, overall synaptic strength can be maintained at least in some cases through an adaptive enhancement of postsynaptic GluR abundance. Second, when innervation by a single motor neuron is biased at adjacent muscles, stable synaptic strength is maintained through both pre- and postsynaptic mechanisms, with a homeostatic increase in postsynaptic GluR abundance necessary at hypo-innervated NMJs. Third, activation of injury-related signaling in motor neurons induces a downregulation in postsynaptic GluR abundance to adaptively reduce the set point of synaptic strength (Han, 2023).

These examples suggest an underappreciated level of postsynaptic plasticity exists at the Drosophila NMJ, which when combined with the sophisticated genetic and functional tools available, highlights the great potential for this system to illuminate how GluR subtype competition and presynaptic function target GluRs for adaptive modulation (Han, 2023).

There are several limitations regarding understanding of the timing and dynamics of the GluR plasticity described in this study. Because GluR composition, abundance, and function were assessednthrough imaging and electrophysiology at fixed times in later stages of development (third-instar larvae), the time course of the changes in GluR levels (minutes, hours, days) is uncertain. It is also not clear whether the reduction in GluRIIA levels, in response to excess glutamate release, is due to reduced transcription, translation, trafficking, and/or enhanced degradation. Finally, it is not clear to what extent action-potential-driven patterns of activity are altered during development in vGlut-OE NMJs and whether these potential differences contribute to GluR plasticity in addition to the enhanced glutamate emitted from individual synaptic vesicles. Future studies, including intravital live imaging of GluRs, will help to address these limitations (Han, 2023).

Regulation of postsynaptic retrograde signaling by presynaptic exosome release

Retrograde signals from postsynaptic targets are critical during development and plasticity of synaptic connections. These signals serve to adjust the activity of presynaptic cells according to postsynaptic cell outputs and to maintain synaptic function within a dynamic range. Despite their importance, the mechanisms that trigger the release of retrograde signals and the role of presynaptic cells in this signaling event are unknown. This study shows that a retrograde signal mediated by Synaptotagmin 4 (Syt4) is transmitted to the postsynaptic cell through anterograde delivery of Syt4 via exosomes. Thus, by transferring an essential component of retrograde signaling through exosomes, presynaptic cells enable retrograde signaling (Korkut, 2013).

This study shows that Syt4 protein functions in postsynaptic muscles to mediate activity-dependent presynaptic growth and potentiation of quantal release. However, to mediate this function Syt4 needs to be transferred from presynaptic terminals to postsynaptic muscle sites. Evidence is presented that, most likely, the entire pool of postsynaptic Syt4 is derived from presynaptic cells. Like the Wnt binding protein, Evi, Syt4 is packaged in exosomes, which provides a mechanism for the unusual transfer of transmembrane proteins across cells. Taken together, these studies support a novel mechanism for the presynaptic control of a retrograde signal, through the presynaptic release of exosomes containing Syt4 (Korkut, 2013).

Larval NMJs continuously generate new synaptic boutons and their corresponding postsynaptic specializations, ensuring constant synaptic efficacy despite the continuous growth of muscle cells. This precise matching of pre- and postsynaptic compartments is regulated by electrical activity, which induces a retrograde signal in muscle to stimulate new presynaptic growth. This process is likely to fine-tune the magnitude of the retrograde signal in specific nerve terminal-muscle cell pairs, each with a characteristic size. Given that most larval muscle cells are innervated by multiple motorneurons, this mechanism may also enable spatial coincidence to ensure the synaptic specificity of plasticity, making certain that only those activated synapses within a cell become structurally regulated (Korkut, 2013).

Macros to Quantify Exosome Release and Autophagy at the Neuromuscular Junction of Drosophila Melanogaster

Automatic quantification of image parameters is a powerful and necessary tool to explore and analyze crucial cell biological processes. This article describes two ImageJ/Fiji automated macros to approach the analysis of synaptic autophagy and exosome release from 2D confocal images. Emerging studies point out that exosome biogenesis and autophagy share molecular and organelle components. Indeed, the crosstalk between these two processes may be relevant for brain physiology, neuronal development, and the onset/progression of neurodegenerative disorders. In this context, this study studied the macros "Autophagoquant" and "Exoquant" to assess the quantification of autophagosomes and exosomes at the neuronal presynapse of the Neuromuscular Junction (NMJ) in Drosophila melanogaster using confocal microscopy images. The Drosophila NMJ is a valuable model for the study of synapse biology, autophagy, and exosome release. By use of Autophagoquant and Exoquant, researchers can have an unbiased, standardized, and rapid tool to analyze autophagy and exosomal release in Drosophila NMJ (Sanchez-Mirasierra, 2021).

A distinct perisynaptic glial cell type forms tripartite neuromuscular synapses in the Drosophila adult

Studies of Drosophila flight muscle neuromuscular synapses have revealed their tripartite architecture and established an attractive experimental model for genetic analysis of glial function in synaptic transmission. This study defined a new Drosophila glial cell type, designated peripheral perisynaptic glia (PPG), which resides in the periphery and interacts specifically with fine motor axon branches forming neuromuscular synapses. Identification and specific labeling of PPG was achieved through cell type-specific RNAi-mediated knockdown (KD) of a glial marker, Glutamine Synthetase 2 (GS2). In addition, comparison among different Drosophila neuromuscular synapse models from adult and larval developmental stages indicated the presence of tripartite synapses on several different muscle types in the adult. In contrast, PPG appear to be absent from larval body wall neuromuscular synapses, which do not exhibit a tripartite architecture but rather are imbedded in the muscle plasma membrane. Evolutionary conservation of tripartite synapse architecture and peripheral perisynaptic glia in vertebrates and Drosophila suggests ancient and conserved roles for glia-synapse interactions in synaptic transmission (Strauss, 2015).

The extracellular-regulated kinase effector Lk6 is required for Glutamate receptor localization at the Drosophila neuromuscular junction

The proper localization and synthesis of postsynaptic glutamate receptors are essential for synaptic plasticity. Synaptic translation initiation is thought to occur via the target of rapamycin (TOR) and mitogen-activated protein kinase signal-integrating kinase (Mnk) signaling pathways, which is downstream of extracellular-regulated kinase (ERK). This study used the model glutamatergic synapse, the Drosophila neuromuscular junction, to better understand the roles of the Mnk and TOR signaling pathways in synapse development. These synapses contain non-NMDA receptors that are most similar to AMPA receptors. The data show that Lk6, the Drosophila homolog of Mnk1 and Mnk2, is required in either presynaptic neurons or postsynaptic muscle for the proper localization of the GluRIIA glutamate receptor subunit. Lk6 may signal through eukaryotic initiation factor (eIF) 4E to regulate the synaptic levels of GluRIIA as either interfering with eIF4E binding to eIF4G or expression of a nonphosphorylatable isoform of eIF4E resulted in a significant reduction in GluRIIA at the synapse. It was also found that Lk6 and TOR may independently regulate synaptic levels of GluRIIA. (Hussein, 2016).

This study is the first to provide information on the properties and regulation of the Drosophila protein kinase LK6. Its catalytic domain is strikingly similar to those of mammalian Mnks; similar to them, in mammalian cells LK6 can bind to ERK, can be activated by ERK signalling and can phosphorylate eIF4E. This occurs at the physiological site, Ser²⁰⁹. The MAPK-binding motif of LK6 is of the type previously shown to bind ERK but not p38 MAPK. Consistent with this, when expressed in mammalian cells, LK6 is not activated by stimuli that turn on p38 MAPK (Hussein, 2016).

It is more challenging to perform similar experiments in Drosophila cells owing to the difficulty in transfecting, e.g. S2 cells with high efficiency. However, importantly, this study shows that LK6 also interacts with the ERK homologue Rolled, but not with the Drosophila p38 homologue. The results, furthermore, show that LK6 is activated by Phorbol myristate acetate (PMA), but not by arsenite, which activates p38 MAPK. The regulatory properties of LK6 thus appear to be similar in mammalian and Drosophila cells, indicating that the specificity of the MAPK-interaction motifs is probably similar in both mammals and Diptera. Similar to Mnk1 and Mnk2a, LK6 is primarily, if not exclusively, cytoplasmic. It does contain a basic region of the type that, in Mnk1 and Mnk2, can bind to the nuclear shuttling protein importin-α. It therefore seems probable that either (1) it contains an NES, which ensures its efficient re-export from the nucleus, or (2) the basic region is not accessible to importin-α. The lack of effect of LMB on the localization of LK6 rules out the operation of a CRM1-type NES of the kind found in Mnk1, although the very long C-terminal extension of LK6 might contain an LMB-insensitive NES (Hussein, 2016).

By analogy with the Mnks, it is probable that the N-terminal polybasic region of LK6 mediates its binding to eIF4G and could also interact with importin-α. Given that full-length LK6 shows less efficient binding to eIF4G when compared with Mnk1, it also seems possible that it binds importin-α less efficiently, which may contribute to the finding that LK6 is cytoplasmic. It has been shown previously that even the much shorter C-terminus of Mnk2a impedes access to the N-terminal basic region in that protein, so it is entirely possible that the much larger C-terminal part of LK6 has a similar effect. This could explain why the fragment of LK6 that lacks the C-terminus bound better to eIF4G than did the full-length protein. It may also be that the low degree of binding reflects the fact that the association of LK6 with the heterologous human protein was being studied, rather than with Drosophila eIF4G. Repeated attempts have been made to use the available antisera to examine the association of LK6 with eIF4G in S2 cells, but without success. Comparison of the polybasic region of LK6 with those of Mnk1 and Mnk2a (which do bind eIF4G and importin-α), and recent results for mutants with alterations in these features, do not reveal any difference that might obviously explain the decreased ability of LK6 to bind mammalian eIF4G. As argued above, the C-terminus of LK6 may also impair its activation by ERK, based on the observation that the catalytic domain is more effectively activated than a mutant of the full-length protein that also lacks the ERK-binding motif (Hussein, 2016).

The results support the idea that LK6 is a Drosophila eIF4E kinase. LK6 can phosphorylate eIF4E in vitro and its overexpression in cells leads to increased phosphorylation of endogenous eIF4E. Furthermore, the activation of LK6 by ERK signalling but not by p38 MAPK signalling correlates well with the observed behaviour of the phosphorylation of eIF4E in PMA- or arsenite-treated Drosophila cells, and the fact that LK6 is activated by stimuli that stimulate ERK but is not activated by stimuli that activate p38 MAPK, in HEK-293 cells. The ability of LK6 to bind eIF4G also supports the contention that it can act as an eIF4E kinase in vivo (Hussein, 2016).

The observation that phosphorylation of the endogenous eIF4E in S2 cells is increased by PMA but not by arsenite is consistent with the regulatory properties of LK6 and with the notion that LK6 may phosphorylate eIF4E in these cells. The fact that it is the only close homologue of the Mnks in the fruitfly genome is also consistent with this notion. Phosphorylation of eIF4E has previously been shown to play an important role in growth in this organism and in its normal development. The current data show that LK6 can phosphorylate Drosophila eIF4E in vitro, consistent with the idea that LK6 acts as an eIF4E kinase in this organism. The dsRNAi data that was obtained, which show that two different interfering dsRNAs directed against LK6 each markedly decrease eIF4E phosphorylation in S2 cells, offer strong support to the conclusion that LK6 acts as an eIF4E kinase in Drosophila. Unfortunately, the poor quality of the available anti-LK6 antisera prevented assessing whether the incomplete nature of the loss of phosphorylation of eIF4E reflects incomplete elimination of LK6 expression (Hussein, 2016).

Previous genetic studies have linked LK6 to Ras signalling in Drosophila. This agrees very well with the finding that LK6 is activated by ERK signalling, since ERK lies downstream of Ras. LK6 was first identified as interacting with microtubules and centrosomes. Overexpression of LK6 led to defects in microtubule organization, indicative of their increased stability. The connections between the phosphorylations of eIF4E and microtubules are not immediately obvious. However, it is entirely possible that LK6 has additional substrates that interact with microtubules or are components of centrosomes and their phosphorylation may be important in the regulation of, for example, mitosis. Numerous microtubule-associated proteins are indeed phosphorylated. Microtubules undergo massive reorganization during mitosis and this involves an array of phosphorylation events and protein kinases. It may therefore be relevant that LK6 is activated by mitogenic signalling (i.e. through ERK and thus Ras) (Hussein, 2016).

Drosophila ortholog of intellectual disability-related ACSL4, inhibits synaptic growth by altered lipids

Nervous system development and function are tightly regulated by metabolic processes, including the metabolism of lipids such as fatty acids (FAs). Mutations in long-chain acyl-CoA synthetase 4 (ACSL4) are associated with non-syndromic intellectual disabilities. A previous study reported that Acsl, the Drosophila ortholog of mammalian ACSL3 and ACSL4, inhibits neuromuscular synapse growth by suppressing transforming growth factor-beta/bone morphogenetic protein (BMP) signaling. This study reports that Acsl regulates the composition of FAs and membrane lipid, which in turn affect neuromuscular junction (NMJ) synapse development. Acsl mutant brains had decreased abundance of C16:1 fatty acyls; restoration of Acsl expression abrogated NMJ overgrowth and the increase in BMP signaling. A lipidomic analysis revealed that Acsl suppressed the levels of three lipid raft components in the brain, including mannosyl glucosylceramide (MacCer), phosphoethanolamine ceramide, and ergosterol. MacCer level was elevated in Acsl mutant NMJs and along with sterol promoted NMJ overgrowth but was not associated with the increase in BMP signaling in the mutants. These findings suggest that Acsl inhibits NMJ growth by stimulating C16:1 and concomitantly suppressing raft-associated lipid levels (Huang, 2016).

Lipids are essential membrane components that have crucial roles in neural development and function. Dysregulation of lipid metabolism underlies a wide range of human neurological diseases including neurodegeneration and intellectual disability. Acyl-CoA synthetase long-chain family member 4 (ACSL4) is the first gene in fatty acid metabolism associated with non-syndromic intellectual disability. ACSL4 protein has two variants: a ubiquitously expressed short form, and a brain-specific long form that is highly expressed in the hippocampus, a crucial region for memory. Indeed, ACSL4 has been shown to play an important role in synaptic spine formation. However, it is unclear how mutations in ACSL4 lead to intellectual disability (Huang, 2016).

There are 26 genes encoding acyl-CoA synthetases (ACSs) in humans. Each of the enzymes has distinct substrate preferences for fatty acid with various lengths of aliphatic carbon chains. In contrast, there are 13 ACS genes in the Drosophila genome. ACSs convert free fatty acids into acyl-CoAs for lipid synthesis, fatty acid degradation or membrane lipid remodeling. For example, ACSL4 converts long-chain fatty acids (LCFAs; aliphatic tails longer than 12 carbons), preferentially arachidonic acid (C20:4), into LCFA-CoAs that are incorporated into glycerol-phospholipids (GPLs) and neutral lipids in non-neuronal cells. The mechanism of how fatty acids and fatty-acid-modifying enzymes affect lipid composition, and thereby modulate development processes, is beginning to be understood in lower model organisms (Huang, 2016 and references therein).

Synaptic growth is required for normal brain function such as learning and memory. Many neurological disorders including intellectual disability are associated with synaptic defects. The Drosophila neuromuscular junction (NMJ) is a powerful system for studying the mechanisms that regulate synaptic growth. Drosophila Acsl, also known as dAcsl, is the ortholog of mammalian ACSL3 and ACSL4 (Zhang, 2009). It has been reported that Acsl affects axonal transport of synaptic vesicles and inhibits NMJ growth by inhibiting bone morphogenetic protein (BMP) signaling However, how Acsl affects lipid metabolism and the role of Acsl-regulated lipid metabolism in synapse development are largely unknown (Huang, 2016).

This study demonstrates that Acsl positively regulates the abundance of the LCFA palmitoleic acid (C16:1) in the brain. Reduced levels of C16:1 in Acsl mutants led to NMJ overgrowth and enhanced BMP signaling. A lipidomic analysis revealed that mannosyl glucosylceramide (MacCer), phosphoethanolamine ceramide (CerPE, the Drosophila analog of sphingomyelin) and ergosterol levels were increased in Acsl mutant brains. Genetic and pharmacological analyses further showed that the increased level of MacCer and sterol underlie the NMJ overgrowth in Acsl mutants in a pathway parallel to BMP signaling. These results indicate that Acsl regulates fatty acid and sphingolipid levels to modulate growth signals and NMJ growth, providing insight into the pathogenesis of ACSL4-related intellectual disability (Huang, 2016).

Like ACSL4, Acsl primarily associates with the ER and facilitates fatty acid incorporation into lipids ; impairment of Acsl activity reduces the abundance of its substrate LCFAs in lipids. Although this study did not directly determine the substrate preference of Acsl, fatty acid analysis suggests that both Acsl and ACSL4 positively regulate C16:1 abundance in Drosophila brain. The two proteins also have conserved functions in other processes, such as lipid storage, axonal transport and synaptic development (Huang, 2016 and references therein).

The similar NMJ overgrowth in desat1 and Acsl mutants suggests that the normal fatty acid composition is essential for proper development of synapses. The rescue effect of C16:1, together with the genetic interaction between Acsl and desat1, indicates that reduced C16:1 contributes to NMJ overgrowth in Acsl mutants. It has been previously reported that the synaptic overgrowth in Acsl mutants is due in part to an elevation of BMP signaling resulting from defects in endocytic recycling and BMP receptor inactivation. Endosomes are membrane compartments that are regulated by various membrane lipids, particularly the conversion between different PtdIns species, such as PI(3)P, PI(4,5)P₂, PI(3,4,5)P₃ and so on. The current findings suggest that increased BMP signaling in Acsl mutants is associated with an imbalance in fatty acid composition, specifically, a decrease in C16:1. It is thus possible that proper fatty acid composition is necessary for the normal conversion and localization of endosomal lipids (e.g., PtdIns species), affecting endosomal recycling and BMP receptor inactivation. Future studies will examine the regulation of specific fatty acids such as C16:1 in endosomal recycling and BMP signaling (Huang, 2016).

Acsl primarily associates with the ER in multiple cell types including motor neurons and participates in lipid synthesis. In Drosophila, most of acyl chains in GPLs are C16 and C18 LCFAs without VLCFAs. In contrast, sphingolipids contain higher levels of VLCFAs than LCFAs as acyl chains. Thus, most LCFA-CoAs are channeled into GPLs whereas VLCFA-CoAs are mainly incorporated into sphingolipids in Drosophila. This study found that Acsl positively regulates the production of C16:1-containing GPLs, as well as the level of PtdEth, the most abundant GPL in the brain. Presumably, fatty acids that are less preferred by Acsl could be channeled into lipids by other ACSs, and might show increased abundance because of a compensatory mechanism in Acsl mutants, which could contribute to the elevation in VLCFA-containing sphingolipids (Huang, 2016).

In addition to ER localization, ACSL4 and Acsl also localize to peroxisomes in a few non-neuronal cells, suggesting a role for these proteins in the activation of VLCFAs for peroxisomal degradation. Indeed, in animal models and patients with impaired peroxisomal function, accumulation of VLCFAs or increased levels of lipid species with longer fatty acid chains is observed. Thus, a defect in peroxisomal VLCFA degradation might underlie the elevation in sphingolipid species with VLCFA chains in Acsl mutant brains (Huang, 2016).

Alternatively, Acsl might affect lipid composition through a pathway that is independent of fatty acid incorporation. For example, as degradation of sphingolipids occurs primarily within lysosomes, a defect in lysosomal degradation might lead to an accumulation of sphingolipids and sterols in Acsl mutants. In addition, fatty acids and acyl-CoAs are ligands of many transcription factors, and ACSL3 activates the transcription of lipogenic genes in rat hepatocytes. Thus, Acsl might transcriptionally regulate genes encoding enzymes involved in the metabolism of lipids such as MacCer, CerPE and PtdEth. However, the detailed mechanism of how Acsl affects lipid class composition, especially the downregulation of raft-related MacCer and CerPE by Acsl in the nervous system, remains to be elucidated (Huang, 2016).

The data showed that elevation of the raft-related lipids MacCer and sterol facilitate NMJ overgrowth in Acsl mutants. Moreover, MacCer promotes bouton formation in a pathway parallel to BMP signaling, at least in part. It is unclear how these raft-related lipids regulate synaptic growth. It is likely that MacCer and sterol might interact with raft-associated growth signaling pathways. Larval NMJ development is mediated by multiple growth factors and downstream signaling cascades. For instance, Wingless (Wg) (Wnt1 in mammals) is a raft-associated protein that activates signaling pathways essential for NMJ growth and synaptic differentiation. It is therefore possible that the level or activity of some raft-associated growth factors is increased in Acsl mutants, thereby promoting NMJ overgrowth. Further investigation is needed to dissect the regulatory mechanisms of raft-related lipids, particularly MacCer, in promoting synaptic growth and bouton formation. It also would be of interest to address how Acsl-regulated lipids regulate neurotransmission in conjunction with synapse development (Huang, 2016).

Activity-induced synaptic structural modifications by an activator of integrin signaling at the Drosophila neuromuscular junction

Activity-induced synaptic structural modification is crucial for neural development and synaptic plasticity, but the molecular players involved in this process are not well defined. This study reports that a protein named Shriveled, Shv, regulates synaptic growth and activity-dependent synaptic remodeling at the Drosophila neuromuscular junction. Depletion of Shv causes synaptic overgrowth and an accumulation of immature boutons. Shv physically and genetically interacts with βPS integrin. Furthermore, Shv is secreted during intense, but not mild, neuronal activity to acutely activate integrin signaling, induce synaptic bouton enlargement, and increase postsynaptic glutamate receptor abundance. Consequently, loss of Shv prevents activity-induced synapse maturation and abolishes post-tetanic potentiation, a form of synaptic plasticity. These data identify Shv as a novel trans-synaptic signal secreted upon intense neuronal activity to promote synapse remodeling through integrin receptor signaling (Yeun Lee, 2017)

Two algorithms for high-throughput and multi-parametric quantification of Drosophila neuromuscular junction morphology

The Drosophila larval neuromuscular junction (NMJ), a well-established model for glutamatergic synapses, has been extensively studied for decades. Identification of mutations causing NMJ morphological defects revealed a repertoire of genes that regulate synapse development and function. Many of these were identified in large-scale studies that focused on qualitative approaches to detect morphological abnormalities of the Drosophila NMJ. This protocol describes in detail two image analysis algorithms "Drosophila NMJ Morphometrics" and "Drosophila NMJ Bouton Morphometrics", available as Fiji-compatible macros, for quantitative, accurate and objective morphometric analysis of the Drosophila NMJ. This methodology is developed to analyze NMJ terminals immunolabeled with the commonly used markers Dlg-1 and Brp. Additionally, its wider application to other markers such as Hrp, Csp and Syt is presented in this protocol. The macros are able to assess nine morphological NMJ features: NMJ area, NMJ perimeter, number of boutons, NMJ length, NMJ longest branch length, number of islands, number of branches, number of branching points and number of active zones in the NMJ terminal (Castells-Nobau, 2017).

Myostatin-like proteins regulate synaptic function and neuronal morphology

Growth factors of the TGF-beta superfamily play key roles in regulating neuronal and muscle function. Myostatin (or GDF8) and GDF11 are potent negative regulators of skeletal muscle mass. However, expression of both Myostatin and its cognate receptors in other tissues, including brain and peripheral nerves, suggests a potential wider biological role. This study shows that Myoglianin (MYO), the Drosophila homolog of Myostatin and GDF11, regulates not only body weight and muscle size, but also inhibits neuromuscular synapse strength and composition in a Smad2-dependent manner. Both Myostatin and GDF11 affected synapse formation in isolated rat cortical neuron cultures, suggesting an effect on synaptogenesis beyond neuromuscular junctions. This study also shows that Myoglianin acts in vivo to inhibit synaptic transmission between neurons in the escape response neural circuit of adult flies. Thus, these anti-myogenic proteins act as important inhibitors of synapse function and neuronal growth (Augustin, 2017).

Growth factors regulate many aspects of tissue development, growth and metabolism. Myostatin and GDF11 are highly homologous members of the TGF-β superfamily of growth factors. While GDF11 plays a role in a variety of systems, the role of Myostatin appears to be confined to skeletal and cardiac muscles (Augustin, 2017).

Despite the previously described roles of MYO in neural remodelling and synapse refinement (Awasaki, 2011; Yu, 2013) very little is known about the impact of Myoglianin on synaptic physiology. This study first established muscle-derived MYO as a negative regulator of both spontaneous and evoked response at the NMJ, demonstrating its role as a broad regulator of synaptic transmission. The highly coordinated apposition of active zones and glutamate receptors underlies their ability to regulate synaptic strength and plasticity of the larval NMJ. This study has shown that muscle expression of myo inversely affects the NMJ quantity of Brp and GluRIIA, critical pre-and post-synaptic proteins, and determinants of evoked neurotransmitter release and quantal size (i.e., postsynaptic sensitivity to presynaptically released transmitter), respectively. While it is possible that MYO exerts its influence on synaptic strength through other mediators, GluRIIA and Brp are their likely downstream effectors. The electrophysiological results, obtained using the GAL4-UAS system for targeted manipulation of myo, differ from the ones obtained recently using a genetic null myo mutant showing slightly reduced miniature amplitudes (Kim, 2014). The likely explanation is that compensatory effects happen in other tissues in the tissue-specific knockdown animals that cannot occur in genetic nulls, especially for systemic type factors. The other possible explanation is differential cross regulation between different (MYO-like) ligands in genetic null vs tissue knockdown animals. These results thus indicate the relevance of tissue specificity of MYO action, and of myo expression levels, in regulating synaptic function, and emphasize the need for caution when interpreting results from different types of gene manipulations (Augustin, 2017).

myo expression was detected in the glial cells of the larval neuromuscular junction. While Drosophila NMJ contains at least 2 subtypes of glia, myo expression appears confined to the 'repo-positive' subtype both in the central (Awasaki, 2011) and peripheral nervous system. The dual muscle and glial presence makes MYO ideally positioned for regulating NMJ function. Due to the small size of the compartment, however, glia-derived MYO likely has a modulatory role at the neuromuscular junction (Augustin, 2017).

This study also found that muscle suppression of Myoglianin, a Drosophila homolog of Myostatin and GDF11, promotes increased larval weight and body-wall muscle size in developing larvae, resembling the effect of Myostatin knockdown in mammals. Interestingly, pan-glial expression of myo negatively affected larval wet weight, but not the size of somatic myofibers, suggesting previously unsuspected systemic roles for glial cells (Augustin, 2017).

Smad2 is a mediator of MYO action on both evoked response and postsynaptic sensitivity, with MAD having a minor effect on the latter. While MAD primarily functions as a cytoplasmic transducer of BMP signalling, it has been demonstrated that, under certain conditions, MAD can be phosphorylated in response to Activin pathway activation (Peterson, 2012; Augustin, 2017 and references therein).

This study detected elevated levels of phosphorylated Akt and GSK-3/Shaggy in larval somatic muscles of animals with reduced myo expression in this tissue. In flies and mammals, the Akt- mTOR axis promotes skeletal muscle growth, and phosphorylation-induced inhibition of GSK-3/Shaggy induces hypertrophy in skeletal myotube. The effects of attenuated myo expression on larval tissue size, however, do not appear to be mediated by Smad2 (or MAD) activation as their overexpression does not reverse the weight phenotype in 'low myo' background. Indeed, 'non-Smad' signalling pathways have been demonstrated for various TGF-β ligands in vertebrates and Drosophila. In addition to its role as an inhibitor of the NMJ growth and active zone formation in developing Drosophila larvae, GSK-3β is also a critical promoter of synaptic plasticity, possibly through regulation of glutamate receptor function or trafficking. This work has revealed Shaggy as a mediator of reduced MYO action, and as a negative regulator of synaptic strength at the larval NMJ. While MYO likely affects both sides of the synapse directly, an unlikely but possible scenario is that presynaptic motoneuron responds to a retrograde signal released from muscle/glial cells at the NMJ in response to an induction by MYO. An attractive hypothesis is that MYO negatively regulates presynaptic release directly, in conjunction with muscle-secreted Gbb, a positive regulator of neuromuscular synapse development and growth. The effects of MYO could also be mediated through the transmembrane protein Plum, previously proposed to regulate connectivity at the larval NMJ by sequestrating Myoglianin (Yu, 2013; Augustin, 2017 and references therein).

Myostatin negatively regulates synaptic function and neuronal morphology This study found that injections of Myostatin into rapidly growing larvae abolish the positive effect of myo down-regulation on NMJ strength and composition, and reverse the elevated muscle p-Akt levels. Furthermore, both Myostatin and GDF11 surpressed the growth of neuronal processes and perturbed the formation of synapses in cultured brain neurons, suggesting a direct action on neurons and regulation of synaptogenesis beyond neuromuscular junctions. Recently, Myostatin transcript and protein were detected in the mouse hippocampus and olfactory system neurons, respectively, and Myostatin type I (Alk4/5) and type II (ActIIB) receptors were found to be expressed in the mammalian nervous system. The current results therefore expand on these findings, suggesting functional relevance for Myostatin in both peripheral and central nervous system, and beyond its action as a canonical regulator of skeletal muscle growth. These novel roles remain to be further explored (Augustin, 2017).

This study expanded analysis of the functional relevance of MYO in the nervous system by demonstrating its importance in a non-NMJ synapse. Specifically, Myoglianin plays a role in the development of a mixed electro-chemical synapse in the Drosophila escape response pathway, likely by regulating the density of shakB-encoded gap junctions at the GF-TTMn synapse (Blagburn, 1999). These findings implicate MYO as a broad negative regulator of neuronal function across the nervous system and developmental stages (Augustin, 2017).

