InteractiveFly: GeneBrief

bruchpilot: Biological Overview | References

BIOLOGICAL OVERVIEW

Neurotransmitters are released at presynaptic active zones (AZs). In Drosophila, monoclonal antibody (MAB) nc82 specifically labels AZs. Nc82 was used to identify Bruchpilot protein (Brp), a previously unknown AZ component. BRP shows homology to human AZ protein ELKS/CAST/ERC (Note: CAST stands for cytomatrix at the active zone associated structural protein), which binds RIM1 in a complex with Bassoon and Munc13-1. The C terminus of Brp displays structural similarities to multifunctional cytoskeletal proteins. During development, transcription of bruchpilot coincides with neuronal differentiation. Panneural reduction of Brp expression by RNAi constructs permits a first functional characterization of this large AZ protein: larvae show reduced evoked but normal spontaneous transmission at neuromuscular junctions. In adults, loss of T bars at active zones, absence of synaptic components in electroretinogram, locomotor inactivity, and unstable flight (hence 'bruchpilot' - German for crash pilot), were observed. It is proposed that Brp is critical for intact AZ structure and normal-evoked neurotransmitter release at chemical synapses of Drosophila (Wagh, 2007). Further studies show that in brp mutants Ca2+ channels are reduced in density, evoked vesicle release is depressed, and short-term plasticity is altered. Brp-like proteins seem to establish proximity between Ca2+ channels and vesicles to allow efficient transmitter release and patterned synaptic plasticity (Kittel, 2006)

Neurotransmitter release during chemical communication between nerve cells takes place at synaptic active zones, specific sites characterized ultrastructurally by presynaptic membrane thickenings decorated with synaptic vesicles and surrounded by additional synaptic vesicle accumulations. Often, electron-dense projections of various shapes (plaques, pyramids, T-shaped structures, ribbons) extend from the active zone into the presynaptic cytoplasm. Considerable efforts have been undertaken in recent years to identify and functionally characterize the protein components of these projections and the cytoskeletal matrix associated with the active zone (CAZ). This complex meshwork of proteins most likely constitutes an essential part of the molecular machinery mediating neurotransmitter release. The fine regulation of this process is believed to be central to nervous system operation including higher functions such as learning, memory, and cognition (Wagh, 2007)

In vertebrates, several components associated with the presynaptic active zone have been characterized. In addition to the general cytoskeletal proteins actin and spectrin, the large protein Bassoon (420 kDa) (tom Dieck, 1998; Shapira, 2003) is specifically found at the CAZ. This protein has been shown to be required for structural active zone formation and/or maintenance. Piccolo (530 kDa) (Fenster, 2000) contains several putative protein-protein interaction domains and together with Bassoon is assumed to organize components of the active zone, including Rab3-interacting molecule (RIM1), Munc-13, and the CAZ-associated structural protein (ELKS/CAST/ERC) (Wagh, 2007)

Vertebrate ELKS/CAST/ERC proteins were identified as AZ components by purifying synaptic densities from rat brain followed by electrophoresis and mass spectrometry (Ohtsuka, 2002) and, independently, in a yeast-two-hybrid screen of a rat-brain cDNA library as proteins interacting with the RIM1α PDZ domain (Wang, 2002). Several isoforms have been reported to be transcribed from two genes (Wang, 2002; Deguchi-Tawarada, 2004). Two isoforms (CAST1/ERC2 and CAST2α/ERC1b) are brain-specific and contain several coiled-coil domains as well as a C-terminal IWA motif essential for binding the PDZ domain of RIM1 (Ohtsuka, 2002). ELKS/CAST/ERCs form large oligomeric protein complexes with the other known proteins of the CAZ (Munc-13, RIM1, Piccolo, Bassoon) and are believed to be involved in the molecular organization of presynaptic active zones (Ko, 2003) where they regulate the release of neurotransmitter (Takao-Rikitsu, 2004; Wagh, 2007 and references therein).

Although most proteins shown to be relevant for structure and/or function of the vertebrate nervous system are conserved in invertebrates, apparently no Bassoon or Piccolo homologs are encoded in the Drosophila genome. This study identified the Drosophila bruchpilot gene (brp) coding for a protein (Brp) that contains an N-terminal domain with significant sequence homology to vertebrate ELKS/CAST/ERC and a large C-terminal domain rich in coiled-coil structures similar to several cytoskeletal proteins. The structure of this gene was analyzed; the Drosophila Brp protein localizes at the presynaptic active zone. By analyzing various transgenic RNAi lines, the effects were characterized of reduction of Brp expression on synaptic ultrastructure, as well as neurotransmitter release and observe behavioral defects, including unstable flight (hence bruchpilot, crash pilot, named after an old German movie about a pilot who always crashes his planes but survives). It is speculated that Brp might combine functions of ELKS/CAST/ERC and a cytoskeletal structural protein in a single polypeptide that is highly conserved among insects (Wagh, 2007).

Over the last years, some insight into assembly and molecular composition of vertebrate presynaptic active zones has been gained. Cytoskeletal elements like actin and spectrin, as well as large active-zone-specific proteins like Piccolo and Bassoon, seem to form a structural meshwork organizing various components of the active zone. The third coiled-coil domain of Bassoon contains a motif that is highly homologous to the corresponding region of Piccolo and binds in competition to Piccolo the fifth predicted coiled-coil domain of ELKSα/CAST1. This binding of ELKSα/CAST1 to Bassoon seems to be involved in neurotransmitter release (Takao-Rikitsu, 2004). ELKSα/CAST1 itself has been shown to bind RIM1 via the C-terminal PDZ binding motif IWA (Ohtsuka, 2002). RIM1 is a target of the Rab3A small G protein (Wang, 1997) and interacts with Munc13-1. Together with vesicular proteins, this complex might control the recruitment of vesicles and regulate their subsequent fusion with the presynaptic membrane. Recently, ELKS has been shown to function in insulin exocytosis of pancreatic β cells (Ohara-Imaizumi, 2005). Deletion of the elks gene in C. elegans neither relocates RIM nor produces an obvious structural or functional phenotype, although C. elegans ELKS (Deken, 2005) like its mammalian homologs binds to the PDZ domain of RIM (Wagh, 2007)

Although there is a wealth of information on the ultrastructure of insect synapses, the molecular composition of their synaptic active zones is almost completely unknown. This work shows that in Drosophila, a protein with homology to the ELKS/CAST/ERC protein family of vertebrates localizes at the presynaptic active zones of neuronal terminals. The rather uniform distribution of tiny nc82 stained spots in all adult and larval neuropil regions suggests that Brp is present at active zones of most if not all synapses of Drosophila. Thus, the Brp protein provides an entry point to study general active-zone formation and function in this species (Wagh, 2007)

The open-reading frame of the cDNA identified by RT-PCR corresponds in size to the protein recognized by MAB nc82. The fact that the prominent Northern blot signal is about 5.5 kb larger than the known cDNA could indicate that the brp mRNA abundantly expressed in heads likely contains a long 3'UTR. Long 3'UTRs are found in several brain mRNAs (e.g., elav, fne). Possibly, the gene contains alternative polyadenylation sites giving rise to an abundant 11 kb mRNA and a less-abundant mRNA that contains the 3'end of cDNA AT09405 but is not detected in the Northern blot. This hypothesis is supported by two RT-PCR experiments with independent primer pairs downstream of the 3'end of cDNA AT09405. The signal at 2.0 kb cannot be interpreted with present cDNA information. Both signals were identically reproduced in two independent head mRNA blots hybridized with probes specific for either CG12933 and CG30336 or CG30337, or containing the entire cDNA sequence. No difference was observed with these three probes. Transcripts containing ORF CG12932, on the other hand, apparently are not abundantly expressed in adult heads and may or may not belong to the brp transcription unit (Wagh, 2007)

The combined evidence from MS analysis, bacterial cDNA expression, ectopic expression of GFP-labeled Brp, and the RNAi experiments proves that Brp represents the active-zone protein recognized by MAB nc82. Analysis of the amino acid sequence of Drosophila Brp predicts two leucine zipper domains and a glutamine-rich C terminus but no transmembrane domains. High homologies among human ELKSα, C. elegans CAST, and Brp are found in three regions of the proteins. However, no PDZ interaction motif (IWA) for RIM interaction as seen in several mammalian ELKS/CAST isoforms and, interestingly, in the C. elegans homolog seems to be present in insect Brp. No Drosophila protein is detected by BLAST analysis with conserved C-terminal sequences of worm or human. However, for the large C terminus of Brp (1260 aa) significant sequence similarities to cytoskeletal proteins such as plectin, myosin heavy chain, and restin are observed, suggesting a possible cytoskeletal role or interaction of the C terminus of Brp (Wagh, 2007)

The expression of Brp is not restricted to the glutamatergic type I boutons of the NMJ, but Brp is present in active zones of presumably all synapses. Consistently, those layers in the visual neuropil that contain high levels of either choline acetyltransferase or GABA/glutamic acid decarboxylase do not show reduced nc82 staining, and nc82 staining is found in both glutamatergic and nonglutamatergic synaptic boutons of larval NMJs. It was furthermore demonstrated that normal Brp levels are required for normal synaptic transmission at histaminergic synapses (Wagh, 2007)

This study directly address the structural, physiological, and systemic function of Brp, a member of the ELKS/CASTl/ERC family. The ultrastructure of synaptic active zones in terminals of larval motorneurons and adult photoreceptors is impaired when Brp protein levels are severely reduced. The loss of T bars in the lamina is compatible with the hypothesis that Brp may be required for anchoring the T bars to the presynaptic membrane. The fact that similar frequencies of T bars is seen in wild-type photoreceptor terminals (16.8%) and of conspicuous membrane densities in brp-RNAi^C8-expressing terminals (19.5%) supports the proposition that these membrane thickenings may in fact represent presynaptic sites without typical T bars. However, it is unlikely that Brp is restricted to T bars because about 13%-37% of the active zones of type Ib boutons at NMJs of larval muscles 6/7 have no T bar, whereas in light microscopical preparations, only 3% of the active zones identified by their postsynaptic receptor fields contained no Brp label. Unfortunately, in these experiments the epitope recognized by MAB nc82 did not tolerate tissue fixation conditions required for immunoelectron microscopical analysis of T bars (Wagh, 2007)

Regarding a role of Brp in synaptic function, two RNAi lines with panneural expression were tested. Both showed a reduction in Brp protein levels and a decrease in evoked transmitter release as reflected by reduced EJC amplitude at larval NMJs, whereas miniature EJC amplitude and frequency were indistinguishable from wild-type controls (Wagh, 2007)

The genotype brp-RNAi^B12/elav-Gal4 caused embryonic lethality. When Brp was suppressed only in the eye, adult brp-RNAi^B12/gmr-Gal4 flies had a strong effect on eye development ('rough eye') and in some cases, in addition to the lack of ON/OFF transients, showed a much smaller ERG receptor potential than the other three lines. However, because this lethal and rough eye phenotype is observed only for a single RNAi line, it cannot be excluded that unspecific side effects might play a role. The description of the true loss-of-function phenotype will therefore have to await the generation of a genuine null mutant. A special advantage in this context is that Brp presumably is present at all synapses. Therefore, it is possible not only to asses the functionality of transgenic constructs at the electrophysiological level but also to score their effects at the behavioral level by genetically controlled, spatially, and/or temporally selective expression or suppression (Wagh, 2007)

In mammals ELKS/CAST/ERC isoforms apparently have both neuronal and nonneuronal roles. Drosophila Brp seems to correspond to the neuronal CAST isoforms, whereas the nonneuronal functions might be specific to vertebrates. Vesides the protein described here, the only published molecule localized specifically at Drosophila presynaptic active zones is the Ca²⁺ channel encoded by the cacophony gene. This channel seems to be responsible for providing the calcium trigger for evoked neurotransmitter release. The findings indicate, however, that the molecular structure of the presynaptic active zone might be more conserved between vertebrate and insect synapses than thought previously because of the lack of Piccolo and Bassoon homologs in insects. In the future, studying active-zone formation and function in Drosophila will be a valuable addition to similar studies in vertebrates, especially considering the powerful genetic tools available for Drosophila (Wagh, 2007)

Maturation of active zone assembly by Drosophila Bruchpilot

Synaptic vesicles fuse at active zone (AZ) membranes where Ca2+ channels are clustered and that are typically decorated by electron-dense projections. Recently, mutants of the Drosophila ERC/CAST family protein Bruchpilot (BRP) were shown to lack dense projections (T-bars) and to suffer from Ca2+ channel-clustering defects. This study used high resolution light microscopy, electron microscopy, and intravital imaging to analyze the function of BRP in AZ assembly. Consistent with truncated BRP variants forming shortened T-bars, BRP was identified as a direct T-bar component at the AZ center with its N terminus closer to the AZ membrane than its C terminus. In contrast, Drosophila Liprin-α, another AZ-organizing protein, precedes BRP during the assembly of newly forming AZs by several hours and surrounds the AZ center in few discrete punctae. BRP seems responsible for effectively clustering Ca2+ channels beneath the T-bar density late in a protracted AZ formation process, potentially through a direct molecular interaction with intracellular Ca2+ channel domains (Fouquet, 2009).

This study addressed whether BRP signals T-bar formation (without being a direct component of the T-bar) or whether the protein itself is an essential building block of this electron-dense structure. Evidence is provided that BRP is a direct T-bar component. Immuno-EM identifies the N terminus of BRP throughout the whole cross section of the T-bar, and genetic approaches show that a truncated BRP, lacking the C-terminal 30% of the protein's sequence, forms truncated T-bars. Immuno-EM and light microscopy consistently demonstrate that N- and C-terminal epitopes of BRP are segregated along an axis vertical to the AZ membrane and suggest that BRP is an elongated protein, which directly shapes the T-bar structure (Fouquet, 2009).

In brp^5.45 (predicted as aa 1-866), T-bars were not detected, whereas brp^1.3 (aa 1-1,389) formed T-bar-like structures, although fewer and smaller than normal. Moreover, the BRP^D1-3GFP construct (1-1,226) did not rescue T-bar assembly. Thus, domains between aa 1,226 and 1,390 of BRP may also be important for the formation of T-bars. Clearly, however, the assembly scheme for T-bars is expected to be controlled at several levels (e.g., by phosphorylation) and might involve further protein components. Nonetheless, it is highly likely that the C-terminal half of BRP plays a crucial role (Fouquet, 2009).

Since BRP represents an essential component of the electron-dense T-bar subcompartment at the AZ center, it might link Ca²⁺ channel-dependent release sites to the synaptic vesicle cycle. Interestingly, light and electron microscopic analysis has located CAST at mammalian synapses both with and without ribbons. Overall, this study is one of the first to genetically identify a component of an electron-dense synaptic specialization and thus paves the way for further genetic analyses of this subcellular structure (Fouquet, 2009).

The N terminus of BRP is found significantly closer to the AZ membrane than the C terminus, where it covers a confined area very similar to the area defined by the Cac^GFP epitope. Electron tomography of frog NMJs suggested that the cytoplasmic domains of Ca²⁺ channels, reminiscent of pegs, are concentrated directly beneath a component of an electron-dense AZ matrix resembling ribs. In addition, freeze-fracture EM identified membrane-associated particles at flesh fly AZs, which, as judged by their dimensions, might well be Ca²⁺ channels. Peg-like structures were observed beneath the T-bar pedestal. Similar to fly T-bars, the frog AZ matrix extends up to 75 nm into the presynaptic cytoplasm. Based on the amount of cytoplasmic Ca²⁺ channel protein it has been concluded that Ca²⁺ channels are likely to extend into parts of the ribs. Thus, physical interactions between cytoplasmic domains of Ca²⁺ channels and components of ribs/T-bars might well control the formation of Ca²⁺ channel clusters at the AZ membrane. However, a short N-terminal fragment of BRP (aa 1-320) expressed in the brp-null background was unable to localize to AZs efficiently and consistently failed to restore Cac clustering (unpublished data) (Fouquet, 2009).

The mean Ca²⁺ channel density at AZs is reduced in brp-null alleles. In vitro assays indicate that the N-terminal 20% of BRP can physically interact with the intracellular C terminus of Cacaphony (Cac). Notably, it was found that the GFP epitope at the very C terminus of Cac^GFP was closer to the AZ membrane than the N-terminal epitope of BRP. It is conceivable that parts of the Cac C terminus extend into the pedestal region of the T-bar cytomatrix to locally interact with the BRP N terminus. This interaction might play a role in clustering Ca²⁺ channels beneath the T-bar pedestal (Fouquet, 2009).

Clearly, additional work will be needed to identify the contributions of discrete protein interactions in the potentially complex AZ protein interaction scheme. This study should pave the way for a genetic analysis of spatial relationships and structural linkages within the AZ organization. Moreover, the current findings should integrate in the framework of mechanisms for Ca²⁺ channel trafficking, clustering, and functional modulation (Fouquet, 2009).

The imaging assays allowed a temporally resolved analysis of AZ assembly in vivo. BRP is a late player in AZ assembly, arriving hours after DLiprin-α and also clearly after the postsynaptic accumulation of DGluRIIA. Accumulation of Cac was late as well, although it slightly preceded the arrival of BRP, and impaired Cac clustering at AZs lacking BRP became apparent only from a certain synapse size onwards, form at sites distant from preexisting ones and grow to reach a mature, fixed size. Thus, the late, BRP-dependent formation of the T-bar seems to be required for maintaining high Ca²⁺ channel levels at maturing AZs but not for initializing Ca²⁺ channel clustering at newly forming sites. As the dominant fraction of neuromuscular AZs is mature at a given time point, the overall impression is that of a general clustering defect in brp mutants. In reverse, it will be of interest to further differentiate the molecular mechanisms governing early Ca²⁺ channel clustering. Pre- to postsynaptic communication via neurexin-neuroligin interactions might well contribute to this process. A further candidate involved in early Ca²⁺ channel clustering is the Fuseless protein, which was recently shown to be crucial for proper Cac localization at AZs (Fouquet, 2009).

In summary, during the developmental formation of Drosophila NMJ synapses, the emergence of a presynaptic dense body, which is involved in accumulating Ca²⁺ channels, appears to be a central aspect of synapse maturation. This is likely to confer mature release probability to individual AZs and contribute to matching pre- and postsynaptic assembly by regulating glutamate receptor composition. Whether similar mechanisms operate during synapse formation and maturation in mammals remains an open question (Fouquet, 2009).

This study concentrated on developmental synapse formation and maturation. The question arises whether similar mechanisms to those relevant for AZ maturation might control activity-dependent plasticity as well and whether maturation-dependent changes might be reversible at the level of individual synapses. Notably, experience-dependent, bidirectional changes in the size and number of T-bars (occurring within minutes) were implied at Drosophila photoreceptor synapses by ultrastructural means. Moreover, at the crayfish NMJ, multiple complex AZs with double-dense body architecture were produced after stimulation and were associated with higher release probability. In fact, a recent study has correlated the ribbon size of inner hair cell synapses with Ca²⁺ microdomain amplitudes. Thus, a detailed understanding of the AZ architecture might permit a prediction of functional properties of individual AZs (Fouquet, 2009).

A high affinity RIM-binding protein/Aplip1 interaction prevents the formation of ectopic axonal active zones

Synaptic vesicles (SVs) fuse at active zones (AZs) covered by a protein scaffold, at Drosophila synapses comprised of ELKS family member Bruchpilot (BRP) and RIM-binding protein (RBP). This study demonstrates axonal co-transport of BRP and RBP using intravital live imaging, with both proteins co-accumulating in axonal aggregates of several transport mutants. RBP, via its C-terminal Src-homology 3 (SH3) domains, binds Aplip1/JIP1, a transport adaptor involved in kinesin-dependent SV transport. RBP C-terminal SH3 domains were shown in atomic detail to bind a proline-rich (PxxP) motif of Aplip1/JIP1 with submicromolar affinity. Point mutating this PxxP motif provoked formation of ectopic AZ-like structures at axonal membranes. Direct interactions between AZ proteins and transport adaptors seem to provide complex avidity and shield synaptic interaction surfaces of pre-assembled scaffold protein transport complexes, thus, favouring physiological synaptic AZ assembly over premature assembly at axonal membranes (Siebert, 2015).

Large multi-domain scaffold proteins such as BRP/RBP are ultimately destined to form stable scaffolds, characterized by remarkable tenacity and a low turnover, likely due to stabilization by multiple homo- and heterotypic interactions simultaneously. How these large and 'sticky'; AZ scaffold components engage into axonal transport processes to ensure their 'safe'; arrival at the synaptic terminal remains to be addressed. This study found that the AZ scaffold protein RBP binds the transport adaptor Aplip1 using a 'classic'; PxxP/SH3 interaction. Notably, the same RBP SH3 domain (II and III) interaction surfaces are used for binding the synaptic AZ ligands of RBP, that is, RIM and the voltage gated Ca²⁺ channel, though with clearly lower affinity than for Aplip1. A point mutation which disrupts the Aplip1-RBP interaction provoked a 'premature'; capture of RBP and the co-transported BRP at the axonal membrane, thus forming ectopic but, concerning T-bar shape and BRP/RBP arrangement, WT-like AZ scaffolds. The Aplip1 orthologue Jip1 has been shown to homo-dimerize via interaction of its SH3 domain. Thus, the multiplicity of interactions, with Aplip1 dimers binding to two SH3 domains of RBP as well as to KLC, might form transport complexes of sufficient avidity to ensure tight adaptor–cargo interaction and prevent premature capture of the scaffold components (Siebert, 2015).

Intravital imaging experiments showed that within axons RBP and BRP are co-transport in shared complexes together with Aplip1, whereas, despite efforts, no any co-transport of other AZ scaffold components, that is, Syd-1 or Liprin-α with BRP/RBP, were detected. In addition, STED analysis of axonal aggregates in srpk79D mutants showed BRP/RBP in stoichiometric amounts, but also failed to detect other AZ scaffold components. Moreover, BRP and RBP co-aggregated in the axoplasm of several other transport mutants tested (acsl, unc-51, appl, unc-76), consistent with both proteins entering synaptic AZ assembly from a common transport complex. Of note, during AZ assembly at the NMJ, BRP incorporation is invariably delayed compared to the 'early assembly'; phase which is driven by the accumulation of Syd-1/Liprin-α scaffolds. As the early assembly phase is, per se, still reversible, the transport of 'stoichiometric RBP/BRP complexes'; delivering building blocks for the 'mature scaffold'; might drive AZ assembly into a mature, irreversible state, and seems mechanistically distinct from early scaffold assembly mechanisms (Siebert, 2015).

Previous work suggested that AZ scaffold components (Piccolo, Bassoon, Munc-13 and ELKS) in rodent neurons are transported to assembling synapses as 'preformed complexes';, so-called Piccolo-Bassoon-Transport Vesicles (PTVs). The PTVs are thought to be co-transported with SV precursors anterogradely mediated via a KHC(KIF5B)/Syntabuli/Syntaxin-1 complex and retrogradely via a direct interaction between Dynein light chain and Bassoon. Since their initial description, however, further investigations of PTVs have been hampered by the apparent relative scarcity of PTVs, and by the lack of genetic or biochemical options for specifically interfering with their transport or final incorporation into AZs (Siebert, 2015).

A direct interaction of Aplip1 and BRP was not detected although their common transport can be uncoupled from the presence of RBP. One possible explanation could be a direct interaction of Aplip1 to other AZ proteins that are co-transported together with BRP and RBP. It is interesting that the very C-terminus of BRP is essential for SV clustering around the BRP-based AZ cytomatrix. Thus, it is tempting to speculate that adaptor/transport complex binding might block premature AZ protein/SV interactions before AZ assembly, but further analysis will have to await more atomic details than were obtained for the RBP::Aplip1 interaction (Siebert, 2015).

The down-regulation of the motor protein KHC also provoked severe axonal co-accumulations of BRP and RBP but per se should leave the adaptor protein-AZ cargo interaction intact. In contrast to aplip1, the axonal aggregations in khc mutants adapted irregular shapes most of the time, likely not representing T-bar-like structures. Thus, the data suggest a mechanistic difference when comparing the consequences between eliminating adaptor cargo interactions with a direct impairment of motor functions. Still, it cannot be excluded that trafficking of AZ complexes naturally antagonizes their ability to assemble into T-bars (Siebert, 2015).

The idea that proteins/molecules are held in an inactive state till they reach their final target has been observed in many other cell types. For example, in the context of local translation control, mRNAs are shielded or hidden in messenger ribonucleoprotein particles during transport so that they are withheld from cellular processing events such as translation and degradation. Shielding is thought to operate through proteins that bind to the mRNA and alter its conformation while at the correct time or place the masking protein is influenced by a signal that alleviates its shielding effect. As another example, hydrolytic enzymes, for example, lysosomes, are transported as proteolytically inactive precursors that become matured by proteolytic processing only within late endosomes or lysosomes. Particularly relevant in the context of AZ proteins involved in exocytosis, the H_abc domain of Syntaxin-1 folds back on the central helix of the SNARE motif to generate a closed and inactive conformation which might prevent the interaction of Syntaxin-1 with other AZ proteins during diffusion (Siebert, 2015).

Previous genetic analysis of C. elegans axons forming en passant synapses suggested a tight balance between capture and dissociation of protein transport complexes to ensure proper positioning of presynaptic AZs. In this study, overexpression of the kinesin motor Unc-104/KIF1A reduced the capture rate and could suppress the premature axonal accumulations of AZ/SV proteins in mutants of the small, ARF-family G-protein Arl-8. Interestingly, large axonal accumulations in arl-8 mutants displayed a particularly high capture rate. Similarly, both aplip1 alleles exhibited enlarged axonal BRP/RBP accumulations. Thus, the capture/dissociation balance for AZ components might be shifted towards 'capture'; in these mutants, consistent with the ectopic axonal T-bar formation. It is tempting to speculate that loss of Aplip1-dependent scaffolding and/or kinesin binding provokes the exposure of critical 'sticky'; patches of scaffold components such as RBP and BRP. Such opening of interaction surfaces might increase 'premature'; interactions of cargo proteins actually destined for AZ assembly, thus increase overall size of the cargo complexes by oligomerization between AZ proteins and, finally, promote premature capture and ultimately ectopic AZ-like assembly. On the other hand, the need for the system to unload the AZ cargo at places of physiological assembly (i.e., presynaptic AZ) might pose a limit to the 'wrapping'; of AZ components and ask for a fine-tuned capture/dissociation balance (Siebert, 2015).

Several mechanisms for motor/cargo separation such as (1) conformational changes induced by guanosine-5′-triphosphate hydrolysis, (2) posttranslational modification as de/phosphorylation, or (3) acetylation affecting motor-tubulin affinity, have been suggested for cargo unloading. Notably, Aplip1 also functions as a scaffold for JNK pathway kinases, whose activity causes motor-cargo dissociation. JNK probably converges with a mitogen-activated protein kinase (MAPK) cascade (MAPK kinase kinase Wallenda phosphorylating MAPK kinase Hemipterous) in the phosphorylation of Aplip1, thereby dissociating Aplip1 from KLC. Thus, JNK signaling, co-ordinated by the Aplip1 scaffold, provides an attractive candidate mechanism for local unloading of SVs and, as shown in this study, AZ cargo at synaptic boutons. This study further emphasizes the role of the Aplip1 adaptor, whose direct scaffolding role through binding AZ proteins might well be integrated with upstream controls via JNK and MAP kinases. Intravital imaging in combination with genetics of newly assembling NMJ synapses should be ideally suited to further dissect the obviously delicate interplay between local cues mediating capturing and axonal transport with motor-cargo dissociation (Siebert, 2015).

Active zone scaffolds differentially accumulate Unc13 isoforms to tune Ca²⁺ channel-vesicle coupling

Brain function relies on fast and precisely timed synaptic vesicle (SV) release at active zones (AZs). Efficacy of SV release depends on distance from SV to Ca²⁺ channel, but molecular mechanisms controlling this are unknown. This study found that distances can be defined by targeting two unc-13 (Unc13) isoforms to presynaptic AZ subdomains. Super-resolution and intravital imaging of developing Drosophila melanogaster glutamatergic synapses revealed that the Unc13B isoform was recruited to nascent AZs by the scaffolding proteins RhoGAP100F/Syd-1 and Liprin-α, and Unc13A was positioned by Bruchpilot and Rim-binding protein complexes at maturing AZs. Unc13B localized 120 nm away from Ca²⁺ channels, whereas Unc13A localized only 70 nm away and was responsible for docking SVs at this distance. Unc13Anull mutants suffered from inefficient, delayed and EGTA-supersensitive release. Mathematical modeling suggested that synapses normally operate via two independent release pathways differentially positioned by either isoform. Isoform-specific Unc13-AZ scaffold interactions were identified, regulating SV-Ca²⁺-channel topology whose developmental tightening optimizes synaptic transmission (Bohme, 2016).