This work thus reveals broad and novel roles for anti-myogenic TGF-β superfamily of proteins in the nervous system and suggests new targets for interventions into synaptic function across species (Augustin, 2017).

Secreted tissue inhibitor of matrix metalloproteinase restricts trans-synaptic signaling to coordinate synaptogenesis

Synaptogenesis is coordinated by trans-synaptic signals that traverse the specialized synaptomatrix between pre- and postsynaptic cells. Matrix metalloproteinase (Mmp) activity sculpts this environment, balanced by secreted Tissue inhibitors of Mmp (Timp). This study used the reductionist Drosophila matrix metalloproteome to test consequences of eliminating all Timp regulatory control of Mmp activity at the neuromuscular junction (NMJ). Using in situ zymography, Timp was found to limit Mmp activity at the NMJ terminal and shape extracellular proteolytic dynamics surrounding individual synaptic boutons. In newly-generated timp null mutants, NMJs exhibit architectural overelaboration with supernumerary synaptic boutons. With cell-targeted RNAi and rescue studies, postsynaptic Timp was found to limit presynaptic architecture. Functionally, timp nulls exhibit compromised synaptic vesicle cycling, with reduced, lower fidelity activity. NMJ defects manifest in impaired locomotor function. Mechanistically, Timp was found to limit BMP trans-synaptic signaling and the downstream synapse-to-nucleus signal transduction. Pharmacologically restoring Mmp inhibition in timp nulls corrects BMP signaling and synaptic properties. Genetically restoring BMP signaling in timp nulls corrects NMJ structure and motor function. Thus, Timp inhibition of Mmp proteolytic activity restricts BMP trans-synaptic signaling to coordinate synaptogenesis (Shilts, 2017).

The synaptic cleft is populated with a complex extracellular network of secreted and transmembrane proteins, yet little is known about the extracellular mechanisms that act to shape this critical cellular interface. This synaptomatrix contains integrin, heparan sulfate proteoglycan and cognate receptors for a host of known secreted and transmembrane ligands. Extracellular proteins in the cleft are extensively remodeled in parallel with intercellular changes that accompany synaptic maturation and activity-dependent plasticity. Extracellular matrix metalloproteinase (Mmp) enzymes catalyze synaptic remodeling by proteolytically cleaving the secreted and transmembrane substrates regulating synapse structural integrity, and modulating intercellular signaling between presynaptic and postsynaptic partners. Given the powerful organizing effects of these proteases, their activity must be tightly regulated. One key mechanism is secretion of tissue inhibitors of Mmps (Timps), which restrict Mmp activity to proper spatial and temporal windows. Whenever Mmp regulation is disrupted, developmental abnormalities and disease often result. Improper Mmp expression and activity is implicated in a range of neurological disorders, including schizophrenia, addiction, epilepsy and autism spectrum disorder (ASD). As the most common heritable ASD and intellectual disability, Fragile X syndrome (FXS) underscores the importance of preserving Mmp balance to control proper synaptic structure and function. Importantly, the Mmp inhibitor minocycline alleviates synaptic and behavioral phenotypes in FXS disease models, and has shown promise in clinical trials for human patients, showing that elevated Mmp activity is causally linked to FXS neuropathology (Shilts, 2017).

Since Mmp dysregulation produces pronounced synaptic defects in disease states, it was hypothesized that loss of the endogenous Mmp control mediated by Timp would disrupt synapse architecture and function. This study took advantage of the simplified Drosophila melanogaster matrix metalloproteome to test consequences of genetically ablating all Timp regulatory control over Mmp activity. In contrast to mammals, which have at least 24 Mmps and four partially redundant Timps, Drosophila has a single secreted Mmp (Mmp1), a single membrane-anchored Mmp (Mmp2) and a single Timp, all of which are highly conserved and can interact with their respective human homologs. In the Drosophila nervous system, Mmps direct both axonal targeting and dendritic remodeling. Recently, Mmp1 and Mmp2 were found to regulate trans-synaptic signaling at the neuromuscular junction (NMJ) to modulate synaptic structure and function. Moreover, trans-synaptic signaling dysregulation has been causally linked to synaptic defects in the Drosophila FXS model. One trans-synaptic pathway important for both synaptic structure and function involves bone morphogenetic protein (BMP) signaling via the Glass Bottom Boat (Gbb) ligand. Gbb secreted from the muscle regulates NMJ structure, whereas Gbb secreted from the motor neuron regulates neurotransmission. Gbb ligand in the extracellular space surrounding synaptic boutons activates downstream phosphorylated Mothers Against Decapentaplegic (pMAD) signal transduction locally at the synapse and distantly within motor neuron nuclei of the central nervous system. Synaptic pMAD is associated with assembly of functional neurotransmission machinery at the NMJ, whereas the accumulation of nuclear pMAD promotes NMJ growth. It is hypothesized that the balance of Mmp proteolytic activity controlled by Timp guides trans-synaptic signaling pathways to modulate both NMJ synaptic structure and function (Shilts, 2017).

To test this hypothesis, the first ever timp-specific loss-of-function null alleles were generated using CRISPR/Cas9 genome editing. Generation of specific mutations was previously intractable due to a conserved nested relationship that places the timp gene within an intron of the important synaptic synapsin gene. Previously work has shown that Timp localizes in the NMJ perisynaptic space (Dear, 2016), where it shows a co-dependent relationship with both secreted Mmp1 and membrane-anchored Mmp2. The current study employed in situ zymography in living NMJs to show that Timp inhibits synaptic Mmp function and regulates the dynamics of Mmp proteolytic activity in the extracellular space surrounding synaptic boutons. Loss of Timp regulation removes a restraint on synaptic architecture, resulting in the overelaboration of boutons. Using transgenic RNA interference (RNAi) and rescue, Timp secretion from the postsynaptic muscle was shown to be required to regulate the presynaptic motor neuron architecture. In parallel, Timp was also found to control synaptic function. By employing FM1-43 dye imaging, this study found Timp modulates the speed and fidelity of the synaptic vesicle (SV) cycle driving synaptic neurotransmission, and impairs the coordinated muscle peristalsis output of neuromuscular activity. In testing trans-synaptic signaling pathways, Timp function was found to act to restrict BMP signaling, with timp null mutants showing elevated Gbb ligand levels in the extracellular space surrounding synaptic boutons and increased downstream pMAD signal transduction at both the synapse and within motor neuron nuclei. Inhibiting proteolytic activity with minocycline treatment in timp null mutants restores normal BMP signaling and significantly corrects NMJ properties and output motor function. Genetically restoring normal BMP signaling in timp null mutants corrects NMJ architecture and functional motor output, indicating that aberrant trans-synaptic signaling is the causal mechanism. Taken together, these results show that neuromuscular synapses require a responsive balance of Mmp activity controlled by Timp inhibition to restrict the BMP trans-synaptic signaling that modulates NMJ structure and function (Shilts, 2017).

Remodeling of the synaptic extracellular environment is a highly dynamic process, demanding precise spatiotemporal control in response to specific developmental and activity-dependent signals. Mmp proteolytic activity is an ideal node of regulation for the necessary responsive kinetics and specificity, with Timps controlling the timing, duration and spatial specificity of enzyme function. Taking advantage of the simplified matrix metalloproteome of Drosophila, with only a single functionally conserved Timp, it was possible to eliminate all Timp function with one mutation. Using site-directed CRISPR/Cas9, the first timp null allele was generated, with targeted mutation of the timp gene, without disrupting the synapsin gene in which it is nested. Although nested genetic placement does not imply a functional relationship, the highly conserved nesting of timp in synapsin occurs across vertebrates and invertebrates, which are separated by hundreds of millions of years of evolution. The evolutionary conservation of timp nesting in synapsin is interesting, since synapsin encodes a key synaptic regulator and there is evidence of co-regulation of genes nested with Timps. In addition to timp loss-of-function mutants, no viable Mmp gain-of-function has yet been reported in Drosophila. Thus, the new CRISPR-induced timp null is a tool to characterize total Timp function as well as generally elevated Mmp activity as seen in the nervous system. Currently, there are relatively few reports concerning Timp loss in the nervous system. In mice, TIMP-2 knockout causes motor deficits and expanded NMJ branching, and TIMP-1 overexpression reduces outgrowth in cortical cells, supporting findings of this tudy. In Drosophila, Timp overexpression inhibits NMJ growth, which again complements the current findings (Shilts, 2017).

This study uncovered key roles for Timp in controlling synaptic Mmp activity, thereby regulating NMJ structure, function and output. Muscle-secreted Timp limits synaptic Mmp proteolytic activity and shapes the distribution of Mmp activity within the synaptomatrix. This local regulation of Mmp functional dynamics has not been reported in neuronal synapses, but is consistent with known roles of Timp in non-neuronal contexts. This study found that postsynaptic Timp limits presynaptic NMJ architecture and bouton formation. This is surprising given that individual Mmp knockdown similarly limits synaptic structure in flies and mice, but may suggest that both loss and gain of Mmp function converge phenotypically or that, collectively, Timp repression of multiple Mmp activities acts as a brake on synaptic growth. Timp also was found to regulate synaptic function, by facilitating SV endocytosis and maintaining SV cycle fidelity. In comparison, mmp mutants elevate transmission strength, also by altered SV cycling dynamics, consistent with Timp repression of Mmp function. Timp enables faster and higher fidelity muscle contraction peristalsis, driving coordinated locomotion. Motor defects have consistently been found across a range of Mmp manipulations, although molecular mechanisms had not been identified. Taken together, these results complete a characterization of the entire Drosophila matrix metalloproteome in controlling neuromuscular synapse structure and function. The timp null synaptic phenotypes prompt a re-assessment: Mmps are not simply negative regulators of synaptic differentiation, but can promote structural development within a context-dependent mechanism. This work shows Timp and Mmps interact to sculpt synapse form and function (Shilts, 2017).

Timp limits BMP trans-synaptic signals mediating communication between the muscle and motor neuron, with Timp loss elevating Gbb ligand levels. BMP ligands are well known to be sequestered by extracellular molecules, and proteolytic cleavage of these extracellular antagonists controls the distributions of signaling activity in multiple cellular contexts. In Drosophila neurons, Mmp2 regulates motor axon pathfinding and fasciculation via Mmp2-mediated proteolytic cleavage of the ECM Fibrillin/Fibulin-related Faulty Attraction (Frac) protein to enable BMP signaling. Similarly, this study found elevated BMP trans-synaptic signaling in timp mutants with Mmp proteolytic hyperactivity. Gbb secretion from the postsynaptic muscle regulates NMJ architecture, whereas Gbb released from the presynaptic motor neuron regulates neurotransmission function. These roles are consistent with the misregulation of synaptic structure and SV cycle function, respectively, seen in timp mutants with elevated Gbb signaling. The accumulation of Gbb in the perisynaptic synaptomatrix of timp null mutants drives downstream activation of pMAD signal transduction in both motor neuron synaptic terminals and motor neuron nuclei. This is consistent with pMAD activation of transcriptional programs for coordinating synapse structural and functional differentiation. Gbb secreted from the postsynaptic muscle is regulated by Timp that is also secreted from the muscle, which provides control for motor neuron terminals to expand in response to muscle growth and activity-dependent plasticity. In contrast, Mmps come from both presynaptic and postsynaptic cells. Thus, directional Timp control acts as a specific muscle-derived mechanism to regulate Gbb trans-synaptic signaling (Shilts, 2017).

Elevated BMP Gbb signaling in a Drosophila model of Troyer syndrome, a hereditary spastic paraplegia (HSP) disease, causes strikingly similar NMJ synaptic structural and functional defects to those seen upon loss of Timp. Like the timp null mutants, spartin mutants that are causatively associated with Troyer syndrome exhibit expanded synaptic arbors and decreased FM1-43 dye SV endocytic loading with impaired motor function. Importantly, Fragile X Mental Retardation Protein (FMRP) is a downstream effector of Spartin function, limiting BMP Gbb signaling. Loss of FMRP causes Fragile X syndrome (FXS), and reducing non-canonical BMP signaling alleviates the synaptic defects in Drosophila and mouse FXS disease models. Likewise, targeted mutation of the FXS-related S6 kinase (S6K) similarly results in both expanded synaptic architecture and decreased SV endocytosis at the Drosophila NMJ, once again resembling timp null phenotypes. As in timp mutants, there are also clear precedents for mutations of other key regulatory proteins increasing NMJ functional variability to compromise motor output function. These findings with timp demonstrate the utility of variability as a metric to uncover regulatory nodes that preserve the functional resiliency of the nervous system (Shilts, 2017).

By pharmacologically correcting timp null phenotypes with the characterized Mmp inhibitor minocycline, this study has shown that mutant defects are causally linked to Mmp hyperactivity. Alleviation of timp null phenotypes is robust, albeit partial, which may reflect experimental limitations of the drug administration, or possibly reveal other Mmp-independent Timp functions. In particular, behavioral assays of motor function show conspicuous, albeit partial, rescue, which may be evidence of an Mmp-independent contribution to motor function or, more likely, that the precise spatiotemporal dynamics of Timp at the NMJ are necessary for proper motor function. In rats, transient proteolytic activity in the synaptomatrix accompanies long-term potentiation and dendrite maturation , which corroborates the current model that Timp dynamically restricts synaptic modulation through localized ECM proteolysis. Crucially, pharmacologically balancing Mmp activity in timp null mutants with minocycline treatment restores BMP trans-synaptic signaling, and genetically correcting BMP signaling prevent synaptic and movement defects. These findings support the model that Mmp activity in the synaptomatrix, under regulation by Timp, limits BMP trans-synaptic signals, thereby controlling NMJ synaptogenesis and functional motor output (Shilts, 2017).

These studies provide an avenue for possible therapeutic treatments in a range of neurological disease states with elevated Mmp activity. In particular, Mmp hyperactivity has been causally implicated in FXS and related ASD conditions. The synaptic cytoarchitectural phenotypes of timp mutants phenocopy the Drosophila FXS model, and trans-synaptic signaling defects are causative in synaptic structural and functional defects in this disease model, including BMP signaling. By re-creating the elevated Mmp activity characterizing neurological disease conditions such as FXS, the timp genetic tools developed in this study provide insights into fundamental synaptic mechanisms with direct clinical relevance. In future studies, timp manipulations will be combined with established Drosophila disease models in order to more fully dissect contributions of Mmp-dependent trans-synaptic signaling impairments in different neurological disease states (Shilts, 2017).

Notum coordinates synapse development via extracellular regulation of Wnt Wingless trans-synaptic signaling

Synaptogenesis requires orchestrated communication between pre- and postsynaptic cells via coordinated trans-synaptic signaling across the extracellular synaptomatrix. The first discovered Wnt signaling ligand Drosophila Wingless (Wg; Wnt-1 in mammals) plays critical roles in synaptic development, regulating synapse architecture as well as functional differentiation. This study investigated synaptogenic functions of the secreted extracellular deacylase Notum, which restricts Wg signaling by cleaving an essential palmitoleate moiety. At the glutamatergic neuromuscular junction (NMJ) synapse, Notum secreted from the postsynaptic muscle was found to act to strongly modulate synapse growth, structural architecture, ultrastructural development and functional differentiation. In notum nulls, upregulated extracellular Wg ligand and nuclear trans-synaptic signal transduction was found, as well as downstream misregulation of both pre- and postsynaptic molecular assembly. Structural, functional and molecular synaptogenic defects are all phenocopied by Wg over-expression, suggesting Notum acts solely through inhibiting Wg trans-synaptic signaling. Moreover, these synaptic development phenotypes are suppressed by genetically correcting Wg levels in notum null mutants, indicating that Notum normally functions to coordinate synaptic structural and functional differentiation via negative regulation of Wg trans-synaptic signaling in the extracellular synaptomatrix (Kopke, 2017).

In the developing nervous system, Wnt signaling ligands act as potent regulators of multiple stages of neuronal connectivity maturation, stabilization and synaptogenesis, including sculpting structural architecture and determining neurotransmission strength. Drosophila Wingless is secreted from presynaptic neurons and glia at the developing glutamatergic neuromuscular junction (NMJ), to bind Frizzled-2 (Fz2) receptors in both anterograde and autocrine signaling. In the postsynaptic muscle, Wg binding to Fz2 activates the Frizzled Nuclear Import (FNI) signaling pathway, which involves Fz2 endocytosis followed by Fz2 cleavage and Fz2 C-terminus nuclear import (Mathew, 2005). Fz2-C trafficked in nuclear ribonucleoprotein (RNP) granules regulates translation of synaptic mRNAs, thereby driving expression changes that modulate synapse structural and functional differentiation (Speese, 2012). In the presynaptic neuron, Wg binding to Fz2 activates a divergent canonical pathway inhibiting the Glycogen Synthase Kinase 3β (GSK3β) homolog Shaggy (Sgg) to regulate microtubule cytoskeleton dynamics via Microtubule-Associated Protein 1B (MAP1B) homolog Futsch. Futsch binding to microtubules regulates architectural changes in synaptic branching and bouton formation. Such multifaceted Wg functions require tight management throughout synaptic development (Kopke, 2017).

A highly conserved extracellular Wg regulator is the secreted deacylase Notum. The notum gene was discovered in a Drosophila gain-of-function (GOF) mutant screen targeting wing development. Under scalloped-Gal4 control, notum GOF causes loss of the wing and duplication of the dorsal thorax. In the developing wing disc, Notum acts as a secreted, extracellular feedback inhibitor of Wg signaling. Notum function was recently re-defined as a carboxylesterase that cleaves an essential Wg lipid moiety (palmitoleic acid attached to conserved serine), leaving it unable to bind to Fz2 and activate downstream signaling (Kakugawa, 2015). This Wnt palmitoleate moiety is similarly cleaved by human Notum acting as a highly conserved secreted feedback antagonist in the extracellular space to inactivate Wnt signaling (Langton, 2016; Kakugawa, 2015). At the Drosophila NMJ, extracellular regulation of Wg trans-synaptic signaling has been found to play key roles in synaptogenesis (Dani, 2012b; Parkinson et al., 2013). For example, extracellular matrix metalloproteinase (MMP) enzymes cleave heparan sulfate proteoglycan (HSPG) co-receptors to regulate Wg trans-synaptic signaling that controls structural and functional synaptic development. Impairment of this mechanism is causative for Fragile X syndrome (FXS) synaptogenic defects. Similarly, misregulated extracellular mechanisms impair Wg trans-synaptic signaling in both Congenital Disorder of Glycosylation (CDG) and Galactosemia disease states, causing NMJ synaptogenic defects underlying coordinated movement disorders. Given these insights, this study investigated the putative roles for Notum as a new secreted Wg antagonist regulating synaptogenesis (Kopke, 2017).

This study utilized the well-characterized Drosophila NMJ glutamate synapse model to study Notum requirements in synaptic development. Notum, secreted from muscle and glia, is resident in the extracellular space surrounding developing synaptic boutons, where it negatively regulates Wg trans-synaptic signaling. In notum mutants, extracellular Wg ligand levels and downstream Wg signaling are elevated. Null mutants display both increased synapse number and strength, altered synaptic vesicle cycling, and synaptic ultrastructural defects including a decrease in SSR/bouton ratio, decreased synaptic vesicle density and an increase in the size of vesicular organelles. Cell-targeted RNAi studies reveal both postsynaptic and perisynaptic requirements, with muscle and glial notum knockdown resulting in overelaborated NMJ architecture, but neuronal-driven notum knockdown causing no detectable effects on synaptogenesis. Null notum defects are all phenocopied by neuronal Wg overexpression, suggesting that synaptogenic phenotypes arise from lack of Wg inhibition. Consistently, genetically correcting Wg levels at the synapse in notum nulls alleviates synaptogenic phenotypes, demonstrating that Notum functions solely as a negative regulator of Wg signaling. Taken together, these results identify Notum as a secreted Wnt inhibitor resident in the extracellular synaptomatrix with critical functions regulating trans-synaptic Wnt signaling to coordinate structural and functional synaptogenesis (Kopke, 2017).

Tightly coordinated trans-synaptic signals are required for proper development of the pre- and postsynaptic apparatus to ensure efficient communication at the synapse. This signaling is both coordinated and controlled in the extracellular space through the actions of secreted and transmembrane glycans, heparan sulfate proteoglycan (HSPG) co-receptors and secreted enzymes, such as matrix metalloproteinase (Mmp) classes. Wg (Wnt-1) mediates a critical trans-synaptic signaling pathway regulated by these extracellular synaptic mechanisms, with key roles in both structural and functional synaptogenesis. This study proposes that Notum is a novel extracellular regulator limiting Wg trans-synaptic signaling to control NMJ synaptogenesis. Wg is post-translationally modified by addition of palmitoleate on a conserved serine (S239) by membrane-bound O-acyltransferase (MBOAT) Porcupine. This lipidation event is required for Fz2 receptor binding and essential for signaling. At the synaptic interface, the GPI-anchored glypican Dally-like Protein (Dlp) regulates Wg trans-synaptic signaling, and Notum was initially described as cleaving such GPI-anchored glypicans from the cell surface, affecting their ability to interact with the Wg ligand. However, Notum was recently redefined as a secreted carboxylesterase, not a phospholipase (Kakugawa, 2015), with structural studies showing a hydrophobic pocket that binds and then cleaves palmitoleate (Kopke, 2017).

Notum is consistently reported to act primarily as an extracellular Wg feedback inhibitor. The current studies support this function within the synaptomatrix during synaptogenesis. At the Drosophila NMJ, Wg is secreted from both presynaptic neurons and associated peripheral glia (Kerr, 2014), with the glial function specifically regulating synaptic transmission strength and postsynaptic glutamate receptor clustering. This analyses suggest that Notum is secreted from both postsynaptic muscle and peripheral glia, establishing a dynamic, Wg-like expression pattern surrounding synaptic boutons. In notum null mutants, Wg signaling is increased at the developing NMJ, revealed by both decreased Fz2 receptor in the synaptic membrane (Wg-driven endocytosis) and an increase in nuclear Fz2-C punctae (FNI pathway). These findings are consistent with Notum function limiting Wg signaling, as established in other developmental contexts. Notum appears to provide a fascinating directional regulation of Wg trans- synaptic signaling, affecting the anterograde FNI signaling pathway in muscles, but not the autocrine divergent canonical pathway in neurons. Despite the strong elevation in synaptic Wg ligand levels in notum null mutants, no evidence is seen of altered presynaptic MAP1B homolog Futsch or changes in the microtubule cytoskeleton. However, Notum strongly limits Fz2 C-terminus nuclear import into the postsynaptic nuclei, which is known to drive ribonucleoprotein (RNP) translational regulation of synaptic mRNAs to control synapse structural and functional differentiation (Kopke, 2017).

Synaptic morphogenesis and architectural development is strongly perturbed in notum null mutants, including increased NMJ area, branching and bouton formation, consistent with Notum function inhibiting Wg trans-synaptic signaling. Elevating presynaptic Wg closely phenocopies notum synaptic defects, including expanded innervation area, more branching and supernumerary synaptic boutons. The results show that Notum secreted from muscle and peripheral glia controls Wg in the extracellular space, with targeted notum RNAi resulting in a similar NMJ expansion to notum nulls, whereas neuronal notum knockdown produces no effects. Interestingly, the glial-targeted RNAi increases boutons with no change in branching, whereas muscle knockdown has a stronger impact also affecting branching. Presynaptic Futsch/Map1B microtubule loops have been proposed to mediate Wg-dependent branching and bouton formation. However, neuronal Wg overexpression has no discernable effect on Futsch-positive microtubule loops. Consistently, Notum LOF also does not impact this pathway, with notum mutants displaying no change in Futsch-labeled looped, bundled, punctate or splayed microtubules. Wg binding to the presynaptic Fz2 receptor may activate another divergent Wnt pathway that does not involve Futsch. Alternatively, Wg signaling via muscle Fz2 may produce a retrograde signal back to the neuron to alter presynaptic development. To test these two possibilities, future studies will employ cell-targeted Fz2 knockdown in notum nulls to assay for suppression of the synaptic overgrowth phenotypes (Kopke, 2017).

Measures of functional synaptic differentiation reveal elevated neurotransmission and faster motor output function with both notum knockout and Wg over-expression. These results are consistent with Notum function inhibiting Wg trans-synaptic signaling, and consistent with previously characterized roles of Wg in NMJ functional development. Notum LOF increases presynaptic function selectively with an elevated mEJC frequency, greater EJC quantal content and heightened synaptic vesicle release during maintained high- frequency stimulation. Some of these effects may map to the increased synaptic bouton numbers. Both Notum LOF and Wg GOF also cause NMJ boutons to spatially clump together, with ultrastructural studies showing multiple boutons sharing one SSR profile. These are not satellite boutons, but rather aberrantly developing boutons that may result in functional defects. Notum knockdown in glia does not cause detectable mEJC/EJC changes, although Wg from glia regulates NMJ functional properties. Interestingly, loss of Notum appears to improve motor performance, and repo-targeted notum RNAi shows that glial Notum contributes to this function. This is an unusual outcome in a mutant condition, and it is assumed that there must be a counter-balancing cost for increasing neuromuscular function. Live FM dye imaging reveals that notum mutants load less dye into synaptic boutons upon nerve stimulation, indicating a role in synaptic vesicle endocytosis and/or the developmental regulation of synaptic vesicle pool size. These results show Notum function limits Wg trans-synaptic signaling to control presynaptic differentiation critical for synapse function and motor output. As with Wg, the source of Notum (muscle vs. glia) appears to be important for distinct synaptogenic functions. Notum from peripheral glia regulates only bouton formation, whereas Notum from muscle regulates both NMJ growth and function (Kopke, 2017).

Electron microscopy reveals a very strong decrease in synaptic vesicle density in notum null boutons, providing an explanation for the live FM1-43 dye imaging defects. One of the most striking ultrastructural phenotypes is numerous, enlarged synaptic vesicular bodies. These organelles are highly reminiscent of bulk endosomes, in which a large area of presynaptic membrane is internalized, and will subsequently bud off synaptic vesicles. This pathway is usually driven by intense stimulation during activity-dependent bulk endocytosis (ADBE), as first observed at the frog neuromuscular junction. This pathway is induced by high frequency trains of stimulation, and several proteins have been identified that affect the formation of bulk endosomes, including Syndapin and Rolling Blackout (RBO). At the Drosophila NMJ, conditional rbots mutants block ADBE, reducing the number and size of bulk endosomes (Vijayakrishnan, 2009). It will be interesting to test Wg GOF for enlarged endosomal structures, and study their involvement in Wg-dependent synaptic maturation. On the postsynaptic side, Notum also drives proper differentiation. Notum LOF reduces the postsynaptic DLG scaffold and postsynaptic SSR layering. The reduced SSR area in notum mutants is surprising, given that a reduction in postsynaptic Wg signaling also results in fewer SSR layers. However, SSR architecture has not been studied following Wg over-expression. Postsynaptic SSR formation may be sensitive to bidirectional Wg changes, and may be reduced if Wg is tipped in either direction (Kopke, 2017).

Mechanistically, Notum controls both pre- and postsynaptic molecular assembly, with LOF defects phenocopied by Wg over-expression. The results are consistent with Notum function inhibiting Wg trans-synaptic signaling, and consistent with previously characterized roles for Wg in synaptic molecular development. This study analyzed both the presynaptic active zone protein Bruchpilot and the two postsynaptic GluR classes. Both presynaptic Brp and postsynaptic GluRs are misregulated in notum nulls, with an increase in synapse number but not density. Importantly, both Notum LOF and Wg GOF elevates synapse number. Consistently, Wnt7a over-expression in mouse cerebellar cells also increases the number of synaptic sites and causes accumulation of presynaptic proteins required for synaptic vesicle function. The increased synapse density per NMJ may compensate for reduced neurotransmission per bouton, leading to a net stronger overall NMJ function. In notum mutants, this could reconcile the elevated synaptic strength measured by electrophysiology compared to compromised single bouton function measured by FM dye imaging and impaired TEM ultrastructure. In any case, synaptic assembly during development is regulated by Notum function limiting Wg trans-synaptic signaling (Kopke, 2017).

Genetically reducing Wg by combining a heterozygous wg null mutation into the homozygous notum null background reduces extracellular synaptic Wg back to control levels. Wg reduction suppresses synaptogenic defects, restoring increased NMJ area, branching and bouton numbers completely back to normal. Both notumKO and Wg GOF causes hyperactive movement, with roll-over speeds supporting synaptogenic defects of larger, stronger NMJs in both mutant conditions. However, notumKO motor function is only partially restored by correcting Wg levels. One explanation for incomplete rescue is that multiple Wnts may contribute to motor behavior. Serine lipidation is conserved for all Wnts, and at least two other Wnts have been suggested to act at the Drosophila NMJ (Wnt2, Wnt5). Wnts are the only secreted ligands suggested to be O-palmitoleated on a serine to function as Notum substrates (Kopke, 2017).