All presynaptic AZs accumulate scaffold proteins from a canonical set of few protein families, which are characterized by extended coiled-coil stretches, intrinsically unstructured regions and a few classical interaction domains, particularly PDZ and SH3 domains. These multidomain proteins collectively form a compact 'cytomatrix' often observable by electron-dense structures covering the AZ membrane, which have been found to physically contact SVs, and thus have been suggested to promote SV docking and priming as well as to recruit Ca²⁺ channels. Still, how the structural scaffold components (ELKS, RBP, RIM and Liprin-α) tune the functionality of the SV-release machinery has remained largely enigmatic. Liprin-α is crucial for the AZ assembly process and at Drosophila NMJ AZs, Liprin-α-Syd-1 cluster formation initializes the assembly of an 'early' scaffold complex, which subsequently guides the accumulation of a 'late' RBP-BRP scaffold complex. This study provides evidence that these scaffold complexes together operated as 'molecular rulers' that confer a remarkable degree of order, patterning AZ composition and function in space and time: the 'early' Liprin-α-Syd-1 clusters recruited Unc13B, and this scaffold served as a template to accumulate the 'late' BRP-RBP scaffold, which recruited Unc13A. Unc13 isoforms were precisely organized in the tens of nanometers range, which the data suggest to be instrumental to control SV release probability and SV-Ca²⁺ channel coupling. As a molecular basis of this patterning and recruitment, a multitude of molecular contacts was identified between the Unc13 N termini and the respective scaffold components using systematic Y2H analysis. As one out of several interactions, this study identified a cognate PxxP motif in the N terminus of Unc13A to interact with the second and third SH3 domains of RBP. Point mutants within the PxxP motif interfered with the binding of the RBP-SH3 domains II and III on the Y2H level but did not have a major impact on Unc13A localization and function when introduced into an Unc13 genomic transgene. Nonetheless, elimination of the scaffold components BRP and RBP on the one hand or Liprin-α on the other hand drastically impaired the accumulation of Unc13A or Unc13B. It is suggested that these results are explained by a multitude of parallel interactions that provide the avidity needed to enrich the respective Unc13 isoforms in their specific 'niches' and may cause a functional redundancy among interaction motifs, as was likely observed in the case of the Unc13A PxxP motif. Future analysis will be needed to investigate these interaction surfaces in greater detail, and address how exactly 'early' and 'late' scaffolds coordinate AZ assembly (Bohme, 2016).

Unc13 proteins have well-established functions in SV docking and priming. Accordingly, it was observed that loss of Unc13A resulted in overall reduced SV docking without affecting T-bar-tethered SVs, which is qualitatively opposite to a function of BRP in SV localization, whose C-terminal amino acids function in T-bar-tethering, but not docking. Variants lacking these residues suffer from increased synaptic depression, suggesting a role in SV replenishment. Therefore, in addition to its role in localizing Unc13A to the AZ reported here, BRP may also cooperate functionally with Unc13A by facilitating SV delivery to docking sites (Bohme, 2016).

Synapses are highly adapted to their specific features, varying widely concerning their release efficacy and short-term plasticity. These features impact information transfer and may provide neurons with the ability to detect input coherence, maintain stability and promote synchronization. Differences in the biochemical milieu of SVs can tune priming efficacy and release probability, which largely affects short-term plasticity. In the current experiments, it was found that loss of Unc13A resulted in dramatically (~90%) reduced synaptic transmission, which exceeded the (~50%) reduction in SV docking, pointing to an additional function in enhancing release efficacy. These changes were paralleled by drastically increased short-term facilitation as well as EGTA hypersensitivity and could be due to decreased Ca²⁺ sensitivity of the molecular release machinery, for example, mediated by different Synaptotagmin-type Ca²⁺ sensors, or different numbers of SNARE complexes. However, although a rightward shift was observed of the dependence of normalized release amplitudes on extracellular Ca²⁺ concentration at Unc13A-deficient synapses, its slope and thus Ca²⁺ cooperativity was unaltered, arguing against fundamentally different Ca²⁺-sensing mechanisms. Instead a scenario is favored in which SV Ca²⁺ sensing is conserved, but local Ca²⁺ signals at SV positions are attenuated because of their larger distances to Ca²⁺ channels upon loss of Unc13A. Both Unc13 isoforms were clearly segregated physically with different distances to the Ca²⁺ channel cluster, and loss of Unc13A selectively reduced the number of docked SVs in the AZ center. These findings are best explained by Unc13A promoting the docking and priming of SVs closer to Ca²⁺ channels than Unc13B. In fact, mathematical modeling reproduced the data by merely assuming release from two independent pathways with identical Ca²⁺ sensing and fusion mechanisms that only differed in their physical distance to the Ca²⁺ source in the AZ center. The distances estimated by the model were in very good agreement with the positions of the two Unc13 isoforms defined by STED microscopy. Thus, the data suggest that differences in the distance of SVs in the tens of nanometer range to the Ca²⁺ channels mediated by the two Unc13 isoforms likely contributed profoundly to the observed phenotypes. It is proposed that the role of the N terminus is to differentially target the isoforms into specific zones of the AZ, while the conserved C terminus confers identical docking and priming functions at both locations. Notably, recent work in Caenorhabditis elegans also characterized two Unc13 isoforms, with fast release being mediated by UNC-13L, whereas slow release required both UNC-13L and UNC-13S44. The proximity of the UNC-13L isoform to Ca²⁺ entry sites was mediated by the protein's N-terminal C2A-domain (not present in Drosophila) and was critical for accelerating neurotransmitter release, and for increasing/maintaining the probability of evoked release assayed by the fraction of AP- to sucrose-induced release. In contrast, the slow SV release form dominantly localized outside AZ regions. Thus it would be interesting to investigate the sub-AZ distribution of C. elegans Unc-13 isoforms and test whether the same scaffold complexes as in Drosophila mediate the localization of the different Unc-13 isoforms (Bohme, 2016).

Notable differences in short-term plasticity have been reported for mammalian Unc13 isoforms. The mammalian genome harbors five Munc13 genes. Of those, Munc13-1, -2 and -3 are expressed in the brain, and function in SV release; differential expression of Munc13 isoforms at individual synapses may represent a mechanism to control short-term plasticity. Thus, it might be warranted to analyze whether differences in the sub-active zone distribution of Munc13 isoforms contribute to these aspects of synapse diversity in the rodent brain (Bohme, 2016).

Fast and slow phases of release have recently been attributed to parallel release pathways operating in the calyx of Held of young rodents (56 nm and 135 nm) qualitatively matching the coexistence of two differentially positioned release pathways described in this study. The finding of discretely localized release pathways with distances larger than 60 nm is further in line with the recent suggestion that, at some synapses, SVs need to be positioned outside an 'exclusion zone' from the Ca²⁺ source (~50 nm distance to the center of the SV for the calyx of Held). At mammalian synapses, developmental changes in the coupling of SVs and Ca²⁺ channels have been described, which qualitatively matches the sequential arrival of loosely and tightly coupled Unc13B and Unc13A isoforms during synaptogenesis described here. Thus, this work suggests that differential positioning of Unc13 isoforms couples functional and structural maturation of AZs. To what degree modulation of this process contributes to the functional diversification of synapses is an interesting subject of future analysis (Bohme, 2016).

Levels of Par-1 kinase determine the localization of Bruchpilot at the Drosophila neuromuscular junction synapses

Functional synaptic networks are compromised in many neurodevelopmental and neurodegenerative diseases. While the mechanisms of axonal transport and localization of synaptic vesicles and mitochondria are relatively well studied, little is known about the mechanisms that regulate the localization of proteins that localize to active zones. Recent finding suggests that mechanisms involved in transporting proteins destined to active zones are distinct from those that transport synaptic vesicles or mitochondria. This study reports that localization of BRP-an essential active zone scaffolding protein in Drosophila, depends on the precise balance of neuronal Par-1 kinase. Disruption of Par-1 levels leads to excess accumulation of BRP in axons at the expense of BRP at active zones. Temporal analyses demonstrate that accumulation of BRP within axons precedes the loss of synaptic function and its depletion from the active zones. Mechanistically, it was found that Par-1 co-localizes with BRP and is present in the same molecular complex, raising the possibility of a novel mechanism for selective localization of BRP-like active zone scaffolding proteins. Taken together, these data suggest an intriguing possibility that mislocalization of active zone proteins like BRP might be one of the earliest signs of synapse perturbation and perhaps, synaptic networks that precede many neurological disorders (Barber, 2018).

Par-1 is an evolutionarily conserved serine threonine kinase that has many diverse roles, including important roles in regulating cell polarity and regulating microtubule stability. Genome-wide association studies have implicated Par-1 (MARK) in Alzheimer's disease (AD). While accumulations of Aβ and tau are implicated in the widespread neuronal death found in late stages of AD, synapse instability is often associated with early stages during the progression of AD. Indeed, animal models of tauopathy show an increase in synapse instability. Therefore, it is proposed that synapse instability might be one of the early events in neurodegenerative diseases like AD and that increase in Par-1/ MARK4 could facilitate the instability and hasten the demise of synapses (Barber, 2018).

Synaptic plasticity is determined by its ability to modulate its response to stimulation. Generally, activity leads to strengthening of synapses, which is bigger response to stimulation. Therefore, maintenance of synapses is important in maintaining the synaptic networks, which are disrupted in both neurodevelopmental and neurodegenerative diseases. Indeed, mutations in cysteine string protein (CSP), which plays an important role in synaptic maintenance, causes a progressive motor neuron disorder characterized by neurodegeneration. Thus, maintaining stable synapses might be important to avoid the failure of synaptic networks (Barber, 2018).

At the Drosophila NMJ synapses, active zones can be rapidly modified to induce synaptic homeostatic changes, which are partly dependent on BRP. Interestingly, in a Drosophila model of ALS, disruption of shape and size of T-bars, which consists primarily of BRP, precedes synapse degeneration. These data suggest that disruption of T-bars might be an early marker for synapse breakdown. The current data support this hypothesis because it was found that the doughnut shape of T-bars is dramatically altered in flies overexpressing Par-1 and this happens before the decrease in the number of AZs marked by BRP. Finally, it is posited that loss of BRP from synapses could lead to a failure of synaptic homeostasis because BRP plays an important role in synaptic vesicle release. Interestingly, loss of synaptic homeostasis has been implicated in early phases of neurodegeneration and, restoring synaptic homeostasis can restore synaptic strength in a Drosophila model of ALS. Thus, gradual loss of BRP from synapse may impair the ability of a synapse to efficaciously respond to changes that perturb synaptic homeostasis leading to catastrophic failure of neural networks (Barber, 2018).

One of the vital functions performed by axonal transport is to maintain steady state levels of synaptic proteins required for the efficacious release of neurotransmitter release. Disruption of axonal transport has been implicated in neurodegenerative diseases. Indeed, mutations that affect axonal transport lead to neurodegenerative diseases. A recent study suggests that active zone density is maintained during the developmental stages but is significantly decreased with aging. Interestingly, axonal transport also declines with aging suggesting that a combination of decreased axonal transport of active zone proteins along with aging may lead to a gradual decrease in the maintenance of active zones. This may eventually lead to a failure to maintain synaptic function and ultimately lead to synapse degeneration. While this hypothesis is generally accepted, it has proven difficult to determine whether axonal transport is a cause or consequence of synapse loss. Temporal analysis suggests that following sequence of events: Par-1 localizes to the axons followed by BRP accumulation in axons likely leading to the decreased synaptic function and finally the reduction of BRP from synaptic active zones likely leading to synapse instability. Together, these findings support the hypothesis that defects in axonal transport cause synapse degeneration (Barber, 2018).

While so far it is not precisely understandood how active zone scaffold proteins like BRP are localized, based on the present study, it is speculated that phosphorylation of Par-1 substrate may be important in determining the localization of BRP. This is because while the expression of WT Par-1 causes accumulation of BRP within axons, expression of inactive Par-1 does not lead to show any aberrant localization of BRP. The data suggest that defects in BRP localization are not mediated either by tau or Futsch but BRP may be a possible substrate of Par-1. This is because the data indicate that BRP and Par-1 may be in the same molecular complex. However, it remains to be determined whether Par-1 can phosphorylate BRP and whether phosphorylation of BRP is required for its localization. Previous studies have shown that BRP can be acetylated, and that this posttranslational modification is important in regulating the structure of T-bars but whether BRP can be phosphorylated remains to be studied. Finally, the data indicate that presynaptic Par-1 levels are important in determining BRP localization because Par-1 knockdown also results in the accumulation of BRP within the axons. Thus, Par-1 not only has an important role in postsynaptic compartment but also has an important function on the presynaptic side. Finally, it should be noted that this study is a limited but an important extension of a previous study of how Par-1 regulates the localization of important active zone proteins such as BRP. This study also opens up a lot of questions. For example, what is the half life of BRP at the AZs? Does BRP get replaced? If so, at what rate? These are some important questions that should be addressed by future studies but this study opens up the possibility to study these processes in much more detail (Barber, 2018).

Drosophila Syd-1 has RhoGAP activity that is required for presynaptic clustering of Bruchpilot/ELKS but not Neurexin-1

Syd-1 proteins are required for presynaptic development in worm, fly, and mouse. Syd-1 proteins in all three species contain a Rho GTPase activating protein (GAP)-like domain of unclear significance: invertebrate Syd-1s are thought to lack GAP activity, and mouse mSYD1A has GAP activity that is thought to be dispensable for its function. This study shows that Drosophila melanogaster Syd-1 can interact with all six fly Rhos and has GAP activity toward Rac1 and Cdc42. During development, fly Syd-1 clusters multiple presynaptic proteins at the neuromuscular junction (NMJ), including the cell adhesion molecule Neurexin (Nrx-1) and the active zone (AZ) component Bruchpilot (Brp), both of which Syd-1 binds directly. A mutant form of Syd-1 that specifically lacks GAP activity localizes normally to presynaptic sites and is sufficient to recruit Nrx-1 but fails to cluster Brp normally. Evidence is provided that Syd-1 participates with Rac1 in two separate functions: (1) together with the Rac guanine exchange factor (RacGEF) Trio, GAP-active Syd-1 is required to regulate the nucleotide-bound state of Rac1, thereby promoting Brp clustering; and (2) Syd-1, independent of its GAP activity, is required for the recruitment of Nrx-1 to boutons, including the recruitment of Nrx-1 that is promoted by GTP-bound Rac1. It is concluded that, contrary to current models, the GAP domain of fly Syd-1 is active and required for presynaptic development; it is suggested that the same may be true of vertebrate Syd-1 proteins. In addition, the data provide new molecular insight into the ability of Rac1 to promote presynaptic development (Spinner, 2018).

This paper has shown that Syd-1^wt and Syd-1^RA promote NMJ growth to similar degrees in wild-type animals and recruit similar levels of Nrx-1 to presynaptic boutons in both wild type and syd-1 mutants. However, unlike Syd-1^wt, Syd-1^RA is unable to restore Brp clustering in syd-1 mutants, indicating that Syd-1's GAP activity is required for this process. The results further suggest that Syd-1's GAP activity promotes Brp clustering by working together with the RacGEF Trio to potentiate Rac1. However, while loss of a GEF for Rac1 would be predicted to increase the proportion of GDP-bound, and therefore inactive, Rac1, it is noted that loss of a GAP for Rac1 should instead increase the proportion of GTP-bound, and therefore active, Rac1. Why, instead, might loss of Syd-1 GAP activity impair Rac1 function at NMJ (Spinner, 2018)?

Given the finding that Syd-1's GAP domain also increases the GTPase activity of Cdc42, the possibility is considered that Syd-1 might potentiate Rac1 only indirectly, by acting upon Cdc42. The phenotype caused by Cdc42 loss from motorneurons (an increase in NMJ bouton number) is the same as that caused by Rac1 gain, suggesting that Cdc42 and Rac1 might antagonize one another during NMJ development. Syd-1 loss might then impair Rac1 by increasing the proportion of GTP-bound, active Cdc42. However, this model is unlikely for three reasons. First, presynaptic loss of cdc42 significantly enhances the formation of abnormally positioned 'satellite' boutons, a hallmark of increased BMP signaling, and no increase is observed in satellite boutons in animals overexpressing Syd-1^wt (or in syd-1 mutants), suggesting that Syd-1 does not normally regulate Cdc42 at NMJ. Second, whether coexpressing Cdc42 with Rac1 would impair the latter's ability to increase NMJ bouton number was directly tested, and it does not. Third, a model in which decreasing syd-1 dosage potentiates a Rho GTPase with antagonistic effects on Rac1 does not explain the specific sensitivity of Rac1^wt and not Rac1^V12 to this manipulation. Finally, it is noted that Syd-1 could also theoretically potentiate Rac1 indirectly by acting upon one of the other two fly Racs, if either of the latter were antagonistic to Rac1. However, there is no evidence of such antagonism: reducing the levels of one, two, or all three fly Racs has previously been shown either to have no effect or to decrease NMJ bouton number, and the third point (above) applies to this model too (Spinner, 2018).

The data are therefore more consistent with Syd-1 directly regulating the nucleotide-bound state of Rac1. In previous cases in which loss of the RhoGAP or RhoGEF for a single Rho GTPase causes the same defect, the explanation has been that the ability of the GTPase to cycle between GDP- and GTP-bound states is critical; loss of the regulatory RhoGAP or RhoGEF then causes the same defect because either manipulation reduces the rate of Rho cycling. While it is theoretically possible that, instead, both GDP-bound and GTP-bound forms of Rac1 are separately required to promote Brp clustering at NMJ, there is no precedent for such a model: GDP-bound Rac1 is thought to be inactive. Therefore a model is favored in which Rac1 cycling is important for Brp clustering at NMJ (Spinner, 2018).

How might Rac1 cycling affect Brp recruitment? Brp has previously been shown to directly bind in vitro to N- and C-terminal fragments of Syd-1 that lack the RhoGAP domain. One possibility is that the ability of Syd-1 to bind Brp in vivo is somehow regulated intramolecularly by the process of transforming a GTP-bound Rho GTPase into a GDP-bound form (simply binding the GTP-bound form would not be sufficient, since R979A Syd-1 binds all six Rhos yet cannot cluster Brp). Alternatively, Syd-1's RhoGAP activity may have an indirect effect on Brp by, in parallel, promoting a Rho GTPase-dependent change in presynaptic structure that facilitates the ability of Syd-1 to cluster Brp properly. Rho GTPases are classically involved in regulating actin assembly, and presynaptic development is characterized by the early appearance of actin-rich structures to which other molecules, including Syd-1, are recruited. Perhaps Rac1, regulated by Syd-1 and Trio, sculpts the local actin environment at presynaptic sites, creating a permissive environment for Syd-1 to recruit additional presynaptic components, including Brp (Spinner, 2018).

In contrast to the evidence that Rac1 cycling may be important for Brp clustering, this study found that Rac1^V12, which stably mimics the GTP-bound state, increases Nrx-1 levels in wild type, and that Syd-1 lacking GAP activity is sufficient to increase Nrx-1 levels, even in the absence of endogenous Syd-1. These results suggest that Rac1 does not need to enter the GDP-bound state in order to promote Nrx-1 recruitment and that Rac1 cycling is therefore not required for this process. Nonetheless, complete loss of syd-1 prevents Nrx-1 recruitment, even by Rac1^V12. Together, these results indicate that Syd-1 is required downstream of or in parallel to GTP-bound Rac1 to recruit Nrx-1 to boutons. Syd-1 and Nrx-1 have previously been shown to bind via an interaction between the former's PDZ domain and the latter's PDZ-binding domain; each protein depends on the other for its localization. One possibility is that Syd-1 localization or its ability to recruit Nrx-1 is potentiated by direct binding between Syd-1 and GTP-bound Rac1, an interaction of which Syd-1^RA remains capable (Spinner, 2018).

This study has shown that fly Syd-1 requires its GAP activity to cluster the ELKS protein Brp. However, worm Syd-1 apparently lacks GAP activity and yet is able to cluster ELKS. How might this work? Both worm and fly Syd-1 also recruit the scaffolding protein Liprin-alpha (Syd-2 in worm), which binds ELKS. Perhaps in worm this parallel mechanism of ELKS recruitment is sufficient for normal ELKS localization, alleviating the need for Rac cycling. Alternatively, some other RacGAP in worm might promote Rac cycling during synaptogenesis, creating a permissive environment for the GAP-dead worm Syd-1 to recruit ELKS (Spinner, 2018).

By contrast, mouse mSYD1A does have GAP activity toward RhoA, but this activity is not required for its ability to rescue the presynaptic defects caused by mSYD1A loss. An obvious possibility is that the molecular mechanisms that promote presynaptic assembly differ substantially between vertebrates and invertebrates. However, it is noted that mice have a second Syd-1 homolog, mSYD1B, which has not yet been analyzed but which may have assumed some of the functions that depend on the single Syd-1 in invertebrates. Consistent with this possibility, the presynaptic defects caused by mSYD1A loss are far milder than those of the invertebrate syd-1 mutants. Because fly Syd-1^RA can promote increased synaptogenesis in the presence of wild-type endogenous Syd-1, one possibility is that mouse mSYD1A lacking the RhoGAP domain can promote SV clustering in mSYD1A^KO knockout cells because wild-type mSYD1B is present. It will be interesting to examine the effects of deleting both mouse Syd-1 proteins and to test the functionality of mutant versions of those proteins in the double mutant animals. Fly Syd-1 was previously thought to lack RhoGAP activity in part because mouse mSYD1A GAP activity toward RhoA is eliminated by changing a single one of its residues into the corresponding fly residue. The current results confirm that fly Syd-1's RhoGAP domain does not have GAP activity toward RhoA, but instead it can regulate Rac1 and Cdc42. It will be interesting to see whether the GAP domain of mouse mSYD1B might share fly Syd-1's specificity (Spinner, 2018).

Nicotinamide mononucleotide adenylyltransferase maintains active zone structure by stabilizing Bruchpilot

Active zones are specialized presynaptic structures critical for neurotransmission. A neuronal maintenance factor, nicotinamide mononucleotide adenylyltransferase (NMNAT), is required for maintaining active zone structural integrity in Drosophila by interacting with the active zone protein, Bruchpilot (BRP), and shielding it from activity-induced ubiquitin-proteasome-mediated degradation. NMNAT localizes to the peri-active zone and interacts biochemically with BRP in an activity-dependent manner. Loss of NMNAT results in ubiquitination, mislocalization and aggregation of BRP, and subsequent active zone degeneration. It is proposed that, as a neuronal maintenance factor, NMNAT specifically maintains active zone structure by direct protein-protein interaction (Zang, 2013).

The findings of ubiquitinated, clustered and mislocalized BRP in loss-of-NMNAT neurons, and that increased activity leads to increased NMNAT-BRP interaction, together with the observation that active zone structure is maintained in nmnat-null neurons when neuronal activity is reduced, suggest the following model of the activity-dependent role of NMNAT in active zone maintenance. Under normal activity conditions, NMNAT is required to maintain active zone structure by interacting with BRP and to prevent the ubiquitination of BRP, inasmuch as loss of NMNAT results in BRP ubiquitination, mislocalization, aggregation and reduced active zone size. When neuronal activity is minimized (for example, by blocking light stimulation (dark rearing), or by blocking phototransduction (NorpA)), the demand on maintenance by NMNAT is reduced. These studies have revealed a specific role of NMNAT in regulating homeostasis of the active zone protein BRP. The role of NMNAT in protein-protein interaction is consistent with the chaperone function of NMNAT. Chaperones, such as CSP, have been implicated in maintaining synaptic integrity. Moreover, recent studies have shown that an elevated activity level poses stress to synaptic proteins by highlighting the effect of CSP in maintaining synaptic function. It is expected that increased neuronal and/or synaptic activity will lead to increased protein misfolding and turnover, and therefore to an increase in the load/demand of maintenance of synaptic protein homeostasis. This notion is supported by a study showing that the level of ubiquitin conjugation of synaptic proteins is altered by the level of synaptic activity. These studies describe NMNAT as a synapse maintenance factor under normal activity conditions post assembly, when most of the BRP protein is present at the active zone and NMNAT protein is localized to the active zone area to carry out its maintenance function. The interesting observation of clustered BRP protein in the cell body away from the synapse in loss-of-NMNAT neurons indicates a possible defect in the transport of BRP. Two possibilities might explain this phenotype. One, NMNAT facilitates the anterograde transport of BRP during activity. Reduced NMNAT level leads to inefficient transport and subsequent clustering of BRP in the cell body. Two, these BRP clusters are retrogradely transported from the active zone en route to degradation in the cell body. Further work will be required to determine the direction of transport. In summary, this work has identified NMNAT as a chaperone for maintaining active zones, and for facilitating their maintenance during neuronal activity by binding to active zone structural protein BRP, adding NMNAT to the list of synaptic chaperones that are required to maintain functional and structural integrity in neurons (Zang, 2013).

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 here 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).

CRY-dependent plasticity of tetrad presynaptic sites in the visual system of Drosophila at the morning peak of activity and sleep

Tetrad synapses are formed between the retina photoreceptor terminals and postsynaptic cells in the first optic neuropil (lamina) of Drosophila. They are remodelled in the course of the day and show distinct functional changes during activity and sleep. These changes result from fast degradation of the presynaptic scaffolding protein Bruchpilot (BRP) by Cryptochrome (CRY) in the morning and depend on BRP-170, one of two BRP isoforms. This process also affects the number of synaptic vesicles, both clear and dense-core, delivered to the presynaptic elements. In cry⁰¹ mutants lacking CRY and in brp^Δ170, the number of synaptic vesicles is lower in the morning peak of activity than during night-sleep while in wild-type flies the number of synaptic vesicles is similar at these two time points. CRY may also set phase of the circadian rhythm in plasticity of synapses. The process of synapse remodelling stimulates the formation of clear synaptic vesicles in the morning. They carry histamine, a neurotransmitter in tetrad synapses and seem to be formed from glial capitate projections inside the photoreceptor terminals. In turn dense-core vesicles probably carry synaptic proteins building the tetrad presynaptic element (Damulewicz, 2020).

The results confirmed earlier studies that the presynaptic element (T-bar) of tetrad synapses is remodelled during the day and night and this rhythm is regulated by light and circadian clock (Gorska-Andrzejak, 2013; Woznicka, 2015). In the present study, it was found additionally that cyclic changes occur in the T-bar ultrastructure and its volume. Ultrastructural changes in T-bars and synaptic vesicles were possible because of using high resolution electron microscope tomography (EMT). In the present study it was also found that the number of synaptic vesicles cycles during the day, but this rhythm is masked by light. This result indicates that in the case of tetrad synapses, which are sites of fast neurotransmission between photoreceptors and the first order interneurons, intense neurotransmission occurs during the morning peak of locomotor activity, when more synaptic vesicles are attached to the T-bar platform than during sleep (ZT16) and are transported from capitate projections located next to the T-bars. At ZT16, tetrad synapses are ready for neurotransmission, since the total number of synaptic vesicles near tetrad synapses is similar at ZT1 and ZT16, but it is in a standby mode with lower frequency of synapses and vesicles, which are not attached to the T-bar platform and are not delivered from glial cells, respectively. However, transmission can be activated and is efficient because synapses during sleep have larger volume, and synaptic vesicles can be transported to the T-bar platform, if necessary, in response to an unexpected light pulse. Cyclic remodelling of tetrad presynaptic sites depends on BRP, which must be delivered to T-bars and degraded after light exposure after binding to CRY. This fast remodelling of synapses affects the number of vesicles transported to the presynaptic element (Damulewicz, 2020).

The present study was carried out only in light/dark (LD 12:12) conditions because in earlier studies it was found that the rhythm of the changes in BRP abundance in tetrad synapses of D. melanogaster is circadian. The rhythm is maintained in constant darkness (DD) and abolished in the per⁰¹ clock null mutant. On the basis of these results, it is assumed that rhythmic changes during the 24 h cycle reported is this paper are also circadian; however, in DD, the morning peak in BRP is not present because it depends on light (Damulewicz, 2020).

Electron microscope tomography (EMT) used in this study showed that synaptic vesicles are attached to the platform of the T-bar with filamentous proteins that are reduced in brp^Δ170 and brp^Δ190 mutants, which have fewer synaptic vesicles compared with wild-type flies. Two types of vesicles, clear and dense-core were detected. Dense-core synaptic vesicles were less numerous than clear ones. It is known that synaptic vesicles of tetrad synapses contain histamine, while the content of dense-core vesicles is unknown. It is possible that they carry presynaptic proteins to the T-bar. The comparison of ultrastructure of tetrad synapses in the morning peak of activity (ZT1) and during sleep (ZT16) indicates that more vesicles are attached to the T-bar platform and to capitate projections at ZT1, but this pattern is not present in brpΔ170, brp^Δ190 and cry⁰¹ . This result confirmed an earlier study that both high motor activity in the morning and light exposure increase activity of the visual system. In the morning, there is intense transport of synaptic vesicles to T-bars and delivery of histamine in clear vesicles from the epithelial glial cells through capitate projections. The evidence for a role of capitate projections in neurotransmitter recycling has already been reported and now this study showed, using EMT, that vesicles are produced from capitate projections and directly delivered to T-bars. This intense transport is damaged in all mutants studied, suggesting that both BRP isoforms and CRY are needed for this process. In addition, in the brp^Δ190 mutant lacking BRP-190, the T-bar structure is less dense than in the other strains studied. In a previous study, it was found that there are approximately 50% fewer synapses in brp^Δ190 than in Canton S and brp^Δ170. Another study reported that BRP isoforms are important for the formation of T-bars in neuromuscular junctions, and in brp mutants T-bars are smaller than in controls. T-bar height was reduced in brp^Δ190, whereas the widths of pedestal and platform were reduced in both mutants. They also decrease transmission since the active zone was smaller in both mutants and the number of synaptic vesicles was reduced. These ultrastructural changes are correlated with cell physiology since the amplitude of evoked excitatory junctional current was decreased in both mutants with a stronger effect in brp^Δ190 (Damulewicz, 2020).

The obtained reconstructions of tetrad T-bars from TEM serial sections of the lamina showed that although there were fewer synapses during the night (ZT16), the volume of the T-bar was larger at that time than in the morning (ZT1), while the total number of synaptic vesicles was similar. In contrast, a day/night difference (ZT1 vs. ZT16) in the number of vesicles was observed in brp^Δ170 and cry⁰¹ . This suggests that the CRY protein and BRP-170 are responsible for an increase in the number of synaptic vesicles during the morning peak of activity. Since CRY co-localizes with BRP and is involved in BRP degradation in the morning, CRY is probably also involved in the degradation of other proteins of synaptic vesicle organization in the morning since the number of vesicles in cry⁰¹ was low in the morning but high at night (ZT16). It seems that in cry⁰¹, synaptic vesicles are not delivered to the photoreceptor terminals from capitate projections and tethered to the cytomatrix in the morning. It is also interesting that the daily rhythm in the number of vesicles is not maintained in BRP mutants, which confirms an earlier study, and the BRP N-terminus, which lacks brp^Δ190, is necessary to maintain daily remodelling of the T-bar structure. Although both isoforms participate in building the cytomatrix their functions seem to be different in the course of the day. It is also possible that CRY is not only responsible for degradation of synaptic proteins but also as a protein, what is known, affecting the clock. In another cell types, in clock neurons l-LNvs, CRY, except interaction with TIM, is responsible for blue light response and firing of the l-LNvs. In the lamina it was found that in cry⁰¹ mutant the daily rhythm in synapse number and their remodelling was delayed in phase and the day/night difference in Canton S increased when peaks in the number of synapses were shift to ZT4 and ZT16. In result the difference between ZT1 and ZT16 was increased in cry⁰¹ (Damulewicz, 2020).

BRP is also responsible for rapid remodelling of the presynaptic active zone (AZ), and as reported in Drosophila NMJ, presynaptic homeostatic potentiation increases the number of BRP molecules and other AZ proteins, Unc13A and RBP, within minutes (Damulewicz, 2020).

When synaptic vesicles were counted at two different distances from the T-bar, to 200 nm and above 200 nm, there were differences between clear vesicles containing histamine and dense-core ones located in both areas. More clear vesicles were located near the T-bar and fewer above 200 nm. In the case of dense-core vesicles, their number was similar in both areas. This difference was not so striking in mutants in the case of vesicles located next to the platform, but in brp^Δ170 and cry⁰¹, there were more dense-core vesicles at ZT16 in the distance above 200 nm than closer to the T-bar. This result indicates that BRP-170 and CRY are important for the distribution of clear synaptic vesicles next to the T-bar as well as dense-core vesicles located above 200 nm from the presynaptic element. It is possible that dense-core vesicles contain T-bar proteins, probably BRP. When transport along the actin cytoskeleton is disrupted, the number of tetrad synapses decreases, and rapid AZ remodelling also fails (Damulewicz, 2020).

The above mentioned ultrastructural changes depend on the level of the presynaptic scaffolding protein BRP, which changes in abundance during the day and night. These changes are controlled by the daily expression of CRY, which seems to have many functions in photoreceptors in addition to being the circadian clock photoreceptor. In an earlier study, it was found that CRY interacts with BRP but only during light exposure and leads to the degradation of BRP during the day/light phase of the 24 h cycle. This seems to be responsible for the decrease in BRP level in the middle of the day after its peak at the beginning of the day. The lack of CRY in cry⁰¹ mutants changes the pattern of the tetrad presynaptic profile frequency during the day and the size of the T-bar. However, the rhythm is not completely abolished, which indicates that other proteins are also involved in the daily remodelling of tetrad synapses. Since CRY plays several functions in photoreceptors, changes in the number of tetrad synapse and T-bar size in cry⁰¹ may result from different processes and lack of interactions of CRY with TIM and BRP. CRY is a component of the molecular clock and interacts with TIM, and this may affect daily changes in the number and size of T-bars. In addition, light-activated CRY binds BRP and targets it to degradation. Previous work showed that flies with constitutively active CRY have low BRP level. In turn, cry⁰¹ mutants show changes in the pattern of BRP expression, with higher BRP level during the day (ZT4), at the time when wild-type flies have minimum of BRP expression. The pattern of BRP expression is similar to the pattern of daily changes in tetrad synapse number, so it is possible that the number of synapses or T-bar size is directly dependent on the amount of BRP which oscillates during the day. However, CRY in the retina photoreceptors binds also actin and is involved in the organization of phototransduction cascade of proteins in rhabdomeres38. This may be also involved in the regulation of T-bar structure. The differences in T-bar size of cry⁰¹ are shown at time when in Canton S CRY is active (during the day) or its level increases (ZT16). At the beginning of the night the level of CRY is very low, so there was no effect on T-bar structure and no difference between CS and cry⁰¹ was observed (Damulewicz, 2020).

The epithelial glial cells are important for many processes during phototransduction and in recycling neurotransmitters and other compounds during the night. Glia take up histamine and metabolize it to carcinin, which is next delivered to the photoreceptor terminals, and capitate projections are involved in this process. Activity of glial cells is also controlled by the circadian clock. During the night, glial cells seem to be more active than neurons to recycle neurotransmitters, and many proteins, including proteins of ion pumps, are found at higher concentrations at that time. The high number of synaptic vesicles near the tetrad T-bar during the morning peak of activity in Drosophila seems to depend on capitate projections invaginating from the epithelial glia to the photoreceptor terminals in the lamina of Drosophila (Damulewicz, 2020).

Although the presynaptic cytomatrix can be rapidly remodelled with transmission strength, it is also affected by motor and visual system activity, external factors, such as light in the case of the visual system, and the circadian clock, showing plasticity and correlation to changes in behaviour during the day/night cycle. As was shown in the present study synaptic plasticity and synapse remodelling during the day is a complex process which involves presynaptic proteins of the T-bar as well as two types of synaptic vesicles, clear and dense-core, and glial cells. It was also found that fast degradation of proteins involved in transmission is as important as pre- and postsynaptic protein synthesis (Damulewicz, 2020).

Sleep deprivation results in diverse patterns of synaptic scaling across the Drosophila mushroom bodies

Sleep is essential for a variety of plastic processes, including learning and memory. However, the consequences of insufficient sleep on circuit connectivity remain poorly understood. To better appreciate the effects of sleep loss on synaptic connectivity across a memory-encoding circuit, changes were examined in the distribution of synaptic markers in the Drosophila mushroom body (MB). Protein-trap tags for active zone components indicate that recent sleep time is inversely correlated with Bruchpilot (BRP) abundance in the MB lobes; sleep loss elevates BRP while sleep induction reduces BRP across the MB. Overnight sleep deprivation also elevated levels of dSyd-1 and Cacophony, but not other pre-synaptic proteins. Cell-type-specific genetic reporters show that MB-intrinsic Kenyon cells (KCs) exhibit increased pre-synaptic BRP throughout the axonal lobes after sleep deprivation; similar increases were not detected in projections from large interneurons or dopaminergic neurons that innervate the MB. These results indicate that pre-synaptic plasticity in KCs is responsible for elevated levels of BRP in the MB lobes of sleep-deprived flies. Because KCs provide synaptic inputs to several classes of post-synaptic partners, a fluorescent reporter for synaptic contacts was used to test whether each class of KC output connections is scaled uniformly by sleep loss. The KC output synapses that were observed in this study can be divided into three classes: KCs to MB interneurons; KCs to dopaminergic neurons, and KCs to MB output neurons. No single class showed uniform scaling across each constituent member, indicating that different rules may govern plasticity during sleep loss across cell types (Weiss, 2021).

This study used genetic reporters to quantify the effects of sleep loss on pre-synaptic active zone markers and putative synaptic contacts in the Drosophila MB lobes. Abundance of Brp, dSyd-1, and Cacophony broadly increase across all MB lobes after overnight sleep deprivation and that acutely increasing sleep for 6 h is sufficient to reduce Brp levels across the α, 'β, γ, and 'β' lobes. KCs strongly contribute to the increase in Brp across each MB lobe following sleep loss, while pre-synapses of other MB cell types are less sensitive to sleep disruption. Because release of Drosophila neuromodulators likely occurs through a combination of classical neurotransmission and extrasynaptic release, these studies do not rule out the possibility that BRP-independent secretion of dopaminergic dense core vesicles might be altered in the MB lobes by sleep loss. The elevated levels of Brp present in KCs of sleep-deprived flies return to control levels within 48 h of ab libitum recovery sleep. While associative learning can recover within only a few hours after sleep deprivation, these studies indicate that some synaptic consequences of prolonged waking may persist for at least 24 h of recovery. These findings parallel those from humans and rodents, suggesting that some measures of cognition and neurophysiology recover rapidly after acute sleep loss while others last much longer, even for several days in some cases. The tractability of Drosophila may provide opportunities for future studies to investigate the processes that mediate recovery from sleep loss and to test whether similar trends in plasticity occur in other neuropil regions across the brain (Weiss, 2021).

Interestingly, sleep deprivation does not seem to increase other active zone components: Rim and Syt1 only show localized changes in some MB lobes, and the primarily post-synaptic marker Dlg shows no significant changes across the MB after sleep loss. Additionally, it was found that the abundance of vesicular proteins Rab3 and nSyb decreases across all MB lobes following overnight sleep deprivation. The varying responses between pre-synaptic components may indicate that sleep deprivation may alter the abundance of some active zone constituents along differing time courses or that active zone release machinery may be regulated differently from synaptic vesicle pools. The varied responses of each synaptic reporter that was observed suggests that Brp, dSyd-1, and Cac levels may underlie the consequences of sleep loss on MB functioning, but the precise physiological consequences of these changes on KC neurotransmitter release are unclear. Previous work finds that increasing BRP gene copy number drives changes in other active zone proteins that recapitulate protein levels observed in short sleeping mutants and also increases sleep in a dose-dependent manner (Weiss, 2021).

It is tempting to speculate that increases in Brp with sleep loss may drive concomitant increases in some core active zone scaffolding components and compensatory decreases in some proteins regulating synaptic vesicle release. Experiments at the Drosophila larval NMJ indicate that elevated Brp levels increase the rate of spontaneous release and enhance facilitation with pairs of stimuli, while other markers of synapse strength, including the amplitudes of evoked and spontaneous junction potentials, remained unchanged (Weiss, 2021).

It is unclear whether acute changes in Brp with sleep loss induce the same physiological changes at MB-output synapses, and additional studies will be required to understand how plastic mechanisms that contribute to memory formation might be altered by the pre-synaptic changes described above. Recent work finds that pan-neuronal knockdown of dSyd-1 can reduce sleep and dampen homeostatic rebound, even in flies with elevated BRP (Weiss, 2021).

Consistent with the idea that dSyd-1 levels may influence sleep pressure, decreased dSyd-1MI05387-GFSTF abundance was found in previously sleep-deprived flies after 48 h of recovery. While the MB contains several different cell types, pre-synapses in the axons of KCs appear to be uniquely plastic during sleep loss. Use of an activity-dependent fluorescent GRASP reporter of synaptic contacts observed that sleep loss altered synaptic contacts between KCs and distinct post-synaptic partners in different ways (Weiss, 2021).

Among these changes, it was found that GRASP fluorescence reporting contacts from KCs to PPL1 DANs is strongly decreased after sleep loss, indicating a weakening of the KC > PPL1 DAN contacts. Interestingly, these connections may be vital for recurrent activation within MB compartments during learning and could contribute to prediction error signals (Weiss, 2021).

While further studies will be required to examine the contribution of these particular connections to learning deficits after sleep loss, human subjects have been reported to exhibit impaired error prediction and affective evaluation in learning tasks following sleep loss (Weiss, 2021).

Because reduced GRASP signal was observed in KC > PPL1 DAN connections, which mediate aversive reinforcement, and not in KC > PAM DAN connections, which influence appetitive reinforcement, it is also possible that sleep loss may not equally degrade the encoding of reinforcement signals across all valences or modalities. Recent findings also suggest that not all forms of memory require sleep for consolidation; appetitive olfactory memories can be consolidated without sleep when flies are deprived of food, and sleep-dependent and independent memory traces in these conditions are stored in separate MB zones (Weiss, 2021).

The KC > MBON connections that contribute to sleep-dependent memory (KC > γ2α'1) also show an overall decrease in GRASP signal with sleep loss, while those that are vital for sleep-independent memory (MBON-γ1pedc) show no GRASP change after sleep deprivation. These compartment-specific variations in the effects of sleep on both memory and synaptic distribution further indicate that local MB zones may follow distinct plasticity rules under physiological stressors, including sleep loss (Weiss, 2021).

Additionally, GRASP signal from KCs to APL is significantly elevated following sleep loss, suggesting a strengthening of KC > APL connections. KCs and APL form a negative feedback circuit, where KCs activate APL and APL inhibits KCs: this feedback inhibition maintains sparseness of odor coding and odor specificity of memories (Weiss, 2021).

It is possible that KCs compensate for increased synaptic abundance accumulated during sleep loss by recruiting inhibition from APL. While further experimentation is needed to examine the role of these connections in the regulation of net synaptic strength during sleep loss, sleep deprivation results in increased cortical excitability in humans and rodents, and hyperexcitability is often counteracted by increased synaptic inhibition (Weiss, 2021).

Conversely, sleep loss reduces connectivity between KCs and DPM, a second large interneuron that may facilitate recurrent activity in the MB lobes. The current results also indicate that KC > MBON synaptic contacts exhibit a variety of changes in response to sleep deprivation. The specific KC > MBON connections that show significantly elevated or reduced GRASP signal in this study are not clearly assorted based on valence encoding, contribution to specific associative memory assays, or influence on sleep/wake regulation (Weiss, 2021).

Activity in several MB cell types, including α'/'β' KCs, MBON-γ5'β'2, MBON-γ2 α'1, DPM, and PAM DANs regulate sleep. The observation that KC > MBON-γ5'β'2a labeling is reduced with sleep loss complements previous observations of reduced electrical activity in MBON-γ5'β'2 following sleep deprivation (Weiss, 2021).

Other sleep-promoting MB neurons, however, such as DPM, do not show an overall increase in BRP abundance, suggesting either that other changes in excitability, synaptic drive, or post-synaptic adaptations might drive homeostatic sleep regulation in these cells or that distinct subsets of connections within the populations that were labelled in this study might be sleep regulatory. The compartment-to-compartment variance in KC > MBON responses to sleep loss also parallels previous findings that plasticity rules can vary between MBONs during heterosynaptic plasticity (Weiss, 2021).

While GRASP results suggest diverse changes in putative synaptic contacts with sleep loss, the functional effects of these changes require further study. It is important to note that a significant portion of MB synapses are composed of connections between either pairs or groups of KCs. The genetic strategies that were used in this study have prevented reliable visualization and quantification of these connections. As a result, the effect of sleep loss on KC > KC synapses has not been examined in this study but may comprise a portion of the increase in KC pre-synaptic abundance that was observed in this study. While these studies identify synaptic classes that exhibit altered GRASP labeling across sleep loss, future studies using super resolution imaging and/or physiology could examine the structural and molecular changes that underlie this plasticity. Connections between neurons in the MB may be also influenced by non-neuronal cell types, including astrocytes. Astrocytic contact with KCs can be reduced by sleep loss and astrocytic calcium levels correlate with sleep pressure, which suggests that astrocytic processes could be positioned to mediate sleep-dependent plasticity in the MB (Weiss, 2021).

The broad conservation of release machinery across active zones within and between cell types has simplified examination of pre-synaptic plasticity during sleep loss. Assays of both Hebbian and homeostatic plasticity have also identified a variety of post-synaptic adaptations. Interestingly, post-synaptic densities isolated from rodent cortex show significant reorganization of post-synaptic GluR5 receptors. This depends upon the activity of Homer and sleep-dependent phosphorylation of CaMKII and GluR1, that contribute to consolidation of visual cortex plasticity (Weiss, 2021).

Because MBONs exhibit post-synaptic plasticity during other contexts, including the formation of associative memories, sleep deprivation may also alter post-synaptic organization of MBONs or other cell types in the MB. Although the distribution of Dlg is not significantly changed by sleep loss, the rich variety of post-synaptic receptors for acetylcholine, dopamine, GABA, and other signals in the MB requires development of additional reporters to examine these post-synaptic consequences of insufficient sleep in MB neurons. Additionally, while the data outline changes in pre-synaptic protein abundance and pre-synaptic KC contacts that result from sleep loss, the possibility that these synaptic changes may be accompanied by homeostatic compensation in neuronal excitability or firing patterns remains to be tested. Because sleep-deprived flies can recover the capacity to learn after only a brief nap, homeostatic adjustments in post-synaptic strength and/or excitability may permit MBs to compensate for pre-synaptic changes that appear to persist for at least 24 h after sleep deprivation. Further, recovery sleep or pharmacological sleep enhancement may not simply reverse the effects of sleep loss, and it is unclear how particular subsets of synaptic proteins or connections may be selected for removal during times of elevated sleep (Weiss, 2021).

The consequences of sleep loss on synaptic connectivity are not clearly understood, but previous work has found net changes in synaptic abundance or size across brain regions. This study characterized a diverse array of synaptic responses to sleep loss among different cell types within the same circuit. These findings may suggest that distinct cell types and connections within the MB are governed by heterogeneous plasticity rules during sleep disruption. While previous studies have characterized the synaptic effects of sleep history on individual cell types within plastic circuits, the data provide a more comprehensive understanding of the consequences of sleep loss on MB circuits. While this project outlines the local effects of sleep loss on MB connectivity, it is unclear whether specific neural subsets also drive BRP increases within other neuropil compartments of sleep-deprived brains (Weiss, 2021).

This study found an overall increase in the abundance of reporters for some, but not all, pre-synaptic proteins. These pre-synaptic changes are not distributed equally across all cell types: they are most pronounced in MB-intrinsic KCs. Further, output connections from KCs to different classes of synaptic partners show varying patterns of plasticity in MB sub-circuits that contribute to encoding odor valence, comprise recurrent feedback loops, or relay reinforcement signals. The results indicate that sleep loss may degrade MB-dependent memory by altering several different classes of synapses, but future studies will be required to test the specific roles of changes at individual synapse types and the mechanisms by which prolonged waking reorganizes MB connectivity (Weiss, 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).

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).

Circadian programming of the ellipsoid body sleep homeostat in Drosophila

Homeostatic and circadian processes collaborate to appropriately time and consolidate sleep and wake. To understand how these processes are integrated, brief sleep deprivation was scheduled at different times of day in Drosophila, and elevated morning rebound was compared to evening. These effects depend on discrete morning and evening clock neurons, independent of their roles in circadian locomotor activity. In the R5 ellipsoid body sleep homeostat, this study identified elevated morning expression of activity dependent and presynaptic gene expression as well as the presynaptic protein BRUCHPILOT consistent with regulation by clock circuits. These neurons also display elevated calcium levels in response to sleep loss in the morning, but not the evening consistent with the observed time-dependent sleep rebound. These studies reveal the circuit and molecular mechanisms by which discrete circadian clock neurons program a homeostatic sleep center (Andreani, 2022).

Sleep is characterized by quiescence, increased arousal thresholds, changes in neuronal activity, and circadian and homeostatic regulation. Flies display each of these hallmarks and have simple, well-characterized circadian and sleep neural networks. About 150 central pacemaker neurons that express molecular clocks. Of these, four small ventral lateral neurons (sLNvs) (per hemisphere) that express pigment dispersing factor (PDF) are necessary for driving morning activity in anticipation of lights on and exhibit peak levels of calcium around dawn (~ZT0). The dorsal lateral neurons (LNds) and a 5th PDF- sLNv are necessary for evening anticipation of lights off and show a corresponding evening calcium peak (ZT8-ZT10). The posterior DN1 (DN1ps) consist of glutamate-positive (Glu+) subsets necessary for morning anticipation and Glu- necessary for evening anticipation under low light conditions. Lateral posterior neurons (LPN) are not necessary for anticipation but are uniquely sensitive to temperature cycling. Specific pacemaker subsets have been linked to wake promotion [PDF+ large LNv, diuretic hormone 31 (DH31+) DN1ps] and sleep promotion (Glu+ DN1ps, Allostatin A+ LPNs), independently of their clock functions. How these neurons regulate homeostatic sleep drive itself remains unsettled (Andreani, 2022).

Timed signaling from these clock neurons converges on the neuropil of the ellipsoid body (EB). The sLNvs and LNds appear to communicate to R5 EB neurons through an intermediate set of dopaminergic PPM3 neurons based largely on correlated calcium oscillations. The anterior projecting subset of DN1ps provide sleep promoting input to other EB neurons (R2/R4M) via tubercular bulbar (TuBu) interneurons. Activation of a subset of these TuBu neurons synchronizes the activity of the R5 neurons which is important for sleep maintenance. Critically, the R5 neurons are at the core of sleep homeostasis in Drosophila. R5 neuronal activity is both necessary and sufficient for sleep rebound. Extended sleep deprivation (12-24 hr) elevates calcium, the critical presynaptic protein BRUCHPILOT (BRP), and action potential firing rates in R5 neurons. The changes in BRP in this region not only reflect increased sleep drive following sleep deprivation (SD) but also knockdown (KD) of brp in R5 decreases rebound suggesting it functions directly in regulating sleep homeostasis. R5 neurons stimulate downstream neurons in the dorsal fan-shaped body (dFB), which are sufficient to produce sleep. Yet how the activity of key clock neurons are integrated with signals from the R5 homeostat to determine sleep drive remains unclear (Andreani, 2022).

This study dissect the link between the circadian and homeostatic drives by examining which clock neural circuits regulate sleep rebound at different times of day in Drosophila. Akin to the forced desynchrony protocols, wakefulness was enforced at different times of day and sleep rebound was assessed. Flies were exposed to 7 hr cycles of sleep deprivation and recovery, enabling assessment of homeostasis at every hour of the day. It was found that rebound is suppressed in the evening in a Clk-dependent manner. Time-dependent rebound was demonstrated to be is mediated by specific subsets of pacemaker neurons, independently of their effects on locomotor activity. Moreover, homeostatic R5 EB neurons integrate circadian timing and homeostatic drive; it was demonstrated that activity dependent and presynaptic gene expression, BRP expression, neuronal output, and wake sensitive calcium levels are all elevated in the morning compared to the evening, providing an underlying mechanism for clock programming of time-of-day dependent homeostasis (Andreani, 2022).

This study describes the neural circuit and molecular mechanisms by which discrete populations of the circadian clock network program the R5 sleep homeostat to control the homeostatic response to sleep loss. A novel protocol was developed to administer brief duration SD and robustly measure homeostatic rebound sleep. Using this strategy, it was demonstrated that homeostatic rebound is significantly higher in the morning than in the evening. Distinct subsets were identified of the circadian clock network and their downstream neural targets that mediate the enhancement and suppression of morning and evening rebound respectively. Using unbiased transcriptomics very little gene expression was observed that was significantly altered in response to the 2.5 hr sleep deprivation. On the other hand,elevated expression of activity-dependent and presynaptic genes were identified in the morning, independent of sleep deprivation. Consistent with this finding, elevated levels of the presynaptic protein BRP were observed that was absent in the absence of Clk. These baseline changes are accompanied by an elevated calcium response to sleep deprivation in the morning mirroring the enhanced behavioral rebound in the morning. Taken together, these data support the model of a circadian regulated homeostat that turns the homeostat up late at night to sustain sleep and down late in the day to sustain wake (Andreani, 2022).

These studies suggest that homeostatic drive in the R5 neurons is stored post-transcriptionally. As part of these studies, a novel protocol was developed using minimal amounts of SD which could be useful for minimizing mechanical stress effects and isolating underlying molecular processes crucial for sleep homeostasis. Six to 24 hr of SD in Drosophila is commonly used despite the potential stressful or even lethal effects. This study demonstrates that shorter 2.5 hr deprivations not only induce a robust rebound sleep response, but also the percent of sleep lost recovered at ZT0 is close to 100% versus 14-35% seen in 12 hr SD protocols. Using this shorter SD, it was found that many effects observed in R5 neurons with 12 hr SD (e.g. increased BRP and upregulation of Nmdar subunits) are no longer observed with shorter SD, even though the necessity of R5 neurons for rebound is retained after 2.5 hr SD. Previously, translating ribosome affinity purification (TRAP) was used to show upregulation of nmdar subunits following 12 hr SD.FACS and TRAP are distinct methodologies for targeted collection of RNA for sequencing and can yield unique gene lists. One possibility is that upregulation of nmdar subunits is occurring locally in neuronal processes, which are often lost during FACS, and/or is at the level of translation initiation or elongation. Nonetheless, in agreement with previous work, this study observed SD-induced increases in calcium correlated with behavioral rebound in the morning, suggesting that this process is a core feature of the cellular homeostatic response (Andreani, 2022).

Using genetically targeted 'loss-of-function' manipulations, this study has defined small subsets of circadian clock neurons and downstream circuits that are necessary for intact clock modulation of sleep homeostasis. The use of intersectional approaches enabled highly resolved targeting not possible with traditional lesioning experiments in the SCN. Collectively these studies defined a potential Glu+ DN1p-TuBu-R4m circuit important for enhancing morning rebound as well as a discrete group of LNds important for suppressing evening rebound. Importantly, most of these effects on sleep rebound are evident in the absence of substantial changes in baseline activity, despite other studies indicating their necessity for normal circadian behavior. Of note, the proposed roles of the DN1p and LNd clock neurons are sleep and wake promotion consistent with the current findings after sleep deprivation. It is hypothesized that by using chronic silencing methods, baseline effects may not be evident due to compensatory changes but that these effects are only revealed when the system is challenged by sleep deprivation. Similar genetic strategies in mammals may be useful in uncovering which SCN neurons are driving circadian regulation of sleep homeostasis given the comparable suppression of sleep rebound in the evening in humans. Nonetheless, the finding of sleep homeostasis phenotypes in the absence of significant baseline effects suggests that a major role of these clock neuron subsets may be to manage homeostatic responses (Andreani, 2022).

These studies suggest that circadian and homeostatic processes do not compete for influence on a downstream neural target but that the circadian clock programs the homeostat itself. Using an unbiased transcriptomic approach, this study discovered time-dependent expression of activity dependent and presynaptic genes, consistent with previous data that the R5 neurons exhibit time-dependent activity. Significant upregulation was observed of several genes involved in synaptic transmission (Syx1a, Rim, nSyb, unc-104, Srpk79D, para, CG5890) evincing a permissive active state for R5 neurons in the morning. This is accompanied by elevated levels of the key presynaptic protein BRP in the morning compared to evening. It is notable that elevated BRP in the morning is the opposite of what would be expected based on a sleep-dependent reduction in BRP proposed by the synaptic homeostasis hypothesis, suggesting a sleep-wake independent mechanism. Previous studies have shown that modulation of BRP levels in the R5 are important for its sleep function, suggesting that changes in BRP levels impact R5 function. It is hypothesized that these baseline transcriptomic changes underlie the differential R5 sensitivity to sleep deprivation is evident as calcium increases in the morning and not the evening. Indeed, trancriptomic and proteomic studies of the mouse forebrain across time and after sleep deprivation are consistent with the model that the circadian clock programs the transcriptome while homeostatic process function post-trranscriptionally , paralleling what this study has found for R5. It will be of great interest to understand the circuit and molecular mechanisms by which circadian clocks regulate the R5 neuronal calcium and synaptic properties and whether similar circuit architectures underlie daily mammalian sleep-wake (Andreani, 2022).