Carrier of Wingless (Cow) regulation of Drosophila neuromuscular junction development

The first Wnt signaling ligand discovered, Drosophila Wingless (Wg; Wnt1 in mammals), plays critical roles in neuromuscular junction (NMJ) development, regulating synaptic architecture and function. Heparan sulfate proteoglycans (HSPGs), consisting of a core protein with heparan sulfate (HS) glycosaminoglycan (GAG) chains, bind to Wg ligands to control both extracellular distribution and intercellular signaling function. Drosophila HSPGs previously shown to regulate Wg trans-synaptic signaling at the NMJ include the glypican Dally-like Protein (Dlp) and perlecan Terribly Reduced Optic Lobes (Trol). This study investigated synaptogenic functions of the most recently described Drosophila HSPG, secreted Carrier of Wingless (Cow), which directly binds Wg in the extracellular space. At the glutamatergic NMJ, Cow secreted from the presynaptic motor neuron was found to act to limit synaptic architecture and neurotransmission strength. In cow null mutants, this study found increased synaptic bouton number and elevated excitatory current amplitudes, phenocopying presynaptic Wg overexpression. cow null mutants exhibit an increased number of glutamatergic synapses and increased synaptic vesicle (SV) fusion frequency based both on GCaMP imaging and electrophysiology recording. Membrane-tethered Wg prevents cow null defects in NMJ development, indicating that Cow mediates secreted Wg signaling. It has been shown previously that the secreted Wg deacylase Notum restricts Wg signaling at the NMJ. This study shows that Cow and Notum work through the same pathway to limit synaptic development. It is concluded Cow acts cooperatively with Notum to coordinate neuromuscular synapse structural and functional differentiation via negative regulation of Wg trans-synaptic signaling within the extracellular synaptomatrix (Kopke, 2020).

The developing nervous system requires the coordinated action of many signaling molecules to ensure proper synapse formation and function. One key class of signals is the Wnt ligands. The first discovered Wnt, Drosophila Wingless (Wg), is secreted from presynaptic neurons and glia at the developing glutamatergic neuromuscular junction (NMJ) to bind to the Frizzled-2 (Fz2) receptor in both anterograde and autocrine signaling. In the postsynaptic muscle, Wg binding to Fz2 activates the noncanonical Frizzled Nuclear Import (FNI) pathway, which leads to Fz2 endocytosis and cleavage of the Fz2 C terminus (Fz2-C). The Fz2-C fragment is trafficked to the nucleus to control translation of synaptic mRNAs and glutamate receptors (GluRs). In presynaptic neurons, Wg binding to Fz2 activates a divergent canonical pathway inhibiting glycogen synthase kinase 3β (GSK3β) homolog Shaggy (Sgg) to control microtubule cytoskeletal dynamics via the microtubule-associated protein 1B (MAP1B) homolog Futsch, resulting in synaptic bouton growth. The Wg signaling ligand must be tightly regulated in the synaptic extracellular space (synaptomatrix) to ensure proper NMJ development (Kopke, 2020).

One critical category of proteins regulating Wg ligand in the synaptomatrix is heparan sulfate proteoglycans (HSPGs). HSPGs consist of a core protein to which heparan sulfate (HS) glycosylphosphatidylinositol (GAG) chains are covalently attached. HS GAG chains are composed of repeating disaccharide subunits expressing variable sulfation patterns (the "sulfation code"). These GAG chains bind secreted extracellular ligands to regulate intercellular signaling. There are three HSPG families: transmembrane; glycerophosphatidylinositol (GPI) anchored; and secreted. The Drosophila genome encodes only five HSPGs, with the following three known to affect NMJ development: transmembrane syndecan; GPI-anchored Dally-like protein (Dlp); and secreted perlecan. A second secreted HSPG recently characterized in Drosophila was named Carrier of Wingless (Cow; Chang, 2014). In the developing wing disk, Cow directly binds secreted Wg and promotes its extracellular transport in an HS-dependent manner. Cow shows a biphasic effect on Wg target genes. Removing Cow results in a Wg overexpression (OE) phenotype for short-range targets, and a loss-of-function phenotype for long-range targets (Chang, 2014; Kopke, 2020 and references therein).

The mammalian homolog of Cow, Testican-2, is highly expressed within the developing mouse brain, and inhibits neurite extension in cultured neurons, although the mechanism of action is not known. This study therefore set out to characterize Cow functions at the developing Drosophila NMJ. The larval NMJ model is used because it is large, accessible and particularly well characterized for HSPG-dependent Wg trans-synaptic signaling (Sears, 2018). Each NMJ terminal consists of a relatively stereotypical innervation pattern, with consistent axonal branching and synaptic bouton formation. Boutons are the functional unit of the NMJ, containing presynaptic components required for neurotransmission including glutamate-containing synaptic vesicle (SV) pools and specialized active zone (AZ) sites for SV fusion. AZs contain Bruchpilot (Brp) scaffolds, which both cluster Ca2+ channels and tether SVs. AZs are directly apposed to GluR clusters in the postsynaptic muscle membrane. This spatially precise juxtaposition is critical for high-speed and efficient synaptic communication between neuron and muscle (Kopke, 2020).

This study sought to test Cow functions at the NMJ, with the hypothesis that Cow should facilitate extracellular Wg transport across the synapse. Structurally, cow null mutants display overelaborated NMJs with more boutons and more synapses, phenocopying Wg overexpression. This phenotype is replicated with targeted neuronal Cow knockdown, but not muscle Cow knockdown, which is consistent with Cow secretion from the presynaptic terminal. Functionally, cow null mutants display increased synaptic transmission strength. Both electrophysiology recording and postsynaptically targeted GCaMP imaging show increased SV fusion, indicating elevated presynaptic function. Replacing native Wg with a membrane-tethered Wg blocks secretion. Tethered Wg has little effect on NMJ development, but when combined with the cow null suppresses the synaptic bouton increase, indicating that Cow mediates only secreted Wg signaling. It was recently shown that Notum, a secreted Wg deacylase, also restricts Wg signaling at the NMJ (Kopke, 2017). This study shows that combining null cow and notum heterozygous mutants causes a synergistic increase in NMJ development, indicating nonallelic noncomplementation. Moreover, combining null cow and notum homozygous mutants did not cause an increase in NMJ development compared with the single nulls, indicating an interaction within the same pathway. It is concluded that Cow functions via negative regulation of Wg trans-synaptic signaling (Kopke, 2020).

The function of signaling ligands in the extracellular space is tightly regulated to ensure coordinated intercellular development, often via glycan-dependent mechanisms. The most recently discovered Drosophila HSPG, secreted Cow, was characterized with this role (Chang, 2014). In the developing wing disk, the Wnt Wg is produced in a stripe of cells at the dorsal/ventral margin boundary, and acts as an intercellular morphogen through Fz2 receptor signaling. The glypican HSPGs Dally and Dlp, bound to outer plasma membrane leaflets via GPI anchors, bind Wg to regulate both ligand distribution and intercellular signaling. It has been proposed that Dally/Dlp HSPGs are involved in the movement of extracellular Wg to form a morphogen gradient. However, in dally dlp double mutant clones, extracellular Wg is detected far away from Wg-secreting cells, suggesting that another extracellular factor can transport Wg. Cow was shown to fill this role by binding extracellular Wg to increase stability and rate of movement from producing to receiving cells (Chang, 2014). Supporting this model, cow mutants manifest Wg ligand gain-of-function/overexpression phenotypes for short-range targets, and loss-of-function phenotypes for long-range targets (Kopke, 2020).

At the NMJ, such a long-range Wg morphogen transport function is not seemingly required, except perhaps as a clearance mechanism, but Wg extracellular regulation and short-range Wg transport to cross the synaptic cleft is critical for NMJ development. At the forming of NMJ, Wg from neurons and glia signals both presynaptically (neuronal) and postsynaptically (muscle) via Fz2 receptors. In the motor neuron, Wg signaling inhibits the GSK3β homolog Sgg to regulate the MAP1B homolog Futsch to modulate microtubule dynamics controlling NMJ bouton formation. However, Futsch distribution and microtubule dynamics do not change with elevated Wg signaling, so this pathway alone does not explain the increased bouton formation with increased Wg signaling. In the postsynaptic muscle, Wg signaling drives Fz2 endocytosis and C-terminus cleavage, with transport to the nucleus regulating mRNAs involved in synaptogenesis, including postsynaptic GluR distribution. In wg mutants, GluRs are more diffuse; with clusters irregular in size/shape, increased receptor numbers and a larger postsynaptic volume. Thus, Wg trans-synaptic signaling controls both NMJ structure and function (Kopke, 2020).

Based on the findings from Chang (2014), it was hypothesized that Cow binds Wg to facilitate the transport across the synapse to Fz2 receptors on the muscle. If this is correct, a presynaptic Wg OE phenotype would be expected in the absence of Cow (Wg buildup at the source), and a postsynaptic Wg decrease/loss phenotype (failure of Wg transport). Presynaptically, increased synaptic bouton number was found in cow null mutants phenocopying the Wg OE condition (Kopke, 2017), consistent with this hypothesis. These results indicate that Cow normally inhibits NMJ bouton formation, consistent with the effects of inhibiting presynaptic Wg signaling. Postsynaptically, an increased number of GluR clusters were found due to elevated synapse formation in cow null mutants, but no evidence of diffuse GluR clusters of irregular size/shape and larger volume, as has been reported in wg mutants. Therefore, no strong support for the second prediction of the hypothesis. GluR changes within single postsynaptic domains are challenging to see even with enhanced resolution microscopy , but future studies could focus more on GluRIIA cluster size/shape/intensity in cow mutants. If GluR defects are detected in cow nulls, it would be interesting to test the Frizzled Nuclear Import (FNI) pathway (Kopke, 2020).

Wg signaling regulates multiple steps of NMJ development including branching, satellite bouton budding, and synaptic bouton maturation. None of the cow manipulations cause changes in branching, indicating that Cow does not regulate this Wg signaling, likely working in concert with other Wg regulators. Wg loss (wg^ts) decreases bouton formation, while neural Wg OE increases branching, satellite, and total bouton numbers. Satellite boutons represent an immature stage of development, with small boutons connected to the mature (parent) bouton or adjacent axon. Neuronal Cow OE does not change mature bouton number, but increases satellite bouton budding. Neuronal Cow RNAi also increases satellite boutons. Thus, changing neural Cow levels in either direction elevates satellite bouton numbers, suggesting different consequences on budding versus developmental arrest. It also appears that the cellular source of secreted Cow, or the balance between sources, may be important for proper Wg regulation. Importantly, glia-secreted Wg regulates distinct aspects of synaptic development, with loss of glial-derived Wg accounting for some, but not all, of wg mutant phenotypes. Similarly, cell-targeted cow manipulations cause different NMJ phenotypes. There is no evidence for normal Cow function in postsynaptic muscle, but it remains possible that Cow secreted from glia could regulate Wg trans-synaptic signaling (Kopke, 2020).

Increasing Wg signaling elevates evoked transmission strength and functional synapse number (Kopke, 2017), which is phenocopied in cow null mutants. Block of postsynaptic Wg signaling causes increased SV fusion frequency and amplitude of miniature excitatory junctional potentials (Speese, 2012). With neuronal cow RNAi, there is a similar increase in event frequency and amplitude. These results suggest a decrease in postsynaptic Wg signaling when cow is lost, supporting the Wg transport hypothesis. Blocking Wg secreted from neurons or glia increases muscle GluR cluster size, albeit with differential effects on neurotransmission efficacy. Reducing neuronal Wg has no effect on mEJC frequency, but reducing glial-derived Wg increases SV fusion frequency. Both nerve-evoked and spontaneous neurotransmission are increased in cow null mutants, together with increased Brp active zones and postsynaptic GluR clusters forming supernumerary synapses. SynapGCaMP is an exciting new tool to test function at individual synapses. With targeted neuronal cow RNAi, there is an increase in both the number of SV fusion events and the postsynaptic Ca2+ signal amplitude, which is consistent with both presynaptic and postsynaptic regulation of Wg signaling. These functional phenotypes, combined with coordinated changes in presynaptic and postsynaptic formation suggest Cow regulates trans-synaptic Wg transport (Kopke, 2020).

There were differences between spontaneous synaptic vesicle fusion findings between TEVC electrophysiological recordings and SynapGCaMP reporter (MHC-CD8-GCaMP6f-Sh) Ca2+ imaging. Motor neurons that presynaptically targeted cow RNAi showed stronger impacts on SV fusion frequency with imaging in contrast to recordings, comparable to effects in the cowGDP null mutants. Moreover, SynapGCaMP imaging revealed significantly larger SV fusion event magnitudes in contrast to the lack of change found with TEVC recording. While the basis of these differences is unknown, it is speculated that it is due to the differential nature or sensitivity of these two methods. The Ca2+ imaging is based on measuring the change in the fluorescence signal over the baseline NMJ fluorescence, and it may be that glutamate receptor Ca2+ permeability or intracellular Ca2+ signaling dynamics is changed in a way not directly related to detectable membrane current changes in the cow mutants. TEVC recordings capture whole NMJ activity, whereas with imaging type 1b bouton activity was only captured normalized to area. In future studies, SynapGCaMP imaging can be used to map spatial changes in synapse function by assaying quantal activity separately in convergent type 1s and 1b motor neuron inputs and within discrete synaptic boutons. Moreover, differences between cowGDP and cowGDP/Df conditions could be influenced by second site-enhancing mutations on the Df chromosome. Overall, it should be noted that the changes in spontaneous SV fusion frequency and amplitude in cow mutants are subtle and variable, and need to be further studied in the future (Kopke, 2020).

Wg is lipid modified via palmitoylation to become strongly membrane associated. The hydrophobic moiety is located at the interface of Wg and Fz2 binding, shielded from the aqueous environment by multiple extracellular transporters until signaling interaction with the receptor. There have been many modes of extracellular Wg transport demonstrated, primarily from work in the wing disk, including microvesicles, lipoproteins, exosomes, and cytoneme membrane extensions. These multiple mechanisms of transport are much less studied at the synapse; however, exosome-like vesicles containing the Wg-binding protein Evenness Interrupted (Evi) have been demonstrated at the Drosophila NMJ. Cow could be considered an alternative extracellular Wg transport method, acting to shield Wg while facilitating transport through the extracellular synaptomatrix. In addition, HSPGs have been shown to regulate ligands by stabilizing, degrading, or sequestering the ligand, or as bifunctional coreceptors, or as facilitators of transcytosis. Results presented in this study are consistent with the hypothesis that Cow is mediating Wg transport across the NMJ synapse, but also that Cow has an additional role in the negative regulation of Wg synaptic signaling (Kopke, 2020).

The need for secreted Wg has been recently challenged, with Wg tethering to the membrane (NRT-wg) showing Wg secretion to be largely dispensable for development. In contrast, other recent studies suggest that Wg release and spreading is necessary. This study finds that tethering Wg at the NMJ synapse increases extracellular Wg ligand levels, with no change in mature bouton numbers. This Wg accumulation shows that NRT-wg is more stable at the synaptic signaling interface, consistent with other studies. However, although Wg levels increase, Wg signaling is less effective. With NRT-wg, only the budding of new satellite bouton is increased, with no increase in mature bouton formation. Reducing Wg function causes Fz2 upregulation, so this study hypothesized that Wg signaling could be maintained by increased presynaptic Fz2 receptors. When Wg is tethered, Cow cannot mediate intercellular transport, so the hypothesis predicts a similar phenotype with Cow (NRT-wg) or without Cow (NRT-wg; cowGDP). Indeed, Cow removal in the NRT-wg condition does not impact synaptic bouton number, although it does block the increase in satellite boutons, consistent with a Cow role in greater Wg stability (Chang, 2014). These results show that Wg secretion is required for the elevated NMJ development characterizing cow mutant animals (Kopke, 2020).

To further test how Cow is working through the Wg pathway to negatively regulate NMJ development, genetic interaction tests were performed with the Wg-negative regulator Notum. At the NMJ, Wg trans-synaptic signaling is elevated in the absence of Notum, and null notum mutants display larger NMJs with more synaptic boutons, increased synapse number and elevated neurotransmission (Kopke, 2017). All these defects are phenocopied by neuronal Wg OE, showing that the positive synaptogenic phenotypes arise from lack of Wg signaling inhibition. Consistently, genetically correcting Wg levels at the synapse in notum nulls alleviates synaptogenic phenotypes (Kopke, 2017). This study shows that cow null mutants have the same phenotypes of expanded NMJs, supernumerary synaptic boutons, greater synapse number/function, and strengthened transmission, suggesting that Cow acts like Notum in regulating Wg signaling. A genetic test was performed to ask whether Cow and Notum work in this same pathway. While cow and notum null heterozygotes do not exhibit NMJ defects, cow/notum trans-heterozygotes display grossly expanded NMJs with excess boutons. This combined haplo-insufficiency (type 3 SSNC) of nonallelic noncomplementation suggests that Cow and Notum share related roles. When full double mutants were tested, there is no additive effect, showing that Cow and Notum restrict Wg signaling in the same pathway. However, this pathway convergence appears restricted only to the control of structural synaptogenesis but not of functional neurotransmission, although the control neurotransmission amplitude was elevated in these studies (Kopke, 2020).

Cow now joins the list of synaptic HSPGs with key roles in NMJ development. HSPGs have been implicated in vertebrate NMJ synapse formation for over 3 decades. The Agrin HSPG is secreted from presynaptic terminals to maintain postsynaptic acetylcholine receptor clustering. Another secreted HSPG, perlecan, regulates acetylcholinesterase localization. Drosophila NMJ analyses have begun to more systematically elucidate HSPG roles in NMJ formation and function. In particular, the glypican HSPG Dlp regulates Wg signaling to modulate both NMJ structure and function, including the regulation of active zone formation and SV release. Wg binds the core Dlp, with HS chains enhancing this binding, to retain Wg on the cell surface, where it can both compete with Fz2 receptors and facilitate Wg-Fz2 binding. This biphasic activity depends on the ratio of Wg, Fz2, and Dlp HSPG as expounded in the 'exchange factor model'. Cow may impact this exchange factor mechanism as a fourth player, acting with Dlp to modulate Wg transport and Wg-Fz2 binding at the synaptic interface. It will be important to test Dlp levels and distribution in cow nulls to see how Cow fits into this model (Kopke, 2020).

In addition to Cow, perlecan (Trol) is another secreted HSPG reported to regulate bidirectional Wg signaling at the Drosophila NMJ. Trol has been localized near the muscle membrane, where it promotes postsynaptic Wg accumulation. In the absence of Trol, Wg builds up presynaptically, causing excess satellite bouton formation. It is interesting to note that cow mutants enhance Wg signaling without increasing satellite boutons. In trol mutants, ghost boutons increase due to decreased postsynaptic Wg signaling. Note that cow mutants do not exhibit ghost boutons, which fails to support decreased postsynaptic Wg signaling. Other postsynaptic defects in trol mutants (e.g., reduced SSR, increased postsynaptic pockets) are NMJ ultrastructural features that could be a future focus using electron microscopy studies. Similar to cow mutants, extracellular Wg levels are decreased in the absence of Trol, speculated due to increased Wg proteolysis, since HS protects HS-binding proteins from degradation. In cow mutants, it is not yet known whether Wg is decreased due to elevated signaling (ligand/receptor endocytosis) or to increased degradation due to Cow no longer protecting/stabilizing the ligand. Given that synaptic Fz2 is internalized with Wg binding, future experiments could test internalized Fz2 levels in cow mutants as a proxy of Wg signaling (Kopke, 2020).

In summary, this study has confirmed new tools to study Cow HSPG function and has discovered that Cow from presynaptic motor neurons restricts NMJ bouton formation, glutamatergic synapse number, and NMJ functional differentiation. Cow acts within the same Wg trans-synaptic signaling pathway as Notum by regulating the Wg ligand in the extracellular synaptomatrix. Secreted Cow modulates extracellular Wg ligand levels, with additional functions controlling Wg signaling efficacy, which may be independent of or dependent on Wg transport. It will be interesting to determine whether Cow core protein and/or its HS chains are important for the synaptic structural and functional phenotypes. Wg must be secreted for Cow to act on it, as shown by the membrane-tethered interaction studies, showing that secreted Cow must work on the freely diffusible Wg ligand. Perhaps most informative for future studies will be dissection of the interactions, coordination or redundancy of the multiple synaptic HSPGs at the NMJ, to further the understanding of extracellular Wg trans-synaptic signaling regulation during synaptic development. Drosophila is a particularly well suited model to study HSPGs because of the relatively reduced complexity in this system (Kopke, 2020).

Retrograde semaphorin-plexin signalling drives homeostatic synaptic plasticity

Homeostatic signalling systems ensure stable but flexible neural activity and animal behaviour. Presynaptic homeostatic plasticity is a conserved form of neuronal homeostatic signalling that is observed in organisms ranging from Drosophila to human. Defining the underlying molecular mechanisms of neuronal homeostatic signalling will be essential in order to establish clear connections to the causes and progression of neurological disease. During neural development, semaphorin-plexin signalling instructs axon guidance and neuronal morphogenesis. However, semaphorins and plexins are also expressed in the adult brain. This study shows that semaphorin 2b (Sema2b) is a target-derived signal that acts upon presynaptic plexin B (PlexB) receptors to mediate the retrograde, homeostatic control of presynaptic neurotransmitter release at the neuromuscular junction in Drosophila. Further, Sema2b-PlexB signalling regulates presynaptic homeostatic plasticity through the cytoplasmic protein Mical and the oxoreductase-dependent control of presynaptic actin. It is proposed that semaphorin-plexin signalling is an essential platform for the stabilization of synaptic transmission throughout the developing and mature nervous system. These findings may be relevant to the aetiology and treatment of diverse neurological and psychiatric diseases that are characterized by altered or inappropriate neural function and behaviour (Orr, 2017b).

Semaphorins are a large family of secreted or membrane-associated signalling proteins and plexins serve as signal-transducing semaphorin receptors. Semaphorin-plexin signalling was initially described as mediating growth cone collapse. But, semaphorin-plexin signalling is far more diverse. Notably, semaphorins and plexins continue to be expressed in the mature brain, where their function remains mostly unknown. Semaphorins have been shown to be synaptic signalling proteins, but the activity of semaphorins has been limited to the control of neuroanatomical synapse formation and elimination. This study demonstrates that semaphorin-plexin signalling achieves retrograde, trans-synaptic control of presynaptic neurotransmitter release and homeostatic plasticity (Orr, 2017b).

A well-documented assay was used to induce presynaptic homeostatic plasticity (PHP), applying a sub-blocking concentration of the glutamate-receptor antagonist philanthotoxin-433 (PhTx; 15 μM) to significantly decrease the amplitude of average miniature excitatory postsynaptic potentials (mEPSPs; 0.3 μM [Ca2+]e) or miniature excitatory postsynaptic currents (mEPSCs; 1.5 μM [Ca2+]e). This postsynaptic perturbation induces a significant increase in presynaptic neurotransmitter release (the quantal content) that offsets the postsynaptic perturbation and restores normal muscle excitation. This offsetting increase in presynaptic neurotransmitter release is characteristic of PHP1. When this assay was used in larvae containing a null mutation in either the sema2b gene (sema2bC4) or the PlexB gene (PlexB^KG0088), PHP was blocked. Consistent with this being a loss-of-function phenotype, heterozygous mutations (either sema2b/+ or PlexB/+) have normal PHP. Remarkably, a double-heterozygous mutant combination of sema2b/+ and PlexB/+ blocks PHP, consistent with both genes acting in concert to drive the expression of PHP (Orr, 2017b).

The long-term maintenance of PHP was investigated and the involvement of other semaphorin or Plexin gene family members. Deletion of a non-essential glutamate-receptor subunit (GluRIIA) induces a long-lasting form of PHP1. Long-term PHP is blocked in a sema2b;GluRIIA double mutant as well as in GluRIIA larvae expressing transgenic RNA interference (RNAi) to knockdown PlexB selectively in motor neurons. Next, the effect of mutations was separately tested in all of the remaining semaphorin and Plexin genes encoded in the Drosophila genomet. The sema2b and PlexB mutants are the only mutants that show disruption of PHP (Orr, 2017b).

Tissue-specific RNAi and transgenic rescue experiments were performed. Expression of UAS-Sema2b-RNAi in motor neurons (OK371-Gal4) had no effect on PHP, whereas expression in muscle (BG57-Gal4) blocked PHP. In addition, expression of UAS-sema2b in muscle rescues PHP in the sema2b-mutant background. Consistent with these data, sema2b was found to be expressed in muscle and Sema2b protein, expressed under endogenous promoter sequences, concentrates at postsynaptic membranes. Next, it was shown that motor neuron-specific expression of UAS-PlexB-RNAi blocks PHP, whereas muscle-specific expression does not. Motor neuron-specific expression of a previously characterized UAS-PlexBDN dominant-negative transgene, lacking the intracellular signalling domain, blocks PHP. RNA-sequencing analysis of purified motor neurons demonstrates PlexB expression in motor neurons. Finally, motor neuron-specific expression of a PlexB-myc transgene shows that PlexB traffics to the presynaptic nerve terminal. Taken together, these data indicate that Sema2b is a ligand originating in the muscle that acts via presynaptic PlexB to drive expression of PHP (Orr, 2017b).

If Sema2b is a retrograde signal that acts upon the presynaptic PlexB receptor, then it should be possible to reconstitute this retrograde signalling by acute application of Sema2b protein. Purified Sema2b protein was acutely applied to the neuromuscular junction (NMJ) of sema2b mutants following PhTx treatment to induce PHP. Sema2b protein (100 nM) was found to completely restores PHP in the sema2b mutant, but fails to restore PHP in the PlexB mutant. In addition, application of Sema2b protein is sufficient to potentiate baseline release, and this effect is also dependent upon PlexB. Finally, a membrane-tethered UAS-sema2b transgene, expressed in muscle, fails to rescue PHP, even though it is concentrated on the postsynaptic membranes. Together, these results indicate that Sema2b is a secreted, postsynaptic ligand that acts upon presynaptic PlexB to enable the expression of PHP. The possibility is acknowledged that PlexB could require a presynaptic co-receptor of, as yet, unknown identity (Orr, 2017b).

Given that acute application of Sema2b protein rescues PHP in the sema2b mutant, the failure of PHP in sema2b-mutant larvae cannot be a secondary consequence of altered NMJ development. Nonetheless, Sema2b-PlexB signalling is required for normal NMJ growth. Axon-targeting errors are rare at muscles 6/7, analysed at the third instar larval stage. This study demonstrated that the NMJs in sema2b and PlexB mutants are composed of fewer, larger synaptic boutons with no change in total NMJ area. The abundance of the active-zone-associated protein Bruchpilot (Brp) is unaltered in the sema2b mutant and the sema2b/+;;PlexB/+ double-heterozygous larvae, both of which block PHP. There is a significant decrease in total Brp staining in the PlexB mutant, an effect of unknown consequence. Qualitatively, the ring-like organization of Brp staining was similar across all genotypes, indicative of normal active-zone organization. Finally, there is no consistent difference in synapse ultrastructure across genotypes. Therefore, the Sema2b-PlexB-dependent control of bouton size may be a separate function of Sema2b-PlexB signalling, analogous to anatomical regulation by semaphorins in mammalian systems (Orr, 2017b).

PHP occurs through the potentiation of the readily releasable pool (RRP) of synaptic vesicles. Application of PhTx induces a doubling of the apparent RRP in wild-type larvae, an effect that is disrupted in both sema2b and PlexB mutants. Failure to potentiate the RRP is also shown as a failure to maintain the cumulative EPSC amplitude after PhTx application. A strong genetic interaction was subsequently shown with a mutation in the presynaptic scaffolding gene rab3-interacting molecule (rim), a PHP gene. Heterozygous mutations in rim, or in sema2b or PlexB have no effect on PHP. However, double-heterozygous combinations of rim/+ with either sema2b/+ or PlexB/+ strongly impaired the expression of PHP (sema2b/+,rim/+) or abolished PHP (rim/+;;PlexB/+). These data do not, however, reflect direct signalling between PlexB and Rim (Orr, 2017b).

To define how PlexB could modulate the RRP, known downstream signalling elements were tested. Mical is necessary for PHP. In Drosophila a single mical gene encodes a highly conserved multi-domain cytoplasmic protein that mediates actin depolymerization, achieved through redox modification of a specific methionine residue (Met44) in actin. Notably, prior genetic evidence has placed Mical downstream of both PlexA and PlexB signalling during axon guidance (Orr, 2017b).

An analysis of multiple mical mutations in larvae as well as transgenic rescue animals demonstrates that mical is necessary presynaptically for PHP. Mical protein is present presynaptically and presynaptic expression of a Mical-resistant UAS-Actin5C transgene, which interferes with Mical-mediated actin depolymerization, blocks PHP. This transgenic protein also concentrates within presynaptic boutons. Additional experiments reveal that the homeostatic expansion of RRP is blocked in mical mutants and when Mical-resistant UAS-Act5 is expressed presynaptically. Strong genetic interactions were found between mical and both the PlexB and rim mutants. Finally, anatomical experiments demonstrate that active zones are normal in the mical mutant, including in both light and electron microscopy experiments. It is proposed that Mical enables PlexB-mediated control of the RRP through the regulation of presynaptic actin (Orr, 2017b).

For half a century, evidence has underscored the importance of target-derived, retrograde signalling that controls presynaptic neurotransmitter release1. Gene discovery, based on forward genetics, indicates that PHP is controlled by the coordinated action of at least three parallel signalling systems. These data regarding Sema2b, PlexB and Mical can be generalized, then semaphorin-plexin signalling could represent a platform for retrograde, trans-synaptic, homeostatic control of presynaptic release, thereby stabilizing synaptic transmission and information transfer throughout the nervous systems of organisms ranging from Drosophila to humans (Orr, 2017b).

Synapse-specific and compartmentalized expression of presynaptic homeostatic potentiation

Postsynaptic compartments can be specifically modulated during various forms of synaptic plasticity, but it is unclear whether this precision is shared at presynaptic terminals. Presynaptic Homeostatic Plasticity (PHP) stabilizes neurotransmission at the Drosophila neuromuscular junction, where a retrograde enhancement of presynaptic neurotransmitter release compensates for diminished postsynaptic receptor functionality. To test the specificity of PHP induction and expression, this study has developed a genetic manipulation to reduce postsynaptic receptor expression at one of the two muscles innervated by a single motor neuron. PHP can be induced and expressed at a subset of synapses, over both acute and chronic time scales, without influencing transmission at adjacent release sites. Further, homeostatic modulations to CaMKII, vesicle pools, and functional release sites are compartmentalized and do not spread to neighboring pre- or post-synaptic structures. Thus, both PHP induction and expression mechanisms are locally transmitted and restricted to specific synaptic compartments (Li, 2018a).