Active forgetting requires Sickie function in a dedicated dopamine circuit in Drosophila

Forgetting is an essential component of the brain's memory management system, providing a balance to memory formation processes by removing unused or unwanted memories, or by suppressing their expression. However, the molecular, cellular, and circuit mechanisms underlying forgetting are poorly understood. This study shows that the memory suppressor gene, sickie, functions in a single dopamine neuron (DAn) by supporting the process of active forgetting in Drosophila. RNAi knockdown (KD) of sickie impairs forgetting by reducing the Ca(2+) influx and DA release from the DAn that promotes forgetting. Coimmunoprecipitation/mass spectrometry analyses identified cytoskeletal and presynaptic active zone (AZ) proteins as candidates that physically interact with Sickie, and a focused RNAi screen of the candidates showed that Bruchpilot (Brp)-a presynaptic AZ protein that regulates calcium channel clustering and neurotransmitter release-impairs active forgetting like sickie KD. In addition, overexpression of brp rescued the impaired forgetting of sickie KD, providing evidence that they function in the same process. Moreover, this study showed that sickie KD in the DAn reduces the abundance and size of AZ markers but increases their number, suggesting that Sickie controls DAn activity for forgetting by modulating the presynaptic AZ structure. These results identify a molecular and circuit mechanism for normal levels of active forgetting and reveal a surprising role of Sickie in maintaining presynaptic AZ structure for neurotransmitter release (Zhang, 2022).

Forgetting, the flip side of memory acquisition and consolidation, is an essential component of the brain's memory management system that provides a balance to memory formation processes by removing unused or unwanted memories, or by suppressing their expression. However, the molecular, cellular, and circuit mechanisms underlying forgetting are poorly understood (Zhang, 2022).

Previous studies showed that dopamine (DA) and its downstream signaling molecules in postsynaptic neurons are essential for active forgetting and transient forgetting in Drosophila. Small subsets of DA neurons (DAn) within the PPL1 cluster of 12 DAn that innervate the Drosophila mushroom body neurons (MBn) mediate forgetting. Blocking the synaptic output from these DAn after learning inhibits forgetting, whereas stimulating the DAn increases forgetting. Moreover, external factors and internal states, such as locomotor activity, stress, and arousal increase the ongoing activity of these DAn and accelerate forgetting. Conversely, sleep or rest after learning, which decreases the ongoing activity of these DAn, inhibits forgetting (Berry, 2015). This DA-based forgetting is mediated by a DA receptor, DAMB, expressed on the postsynaptic MBn, and requires a downstream signaling pathway involving Scribble, Rac1, and Cofilin for actin remolding (Zhang, 2022 and references therein).

A large RNA interference (RNAi) screen of ∼3,500 genes identified sickie as a memory suppressor genes in Drosophila. It was classed as such because knockdown (KD) of sickie led to increased memory expression. Sickie was initially found to be required for the nuclear translocation of Relish for normal innate immune responses using cultured Drosophila S2 cells. Its homologs, NAV2 in humans and Unc53 in Caenorhabditis elegans, were reported to control neurite outgrowth and the anteroposterior directional guidance of some migratory cells. Other studies also suggested that NAV2 is an oncogene whose expression level is closely related to several human tumors. A recent study found that Sickie regulates F-actin-mediated axon growth of Drosophila MBn (Abe, 2014). However, sickie's role in learning and memory was not explored, and its mechanism for memory suppression was unknown (Zhang, 2022).

This study shows that sickie is required in a single DAn for active forgetting, but not for memory acquisition or consolidation. Sickie KD impairs forgetting by reducing the ongoing activity of DAn. Coimmunoprecipitation and mass spectrometry (co-IP/MS) experiments identify presynaptic active zone (AZ) proteins as the top candidates that interact with Sickie. An RNAi screen of the top candidates along with additional experiments reveal that Sickie interacts physically and genetically with Bruchpilot (Brp) to mediate forgetting through the DAn. Moreover, sickie KD was shown to alter the structure of the presynaptic AZ. Taken together, these results suggest a model whereby Sickie maintains the normal structure and function of presynaptic AZ of a single DAn for DA-based forgetting, through its interaction with presynaptic AZ protein Brp and regulating neurotransmitter release (Zhang, 2022).

This study presents data showing that sickie function is required in a single DAn for the active forgetting of olfactory memory. It does this by regulating the ongoing release of DA, by interacting with and altering the function of the important presynaptic protein, Brp, in the maturation or stability of T-bars at presynaptic AZ. It is most interesting that of the dozen or more DAn that innervate the axons of the MBn in defined physical segments, it is the MP1 DAn and its associated target-the heel of the MB neuropil-that has the most pronounced role in the process of active forgetting. This reinforces the conclusion that the 12 DAn in the PPL1 cluster have distinct and specialized functions. Supporting this conclusion, a prior study has shown that TrpA1 activation of MP1 DAn, but not other DAn in the PPL1 cluster, after training is sufficient to induce forgetting. Thus, the specific role for sickie in MP1 DAn for forgetting results from the intersection of sickie's role in regulating ongoing DAn activity and the unique requirement of MP1 DAn for active forgetting. Most importantly, the results identify a player in the process of active forgetting and in the AZ protein machinery that regulates neurotransmitter release (Zhang, 2022).

The results also open the question about the developmental and physiological roles that have been described for sickie. sickie was previously reported to interact genetically with rac1, slingshot, and cofilin to regulate F-actin-mediated axon growth of Drosophila MBn. However, behavioral data after temporal KD and structural data argue against a similar developmental role for sickie in DAn, and point to an additional, physiological role in adult DAn after axon extension. Nevertheless, it is intriguing that sickie genetically interacts with rac1 and cofilin for developmental processes. These two genes are also involved in the MBn for active forgetting. Thus, the genes and their protein products can exhibit functional interactions that depend on cell type and developmental state. Neither Rac1, Slingshot, or Cofilin were observed among the candidates from co-IP/MS data, indicating that either Sickie has no direct physical interaction with these proteins in the adult head, or the interaction is too weak, sparse, or transient to be captured through antibody pulldowns (Zhang, 2022).

However, it is possible that Sickie may mediate more subtle morphological changes in synapse structure. Indeed, putative physical interactors and protein clusters were discovered that point to possible roles in regulating fine synapse morphology. For example, the actin cross-linking protein Actn is among the top 10% of our MS candidates along with other cytoskeletal proteins like α- and &beta-Spec. This observation suggests a role for Sickie in interacting with cytoskeletal proteins to control synapse structure. RNAi KD experiments of these cytoskeletal protein-encoding genes did not uncover a behavioral phenotype, but this could be due to lack of potency of the RNAi used for this focused screen (Zhang, 2022).

Although it cannot be ruled out that sickie KD causes mild morphological changes of the DAn presynaptic terminals by interacting with cytoskeletal proteins to decrease the ongoing activity of the neuron, the results point to altered DA release through Sickie's interaction with Brp. Brp was a top candidate from co-IP/MS experiments; its KD in MP1 DAn produced the same elevated memory phenotype as sickie KD. Brp abundance was reduced in sickie KD flies, and its overexpression rescued the forgetting phenotype of sickie KD. Brp is a component of the T-bar in the presynaptic AZ and is required for the proper clustering of Ca2+ channels at the AZ. It could be that the lack of proper clustering of the Ca2+ channels leads to the decrease in ongoing Ca2+ influx detected in MP1 DAn terminals. Complete loss-of-function of brp dramatically decreases the evoked release at low stimulation frequency, but does not abolish the release, indicating that the protein participates in release but is not an absolute requirement. In a parallel way, sickie KD impairs ongoing DAn release but not electric shock-induced activity. These observations align with one another, leading to the model that sickie KD reduces Brp abundance in MP1 DAn, reducing Ca2+ influx from ongoing activity, reducing DA release during ongoing activity, and impairing forgetting (Zhang, 2022).

In addition to its role in Ca2+ channel clustering, Brp is required for the tethering of synaptic vesicles in the AZ cytomatrix via its C-terminal sequence at the neuromuscular junction of Drosophila larvae, potentially through its genetic interaction with the SNARE regulator, Complexin. This protein network and gene ontology analysis also uncovered a protein cluster for synaptic transmission involving Brp, Syt1, Syn, Dlg1, and RhoGAP100F. Syt1 is a synaptic vesicle protein that is essential for Ca2+-dependent release, whereas Syn functions for synaptic vesicle clustering and synaptic transmission. Thus, these studies may also suggest a role for Sickie in synaptic vesicle trafficking and tethering for neurotransmitter release, either by interacting with Brp or other proteins. Because of the highly conserved structure of the presynaptic AZ and neurotransmitter release machinery across species, the findings suggest a possible but unexplored role for sickie's homologs in neurotransmitter release and forgetting in other species (Zhang, 2022).

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).

Transient active zone remodeling in the Drosophila mushroom body supports memory

Elucidating how the distinct components of synaptic plasticity dynamically orchestrate the distinct stages of memory acquisition and maintenance within neuronal networks remains a major challenge. Specifically, plasticity processes tuning the functional and also structural state of presynaptic active zone (AZ) release sites are widely observed in vertebrates and invertebrates, but their behavioral relevance remains mostly unclear. This study provides evidence that a transient upregulation of presynaptic AZ release site proteins supports aversive olfactory mid-term memory in the Drosophila mushroom body (MB). Upon paired aversive olfactory conditioning, AZ protein levels (ELKS-family BRP/(m)unc13-family release factor Unc13A) increased for a few hours with MB-lobe-specific dynamics. Kenyon cell (KC, intrinsic MB neurons)-specific knockdown (KD) of BRP did not affect aversive olfactory short-term memory (STM) but strongly suppressed aversive mid-term memory (MTM). Different proteins crucial for the transport of AZ biosynthetic precursors (transport adaptor Aplip1/Jip-1; kinesin motor IMAC/Unc104; small GTPase Arl8) were also specifically required for the formation of aversive olfactory MTM. Consistent with the merely transitory increase of AZ proteins, BRP KD did not interfere with the formation of aversive olfactory long-term memory (LTM; i.e., 1 day). These data suggest that the remodeling of presynaptic AZ refines the MB circuitry after paired aversive conditioning, over a time window of a few hours, to display aversive olfactory memories (Turrel, 2022).

Synapses are key sites of information processing and storage in the brain. Notably, synaptic transmission is not hardwired but adapts through synaptic plasticity to provide appropriate input-output relationships as well as to process and store information on a circuit level. Still, there are fundamental gaps in understanding of exactly how the dynamic changes of synapse performance intersect with circuit operation and consequently define behavioral states. This is partly due to the inherent complexity of synaptic plasticity mechanisms, which operate across a large range of timescales (sub-second to days) and use a rich spectrum of both pre- and post-synaptic molecular and cellular mechanisms. Lately, refinement processes following the immediate engram formation have been described, which might promote specific neuronal activity patterns to select neurons for longer-term information display and storage (Turrel, 2022).

Synaptic transmission across chemical synapses is evoked by action potentials that activate presynaptic Ca2+ influx through voltage-gated Ca2+ channels to trigger the fusion of synaptic vesicles (SVs) containing neurotransmitter at sites called active zones (AZs). AZs assemble from conserved scaffold proteins, including ELKS (Drosophila ortholog: BRP), RIM, and the RIM-binding protein (RBP) family. Recent work in Drosophila showed that discrete SV release sites form at AZ. In the AZ, the ELKS-family BRP master scaffold protein localizes the critical Munc13 family release factor Unc13A in defined nanoscopic clusters around Ca2+ channels (BRP/Unc13A nanomodules). This AZ architecture of the nanoscale organization between BRP/Unc13 release machinery and the AZ-centric Ca2+ channels is present across all Drosophila synapses, including Kenyon cell (KC) derived AZs, and munc13-clusters also define release sites at central mammalian synapses. Importantly, AZ structure and function is dynamic and can remodel within 10 min, as shown at Drosophila neuromuscular junction (NMJ) synapses (Turrel, 2022).

The Drosophila mushroom body (MB) forms and subsequently stores olfactory memories. Importantly, a depression of SV release from the AZ of intrinsic KCs within specific compartments of the MB lobes was found to promote the formation of olfactory memories within a few minutes of paired conditioning. Indeed, Ca2+ in vivo imaging experiments indicate that dopamine bidirectionally tunes the strength of KC synapses to output neurons, with forward conditioning driving depression of those synapses and backward conditioning generally driving potentiation. How this tuning is executed at AZ level is not yet known (Turrel, 2022).

This study present evidence for AZ remodeling (BRP, Syd1, and Unc13A) to take place within MB lobes after paired conditioning for a few hours and provide genetic evidence that this AZ remodeling within the MB-intrinsic KCs is crucial for mid-term aversive olfactory memories. To identify candidate mechanisms of presynaptic remodeling to then be tested in MB-dependent olfactory memory, the role of AZ remodeling was studied during extended larval NMJ plasticity and relevant transport factors were identified. These data suggest that broad but transient changes of presynaptic AZs depending on the transport of new biosynthetic material support refinement processes within KC and MB circuitry and are specifically needed for stable formation of mid-term olfactory memories (Turrel, 2022).

Historically, postsynaptic plasticity mechanisms have been analyzed extensively, and molecular and cellular processes targeting postsynaptic neurotransmitter receptors have been convincingly connected to learning and memory. At the same time, the necessity of using postsynaptic neurons as reporters of presynaptic activity (and, thus, setup paired recordings) has imposed an additional obstacle specific to the functional study of presynaptic forms of mid- and long-term plasticity. Furthermore, the cellular and molecular processes remodeling presynaptic AZs are not characterized as extensively as those at the postsynapse. Consequently, although widely expressed by excitatory and inhibitory synapses of mammalian brains, the behavioral relevance of longer-term presynaptic plasticity remains largely obscure (Turrel, 2022).

This study combined the possibility of genetically analyzing memory formation and stabilization within discrete neuron populations of the Drosophila MB with the identification of molecular machinery remodeling presynaptic AZs in vivo. Evidence is provided for an extended but temporally restricted (a few hours post training) upregulation of presynaptic AZ proteins across the MB lobes, a process seemingly needed in MB intrinsic neurons to display olfactory MTM (Turrel, 2022).

Notably, the acute formation of aversive STM was previously shown to trigger synaptic depression at the KC::MBON synapse in the respective MB compartments. It is emphasized that the exact relation of the AZ remodeling described in this study to this STM-controlling short-term depression is presently unknown. Particularly, it is not possible to tell whether the conditioning-associated presynaptic remodeling described in this study is indeed potentiating KCs and MB AZs or whether overlapping sets of synapses are involved in STM and MTM formation and display. What can be concluded, however, is that molecular machinery that executes structural remodeling at NMJ AZs is critically needed for MTM within the MB intrinsic neurons. Establishing the degree to which synaptic weight changes are associated with the mechanism of MB presynaptic remodeling will have to await the development of protocols to directly follow synapses in vivo for hours after conditioning. Different from presynaptic remodeling being part of the memory trace or engram itself, the idea is favored that synaptic upregulation might instead execute a refinement function extending over larger parts of the MB AZ populations. Refinement is an emerging concept stating that stable propagation and maintenance of memory traces might depend on homeostatic regulations of neuronal circuitry. Sleep-dependent synaptic plasticity is suggested to similarly play an important role in neuronal circuit refinement after learning (Turrel, 2022).

Notably, it has been recently shown that a similar upregulation of AZ proteins (BRP/Unc13A) is indeed a functional part of Drosophila sleep homeostasis, where it suffices to trigger rebound sleep patterns. It thus appears conceivable that the AZ changes associated with conditioning reported in this study might promote specific MB activity patterns instrumental for MTM. An alternative, not mutually exclusive, option is that the initial synaptic depression associated with aversive conditioning must, on a longer term, be compensated by the MB AZ changes (and potential potentiation) described in this study (Turrel, 2022).

Notably, compartment-specific synaptic changes occur in the MB in response to sheer odor presentation or DAN activity although AZ remodeling in this study behaved strictly conditioning dependent, meaning it was not observed after unpaired conditioning, and appeared broadly distributed. It cannot be excluded, however, that smaller size, compartment-specific AZ changes, have been missed, given the limited resolution of the staining assays (Turrel, 2022).

Cell biological processes remodeling presynaptic AZs at larval NMJ synapses can also be of relevance for memory formation in the adult fly KCs. Concretely, this study found that the MB KC-specific KD of transport factors, which at the NMJ level provoked plasticity profiles similar to BRP, also specifically affected MTM but spared STM. Given that several molecular factors, including transport proteins not directly physically associated with the AZ, fulfilled this relation, it indeed appears likely that retrieving axon-transported biosynthetic AZ precursor material is what is critical here (Turrel, 2022).

Speaking of the specificity of rthe MTM phenotypes in relation to AZ remodeling, this study found STM formation undisturbed, but at the same time, MTM to be severely affected after BRP and transport factor KD. This is strong evidence against the possibility of baseline synaptic defects being responsible for the observed MTM deficits. It is also emphasized that this study achieved behavioral phenotypes by comparatively mild and strictly post-developmental KD and that odor Ca2+ responses in MBON neurons postsynaptic to KC appeared normal in BRP KD flies (Turrel, 2022).

When analyzing in a MB-lobe-specific manner, α/&betal and α'/β' neurons showed stronger and more sustained upregulation of BRP/Unc13A than the γ lobes. This might indicate that the extent and role of refinement across the MB lobes is adapted to their specific roles in memory acquisition and retrieval. This is also in accordance with previous observations showing heterogeneity in the exact AZ protein composition across synapses of the Drosophila brain (Turrel, 2022).

Interestingly, Syd-1 levels are significantly increased 1 h after conditioning in the α/β and α'/β' lobes, whereas it has been shown that Syd-1 levels are not increased 10 min after PhTx treatment at the NMJ. This finding indicates that some of the AZ proteins may be affected differently in those two plasticity processes (Turrel, 2022).

Given the generally observed sparse representation of odors within the MB KCs, one might expect initial synaptic changes to be specific to only a few odor-response KCs. Still, this analysis apparently reveals more extended changes of synaptic AZs across the lobes. Potentially, upon successful conditioning, the initial, more restricted, synaptic changes might be followed by an extended communication between the neurons involved in the memory circuit, potentially including KC::KC communication. Indeed, there is ample evidence for a transfer of requirement between different subsets of KCs in the temporal evolution of olfactory memory. This communication seemingly involves gap junctions between KCs but might in parallel also use chemical synapses and their AZs. Concerning the broad distribution of the AZ changes across compartments, it is interesting to mention that KC-global, conditioning-dependent metabolic changes have been observed, being critical for LTM but also MTM (Turrel, 2022).

It is tempting to speculate that the initial, compartment-specific changes, confined to a few odor-responding KCs, might overcome a threshold to also trigger more global synaptic changes. Also interesting in this context, dorsal paired medial (DPM) neurons' odor response increase following spaced conditioning, also indicating that opposite synaptic strength changes might counterbalance the initial synaptic changes occurring in the memory-relevant compartment or depending on post-synaptic partner neurons provoke either potentiation or depression (Turrel, 2022).

As mentioned above, this study found that KD of BRP in the adult MB lobes did not affect LTM, whereas MTM was decreased both at 1 and 3 h. Such a phenotype, a deficit of MTM but subsequent memory phases being intact, was only rarely observed before (Nep2-RNAi in adult DPM neurons, synapsin mutants with memory deficits up to 1 h but normal memory later on). On one hand, this reinforces the idea that MTM and LTM might form using separate circuits, and on the other hand, that cell types other than KCs might contribute to aversive olfactory LTM formation. Different sets of proteins in the same lobes might operate in parallel circuits similar to what has been observed in the honeybee. However, it might also well be that the presynaptic AZ remodeling observed in this study is indeed specific for the display of MTM and that the synaptic memory traces orchestrating the later recall of LTM are mediated by independent parallel molecular/synaptic mechanisms or distinct circuit (Turrel, 2022).

HDAC6 is a Bruchpilot deacetylase that facilitates neurotransmitter release

Presynaptic densities are specialized structures involved in synaptic vesicle tethering and neurotransmission; however, the mechanisms regulating their function remain understudied. In Drosophila, Bruchpilot is a major constituent of the presynaptic density that tethers vesicles. This study shows that HDAC6 is necessary and sufficient for deacetylation of Bruchpilot. HDAC6 expression is also controlled by TDP-43, an RNA-binding protein deregulated in amyotrophic lateral sclerosis (see Drosophila as a Model for Human Diseases: Amyotrophic lateral sclerosis). Animals expressing TDP-43 harboring pathogenic mutations show increased HDAC6 expression, decreased Bruchpilot acetylation, larger vesicle-tethering sites, and increased neurotransmission, defects similar to those seen upon expression of HDAC6 and opposite to hdac6 null mutants. Consequently, reduced levels of HDAC6 or increased levels of ELP3, a Bruchpilot acetyltransferase, rescue the presynaptic density defects in TDP-43-expressing flies as well as the decreased adult locomotion. This work identifies HDAC6 as a Bruchpilot deacetylase and indicates that regulating acetylation of a presynaptic release-site protein is critical for maintaining normal neurotransmission (Miskiewicz, 2014).

This study finds that HDAC6 controls vesicle tethering and synaptic transmission by regulating BRP deacetylation, thereby antagonizing ELP3, a BRP acetyltransferase (Miśkiewicz, 2011). This work defines BRP as a deacetylation target of HDAC6. Acetylation of the C-terminal end of BRP results in more condensed T-bars, while deacetylation leads the protein to send excessive tentacles into the cytoplasm to contact more synaptic vesicles. Similar to chromatin structure being regulated by electrostatic mechanisms at the level of histone acetylation, it is proposed that electrostatic interactions between acetylated and deacetylated lysines in individual BRP strands regulate presynaptic density structure and function (Miskiewicz, 2014).

While many HDAC-like proteins are present in the nucleus to deacetylate histones, HDAC6 predominantly locates to the cytoplasm, where it has been implicated in the modification of different proteins, including α-tubulin, contractin, and HSP90. In neurons, HDAC6-dependent α-tubulin deacetylation may affect axonal transport by promoting kinesin-1 and dynein binding to microtubules. However, hdac6 null mutant flies did not show overt changes in synaptic features other than T-bar morphology as gauged by electron microscopy, suggesting that axonal transport as a consequence of tubulin defects was not massively affected, although more subtle transport defects cannot be excluded (Miskiewicz, 2014).

BRP is a presynaptic density structural component important to cluster calcium channels at release sites while tethering synaptic vesicles at its C-terminal end. The regulation of BRP by HDAC6-dependent deacetylation indicates the BRP C-terminal end is important to sustain neurotransmitter release during intense (60 Hz) stimulation by orchestrating vesicle tethering. Corroborating these results, mutations in the BRP C-terminal end (brp^nude) cause defects in vesicle tethering and the maintenance of release during intense 60 Hz stimulation (Hallermann et al., 2010a). Similarly brp-isoform mutations that leave calcium channel clustering intact but result in a much more condensed T-bar top show a smaller readily releasable vesicle pool, very similar to the defects when BRP is excessively acetylated. The brp^nude mutation shows somewhat less severe defects to maintain synaptic transmission, possibly because more vesicles still manage to tether in these mutants during stimulation compared to the conditions that result in strong shrinking of the T-bar top. Nonetheless, the data indicate that in flies, BRP orchestrates efficient synaptic transmission during intense activity (Miskiewicz, 2014).

In the model presented in this study, ELP3 and HDAC6 antagonistically control presynaptic function. TDP-43, a gene mutated in ALS, positively regulates HDAC6 expression, and in flies, increased HDAC6 activity or expression of pathogenic TDP-43 results in the deacetylation of active zone material and increased synaptic release. Remarkably, the presence of an ALS risk-associated ELP3 allele in humans correlates with reduced ELP3 expression in ALS patient spinal cords. In flies, elp3 mutants also cause active zone deacetylation and more synaptic release. Together with genetic interactions in fruit flies, the data suggest that decreased HDAC6 function and increased ELP3 function act antagonistically, both in flies and humans. However, the target(s) on which these enzymes converge in humans remains to be discovered. In flies, the data are consistent with ELP3-dependent acetylation to occur at the C-terminal tail of the BRP protein. However, the mammalian BRP counterpart, ELKS/CAST, that resides in the presynaptic density, does not contain a long C-terminal tail. ELKS/CAST in mammals has been found to be associated with filamentous structures, and the activity to concentrate synaptic vesicles near release sites may thus be executed by binding partners of ELKS/CAST such as Picollo or Bassoon. Hence, it will be interesting to test if ELP3 and HDAC6 regulate acetylation at the much shorter ELKS/CAST tail or whether ELKS/CAST binding partners are acetylated also in the context of ALS. It is in this perspective interesting to note that another active zone-associated protein, UNC13A, is implicated in ALS as well, but the pathomechanism of how UNC13A is implicated remains to be elucidated (Miskiewicz, 2014).

Quantitative super-resolution imaging of Bruchpilot distinguishes active zone states

The precise molecular architecture of synaptic active zones (AZs) gives rise to different structural and functional AZ states that fundamentally shape chemical neurotransmission. However, elucidating the nanoscopic protein arrangement at AZs is impeded by the diffraction-limited resolution of conventional light microscopy. This study introduces new approaches to quantify endogenous protein organization at single-molecule resolution in situ with super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM). Focusing on the Drosophila neuromuscular junction (NMJ), the AZ cytomatrix (CAZ) was found to be composed of units containing ~137 Bruchpilot (Brp) proteins, three quarters of which are organized into about 15 heptameric clusters. Tests were performed for a quantitative relationship between CAZ ultrastructure and neurotransmitter release properties by engaging Drosophila mutants and electrophysiology. The results indicate that the precise nanoscopic organization of Brp distinguishes different physiological AZ states and link functional diversification to a heretofore unrecognized neuronal gradient of the CAZ ultrastructure (Ehmann, 2014).

External and circadian inputs modulate synaptic protein expression in the visual system of Drosophila melanogaster

In the visual system of Drosophila the retina photoreceptors form tetrad synapses with the first order interneurons, amacrine cells and glial cells in the first optic neuropil (lamina), in order to transmit photic and visual information to the brain. Using the specific antibodies against synaptic proteins; Bruchpilot (BRP), Synapsin (SYN), and Disc Large (DLG), the synapses in the distal lamina were specifically labeled. Then their abundance was measured as immunofluorescence intensity in flies held in light/dark (LD 12:12), constant darkness (DD), and after locomotor and light stimulation. Moreover, the levels of proteins (SYN and DLG), and mRNAs of the brp, syn, and dlg genes, were measured in the fly's head and brain, respectively. In the head, SYN and DLG oscillations were not detected. It was found, however, that in the lamina, DLG oscillates in LD 12:12 and DD but SYN cycles only in DD. The abundance of all synaptic proteins was also changed in the lamina after locomotor and light stimulation. One hour locomotor stimulations at different time points in LD 12:12 affected the pattern of the daily rhythm of synaptic proteins. In turn, light stimulations in DD increased the level of all proteins studied. In the case of SYN, however, this effect was observed only after a short light pulse (15 min). In contrast to proteins studied in the lamina, the mRNA of brp, syn, and dlg genes in the brain was not cycling in LD 12:12 and DD, except the mRNA of dlg in LD 12:12. The abundance of BRP, SYN and DLG in the distal lamina, at the tetrad synapses, is regulated by light and a circadian clock while locomotor stimulation affects their daily pattern of expression. The observed changes in the level of synaptic markers reflect the circadian plasticity of tetrad synapses regulated by the circadian clock and external inputs, both specific and unspecific for the visual system (Krzeptowski, 2014).

CK2-alpha regulates the transcription of BRP in Drosophila

Development and plasticity of synapses are brought about by a complex interplay between various signaling pathways. Typically, either changing the number of synapses or strengthening an existing synapse can lead to changes during synaptic plasticity. Altering the machinery that governs the exocytosis of synaptic vesicles, which primarily fuse at specialized structures known as active zones on the presynaptic terminal, brings about these changes. Although signaling pathways that regulate the synaptic plasticity from the postsynaptic compartments are well defined, the pathways that control these changes presynaptically are poorly described. In a genetic screen for synapse development in Drosophila, this study found that mutations in CK2α lead to an increase in the levels of Bruchpilot (Brp), a scaffolding protein associated with the active zones. Using a combination of genetic and biochemical approaches, this study found that the increase in Brp in ck2α mutants is largely due to an increase in the transcription of brp. Interestingly, the transcripts of other active zone proteins that are important for function of active zones were also increased, while the transcripts from some other synaptic proteins were unchanged. Thus, these data suggest that CK2α might be important in regulating synaptic plasticity by modulating the transcription of Brp. Hence, it is proposed that CK2α is a novel regulator of the active zone protein, Brp, in Drosophila (Wairkar, 2013).