Although the genes and mechanisms that mediate retrograde homeostatic potentiation have been intensively investigated, whether this process can be expressed and restricted to a subset of synapses within a single neuron has not been determined. This study has developed a manipulation that enables the loss of GluRs on only one of the two postsynaptic targets innervated by a Type Ib motor neuron at the Drosophila NMJ. The analysis of synaptic structure and function in this condition has revealed the spectacular degree of compartmentalization in postsynaptic signaling and presynaptic expression that ultimately orchestrate the synapse- specific modulation of presynaptic efficacy (Li, 2018a).

Compartmentalization of postsynaptic PHP signaling GluRs are dynamically trafficked in postsynaptic compartments where they mediate the synapse-specific expression of Hebbian plasticity such as LTP and homeostatic plasticity, including receptor scaling. In contrast, homeostatic plasticity at the human, mouse, and fly NMJ is expressed through a presynaptic enhancement in neurotransmitter release, but is induced through a diminishment of postsynaptic neurotransmitter receptor functionality. Using biased expression of Gal4 to reduce GluR levels on only one of the two muscle targets innervated by a single motor neuron, this study demonstrates that the inductive signaling underlying PHP is compartmentalized at the postsynaptic density, and does not influence activity at synapses innervating the adjacent muscle (Li, 2018a).

Postsynaptic changes in CaMKII function and activity have been associated with PHP retrograde signaling. Consistent with this compartmentalized inductive signaling, this study observed pCaMKII levels to be specifically reduced at postsynaptic densities of Ib boutons in which GluR expression is perturbed, while pCaMKII was unchanged at postsynaptic compartments opposite to Is boutons and at NMJs in the adjacent muscle with normal GluR expression. Further, postsynaptic overexpression of the constitutively active CaMKII occludes the expression of PHP. Similar synapse-specific control of postsynaptic CaMKII phosphorylation, modulated by activity, has been previously observed. As noted in other studies, this localized reduction in pCaMKII provides a plausible mechanism for the inductive PHP signaling restricted to and compartmentalized at Ib synapses (Li, 2018a).

How does a perturbation to GluR function lead to a reduction in CaMKII activity that is restricted to postsynaptic densities opposing Type Ib boutons? Recent evidence suggests that distinct mechanisms regulate pCaMKII levels during retrograde PHP signaling depending on pharmacologic or genetic perturbation to glutamate receptors and the role of protein synthesis. Scaffolds at postsynaptic densities are associated in complexes with GluRs and CaMKII. Intriguingly, the scaffold dCASK is capable of modulating CaMKII activity at specific densities in an activity-dependent fashion. Further, CaMKII activity can regulate plasticity with specificity at subsets of synapses in Drosophila and other systems. Although intra-cellular 'cross talk' between Is and Ib boutons cannot be ruled out, as GluRIIA is reduced at postsynaptic sites of both neuronal subtypes, it is striking that reductions in pCaMKII are restricted to Ib postsynaptic compartments. An attractive model, therefore, is that the postsynaptic density isolates calcium signaling over chronic time scales to compartmentalize PHP induction. The membranous complexity and geometry of the SSR at the Drosophila NMJ may be the key to restricting calcium signaling at these sites, as this structure can have major impacts on ionic signaling during synaptic transmission. These properties, in turn, may lead to local modulation of CaMKII function. Interestingly, Drosophila mutants with defective SSR elaboration and complexity have been associated with defects in PHP expression. In the mammalian central nervous system, it is well established that dendritic spines function as biochemical compartments that isolate calcium signaling while enabling propagation of voltage changes, and it is tempting to speculate that the SSR may subserve similar functions at the Drosophila NMJ to enable synapse-specific retrograde signaling (Li, 2018a).

The homeostatic modulation of presynaptic neurotransmitter release is compartmentalized at the terminals of Type Ib motor neurons. It was previously known that PHP can be acutely induced and expressed without any information from the cell body of motor neurons. The current data suggests that the signaling necessary for PHP expression is even further restricted to specific postsynaptic densities and presynaptic boutons, demonstrated through several lines of evidence. First, quantal content is specifically enhanced at boutons innervating muscle 6 in M6>GluRIIARNAi without measurably impacting transmission on the neighboring boutons innervating muscle 7. In addition, PHP can be acutely induced at synapses innervating muscle 7 despite PHP having been chronically expressed at muscle 6. Finally, the homeostatic modulation of the RRP and enhancement of the functional number of release sites is fully expressed regardless of whether PHP is induced at all Type Ib boutons or only a subset. Thus, PHP signaling is orchestrated at specific boutons according to the state of GluR functionality of their synaptic partners and does not influence neighboring boutons within the same motor neuron. Although the compartmentalized expression of PHP was not unexpected, there was precedent to suspect inter-bouton crosstalk during homeostatic signaling. In the dynamic propagation of action potentials along the axon, the waveform could, in principle, change following PHP expression to globally modulate neurotransmission at all release sites in the same neuron. However, voltage imaging did not identify any change in the action potential waveform at individual boutons following PHP signaling, and this study did not observe any impact on neighboring boutons despite PHP being induced at a subset of synapses in the same motor neuron. Further, mobilization of an enhanced readily releasable synaptic vesicle pool is necessary for the expression of PHP, and synaptic vesicles and pools are highly mobile within and between presynaptic compartments. Hence, it was conceivable that a mobilized RRP, induced at some presynaptic compartments, may be promiscuously shared between other boutons. However, while a large enhancement was observed in the RRP at synapses innervating muscle 6 in M6>GluRIIARNAi, this adaptation had no impact on the RRP at adjacent presynaptic compartments innervating muscle 7. Thus, PHP signaling is constrained to boutons innervating one of two postsynaptic targets and does not 'spread' to synapses innervating the adjacent target despite sharing common cytosol, voltage, and synaptic vesicles (Li, 2018a).

What molecular mechanisms mediate the remarkable specificity of PHP expression at presynaptic compartments? One attractive possibility is that active zones themselves are fundamental units and act as substrates for the homeostatic modulation of presynaptic function. The active zone scaffold BRP remodels during both acute and chronic PHP expression (Weyhersmuller, 2011), and other active zone proteins are likely to participate in this remodeling. Indeed, many genes encoding active zone components are required for PHP expression, including the calcium channel cac and auxiliary subunit α2-δ, the piccolo homolog fife, the scaffolds RIM (Rab3-interacting Molecule) and RIM-binding protein (RBP), and the kainite receptor DKaiR1D. If individual active zones can undergo the adaptations necessary and sufficient for PHP expression, this would imply that PHP can be induced and expressed with specificity at individual active zones. Indeed, the BRP cytomatrix stabilizes calcium channel levels at the active zone, and also controls the size of the RRP, two key presynaptic expression mechanisms that drive PHP. Further, the recruitment of new functional release sites have been observed following both chronic and acute PHP expression, suggesting that previously silent active zones become 'awakened' and utilized to potentiate presynaptic neurotransmitter release (Li, 2018a).

Interestingly, presynaptic GluRs, localized near active zones, are necessary for PHP expression and have the capacity to modulate release with specificity at individual active zones. Thus, active zones have the capacity to remodel with both the specificity and precision necessary and sufficient for compartmentalized PHP expression. If each active zone operates as an independent homeostat to adjust release efficacy in response to target-specific changes, how is information transfer at individual sites integrated to ensure stable and stereotypic 'global' levels of neurotransmission? One speculative possibility is that active zones at terminals of each neuron are endowed with a total abundance of material that is tightly controlled and sets stable global levels of presynaptic neurotransmitter release. Such active zone material may be 'sculpted' with considerable heterogeneity within presynaptic terminals, varying in number, size, and density. Consistent with such a possibility, mutations in the synaptic vesicle component Rab3 exhibit extreme changes in active zone size, number, and density, but stable global levels of neurotransmission. Within this global context, plasticity mechanisms may operate at individual active zones, superimposed as independent homeostats to adaptively modulate synaptic strength. In addition, there is intriguing evidence for the existence of 'nanocolumns' between presynaptic active zones and postsynaptic GluRs that form structural and functional signaling complexes (Biederer, 2017; Tang, 2016). One particularly appealing possibility, therefore, is that a dialogue traversing synaptic nanocolumns functions to convey the retrograde signaling and active zone remodeling necessary for PHP expression at individual release sites. Studies in mammalian neurons have revealed parallel links between the functional plasticity of active zones, including their structure and size, and the homeostatic modulation of neurotransmitter release. Such intercellular signaling systems are likely to modify synaptic structure and function to not only establish precise pre- and post-synaptic apposition during development, but also to maintain the plasticity necessary for synapses to persist with the flexibility and stability to last a lifetime (Li, 2018a).

Distinct homeostatic modulations stabilize reduced postsynaptic receptivity in response to presynaptic DLK signaling

Synapses are constructed with the stability to last a lifetime, yet sufficiently flexible to adapt during injury. Although fundamental pathways that mediate intrinsic responses to neuronal injury have been defined, less is known about how synaptic partners adapt. This study investigated responses in the postsynaptic cell to presynaptic activation of the injury-related Dual Leucine Zipper Kinase pathway at the Drosophila neuromuscular junction. The postsynaptic compartment reduces neurotransmitter receptor levels, thus depressing synaptic strength. Interestingly, this diminished state is stabilized through distinct modulations to two postsynaptic homeostatic signaling systems. First, a retrograde response normally triggered by reduced receptor levels is silenced, preventing a compensatory enhancement in presynaptic neurotransmitter release. However, when global presynaptic release is attenuated, a postsynaptic receptor scaling mechanism persists to adaptively stabilize this diminished neurotransmission state. Thus, the homeostatic set point of synaptic strength is recalibrated to a reduced state as synapses acclimate to injury (Goel, 2018).

Neurons are endowed with robust surveillance systems that detect injury and initiate latent plasticity programs involving regenerative and degenerative responses. A fundamental signaling system induced after neuronal injury is mediated by an evolutionarily conserved mitogen-activated protein kinase called Dual Leucine Zipper Kinase (DLK). DLK signaling initiates translational changes in axons and transcriptional responses in the nucleus that ultimately promotes degeneration at the distal axon and regeneration proximal to the site of injury. Members of the Phr1/Highwire/Rpm-1 (PHR) protein family control DLK signaling to gate neuronal injury-related signaling programs. At the Drosophila neuromuscular junction, highwire (hiw) encodes an E3 ubiquitin ligase that constitutively degrades the DLK homolog Wallenda (Wnd). However, after axonal injury, Wnd is no longer degraded by Hiw, leading to increased Wnd protein levels and activation of regenerative and degenerative signaling programs. Genetic loss of hiw in neurons constitutively activates Wnd signaling, while neuronal overexpression of wnd can overcome Hiw-mediated degradation to activate this same program. This relationship between PHR proteins, DLK activity, and injury-related signaling is conserved in other invertebrate and mammalian systems. Thus, loss of hiw or overexpression of wndf in neurons activates an intrinsic signaling system that transforms the cell into a state of persistent degenerative and regenerative adaptations related to a programmed response to injury (Goel, 2018).

Although, Wnd/DLK signaling and other injury-related responses occur cell autonomously and are intrinsic to the specific neuron, there is emerging evidence that synaptically connected cells may sense this perturbation and adapt in response. For example, foundational studies at the mouse NMJ have demonstrated that motor neuron injury, denervation, or synapse elimination can provoke disassembly and remodeling of the postsynaptic specialization, including a diminution of neurotransmitter receptor levels, which can precede obvious changes in the overlying nerve terminal. There is also evidence for neuronal and synaptic remodeling in the spinal cord and other areas in the central nervous system following injury. At the glutamatergic Drosophila NMJ, an apparent reduction in the synaptic response to glutamate (quantal size) was shown in hiw mutants as well as following a 'nerve crush' injury to otherwise wild-type NMJs. Neuronal expression of hiw restores normal synaptic strength and quantal size in hiw mutants, indicating that while the postsynaptic muscle does not itself experience hiw-related signaling, a reduction in either the amount of glutamate released and/or the postsynaptic sensitivity to neurotransmitter occurs in response. However, it remains unclear how synaptic function and plasticity change in response to presynaptic Wnd/DLK signaling, a state in which injury-related signaling is active but before complete degeneration or loss of the presynaptic terminal has occurred (Goel, 2018).

At the Drosophila NMJ, two forms of homeostatic plasticity have been described that stabilize synaptic strength in response to perturbations that would otherwise disrupt functionality. First, pharmacological or genetic manipulations that diminish postsynaptic glutamate receptor (GluR) function initiate a retrograde signaling system. Specifically, a retrograde signal emitted from the muscle instructs the neuron to compensate by increasing presynaptic glutamate release to restore set point levels of synaptic strength. This process is conserved in rodents and humans, and is termed presynaptic homeostatic potentiation (PHP) because the expression mechanism of this form of plasticity is a presynaptic increase in neurotransmitter release. Second, hypo-innervation of the NMJ induces a distinct form of adaptive plasticity. In this situation, a reduction in presynaptic neurotransmitter release is observed proportional to the reduction in synapse number. In response, a homeostatic increase in quantal size is induced that stabilizes synaptic strength. However, to what extent these homeostatic mechanisms operate at synapses that have adapted to injury-related signaling is unknown (Goel, 2018).

This study has characterized synaptic structure, function, and plasticity at NMJs with active Wnd signaling in presynaptic neurons, with a particular focus on how the postsynaptic muscle responds. This analysis has revealed that the postsynaptic muscle responds by diminishing GluR abundance and by silencing the retrograde homeostatic signaling system that would normally enhance presynaptic release following reduced GluR levels. However, this subdued state of synaptic strength is homeostatically maintained through adaptive modulations to postsynaptic GluR levels following hypo-innervation. Together, this illuminates the distinct signaling systems targeted for modulation in the postsynaptic cell that stabilize a muted synaptic state in response to presynaptic Wnd signaling (Goel, 2018).

The conserved hiw/PHR gene family regulates Wnd/DLK signaling and has diverse roles in synaptic development, degeneration and regeneration, and neurotransmission. Although there are clearly myriad changes induced in neurons following Wnd activity, at the Drosophila NMJ, no evidence was found for hiw or wnd having any functions in the postsynaptic muscle. Although Wnd has no apparent function in regulating presynaptic neurotransmitter release, its negative regulator, Hiw, does have an independent function in promoting neurotransmitter release. The putative substrate that mediates this role for Hiw is unknown, but must be separate from Wnd and the downstream pathways orchestrated through Wnd signaling. Therefore, following injury, inhibition of Hiw exerts two separate influences that mutes functionality at both synaptic compartments: First, Wnd is activated to provoke a diminished state of responsiveness in the muscle, and second, presynaptic neurotransmitter release is reduced. The substrate that mediates the Wnd-independent role of Hiw to reduce presynaptic efficacy is unknown, but will be an interesting target to define in future studies (Goel, 2018).

It is remarkable that although Wnd signaling is driven solely in the presynaptic compartment at the NMJ, this process is sensed and transforms the postsynaptic muscle into a novel state characterized by subdued responsiveness to presynaptic activity. Neuronal Wnd signaling must therefore impart anterograde information to the muscle. Importantly, this information is communicated independently of altered synaptic development and structure, as well as evoked neurotransmission. An attractive possibility is that trans-synaptic adhesion proteins may mediate the activity-independent anterograde signaling following presynaptic Wnd activation. Indeed, multiple extracellular cues and trans-synaptic signals interpose a rich dialog between presynaptic terminals, postsynaptic compartments, and the extracellular matrix at synapses. Trans-synaptic 'nanocolumns' have recently emerged as inter-cellular signaling complexes that orchestrate synaptic remodeling and plasticity in addition to development, maturation, and structural alignment at synapses. It is tempting to speculate that direct interactions through these nanocolumns may communicate injury-related signaling from presynaptic terminals to postsynaptic partners to remodel receptor fields and plasticity pathways. Intriguingly, there is evidence that many new proteins are expressed in neurons following activation of Wnd/DLK signaling, providing possible candidates to test for roles in this process (Goel, 2018).

One major outcome of the response in muscle to presynaptic Wnd signaling is a diminution of postsynaptic neurotransmitter receptors. This parallels observations at the mouse NMJ following denervation or synapse elimination, in which a reduction in neurotransmitter receptor levels and protein synthesis in muscle has been demonstrated. This selective loss-of-acetylcholine receptors at synaptic sites is a result of removal of receptors from these areas coupled with a lack of insertion of new receptors. It is likely that similar mechanisms work to reduce GluR levels at the Drosophila NMJ following neuronal Wnd signaling. In addition, the decreased synaptic protein levels observed in the muscle following neuronal Wnd signaling may be result from modulation of Tor activity, as postsynaptic overexpression of Tor globally elevates protein synthesis and partially restores receptor levels. Finally, an intriguing study in the rodent visual system revealed that ablation of presynaptic photoreceptors leads to remodeling of the postsynaptic apparatus, including the rapid and localized disappearance of GluRs. Together, these studies demonstrate that postsynaptic targets adapt to injury, disease, and loss-of-presynaptic inputs by remodeling postsynaptic neurotransmitter receptor complexes to reduce sensitivity, suggesting a conserved response (Goel, 2018).

While beneficial effects of fasting on organismal function and health are well appreciated, little is known about the molecular details of how fasting influences synaptic function and plasticity. Genetic and electrophysiological experiments demonstrate that acute fasting blocks retrograde synaptic enhancement that is normally triggered as a result of reduction in postsynaptic receptor function at the Drosophila larval neuromuscular junction (NMJ). This negative regulation critically depends on transcriptional enhancement of eukaryotic initiation factor 4E binding protein (4E-BP) under the control of the transcription factor Forkhead box O (Foxo). Furthermore, the findings indicate that postsynaptic 4E-BP exerts a constitutive negative input, which is counteracted by a positive regulatory input from the Target of Rapamycin (TOR). This combinatorial retrograde signaling plays a key role in regulating synaptic strength. These results provide a mechanistic insight into how cellular stress and nutritional scarcity could acutely influence synaptic homeostasis and functional stability in neural circuits (Goel, 2018).

One mechanism that stabilizes the reduced state of synaptic strength following neuronal Wnd signaling is a selective occlusion of postsynaptic PHP transduction. Neurons experiencing Wnd signaling are competent to homeostatically modulate presynaptic neurotransmitter release, but apparently do not receive the retrograde information necessary from the muscle, even following additional perturbations to or restorations of postsynaptic GluRs. Interestingly, perturbations to Cap-dependent protein synthesis in the postsynaptic muscle have been shown to disrupt PHP retrograde signaling, and metabolic changes in the muscle can also impinge on this pathway to modulate PHP signaling (Kauwe, 2016). This indicates that one mechanism utilized by the muscle to respond to neuronal Wnd signaling, distinct from a general reduction in protein synthesis, may be a selective inhibition of Tor-dependent protein synthesis which, in turn, contributes to the occlusion of PHP signaling. It is interesting to note that while PHP signaling is highly compartmentalized, it can be still be expressed in neurons despite perturbations to a variety of processes that disrupt synaptic structure and function independently of injury-related signaling. Thus, an instructive cue mediated by Wnd signaling selectively impairs retrograde PHP communication in the muscle to stabilize a reduced level of synaptic strength (Goel, 2018).

Although PHP signaling appears to be inhibited, a second mechanism stabilizes the reduced set point of synaptic strength following neuronal Wnd signaling, GluR scaling. This postsynaptic form of homeostatic plasticity parallels the postsynaptic scaling of GluRs observed following silencing of neuronal activity in mammalian central synapses. At the Drosophila NMJ, the induction of this form of homeostatic plasticity was known to require hypo-innervation, and this study has shown that a selective increase in postsynaptic GluRIIA-containing receptors compensates for reduced neurotransmitter release and maintains stable synaptic strength. Although the signal transduction system that mediates this form of plasticity is enigmatic, it is clearly distinct from the retrograde signaling system that drives PHP. The homeostatic set point of synaptic strength has been demonstrated to be plastic, and can be stabilized at levels distinct from baseline values in mutations that disrupt synaptic function and during aging of the synapse. The current results define injury-related signaling as an additional process that has the capacity to adjust the homeostatic set point of synaptic strength (Goel, 2018).

Why might synaptic strength be adjusted to a reduced level following injury? The coordinated reduction in Wnd-mediated postsynaptic responsiveness and Hiw-mediated presynaptic efficacy may promote sufficient time for the process of repair vs. degeneration to be adjudicated within an injured neuron, while still maintaining synaptic communication at a reduced level. Loss or further reductions to neurotransmission may destabilize synaptic integrity, impairing the series of subsequent steps necessary to restore normal synaptic strength and structure should the injury be successfully overcome. In addition, a further reduction in postsynaptic receptivity may deprive the neuron of necessary trophic support and promote neuronal degeneration. Indeed, a lack of trophic support from the muscle due to weakened synaptic activity contributes to neuromuscular disease pathogenesis. In the central nervous system, there is evidence that synaptically coupled cells sense and respond to injury. Hence, injury to an individual neuron can propagate and destabilize an entire neural circuit without adaptive counter measures. The current findings illustrate the acclimations that occur in postsynaptic targets to neurons experiencing injury-related signaling and highlight the adaptations to synaptic plasticity that maintain stable functionality around a reduced set point of synaptic strength (Goel, 2018).

A glutamate homeostat controls the presynaptic inhibition of neurotransmitter release

This study has interrogated the synaptic dialog that enables the bi-directional, homeostatic control of presynaptic efficacy at the glutamatergic Drosophila neuromuscular junction (NMJ). Homeostatic depression and potentiation use disparate genetic, induction, and expression mechanisms. Specifically, homeostatic potentiation is achieved through reduced CaMKII activity postsynaptically and increased abundance of active zone material presynaptically at one of the two neuronal subtypes innervating the NMJ, while homeostatic depression occurs without alterations in CaMKII activity and is expressed at both neuronal subtypes. Furthermore, homeostatic depression is only induced through excess presynaptic glutamate release and operates with disregard to the postsynaptic response. It is proposed that two independent homeostats modulate presynaptic efficacy at the Drosophila NMJ: one is an intercellular signaling system that potentiates synaptic strength following diminished postsynaptic excitability, while the other adaptively modulates presynaptic glutamate release through an autocrine mechanism without feedback from the postsynaptic compartment (Li, 2018b).

The Drosophila neuromuscular junction (NMJ) is a powerful model system to study the bi-directional, homeostatic control of synaptic strength. At this glutamatergic synapse, acute pharmacological and chronic genetic manipulations that reduce postsynaptic glutamate receptor (GluR) function activate a retrograde, trans-synaptic signaling system that triggers a compensatory increase in presynaptic glutamate release, restoring baseline levels of synaptic strength. Because the expression of this form of plasticity requires a presynaptic increase in neurotransmitter release, this process is referred to as presynaptic homeostatic potentiation (PHP). Multiple lines of evidence have established that the homeostat that governs PHP is exquisitely sensitive to diminished postsynaptic excitability and operates through a retrograde enhancement of presynaptic efficacy, stabilizing overall synaptic strength. Parallel phenomena have been observed at cholinergic NMJs in rodents and humans, suggesting this is a fundamental and conserved form of synaptic plasticity that does not depend on the neurotransmitter system (Li, 2018b).

In contrast to PHP, far less is known about the homeostat that governs an inverse process at the Drosophila NMJ, referred to as presynaptic homeostatic depression (PHD). The first evidence for PHD, although not appreciated as such, was discovered while characterizing mutations in synaptic vesicle endocytosis genes, in which increased synaptic vesicle size was found to result from defects in vesicle re-formation mechanisms. Independently, evidence for PHD was found using a separate manipulation that also increased synaptic vesicle size through overexpression of the vesicular glutamate transporter (vGlut; vGlut-OE). Both defective endocytosis and vGlut-OE result in enlargement of individual synaptic vesicles, leading to excess glutamate emitted from each synaptic vesicle and enhanced postsynaptic responsiveness (quantal size). However, normal levels of synaptic strength (excitatory postsynaptic potential [EPSP] amplitude) were observed due to a homeostatic reduction in the number of synaptic vesicles released (quantal content). When the phenomenon of PHD was initially defined, one hypothesis put forward was that PHD may be induced as an adaptive response to excess glutamate. More recently, PHD has been considered a mechanism that stabilizes neurotransmission in the same way that PHP operates, implying that PHD is calibrated as a homeostat that responds to overall synaptic strength. Despite these studies, the nature of the homeostat that controls PHD, as well as the genes and mechanisms involved, remains much less understood relative to PHP. It is not even clear whether trans-synaptic communication is required to induce, express, or modulate PHD (Li, 2018b).

This study has characterized the adaptations to synaptic physiology, growth, structure, and plasticity when PHP and PHD are induced and expressed alone and in conjunction at an individual synapse. Several lines of evidence demonstrate that PHP and PHD are independent processes that use distinct mechanisms to modulate presynaptic efficacy in opposing directions and operate at separate neuronal subtypes. However, PHP and PHD are not simply independent signaling systems that each tune presynaptic efficacy to maintain stable levels of synaptic strength. Rather, the data indicate that PHP is indeed a homeostat dedicated to maintaining synaptic strength, induced through retrograde signaling in the postsynaptic compartment. In contrast, PHD operates with indifference to the state of the postsynaptic cell and is oblivious to overall synaptic strength, instead functioning cell autonomously in the presynaptic neuron as a negative feedback system to homeostatically modulate glutamate release (Li, 2018b).

Clearly, distinct genetic mechanisms underlie PHP and PHD signaling, because genes necessary for PHP have no role in PHD. This is illustrated by loss of the gene dysbindin, which is required for PHP expression but has no impact on PHD, consistent with findings that other genes necessary for PHP do not impact PHD. Thus, while PHP and PHD appear to be parallel processes that modulate presynaptic neurotransmitter release in inverse directions, they do not employ overlapping genetic machinery (Li, 2018b).

PHP and PHD also use distinct physiological expression mechanisms. PHD reduces probability of release at both type Ib and Is motor neurons through an apparent reduction in Ca2+ influx yet without a change in BRP or the size of the RRP (Gavino, 2015). In contrast, the current study and others have found PHP adaptations involve an increase in presynaptic Pr mediated through increased Ca2+ influx, active zone scaffolding, Ca2+ channel abundance, and enhancement of the RRP. Indeed, remodeling of BRP at active zones appears to be unique to PHP and to terminals of type Ib boutons. The BRP scaffold controls the size of the RRP and stabilizes Cac channels at the active zone. Therefore, an attractive hypothesis is that PHP requires enhancements in Cac and BRP abundance to promote both Ca2+ influx and increase RRP size. Ca2+ channels and active zone scaffolds are also homeostatically regulated to control presynaptic neurotransmitter release in mammalian neurons, suggesting that such plasticity mechanisms may be evolutionarily conserved (Li, 2018b).

The postsynaptic induction mechanisms that orchestrate PHP signaling are enigmatic. However, it is clear that PHP signaling is extremely sensitive to reductions in postsynaptic excitability, which triggers a compartmentalized intercellular signaling system that originates in the postsynaptic muscle and requires a reduction in CaMKII activity to potentiate neurotransmitter release in the presynaptic neuron (Haghighi, 2003, Li, 2018b, Newman, 2017). If PHD were a homeostat designed to stabilize synaptic strength in a way that parallels PHP, then enhanced muscle excitability should induce a retrograde signaling system to depress presynaptic glutamate release. However, the data and previous work demonstrate that homeostatic depression is not induced when quantal size is increased. Rather, excess glutamate release from the motor neuron appears to be necessary and sufficient to induce and express PHD. This suggests that an autocrine mechanism triggers PHD signaling, in which excess glutamate is sensed and transduced into a reduction in presynaptic efficacy. Such an autocrine mechanism was astutely proposed as a possibility in the original vGlut-OE study (Li, 2018b).

If an autocrine mechanism mediates PHD induction, this would imply the existence of presynaptic GluRs that can sense excess glutamate and initiate presynaptic inhibition in response. Presynaptic autoreceptors are present and modulate presynaptic function at glutamatergic NMJs of invertebrates and vertebrates. One attractive candidate is the lone metabotropic GluR encoded in the Drosophila genome, mGluRA. mGluRA is present at presynaptic terminals of motor neurons at the larval NMJ and promotes presynaptic inhibition following excess glutamate released during high-frequency stimulation (Bogdanik, 2004). Other possibilities include presynaptic NMDA receptors, which mediate presynaptic inhibition in response to excess glutamate release in the mammalian hippocampus. Notably, NMDA receptors have been reported to be present at the Drosophila NMJ. The nature of the glutamate sensor and autocrine signaling system that govern the induction and expression of PHD remains to be defined (Li, 2018b).

Why doesn't a homeostat governing synaptic strength exist at the NMJ that is responsive to enhanced postsynaptic excitability? Proper control of muscle contraction is essential to life, and NMJs in many systems use a safety factor that ensures neurotransmitter is released in excess to stably promote muscle contraction. Hence, given this safety factor, it is not clear that a postsynaptic signaling system at the NMJ is necessary to detect and respond to heightened neurotransmitter release or sensitivity. Pharmacological perturbations to cholinergic NMJs that inhibit the enzymatic breakdown of neurotransmitter in worms and mammals lead to rapid paralysis and death, and there is no evidence that homeostatic retrograde signaling systems are initiated to inhibit presynaptic neurotransmitter release during these challenges. Thus, a retrograde homeostatic signaling system to depress presynaptic efficacy in response to increased postsynaptic excitability may not have developed due to a lack of evolutionary pressure (Li, 2018b).

Why, then, does a process like PHD exist at the Drosophila NMJ, designed to inhibit glutamate release through an autocrine presynaptic signaling system? One attractive possibility is that PHD may be a process that maintains stable glutamate levels at the larval NMJ of Drosophila in lieu of classical glutamate reuptake mechanisms. In the CNS, various clearance mechanisms homeostatically maintain ambient glutamate levels to prevent excitotoxicity, and excitatory GluRs are present at presynaptic terminals of the larval Drosophila NMJ. The major mechanism for glutamate clearance in the mammalian brain requires glutamate transporter proteins in the plasma membrane of both glial cells and neurons. In Drosophila, there a single excitatory amino acid transporter specific for glutamate reuptake encoded in the genome, dEAAT1. dEAAT1 is expressed in the central nervous system and in peripheral glia at the adult NMJ, where it is involved in glutamate clearance. However, dEAAT1 is not expressed at the embryonic or larval NMJ, and it is unclear how glutamate is controlled in this system. Accordingly, PHD may serve as an adaptive cell autonomous mechanism that responds to excess glutamate and inhibits release to maintain glutamate homeostasis, a process that may have parallels in the mammalian CNS (Li, 2018b).

Neuronal glutamatergic synaptic clefts alkalinize rather than acidify during neurotransmission

This study used genetically-encoded fluorescent pH indicators to examine synaptic cleft pH at conventional neuronal synapses. At the neuromuscular junction of female Drosophila larvae, alkaline spikes of over 1 log unit were observed during fictive locomotion in vivo. Ex vivo, single action potentials evoked alkalinizing pH transients of only approximately 0.01 log unit, but these transients summated rapidly during burst firing. A chemical pH indicator targeted to the cleft corroborated these findings. Cleft pH transients were dependent on Ca(2+) movement across the postsynaptic membrane, rather than neurotransmitter release per se, a result consistent with cleft alkalinization being driven by the Ca(2+)/H(+) antiporting activity of the plasma membrane Ca(2+)-ATPase at the postsynaptic membrane. Targeting the pH indicators to the microenvironment of the presynaptic voltage-gated Ca(2+) channels revealed that alkalinization also occurred within the cleft proper at the active zone and not just within extra-synaptic regions. Application of the pH indicators at the mouse calyx of Held, a mammalian central synapse, similarly revealed cleft alkalinization during burst firing in both males and females. These findings, made at two quite different non-ribbon type synapses, suggest that cleft alkalinization during neurotransmission, rather than acidification, is a generalizable phenomenon across conventional neuronal synapses (Stawarski, 2020).

Distinct Target-Specific Mechanisms Homeostatically Stabilize Transmission at Pre- and Post-synaptic Compartments

Neurons must establish and stabilize connections made with diverse targets, each with distinct demands and functional characteristics. At Drosophila neuromuscular junctions (NMJs), synaptic strength remains stable in a manipulation that simultaneously induces hypo-innervation on one target and hyper-innervation on the other. However, the expression mechanisms that achieve this exquisite target-specific homeostatic control remain enigmatic. This study identified the distinct target-specific homeostatic expression mechanisms. On the hypo-innervated target, an increase in postsynaptic glutamate receptor (GluR) abundance is sufficient to compensate for reduced innervation, without any apparent presynaptic adaptations. In contrast, a target-specific reduction in presynaptic neurotransmitter release probability is reflected by a decrease in active zone components restricted to terminals of hyper-innervated targets. Finally, loss of postsynaptic GluRs on one target induces a compartmentalized, homeostatic enhancement of presynaptic neurotransmitter release called presynaptic homeostatic potentiation (PHP) that can be precisely balanced with the adaptations required for both hypo- and hyper-innervation to maintain stable synaptic strength. Thus, distinct anterograde and retrograde signaling systems operate at pre- and post-synaptic compartments to enable target-specific, homeostatic control of neurotransmission (Goel, 2020).

Antagonistic interactions between two Neuroligins coordinate pre- and postsynaptic assembly

As a result of developmental synapse formation, the presynaptic neurotransmitter release machinery becomes accurately matched with postsynaptic neurotransmitter receptors. Trans-synaptic signaling is executed through cell adhesion proteins such as Neurexin::Neuroligin pairs but also through diffusible and cytoplasmic signals. How exactly pre-post coordination is ensured in vivo remains largely enigmatic. This study identified a 'molecular choreography' coordinating pre- with postsynaptic assembly during the developmental formation of Drosophila neuromuscular synapses. Two presynaptic Neurexin-binding scaffold proteins, Syd-1 and Spinophilin (Spn), spatio-temporally coordinated pre-post assembly in conjunction with two postsynaptically operating, antagonistic Neuroligin species: Nlg1 and Nlg2. The Spn/Nlg2 module promoted active zone (AZ) maturation by driving the accumulation of AZ scaffold proteins critical for synaptic vesicle release. Simultaneously, these regulators restricted postsynaptic glutamate receptor incorporation. Both functions of the Spn/Nlg2 module were directly antagonized by Syd-1/Nlg1. Nlg1 and Nlg2 also had divergent effects on Nrx-1 in vivo motility. Concerning diffusible signals, Spn and Syd-1 antagonistically controlled the levels of Munc13-family protein Unc13B at nascent AZs, whose release function facilitated glutamate receptor incorporation at assembling postsynaptic specializations. As a result, this study has provided direct in vivo evidence illustrating how a highly regulative and interleaved communication between cell adhesion protein signaling complexes and diffusible signals allows for a precise coordination of pre- with post-synaptic assembly. It will be interesting to analyze whether this logic also transfers to plasticity processes (Ramesh, 2021).

Synaptic vesicle (SV) release at chemical synapses depends on the formation of active zone (AZ) scaffolds composed of a canonical apparatus of proteins including Unc13, RIM-binding protein (RIM-BP), Liprin-α and CAST/ELKS (called Bruchpilot [BRP] in Drosophila). The size of individual AZ scaffolds scales with SV release probability. Once matured, each AZ apparently forms an integer number of release sites apposed by postsynaptic glutamate receptors (GluRs), likely spatially coordinated through a trans-synaptic micropattern ('nanocolumns'). Importantly, in the course of maturation, AZ size becomes closely matched to the size of the postsynaptic density (PSD) scaffold clustering neurotransmitter (NT) receptors (Ramesh, 2021).

How the in vivo synapse assembly process and associated regulatory steps achieve this precise pre-post matching during developmental assembly is not fully understood. Notably, trans-synaptic cell-adhesion molecules (CAMs) have the capacity to bidirectionally tune synapse assembly, and Neurexin (Nrx) and Neuroligin (Nlg) interactions represent a regulatory principle conserved across vertebrate and invertebrate synapses. Although many synaptic CAMs and cytoplasmic proteins have been studied in isolation, how different CAMs selectively engage with each other and their cytoplasmic partners to ensure pre-post matching during synapse assembly has remained enigmatic, partly because of the high genetic redundancy among mammalian CAMs. Besides CAM signaling, diffusible signals including NT release at nascent AZs might play a regulatory role in postsynaptic assembly (Ramesh, 2021).

This study characterize mechanisms that ensure pre-post matching during the assembly of individual glutamatergic synapses at the Drosophila neuromuscular junction (NMJ). In developing larvae, synapse maturation ultimately establishes a precisely defined pre-post stoichiometry over the course of several hours. To interrogate these mechanisms, the unique advantages were used of the larval NMJ system, which allows for a synergy of reduced genetic redundancy, super-resolution, and dynamic intravital microscopy and electrophysiology. Nlg1 and Nlg2, two Nlg species previously shown to functionally interact with the only Nrx family protein in Drosophila, Nrx-1. The current results reveal that these two Nlgs serve antagonistic roles and operate in conjunction with two antagonistic presynaptic proteins that bind Nrx-1: Syd-1 cooperating with Nlg1 and Spinophilin (Spn) with Nlg2. Whereas the Spn/Nlg2 functional module promoted AZ maturation (BRP/RIM-BP/Unc13A incorporation) but restrained GluRIIA-containing receptor incorporation, Syd-1/Nlg1 initiated AZ assembly and promoted GluRIIA receptor incorporation through Unc13B recruitment and its glutamate release function. Genetic interaction experiments identified a remarkable degree of crosstalk between these modules, exemplifying a regulatory principle obviously evolved to ensure precise pre-post matching, and integrating Unc13B-dependent glutamate release acting as a diffusible signal. Altogether, these data indicate that synaptic matching is not established via a trans-synaptic 'stoichiometric building principle' to continuously accumulate synaptic components, but via a regulatory crosstalk between antagonistic assembly modules (Ramesh, 2021).

Synapses form out of three interdependent molecular assemblies, each precisely crafted to execute fast and precise information transfer between two cells: the presynaptic AZ where SVs fuse at defined release sites, the synaptic cleft through which NT diffuses, and the postsynaptic compartment where the NT binds its receptors. Importantly, these compartments do not form in isolation, but the size of the AZ (and thus the number of presynaptic release sites per AZ) must closely scale with the number of postsynaptic NT receptors. Super-resolution microscopy identified presynaptic AZ protein nanoclusters to align with concentrated postsynaptic receptors and scaffolding proteins, suggesting the existence of trans-synaptic molecular 'nanocolumns'. Indeed, the exact nanometer location of vesicular release in relation to receptors might be a critical determinant of synaptic strength, which might also contribute to synaptic plasticity (Ramesh, 2021).

A central question now pertains to how trans-synaptic signaling is precisely executed in molecular terms to coordinate pre- with post-synaptic assembly. Candidate molecular scenarios include interactions that directly bridge pre- and postsynaptic membranes like trans-synaptic CAMs, which bidirectionally control synapse formation, remodeling, and elimination. This study exploited the unique features of the Drosophila NMJ system: unique accessibility to intravital imaging to accurately analyze the assembly path, a cytoarchitecture ideal for super-resolution analysis, high-resolution electrophysiological measurements, and a low level of genetic redundancy, to address how presynaptic AZs are matched to postsynaptic GluRs. Moreover, the amount of ELKS protein BRP, easily accessible for STED microscopy, directly scales with presynaptic release at AZs, making it an ideal readout to assess both structural and functional assembly (Ramesh, 2021).

In principle, a strategy of continuously accumulating stoichiometric amounts of pre- and postsynaptic material along the assembly trajectory, potentially via a single transcellular bridge connecting to nucleation processes on both sides, might appear the easiest way to establish pre-post matching. Indeed, such an idea has recently been proposed, where the age of AZs determines their size and strength at the Drosophila NMJ. However, such a solution might lack regulatory flexibility and is also not what was find in this work. Instead, this analysis identifies antagonistic regulatory inputs to be executed by two postsynaptically active Nlg species operating synergistically with their respective 'cognate' presynaptic scaffold proteins, Syd-1 and Spn, previously shown to steer synapse assembly via their Nrx-1-binding function. It here appears likely that autonomy over the presynaptic versus the postsynaptic compartment might be particularly relevant during plasticity processes, shown to involve the specific incorporation of BRP at NMJ synapses. This antagonistic operation might serve to embed contextual information while steering the assembly process and could be particularly robust when utilized in such a highly regulative scheme (Ramesh, 2021).

A model is provided for the functional relations analyzed in this study. Syd-1 and Nlg1 form new AZs in the seeding phase, whereas Spn and Nlg2 promote incorporation of BRP to appropriate levels in the maturation phase. Notably, BRP is the rate-limiting building block of the AZ scaffold, determining the size and functional strength of the AZ specialization. Overactivity of Syd-1/Nrx-1/Nlg1 signaling likely is directly responsible for the Spn AZ phenotype, given that it could be suppressed by lowering the dose of any of these molecules. The same is true for the Nlg2 phenotype as well, suggesting that the Nlg2 AZ phenotype similarly reflects Syd-1/Nlg1 axis overactivity. Furthermore, reduction of Spn efficiently suppressed the normally excessive BRP incorporation at the AZs remaining in Syd-1, Nrx-1, and Nlg-1 mutants. Mechanistically, future analysis will have to clarify whether direct physical interactions of Spn with BRP complexes, co-clustering RIM-BP and Unc13A are of relevance here. Alternatively, the Syd-1 and Spn modules might antagonistically control a downstream process such as the status of F-actin (Ramesh, 2021).

Although in the past, Spn was interpreted as functioning after Syd-1 during the AZ development process, the current data now suggest that Syd-1 and Spn in fact continuously antagonize each other throughout assembly to tune final AZ size and function. Still, intravital imaging of nascent AZs showed that the peak of Syd-1 accumulation precedes the peak of BRP accumulation by hour. The fact that the Syd-1 scaffold is favored over the Spn scaffold during the seeding phase might be explained via a 'quasi-epistatic' relation between these regulators: Syd-1 mutants show lower amounts of Spn, whereas Spn mutants show elevated amounts of Syd-1 suggesting that Syd-1 is needed for Spn accumulation at the AZ, potentially allowing Syd-1-mediated AZ seeding to precede Spn-mediated BRP accumulation. Spn and Syd-1 were shown to interact with each other in Drosophila and in C. elegans. It would be interesting to investigate whether the Spn/Syd-1 interaction plays a role in regulating access to Nrx-1, thereby contributing to define the actual 'assembly mode:' seeding or maturation. Obviously, the assembly modules must communicate to ultimately ensure a well-defined assembly product, e.g., via associated kinase and/or phosphatase activities. For example, the phosphorylation status of BRP can control transport. Furthermore, although Spn attenuation did efficiently suppress the Syd-1, Nrx-1, and Nlg1 AZ phenotypes, Nlg2 attenuation did not suppress the Nrx-1 and Nlg1 phenotypes. This suggests that the trans-synaptic signaling through Nrx-Nlgs might ensure that assembly proceeds from seeding toward maturation during development. This also opens up the possibility that Nlg2 attenuates Syd-1/Nrx-1/Nlg1 function by removing Nrx-1 from the seeding module and/or suppressing Nlg1 activity through cis-heteromerization. FRAP data also indicate that the postsynaptic binding partner identity (Nlg1 or Nlg2) has differential effects on Nrx-1 mobility. Lack of Nlg2 likely boosts the Nrx-1::Nlg1 seeding activity, directly explaining the supernumerary AZs typical for Nlg2 mutant (Ramesh, 2021).

Nlg1 promoted but Nlg2 blocked GluRIIA incorporation, which precedes BRP accumulation. Previous analysis showed that Syd-1 seemingly instructs Nrx-1 to interact with Nlg1 and promotes GluRIIA incorporation before BRP incorporation. Genetic interaction analysis showed that Syd-1/Nlg1 and Spn/Nlg2 execute a mutual regulatory counterplay here. This study now extends the understanding of GluRIIA incorporation to involve the release function of Unc13B, enriched at nascent AZs by Syd-1, a process antagonized by Spn. Spn and Nlg2 functionally cooperate to limit the amount of GluRIIA incorporation in the nascent postsynaptic specialization and match receptor amounts to the AZ size. However, although the Spn mutant phenotype was rescued by Syd-1, Nrx-1, and Nlg1 heterozygosity, the Nlg2 mutant phenotype was only rescued by Syd-1 heterozygosity, suggesting that Nlg1 and Nlg2 have an additional function in mediating GluRIIA incorporation independent of Unc13B. Mechanistically, it might well be that Nlg2 at the nascent postsynaptic compartment directly competes with Nlg1 for the binding of a critical effector, e.g., the ectodomain of the GluR complex or other membrane proteins such as Neto (Ramesh, 2021).

In mice, most synapses formed normally in the absence of NT release during development, but the synapses did not persist as they matured. Experiments in mice have shown in the past that massive local glutamate release could induce spine formation at the postsynapse. However, whether vesicular transmitter release tunes the incorporation dynamics of GluRs during developmental synapse assembly remains inconclusive (Ramesh, 2021).

Unc13B arrives early at nascent NMJ AZs. This recruitment of Unc13B is antagonistically controlled by the two complexes, given that Syd-1 mutants showed reduced but Spn mutants strongly increased synaptic Unc13B amounts. Importantly, the excessive GluRIIA incorporation in Spn mutants critically depended on Unc13B. Notably, treatment of cell cultures with BoNT-C and TNT-E previously was shown to prevent effective postsynaptic insertion of glutamatergic receptors in cultivated hippocampal neurons. However, it cannot be exclude that once Unc13A accumulates at the AZ, Unc13B might continue to mediate GluRIIA incorporation into later stages of synapse assembly (Ramesh, 2021).

Concerning the mode of Unc13B action, the data suggest that evoked Unc13B-mediated glutamate release at nascent sites attracts GluRIIA receptors, which are recruited from diffuse pools at the plasma membrane. Notably, proper gating behavior of GluRIIA in response to presynaptic glutamate release previously was shown to be essential for matching pre- with post-assembly. Unc13B-mediated release is coupled more loosely to Ca2+ channel activity compared with release mediated by the functionally dominant isoform, Unc13A. Likely, sensing glutamate at nascent sites renders GluRIIA into an active state, which allows for postsynaptic incorporation, previously shown to be nearly irreversible. Whether the GluRIIA incorporation subsequent to the glutamate sensing is truly stage dependent, e.g., via specific scaffold or cleft proteins, or whether differences in the spatio-temporal detail of glutamate release between Unc13B and Unc13A are more important here remains to be addressed (Ramesh, 2021).

Nrx-1, Nlg1, and Syd-1 mutants all show reduced NMJ area, whereas Spn and Nlg2 mutants showed normal NMJ sizes, and all of them showed reduced evoked potentials. Previous studies have shown that synaptic terminals can compensate for a change in size by adjusting NT output. A recent study showed that spontaneous neurotransmission is needed for the normal structural maturation of Drosophila NMJ synapses exclusive from the role of evoked neurotransmission. Increasing miniature events was sufficient to induce synaptic terminal growth, and this synapse maturation was locally regulated via a Trio guanine nucleotide exchange factor (GEF) and Rac1 GTPase molecular signaling pathway. Interestingly, Syd-1 was found to interact with Trio signaling. Together with the Rac guanine exchange factor (RacGEF) Trio, Syd-1 GAP activity promotes BRP clustering and independent of its GAP activity, Syd-1 recruits Nrx-1 to boutons. Additionally, mammalian Spn forms a complex with Rac1-GEF Kalirin-7 and, along with Rho-GEF Lfc, control dendritic spine morphology and function. Therefore, it will be interesting to study how Syd-1 and Spn antagonism translates into GAP/GEF signaling, which in turn might control the synapse assembly at Drosophila NMJs (Ramesh, 2021).

Dichotomous cis-regulatory motifs mediate the maturation of the neuromuscular junction by retrograde BMP signaling

Retrograde bone morphogenetic protein (BMP) signaling at the Drosophila neuromuscular junction (NMJ) has served as a paradigm to study TGF-β-dependent synaptic function and maturation. Yet, how retrograde BMP signaling transcriptionally regulates these functions remains unresolved. This study uncovered a gene network, enriched for neurotransmission-related genes, that is controlled by retrograde BMP signaling in motor neurons through two Smad-binding cis-regulatory motifs, the BMP-activating (BMP-AE) and silencer (BMP-SE) elements. Unpredictably, both motifs mediate direct gene activation, with no involvement of the BMP derepression pathway regulators Schnurri and Brinker. Genome editing of candidate BMP-SE and BMP-AE within the locus of the active zone gene bruchpilot, and a novel Ly6 gene witty, demonstrated the role of these motifs in upregulating genes required for the maturation of pre- and post-synaptic NMJ compartments. These findings uncover how Smad-dependent transcriptional mechanisms specific to motor neurons directly orchestrate a gene network required for synaptic maturation by retrograde BMP signaling (Vuilleumier, 2022).

Mayday sustains trans-synaptic BMP signaling required for synaptic maintenance with age

Maintaining synaptic structure and function over time is vital for overall nervous system function and survival. The processes that underly synaptic development are well understood. However, the mechanisms responsible for sustaining synapses throughout the lifespan of an organism are poorly understood. This study demonstrates that a previously uncharacterized gene, CG31475, regulates synaptic maintenance in adult Drosophila NMJs. CG31475 was named mayday, due to the progressive loss of flight ability and synapse architecture with age. Mayday is functionally homologous to the human protein Cab45 (SDF4 - stromal cell derived factor 4), which sorts secretory cargo from the Trans Golgi Network (TGN). Mayday was found to be required to maintain trans-synaptic BMP signaling at adult NMJs in order to sustain proper synaptic structure and function. Finally, mutations in mayday were shown to result in the loss of both presynaptic motor neurons as well as postsynaptic muscles, highlighting the importance of maintaining synaptic integrity for cell viability (Sidisky, 2021).

Among the most prominent neuromuscular synapses in adult Drosophila are those of the indirect flight muscles. One set of IFMs, the Dorsal Longitudinal Muscles (DLMs), are composed of six large muscle fibers innervated by five motor neurons on each side of the thorax. Once the DLM NMJs are established, these stable structures are present throughout the lifespan of the organism. These NMJs are part of the Giant Fiber (GF) pathway that propels flight behavior. Thus, the activity of DLMs can be monitored by assaying flight behavior as a readout of synaptic integrity. Additionally, the DLM NMJs form a tripartite synapse composed of a presynaptic motor neuron, postsynaptic muscle cell, and associated glial cell, that provide the ability to understand synaptic function at the cellular and molecular level. This model also allows for expression of transgenes in non-essential tissue, particularly the DLM motor neurons that are easily accessible. Together, it is possible assess the morphological and functional properties of adult DLM NMJs to elucidate the mechanisms responsible for sustaining synapses in aging adults, then apply this to understand how synapses deteriorate in neurodegenerative diseases (Sidisky, 2021).

Although the processes involved in maintaining synaptic structure and function may not be understood, there are a few key pathways that are crucial for regulating synaptic growth, organization and stability during synaptic development. Specifically, in Drosophila one key signaling cascade that involves coordination between the presynaptic motor neurons and postsynaptic muscle cells is the bone morphogenic protein (BMP) signaling cascade. The morphogen glass bottom boat (Gbb), the Drosophila ortholog to mammalian BMP7, is secreted in a retrograde manner from the postsynaptic muscle cell to the presynaptic motor neuron. Currently, it is not understood how this pathway could function past development. This suggests that this signaling cascade could play a role in maintaining synaptic integrity (Sidisky, 2021).

Gaining a better understanding of synaptic dysfunction should help to identify strategies involved in maintaining synaptic integrity with age. This study identified Mayday, a resident Golgi protein that is required to maintain trans-synaptic signaling across adult NMJs. Mutations in mayday impair retrograde BMP signaling, resulting in degradation of synaptic structure and function. Finally, this study demonstrates that this sustained trans-synaptic signaling is required to maintain the viability of both presynaptic motor neurons and postsynaptic muscles (Sidisky, 2021).

The current study describes mayday (myd), a previously uncharacterized gene that plays a role in maintaining synaptic integrity with age by promoting trans-synaptic signaling. We found that myd^3PM71 mutants have structural and functional deficits in adult DLM NMJs. Through tissue-specific RNAi and rescue experiments, it was determined that Myd is necessary in both postsynaptic muscle tissue and presynaptic motor neurons to maintain synaptic integrity. Myd localizes to the TGN and shares functional homology with human Cab45. Myd sustains retrograde BMP signaling in adult DLM NMJs through genetic interactions with gbb, tkv, wit, and mad mutants and staining of Gbb and pMad markers. Finally, myd sustains the viability of presynaptic motor neurons and postsynaptic muscles (Sidisky, 2021).

From developmental studies, it was learned that Gbb is a morphogen secreted in a retrograde manner trans-synapticaly from postsynaptic muscle tissue to the presynaptic motor neuron in larval NMJs to promote synaptic growth. However, relatively little is known regarding the roles of this pathway in fully developed organisms. Recent evidence demonstrates that sustained BMP signaling is required to maintain FMRFamide expression in a subset of neurons in the Drosophila brain. The current results here further demonstrate that retrograde BMP signaling that regulates NMJ development is required in adult NMJs to sustain synaptic integrity with age. It is also possible that several other signaling pathways crucial for organism development may be required throughout the life of the organism (Sidisky, 2021).

Knockdown and rescue experiments using myd demonstrate that it maintains synaptic integrity through roles in both pre- and postsynaptic tissue. While most studies involving BMP in synaptic growth report a retrograde signaling mechanism, recent evidence suggests that this pathway could also signal in an anterograde fashion. While genetic studies provide support for retrograde BMP signaling, it cannot be ruled out that anterograde BMP signaling also plays an important role in maintaining synaptic integrity. In particular, the levels of pMAD that were observed were present within both presynaptic motor neuron terminals as well as postsynaptic muscles. Further studies aimed at characterizing BMP signal activation within muscle cells should help with our understanding of the mechanisms responsible for synaptic maintenance (Sidisky, 2021).

While trans-synaptic BMP signaling plays a clear role in maintaining synapses, myd mutations likely impair other pathways associated with cargo trafficking. In addition to secretory cargo, Cab45 also has a role in trafficking lysosomal proteases. Given the functional homology shared between Cab45 and Myd, it is possible that the trafficking of these lysosomal hydrolases needed for autophagy could be disrupted. Defects in autophagy have been strongly linked with neurodegenerative diseases. Therefore, it is possible that myd mutants have disruptions in autophagy that lead to the loss of synaptic integrity. It will be interesting to investigate how Myd impacts these other processes that are associated with neuronal dysfunction (Sidisky, 2021).

This assessment of synaptic dysfunction in the current study includes flight performance as a readout of functional integrity, as well as morphological measurements of branch length, branch number, and bouton number using a presynaptic membrane marker. In future studies, it will be helpful to further evaluate synaptic integrity in mayday mutants. Additional functional assays may include electrophysiological measurements of activity across these NMJs, and more structural data could be obtained through the use of a wide array of synaptic markers, as well as ultrastructural analysis using Transmission Electron Microscopy. Together, these types of studies should allow for an even greater understanding of synaptic dysfunction and the mechanisms required to maintain these critical structures (Sidisky, 2021).

The calcineurin regulator Sarah enables distinct forms of homeostatic plasticity at the Drosophila neuromuscular junction

The ability of synapses to maintain physiological levels of evoked neurotransmission is essential for neuronal stability. A variety of perturbations can disrupt neurotransmission, but synapses often compensate for disruptions and work to stabilize activity levels, using forms of homeostatic synaptic plasticity. Presynaptic homeostatic potentiation (PHP) is one such mechanism. PHP is expressed at the Drosophila melanogaster larval neuromuscular junction (NMJ) synapse, as well as other NMJs. In PHP, presynaptic neurotransmitter release increases to offset the effects of impairing muscle transmitter receptors. Prior Drosophila work has studied PHP using different ways to perturb muscle receptor function-either acutely (using pharmacology) or chronically (using genetics). Some previous data suggested that cytoplasmic calcium signaling was important for expression of PHP after genetic impairment of glutamate receptors. This study followed up on that observation. This study used a combination of transgenic Drosophila RNA interference and overexpression lines, along with NMJ electrophysiology, synapse imaging, and pharmacology to test if regulators of the calcium/calmodulin-dependent protein phosphatase calcineurin are necessary for the normal expression of PHP. It was found that either pre- or postsynaptic dysregulation of a Drosophila gene regulating calcineurin, sarah (sra), blocks PHP. Tissue-specific manipulations showed that either increases or decreases in sra expression are detrimental to PHP. Additionally, pharmacologically and genetically induced forms of expression of PHP are functionally separable depending entirely upon which sra genetic manipulation is used. Surprisingly, dual-tissue pre- and postsynaptic sra knockdown or overexpression can ameliorate PHP blocks revealed in single-tissue experiments. Pharmacological and genetic inhibition of calcineurin corroborated this latter finding. These results suggest tight calcineurin regulation is needed across multiple tissue types to stabilize peripheral synaptic outputs (Armstrong, 2022).

A targeted glycan-related gene screen reveals heparan sulfate proteoglycan sulfation regulates WNT and BMP trans-synaptic signaling

A Drosophila transgenic RNAi screen targeting the glycan genome, including all N/O/GAG-glycan biosynthesis/modification enzymes and glycan-binding lectins, was conducted to discover novel glycan functions in synaptogenesis. As proof-of-product,functionally paired heparan sulfate (HS) 6-O-sulfotransferase (hs6st) and sulfatase (sulf1), which bidirectionally control HS proteoglycan (HSPG) sulfation, were characterized. RNAi knockdown of hs6st and sulf1 causes opposite effects on functional synapse development, with decreased (hs6st) and increased (sulf1) neurotransmission strength confirmed in null mutants. HSPG co-receptors for WNT and BMP intercellular signaling, Dally-like Protein and Syndecan, are differentially misregulated in the synaptomatrix of these mutants. Consistently, hs6st and sulf1 nulls differentially elevate both WNT (Wingless; Wg) and BMP (Glass Bottom Boat; Gbb) ligand abundance in the synaptomatrix. Anterograde Wg signaling via Wg receptor dFrizzled2 C-terminus nuclear import and retrograde Gbb signaling via synaptic MAD phosphorylation and nuclear import are differentially activated in hs6st and sulf1 mutants. Consequently, transcriptional control of presynaptic glutamate release machinery and postsynaptic glutamate receptors is bidirectionally altered in hs6st and sulf1 mutants, explaining the bidirectional change in synaptic functional strength. Genetic correction of the altered WNT/BMP signaling restores normal synaptic development in both mutant conditions, proving that altered trans-synaptic signaling causes functional differentiation defects (Dani, 2012b).