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).

Unc-51 controls active zone density and protein composition by downregulating ERK signaling

Efficient synaptic transmission requires the apposition of neurotransmitter release sites opposite clusters of postsynaptic neurotransmitter receptors. Transmitter is released at active zones, which are composed of a large complex of proteins necessary for synaptic development and function. Many active zone proteins have been identified, but little is known of the mechanisms that ensure that each active zone receives the proper complement of proteins. This study used a genetic analysis in Drosophila to demonstrate that the serine threonine kinase Unc-51 (see Atg1) acts in the presynaptic motoneuron to regulate the localization of the active zone protein Bruchpilot opposite to glutamate receptors at each synapse. In the absence of Unc-51, many glutamate receptor clusters are unapposed to Bruchpilot, and ultrastructural analysis demonstrates that fewer active zones contain dense body T-bars. In addition to the presence of these aberrant synapses, there is also a decrease in the density of all synapses. This decrease in synaptic density and abnormal active zone composition is associated with impaired evoked transmitter release. Mechanistically, Unc-51 inhibits the activity of the MAP kinase ERK to promote synaptic development. In the unc-51 mutant, increased ERK activity leads to the decrease in synaptic density and the absence of Bruchpilot from many synapses. Hence, activated ERK negatively regulates synapse formation, resulting in either the absence of active zones or the formation of active zones without their proper complement of proteins. The Unc-51-dependent inhibition of ERK activity provides a potential mechanism for synapse-specific control of active zone protein composition and release probability (Wairkar, 2009).

A large-scale anatomical screen was performed to identify mutants where not every glutamate receptor cluster is apposed to Bruchpilot. Mutants with a global decrease in Brp or DGluRIII across the NMJ were put aside, and instead focus was placed on mutants in which Brp was absent from a subset of synapses. Such mutants were identified by the presence of glutamate receptor clusters unapposed to Bruchpilot puncta. In this screen, mutants were identified in unc-51 (Wairkar, 2009).

In the unc-51 mutant many DGluRIII clusters are unappposed to Brp. Such misapposition could reflect either DGluRIII clusters unapposed to active zones, or receptor clusters apposed to abnormal active zones that do not contain Brp. The ideal experiment to distinguish between these possibilities would be to stain for other presynaptic active zone proteins. Unfortunately the only other such protein that can be visualized in Drosophila is the calcium channel Cacophony, and since its localization depends on Brp this experiment is not be informative. Nonetheless, two results strongly suggest that a subset of glutamate receptors is apposed to abnormal active zones. First, the decreased density of DGluRIII clusters observed via confocal microscopy approximates the decrease in active zone density observed via electron microscopy. If many DGluRIII clusters were unapposed to active zones, then a more dramatic decrease in active zone density would be expected. Second, ultrastructural analysis demonstrates a decrease in the proportion of active zones containing T-bars. Brp is not necessary for the formation of active zones, but is required for the localization of T-bars to active zones. If the absence of Brp were due to the absence of the entire active zone, then each active zone would contain Brp and a normal ratio of T-bars/active zones would be predicted. Instead, the decrease in T-bars/active zone is consistent with the presence of active zones missing Brp and, hence, lacking T-bars. Therefore, it is concluded that Unc-51 is required for the high fidelity of active zone assembly, ensuring that Brp is present at every active zone (Wairkar, 2009).

In addition to the presence of abnormal synapses in the unc-51 mutant, there is also a decrease in the number and density of synapses. It is speculated that the decrease in synaptic density and the presence of abnormal synapses may be related phenotypes that differ in severity. In this view, Unc-51 promotes synapse formation. In its absence, active zone assembly would be less efficient, resulting in either the formation of abnormal active zones missing crucial proteins such as Brp, or in more severe cases leading to complete failure of active zone assembly and, hence, the absence of a synapse. The complete suppression of both the synaptic density and apposition phenotypes by mutation of the downstream target ERK is consistent with these phenotypes sharing an underlying mechanism. As expected, this defect in the number and proper assembly of synapses leads to a dramatic decrease in synaptic efficacy (Wairkar, 2009).

In addition to these synaptic defects, the unc-51 mutant also has a smaller NMJ and accumulations of synaptic material in the axons, suggesting defects in axonal transport. One mechanism that could link a small NMJ with defective transport is synaptic retraction, in which entire presynaptic boutons or branches retract leaving a footprint of postsynaptic proteins. However, no such footprints were observed in the unc-51 mutant, so this is not the cause of the small NMJ. Synaptic growth requires the retrograde transport of a BMP signal to the nucleus, however this study no change in the levels of phosphorylated MAD in motoneuron nuclei, suggesting that this is not a likely cause of the growth defect. Finally, in worms and mice Unc-51 is required for axon outgrowth, which may be somewhat analogous to defects in NMJ growth in Drosophila. However, to form an NMJ the axon must navigate out of the ventral nerve cord and cross a wide expanse of muscle before reaching its target and forming a junction. Since no defects were observed in the pattern of neuromuscular innervation, it is unlikely that a generic defect in axon outgrowth is responsible for the small NMJs. The apparent axonal transport defect is consistent with findings from mammals suggesting a function for Unc-51 in regulating axon transport. The role of Unc-51 for transport was not investigated, but note that it was possible to genetically separate the axonal transport and synapse development phenotypes, so the transport phenotypes may not be primary cause of the synaptic defects (Wairkar, 2009).

These data support the model that Unc-51 inhibits ERK activation to promote proper active zone development. In the unc-51 mutant a modest increase was observed in the levels of activated ERK, demonstrating that Unc-51 is a negative regulator of ERK activation in vivo. This increased ERK activity is responsible for the defects in active zone formation. Double mutants between unc-51 and the ERK hypomorph rl¹ completely suppress the synapse density and apposition phenotypes of the unc-51 mutant, and restore synaptic strength to wild type levels. Hence, ERK is required for the synaptic phenotypes observed in the unc-51 mutant. The axonal transport defects were not suppressed in the double mutant, so Unc-51 must act through other pathways as well. In mammalian cells Unc-51 can downregulate ERK by inhibiting the binding of a scaffolding protein to the FGF receptor. To date, no receptor tyrosine kinase has been identified that regulates active zone formation in Drosophila. Future studies to characterize the mechanism by which Unc-51 inhibits ERK in Drosophila motoneurons may provide clues towards identification of such a pathway. In addition, it is unclear how ERK regulates active zone formation. A previous study demonstrated that phospho-ERK localizes to the active zone, which would suggest a direct mechanism. Unfortunately, these localization findings could not be replicated. The same study demonstrated that the transgenic expression of a constitutively active ras or a gain-of-function ERK allele both lead to an increase in the number of synaptic boutons, which is not consistent with the current finding of a smaller NMJ. Active zone structure and number were not assessed. It is speculated that the global activation of ERK may result in different phenotypes than relief of Unc-51 inhibition of ERK, which could show temporal and spatial specificity (Wairkar, 2009).

In mammalian and Drosophila neurons, release probability varies across release sites formed by a single neuron. One potential mechanism would be the differential localization or activity of core active zone proteins. In Drosophila, Bruchpilot is an excellent candidate for such a protein. It is required for the localization of calcium channels to the active zone, so changes in its localization or function would impact calcium influx and, hence, release probability at an active zone. The unc-51 mutant demonstrates that signaling pathways can differentially regulate the localization of Brp to individual release sites within a single neuron. As such, the Unc-51/Erk signaling pathway is a candidate mechanism to regulate active zone protein composition and release probability in a synapse-specific manner (Wairkar, 2009).

A Syd-1 homologue regulates pre- and postsynaptic maturation in Drosophila

Active zones (AZs) are presynaptic membrane domains mediating synaptic vesicle fusion opposite postsynaptic densities (PSDs). At the Drosophila neuromuscular junction, the ELKS family member Bruchpilot (BRP) is essential for dense body formation and functional maturation of AZs. Using a proteomics approach, Drosophila Syd-1 (DSyd-1: RhoGAP100F), homolog of Syd-1 (synapse defective 1), a multidomain RhoGAP-like protein, that is required for C. elegans HSNL synapse assembly (Dai, 2006; Patel, 2006). was identified as a BRP binding partner. In vivo imaging shows that DSyd-1 arrives early at nascent AZs together with DLiprin-alpha, and both proteins localize to the AZ edge as the AZ matures. Mutants in dsyd-1 form smaller terminals with fewer release sites, and release less neurotransmitter. The remaining AZs are often large and misshapen, and ectopic, electron-dense accumulations of BRP form in boutons and axons. Furthermore, glutamate receptor content at PSDs increases because of excessive DGluRIIA accumulation. The AZ protein DSyd-1 is needed to properly localize DLiprin-alpha at AZs, and seems to control effective nucleation of newly forming AZs together with DLiprin-alpha. DSyd-1 also organizes trans-synaptic signaling to control maturation of PSD composition independently of DLiprin-alpha (Owald, 2010).

Mechanisms which regulate assembly and maturation of presynaptic AZs are not well understood. This study identified the Drosophila Syd-1 homologue (DSyd-1) as a binding partner of BRP. DLiprin-α and DSyd-1 mark presynaptic sites where, subsequently, AZs (and adjunct PSDs) originate and mature, whereas BRP and Ca²⁺ channels accumulate at later time points than DLiprin-α and DSyd-1. DLiprin-α previously has been shown to be important for proper AZ formation. Thus, consistent with reduced numbers of AZs forming at NMJs of dsyd-1 and dliprin-α mutants and with both proteins being localized to AZs, the accumulation of DLiprin-α and DSyd-1 at nascent AZs may be instrumental for transforming selected sites into AZs, a process referred to as 'AZ nucleation activity.' However, as the morphological size of dsyd-1 NMJs is reduced, as is the AZ number, in principle, other growth processes might also become rate-limiting at dsyd-1 mutant NMJs. In other words, reduced AZ numbers could also be a consequence of a reduction in morphological NMJ growth. Studying the coupling between morphological growth and AZ formation will be important for determining the relevance of morphological size to total AZ number (Owald, 2010).

Work on en passant synapses of the C. elegans HSNL motor neuron implies that, in genetic terms, Syd-1 operates upstream of Syd-2/Liprin-α. This is based on the fact that a Syd-2/Liprin-α; dominant allele can bypass the requirement of syd-1, which indicates that the protein's essential role in AZ assembly at HSNL synapses is mediated via Syd-2/Liprin-α. This study provides evidence that DSyd-1 is required to properly target DLiprin-α to AZs. In the absence of DSyd-1, DLiprin-α distributes unevenly at NMJ terminals, sparing many AZs. Thus, direct evidence is provided that the RhoGAP DSyd-1 operates upstream in AZ assembly in vivo: DSyd-1 seemingly stalls DLiprin-α to developing AZs in order to allow for the AZ nucleation function of DLiprin-α to effectively operate (Owald, 2010).

DLiprin-α seems to be a direct substrate of DSyd-. The data imply that other presynaptic substrate proteins of DSyd-1 might exist at nascent synapses, a finding that is unexpected based on analysis of AZ formation in C. elegans. Therefore, it is deduced from these findings that presynaptic DSyd-1 (but apparently not DLiprin-α) plays an important role in shaping the PSD assembly. Embryos and larvae mutants for dsyd-1, and importantly, dliprin-α; dsyd-1 double mutant embryos (the double mutant is embryonic lethal), showed increased overall amounts of postsynaptic GluRs, whereas dliprin-α single mutant embryos and larvae did not. These increased amounts of GluRs in dsyd-1 mutants vanished after presynaptic reexpression of UAS–dsyd-1^cDNA. It is tempting to speculate that the presynaptic DSyd-1 protein helps the AZ localization of an adhesion protein, which via trans-synaptic interaction might steer the incorporation of postsynaptic GluRs. A potential role of the Neurexin–Neuroligin axis should be evaluated in this context (Owald, 2010).

Drosophila NMJs express two functionally distinct GluR complexes, DGluRIIA and IIB, which influence the number of release sites formed. Individual PSDs form distinctly from preexisting ones, and mature over hours, switching from DGluRIIA to IIB incorporation throughout maturation in a manner dependant on presynaptic signaling. DSyd-1 might mediate such a maturation signal, as dsyd-1 mutants show excessive amounts of DGluRIIA incorporation at PSDs. This regulation is likely not (or only partially) due to compensation for reduced presynaptic glutamate release, as dliprin-α mutants (with similarly reduced transmission levels) do not show this dramatic increase in GluR levels (Owald, 2010).

Despite enlarged receptor fields and specifically elevated DGluRIIA levels, average miniature event amplitudes were comparable between dsyd-1 animals and controls, which currently cannot be accounted for. A possible explanation might comprise regulatory processes rendering populations of receptors non-/partially functional. Nonetheless, EJC decay time constants of dsyd-1 mutants resemble those found at dgluRIIB-deficient (and thus GluRIIA dominated) NMJs (Owald, 2010).

Which processes are downstream of the DSyd-1–mediated DLiprin-α activity at nascent AZs? Liprin family proteins steer transport in axons and dendrites (e.g., of AMPA receptors) to support synaptic specializations. Notably, in dsyd-1 mutants, although many AZs lacked proper amounts of DLiprin-α, large ectopic accumulations of DLiprin-α were observed. At the same time, ectopic accumulations of BRP/electron density were observed in the absence of DSyd-1. It is tempting to speculate that these ectopic pools of DLiprin-α provoke the aberrant accumulation of electron densities in dsyd-1 mutants, which is consistent with the transport function of DLiprin-α and the direct interaction of DLiprin-α/Syd-2 and ELKS/BRP. Consistently, large BRP accumulations observed in dsyd-1 embryos were no longer present in dsyd-1; dliprin-α double mutants, which indicates that the presence of DLiprin-α is needed to provoke these overaccumulations of BRP when DSyd-1 is missing (Owald, 2010).

In the absence of DSyd-1, BRP was inappropriately localized, even within the cytoplasm, forming ectopic electron-dense material (which is consistent with its role as building block for the electron-dense T bars). Such 'precipitates' also occurred at and close to non-AZ membranes. Moreover, at dsyd-1 AZs, large malformed T bars formed. Thus, it appears plausible that DSyd-1 keeps BRP 'in solution' to organize its proper consumption at AZs. An alternate and not mutually exclusive explanation may be that axonal BRP precipitates also reflect defects in axonal transport due to the absence of DSyd-1. The presence of several binding interfaces between BRP and DSyd-1 may be considered as a basis for regulating their interplay (Owald, 2010).

BRP accumulation in the center of the AZ is also in the center of the functional and structural AZ assembly process. It appears likely that BRP assembly is regulated on multiple levels. Notably, although BRP accumulation is severely compromised in mutants for the kinesin imac, it is not fully eliminated. Moreover, the serine/arginine protein kinase SRPK79D was recently shown to associate with BRP and to repress premature 'precipitation' of BRP in the axons. Furthermore, mutants for the serine/threonine kinase unc51 have recently been shown to suffer from BRP targeting defects. Phosphorylation of DSyd-1 (e.g., within serine-rich stretches toward the C terminus) might be involved in regulating proper longer-range transport ('blocking precipitation on the way') as well as proper delivery of BRP at nascent AZ sites (Owald, 2010).

Recently, the Rab3 GTPase has been shown to be crucial for effective nucleation of BRP at AZs (Graf, 2009). In an interesting parallel to dsyd-1 defects, rab3 mutant NMJs showed fewer BRP-positive AZs; however, if present, BRP levels were increased. Nonetheless, instead of overgrown T bars, as observed in dsyd-1 mutants, rab3 mutants rather showed multiple T bar AZs (Graf, 2009). It will be interesting to investigate whether these pathways act in parallel or converge, along with their relationships to other synaptogenic signals (Owald, 2010).

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, 2012). 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, 2012), 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).

Negative regulation of active zone assembly by a newly identified SR protein kinase

Presynaptic, electron-dense, cytoplasmic protrusions such as the T-bar (Drosophila) or ribbon (vertebrates) are believed to facilitate vesicle movement to the active zone (AZ) of synapses throughout the nervous system. The molecular composition of these structures including the T-bar and ribbon are largely unknown, as are the mechanisms that specify their synapse-specific assembly and distribution. In a large-scale, forward genetic screen, a mutation was identified termed air traffic controller (atc) that causes T-bar-like protein aggregates to form abnormally in motoneuron axons. This mutation disrupts a gene that encodes for a serine-arginine protein kinase (SRPK79D). This mutant phenotype is specific to SRPK79D and is not secondary to impaired kinesin-dependent axonal transport. The srpk79D gene is neuronally expressed, and transgenic rescue experiments are consistent with SRPK79D kinase activity being necessary in neurons. The SRPK79D protein colocalizes with the T-bar-associated protein Bruchpilot (Brp) in both the axon and synapse. It is proposed that SRPK79D is a novel T-bar-associated protein kinase that represses T-bar assembly in peripheral axons, and that SRPK79D-dependent repression must be relieved to facilitate site-specific AZ assembly. Consistent with this model, overexpression of SRPK79D disrupts AZ-specific Brp organization and significantly impairs presynaptic neurotransmitter release. These data identify a novel AZ-associated protein kinase and reveal a new mechanism of negative regulation involved in AZ assembly. This mechanism could contribute to the speed and specificity with which AZs are assembled throughout the nervous system (Johnson, 2009).

SRPK79D is one of very few proteins known to localize to T-bars or ribbon-like structures at the AZ and is the only known kinase to localize to this site. Genetic evidence is provided that SRPK79D functions to represses the premature assembly of T-bars in axons. In particular, it was shown that loss-of-function mutations in srpk79D cause the appearance of T-bar–like protein aggregates throughout peripheral axons, and the possibility was ruled out that this is an indirect consequence of impaired axonal transport. The appearance of ectopic T-bars is highly specific since numerous other synaptic proteins and mitochondria are normally distributed in the neuron and are normally trafficked to the presynaptic nerve terminal in the srpk79D mutant background. Thus, SRPK79D appears to have a specific function in repressing T-bar assembly prior to the AZ, consistent with the strong colocalization of SRPK79D protein with Brp and T-bar structures (Johnson, 2009).

Finally, a potential function was uncovered for SRPK79D at the active zone (AZ) where it is observed to colocalize with Brp. SRPK79D loss-of-function mutations do not alter the number, density, or organization of Brp puncta at the synapse and do not alter synaptic function. This is consistent with a negative regulatory role for SRPK79D during T-bar assembly and indicates that once SRPK79D-dependent repression of T-bar assembly is relieved, AZ assembly proceeds normally. Overexpression of SRPK79D, however, severely disrupts neurotransmission. The defect in presynaptic release is correlated with a disruption of Brp puncta organization and integrity. These phenotypes are consistent with a function for SRPK79D as a negative regulator of T-bar assembly and AZ maturation (Johnson, 2009).

SRPK79D is a member of the SRPK family of constitutively active cytoplasmic serine-threonine kinases that target serine-arginine–rich domains of SR proteins. Thus, it is interesting to postulate what the relevant kinase target might be. Given that SRPK79D and Brp colocalize, an obvious candidate is the Brp protein itself. However, the Brp protein does not have a consensus SR domain, and decreasing the genetic dosage of srpk79D does not potentiate axonal Brp accumulations that appear upon Brp overexpression. As such, Brp may not be the direct target of SRPK79D kinase activity. It is hypothesized, therefore, that SRPK79D colocalizes with Brp and another putative SR protein that is the direct target of SRPK79D kinase activity (Johnson, 2009).

The best-characterized role for SRPKs is in controlling the subcellular localization of SR proteins, thereby regulating their nuclear pre-mRNA splicing activity. SR protein involvement in several cytoplasmic mRNA regulatory roles has been reported. In particular, a phosphorylation-dependent role for SR proteins has been reported in both Drosophila and mammalian cell culture (Johnson, 2009).

It is interesting to speculate that the function of SRPK79D to prevent premature T-bar assembly might be related to the established function of SRPKs and SR-domain-containing proteins during RNA binding, processing, and translation. One interesting possibility is that RNA species are resident at the T-bar. In such a scenario, SRPK79D-dependent repression of RNA translation could prevent T-bar assembly in the axon, and relief of this repression would enable T-bar assembly at the AZ. The continued association of SRPK79D with the AZ could allow regulated control of further T-bar assembly during development, aging, and possibly as a mechanism of long-term synaptic plasticity. Several results provide evidence in support of such a possibility. First, local translation has been proposed to control local protein concentration within a navigating growth cone. There is also increasing evidence in support of local translation in dendrites and for the presence of Golgi outposts that could support local protein maturation. A specific role for RNA binding proteins at the presynaptic AZ is supported by the prior identification of the RIBEYE protein, which is a constituent of the vertebrate ribbon structure. RIBEYE contains a CtBP domain previously shown to bind RNA. The discovery of a different RNA binding protein (CtBP1) at the ribbon and this description of a putative RNA regulatory protein at the Drosophila T-bar further suggest that RNA processing might be involved in the formation or function of these presynaptic electron dense structures (Johnson, 2009).

In light of these data, the possibility was explored that SRPK79D might participate in translational control related to T-bar assembly. Therefore, mutations in genes that could represent SRPK79D-dependent negative regulators of translation, such as aret (bru), cup, pum, nos, and sqd were examined, reasoning that the loss of such a translational inhibitor might result in the ectopic synthesis of AZ proteins, ultimately leading to a phenotype similar to that observed in srpk79D mutants. Also, genomic deletions were generated for bru2 and bru3. However, no evidence was found of axonal Brp aggregation in any of these mutants. Next, mutations previously shown to be required for mRNA transport and local protein synthesis were assayed. If necessary for T-bar assembly, these mutations might disrupt synaptic Brp-dependent T-bar formation. These mutations, including orb, vas, and stau, have phenotypes at earlier stages of development, but show no defect in synaptic Brp staining. Thus, although these experiments do not rule out a function for SRPK79D in local translation, mutations were examined in several additional candidates and no evidence was uncovered in support of this model (Johnson, 2009).

Another possibility is that SRPK79D inhibits T-bar assembly through the constitutive phosphorylation-dependent control of a putative SR protein that colocalizes with SRPK79D and Brp within a nascent T-bar protein complex. Upon arrival of this nascent T-bar protein complex at the presynaptic nerve terminal, T-bar assembly could be initiated in a site-specific manner through the action of a phosphatase that is concentrated at a newly forming synapse. There are several examples of phosphatases that can be localized to sites of intercellular adhesion, some of which have been implicated in the mechanisms of synapse formation and remodeling. This model, therefore, proposes that negative regulation of T-bar assembly, via SRPK79D, is a critical process required for the rapid and site-specific assembly of the presynaptic AZ-associated T-bar structure. Finally, the possibility cannot be ruled out that SRPK79D normally functions to prevent T-bar superassembly as opposed to T-bar assembly per se. Consistent with this idea is the observation of T-bar aggregates in axons and prior observation that detached ribbon structures coalesce into large assemblies in vertebrate neurons (Johnson, 2009).

Synapse assembly is a remarkably rapid event. There is evidence that the initial stages of synapse assembly can occur in minutes to hours, followed by a more protracted period of synapse maturation. Synapses are also assembled at specific sites. In motoneurons and some central neurons, synapses are assembled when the growth cone reaches its muscle or neuron target. However, many central neurons form en passant synapses that are rapidly assembled at sites within the growing axon, behind the advancing growth cone. Current evidence supports the conclusion that intercellular signaling events mediated by cell adhesion and transmembrane signaling specify the position of the nascent synapse. The subsequent steps of presynaptic AZ assembly remain less clear. Calcium channels and other transmembrane and membrane-associated proteins appear to be delivered to the nascent synaptic site via transport vesicles that fuse at the site of synapse assembly. It has been proposed that cytoplasmic scaffolding molecules then gradually assemble at the nascent synapse by linking to the proteins that have been deposited previously. This model assumes, however, that the protein–protein interactions between the numerous scaffolding molecules that comprise the presynaptic particle web do not randomly or spontaneously occur in the cytoplasm prior to synapse assembly. What prevents these scaffolds from spontaneously assembling in the small volume of an axon, prior to synapse formation at the nerve terminal and between individual en passant synapses? Currently, nothing is known about how premature scaffold assembly is prevented. It is proposed that these studies of srpk79D identify one such mechanism of negative regulation that prevents premature, inappropriate assembly of a presynaptic protein complex. It is further proposed that such a mechanism of negative regulation, when relieved at a site of synapse assembly, could contribute to the speed with which presynaptic specializations are observed to assemble (Johnson, 2009).

Bruchpilot in ribbon-like axonal agglomerates, behavioral defects, and early death in SRPK79D kinase mutants of Drosophila

Defining the molecular structure and function of synapses is a central theme in brain research. In Drosophila the Bruchpilot (BRP) protein is associated with T-shaped ribbons ('T-bars') at presynaptic active zones (AZs). BRP is required for intact AZ structure and normal evoked neurotransmitter release. By screening for mutations that affect the tissue distribution of Bruchpilot, a P-transposon insertion was identified in gene CG11489 (location 79D) which shows high homology to mammalian genes for SR protein kinases (SRPKs). SRPKs phosphorylate serine-arginine rich splicing factors (SR proteins). Since proteins expressed from CG11489 cDNAs phosphorylate a peptide from a human SR protein in vitro, CG11489 was renamed the Drosophila Srpk79D (serine-arginine protein kinase at 79D) gene. Srpk79D transcripts and generated a null mutant. Mutation of the Srpk79D gene causes conspicuous accumulations of BRP in larval and adult nerves. At the ultrastructural level, these correspond to extensive axonal agglomerates of electron-dense ribbons surrounded by clear vesicles. Basic synaptic structure and function at larval neuromuscular junctions appears normal, whereas life expectancy and locomotor behavior of adult mutants are significantly impaired. All phenotypes of the mutant can be largely or completely rescued by panneural expression of SRPK79D isoforms. Isoform-specific antibodies recognize panneurally overexpressed GFP-tagged SRPK79D-PC isoform co-localized with BRP at presynaptic active zones while the tagged -PB isoform is found in spots within neuronal perikarya. SRPK79D concentrations in wild type apparently are too low to be revealed by these antisera. It is proposed that the Drosophila Srpk79D gene may be expressed at low levels throughout the nervous system to prevent the assembly of BRP containing agglomerates in axons and maintain intact brain function. The discovery of an SR protein kinase required for normal BRP distribution calls for the identification of its substrate and the detailed analysis of SRPK function for the maintenance of nervous system integrity (Nieratschker, 2009; full text of article).

These results demonstrate an important role of the kinase SRPK79D for the proper distribution of the active zone protein Bruchpilot. In larval and adult nerves the kinase is required for preventing the formation of conspicuous BRP-containing electron-dense ribbon-like agglomerates observed by electron microscopy in the Srpk79D^VN mutant but not in wild-type controls. It is tempting to speculate that these ribbons may be molecularly related to T-bars beyond the association with BRP and that the kinase prevents the premature assembly of T-bars in peripheral axons. Whether BRP is also involved in generating the behavioral and survival defects observed when SRPK79D-PC/PF isoforms or all SRPK79D isoforms are missing is not known. Since BRP does not contain any serine-arginine rich domains it seems unlikely that BRP is a substrate for these kinases. in vitro phosphorylation data suggest that in Drosophila SRPK79D isoforms modify SR proteins and thus may be involved in splicing regulation. It will now be necessary to identify the endogenous substrates of the SRPK79D kinase and study the mechanisms by which the formation of the extensive BRP-containing electron-dense agglomerates in wild-type axons is prevented. The characterization of an SR protein kinase that appears to be localized at presynaptic active zones and has dramatic effects on the distribution of an active zone protein is likely to modify current views on vertebrate SRPK function and may initiate new approaches to the study of active zone assembly and function (Nieratschker, 2009).

Bruchpilot promotes active zone assembly, Ca²⁺ channel clustering, and vesicle release

The molecular organization of presynaptic active zones during calcium influx-triggered neurotransmitter release is the focus of intense investigation. The Drosophila coiled-coil domain protein Bruchpilot (BRP) was observed in donut-shaped structures centered at active zones of neuromuscular synapses by using subdiffraction resolution STED (stimulated emission depletion) fluorescence microscopy. At brp mutant active zones, electron-dense projections (T-bars) are entirely lost, Ca²⁺ channels are reduced in density, evoked vesicle release is depressed, and short-term plasticity is altered. BRP-like proteins seem to establish proximity between Ca²⁺ channels and vesicles to allow efficient transmitter release and patterned synaptic plasticity (Kittel, 2006).

Synaptic communication is mediated by the fusion of neurotransmitter-filled vesicles with the presynaptic membrane at the active zone, a process triggered by Ca²⁺ influx through clusters of voltage-gated channels. The spacing between Ca²⁺ channels and vesicles at active zones is particularly thought to influence the dynamic properties of synaptic transmission (Kittel, 2006).

The larval Drosophila neuromuscular junction (NMJ) is frequently used as a model of glutamatergic synapses. The monoclonal antibody Nc82 specifically stains individual active zones by recognizing a coiled-coil domain protein of roughly 200 kD named Bruchpilot (Brp). Brp shows homologies to the mammalian active zone components CAST [cytoskeletal matrix associated with the active zone (CAZ)-associated structural protein], also called ERC (ELKS, Rab6-interacting protein 2, and CAST). Whereas confocal microscopy recognized diffraction limited spots, the subdiffraction resolution of stimulated emission depletion (STED) fluorescence microscopy revealed donut-shaped Brp structures at active zones. Viewed perpendicular to the plane of synapses, both single and multiple 'rings' were uncovered, of similar size to freeze-fracture-derived estimates of fly active zones. The donuts were up to 0.16 µm high, as judged by images taken parallel to the synaptic plane (Kittel, 2006).

Brp seems to demark individual active zones associated with clusters of Ca²⁺ channels. Transposon-mediated mutagenesis allowed isolation of a mutant chromosome (brp⁶⁹) in which nearly the entire open reading frame of Brp was deleted. brp mutants [brp⁶⁹/df(2R)BSC29] develop into mature larvae but do not form pupae. The Nc82 label is completely lost from the active zones of brp mutant NMJs but can be restored by re-expressing the brp cDNA in the brp mutant background with use of the neuron-specific driver lines ok6-GAL4. This also rescued larval lethality. Mutants had slightly smaller NMJs and somewhat fewer individual synapses. However, individual receptor fields, identified by the glutamate receptor subunit GluRIID, were enlarged in brp mutants. Thus, principal synapse formation occurred in brp mutants, with individual postsynaptic receptor fields increased in size but moderately decreased in number (Kittel, 2006).

In electron micrographs of brp mutant NMJs, synapses with pre- and postsynaptic membranes in close apposition were present at regular density, and consistent with the enlarged glutamate receptor fields postsynaptic densities appeared larger while otherwise normal. However, intermittent rufflings of the presynaptic membrane were noted, and brp mutants completely lacked presynaptic dense projections (T-bars). Occasionally, very little residual electron-dense material attached to the presynaptic active zone membrane was identified. After re-expressing the Brp protein in the mutant background, T-bar formation could be partially restored, although these structures were occasionally somewhat aberrant in shape. Thus, Brp assists in the ultrastructural assembly of the active zone and is essential for T-bar formation (Kittel, 2006).

In brp mutant larvae a drastic decrease was noted in evoked excitatory junctional current (eEJC) amplitudes by using two-electrode voltage clamp recordings of postsynaptic currents at low stimulation frequencies. This drop in current amplitude could be partially rescued through brp re-expression within the presynaptic motoneurons by using either elav-GAL4 or ok6-GAL4. In contrast, the amplitude of miniature excitatory junctional currents (mEJCs) in response to single, spontaneous vesicle fusion events was increased over control levels. This is consistent with the enlarged individual glutamate receptor fields of brp mutants and excludes a lack of postsynaptic sensitivity as the cause of the reduced eEJC amplitudes (Kittel, 2006).

It follows that the number of vesicles released per presynaptic action potential (AP) (quantal content) was severely compromised at brp mutant NMJs and could not be attributed solely to the moderate decrease in synapse number. The ultrastructural defects of brp mutant synapses may interfere with the proper targeting of vesicles to the active zone membrane and thereby impair exocytosis. The number of vesicles directly docked to active zone membranes was slightly decreased in brp mutants. However, the amplitude distribution and sustained frequency of mEJCs showed that brp mutant synapses did not appear to suffer from extrasynaptic release, as would be caused by a misalignment of vesicle fusion sites with postsynaptic receptors. Consistent with the appropriate deposition of exo- and endocytotic proteins, an apparently normal distribution of Syntaxin, Dap160, and Dynamin was observed at brp mutant synapses (Kittel, 2006).

The exact amplitude and time course of AP-triggered Ca²⁺ influx in the nerve terminal governs the amplitude and time course of vesicle . Nerve-evoked responses of brp mutants were delayed when compared with controls, whereas in contrast mEJC rise times were unchanged. Thus, evoked vesicle fusion events were less synchronized with the invasion of the presynaptic terminal by an AP. Spatiotemporal changes in Ca²⁺ influx have a profound effect on short-term plasticity. Whereas at 10 Hz controls exhibited substantial short-term depression of eEJC amplitudes, brp mutants showed strong initial facilitation before stabilizing at a slightly lower but frequency-dependent steady-state current. As judged by the initial facilitation at 10 Hz, neither a reduction in the number of releasable vesicles nor available release sites could fully account for the low quantal content of brp mutants at moderate stimulation frequencies. Furthermore, the altered short-term plasticity of brp mutant synapses suggested a change in the highly Ca²⁺-dependent vesicle release probability. Paired-pulse protocols were applied to the NMJ. Closely spaced stimuli lead to a buildup of residual Ca²⁺ in the vicinity of presynaptic Ca²⁺ channels, enhancing the probability of a vesicle within this local Ca²⁺ domain to undergo fusion after the next pulse. The absence of marked facilitation at control synapses could be explained by a depletion of release-ready vesicles. At brp mutant NMJs, however, the prominent facilitation at short interpulse intervals showed that the enhancement of release probability strongly outweighed the depletion of releasable vesicles. Thus, initial vesicle release probability was low, and release at brp synapses particularly benefited from the accumulation of intracellular Ca²⁺ (Kittel, 2006).

Vesicle fusion is highly sensitive to the spacing between Ca²⁺ channels and vesicles at release sites. It has been calculated that doubling this distance from 25 to 50 nm decreases the release probability threefold, and the larger this distance, the more effective the slow synthetic Ca²⁺ buffer EGTA should become in suppressing release. Indeed, after bath application of membrane permeable EGTA-AM, the reduction of evoked vesicle release was more pronounced at brp mutant than at control NMJs (Kittel, 2006).

The Ca²⁺-channel subunit Cacophony governs release at Drosophila NMJs. By using a fully functional, GFP (green fluorescent protein)-labeled variant (Cac^GFP), Ca²⁺ channels were visualized in vivo. Consistently, Ca²⁺ channel expression was severely reduced over the entire NMJ and at synapses lacking Brp (Kittel, 2006).

Thus, it is concluded that brp mutants suffer from a diminished vesicle release probability due to a decrease in the density of presynaptic Ca²⁺ channel clusters. It is conceivable that Brp tightly surrounds but is not part of the T-bar structure, contained within the unlabeled center of donuts. Brp may establish a matrix, required for both T-bar assembly as well as the appropriate localization of active zone components including Ca²⁺ channels, possibly by mediating their integration into a restricted number of active zone slots. Related mechanisms might underlie functional impairments of mammalian central synapses lacking active zone components (Altrock, 2003) and natural physiological differences between synapse types. Electron microscopy has identified regular arrangements at active zones of mammalian CNS synapses ('particle web') and frog NMJs ('ribs'), where these structures have also been proposed to organize Ca²⁺ channel clustering. At calyx of Held synapses (an axosomatic synapse in the auditory brainstem), both a fast and a slow component of exocytosis have been described. The fast component recovers slowly and is believed to owe its properties to vesicles attached to a matrix tightly associated with Ca²⁺ channels, whereas the slow component recovers faster and is thought to be important for sustaining vesicle release during tetanic stimulation. In agreement with this concept, the absence or impairment of such a matrix at brp synapses has a profound effect on vesicle release at low stimulation frequencies, but this effect subsides as the frequency increases. The sustained frequency of mEJCs at brp synapses could be explained if spontaneous fusion events arise from the slow release component or a pathway independent of evoked vesicle fusion (Kittel, 2006).

Synapses lacking Brp and T-bars exhibited a defective coupling of Ca²⁺ influx with vesicle fusion, whereas the vesicle availability did not appear rate-limiting under low frequency stimulation. The activity-induced addition of presynaptic dense bodies has been proposed to elevate vesicle release probability. This work supports this hypothesis and suggests an involvement of Brp or related factors in synaptic plasticity by promoting Ca²⁺ channel clustering at the active zone membrane (Kittel, 2006).

Fos and Jun potentiate individual release sites and mobilize the reserve synaptic-vesicle pool at the Drosophila larval motor synapse

In all nervous systems, short-term enhancement of transmitter release is achieved by increasing the weights of unitary synapses; in contrast, long-term enhancement, which requires nuclear gene expression, is generally thought to be mediated by the addition of new synaptic vesicle release sites. In Drosophila motor neurons, induction of AP-1, a heterodimer of Fos and Jun, induces cAMP- and CREB-dependent forms of presynaptic enhancement. Light and electron microscopic studies indicate that this synaptic enhancement is caused by increasing the weight of unitary synapses and not through the insertion of additional release sites. Electrophysiological and optical measurements of vesicle dynamics demonstrate that enhanced neurotransmitter release is accompanied by an increase in the actively cycling synaptic vesicle pool at the expense of the reserve pool. Finally, the observation that AP-1 mediated enhancement eliminates tetanus-induced forms of presynaptic potentiation suggests: (1) that reserve-pool mobilization is required for tetanus-induced short-term synaptic plasticity; and (2) that long-term synaptic plasticity may, in some instances, be accomplished by stable recruitment of mechanisms that normally underlie short-term synaptic change (Kim, 2009).

Drosophila larval motor synapses show increased synaptic strength when AP-1 is overexpressed in motor neurons (Sanyal, 2002). This synaptic enhancement is accompanied by increases in the quantal content of neurotransmitter release, and increases in the number of presynaptic varicosities (Sanyal, 2002). This study asked whether AP-1 mediated synapse enhancement can be explained by increases in synapse number, Ca2+ influx, Ca2+ sensitivity of vesicle fusion or synaptic vesicle number. The observations support a model in which: (1) AP-1 induced synaptic enhancement occurs without an accompanying increase in synapse number; (2) AP-1 increases the size of the cycling synaptic vesicle pool through mobilization of the reserve pool; (3) that AP-1 causes persistent synaptic change by stably recruiting a cellular mechanism transiently used for posttetanic potentiation, a ubiquitous but poorly understood form of short-term synaptic facilitation (Kim, 2009).

Previous studies have shown AP-1 overexpression in Drosophila motor neurons enhances glutamate release from motor terminals in a manner that is accompanied by an increase in bouton number (Sanyal, 2002). These conclusions were confirmed using failure frequency analysis, which, under conditions of very low Ca²⁺, measures frequency of 'failure' to release even a single quantum of neurotransmitter. At 0.3 mM Ca²⁺, frequencies of failure events are reduced in C155/+;UAS Fos/+;UAS Jun/+ (hereafter referred to as 'AP-1') compared with control C155/+ hereafter 'control') synapses. Therefore, this analysis confirmed quantal content (m = ln [number of events/number failures]) is significantly increased in motor synapses from AP-1 animals. Similar results were obtained under nonfailure conditions where quantal content is calculated by m = EJP/mEJP. Because quantal amplitude is not increased by AP-1 (SF1Fig. S1), presynaptic mechanisms completely account for the measured synaptic strengthening. These observations, taken together with previous work (Sanyal, 2002), show that AP-1 increases quantal content of transmitter release at both low and physiological Ca²⁺ concentrations (Kim, 2009).

Although AP-1 overexpression increases the number of presynaptic boutons (Sanyal, 2002), the average bouton size is significantly reduced. For this reason, and because individual boutons contain multiple release sites, bouton number is not necessarily a reliable measure of synapse number. The following strategy was used to assess whether AP-1-terminals have more functional synapses, which is defined as presynaptic release sites apposed to postsynaptic receptor clusters. In wild-type neuromuscular junctions (NMJ), ~95% of GluR clusters are coupled to Bruchpilot (brp/CAST) immunopositive presynaptic puncta (Rasse, 2005). This fraction is not altered by AP-1 expression. Thus, the number of Brp-positive puncta provides a measure of synapse number in AP-1 synapses (Kim, 2009).

Since individual Brp spots are clearly resolved, they could be counted and analyzed with a spot-detection/analysis program. This method yielded values that were in good agreement with those derived from previous serial EM studies of wild-type NMJs. Surprisingly, total Brp positive puncta (per NMJ) decreased by 21% in AP-1 synapses. AP-1 induction did not detectably alter the distribution of T-Bar or synapse size assessed by quantitative fluoresence and electron microscopy respectively. Thus, it is concluded that although AP-1 increases total bouton number, the number of functional synapses is significantly reduced. Because the quantal content of neurotransmitter release is N × p (where N is synapse number and p is the average probability of vesicle release per synapse), these observations point to an increase in p at AP-1 terminals (Kim, 2009).

If AP-1 overexpression leads to changes in the probability of release, it was reasoned that forms of short-term plasticity, which also alter p, might be altered at these motor terminals. To test this idea, two separable forms of short-term plasticity observed at the Drosophila larval NMJ at low Ca²⁺ concentrations were measured. The first form, paired-pulse facilitation (PPF) is short-lived and decays within milliseconds. This is easily distinguished from longer-lived presynaptic plasticity, observed during and after tetanic stimulation, which decays more slowly (10s of seconds to minutes). Although multiple processes (e.g., augmentation and posttetanic potentiation) could contribute to this longer-lived form of plasticity, this phenomenon is referred by a single term, tetanus-induced potentiation (TIP) (Kim, 2009).

At interstimulus intervals (ISI) of 25 ms, 50 ms, 100 ms, and 1,000 ms, the paired pulse ratios exhibited by control and AP-1 motor terminals did not differ significantly. The site of action for residual Ca²⁺ during paired pulse facilitation (PPF) has been demonstrated in previous studies to be located in the Ca²⁺ microdomain immediately surrounding clustered Ca²⁺ channels and vesicle release sites (Blatow, 2003; Zucker, 2002). The observation that PPF is normal in AP-1 synapses suggests that Ca²⁺ dynamics in this microdomain are not significantly altered by AP-1 (Kim, 2009).

In contrast, TIP was strikingly altered by AP-1 expression. In control synapses, transmitter release increases during a 2-min train of 10-Hz stimulation, eventually reaching a plateau. Contributions from both facilitation and TIP processes underlie the potentiated response during delivery of the tetanic stimulus train. Facilitation, however, decays within a few hundred milliseconds. Thus, longer-lived components (TIP), which decay on the order of seconds to minutes, can be isolated in the potentiated response after the tetanic train ends. TIP is greatly reduced in AP-1 terminals compared with the control. The potentiation factor immediately after the tetanus is 2.53 ± 0.13 for control and 1.15 ± 0.10 for AP-1. This early potentiation decays with time but lasts for several minutes as evidenced by the values for PF_2.75 measured 2.75 min after stimulation cessation, which are 1.54 ± 0.14 for control and 0.93 ± 0.11 for AP-1. Thus, in AP-1 appears to affect both PF₀ (Kim, 2009).

The absence of TIP components in AP-1 synapses is consistent with a model where individual release sites are 'prepotentiated' in AP-1 motor terminals. Loss of TIP cannot be explained by postsynaptic receptor saturation, because EJPs of twice this magnitude can easily be detected at this motor synapse. The observation that one form of short-term plasticity (PPF) remains unaltered, whereas longer lived forms (TIP) are dramatically diminished argues that AP-1 acts through a selective and relatively specific mechanism normally used for tetanus-induced presynaptic plasticity (Kim, 2009).

To determine the underlying mechanism of synaptic enhancement by AP-1, three key parameters that influence the efficiency of neurotransmitter release were measured: (1) presynaptic Ca²⁺ entry; (2) sensitivity of the exocytotic machinery to Ca²⁺; and (3) the available pool of synaptic vesicles (Kim, 2009).

A simple mechanism for increasing the probability of exocytosis from an active zone is enhanced Ca²⁺ entry, e.g., because of a decreased presynaptic potassium conductance and/or an increased Ca²⁺ current. The highly comparable paired-pulse ratios in AP-1 and control terminals suggest presynaptic Ca²⁺ entry and, particularly, the molecular target of residual Ca²⁺ during PPF, is unchanged in AP-1 expressing motor neurons (Kim, 2009).

Direct Ca²⁺ imaging to support the above argument is difficult, because small changes in single-action potential induced Ca²⁺ entry potentially can account for the observed increase in quantal content. Using an indirect approach, it was instead asked whether summed Ca²⁺ entry during 40-Hz nerve stimulation was increased in AP-1 expressing animals (Kim, 2009).

In motor terminals expressing the genetically encoded Ca²⁺ indicator, GCaMP 1.6, fluorescence was imaged during sustained 40-Hz stimulation. Values for DF/F at a plateau reached in ~2 seconds were similar in AP-1 and control synapses. Unexpectedly, Ca²⁺ rise times in AP-1 terminals were slightly slower than in the control. This cannot be ascribed to faster Ca²⁺ extrusion as GCaMP signal does not decay any faster in AP-1 synapses after stimulation cessation. Instead, these data indicate that less Ca²⁺ enters AP-1 presynaptic terminals per action potential, at least during high-frequency stimulation. Although GCaMP imaging does not provide absolute measurement of presynaptic Ca²⁺ before and after stimulation, these data argue against increased evoked Ca²⁺ entry as being the primary mechanism for AP-1's effect on transmitter release (Kim, 2009).

Another mechanism to enhance transmitter release is to increase sensitivity of the exocytotic machinery to free Ca²⁺. Measurements, however, show Ca²⁺ cooperativity of transmitter release was not significantly altered by AP-1 expression (Kim, 2009).

The last major parameter that influences and often correlates with quantal content is the size of the active cycling vesicle pool (also referred to as exo-endo cycling pool, ECP) available for release (Murthy, 1999). At Drosophila motor synapses, the ECP contributes to transmitter release at low to moderate rates of nerve stimulation, e.g., 3 Hz. A second 'reserve' pool of vesicles (RP) poorly accessed at 3-Hz stimulation, is efficiently mobilized during high frequency stimulation >10 Hz. Two independent approaches, one electrophysiological and the other, optical allow the sizes of the cycling and total synaptic vesicle pools to be compared at the Drosophila NMJ (Kim, 2009).

ECP sizes were compared as follows. First, AP-1 and control synapses were stimulated continuously at 3 Hz in the presence of 1 μM bafilomycin A1, a drug that pharmacologically blocks the refilling of vesicles with neurotransmitter. Initial rates of synaptic depression under these experimental conditions largely reflect depletion of the cycling pool of vesicles. The later phase in the decay plot, after significant ECP depletion, represents vescles that arise from slow mixing between RP and ECP. The initial phase is extended in AP-1 compared with control, consistent with a larger ECP. To quantitatively estimate ECP size, Y-intercept values were determined by linear regression of the points from the later slow phase of depression in a cumulative plot. These ECP estimates were consistent with substantial enlargement of the ECP in AP-1 motor terminals. Because these estimates derive from fitting the observed curves to a specific (previously suggested) model (Delgado, 2000), a second and completely independent technique was used to estimate the ECP. In this technique, optical measurements of styryl dye uptake into individual varicosities were used. Consistent with predictions from electrophysiological measurements, varicosities at AP-1 synapses were more brightly labeled than control synapses when the ECP was loaded with FM1-43 dye by 3-Hz stimulation for 7 min, indicating a larger ECP (Kim, 2009).

To test whether this increased ECP in AP-1 synapses occurs at the expense of the reserve poolwe measured the total vesicle content in AP-1 and control terminals was measured by stimulating them to depletion at 10-Hz frequency in the presence of Bafilomycin. Total vesicle pool size was estimated by integrating the complete depression curve of quantal content versus stimulus number. This direct electrophysiological estimate showed a slightly smaller total pool size in AP-1 terminals. To independently assess the sizes of the total vesicle pool FM1-43 uptake into presynaptic boutons was measured after 7 min of 30-Hz stimulation, conditions that should label both ECP and RP. Remarkably, both control and AP-1 terminals were labeled to very similar levels under these conditions, with AP-1 showing slightly lower labeling. This indicates that the total number of synaptic vesicles is similar in control and AP-1 synapses. Thus, 2 independent approaches-electrophysiological and optical establish that AP-1 increases the actively cycling vesicle pool by partially mobilizing the reserve pool of synaptic vesicles. EM analyses of synaptic-vesicle density in AP-1 and control nerve terminals are also conistent with this conclusion (Kim, 2009).

Based on these observations, AP-1 synapses show 2 major differences from the wild-type. First, they have a larger fraction of actively cycling vesicles. Second, they exhibit highly reduced TIP. These 2 phenotypes can be linked if one proposes that mobilization from the reserve vesicle pool is required for TIP. In such a model, AP-1 synapses cannot be further potentiated because the RP has already been mobilized. It was therefore asked whether tetanus-induced potentiation requires RP mobilization (Kim, 2009).

Previous work has established that RP mobilization depends on activity of the myosin light chain kinase (MLCK) in Drosophila motor terminals (Verstreken, 2005). Blocking the activity of this enzyme results in failure to recruit vesicles from the inactive pool under high frequency stimulation (Verstreken, 2005). Strikingly, the MLCK inhibitor ML-7 also inhibited tetanus-induced potentiation; PF was not examined at later time points because in relevant control preparations, the small amount of DMSO required to dissolve MLCK increased the rate of decay of TIP]. Taken together, the above experiments indicate that (1) TIP requires synaptic-vesicle mobilization from the reserve pool; and, by inference, (2) AP-1 driven prepotentiation of transmitter release is accompanied by a stable expansion of the cycling pool of vesicles through reserve pool mobilization (Kim, 2009).

One important conclusion from this work is that Fos and Jun enhance synaptic strength, not by increasing synapse number, but rather by increasing the average probability of release from individual active zones. This conclusion is based on the following: (1) increases in synaptic strength in AP-1 motor terminals can be completely accounted for by increased transmitter release; and (2) light microscopic studies show no increases in the number of release sites. Thus, there is an increase in the average probability of synaptic vesicle fusion at release sites of AP-1 motor neurons. There are some caveats to this argument. First, the definition of functional synapses as Brp-positive puncta is based on the assumptions that Brp puncta: (1) mark the large majority of release sites; and (2) are mostly capable of transmitter release postsynaptic stimulation. These assumptions are supported by the tight colocalization of presynaptic Ca²⁺ channels and postsynaptic receptors with Brp puncta (Kim, 2009).

Increased vesicle-release probability from active zones could conceivably be explained by several different mechanisms. In AP-1 synapses, a large increase in the size of the actively cycling synaptic vesicle pool (ECP), which arises at the expense of the reserve pool (RP), was demonstrated. An increased ECP can account for the observed synaptic enhancement in AP-1 motor terminals if it increases the number of synaptic vesicles immediately available for fusion. RP mobilization has been associated with specific instances of short-term plasticity: e.g., cocaine-induced increases in dopamine release from rat striatal neurons. This study shows that this process can be initiated by nuclear gene expression (Kim, 2009).

How widely might RP mobilization be deployed for synaptic change in vivo? Studies of the reserve pool in hippocampal synapses do not easily support the idea that vesicle trafficking from this source controls synaptic vesicle availability within these small axonal terminals. However, RP mobilization can regulate the output from larger synapses such as neuromuscular junctions or the calyx of Held, where inhibition of the myosin light chain kinase required for vesicle mobilization has been shown to reduce the stability of the synaptic firing during repetitive stimulation (Verstreken, 2005). Because the actual mechanism of RP mobilization is poorly understood, more experiments will be required to understand exactly how Fos and Jun regulate this process. In one model, phosphorylation of synapsin, which tethers synaptic vesicles to an actin-based cytoskeleton in the central domain of synaptic boutons, may mobilize the reserve pool by triggering the dissociation of vesicles from the cytoskeleton, and their transport/diffusion to peripheral release sites. In Drosophila NMJs, mitochondria within the presynaptic terminal are required for sustained release of neurotransmitter under high frequency stimulation. One study suggests ATP production from mitochondria is required to fuel MLCK-activated, myosin-propelled transport of reserve vesicles from central to peripheral sites (Verstreken, 2005). Although such processes might be involved, it is also possible that different pathways are used for Fos and Jun mediated RP mobilization. For instance, smaller sized boutons, such as those observed in AP-1 animals, may be less efficient at holding a central pool of reserve vesicles. In such a scenario, bouton geometry, rather than a specific regulatory protein, may prove to be the relevant target of AP-1 activity (Kim, 2009).

The simplest interpretation of these observations is that stable reserve pool mobilization underlies the observed loss of tetanus-induced potentiation in AP-1 synapses. Other work at the Drosophila neuromuscular junction has associated mobilization of the reserve pool with the expression of TIP (Kim, 2009).

Cytosolic Ca²⁺ accumulation and signaling is required for the induction of TIP. However, links between Ca²⁺ signaling and the expression of TIP are poorly defined; indeed, tetanus induced potentiation could include multiple Ca²⁺-dependent processes including augmentation and posttetanic potentiation/PTP. If TIP expression requires RP mobilization, then it would be occluded in 1 of 2 ways. Either: (1) by preexisting mobilization of the reserve pool; or (2) by inhibition of reserve pool mobilization. Drosophila dnc mutants with enhanced cAMP signaling, and rut mutants with reduced cAMP signaling respectively illustrate these 2 different mechanisms of inhibition. Both mutants do not show tetanus-induced potentiation. Although dnc mutants show a greatly increased ECP and already enhanced transmitter release, rut mutants show a large, stable reserve pool that cannot be mobilized by tetanic stimulation. These data indicate that AP-1 synapses behave like dnc mutants in which reserve vesicles have already been mobilized (Kim, 2009).

If reserve pool mobilization is required for TIP, then mutations or drugs that inhibit reserve pool mobilization would also be expected to block TIP. Consistent with this prediction, application of an MLCK inhibitor, which blocks reserve pool mobilization, was shown to dramatically inhibit TIP induction in wild-type motor synapses. Thus, although alternative contributing mechanisms cannot be ruled out, this study shows that tetanus induced presynaptic potentiation is tightly linked to reserve pool mobilization (Kim, 2009).

It is possible that many different direct and indirect targets of AP-1 contribute to various observed AP-1 dependent neuronal phenomena: e.g., increased bouton number, reduced bouton size, increased dendritic growth, elevated evoked transmitter release and increased ECP size. In addition, AP-1 may have effects on some phenomena that are not yet measured, e.g., kinetic and spatial features of synaptic Ca²⁺ dynamics. Nonetheless, this work shows functions of Fos and Jun in neurons, and provides substantial evidence for a model in which transcription-dependent changes in synaptic function occur through stable recruitment of mechanisms used in short-term plasticity. Recent observations that short-term forms of presynaptic plasticity are altered following synaptic enhancements induced by either BDNF or postsynaptic PSD-95 overexpression suggest that this could be a viable strategy for long-term information storage in central synapses (Zakharenko, 2003). If long-term plasticity requires stable recruitment of short-term plasticity mechanisms, then the lability of long-term memory traces, as observed in studies of reconsolidation, may not require the elimination of stable synaptic connections representing the stored memory (Kim, 2009).

Rab3 dynamically controls protein composition at active zones

Synaptic transmission requires the localization of presynaptic release machinery to active zones. Mechanisms regulating the abundance of such synaptic proteins at individual release sites are likely determinants of site-specific synaptic efficacy. A role for the small GTPase Rab3 has been identified in regulating the distribution of presynaptic components to active zones. At Drosophila rab3 mutant NMJs, the presynaptic protein Bruchpilot, calcium channels, and electron-dense T bars are concentrated at a fraction of available active zones, leaving the majority of sites devoid of these key presynaptic release components. Late addition of Rab3 to mutant NMJs rapidly reverses this phenotype by recruiting Brp to sites previously lacking the protein, demonstrating that Rab3 can dynamically control the composition of the presynaptic release machinery. While previous studies of Rab3 have focused on its role in the synaptic vesicle cycle, these findings demonstrate an additional and unexpected function for Rab3 in the localization of presynaptic proteins to active zones (Graf, 2009).

Individual neurons can form thousands of discrete synaptic connections with their postsynaptic partners. Each synapse comprises tightly apposed pre- and post-synaptic membranes, a postsynaptic cluster of neurotransmitter receptors, and a presynaptic complex of proteins that promotes neurotransmitter release. For a synapse to function, the proper complement of proteins must localize to the presynaptic release machinery, and the protein composition at the release site is a likely determinant of its synaptic efficacy. While the general properties of synapses formed by a single axon are similar, the release probability of such synapses can vary dramatically. This presynaptic heterogeneity is likely due to mechanisms that control synapse specific plasticity and may represent one aspect of the molecular basis of learning and memory. Thus, identifying mechanisms that control the protein composition and presynaptic release properties of individual synapses will provide insights into plasticity mechanisms in the brain (Graf, 2009).