It is well known that synaptic interfaces harbor heavily-glycosylated membrane proteins, glycolipids and ECM molecules, but understanding of glycan-mediated mechanisms within this synaptomatrix is limited. A genomic screen aimed to systematically interrogate glycan roles in both structural and functional development in the genetically-tractable Drosophila NMJ synapse. 130 candidate genes were screened, classified into 8 functional families: N-glycan biosynthesis, O-glycan biosynthesis, GAG biosynthesis, glycoprotein/proteoglycan core proteins, glycan modifying/degrading enzymes, glycosyltransferases, sugar transporters and glycan-binding lectins. From this screen, 103 RNAi knockdown conditions were larval viable, whereas 27 others produced early developmental lethality. 35 genes had statistically significant effects on different measures of morphological development: 27 RNAi-mediated knockdowns increased synaptic bouton number, 9 affected synapse area (2 increased, 7 decreased) and 2 genes increased synaptic branch number. These data suggest that overall glycan mechanisms predominantly serve to limit synaptic morphogenesis. 13 genes had significant effects on the functional differentiation of the synapse, with 12 increasing transmission strength and only 1 decreasing function upon RNAi knockdown. Thus, glycan-mediated mechanisms also predominantly limit synaptic functional development. A very small fraction of tested genes (CG1597; pgant35A, CG7480; veg, CG6657; hs6st, CG4451; sulf1, CG6725 and CG11874) had effects on both morphology and function. A large percentage of genes (~30%) showed morphological defects with no corresponding effect on function, while only 7% of genes showed functional alterations without morphological defects, and <5% of all genes affect both. These results suggest that glycans have clearly separable roles in modulating morphological and functional development of the NMJ synapse (Dani, 2012b).

A growing list of neurological disorders linked to the synapse are attributed to dysfunctional glycan mechanisms, including muscular dystrophies, cognitive impairment and autism spectrum disorders. Drosophila homologs of glycosylation genes implicated in neural disease states include ALG3 (CG4084), ALG6 (CG5091), DPM1 (CG10166), FUCT1 (CG9620), GCS1 (CG1597), MGAT2 (CG7921), MPDU1 (CG3792), PMI (CG33718) and PPM2 (CG12151). Two of these genes, Gfr (CG9620) and CG1597, showed synaptic morphology phenotypes in the RNAi screen. Given that connectivity defects are clearly implicated in cognitive impairment and autism spectrum disorders, it would be of interest to explore the glycan mechanism affecting synapse morphology in Drosophila models of these disease states. Glycans are well known to modulate extracellular signaling, including ligands of integrin receptors, to regulate intercellular communication. In the genetic screen, several O-glycosyltransferases mediating this mechanism were identified to show morphological (GalNAc-T2, CG6394; pgant35A, CG7480, O-fut2, CG14789; rumi, CG31152) and functional (pgant5, CG31651; pgant35A, CG7480) synaptic defects upon RNAi knockdown. These findings suggest that known integrin-mediated signaling pathways controlling NMJ synaptic structural and functional development are modulated by glycan mechanisms. The screen showed CG6657 RNAi knockdown affects functional differentiation, consistent with reports that this gene regulates peripheral nervous system development. The corroboration of the screen results with published reports underscores the utility of RNAi-mediated screening to identify glycan mechanisms, and supports use of the screen results for bioinformatic/meta-analysis to link observed phenotypes to neurophysiological/pathological disease states and to direct future glycan mechanism studies at the synapse (Dani, 2012b).

From this screen, the two functionally-paired genes sulf1 and hs6st were selected for further characterization. As in the RNAi screen, null alleles of these two genes had opposite effects on synaptic functional differentiation but similar effects on synapse morphogenesis, validating the corresponding screen results. The two gene products have functionally-paired roles; Hs6st is a heparan sulfate (HS) 6-O-sulfotransferase, and Sulf1 is a HS 6-O-endosulfatase. These activities control sulfation of the same C₆ on the repeated glucosamine moiety in HS GAG chains found on heparan sulfate proteoglycans (HSPGs). At the Drosophila NMJ, two HSPGs are known to regulate synapse assembly; the GPI-anchored glypican Dally-like protein (Dlp), and the transmembrane Syndecan (Sdc). In contrast, the secreted HSPG Perlecan (Trol) is not detectably enriched at the NMJ, and indeed appears to be selectively excluded from the perisynaptic domain. In other developmental contexts, the membrane HSPGs Dlp and Sdc are known to act as co-receptors for WNT and BMP ligands, regulating ligand abundance, presentation to cognate receptors and therefore signaling. Importantly, the regulation of HSPG co-receptor abundance has been shown to be dependent on sulfation state mediated by extracellular sulfatases. Consistently, upregulation of Dlp and Sdc was observed in sulf1 null synapses, whereas Dlp was reduced in hs6st null synapses. In the developing Drosophila wing disc, HSPG co-receptors increase levels of the Wg ligand due to extracellular stabilization, and the primary function of Dlp in this developmental context is to retain Wg at the cell surface. Likewise, in developing Drosophila embryos, a significant fraction of Wg ligand is retained on the cell surfaces in a HSPG-dependent manner, with the HSPG acting as an extracellular co-receptor. Syndecan also modulates ligand-dependent activation of cell-surface receptors by acting as a co-receptor. At the NMJ, regulation of both these HSPG co-receptors occurs in the closely juxtaposed region between presynaptic bouton and muscle subsynaptic reticulum, in the exact same extracellular space traversed by the secreted trans-synaptic Wg and Gbb signals. It is therefore proposed that altered Dlp and Sdc HSPG co-receptors in sulf1 and hs6st mutants differentially trap/stabilize Wg and Gbb trans-synaptic signals at the interface between motor neuron and muscle, to modulate the extent and efficacy of intercellular signaling driving synaptic development (Dani, 2012b).

HS sulfation modification is linked to modulating the intercellular signaling driving neuronal differentiation . In particular, WNT and BMP ligands are both regulated via HS sulfation of their extracellular co-receptors, and both signals have multiple functions directing neuronal differentiation, including synaptogenesis. In the Drosophila wing disc, extracellular WNT (Wg) ligand abundance and distribution was recently shown to be strongly elevated in sulf1 null mutants. Moreover, sulf1 has also recently been shown to modulate BMP signaling in other cellular contexts. Consistently, this study has shown increased WNT Wg and the BMP Gbb abundance and distribution in sulf1 null NMJ synapses. The hs6st null also exhibits elevated Wg and Gbb at the synaptic interface, albeit the increase is lower and results in differential signaling consequences. In support of this contrasting effect, extracellular signaling ligands are known to bind HSPG HS chains differentially dependent on specific sulfation patterns. It is important to note that the sulf1 and hs6st modulation of trans-synaptic signals is not universal, as Jelly Belly (Jeb) ligand abundance and distribution was not altered in the sulf1 and hs6st null conditions. This indicates that discrete classes of secreted trans-synaptic molecules are modulated by distinct glycan mechanisms to control NMJ structure and function (Dani, 2012b).

At the Drosophila NMJ, Wg is very well characterized as an anterograde trans-synaptic signal and Gbb is very well characterized as a retrograde trans-synaptic signal. In Wg signaling, the dFz2 receptor is internalized upon Wg binding and then cleaved so that the dFz2-C fragment is imported into muscle nuclei. In hs6st nulls, increased Wg ligand abundance at the synaptic terminal corresponds to an increase in dFz2C punctae in muscle nuclei as expected. In contrast, the increase in Wg at the sulf1 null synapse did not correspond to an increase in the dFz2C-terminus nuclear internalization, but rather a significant decrease. One explanation for this apparent discrepancy is the 'exchange factor' model based on the biphasic ability of the HSPG co-receptor Dlp to modulate Wg signaling. In the Drosophila wing disc, this model suggests that the transition of Dlp co-receptor from an activator to repressor of signaling depends on Wg cognate receptor dFz2 levels, such that a low ratio of Dlp:dFz2 potentiates Wg-dFz2 interaction, whereas a high ratio of Dlp:dFz2 prevents dFz2 from capturing Wg. In sulf1 null synapses, a very great increase was observed in Dlp abundance (~40% elevated) with no significant change in the dFz2 receptor. In contrast, at hs6st null synapses there is a decrease in Dlp abundance (15% decreased) together with a significant increase in dFz2 receptor abundance (~25% elevated). Thus, the higher Dlp:dFz2 ratio in sulf1 nulls could explain the decrease in Wg signal activation, evidenced by decreased dFz2-C terminus import into the muscle nucleus. In contrast, the Dlp:Fz2 ratio in hs6st is much lower, supporting activation of the dFz2-C terminus nuclear internalization pathway. This previously proposed competitive binding mechanism dependent on Dlp co-receptor and dFz2 receptor ratios predicts the observed synaptic Wg signaling pathway modulation in sulf1 and hs6st dependent manner (Dani, 2012b).

At the Drosophila NMJ, Gbb is very well characterized as a retrograde trans-synaptic signal, with muscle-derived Gbb causing the receptor complex Wishful thinking (Wit), Thickveins (Tkv) and Saxaphone (Sax) to induce phosphorylation of the transcription factor mothers against Mothers against decapentaplegic (P-Mad). Mutation of Gbb ligand, receptors or regulators of this pathway have shown that Gbb-mediated retrograde signaling is required for proper synaptic differentiation and functional development. Further, loss of Gbb signaling results in significantly decreased levels of P-Mad in the motor neurons. This study shows that accumulation of Gbb in sulf1 and hs6st null synapses causes elevated P-Mad signaling at the synapse and P-Mad accumulation in motor neuron nuclei. Importantly, sulf1 null synapses show a significantly higher level of P-Mad signaling compared to hs6st null synapses, and this same change is proportionally found in P-Mad accumulation within the motor neuron nuclei. These findings indicate differential activation of Gbb trans-synaptic signaling dependent on the HS sulfation state is controlled by the sulf1 and hs6st mechanism, similar to the differential effect observed on Wg trans-synaptic signaling. Genetic interaction studies show that these differential effects on trans-synaptic signaling have functional consequences, and exert a causative action on the observed bi-directional functional differentiation phenotypes in sulf1 and hs6st nulls. Genetic correction of Wg and Gbb defects in the sulf1 null background restores elevated transmission back to control levels. Similarly, genetic correction of Wg and Gbb in hs6st nulls restores the decreased transmission strength back to control levels. These results demonstrate that the Wg and Gbb trans-synaptic signaling pathways are differentially regulated and, in combination, induce opposite effects on synaptic differentiation (Dani, 2012b).

Both wg and gbb pathway mutants display disorganized and mislocalized presynaptic components at the active zone (e.g. Bruchpilot; Brp) and postsynaptic components including glutamate receptors (e.g. Bad reception; Brec/GluRIID). Consistently, the bi-directional effects on neurotransmission strength in sulf1 and hs6st mutants are paralleled by dysregulation of these same synaptic components. Changes in presynaptic Brp and postsynaptic GluR abundance/distribution causally explain the bi-directional effects on synaptic functional strength between sulf1 and hs6st null mutant states. Alterations in active zone Brp and postsynaptic GluRs also agree with assessment of spontaneous synaptic activity. Null sulf1 and hs6st synapses showed opposite effects on miniature evoked junctional current (mEJC) frequency (presynaptic component) and amplitude (postsynaptic component). Further, quantal content measurements also support the observation of bidirectional synaptic function in the two functionally paired nulls. Genetic correction of Wg and Gbb defects in both sulf1 and hs6st nulls restores the molecular composition of the pre- and postsynaptic compartments back to wildtype levels. When both trans-synaptic signaling pathways are considered together, these data suggest that HSPG sulfate modification under the control of functionally-paired sulf1 and hs6st jointly regulates both WNT and BMP trans-synaptic signaling pathways in a differential manner to modulate synaptic functional development on both sides of the cleft (Dani, 2012b).

This paper has presented the first systematic investigation of glycan roles in the modulation of synaptic structural and functional development. A host of glycan-related genes were identified that are important for modulating neuromuscular synaptogenesis, and these genes are now available for future investigations, to determine mechanistic requirements at the synapse, and to explore links to neurological disorders. As proof for the utilization of these screen results, this study has identified extracellular heparan sulfate modification as a critical platform of the intersection for two secreted trans-synaptic signals, and differential control of their downstream signaling pathways that drive synaptic development. Other trans-synaptic signaling pathways are independent and unaffected by this mechanism, although it is of course possible that a larger assortment of signals could be modulated by this or similar mechanisms. This study supports the core hypothesis that the extracellular space of the synaptic interface, the heavily-glycosylated synaptomatrix, forms a domain where glycans coordinately mediate regulation of trans-synaptic pathways to modulate synaptogenesis and subsequent functional maturation (Dani, 2012b).

Endostatin is a trans-synaptic signal for homeostatic synaptic plasticity

At synapses in organisms ranging from fly to human, a decrease in postsynaptic neurotransmitter receptor function elicits a homeostatic increase in presynaptic release that restores baseline synaptic efficacy. This process, termed presynaptic homeostasis, requires a retrograde, trans-synaptic signal of unknown identity. Multiplexin was identified in a forward genetic screen for homeostatic plasticity genes. Multiplexin is the Drosophila homolog of Collagen XV/XVIII, a matrix protein that can be proteolytically cleaved to release Endostatin, an antiangiogenesis signaling factor. This study demonstrates that Multiplexin is required for normal calcium channel abundance, presynaptic calcium influx, and neurotransmitter release. Remarkably, Endostatin has a specific activity, independent of baseline synapse development, that is required for the homeostatic modulation of presynaptic calcium influx and neurotransmitter release. These data support a model in which proteolytic release of Endostatin signals trans-synaptically, acting in concert with the presynaptic CaV2.1 calcium channel, to promote presynaptic homeostasis (Wang, S. J., 2014).

The nervous system is continually modified by experience. Given the tremendous complexity of the nervous system, it is astounding that robust and reproducible neural function can be sustained throughout life. It is now apparent that homeostatic signaling systems stabilize the excitable properties of nerve and muscle and, thereby, constrain how the nervous system can be altered by experience or crippled by disease. The Drosophila neuromuscular junction (NMJ) has emerged as a powerful model system to dissect the underlying mechanisms that achieve the homeostatic modulation of presynaptic neurotransmitter release. At the Drosophila NMJ, inhibition of postsynaptic glutamate receptor function causes a homeostatic increase in presynaptic neurotransmitter release that precisely restores muscle excitation to baseline levels. This phenomenon is conserved from fly to human. Importantly, presynaptic homeostasis has also been observed at mammalian central synapses in vitro in response to differences in target innervation and altered postsynaptic excitability and following chronic inhibition of neural activity (Wang, S. J.,2014).

Despite progress in identifying presynaptic effector proteins that are required for the expression of presynaptic homeostasis, the identity of the retrograde signaling system remains unknown. Numerous neurotrophic factors, such as nerve growth factor; brain-derived neurotrophic factor (BDNF); and glia-derived neurotrophic factor, as well as nitric oxide, endocannabinoids, and adhesion molecules, are identified as retrograde signals that regulate presynaptic cell survival, differentiation, and biophysical properties in an activity-dependent manner. Among these molecules, BDNF has been implicated in the trans-synaptic control of presynaptic release in cultured hippocampal neurons. Previous work demonstrated that a bone morphogenetic protein (BMP) ligand (Glass bottom boat) is released from muscle, activates a type II BMP receptor at the presynaptic terminal, and is required for the growth of the presynaptic nerve terminal. This BMP signaling system is also necessary for presynaptic homeostasis. However, the BMP signaling system is a permissive signal that acts at the motoneuron cell body (Wang, S. J.,2014).

A large-scale, electrophysiology-based forward genetic screen for mutations that block presynaptic homeostasis identified multiplexin as a candidate homeostatic plasticity gene. Drosophila Multiplexin is the homolog of human Collagen XV and XVIII, matrix molecules that are expressed ubiquitously in various vascular and epithelial basement membranes throughout the body. Mutations in the human COL18A1 gene cause Knobloch syndrome, characterized by retinal detachment, macular abnormalities, and occipital encephalocele. Patients with Knobloch syndrome are also predisposed to epilepsy, highlighting the critical function of Collagen XVIII in the central nervous system. Moreover, the C-terminal of Collagen XVIII, encoding an Endostatin domain, can be cleaved proteolytically and functions as an antiangiogenesis factor to inhibit tumor progression. Endostatin inhibits angiogenesis by interacting with various downstream signaling factors, including vascular endothelial growth factor receptors, integrins, and Wnt signaling molecules. Little is known regarding the function of multiplexin in the nervous system. This study provides evidence that Endostatin, a proteolytic cleavage product of Drosophila Multiplexin, functions as a trans-synaptic signaling molecule that is essential for the homeostatic modulation of presynaptic neurotransmitter release at the Drosophila NMJ (Wang, S. J.,2014).

Loss of Endostatin blocks the homeostatic modulation of presynaptic calcium influx and presynaptic neurotransmitter release. This activity is remarkably specific to presynaptic homeostasis, since loss of Endostatin has no effect on baseline neurotransmission or synapse morphology. Endostatin also interacts genetically with the pore-forming subunit of the CaV2.1 calcium channel and is required for the homeostatic increase of presynaptic calcium influx during synaptic homeostatic plasticity. Finally, transgenic overexpression of Endostatin is sufficient to rescue synaptic homeostasis and baseline neurotransmitter release when it is supplied to either the presynaptic or postsynaptic side of the synapse. Although deletion of Endostatin does not impair baseline transmission, overexpression of Endostatin in the dmpf07253 mutant is sufficient to restore baseline transmission release even in the absence of the Thrombospondin-like domain. As a working model, it is proposed that inhibition of postsynaptic glutamate receptors initiates the proteolytic cleavage of Multiplexin, which resides in the synaptic cleft. It is further proposed that release of Endostatin acts upon presynaptic calcium channels, directly or indirectly, to potentiate calcium influx and presynaptic neurotransmitter release. This model is consistent with data from other systems demonstrating that activation of Endostatin requires proteolytic cleavage of Collagen XVIII by matrix metalloproteases (MMPs) and cysteine cathepsins. Moreover, only free Endostatin released by cleavage functions as an antiangiogenesis factor (Wang, S. J.,2014).

The means by which presynaptic calcium channel function is modulated by Endostatin remains to be elucidated. Recently, it has been shown that a presynaptic Deg/ENaC channel is also necessary for the homeostatic modulation of presynaptic release. In this previous study, a model is presented in which ENaC channel insertion causes a sodium leak and modest depolarization of the presynaptic resting membrane potential that, in turn, potentiates presynaptic calcium influx. One possibility is that the interaction of Endostatin with the presynaptic CaV2.1 channels enables the channels to respond to low-voltage modulation. This would be consistent with both Endostatin and the ENaC channel being strictly necessary for presynaptic homeostasis. It remains formally possible that Endostatin stabilizes presynaptic ENaC channels and, thereby, influences presynaptic calcium influx. For example, it was demonstrated that the interaction between ENaC channels and extracellular collagens mediates the mechanosensory transduction in the touch reception systems (Wang, S. J.,2014).

Activation of Endostatinin in other systems requires proteolytical cleavage of Collagen XVIII by MMPs and cysteine cathepsins. This raises an intriguing possibility that, during synaptic homeostasis, Multiplexin could be cleaved by synaptic MMPs, releasing Endostatin to trigger a homeostatic change in presynaptic release. In this model, inhibition of postsynaptic glutamate receptors would lead to the activation of MMPs within the synaptic cleft. Thus, the retrograde signal would be a multistage system, providing opportunity for both amplification and multilevel control of the signaling event. At glutamatergic synapses in hippocampal neurons, proteolytic cleavage of neuroligin-1, a synaptic adhesion molecule residing in postsynaptic terminals, is triggered by postsynaptic NMDA receptor activation. Cleavage of neuroligin-1 depresses presynaptic transmission by reducing presynaptic release probability in a trans-synaptic manner. Thus, the activity-dependent cleavage of cell adhesion and extracellular matrix proteins could provide a robust and evolutionarily conserved feedback paradigm for trans-synaptic signaling to regulate synaptic efficacy in diverse neuronal circuits (Wang, S. J.,2014).

N-glycosylation requirements in neuromuscular synaptogenesis

Neural development requires N-glycosylation regulation of intercellular signaling, but the requirements in synaptogenesis have not been well tested. All complex and hybrid N-glycosylation requires MGAT1 (UDP-GlcNAc:alpha-3-D-mannoside-beta1,2-N-acetylglucosaminyl-transferase I) function, and Mgat1 nulls are the most compromised N-glycosylation condition that survive long enough to permit synaptogenesis studies. At the Drosophila neuromuscular junction (NMJ), Mgat1 mutants display selective loss of lectin-defined carbohydrates in the extracellular synaptomatrix, and an accompanying accumulation of the secreted endogenous Mind the gap (MTG) lectin, a key synaptogenesis regulator. Null Mgat1 mutants exhibit strongly overelaborated synaptic structural development, consistent with inhibitory roles for complex/hybrid N-glycans in morphological synaptogenesis, and strengthened functional synapse differentiation, consistent with synaptogenic MTG functions. Synapse molecular composition is surprisingly selectively altered, with decreases in presynaptic active zone Bruchpilot (BRP) and postsynaptic Glutamate receptor subtype B (GLURIIB), but no detectable change in a wide range of other synaptic components. Synaptogenesis is driven by bidirectional trans-synaptic signals that traverse the glycan-rich synaptomatrix, and Mgat1 mutation disrupts both anterograde and retrograde signals, consistent with MTG regulation of trans-synaptic signaling. Downstream of intercellular signaling, pre- and postsynaptic scaffolds are recruited to drive synaptogenesis, and Mgat1 mutants exhibit loss of both classic Discs large 1 (DLG1) and newly defined Lethal (2) giant larvae [L(2)gl] scaffolds. It is concluded that MGAT1-dependent N-glycosylation shapes the synaptomatrix carbohydrate environment and endogenous lectin localization within this domain, to modulate retention of trans-synaptic signaling ligands driving synaptic scaffold recruitment during synaptogenesis (Parkinson, 2013).

This study began with the hypothesis that disruption of synaptomatrix N-glycosylation would alter trans-synaptic signaling underlying NMJ synaptogenesis (Dani, 2012a). MGAT1 loss transforms the synaptomatrix glycan environment. Complete absence of the HRP epitope, α1-3-fucosylated N-glycans, is expected to require MGAT1 activity: key HRP epitope synaptic proteins include fasciclins, Neurotactin and Neuroglian, among others. This study shows that HRP epitope modification of the key synaptogenic regulator Fasciclin 2 is not required for stabilization or localization, suggesting a role in protein function. However, complete loss of Vicia villosa (VVA) lectin reactivity synaptomatrix labeling is surprising because the epitope is a terminal β-GalNAc. This result suggests that the N-glycan LacdiNAc is enriched at the NMJ, and that the terminal GalNAc expected on O-glycans/glycosphingolipids may be present on N-glycans in this synaptic context. Importantly, VVA labels Dystroglycan and loss of Dystroglycan glycosylation blocks extracellular ligand binding and complex formation in Drosophila, and causes muscular dystrophies in humans. This study shows that VVA-recognized Dystroglycan glycosylation is not required for protein stabilization or synaptic localization, but did not test functionality or complex formation, which probably requires MGAT1-dependent modification. Conversely, the secreted endogenous lectin MTG is highly elevated in Mgat1 null synaptomatrix, probably owing to attempted compensation for complex and hybrid N-glycan losses that serve as MTG binding sites. MTG binds GlcNAc in a calcium-dependent manner and pulls down a number of HRP-epitope proteins by immunoprecipitation (Rushton, 2012), although the specific proteins have not been identified. It will be of interest to perform immunoprecipitation on Mgat1 samples to identify changes in HRP bands. Importantly, MTG is crucial for synaptomatrix glycan patterning and functional synaptic development. MTG regulates VVA synaptomatrix labeling, suggesting a mechanistic link between the VVA and MTG changes in Mgat1 mutants. The MTG elevation observed in Mgat1 nulls provides a plausible causative mechanism for strengthened functional differentiation (Parkinson, 2013).

Consistent with recent glycosylation gene screen findings (Dani, 2012a), Mgat1 nulls exhibit increased synaptic growth and structural overelaboration. Therefore, complex and hybrid N-glycans overall provide a brake on synaptic morphogenesis, although individual N-glycans may provide positive regulation. Likely players include MGAT1-dependent HRP-epitope proteins (e.g., fasciclins, Neurotactin, Neuroglian), and position-specific (PS) integrin receptors and their ligands, all of which are heavily glycosylated and have well-characterized roles regulating synaptic architecture. An alternative hypothesis is that Mgat1 phenotypes may result from the presence of high-mannose glycans on sites normally carrying complex/hybrid structures, suggesting possible gain of function rather than loss of function of specific N-glycan classes. NMJ branch and bouton number play roles in determining functional strength, although active zones and GluRs are also regulated independently. Thus, the increased functional strength could be caused by increased structure at Mgat1 null NMJs. However, muscle-targeted UAS-Mgat1 rescues otherwise Mgat1 null function, but has no effect on structural defects, demonstrating that these two roles are separable. Presynaptic Mgat1 RNAi also causes strong functional defects, showing there is additionally a presynaptic requirement in functional differentiation. Neuron-targeted Mgat1 causes lethality, indicating that MGAT1 levels must be tightly regulated, but preventing independent assessment of Mgat1 presynaptic rescue of synaptogenesis defects (Parkinson, 2013).

Presynaptic glutamate release and postsynaptic glutamate receptor responses drive synapse function. Using lipophilic dye to visualize SV cycling, this study found Mgat1 null mutants endogenously cycle less than controls, but have greater cycling capacity upon depolarizing stimulation. The endogenous cycling defect is consistent with the sluggish locomotion of Mgat1 mutants, whereas the elevated stimulation-evoked cycling is consistent with electrophysiological measures of neurotransmission. Similarly, mutation of dPOMT1, which glycosylates VVA-labeled Dystroglycan, decreases SV release probability (Wairkar, 2008), although dPOMT1 adds mannose not GalNAc. Null Mgat1 mutants display no change in SV cycle components (e.g. Synaptobrevin, Synaptotagmin, Synaptogyrin, etc.), but exhibit reduced expression of the key active zone component Bruchpilot. Other examples of presynaptic glycosylation requirements include the Drosophila Fuseless (FUSL) glycan transporter, which is critical for Cacophony (CAC) voltage-gated calcium channel recruitment to active zones, and the mammalian GalNAc transferase (GALGT2), whose overexpression causes decreased active zone assembly. Postsynaptically, Mgat1 nulls show specific loss of GLURIIB-containing receptors. Similarly, dPOMT1 mutants exhibit specific GLURIIB loss (Wairkar, 2008), although dystroglycan nulls display GLURIIA loss. Selective GLURIIB loss in Mgat1 nulls may drive increased neurotransmission owing to channel kinetics differences in GLURIIA versus GLURIIB receptors (Parkinson, 2013).

Bidirectional trans-synaptic signaling regulates NMJ structure, function and pre/postsynaptic composition. This intercellular signaling requires ligand passage through, and containment within, the heavily glycosylated synaptomatrix, which is strongly compromised in Mgat1 mutants. In testing three well-characterized signaling pathways, this study found that Wingless (Wg) accumulates, whereas both GBB and JEB are reduced in the Mgat1 null synaptomatrix. WG has two N-glycosylation sites, but these do not regulate ligand expression, suggesting WG build-up occurs owing to lost synaptomatrix N-glycosylation. Importantly, WG overexpression increases NMJ bouton formation similarly to the phenotype of Mgat1 nulls, suggesting a possible causal mechanism. GBB is predicted to be N-glycosylated at four sites, but putative glycosylation roles have not yet been tested. Importantly, GBB loss impairs presynaptic active zone development similarly to Mgat1 nulls, suggesting a separable causal mechanism. JEB is not predicted to be N-glycosylated, indicating that JEB loss is caused by lost synaptomatrix N-glycosylation. Importantly, it has been shown that loss of JEB signaling increases functional synaptic differentiation similarly to Mgat1 nulls (Rohrbough, 2013). In addition, jeb mutants exhibit strongly suppressed NMJ endogenous activity, similarly to the reduced endogenous SV cycling in Mgat1 nulls. Moreover, the MTG lectin negatively regulates JEB accumulation in NMJ synaptomatrix, consistent with elevated MTG causing JEB downregulation in Mgat1 nulls (Parkinson, 2013).

Trans-synaptic signaling drives recruitment of scaffolds that, in turn, recruit pre- and postsynaptic molecular components. Specifically, DLG1 and L(2)GL scaffolds regulate the distribution and density of both active zone components (e.g. BRP) and postsynaptic GluRs, and both of these scaffolds are reduced at Mgat1 null NMJs. Importantly, dlg1 mutants display selective loss of GLURIIB, with GLURIIA unchanged, similar to Mgat1 nulls, suggesting a causal mechanism. Moreover, l(2)gl mutants display both a selective GLURIIB impairment as well as reduction of BRP aggregation in active zones, similarly to Mgat1 nulls, suggesting a separable involvement for this synaptic scaffold. DLG1 and L(2)GL are known to interact in other developmental contexts, indicating a likely interaction at the developing synapse. Although synaptic ultrastructure has not been examined in l(2)gl mutants, dlg1 mutants exhibit impaired NMJ development, including a deformed SSR. These synaptogenesis requirements predict similar ultrastructural defects in Mgat1 mutants, albeit presumably due to the combined loss of both DLG1 and L(2)GL scaffolds. Future work will focus on electron microscopy analyses to probe N-glycosylation mechanisms of synaptic development (Parkinson, 2013).