The Drosophila neuromuscular junction (NMJ) is an excellent system for identifying mechanisms that regulate the protein composition of individual active zones. A single Drosophila motoneuron and single muscle cell form an NMJ comprising hundreds of individual release sites, or presynaptic active zones, each apposed to a postsynaptic glutamate receptor (GluR) cluster. Each release site is akin to a single mammalian central nervous system synapse, and like CNS synapses, there is heterogeneity in their release properties. Drosophila contains orthologs of all of the major vertebrate presynaptic proteins with the exception of Bassoon and Piccolo. Among these, Bruchpilot (Brp), the Drosophila ortholog of CAST, plays an essential role in organizing the presynaptic release machinery. This role is similar in mammals where CAST acts as a molecular scaffold within the cytomatrix at the active zone, interacting with Piccolo, Bassoon, Rim1α, and α-liprins/SYD-2 and in C. elegans where the Brp homolog ELKS-1 acts with SYD-2/α-liprin to promote the assembly of presynaptic active zone components. In Drosophila, Bruchpilot localizes to every active zone, but its distribution is heterogeneous, and the abundance of Brp at an active zone appears to correlate with the release probability of that site. Brp is not required for active zone formation per se, but is an integral component of T bars, electron-dense active zone specializations that likely promote transmitter release, and is required for the continuous accumulation of Ca²⁺-channels at active zones during synapse maturation. These findings with Brp imply that mechanisms exist to (1) ensure that Brp is present at each release site and (2) regulate the level of Brp at each site. Such mechanisms would likely impact site-specific release probability by controlling the protein composition of the release machinery at each site. To identify such mechanisms, a large-scale genetic screen was performed to identify genes required for the proper localization of Brp to active zones (Graf, 2009).

This study found that the small GTPase Rab3 functions to influence the distribution of Brp and other crucial presynaptic active zone components to release sites. In the absence of Rab3, key constituents of the presynaptic release machinery are concentrated at a fraction of available sites, resulting in the formation of a small number of super sites with enhanced release probability and a larger number of sites devoid of key presynaptic release proteins. Rab3 can rapidly recruit Brp to active zones, demonstrating that the protein composition of the release machinery is under dynamic control and that Rab3 is well positioned to participate in synapse-specific plasticity mechanisms. Whereas previous studies have implicated Rab3 in the cycling and docking of synaptic vesicles, this study reports a role for Rab3 in influencing the protein composition of the presynaptic release apparatus at individual active zones (Graf, 2009).

To identify mechanisms that control the molecular composition of individual release sites, a collection of Drosophila mutants was screened for those with defects that differentially affect presynaptic active zones within an NMJ. An anatomical genetic screen was performed on a collection of ∼1500 lines that carry unique insertions of transposable elements in or near genes on the second chromosome. Third-instar homozygous mutant larvae were dissected from each line and stained for the presynaptic active zone protein Bruchpilot (Brp) and the essential glutamate receptor subunit DGluRIII. The immunostained NMJs were stained with fluorescence microscopy and mutants were identified with altered active zones, including changes in Brp puncta size, number, or intensity, as well as those with defects in the apposition of presynaptic Brp and postsynaptic DGluRIII puncta. Within this group, one line, P{SUPor-P}KG07292, was identified that has an active zone phenotype. In this mutant, there is a dramatic loss of Brp-positive active zones, yet the morphology and number of DGluRIII clusters appears grossly normal. As such, most GluR clusters are unapposed to a Brp-positive active zone. The remaining Brp puncta are apposed to GluR clusters, and these Brp puncta are significantly larger than in wild-type. Due to the large number of unapposed GluR clusters, this mutant was named running-unapposed (rup) (Graf, 2009).

While Brp morphology is altered in rup, the gross morphology of the mutant NMJ is normal, and the synaptic terminal area is not significantly different than in wild-type. Staining with an antibody against the vesicular glutamate transporter (DVGLUT) demonstrates that synaptic vesicles are distributed throughout the NMJ, and co-staining for the postsynaptic scaffolding protein Discs-large (Dlg) reveals that the presynaptic terminal is apposed to the postsynaptic specialization across its length. Hence, the presence of unapposed GluR clusters is not due to synaptic retraction of synaptic boutons or branches; instead, affected synapses distribute in a salt-and-pepper pattern throughout the synaptic terminal, suggesting that the defect occurs at the level of individual synapses. Glutamate receptors colocalize with the serine-threonine kinase Pak at the Drosophila NMJ. Pak distribution appears normal in the rup mutant, and like GluR clusters, most Pak clusters are unapposed to Brp-positive active zones. Hence, in rup postsynaptic morphology is relatively normal and the primary morphological defect is likely presynaptic. Despite their abnormal active zones, rup mutant animals are viable and fertile (Graf, 2009).

To investigate the mechanism underlying the defective active zones in rup, it was necessary to identify the responsible gene. Although rup was found in a collection of insertional mutants, the phenotype does not map to the P{SUPor-P}KG07292 transposable element. Instead, rup is a second-site mutation fortuitously present on the chromosome. rup was roughly mapped by meiotic recombination to position 43-48 on the right arm of the second chromosome and a deficiency chromosome (Df(2R)ED2076) was identified that fails to complement the mutation. This deficiency deletes 26 predicted genes in the region between 47A10 and 47C1. The list of candidates was narrowed to 21 by complementation testing with known null mutants in the region. The coding regions of candidate genes was sequenced, ultimately identifying a five base pair deletion near the 3' end of the rab3 gene. This deletion throws rab3 out of frame and would lead to a deletion of the last 35 amino acids of the protein, including the final CXC motif that in other systems is required for lipid modification, the binding of Rab3 to synaptic vesicles, and proper Rab3 localization (Graf, 2009 and references therein).

A single ortholog of Drosophila rab3 was previously cloned and demonstrated to be highly conserved. It was further shown to be expressed throughout the fly nervous system; however, no functional studies were performed. To investigate the localization of Rab3 protein and the nature of the mutant allele, we generated a polyclonal antibody to a peptide epitope in the unique C-terminal region of Drosophila Rab3. This antibody stains synaptic terminals of wild-type NMJs in a pattern similar to synaptic vesicle markers such as synapsin and DVGLUT. However, unlike synapsin and DVGLUT, Rab3 staining is further concentrated in a punctate pattern at active zones, as visualized by costaining with Brp. This punctate localization of Rab3 at active zones is not observed in brp mutant NMJs (brp⁶⁹/Df(2R)BSC29) even though the synaptic vesicle-like distribution of Rab3 staining remains. In addition, the antibody recognizes a single band of the predicted size on immunoblots from wild-type larvae. Both the synaptic staining at the NMJ and the band on the immunoblot are absent in the rup mutant, demonstrating that the antibody is specific for Rab3 and that the rup mutant does not express wild-type Rab3 protein. Since the mutation in rup is located in the C-terminal region of rab3 just upstream of the epitope, it is possible that a truncated protein could be expressed. While such a mutant protein could have residual function, the active zone phenotype of rup homozygotes and transheterozygotes of rup and Df(2R)ED2076 are similar in terms of the percentage of GluR clusters apposed to Brp and the average area of individual Brp punctum. Therefore, rup behaves as a genetic null or a very strong hypomorph (Graf, 2009).

This study shows that the small GTPase Rab3 controls the protein composition and release probability of individual active zones at the Drosophila neuromuscular junction. In a rab3 mutant, key constituents of the presynaptic release machinery are enriched at a subset of active zones while the remaining release sites are apparently devoid of these proteins. Expression of Rab3 rapidly and reversibly rescues this altered protein distribution. Physiological studies are consistent with these morphological findings, demonstrating an increase in release probability from an apparently decreased number of release sites. Mechanistic studies indicate that Rab3 functions to increase the probability that the essential synaptic organizing molecule Bruchpilot will cluster at an active zone. This Rab3-dependent regulation of active zone protein composition and release probability provides a potential mechanism for the synapse-specific control of synaptic efficacy (Graf, 2009).

The Drosophila NMJ consists of a motoneuron axon terminal arranged as a chain of synaptic boutons closely associated with the postsynaptic muscle membrane. Within each string of boutons are hundreds of individual synapses, discrete sites of neurotransmitter release where a presynaptic active zone is directly apposed to a postsynaptic glutamate receptor cluster. Such a synapse comprises (1) the site where the axon and muscle membranes are in closest proximity, likely tethered by trans-synaptic cell adhesion molecules; (2) the presynaptic release apparatus that influences the Ca²⁺-mediated release of the neurotransmitter-filled vesicles; and (3) the neurotransmitter receptors and scaffolding and signaling proteins of the postsynaptic density. This study demonstrates that disrupting rab3 alters the distribution of proteins that make up the presynaptic release machinery without grossly disturbing the other two components of the synapse (Graf, 2009).

In the absence of Rab3, a subset of synapses contain increased amounts of the active zone protein Bruchpilot, higher levels of the calcium channel Cacophony, and more electron dense T bars at the active zone. Since Brp is a component of T bars and influences Ca²⁺-channel accumulation, the altered distribution of these components is likely a direct consequence of changes in Brp distribution. The creation of additional active zone markers will be necessary to determine the full extent of this altered distribution. However, since all three components examined influence the probability of evoked vesicle release, the active zones where they accumulate likely are sites of enhanced vesicle release. Conversely, the remaining sites that are devoid of these components likely exhibit impaired evoked release. Two lines of evidence support this conclusion. First, glutamate receptors preferentially cluster opposite sites with the highest release probability. In the rab3^rup mutant, GluR clusters are larger at Brp-positive than Brp-negative sites, suggesting that those active zones containing Brp have a higher release probability. Second, facilitation resulting from short stimulus trains is reduced in the mutant, consistent with an increased release probability (p). However, since quantal content and quantal size are unchanged, the increase in p must be balanced by a decrease in the number of sites that are firing. Hence, both the morphological and electrophysiological data are consistent with the model that Rab3 controls the distribution of active zone proteins to influence the efficacy of individual release sites (Graf, 2009).

Other Drosophila mutants have active zone phenotypes, but none have the combination of phenotypes described in this study. Mutations in synaptojanin, neurexin, and spectrin affect the size and spacing of the entire array of active zones. Mutations in the Unc-51 kinase and the protein phosphatase PP2A have differential affects on active zones, resulting in a subset of glutamate receptors unapposed to Bruchpilot puncta as in the rab3^rup mutant. However, in the unc-51 and PP2A mutants, the remaining Brp puncta are not enlarged and there is no increase in the proportion of active zones with multiple T bars. Such phenotypes are consistent with defects in active zone formation, rather than in the distribution of proteins across active zones. Finally, GluR clusters unapposed to Brp puncta occurs following synaptic retraction, but in such mutants the active zone defects are secondary to the loss of the entire presynaptic terminal. Hence, Rab3 participates in a previously undescribed mechanism that differentially regulates active zones within an NMJ (Graf, 2009).

These findings demonstrate that Rab3 plays a central role in the localization of Bruchpilot to individual active zones. In the absence of Rab3, approximately 70% of active zones are devoid of Brp while the other 30% contain an excess of Brp. What is the function of Rab3 such that its loss leads to this altered Brp distribution? It is suggested that Brp is present in two pools: one fraction bound in complexes at active zones and a second mobile fraction in the cytosol. It is further suggested that Brp is dynamic and may alternate between these two pools by associating with or dissociating from the active zone complex. As such, unbound Brp in the cytosol may either nucleate a cluster at an active zone, creating a new Brp punctum, or add to a pre-existing Brp punctum making it larger. Given this scenario, the rab3 phenotype may be explained by two alternative models of Rab3 function: (1) Rab3 limits Brp puncta size, or (2) Rab3 increases the ability of Brp to nucleate new Brp clusters at active zones. If Rab3 functions to limit the addition of Brp to already existing sites, disruption of Rab3 would allow Brp clusters to grow to a maximal size, reducing the availability of cytosolic Brp to create new puncta and consequently constraining the number of puncta formed. In such a model, it would be predicted that Brp puncta size would be large at rab3^rup mutant NMJs regardless of Brp expression levels. Instead, decreasing Brp levels in the rab3^rup mutant decreases the size of Brp puncta. Even more telling, increasing Brp levels in the rab3^rup mutant also reduces the size of Brp puncta. These results are inconsistent with the model that primary function of Rab3 is to limit the size of Brp puncta (Graf, 2009).

Instead, it is suggested that Rab3 functions to increase the probability that Brp will nucleate a new cluster at an active zone. The presence of Brp at some active zones demonstrates that Rab3 is not absolutely required for Brp localization. Why then is Rab3 required for Brp localization to the 70% of active zones that are bereft of Brp? Rather than posit that these two classes of active zones are fundamentally different in the rab3^rup mutant, it is suggested that in the absence of Rab3, Brp is much less likely to nucleate a cluster at an active zone (Graf, 2009).

The data presented in this study are consistent with this model. First, late rescue with rab3 leads to the rapid addition of new, small Brp puncta and, on a slower time scale, a decrease in the size of the large Brp puncta. This demonstrates that Brp is dynamic and can move into and out of active zones. Second, reducing the levels of Brp at wild-type synapses leads to a decrease in both the number of Brp puncta formed as well as their size. An increase in Brp expression at a wild-type synapse cannot increase the number of puncta since essentially 100% of active zones already contain a Brp puncta, but it does lead to an increase in the size of the puncta. Hence, Brp levels affect both the likelihood of forming a Brp puncta at an active zone as well as the ultimate size of the Brp puncta. Third, increased Brp expression enhances the ability of Brp to cluster at active zones, overcoming the absence of Rab3 and leading to the formation of more Brp puncta in the rab3^rup mutant. This demonstrates that these mutant active zones do have the capacity to cluster Brp, but that it requires the stronger driving force provided by the additional Brp to overcome the absence of Rab3. Finally, when Brp is overexpressed in the rab3^rup mutant the Brp puncta are smaller than when Brp is expressed at wild-type levels. This apparent paradox suggests that Brp puncta compete for unbound Brp and that the large increase in the number of Brp puncta provides more sites for unbound Brp and so ultimately results in smaller puncta. Hence, this model explains the variation in the number and size of Brp puncta present in the various genetic backgrounds tested above, and highlights a novel role for Rab3 in controlling the protein composition of active zones (Graf, 2009).

Brp appears to play a prominent role in the mechanism by which Rab3 regulates the distribution of active zone components to release sites. However, there is no evidence that Rab3 interacts directly with Brp, and such a direct interaction between Rab3 and members of the CAST/ERC family, of which Brp is an ortholog, has not previously been reported. Other proteins could mediate the interaction between Rab3 and Brp. In other species, Rab3 is known to interact with proteins involved in the Rab3 GTPase cycle such as Rab3-GEF, Rab3-GAP, and GDI, as well as the putative Rab3 effectors Sec15, Rabphilin, and Rim. Among the Rab3 effectors, Rabphilin is an unlikely candidate because Rabphilin knockout mice and worms have no observable morphological or physiological synaptic defects. Rim is a more plausible candidate because it is a constituent of the presynaptic release apparatus and binds to many other presynaptic active zone proteins including orthologs of Brp. Alternatively, Rab3 may act on a yet unidentified target to regulate the molecular properties of Brp. Understanding Rab3 function at the fly NMJ will require the identification of the protein(s) Rab3 interacts with to distribute active zone components among release sites (Graf, 2009).

Previous studies of rab3 knockouts in other organisms suggest that Rab3 is involved in regulating vesicle cycling, docking, and exocytosis. While Rab3 may play a direct role in vesicle dynamics and release at the Drosophila NMJ, this study also suggesta that Rab3 plays a second, separate role in influencing the distribution of the presynaptic release apparatus. Defects at the active zone in the rab3^rup mutant are unlikely to be secondary to altered synaptic vesicle release because (1) other mutants affecting release do not disrupt the composition of the active zone and (2) neither increasing nor decreasing activity in the rab3^rup mutant exacerbates or ameliorates the active zone phenotype (Graf, 2009).

If Rab3 controls the protein composition of the active zone, then why have genetic analyses of rab3 in mice and C. elegans not identified structural abnormalities at the synapse? In Drosophila, loss of rab3 results in a very specific ultrastructural phenotype. The active zone, visualized as an electron dense thickening of tightly apposed pre- and postsynaptic membranes, is normal in Drosophila rab3^rup mutants as it is in worms and mice. However, some synapses, including Drosophila NMJ synapses, contain prominent electron-dense specializations such as T bars that are thought to promote transmitter release. It is the distribution of these T bars that is altered in the Drosophila rab3^rup mutant, which would not be apparent at, for example, hippocampal synapses, where such dense bodies are not readily observed. While structural defects have not been detected in other organisms, the electrophysiological findings in mice show interesting parallels to the fly phenotype. The quadruple knockout of Rab3A, Rab3B, Rab3C, and Rab3D in mice demonstrates that Rab3 increases the release probability of a subset of vesicles in the readily releasable pool. Two hypotheses were proposed to explain these findings. The first stays within the traditional vesicle-centric framework for Rab3, suggesting that Rab3 docks specific vesicles to sites of high release probability. The second hypothesis posits that Rab3 recruits additional proteins to the release machinery at certain synapses, thereby making Ca²⁺-mediated release more efficient. This second possibility is consistent with the findings in Drosophila that Rab3 regulates the distribution of release apparatus proteins to control the efficacy of individual sites (Graf, 2009).

Many neurons differentially regulate the release properties of individual release sites along their axonal lengths through presynaptic, synapse-specific mechanisms. These include the regulation of Ca²⁺-channel localization and function and the selective accumulation of group III metabotropic glutamate receptors to specific presynaptic active zones. It is suggested that Rab3 is well positioned to participate in such synapse-specific plasticity mechanisms. The finding that late expression of Rab3 can rapidly reverse the apposition phenotype of the mutant and redistribute Brp to active zones that previously lacked the protein indicates that (1) Brp is highly mobile and (2) Rab3 can rapidly modulate its distribution among individual sites. Multiple proteins control Rab3 function via its GTPase cycle, so mechanisms that locally activate or inhibit Rab3 could lead to rapid and local changes in active zone structure and function. Thus, Rab3 is a candidate to participate in plasticity mechanisms that regulate the protein composition and efficacy of individual release sites (Graf, 2009).

ELP3 controls active zone morphology by acetylating the ELKS family member Bruchpilot

Elongator protein 3 (ELP3) acetylates histones in the nucleus but also plays a role in the cytoplasm. This study reports that in Drosophila neurons, ELP3 is necessary and sufficient to acetylate the ELKS family member Bruchpilot, an integral component of the presynaptic density where neurotransmitters are released. In elp3 mutants, presynaptic densities assemble normally, but they show morphological defects such that their cytoplasmic extensions cover a larger area, resulting in increased vesicle tethering as well as a more proficient neurotransmitter release. A model is proposed where ELP3-dependent acetylation of Bruchpilot at synapses regulates the structure of individual presynaptic densities and neurotransmitter release efficiency (Miskiewicz, 2011).

Naked dense bodies provoke depression

At presynaptic active zones (AZs), the frequently observed tethering of synaptic vesicles to an electron-dense cytomatrix represents a process of largely unknown functional significance. This study identified a hypomorphic allele, brp^nude, lacking merely the last 1% of the C-terminal amino acids (17 of 1740) of the active zone protein Bruchpilot. In brp^nude, electron-dense bodies were properly shaped, though entirely bare of synaptic vesicles. While basal glutamate release was unchanged, paired-pulse and sustained stimulation provoked depression. Furthermore, rapid recovery following sustained release was slowed. These results causally link, with intramolecular precision, the tethering of vesicles at the AZ cytomatrix to synaptic depression (Hallermann, 2010).

The specific impairment of vesicle tethering reported in this study delivers the first direct demonstration that efficient sustained release relies on the ability of the AZ to tether vesicles. While the overall AZ structure, including the distribution of Ca²⁺ channels, was unaffected, the impairment of vesicle tethering provoked pronounced synaptic depression and a slowed first component of recovery (Hallermann, 2010).

The C-terminal half of BRP consists of ~1000 aa of essentially contiguous coiled-coil sequence, reminiscent of Golgi/ER-resident tethering factors such as, e.g., GM130. These coiled-coils typically form rod-like structures, where 100 aa residues extend ~15 nm when dimerized, and proteins such as Uso1p extend over 150 nm. These rod-like proteins are believed to act before SNARE protein assembly by forming contacts between membranes at a distance, thereby increasing the specificity or efficiency of the initial attachment of vesicles (tethering). This study has provided morphological and functional evidence that BRP filaments tether vesicles, and thus further mechanistic comparisons between AZ and Golgi/ER trafficking, e.g., concerning the role of small GTPases, might well be informative (Hallermann, 2010).

The C-terminal half of BRP is very highly conserved in insects but not elsewhere. Interestingly, the Drosophila genome does not appear to encode homologs of the vertebrate AZ components Piccolo and Bassoon, which are key regulators of the vertebrate cytomatrix. At central vertebrate synapses, CAST and Bassoon immunoreactivities (closer and further from the AZ membrane, respectively) were recently found to be associated with filaments that may connect vesicles to the AZ. It is tempting to speculate that at AZs of central vertebrate synapses, CAST associates with coiled-coil domain proteins, such as bassoon, to perform the dual functions of Ca²⁺ channel clustering and vesicle tethering executed by the N-terminal and the C-terminal domains of BRP, respectively (Hallermann, 2010).

How synapses manage to repetitively release transmitter with high precision is intensely investigated. Vesicles tethered to electron-dense bodies may represent a reservoir of vesicles required for sustained release. Consistent with this hypothesis, synaptic stimulation provokes depletion of vesicles tethered at dense bodies. While the supply of vesicles appears rate limiting during the train and the first component of recovery, the maturation of vesicles closer to Ca²⁺ channels appears rate limiting during the second component of recovery. One may argue that the rapid component of depression observed at brp^nude synapses could be partially attributed to fewer docked vesicles (though not significantl) with a higher initial release probability. However, a functional estimation of the number of readily releasable vesicles using back-extrapolation from the cumulative EPSC amplitudes in the trains revealed similar numbers of readily releasable vesicles in brp^nude and controls. Finally, it is pointed out that the C-term of BRP could be involved in endocytotic mechanisms, which have been shown to be crucial for sustained release. Novel techniques have begun to address the spatial organization of local vesicle reuse within active zones. It will have to be clarified via which routes vesicles move within active zones and in which direction Bruchpilot steers their translocation (Hallermann, 2010).

Genetic dissection of aversive associative olfactory learning and memory in Drosophila larvae

Memory formation is a highly complex and dynamic process. It consists of different phases, which depend on various neuronal and molecular mechanisms. In adult Drosophila it was shown that memory formation after aversive Pavlovian conditioning includes-besides other forms-a labile short-term component that consolidates within hours to a longer-lasting memory. Accordingly, memory formation requires the timely controlled action of different neuronal circuits, neurotransmitters, neuromodulators and molecules that were initially identified by classical forward genetic approaches. Compared to adult Drosophila, memory formation was only sporadically analyzed at its larval stage. This study deconstructed the larval mnemonic organization after aversive olfactory conditioning. After odor-high salt conditioning (establishing an aversive olfactory memory) larvae form two parallel memory phases; a short lasting component that depends on cyclic adenosine 3'5'-monophosphate (cAMP) signaling and synapsin gene function. In addition, this study shows for the first time for Drosophila larvae an anesthesia resistant component, which relies on radish and bruchpilot gene function, protein kinase C (PKC) activity, requires presynaptic output of mushroom body Kenyon cells and dopamine function. Given the numerical simplicity of the larval nervous system this work offers a unique prospect for studying memory formation of defined specifications, at full-brain scope with single-cell, and single-synapse resolution (Widmann, 2016).

Memory formation and consolidation usually describes a chronological order, parallel existence or completion of distinct short-, intermediate- and/or long-lasting memory phases. For example, in honeybees, in Aplysia, and also in mammals two longer-lasting memory phases can be distinguished based on their dependence on de novo protein synthesis. In adult Drosophila classical odor-electric shock conditioning establishes two co-existing and interacting forms of memory--ARM and LTM--that are encoded by separate molecular pathways (Widmann, 2016).

Seen in this light, memory formation in Drosophila larvae established via classical odor-high salt conditioning seems to follow a similar logic. It consist of LSTM (larval short lasting component) and LARM (anesthesia resistant memory). Aversive olfactory LSTM was already described in two larval studies using different negative reinforcers (electric shock and quinine) and different training protocols (differential and absolute conditioning). The current results introduce for the first time LARM that was also evident directly after conditioning but lasts longer than LSTM. LARM was established following different training protocols that varied in the number of applied training cycles and the type of negative or appetitive reinforcer. Thus, LSTM and LARM likely constitute general aspects of memory formation in Drosophila larvae that are separated on the molecular level (Widmann, 2016).

Memory formation depends on the action of distinct molecular pathways that strengthen or weaken synaptic contacts of defined sets of neurons. The cAMP/PKA pathway is conserved throughout the animal kingdom and plays a key role in regulating synaptic plasticity. Amongst other examples it was shown to be crucial for sensitization and synaptic facilitation in Aplysia, associative olfactory learning in adult Drosophila and honeybees, long-term associative memory and long-term potentiation in mammals (Widmann, 2016).

For Drosophila larvae two studies by Honjo (2005) and Khurana (2009) suggest that aversive LSTM depends on intact cAMP signaling. In detail, they showed an impaired memory for rut and dnc mutants following absolute odor-bitter quinine conditioning and following differential odor-electric shock conditioning. Thus, both studies support the interpretation of the current results. It is argued that odor-high salt training established a cAMP dependent LSTM due to the observed phenotypes of rut, dnc and syn mutant larvae. The current molecular model is summarized in A molecular working hypothesis for LARM formation. Yet, it has to be mentioned that all studies on aversive LSTM in Drosophila larvae did not clearly distinguish between the acquisition, consolidation and retrieval of memory. Thus, future work has to relate the observed genetic functions to these specific processes (Widmann, 2016).

In contrast, LARM formation utilizes a different molecular pathway. Based on different experiments, it was ascertained, that LARM formation, consolidation and retrieval is independent of cAMP signaling itself, PKA function, upstream and downstream targets of PKA, and de-novo protein synthesis. Instead it was found that LARM formation, consolidation and/or retrieval depends on radish (rsh) gene function, brp gene function, dopaminergic signaling and requires presynaptic signaling of MB KCs (Widmann, 2016).

Interestingly, studies on adult Drosophila show that rsh and brp gene function, as well as dopaminergic signaling and presynaptic MB KC output are also necessary for adult ARM formation. Thus, although a direct comparison of larval and adult ARM is somehow limited due to several variables (differences in CS, US, training protocols, test intervals, developmental stages, and coexisting memories), both forms share some genetic aspects. This is remarkable as adult ARM and LARM use different neuronal substrates. The larval MB is completely reconstructed during metamorphosis and the initial formation of adult ARM requires a set of MB α/β KCs that is born after larval life during puparium formation (Widmann, 2016).

In addition, this study has demonstrated the necessity of PKC signaling for LARM formation in MB KCs. The involvement of the PKC pathway for memory formation is also conserved throughout the animal kingdom. For example, it has been shown that PKC signaling is an integral component in memory formation in Aplysia, long-term potentiation and contextual fear conditioning in mammals and associative learning in honeybees. In Drosophila it was shown that PKC induced phosphorylation cascade is involved in LTM as well as in ARM formation. Although the exact signaling cascade involved in ARM formation in Drosophila still remains unclear, this study has established a working hypothesis for the underlying genetic pathway forming LARM based on the current findings and on prior studies in different model organisms. Thereby this study does not take into account findings in adult Drosophila. These studies showed that PKA mutants have increased ARM and that dnc sensitive cAMP signaling supports ARM. Thus both studies directly link PKA signaling with ARM formation. (Widmann, 2016).

KCs have been shown to act on MB output neurons to trigger a conditioned response after training. Work from different insects suggests that the presynaptic output of an odor activated KCs is strengthened if it receives at the same time a dopaminergic, punishment representing signal. The current results support these models as they show that LARM formation requires accurate dopaminergic signaling and presynaptic output of MB KCs. Yet, for LARM formation dopamine receptor function seems to be linked with PKC pathway activation. Indeed, in honeybees, adult Drosophila and vertebrates it was shown that dopamine receptors can be coupled to Gαq proteins and activate the PKC pathway via PLC and IP3/DAG signaling. As potential downstream targets of PKC radish and bruchpilot are suggested. Interference with the function of both genes impairs LARM. The radish gene encodes a functionally unknown protein that has many potential phosphorylation sites for PKA and PKC. Thus considerable intersection between the proteins Rsh and PKC signaling pathway can be forecasted. Whether this is also the case for the bruchpilot gene that encodes for a member of the active zone complex remains unknown. The detailed analysis of the molecular interactions has to be a focus of future approaches. Therefore, the current working hypothesis can be used to define educated guesses. For instance, it is not clear how the coincidence of the odor stimulus and the punishing stimulus are encoded molecularly. The same is true for ARM formation in adult Drosophila. Based on the working hypothesis it can be speculated that PKC may directly serve as a coincidence detector via a US dependent DAG signal and CS dependent Ca²⁺ activation (Widmann, 2016).