Three-dimensional imaging of Drosophila motor synapses reveals ultrastructural organizational patterns

This study combined cryo-preservation of intact Drosophila larvae and electron tomography with comprehensive segmentation of key features to reconstruct the complete ultrastructure of a model glutamatergic synapse in a near-to-native state. Presynaptically, a complex network of filaments was detailed that connects and organizes synaptic vesicles. The complexity of this synaptic vesicle network was linked to proximity to the active zone cytomatrix, consistent with the model that these protein structures function together to regulate synaptic vesicle pools. A net-shaped network of electron-dense filaments spanning the synaptic cleft was identified that suggests conserved organization of trans-synaptic adhesion complexes at excitatory synapses. Postsynaptically, a regular pattern of macromolecules was characterized that yields structural insights into the scaffolding of neurotransmitter receptors. Together, these analyses reveal an unexpected level of conservation in the nanoscale organization of diverse glutamatergic synapses and provide a structural foundation for understanding the molecular machines that regulate synaptic communication at a powerful model synapse (Zhan, 2016).

Unraveling synaptic GCaMP signals: differential excitability and clearance mechanisms underlying distinct Ca(2+) dynamics in tonic and phasic excitatory, and aminergic modulatory motor terminals in Drosophila

GCaMP is an optogenetic Ca(2+) sensor widely used for monitoring neuronal activities but the precise physiological implications of GCaMP signals remain to be further delineated among functionally distinct synapses. The Drosophila neuromuscular junction (NMJ), a powerful genetic system for studying synaptic function and plasticity, consists of tonic and phasic glutamatergic and modulatory aminergic motor terminals of distinct properties. This study reports a first simultaneous imaging and electric recording study to directly contrast the frequency characteristics of GCaMP signals of the three synapses for physiological implications. Distinct mutational and drug effects on GCaMP signals indicate differential roles of Na(+) and K(+) channels, encoded by genes including paralytic (para), Shaker (Sh), Shab, and ether-a-go-go (eag), in excitability control of different motor terminals. Moreover, the Ca(2+) handling properties reflected by the characteristic frequency dependence of the synaptic GCaMP signals were determined to a large extent by differential capacity of mitochondria-powered Ca(2+) clearance mechanisms. Simultaneous focal recordings of synaptic activities further revealed that GCaMPs were ineffective in tracking the rapid dynamics of Ca(2+) influx that triggers transmitter release, especially during low-frequency activities, but more adequately reflected cytosolic residual Ca(2+) accumulation, a major factor governing activity-dependent synaptic plasticity. These results highlight the vast range of GCaMP response patterns in functionally distinct synaptic types and provide relevant information for establishing basic guidelines for the physiological interpretations of presynaptic GCaMP signals from in situ imaging studies (Xing, 2018a).

Ca2+ influx on action potential arrival at synaptic terminals triggers neurotransmitter release, and residual Ca2+ accumulation following repetitive action potentials regulates activity-dependent synaptic plasticity. Na+ and K+ channels play fundamental roles in shaping the axonal action potential and its repetitive firing pattern and thus can profoundly influence the amplitudes and kinetics of synaptic Ca2+ elevation. Conversely, Ca2+ clearance mechanisms, including mitochondrial and endoplasmic reticulum (ER) sequestration and energy-dependent extrusion via plasma membrane Ca2+-ATPase (PMCA), are critical in the restoration of synaptic basal Ca2+ levels (Xing, 2018a).

GCaMPs are widely used genetically encoded Ca2+ indicators. Despite the frequent applications of GCaMPs in monitoring neuronal activities in nervous systems of various animal species, it is unclear how differences in membrane excitability and Ca2+ clearance mechanisms determine the amplitude and kinetics of GCaMP Ca2+ signals in functionally distinct categories of synapses (Xing, 2018a).

This analyzed GCaMP signals in the Drosophila larval neuromuscular junction (NMJ), in which both excitatory (glutamatergic tonic type Ib and phasic type Is) as well as modulatory (octopaminergic type II) synapses could be monitored simultaneously within the same optical microscopy field. The glutamatergic type I synapses have been extensively studied for their electrophysiological properties and striking phenotypes caused by ion channel mutations. Octopaminergic type II synaptic terminals are known to modulate the growth and transmission properties of type I synapses and to display remarkable excitability-dependent plasticity. However, differences in excitability control and Ca2+ handling properties among these three distinct synaptic types remain to be determined (Xing, 2018a).

This decade-long study, extended from earlier results, employed different versions of GCaMPs, including GCaMPs 1, 5, and 6, to delineate the distinct frequency characteristics of GCaMP signals from type Ib, Is, and II synapses and their preferential sensitivities to different pharmacological or genetic perturbations. In particular, the results show that type II synapses were most strongly affected by manipulations of channels encoded by ether-a-go-go (eag, Eag, or KV10 ortholog), Shab (KV2 ortholog), and paralytic (para, NaV1) channels, whereas type Is synapses were most severely modified by manipulations of Shaker (Sh, KV1 ortholog). Strikingly, double insults through manipulating Sh together with either eag or Shab could generate extreme hyperexcitability in type Is synapses, leading to greatly enhanced GCaMP signals on individual nerve stimulation. In contrast, type Ib synapses remained largely intact in the above experimentations but could display similar extreme hyperexcitability following triple insults with combinations of mutations or blockers of K+ channels. Simultaneous focal electrical recordings of synaptic activities revealed that such extreme cases of enhanced GCaMP signals actually resulted from supernumerary high-frequency (>100 Hz) repetitive firing in the motor terminals following each single stimulus (Xing, 2018a).

Further kinetic analysis revealed different Ca2+ clearance capacity among three types of synaptic terminals. This study found that Na+ and K+ channel mutations or blockers influence mainly the rise kinetics of GCaMP signals, whereas inhibiting Ca2+ clearance mediated by PMCA (via high pH treatment) slowed the decay phase acutely. In addition, it was discovered that long-term inhibition of mitochondrial energy metabolism by incubation with either 2,4-dinitrophenol (DNP) or azideled to drastically lengthened decay time of the GCaMP signal and significantly altered its frequency responses to repetitive stimulation, over a time course of tens of minutes (Xing, 2018a).

Overall, this study demonstrates a wide range of GCaMP response patterns indicating differential membrane excitability and Ca2+ clearance mechanisms in functionally distinct types of synapses. Although the slow kinetics of GCaMP signals could not adequately resolve the rapid process of Ca2+ influx triggered by individual action potentials, they could nevertheless report cytosolic residual Ca2+ accumulation on repetitive synaptic activities. These data thus provide essential baseline information for refined interpretations of GCaMP signals when monitoring in vivo neural circuit activities that often result from interplay among different categories of synapses (Xing, 2018a).

Genetically encoded GCaMP indicators are widely used for detecting neuronal circuit activities in vivo. However, the analytic power of GCaMP signals has not been fully exploited to extract information regarding basic synaptic physiology. This study took advantage of the special anatomic features of the Drosophila larval NMJ to contrast properties of metabotropic aminergic (type II) and ionotropic glutamatergic (tonic type Ib and phasic type Is) synapses using several GCaMP Ca2+ indicators. Simultaneous monitoring of GCaMP signals from the three synapses within the same microscopic field demonstrates differential excitability control of Ca2+ influx by Na+ and K+ channels. Analyses of both kinetic and amplitude features of GCaMP signals reveal the extreme effects of particular Na+ and K+ channels on each of the three synaptic types, as well as the prominent roles of mitochondria-powered Ca2+ clearance mechanisms in shaping their distinct Ca2+ handling properties (Xing, 2018a).

A summary diagram is presented of how the various genetic and pharmacological manipulations influence Ca2+ influx and clearance, hence the amplitude and kinetics of GCaMP signals (Presynaptic cytosolic residual Ca2+ regulation in Drosophila NMJ synapses). Action potentials, generated and fine-tuned by Na+ and K+ channels, depolarize synaptic terminals and allows Ca2+ influx, which triggers synaptic transmission rapidly in milliseconds. The influx of Ca2+ ions are either actively extruded by PMCA locally, or sequestered by intracellular organelles such as mitochondria and ER, or buffered by Ca2+ binding proteins. The rise of GCaMP signals spans from hundreds of milliseconds up to seconds before peaking, depending on stimulation frequencies and external Ca2+ concentrations. Even with improved sensitivity, GCaMP6 signals are not faster compared to GCaMP1.3, taking at least 100 ms after a single stimulus to reach the peak of fluorescence at high external Ca2+ concentration. Thus, GCaMP signals are several orders slower than individual action potentials and the ensuing postsynaptic potentials. Further, unlike the synthetic Ca2+ indicators such as Oregon Green BAPTA (Hill coefficient 1.48), a GCaMP protein, with calmodulin as the Ca2+ sensor, typically binds four Ca2+ ions allosterically to produce enhanced fluorescence (Hill coefficients of GCaMP1 = 3.3). The magnitude of enhancement is thus limited especially at low levels of Ca2+ elevations evoked by single action potentials. Therefore, GCaMP signals better serve as the readout of a leaky integrator that registers cytosolic residual Ca2+, i.e., the net Ca2+ accumulation as determined by the process of influx and clearance over repetitive firing of action potentials, which can be induced either by trains of stimulation, or hyperexcitability (Xing, 2018a).

Electrophysiological recording of postsynaptic EJCs or EJPs generally detects the ensemble effects of type Ib, Is, and II synapses. Unlike type Ib and Is synapses, electrophysiological characterization of aminergic type II synapses is more technically challenging because they do not generate readily detectable postsynaptic electrical responses. In contrast, GCaMP signals offer the necessary spatial resolution, and thus enabled demonstration for the first time that mutations or blockers of specific ion channels lead to drastically different effects on type II, as well as type Ib and Is, axonal terminals (Xing, 2018a).

These results demonstrated that type Ib synapses were most enriched in the reserve of repolarizing capacity pooled from different K+ channel subtypes and could sustain multiple insults of K+ channel elimination or blockage before exhibiting the 'hallmark' of extreme hyperexcitability (single pulse-evoked giant GCaMP signals at 0.1 mM Ca2+). In comparison, type II synapses had the smallest repertoire of K+ channels and simply knocking down either Shab or eag could induce the hallmark hyperexcitability effect. In type Is synapses, Sh appeared to be the central player for repolarization and perturbing the Sh channel together with either Eag or Shab channels induced the hallmark ceiling effect of extreme hyperexcitability. This finding also resolved type Is but not Ib motor axons as the major source of the striking electrophysiological phenotype, i.e., axonal high-frequency repetitive firing (Xing, 2018a).

Alleles of para also have differential effects on type Ib, Is, and II synapses, possibly reflecting differential expression of the Para product, e.g., different splice isoforms, or posttranslational modifications (Xing, 2018a).

Type II synapses were more prone to conduction failure on high-frequency stimulation, as indicated by GCaMP signals that frequently became intermittent, or even totally missing during 10- to 40-Hz stimulation. This reflects the well-known axonal passive cable properties; thinner axons have proportionally higher longitudinal internal resistance relative to trans-membrane resistance, resulting in a more limited safety margin of axonal conduction and a longer refractory period for action potentials. Therefore, type II terminals are more prone to K+ and Na+ channels modifications (Xing, 2018a).

Morphometric analysis confirms that the differential excitability and distinct Ca2+ dynamics found in this study reflect intrinsic properties of type Ib, Is, and II synapses. The GCaMP responses characteristic of each synaptic type were independent of different sizes of boutons along individual axonal synaptic terminals, implying that differences in the physical dimensions among the three synaptic bouton types do not contribute to the distinct properties of type Ib, Is, and II synapses reported in this study (Xing, 2018a).

Obviously, besides Na+ and K+ channels, other channels may contribute to excitability-controlled Ca2+ influx. In particular, different types of Ca2+ channels await further study. Notably, previous anatomic studies have shown differences in presynaptic density area among different types of boutons. Ca2+ channels are known to be closely associated with active zones embedded within presynaptic density areas. It has been shown that type Is has higher density of active zones than type Ib synapses (Xing, 2018a and references therein).

It should be noted that differences in Ca2+ clearance capacity correlate well with the distinct frequency responses in the Ca2+ dynamics of these synaptic categories. Type II synapses apparently have the slowest rate of Ca2+ clearance, as evidenced by its slowest decay of GCaMP signals after secession of stimulation. The faster Ca2+ clearance in type Ib when compared to type Is synapses (He, 2009) appears to parallel its higher firing frequency (40-60 Hz in Ib vs 10-20 Hz in type Is) during natural bursting activities in semi-intact larval preparations, whereas presynaptic cytosolic Ca2+ elevation during repetitive firing stimulate mitochondrial oxidative phosphorylation so as to meet temporary burst energy needs. It is conceivable that type Ib synapses thus require a more efficient Ca2+ clearance system to avoid intracellular Ca2+ build-up. Interestingly, earlier electron microscopy studies have shown that tonic (type Ib) synapses contain more mitochondria than phasic (type Is) synapses in both Drosophila larval and crayfish NMJs. The current observation using mitochondrial staining confirmed this conclusion and also revealed a far lower density of mitochondria in type II synapses (Xing, 2018a).

This study showed the importance of mitochondria-powered Ca2+ clearance in shaping the distinct dynamics of cytosolic residual Ca2+ build-up in type Ib, Is, and II synapses. Inhibiting mitochondrial function with two different means, incubation with either DNP, a proton ionophore that dissipates mitochondrial proton gradient, or azide, an electron-transport chain inhibitor (complex IV), consistently resulted in slower GCaMP signal decay time course and shifted the frequency dependence in type II, Is, and Ib over a period of tens of minutes (Xing, 2018a).

In contrast to the slow effect of mitochondrial inhibition, high-pH inhibition of PMCA clearly impedes the GCaMP signal decay time course acutely. Ca2+ extrusion via PMCA, a Ca2+-ATPase, has been characterized in the Drosophila NMJ, as well as goldfish retina. Although under in vitro conditions, the fluorescence intensity of GCaMP protein can be affected by pH change, intracellularly expressed GCaMP protein is less likely to be affected by extracellular pH manipulation. This notion was supported by lack of change in presynaptic GCaMP baseline fluorescence intensity on external pH changes. Therefore, impaired ATP production from mitochondria can lead to PMCA-mediated Ca2+ extrusion shut-down, which could account for the striking effect of long-term DNP incubation (Xing, 2018a).

Notably, DNP treatment significantly impeded the GCaMP signal decay time course only after long-term incubation (beyond 20 min). Previous studies employing other proton ionophores such as carbonyl cyanide m-chlorophenyl hydrazine (CCCP) has demonstrated that inhibition of mitochondrial proton gradient does not significantly alter overall cytosolic Ca2+ dynamics acutely (Xing, 2018a).

Besides mitochondria, ER may also actively sequestrate Ca2+ via sarco/ER Ca2+ ATPase (SERCA) in synapses. This study inhibited SERCA with thapsigargin (1-2 µM, 1-h treatment) and found no obviously detectable effects on GCaMP signals comparable to the effect of DNP on any of the three types of synapses (in 4 larvae). Previous publications with a higher thapsigargin concentration (10 µM; Klose et al., 2009) or more sensitive Ca2+ indicator (Oregon Green BAPTA) have demonstrated only mildly increased Ca2+ signal amplitude and slower time course in type Ib synapses. Therefore, the contributions of mitochondrial and ER Ca2+ sequestration may be masked by other high-capacity ATP-dependent Ca2+ clearance mechanisms, such as PMCA. Nevertheless, when ATP production by mitochondria is inhibited, these active Ca2+ clearance mechanisms could be diminished on gradual depletion of ATP reserve (Xing, 2018a).

In this study, synapses remain viable, and Ca2+ clearance system remains functioning for at least tens of minutes, despite mitochondrial inhibition by DNP or azide. Nonmitochondrial sources of ATP such as glycolysis or ATP binding proteins might sustain for some time, until the first sign of depletion, i.e., the appearance of slower GCaMP signal decay kinetics (Xing, 2018a).

In fact, some vertebrate central nervous system synapses are known to operate without local presynaptic mitochondria. Similarly, Drosophila mutant drp1 and dMiro larval NMJs, with greatly reduced numbers of synaptic mitochondria, remain viable and display essentially normal Ca2+ dynamics and buffer capacity unless challenged by prolonged stimulation beyond minutes. In these studies, type II synapses had a lower abundance in mitochondria and thus more limited ATP reserve and in consequence were most vulnerable to DNP treatment. They were the first to show lengthened decay and to become completely nonresponsive subsequently during DNP incubation (Xing, 2018a).

Overall, this study indicates that analysis of GCaMP signals can be extended to extract information about specific synaptic physiologic properties. GCaMP signals offer higher spatial resolution and can complement electrophysiology data to pinpoint critical differences in channel expression and excitability properties among neighboring synaptic terminals (Xing, 2018a).

Systematic kinetic analysis of GCaMP signals revealed the predominant effects of hyperexcitability on the rise kinetics and Ca2+ clearance capacity on the decay kinetics. In conjunction with focal electrophysiological recording, genetic and pharmacological analyses indicate a close relationship between GCaMP signals and cytosolic residual Ca2+ accumulation rather than the rapid process of Ca2+ influx that triggers transmitter release. This approach also revealed the striking hyperexcitable effects caused by insults to multiple K+ channels, leading to the hallmark giant GCaMP signals evoked by single stimuli that generated high-frequency supernumerary firing of nerve action potentials. Thus, GCaMP signals may be further exploited to shed new light on activity-dependent plasticity in synapses of distinct properties. This work may help to establish guidelines for refined interpretations of GCaMP signals beyond the first-order, qualitative indications for gross neuronal activities in neural circuits (Xing, 2018a).

Inter-relationships among physical dimensions, distal-proximal rank orders, and basal GCaMP fluorescence levels in Ca(2+) imaging of functionally distinct synaptic boutons at Drosophila neuromuscular junctions

GCaMP imaging is widely employed for investigating neuronal Ca(2+) dynamics. The Drosophila larval neuromuscular junction (NMJ) consists of three distinct types of motor terminals (type Ib, Is and II). This study investigated whether variability in synaptic bouton sizes and GCaMP expression levels confound interpretations of GCaMP readouts. Analysis of large data sets accumulated over years established the wide ranges of bouton sizes and GCaMP baseline fluorescence, with large overlaps among synaptic categories. Bouton size and GCaMP baseline fluorescence were not confounding factors in determining the characteristic frequency responses among type Ib, Is and II synapses. More importantly, the drastic phenotypes that hyperexcitability mutations manifest preferentially in particular synaptic categories, were not obscured by bouton heterogeneity in physical size and GCaMP expression level. The results illustrate the conditions that disrupt or enhance the distal-proximal gradients. For example, stimulus frequencies just above the threshold level produced the steepest gradient in low Ca(2+) (0.1 mM) saline, while supra-threshold stimulation flattened the gradient. Moreover, membrane hyperexcitability mutations (eag(1) Sh(120) and para(bss1)) and mitochondrial inhibition by dinitrophenol (DNP) disrupted the gradient. However, a novel distal-proximal gradient of decay kinetics appeared after long-term DNP incubation. Focal recording was performed to assess the failure rates in transmission at low Ca(2+) levels, which yielded indications of a mild distal-proximal gradient in release probability (Xing, 2018b).

Regulation of presynaptic Ca2+ is critical for transmitter release as well as short-term and long-term synaptic plasticity. The Drosophila larval body-wall neuromuscular junction (NMJ) is an ideal system to contrast Ca2+ dynamics in synapses of different functional categories, as it contains in close proximity both tonic and phasic (type Ib and Is, respectively) glutamatergic synapses, as well as modulatory octopaminergic (type II) synapses, all of which can be imaged in the same microscopic field. A previous work has demonstrated characteristic frequency dependence of GCaMP signals for type Ib, Is and II synaptic boutons, indicating their distinct Ca2+ dynamics, i.e., type II, Is and Ib synapses being responsive to low, medium and high stimulus frequencies, respectively (Xing, 2018b).

Studies over the past years have accumulated a large body of single bouton records of GCaMP responses, along with quantifications of bouton sizes and GCaMP baseline fluorescence intensities. Despite the nomenclature implying size-related distinctions among these synapses (type Ib also known as 'I big', Is as 'I small'), significant heterogeneity is seen with overlapping bouton sizes between different synaptic categories. Furthermore, for each synaptic category, the level of GCaMP expression (as indicated by baseline fluorescence intensity F) varied significantly in the database. This study set out to determine the ranges of variation in these parameters and examined whether such high levels of heterogeneity confound interpretations from GCaMP measurements for salient physiological properties of the distinct synaptic bouton types (Xing, 2018b).

The database for this study also allowed a re-examination of the previously reported distal-to-proximal gradient in GCaMP response (ΔF/F). Analyses based on genetic and pharmacological manipulations further revealed the various conditions that can obscure or optimize the gradient. Lastly, simultaneous electrophysiological focal recording was carried out to map local synaptic transmission events to investigate the physiological significance of such distal-proximal GCaMP response gradient along the motor terminal branch (Xing, 2018b).

Among different cell types, neurons exhibit the most complex cellular morphology, with many specialized subcellular compartments, including presynaptic boutons and postsynaptic spines in different parts of soma and along axons and dendrites. Ca2+dynamics are crucial in the regulation of synaptic development, function, and plasticity. However, these structures of micrometer scale are highly variable and plastic in their size and location. Furthermore, synapses of different categories of neurons in the nervous systems, e.g. ionotropic and metabotropic, are distinct in morphology and distribution. It is therefore important to determine whether and how variation in geometric factors, such as physical size and location of synapses, sets constraints on local Ca2+ dynamics, and interferes with the readout of Ca2+ indicators in the investigation of intrinsic regulation mechanisms in functionally distinct synapses (Xing, 2018b).

Although a plethora of Drosophila GCaMP imaging studies has been published on CNS neuronal activity and peripheral synaptic function, the inter-relationships between synaptic parameters, such as bouton size, location, GCaMP baseline fluorescence F, and GCaMP signal amplitude ΔF/F, have not been fully established (Xing, 2018b).

The Drosophila NMJ provides a unique opportunity to contrast ionotropic synapses (type Ib and Is) with metabotropic synapses (type II) in close proximity for simultaneous imaging within the same microscopic field. Previous results show that type Ib, Is and II synapses manifest distinct frequency responses of GCaMP Ca2+ signals controlled by different ion channels and clearance mechanisms . In the present study, correlation analysis of large samples of boutons clarifies that neither bouton size nor GCaMP baseline fluorescence represents a confounding factor, when determining the intrinsic distinctions of frequency-dependent responses among type Ib, Is and II synapses (Xing, 2018b).

In principle, bouton size variation can be a contributing factor in determining the dynamics of GCaMP Ca2+ membrane properties, larger boutons should have a slower rate in accumulating cytosolic Ca2+ to produce detectable GCaMP signals. Thus, it can be argued that the size differences could be a possible explanation for the characteristic higher frequency response for type Ib bouton in contrast to the lower frequency ranges for type Is and even lower for type II synapses. In fact, the bouton size effect has been demonstrated in type Ib boutons to exist only for single-stimulus evoked Ca2+ transients, but not for plateau levels of Ca2+ signals evoked by trains of repetitive stimuli, using the fast indicator OGB-1. Theoretically, the rise of Ca2+ transients to a steady plateau level in response to a prolonged stimulus train is a process involving both Ca2+ influx and clearance, kinetically distinct from the measured amplitude of a single-stimulus evoked Ca2+ signal. In the current case, GCaMP signals are generally slow and do not resolve Ca2+ transients evoked by single action potentials. Instead, the plateaus of GCaMP signals report integration of Ca2+ influx and clearance over repetitive stimulus responses. In this study, the results demonstrate that the large overlaps of bouton sizes among the three synaptic types did not obscure their distinct frequency-dependent GCaMP signal characteristics. Therefore, physical dimension of synaptic boutons is not a practical predictor for the dynamics of GCaMP signals for boutons either within or between synaptic categories. Furthermore, it cannot account for the striking preferential effects of hyperexcitability mutations on different synaptic categories. Instead, intrinsic mechanisms such as membrane excitability and Ca2+ clearance capacity play far more important roles in the regulation of presynaptic Ca2+ dynamics (Xing, 2018b).

However, the data indeed confirm that GCaMP baseline fluorescence is significantly correlated with bouton size, as expected from their longer optical path in which GCaMP indicators interact with excitation light. Nonetheless, neither of these two absolute measures (in &mi;m2 and mV) plays a significant role in determining GCaMP signal amplitudes, which are normalized, unit-less quantities (ΔF/F). A direct measurement of bouton width may be an alternative method to estimate bouton thickness and correlate with bouton fluorescence intensity, which can be investigated in further studies (Xing, 2018b).

Earlier Ca2+ imaging studies based on different Ca2+ indicators have led to somewhat different pictures on the distal-proximal gradient along synaptic terminals in the Drosophila NMJ. A relatively weak distal-proximal gradient of presynaptic Ca2+ dynamics has been detected by back-filling type Ib and Is synaptic terminals with the synthetic indicator Oregon Green BAPTA-1 (OGB-1) in muscles 4, 6 and 7 in high Ca2+ saline (1 mM). A different study using presynaptic expression of genetically encoded Ca2+ indicators (Cameleon2.3) has also demonstrated a presynaptic Ca2+ gradient of similar magnitude (Xing, 2018b).

Postsynaptic expression of Ca2+ indicators, including GCaMP and derivatives of Cameleon, has revealed a stronger distal-proximal gradient of nerve-evoked optical signal representing Ca2+ influx through postsynaptic glutamate receptors along type Ib synaptic terminals. However, a more recent study reports relative uniform distribution of evoked postsynaptic myrGCaMP5 signals. It is not known whether the differences in the indicator types and their cellular localization could account for the discrepancy between these observations (Xing, 2018b).

The current work indicates that several factors and conditions may be manipulated to either enhance or obscure the detection of a gradient of GCaMP signals along the synaptic terminals. These include external Ca2+ concentration, stimulus frequency, and larval genotype. The initial choice to use low Ca2+ saline in earlier GCaMP imaging studies was to optimize the striking effects of hyperexcitable ion channel mutations and to avoid muscle contraction without relying on the usage of glutamate for postsynaptic receptor desensitization. Fortuitously, it was found that low Ca2+ saline could better reveal the distal-proximal gradients (a proximal/distal ratio about 30%), in contrast to the previously reported gradient (type Ib: in the range of 60%-80%; type Is: 80%-90%) at high Ca2+concentrations (1-1.5 mM). To sum up, the optimal detection of the distal-proximal gradient falls in the range just above the threshold of effective stimulus frequency (20 Hz for Is, 40-80 Hz for Ib) for GCaMP imaging experiments at 0.1 mM Ca2+ (Xing, 2018b).

For both type Is and type Ib, longitudinal ΔF/F gradients could be demonstrated corresponding to the bouton rank order (Figure 6), or their physical distance from the distal end of the terminals. It should be noted, however, that there were no corresponding longitudinal gradients in bouton sizes or GCaMP baseline fluorescence (Xing, 2018b).

This gradient was disrupted by hyperexcitable mutations, or DNP incubation. Interestingly, after prolonged DNP treatment, which saturated the ΔF/F response and obscured its gradient, a novel gradient in decay kinetics of the GCaMP signals was revealed in both type Ib and Is, i.e. slower half-decay time in distal boutons, suggesting a possible role of mitochondria in gradient formation (Xing, 2018b).

To investigate the functional significance of the GCaMP signal gradient, advantage was taken of the focal recording of efEJP events from distal and proximal boutons to correlate with the optical imaging results from type Ib boutons in muscle 4. With this approach, the failure rate of transmission could be readily quantified at low Ca2+ levels. There were indications for a relatively weak distal-proximal difference in release probability with repetitive 1-Hz stimulation. However, to assess the GCaMP response amplitude, a higher simulation frequency was applied (40 Hz), at which a clear distal-proximal gradient of GCaMP signals could be observed in all terminals that displayed ΔF/F above the noise level (0.1 mM Ca2+). In this case, it was found that GCaMP ΔF/F signals beyond the cut-off level (0.05) were correlated with drastically higher release probability. A clear trend of facilitation was observed during the 2 s stimulus train in both distal and proximal boutons. However, no striking distal-proximal difference of facilitation properties was observed under this condition (Xing, 2018b).

It should be stressed that GCaMP signals reflect cytosolic residual Ca2+ accumulation, which takes place in a time scale of hundreds of milliseconds to a few seconds, and involves both Ca2+ influx and clearance mechanisms. In contrast, Ca2+ entry and subsequent vesicle fusion and transmitter release occur in milliseconds. Therefore, depending on the stimulus protocols and local physiological conditions, the two measurements may yield rather different readouts that reflect the states of two steps in the chain of cellular Ca2+ dynamics, from influx, local actions, cytoplasmic accumulation and clearance. Thus, in type Ib synaptic boutons at low Ca2+condition, the distal-proximal gradient of GCaMP signal gradient does not necessarily imply a similar gradient in transmission strength (Xing, 2018b).

Since GCaMP signals reflect the dynamics of cytosolic residual Ca2+ accumulation, more investigations are needed to fully understand its relationship with short-term synaptic plasticity properties. Furthermore, a more definitive assessment of any gradient in transmitter release, and transmission efficacy of individual boutons along the synaptic terminal requires direct quantification of local synaptic currents at various Ca2+ levels under voltage-clamp conditions. This awaits future studies employing the focal loose-patch clamp, first pioneered in this preparation by Prof. Harold Atwood and associates (Xing, 2018b).

Glucuronylated core 1 glycans are required for precise localization of neuromuscular junctions and normal formation of basement membranes on Drosophila muscles

T antigen (Galβ1-3GalNAcalpha1-Ser/Thr) is an evolutionary-conserved mucin-type core 1 glycan structure in animals synthesized by core 1 β1,3-galactosyltransferase 1 (C1GalT1). Previous studies showed that T antigen produced by Drosophila C1GalT1 (dC1GalT1) was expressed in various tissues and dC1GalT1 loss in larvae led to various defects, including mislocalization of neuromuscular junction (NMJ) boutons, and ultrastructural abnormalities in NMJs and muscle cells. Although glucuronylated T antigen (GlcAβ1-3Galβ1-3GalNAcalpha1-Ser/Thr) has been identified in Drosophila, the physiological function of this structure has not yet been clarified. This study has unraveled biological roles of glucuronylated T antigen. The data show that in Drosophila, glucuronylation of T antigen is predominantly carried out by Drosophila β1,3-glucuronyltransferase-P (dGlcAT-P). dGlcAT-P null mutants were created, and it was found that mutant larvae showed lower expression of glucuronylated T antigen on the muscles and at NMJs. Furthermore, mislocalization of NMJ boutons and a partial loss of the basement membrane components collagen IV (Col IV) and nidogen (Ndg) at the muscle 6/7 boundary were observed. Those two phenotypes were correlated and identical to previously described phenotypes in dC1GalT1 mutant larvae. In addition, dGlcAT-P null mutants exhibited fewer NMJ branches on muscles 6/7. Moreover, ultrastructural analysis revealed that basement membranes that lacked Col IV and Ndg were significantly deformed. It was also found that the loss of dGlcAT-P expression caused ultrastructural defects in NMJ boutons. Finally, a genetic interaction was shown between dGlcAT-P and dC1GalT1. Therefore, these results demonstrate that glucuronylated core 1 glycans synthesized by dGlcAT-P are key modulators of NMJ bouton localization, basement membrane formation, and NMJ arborization on larval muscles (Itoh, 2018).

RNAi-mediated reverse genetic screen identified Drosophila chaperones regulating eye and neuromuscular junction morphology

Accumulation of toxic proteins in neurons have been linked with the onset of neurodegenerative diseases, which in many cases, are characterized by altered neuronal function and synapse loss. Molecular chaperones help protein folding and resolubilization of unfolded proteins thereby reducing the protein aggregation stress. While most of the chaperones are expressed in neurons, their functional relevance largely remains unknown. Using bioinformatics analysis, this study identified 95 Drosophila chaperones and classified them into seven different classes. Ubiquitous actin5C-Gal4 mediated RNAi knockdown revealed that about 50% of the chaperones are essential in Drosophila. Knocking down these genes in eyes revealed that about 30% of the essential chaperones are crucial for eye development. Using neuron-specific knockdown, immunocytochemistry and robust behavioural assays, a new set of chaperones were identified that play critical roles in the regulation of Drosophila NMJ structural organization. Together, these data presents the first classification and comprehensive analysis of Drosophila chaperones. The screen identified new set of chaperones that regulate eye and NMJ morphogenesis. Outcome of the screen reported here provides a useful resource for further elucidating the role of individual chaperones in Drosophila eye morphogenesis and synaptic development (Raut, 2017).

Ultrastructural comparison of the Drosophila larval and adult ventral abdominal neuromuscular junction

Drosophila melanogaster has recently emerged as model system for studying synaptic transmission and plasticity during adulthood, aging and neurodegeneration. However, still little is known about the basic neuronal mechanisms of synaptic function in the adult fly. Per se, adult Drosophila neuromuscular junctions should be highly suited for studying these aspects as they allow for genetic manipulations in combination with ultrastructural and electrophysiological analyses. Although different neuromuscular junctions of the adult fly have been described during the last years, a direct ultrastructural comparison with their larval counterpart is lacking. The present study was designed to close this gap by providing a detailed ultrastructural comparison of the larval and the adult neuromuscular junction of the ventrolongitudinal muscle. Assessment of several parameters revealed similarities but also major differences in the ultrastructural organisation of the two model neuromuscular junctions. While basic morphological parameters are retained from the larval into the adult stage, the analysis discovered major differences of potential functional relevance in the adult: The electron-dense membrane apposition of the presynaptic and postsynaptic membrane is shorter, the subsynaptic reticulum is less elaborated and the number of synaptic vesicles at a certain distance of the presynaptic membrane is higher (Wagner, 2017).

Downregulation of glutamic acid decarboxylase in Drosophila TDP-43-null brains provokes paralysis by affecting the organization of the neuromuscular synapses

Amyotrophic lateral sclerosis is a progressive neurodegenerative disease that affects the motor system, comprised of motoneurons and associated glia. Accordingly, neuronal or glial defects in TDP-43 function provoke paralysis due to the degeneration of the neuromuscular synapses in Drosophila. To identify the responsible molecules and mechanisms, a genome wide proteomic analysis was performed to determine differences in protein expression between wild-type and TDP-43-minus fly heads. The data established that mutant insects presented reduced levels of the enzyme glutamic acid decarboxylase (Gad1) and increased concentrations of extracellular glutamate. Genetic rescue of Gad1 activity in neurons or glia was sufficient to recuperate flies locomotion, synaptic organization and glutamate levels. Analogous recovery was obtained by treating TDP-43-null flies with glutamate receptor antagonists demonstrating that Gad1 promotes synapses formation and prevents excitotoxicity. Similar suppression of TDP-43 provoked the downregulation of GAD67, the Gad1 homolog protein in human neuroblastoma cell lines and analogous modifications were observed in iPSC-derived motoneurons from patients carrying mutations in TDP-43, uncovering conserved pathological mechanisms behind the disease (Romano, 2018).

Miniature neurotransmission is required to maintain Drosophila synaptic structures during ageing

The decline of neuronal synapses is an established feature of ageing accompanied by the diminishment of neuronal function, and in the motor system at least, a reduction of behavioural capacity. This study has investigated Drosophila motor neuron synaptic terminals during ageing. Cumulative fragmentation of presynaptic structures was observed, accompanied by diminishment of both evoked and miniature neurotransmission occurring in tandem with reduced motor ability. Through discrete manipulation of each neurotransmission modality, it was found that miniature but not evoked neurotransmission is required to maintain presynaptic architecture and that increasing miniature events can both preserve synaptic structures and prolong motor ability during ageing. These results establish that miniature neurotransmission, formerly viewed as an epiphenomenon, is necessary for the long-term stability of synaptic connections (Banerjee, 2021).

Deterioration of both central and peripheral synaptic structures and alterations of neurotransmission is a conserved feature of ageing in both rodents and humans. Consistently, using a preparation that was developed to examine adult synapses in Drosophila, this study also observe age-dependent reductions of synaptic structure and neurotransmitter release. Interrogating this system, it was found that age-dependent changes in vesicular neurotransmission, rather than being a consequence of alterations in synaptic structure, may instead precede and promote the decline of synaptic architecture. This finding is surprising and contrary to the generally assumed sequence of age-dependent synapse degeneration events. Moreover, this study established that miniature neurotransmitter release is the key constituent of vesicular neurotransmission that is singularly required to maintain synaptic structures as they age (Banerjee, 2021).

Since their discovery over 60 years ago, miniature events have been observed at every chemical synapse studied, but were often dismissed as 'noise' produced as a by-product of high fidelity evoked neurotransmission. The supposition that miniature neurotransmission is an epiphenomenon has been maintained in part by the difficulty to discretely attenuate presynaptic miniature release in vivo without also simultaneously perturbing evoked neurotransmission. By employing precise amino acid mutants of neurotransmission proteins derived from the intensive investigation of the mechanisms of vesicular release, in combination with adult-specific genetic manipulations, this study has revealed an essential and exclusive function for miniature events to maintain the structural integrity of synaptic terminals. Reducing miniature events results in premature disintegration of synaptic terminals in young animals while increasing these events in older animals can not only preserve terminal morphology, but in addition retard the age-dependent decline of motor ability. Simultaneous inhibition of evoked release does not either further increase terminal degeneration when miniature neurotransmission is inhibited or limit the benefits to terminal structural integrity when miniature events are increased during ageing, indicating a unique and singular requirement for miniature events. It is speculated that miniature release may be uniquely suited for processes necessary for the long-term stability of synapses as these events are produced continuously, in contrast to the stochastic and intermittent nature of evoked release. The finding of the necessity of miniature neurotransmission for the long-term structural stability of synapses extends upon the ongoing re-evaluation of the functional importance of miniature events, in particular for synapse and circuit development (Banerjee, 2021).

While the data support a unique role for miniature neurotransmission in maintaining synaptic structural integrity, it remains to be determined how small amplitude miniature events can be discriminated from much larger evoked release to elicit their unique properties. In developing synaptic terminals, it was previously shown that the effects of postsynaptic depletion of miniature neurotransmission on presynaptic bouton maturation required the ionotropic activity of glutamate receptors. Miniature events could potentially activate spatially distinct subpopulations of postsynaptic receptors to those activated by evoked neurotransmitter release to trigger differentiating signalling cascades. In support of this, postsynaptic functional imaging with active zone resolution of Drosophila larval synaptic terminals has shown that the probability of synaptic vesicle release during evoked or miniature neurotransmission can be spatially segregated to distinct active zones. Similar observations have been made in mammalian neurons. Another possibility is that differences in the release kinetics between evoked and miniature neurotransmission could allow postsynaptic mechanisms to detect and discriminate miniature events. For example, differential activation of Calmodulin can distinguish between local or global Ca2+ signaling acting through voltage-gated Ca2+ channels. The ability to selectively inhibit either miniature or evoked neurotransmission in the presynapse, as described in this study, should enable further interrogation of these potential mechanisms (Banerjee, 2021).

Also described in this study is a Drosophila abdominal neuromuscular synapse preparation suitable to investigate mature adult synapses throughout their lifespan. Similar to intensively studied larval abdominal neuromuscular synapses, adult A2 musculi ventralis interni mediales (mvim) abdominal muscles also have both tonic and phasic innervation, though only the tonic terminal producing motor neurons expresses the HB9 transcription factor. The restriction of HB9 (and HB9 > Gal4) expression to a small subset of adult motor neurons, enables experiments, such as inhibition of neurotransmission throughout ageing as was carried out in this study, without compromising overall animal behaviour or lifespan. A voltage clamp electrophysiological approach was developed for this muscle enabling direct measurement of synaptic currents and thus accurate quantification of neurotransmission properties, particularly important during ageing where potential confounding alterations of membrane potential and electrical resistance have been documented. In this preparation, no age-dependent increase was found in quantal content or 'set-point' as has been described at Drosophila proboscis NMJ terminals. Rather a progressive age-dependent decrease was observed in evoked neurotransmission (in addition to miniature event frequency), quantal content and a reduction in the size of the ready realisable pool of synaptic vesicles. These observations at this terminal are consistent with the age-dependent decline of neurotransmission described in other Drosophila, invertebrate and vertebrate synapses (Banerjee, 2021).

As this study described in Drosophila and as others have observed in mammals, miniature events decline as animals age. Alterations of miniature neurotransmission have also been reported in the context of several neurodevelopmental, neurodegenerative and psychiatric diseases. The results suggest that, in addition to age-dependent synaptic decline, alterations of miniature events should be investigated further for a potential causal role in synaptic structural changes associated with brain disorders (Banerjee, 2021).

Glycosphingolipids are linked to elevated neurotransmission and neurodegeneration in a Drosophila model of Niemann Pick type C

The lipid storage disease Niemann Pick type C (NPC) causes neurodegeneration owing primarily to loss of NPC1. This study employed a Drosophila model to test links between glycosphingolipids, neurotransmission and neurodegeneration. Npc1a nulls had elevated neurotransmission at the glutamatergic neuromuscular junction (NMJ), which was phenocopied in brainiac (brn) mutants, impairing mannosyl glucosylceramide (MacCer) glycosylation. Npc1a; brn double mutants had the same elevated synaptic transmission, suggesting that Npc1a and brn function within the same pathway. Glucosylceramide (GlcCer) synthase inhibition with miglustat prevented elevated neurotransmission in Npc1a and brn mutants, further suggesting epistasis. Synaptic MacCer did not accumulate in the NPC model, but GlcCer levels were increased, suggesting that GlcCer is responsible for the elevated synaptic transmission. Null Npc1a mutants had heightened neurodegeneration, but no significant motor neuron or glial cell death, indicating that dying cells are interneurons and that elevated neurotransmission precedes neurodegeneration. Glycosphingolipid synthesis mutants also had greatly heightened neurodegeneration, with similar neurodegeneration in Npc1a; brn double mutants, again suggesting that Npc1a and brn function in the same pathway. These findings indicate causal links between glycosphingolipid-dependent neurotransmission and neurodegeneration in this NPC disease model (Eberwein, 2023).

Rab11 rescues muscle degeneration and synaptic morphology in the park(13)/+ Parkinson model of Drosophila melanogaster

Mutation in parkin and pink1 is associated with Parkinson's disease (PD), the most common movement disorder characterized by muscular dysfunction. In a previous study, it was observed that Rab11, a member of the small Ras GTPase family, regulates the mitophagy pathway mediated by Parkin and Pink1 in the larval brain of the Drosophila PD model. sThis study describes that the expression and interaction of Rab11 in the PD model of Drosophila is highly conserved across different phylogenic groups. The loss of function in these two proteins, i.e., Parkin and Pink1, leads to mitochondrial aggregation. Rab11 loss of function results in muscle degeneration, movement disorder and synaptic morphological defects. Overexpression of Rab11 in park¹³ heterozygous mutant improves muscle and synaptic organization by reducing mitochondrial aggregations and improving cytoskeleton structural organization. The functional relationship was also shown between Rab11 and Brp, a pre-synaptic scaffolding protein, required for synaptic neurotransmission. Using park¹³ heterozygous mutant and pink1RNAi lines, this study showed reduced expression of Brp and consequently, there were synaptic dysfunctions including impaired synaptic transmission, decreased bouton size, increase in the bouton numbers, and the length of axonal innervations at the larval neuromuscular junction (NMJ). These synaptic alterations were rescued with the over-expression of Rab11 in the park¹³ heterozygous mutants. In conclusion, this work emphasizes the importance of Rab11 in rescuing muscle degeneration, movement dysfunction and synaptic morphology by preserving mitochondrial function in the PD model of Drosophila (Rai, 2023).

Analysis of asymptomatic Drosophila models for ALS and SMA reveals convergent impact on functional protein complexes linked to neuro-muscular degeneration

Spinal Muscular Atrophy (SMA) and Amyotrophic Lateral Sclerosis (ALS) share phenotypic and molecular commonalities, including the fact that they can be caused by mutations in ubiquitous proteins involved in RNA metabolism, namely SMN, TDP-43 (TBPH) and FUS. There is currently no model to explain the resulting motor neuron dysfunction. This study generated parallel set of Drosophila models for adult-onset RNAi and tagged neuronal expression of the fly orthologues of the three human proteins, named Smn, TBPH and Caz, respectively. To unravel the mechanisms underlying the common functional impact of these proteins on neuronal cells, a computational approach was devised based on the construction of a tissue-specific library of protein functional modules, selected by an overall impact score measuring the estimated extent of perturbation caused by each gene knockdown. Transcriptome analysis revealed that the three proteins do not bind to the same RNA molecules and that only a limited set of functionally unrelated transcripts is commonly affected by their knock-down. However, through the integrative approach it was possible to identify a concerted effect on protein functional modules, albeit acting through distinct targets. Most strikingly, functional annotation revealed that these modules are involved in critical cellular pathways for motor neurons, including neuromuscular junction function. Furthermore, selected modules were found to be significantly enriched in orthologues of human neuronal disease genes. These results show that SMA and ALS disease-associated genes linked to RNA metabolism functionally converge on neuronal protein complexes, providing a new hypothesis to explain the common motor neuron phenotype. The functional modules identified represent promising biomarkers and therapeutic targets, namely given their alteration in asymptomatic settings (Garcia-Vaquero, 2023)

Dysregulated glial genes in Alzheimer's disease are essential for homeostatic plasticity: Evidence from integrative epigenetic and single cell analyses

Synaptic homeostatic plasticity is a foundational regulatory mechanism that maintains the stability of synaptic and neural functions within the nervous system. Impairment of homeostatic regulation has been linked to synapse destabilization during the progression of Alzheimer's disease (AD). Various glial cell types play critical roles in modulating synaptic functions both during the aging process and in the context of AD. This study investigated the impact of glial dysregulation of histone acetylation and transcriptome in AD on synaptic homeostatic plasticity, using computational analysis combined with electrophysiological methods in Drosophila. By integrating snRNA-seq and H3K9ac ChIP-seq data from the same AD patient cohort, this study pinpointed cell type-specific signature genes that were transcriptionally altered by histone acetylation. The role of these glial genes in regulating presynaptic homeostatic potentiation was subsequently investigated in Drosophila. Remarkably, nine glial-specific genes, which were identified through computational methods as targets of H3K9ac and transcriptional dysregulation, were found to be crucial for the regulation of synaptic homeostatic plasticity at the NMJ in Drosophila. This genetic evidence connects abnormal glial transcriptomic changes in AD with the impairment of homeostatic plasticity in the nervous system. In summary, these integrative computational and genetic studies highlight specific glial genes as potential key players in the homeostatic imbalance observed in AD (Cai, 2023)

ALS-Associated KIF5A Mutation Causes Locomotor Deficits Associated with Cytoplasmic Inclusions, Alterations of Neuromuscular Junctions, and Motor Neuron Loss

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting motor neurons. Recently, genome-wide association studies identified KIF5A as a new ALS-causing gene. KIF5A encodes a protein of the kinesin-1 family, allowing the anterograde transport of cargos along the microtubule rails in neurons. In ALS patients, mutations in the KIF5A gene induce exon 27 skipping, resulting in a mutated protein with a new C-terminal region (KIF5A Δ27). To understand how KIF5A Δ27 underpins the disease, this study developed an ALS-associated KIF5A Drosophila model. When selectively expressed in motor neurons, KIF5A Δ27 alters larval locomotion as well as morphology and synaptic transmission at neuromuscular junctions in both males and females. The distribution of mitochondria and synaptic vesicles was found to be profoundly disturbed by KIF5A Δ27 expression. That is consistent with the numerous KIF5A Δ27-containing inclusions observed in motor neuron soma and axons. Moreover, KIF5A Δ27 expression leads to motor neuron death and reduces life expectancy. This in vivo model reveals that a toxic gain of function underlies the pathogenicity of ALS-linked KIF5A mutant (Soustelle, 2023).

Disrupted endoplasmic reticulum-mediated autophagosomal biogenesis in a Drosophila model of C9-ALS-FTD

Macroautophagy/autophagy is a major pathway for the clearance of protein aggregates and damaged organelles, and multiple intracellular organelles participate in the process of autophagy, from autophagosome formation to maturation and degradation. Dysregulation of the autophagy pathway has been implicated in the pathogenesis of neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), however the mechanisms underlying autophagy impairment in these diseases are incompletely understood. Since the expansion of GGGGCC (G4C2) repeats in the first intron of the C9orf72 gene is the most common inherited cause of both ALS and FTD (C9-ALS-FTD), this study investigated autophagosome dynamics in Drosophila motor neurons expressing 30 G4C2 repeats (30 R). In vivo imaging demonstrates that expression of expanded G4C2 repeats markedly impairs biogenesis of autophagosomes at synaptic termini, whereas trafficking and maturation of axonal autophagosomes are unaffected. Motor neurons expressing 30 R display marked disruption in endoplasmic reticulum (ER) structure and dynamics in the soma, axons, and synapses. Disruption of ER morphology with mutations in Rtnl1 (Reticulon-like 1) or atl (atlastin) also impairs autophagosome formation in motor neurons, suggesting that ER integrity is critical for autophagosome formation. Furthermore, live imaging demonstrates that autophagosomes are generated from dynamic ER tubules at synaptic boutons, and this process fails to occur in a C9-ALS-FTD model. Together, these findings suggest that dynamic ER tubules are required for formation of autophagosomes at the neuromuscular junction, and that this process is disrupted by expanded G4C2 repeats that cause ALS-FTD (Sung, 2024).

Reduced levels of ALS gene DCTN1 induce motor defects in Drosophila

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neuromuscular disease that has a strong genetic component. Deleterious variants in the DCTN1 gene are known to be a cause of ALS in diverse populations. DCTN1 encodes the p150 subunit of the molecular motor dynactin which is a key player in the bidirectional transport of cargos within cells. Whether DCTN1 mutations lead to the disease through either a gain or loss of function mechanism remains unresolved. Moreover, the contribution of non-neuronal cell types, especially muscle tissue, to ALS phenotypes in DCTN1 carriers is unknown. This study shows that gene silencing of Dctn1, the Drosophila main orthologue of DCTN1, either in neurons or muscles is sufficient to cause climbing and flight defects in adult flies. Dred, a protein with high homology to Drosophila Dctn1 and human DCTN1 was identified, that on loss of function also leads to motoric impairments. A global reduction of Dctn1 induced a significant reduction in the mobility of larvae and neuromuscular junction (NMJ) deficits prior to death at the pupal stage. RNA-seq and transcriptome profiling revealed splicing alterations in genes required for synapse organisation and function, which may explain the observed motor dysfunction and synaptic defects downstream of Dctn1 ablation. These findings support the possibility that loss of DCTN1 function can lead to ALS and underscore an important requirement for DCTN1 in muscle in addition to neurons (Borg, 2023).

Loss of amyotrophic lateral sclerosis risk factor SCFD1 causes motor dysfunction in Drosophila

Amyotrophic lateral sclerosis (ALS) is a progressive neuromuscular disease mostly resulting from a complex interplay between genetic, environmental and lifestyle factors. Common genetic variants in the Sec1 Family Domain Containing 1 (SCFD1) gene have been associated with increased ALS risk in the most extensive genome-wide association study (GWAS). SCFD1 was also identified as a top-most significant expression Quantitative Trait Locus (eQTL) for ALS. Whether loss of SCFD1 function directly contributes to motor system dysfunction remains unresolved. This study shows that moderate gene silencing of Slh, the Drosophila orthologue of SCFD1, is sufficient to cause climbing and flight defects in adult flies. A more severe knockdown induced a significant reduction in larval mobility and profound neuromuscular junction (NMJ) deficits prior to death before metamorphosis. RNA-seq revealed downregulation of genes encoding chaperones that mediate protein folding downstream of Slh ablation. These findings support the notion that loss of SCFD1 function is a meaningful contributor to ALS and disease predisposition may result from erosion of the mechanisms protecting against misfolding and protein aggregation (Borg, 2023).

Parkinsonism mutations in DNAJC6 cause lipid defects and neurodegeneration that are rescued by Synj1

Recent evidence links dysfunctional lipid metabolism to the pathogenesis of Parkinson's disease, but the mechanisms are not resolved. This study generated a new Drosophila knock-in model of DNAJC6/Auxilin and found that the pathogenic mutation causes synaptic dysfunction, neurological defects and neurodegeneration, as well as specific lipid metabolism alterations. In these mutants, membrane lipids containing long-chain polyunsaturated fatty acids, including phosphatidylinositol lipid species that are key for synaptic vesicle recycling and organelle function, are reduced. Overexpression of another protein mutated in Parkinson's disease, Synaptojanin-1, known to bind and metabolize specific phosphoinositides, rescues the DNAJC6/Auxilin lipid alterations, the neuronal function defects and neurodegeneration. This work reveals a functional relation between two proteins mutated in Parkinsonism and implicates deregulated phosphoinositide metabolism in the maintenance of neuronal integrity and neuronal survival (Jacquemyn, 2023).

Identification of atlastin genetic modifiers in a model of hereditary spastic paraplegia in Drosophila

Hereditary spastic paraplegias (HSPs) are a group of neurodegenerative disorders characterized by progressive dysfunction of corticospinal motor neurons. Mutations in Atlastin1/Spg3, a small GTPase required for membrane fusion in the endoplasmic reticulum, are responsible for 10% of HSPs. Patients with the same Atlastin1/Spg3 mutation present high variability in age at onset and severity, suggesting a fundamental role of the environment and genetic background. This study used a Drosophila model of HSPs to identify genetic modifiers of decreased locomotion associated with atlastin knockdown in motor neurons. First, a screen was performed for genomic regions that modify the climbing performance or viability of flies expressing atl RNAi in motor neurons. 364 deficiencies spanning chromosomes two and three were tested; 35 enhancer and four suppressor regions of the climbing phenotype were found. Candidate genomic regions could also rescue atlastin effects at synapse morphology, suggesting a role in developing or maintaining the neuromuscular junction. Motor neuron-specific knockdown of 84 genes spanning candidate regions of the second chromosome identified 48 genes required for climbing behavior in motor neurons and 7 for viability, mapping to 11 modifier regions. atl was found to interact genetically with Su(z)2, a component of the Polycomb repressive complex 1, suggesting that epigenetic regulation plays a role in the variability of HSP-like phenotypes caused by atl alleles. These results identify new candidate genes and epigenetic regulation as a mechanism modifying neuronal atl pathogenic phenotypes, providing new targets for clinical studies (Candia, 2023).

Computational modeling predicts ephemeral acidic microdomains followed by prolonged alkalinization in the glutamatergic synaptic cleft

At chemical synapses, synaptic vesicles release their acidic contents into the cleft leading to the expectation that the cleft should acidify. However, fluorescent pH probes targeted to the cleft of conventional glutamatergic synapses in both fruit flies and mice reveal cleft alkalinization, rather than acidification. It is study, using a reaction-diffusion scheme, pH dynamics were modeled at the Drosophila meuromuscular junction as glutamate, adenosine triphosphate (ATP) and protons (H(+)) are released into the cleft. The model incorporates bicarbonate and phosphate buffering systems as well as plasma membrane calcium-ATPase (PMCA) activity and predicts substantial cleft acidification but only for fractions of a millisecond following neurotransmitter release. Thereafter, the cleft rapidly alkalinizes and remains alkaline for over 100 milliseconds, as the PMCA removes H(+) from the cleft in exchange for calcium ions (Ca(2+)) from adjacent pre- and postsynaptic compartments; thus recapitulating the empirical data. The extent of synaptic vesicle loading and time course of exocytosis has little influence on the magnitude of acidification. Phosphate, but not bicarbonate buffering is effective at suppressing the magnitude and time course of the acid spike, while both buffering systems are effective at suppressing cleft alkalinization. The small volume of the cleft levies a powerful influence on the magnitude of alkalinization and its time course. Structural features that open the cleft to adjacent spaces appear to be essential for alleviating the extent of pH transients accompanying neurotransmission (Feghhi, 2021).

Bisphenol A affects neurodevelopmental gene expression, cognitive function, and neuromuscular synaptic morphology in Drosophila melanogaster

Bisphenol A (BPA) is an environmentally prevalent endocrine disrupting chemical that can impact human health and may be an environmental risk factor for neurodevelopmental disorders. BPA has been associated with behavioral impairment in children and a variety of neurodevelopmental phenotypes in model organisms. This study used Drosophila melanogaster to explore the consequences of developmental BPA exposure on gene expression, cognitive function, and synapse development. Transcriptome analysis indicated neurodevelopmentally relevant genes were predominantly downregulated by BPA. Among the misregulated genes were those with roles in learning, memory, and synapse development, as well as orthologs of human genes associated with neurodevelopmental and neuropsychiatric disorders. To examine how gene expression data corresponded to behavioral and cellular phenotypes, a predator-response behavioral paradigm was used, and it was found that BPA disrupts visual perception. Further analysis using conditioned courtship suppression showed that BPA impairs associative learning. Finally, synapse morphology within the larval neuromuscular junction was examined; BPA significantly increased the number of axonal branches. Given that these findings align with studies of BPA in mammalian model organisms, this data indicates that BPA impairs neurodevelopmental pathways that are functionally conserved from invertebrates to mammals. Further, because Drosophila do not possess classic estrogen receptors or estrogen, this research suggests that BPA can impact neurodevelopment by molecular mechanisms distinct from its role as an estrogen mimic (Welch, 2022).

FM dye cycling at the synapse: Comparing high potassium depolarization, electrical and channelrhodopsin stimulation

FM dyes are used to study the synaptic vesicle (SV) cycle. These amphipathic probes have a hydrophilic head and hydrophobic tail, making them water-soluble with the ability to reversibly enter and exit membrane lipid bilayers. These styryl dyes are relatively non-fluorescent in aqueous medium, but insertion into the outer leaflet of the plasma membrane causes a >40X increase in fluorescence. In neuronal synapses, FM dyes are internalized during SV endocytosis, trafficked both within and between SV pools, and released with SV exocytosis, providing a powerful tool to visualize presynaptic stages of neurotransmission. A primary genetic model of glutamatergic synapse development and function is the Drosophila neuromuscular junction (NMJ), where FM dye imaging has been used extensively to quantify SV dynamics in a wide range of mutant conditions. The NMJ synaptic terminal is easily accessible, with a beautiful array of large synaptic boutons ideal for imaging applications. This study compared and contrastd the three ways to stimulate the Drosophila NMJ to drive activity-dependent FM1-43 dye uptake/release: 1) bath application of high [K(+)] to depolarize neuromuscular tissues, 2) suction electrode motor nerve stimulation to depolarize the presynaptic nerve terminal, and 3) targeted transgenic expression of channelrhodopsin variants for light-stimulated, spatial control of depolarization. Each of these methods has benefits and disadvantages for the study of genetic mutation effects on the SV cycle at the Drosophila NMJ. These advantages and disadvantages are discussed to assist the selection of the stimulation approach, together with the methodologies specific to each strategy. In addition to fluorescent imaging, FM dyes can be photoconverted to electron-dense signals visualized using transmission electron microscopy (TEM) to study SV cycle mechanisms at an ultrastructural level. Comparisons are provided of confocal and electron microscopy imaging from the different methods of Drosophila NMJ stimulation, to help guide the selection of future experimental paradigms (Kopke, 2018).

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