Do the current findings in general apply to learning and memory in Drosophila larvae? To this the most comprehensive set of data can be found on sugar reward learning. Drosophila larva are able to form positive associations between an odor and a number of sugars that differ in their nutritional value. Using high concentrations of fructose as a reinforcer in a three cycle differential training paradigm (comparable to the one used in this study for high salt learning and fructose learning) other studies found that learning and/or memory in syn⁹⁷ mutant larvae is reduced to ~50% of wild type levels. Thus, half of the memory seen directly after conditioning seems to depend on the cAMP-PKA-synapsin pathway. The current results in turn suggest that the residual memory seen in syn⁹⁷ mutant larvae is likely LARM. Thus, aversive and appetitive olfactory learning and memory share general molecular aspects. Yet, the precise ratio of the cAMP-dependent and independent components rely on the specificities of the used odor-reinforcer pairings. Two additional findings support this conclusion. First, a recent study has shown that memory scores in syn⁹⁷ mutant larvae are only lower than in wild type animals when more salient, higher concentrations of odor or fructose reward are used. Usage of low odor or sugar concentrations does not give rise to a cAMP-PKA-synapsin dependent learning and memory phenotype. Second, another study showed that learning and/or memory following absolute one cycle conditioning using sucrose sugar reward is completely impaired in rut¹, rut²⁰⁸⁰ and dnc¹ mutants. Thus, for this particular odor-reinforcer pairing only the cAMP pathway seems to be important. Therefore, a basic understanding of the molecular pathways involved in larval memory formation is emerging. Further studies, however, will be necessary in order to understand how Drosophila larvae make use of the different molecular pathways with respect to a specific CS/US pairing (Widmann, 2016).

Bruchpilot, a synaptic active zone protein for anesthesia-resistant memory

In Drosophila, aversive associative memory of an odor consists of heterogeneous components with different stabilities. This study report that Bruchpilot (Brp), a ubiquitous presynaptic active zone protein, is required for olfactory memory. Brp was shown before to facilitate efficient vesicle release, particularly at low stimulation frequencies. Transgenic knockdown in the Kenyon cells of the mushroom body, the second-order olfactory interneurons, revealed that Brp is required for olfactory memory. It was further demonstrated that Brp in the Kenyon cells preferentially functions for anesthesia-resistant memory. Another presynaptic protein, Synapsin, was shown previously to be required selectively for the labile anesthesia-sensitive memory, which is less affected in brp knockdown. Thus, consolidated and labile components of aversive olfactory memory can be dissociated by the function of different presynaptic proteins (Knapek, 2011).

In Drosophila, middle-term olfactory memory after a single training cycle comprises functionally dissociable forms of memory: the labile ASM and the stable ARM. In contrast to ASM, the molecular basis of ARM formation is poorly understood. Only a few molecules have been shown to be important for ARM so far. This study demonstrates that Brp in the Kenyon cells of the mushroom body is preferentially required for ARM. Although there is no apparent developmental defect in the downregulation of Brp, the requirement for ARM in the adult was not specified in previous studies (Knapek, 2011).

The Brp protein is specifically localized to the active zone at the presynaptic terminals, in which it forms electron dense projections. Interestingly, the Radish protein that is also required for ARM is highly enriched in the lobes of the mushroom body. Thus, Brp and Radish might interact at the active zones to regulate neurotransmission underlying ARM (Knapek, 2011).

Several memory mutants have been shown to have a selective phenotype in ASM. Consistent with the parallel memory formation of ASM and ARM, the brp knockdown and a rutabaga mutation caused an additive memory deficit. Interestingly, Synapsin is required for ASM preferentially, and the null mutation caused no augmentation of the memory phenotype in the rutabaga single mutant (Knapek, 2010). Thus, the complementary forms of memory might recruit differential signaling mechanisms that rely on distinct presynaptic machineries (Knapek, 2011).

In a current model of memory dynamics, ARM gradually develops after training, whereas ASM occupies the largest part of early memory and decays more quickly. Although Radish and Brp are selectively required for ARM measured at 3 h after training, flies lacking either of the proteins are impaired also in immediate memory. By applying cold anesthesia for STM, it was found that STM does contain a significant ARM component. The consistent requirement of Brp for short-term and 3 h ARM may contribute to a synaptic mechanism of memory that is stable against amnesic treatment (Knapek, 2011).

If ARM and ASM were formed at the same synapses of Kenyon cells, how could the two synaptic proteins Brp and Synapsin dissociate these different forms of memory? Notably, Brp and Synapsin are meant to be required for distinct components of action potential-evoked vesicle release. In vertebrates, Synapsin has been shown to be particularly important for recruitment of synaptic vesicles from reserve pools at high stimulation frequencies. Consistently, synapsin mutants show normal quantal content (number of synaptic vesicles released per action potential) at moderate action potential frequencies. Similarly, Drosophila Synapsin maintains the reserve pool of vesicles and mediates mobilization of the reserve pool during intense stimulation. The brp null mutant in contrast shows decreased quantal contents particularly in response to the first arrival of an action potential. Vesicle release after subsequently following high-frequency spikes however is less affected, suggesting the importance in vesicle release at low-frequency stimulation. The two different modes of neurotransmission (e.g., different release probabilities during high- vs low-frequency stimulation) could therefore differentiate ASM and ARM, even if the traces of these different forms of memory resided in the same synapses of the Kenyon cells (Knapek, 2011).

Alternatively, the memory traces of ASM and ARM could be spatially separated within the same neurons, i.e., localized at different synapse populations. In the lobes, Kenyon cell axons have multiple compartments that are intersected by transverse extrinsic neurons. This study found that brp knockdown in the α/β neurons affected ARM. This is consistent with a previous report, in which inhibition of the output of the α/β neurons impaired ARM. Interestingly, inhibition of a specific type of dopaminergic neurons that synapse onto another restricted compartment of the β lobe selectively affected ASM (Aso, 2010). Thus, associative plasticity underlying ASM and ARM could be formed by stimulating different synapses of the same neurons. This hypothesis may be tested in future by the identification of extrinsic neurons that are specifically required for ARM and corresponding functional imaging of memory traces (Knapek, 2011).

Defects in Synapse Structure and Function Precede Motor Neuron Degeneration in Drosophila Models of FUS-Related ALS

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease that leads invariably to fatal paralysis associated with motor neuron degeneration and muscular atrophy. One gene associated with ALS encodes the DNA/RNA-binding protein Fused in Sarcoma (FUS). There now exist two Drosophila models of ALS. In one, human FUS with ALS-causing mutations is expressed in fly motor neurons; in the other, the gene cabeza (caz), the fly homolog of FUS, is ablated. These FUS-ALS flies exhibit larval locomotor defects indicative of neuromuscular dysfunction and early death. The locus and site of initiation of this neuromuscular dysfunction remain unclear. This study shows that in FUS-ALS flies, motor neuron cell bodies fire action potentials that propagate along the axon and voltage-dependent inward and outward currents in the cell bodies are indistinguishable in wild-type and FUS-ALS motor neurons. In marked contrast, the amplitude of synaptic currents evoked in the postsynaptic muscle cell is decreased by >80% in FUS-ALS larvae. Furthermore, the frequency but not unitary amplitude of spontaneous miniature synaptic currents is decreased dramatically in FUS-ALS flies, consistent with a change in quantal content but not quantal size. Although standard confocal microscopic analysis of the larval neuromuscular junction reveals no gross abnormalities, superresolution stimulated emission depletion (STED) microscopy demonstrates that the presynaptic active zone protein Bruchpilot is aberrantly organized in FUS-ALS larvae. The results are consistent with the idea that defects in presynaptic terminal structure and function precede, and may contribute to, the later motor neuron degeneration that is characteristic of ALS (Shahidullah, 2013).

Age-associated increase of the active zone protein Bruchpilot within the honeybee mushroom body

In honeybees, age-associated structural modifications can be observed in the mushroom bodies. Prominent examples are the synaptic complexes (microglomeruli, MG) in the mushroom body calyces, which were shown to alter their size and density with age. It is not known whether the amount of intracellular synaptic proteins in the MG is altered as well. The presynaptic protein Bruchpilot (BRP) is localized at active zones and is involved in regulating the probability of neurotransmitter release in the fruit fly, Drosophila melanogaster. This study explored the localization of the honeybee BRP (Apis mellifera BRP, AmBRP) in the bee brain and examined age-related changes in the AmBRP abundance in the central bee brain and in microglomeruli of the mushroom body calyces. Predominant AmBRP localization is reported near the membrane of presynaptic boutons within the mushroom body MG. The relative amount of AmBRP was increased in the central brain of two-week old bees whereas the amount of Synapsin, another presynaptic protein involved in the regulation of neurotransmitter release, shows an increase during the first two weeks followed by a decrease. In addition, an age-associated modulation was demonstrated of AmBRP located near the membrane of presynaptic boutons within MG located in mushroom body calyces where sensory input is conveyed to mushroom body intrinsic neurons. The observed age-associated AmBRP modulation might be related to maturation processes or to homeostatic mechanisms that might help to maintain synaptic functionality in old animals (Gehring, 2017).

In Drosophila, it has been demonstrated that Bruchpilot (DmBRP) in Kenyon cells plays a critical role in the formation of an anesthesia-resistant memory (Knapek, 2011): a 70% reduction of DmBRP in the Kenyon cells reduces this type of memory significantly. Accordingly, an age-associated increase of BRP, as observed in this experiment, might facilitate memory formation in fruit fly and possibly also in honeybees. However, Gupta (2016) demonstrated that an age-induced increase of DmBRP, which could be mimicked by an increase of the BRP copy number, did not facilitate anesthesia-resistant memory but instead blocked a cold-sensitive, anesthesia-sensitive memory. Based on these results, it was proposed that, in the Drosophila nervous system, aging synapses might steer towards the upper limit of their operational range by increasing BRP levels. This age-dependent process might limit synaptic plasticity and contribute to impairment of memory formation with age (Gehring, 2017).

Previous studies demonstrated that the packing density of boutons in lip and dense collar decreases with age resulting in fewer boutons in a defined area, i.e. a region of interest (ROI), of these neuropils. Thus, one would predict that presynaptic proteins in lip and dense collar are decreasing with age due to the decreased packing density of boutons resulting in fewer boutons per ROI that were analyzed. Indeed, this prediction proves true for Synapsin in the dense collar in this study, since an age-associated reduction was observed of the number of anti-SYNORF1-positive pixels. However, this is not the case for Synapsin in the lip where the number of anti-SYNORF1-positive pixels does not change with age. What might be the reason for this finding? It was shown that, in addition to the decrease in density, the mean volume of individual boutons increases with age in the lip and the dense collar. This increase is stronger in the lip than in the collar. Thus, the decrease in bouton density and the increase in bouton volume most likely counteract each other in the lip and this might be the reason why no change is seen in the amount of Synapsin in the lip (Gehring, 2017).

As it is the case with Synapsin, age-associated alterations in the structural organization of lip and collar boutons might influence the detection of anti-BRP^last200-positive pixels. Thus, the ratio between the median number of anti-BRP^last200-positive pixels to the median number of anti-SYNORF1-positive pixels per ROI was calculated, thereby factoring out the influence of morphological changes in the density and volume of the boutons on the detection of anti-BRP^last200-positive pixels. The ratios, i.e. the relative area, and thus probably the amount, of AmBRP increased in an age-associated manner in both, lip and collar: In the dense collar and the lip, the relative amount of AmBRP is significantly increased in 43-day-old bees. In addition, an increase was observed in the relative amount of AmBRP in the first week after emergence in the lip (Gehring, 2017).

AmBRP is a protein predominately located at presynapses. Due to the age-associated increase in bouton volume, boutons with a larger surface might also have more active zones. Increased numbers of active zones per bouton would lead to increased AmBRP levels which would provide an explanation for the observed age-associated increase in the relative amount of AmBRP. Indeed, this hypothesis could hold true for the collar as it was shown that the number of active zones per bouton is increased in 35-day-old bees compared with 1-day-old bees and that the proportion of ribbon vs. non-ribbon type active zones is increased in 35-day-old bees compared to 1-day-old bees. The latter is interesting, because ribbon-synapses in bees resemble T-bar-shaped synapses in fruit flies, that contain BRP, whereas non-ribbon synapses do not resemble this synapse type. Thus, these data are in line with findings of an increase of AmBRP from day 1, day 8 and day 15 to day 43 (Gehring, 2017).

In contrast to the collar, the number of active zones per bouton remains unchanged between 1- and 35-day-old bees in the lip. However, the same study showed that also in lip boutons the proportion of ribbon vs. non-ribbon type active zones increases. Thus, the AmBRP increase in the lip might not be indicative for the formation of new active zones and thus new synapses. Rather, it is suggested that, in the lip, it is the amount of AmBRP at existing active zones that is altered in an age-associated manner. As mentioned above, this alteration seems to take place twice: Early after emergence and late in the bees' lifetime between day 29 and 43. It might well be that an alteration of the amount of AmBRP at existing synapses shifts the proportion of ribbon vs non-ribbon active zones such that ribbon-active zones are increasing in an age-dependent manner (Gehring, 2017).

What might be the cause of the observed age-associated alterations of AmBRP in the lip and collar? Based on the existing literature, the first increase of AmBRP in the lip could be due to maturation processes in the olfactory system. The lip can be regarded as part of this olfactory system as projection neurons from the antennal lobes convey odor information onto MB Kenyon cells in this region. Neuropils belonging to the olfactory system such as the antennal lobes are not yet fully developed in newly emerged bees and mature during the first days after emergence. These maturation processes occurring in the antennal lobe might also influence synaptic connections, and thereby probably the amount of AmBRP, in upstream odor processing centers such as the lip (Gehring, 2017).

In addition to an AmBRP increase during the first week of a bee's life, increased AmBRP levels were found in very old bees (43-day-old) in the lip, but also in the collar. Similar results were observed at neuromuscular junctions of aged fruit flies (Gupta, 2016). The authors found that, with progression of age, the number of BRP-labeled spots, which indicate active zones, per bouton increased up to an age of 42 days and that this increase is accompanied by an increase in bouton volume. It is known from studies on endocytosis mutants, that an increase in number of boutons and active zones compensates a decrease of synaptic vesicle exocytosis. Thus, increased AmBRP levels at boutons in older insects might represent compensatory mechanisms for age-associated lower synaptic transmission. This hypothesis is in line with the view that age-associated synaptic alterations might be the consequence of adaptive processes due to neuronal plasticity that compensate for age-dependent cognitive impairments. Indeed, it has been demonstrated that a drop in postsynaptic excitability drives an increase of presynaptic scaffolds. According to the authors, this increase of presynaptic scaffolds might lead to an increase of synaptic vesicle release, which has been shown to be age-dependent (Gupta, 2016). In line, in a fruit fly model of Alzheimer's disease, an age-dependent reduction of the amount of BRP and the synaptic vesicle release probability has been observed suggesting that presynaptic β-amyloid plaques in the fruit fly brain might hinder a compensation of age-dependent processes that could be related to the amount of BRP (Gehring, 2017).

A striking feature of honeybee workers is their age-related division of labor. Individual workers perform different tasks within and outside the hive in an age-dependent manner: For the first 2-3 weeks after adult emergence, workers perform in-hive duties such as brood care and food processing, and start to forage for nectar and pollen outside the hive thereafter. This behavioral plasticity has been suggested to have both age- and experience-related determinants. Therefore, it should be taken into account that age-associated processes observed in honeybees are not only due to their chronological age but also due to the task they fulfill because of their age and because of the state of the colony. Thus, the age-associated effects observed in this study could be due to the (unknown) age-dependent signal that triggers the switch between the two tasks, due to experiences made when fulfilling the age-associated task, or due to the internal state of the colony. In the latter case, the observed effects would not be due to the bees' age but to the state of the colony. Since bees of defined ages were observed in a colony that was not manipulated, it is proposed that this study observed 'normally' aging bees and that the effects that were observed are directly or indirectly associated with the bees' age (Gehring, 2017).

This study reports that the level of the presynaptic proteins, Synapsin and AmBRP, are modified in an age-associated manner in the honeybee brain. An early increase was found in the relative amount of AmBRP during the first week after emergence in the MB lip, which was hypothesized to be due to maturation processes in the olfactory system. This study has shown that both MB regions, lip and collar, have increased amounts of AmBRP in 43-day-old bees. Given that BRP is homologous to the vertebrate ELKS/CAST/ERC protein, which is part of the presynaptic active zone, it will be interesting if these proteins are altered in an age-associated manner in vertebrates as well and if an AmBRP increase compensates for age-dependent cognitive impairments (Gehring, 2017).

Vesicle tethering on the surface of phase-separated active zone condensates from rat brains

Tethering of synaptic vesicles (SVs) to the active zone determines synaptic strength, although the molecular basis governing SV tethering is elusive. This study discovered that small unilamellar vesicles (SUVs) and SVs from rat brains coat on the surface of condensed liquid droplets formed by active zone proteins RIM, RIM-BP, and ELKS via phase separation. Remarkably, SUV-coated RIM/RIM-BP condensates are encapsulated by synapsin/SUV condensates, forming two distinct SUV pools reminiscent of the reserve and tethered SV pools that exist in presynaptic boutons. The SUV-coated RIM/RIM-BP condensates can further cluster Ca(2+) channels anchored on membranes. Thus, this study has reconstituted a presynaptic bouton-like structure mimicking the SV-tethered active zone with its one side attached to the presynaptic membrane and the other side connected to the synapsin-clustered SV condensates (see Drosophila Synapsin). The distinct interaction modes between membraneless protein condensates and membrane-based organelles revealed here have general implications in cellular processes, including vesicular formation and trafficking, organelle biogenesis, and autophagy (Wu, 2020).

Active zones in presynaptic terminal boutons of synapses are intimately involved in neurotransmitter release, because active zones are responsible for tethering and docking synaptic vesicles (SVs) to be near release sites at plasma membranes, priming SVs for action potential-evoked fusion and clustering voltage-gated Ca2+channels (VGCCs) for precise spatiotemporal control of Ca2+level at the release sites. Electron microscopy and super-resolution optical imaging experiments have shown that active zone proteins, including RIM, RIM-BP, ELKS, Munc13, and Liprin, are located within a narrow space of <100 nm from the presynaptic terminal membranes. Biochemically, these active zone proteins are all multi-domain scaffold proteins without intrinsic enzymatic activities, and these proteins can form an intricate molecular network by specific protein-protein interactions, suggesting that active zones are large and dense protein-based molecular assemblies. Such active zone assemblies were proposed to be anchored to presynaptic plasma membranes by directly binding to lipids or transmembrane proteins, such as LAR and VGCCs, but none of them alone has been proved to be essential. Genetic and cell biology studies have shown that these proteins function together and in redundant ways in organizing active zones and in modulating neurotransmitter releases. For example, removal of each active zone component in rodents individually showed little impact on the active zone structures and vesicle tethering to active zones, except that removal of RIM led to 50% reduction of tethered vesicles. Combined removals of RIM with RIM-BP or with ELKS led to disintegration of active zone molecular assemblies, near-total loss of vesicle tethering and docking, and dramatic impairments of synaptic release. It had been recently shown that RIM and RIM-BP, when mixed in vitro, can autonomously form condensed molecular assembly via phase separation. The RIM/RIM-BP condensates can cluster the cytosolic tail of VGCC (VGCC-CT) tethered to lipid membranes, forming highly concentrated RIM/RIM-BP/VGCC-CT assembly on the surface of membranes. The formation of condensed RIM/RIM-BP/VGCC-CT on the surface of lipid membranes via phase separation provides an explanation to how condensed active zone protein assemblies, which are not enclosed by any physical barriers, can form at presynaptic boutons. The highly dynamic nature of the RIM/RIM-BP/VGCC-CT condensates may also explain why the dense projection structures in active zones can be observed by electron microscopy (EM) only when synapses are chemically fixed. The presence of dense RIM clusters within the active zone has also been observed in living neurons. Phase separation-mediated formation of condensed molecular assemblies has also been suggested to form the postsynaptic density and for AMPA receptor synaptic clustering and transmission (Zeng, 2019). Formation of condensed molecular assemblies via phase separation is likely to be advantageous for synapse formation and plasticity, although such research is in its early stage (Wu, 2020).

Although tethering of SVs to release sites of presynaptic membranes requires the active zone, how SVs are physically tethered to active zones is not known. Recently, Milovanovic (2018) showed that lipid vesicles can be clustered by coacervation with the synapsin condensates, providing a mechanism for maintaining large reserve pool SVs away from active zones. However, numerous EM studies have shown that tethered SVs are discretely distributed within active zone sheets and the number of tethered SVs is linearly proportional to the areas of active zone sheets. In EM images of chemically fixed synapses, SVs are evenly separated by the dense projections, which are grid-like structures on active zone sheets. In addition, perturbation of synapsin affects only the reserve pool SVs, and the tethered pool remains unchanged. Conversely, disruption of active zone scaffolds impairs only the tethered pool SVs without changing the reserve pool. Thus, tethering of SVs to the active zones is likely mediated by mechanisms that are very different from that seen in the reserve pool SV clustering by synapsin. This study demonstrates that negatively charged liposomes, as well as SVs purified from rat brains, coat on the surface of the phase-separated RIM/RIM-BP condensates. By tethering VGCC-CT to the giant unilamellar vesicle (GUV) membranes, this study shows that the RIM/RIM-BP condensates can bind to and cluster VGCC-CT on GUV membranes on one hand and link SUVs on the other hand, resembling active zone-mediated tethering of SVs to presynaptic membrane release sites. Remarkably, the synapsin-clustered SUV condensates and the SUV-coated RIM/RIM-BP condensates form phase-in-phase assemblies, which recapitulate coexistence of reserved and tethered pools of SVs in presynaptic boutons. The finding that the RIM/RIM-BP condensates can simultaneously link two totally different types of membrane-based cellular organelles suggests a wide range of unanticipated cellular roles of membraneless biological condensates formed by phase separation (Wu, 2020).

Tethered SVs are typically defined by their physical distance(30 nm or less) to the presynaptic plasma membranes. Decades of past research have identified molecules, including RIM, RIM-BP,ELKS, and Munc13, that are critical for SVs to tether and dock to presynaptic membranes. However, how SVs are retained at the active zone remains elusive. This study discovered that SVs coat on the surface of condensed droplets formed by the active zone proteins, including RIM, RIM-BP, and ELKS, via phase separation. This finding may explain several observations made in past decades of studies. First, the number of tethered SVs is proportional to the surface area of the active zone. Second, SVs coated on the surface of RIM/RIM-BP condensates are highly dynamic but are very close to both fusion machineries and presynaptic plasma membranes; thus, such active zone tethered SVs are primed for rapid fusion. Third, the RIM/RIM-BP condensates can cluster Ca2+ channels on the membranes. The direct coating of SVs on the surface of the RIM/RIM-BP droplets can physically link clustered Ca2+ channels with SVs for Ca2+-regulated fusion reactions. Fourth, formation of the RIM/RIM-BP condensates can greatly enhance the weak interaction between RIM/RIM-BP and negatively charged lipids on SVs, and thus allow stable interaction between the active zone molecular assembly and SVs. Fifth, the binding of SVs to the liquid-like active zone may shape the active zone into grid-like structures observed by EM under chemical fixing conditions. The condensed droplets formed by proteins, including RIM, RIM-BP, and ELKS, are highly dense but still very dynamic. Chemical fixation may capture such dynamic but dense assemblies referred to as dense projections. The coating of SVs on the surfaces of active zones likely involves proteins in addition to RIM, RIM-BP, and ELKS examined in this study. Proteins such as Munc13, Munc18, Liprin, Piccolo, Bassoon,SNAREs, the Rab family small GTPases, synaptotagmins, and so on can modulate SV docking and priming. These proteins may also participate in the regulation of active zone assembly and SVs coating. It is noted that the SV-coated RIM/RIM-BP condensates are not physically insulated by SVs. Proteins can readily enter in or escape from the SV-coated active zone protein condensates. Thus, such SV-coated active zone protein condensates can connect with molecular components both near and distal to the presynaptic membranes (Wu, 2020).

Another major finding of this study is that interactions of SVs with different protein condensates formed via phase separation can be radically different. SVs co-phase separate with synapsin, leading to clustering and concentration of SVs. Given that synapsin is massively concentrated in presynaptic boutons, the protein is ideally suited for maintaining the vast majority of SVs as the reserve pool. Strikingly, SVs do not enter the condensed phase formed by the active zone proteins, but instead coat on the surface of the condensates. Remarkably, the synapsin/SUV condensates encapsulate the SUV-coated RIM/RIM-BP droplets when the two phase-separated condensates are mixed, suggesting that the synapsin-clustered reserve SV pool can co-exist with the active zone-tethered SVs, and the two pools of SVs can exchange with each other. Finally, this study has been able to reconstitute a minimalistic presynaptic bouton-like structure, in which the active zone-coated vesicles are attached to the presynaptic plasma membrane and the synapsin-clustered reserve pool SV condensates are situated more distal to the presynaptic plasma membrane but directly interact with the active zone condensates. This reconstituted system recapitulates the basic features of the SV clustering and tethering in presynaptic boutons. In real synapses, the presynaptic plasma membranes together with various membrane proteins likely serve as the starting layer structure to organize the subsequent active zone layer, tethered SVs, and synapsin-clustered reserve pool SVs, forming the elaborate and polarized multiphase organizations. It should be noted that the scale/size of the reconstituted, presynaptic bouton-like assemblies here is much larger than the sizes of real presynaptic boutons. The awkward size of pre-synaptic boutons has presented challenges to investigate whether the layered structures observed in EM studies are indeed formed via the multiphase organization seen in this in vitro study. New technologies will need to be developed to answer this question in the future (Wu, 2020).

In a broader cell biology context, distinct mode of interactions between membraneless organelles and various membrane demarcated organelles, similar to what is observed in this study, may be broadly adopted by cells to modulate processes such as organelle biogenesis, vesicle formation and trafficking, autophagy, and membrane-associated signaling assembly formation (Wu, 2020).

Assembly of synaptic active zones requires phase separation of scaffold molecules

The formation of synapses during neuronal development is essential for establishing neural circuits and a nervous system. Every presynapse builds a core 'active zone' structure, where ion channels cluster and synaptic vesicles release their neurotransmitters. Although the composition of active zones is well characterized, it is unclear how active-zone proteins assemble together and recruit the machinery required for vesicle release during development. This study found that the core active-zone scaffold proteins SYD-2 (also known as liprin-alpha) and ELKS-1 undergo phase separation during an early stage of synapse development, and later mature into a solid structure. The in vivo function of phase separation was directly tested by using mutant SYD-2 and ELKS-1 proteins that specifically lack this activity. These mutant proteins remain enriched at synapses in Caenorhabditis elegans, but show defects in active-zone assembly and synapse function. The defects are rescued by introducing a phase-separation motif from an unrelated protein. In vitro, the SYD-2 and ELKS-1 liquid-phase scaffold was reconstructed and found that it is competent to bind and incorporate downstream active-zone components. The fluidity of SYD-2 and ELKS-1 condensates is essential for efficient mixing and incorporation of active-zone components. These data reveal that a developmental liquid phase of scaffold molecules is essential for the assembly of the synaptic active zone, before maturation into a stable final structure (McDonald, 2020).

The liquid-liquid phase separation (LLPS) motifs that were identified in SYD-2 and ELKS-1 are complex and multivalent. Both motifs are enriched in residues that are capable of pi contacts, but neither is particularly repetitive or of low complexity-properties of certain other LLPS motifs. Although these motifs function in the same cellular structure, they are not interchangeable, and only an exogenous motif from the unrelated FUS protein is able to rescue the syd-2(IDRΔ) defects. It is unsurprising that not all LLPS motifs are interchangeable, but it is not immediately clear from measurable in vitro properties what differentiates them. It is likely that distinct LLPS motifs are tuned for specific properties by complex structural mechanisms that are only beginning to be understood (McDonald, 2020).

Functionally, it was found, surprisingly, that LLPS of SYD-2 and ELKS-1 is not important for their synaptic localization. Instead, LLPS seems to be essential for incorporating binding partners out of the axonal cytoplasm. In addition to the recruitment of active-zone components, in vitro results suggest that the liquid properties of condensates may have an organizational role in homogenizing active-zone components before they solidify into a final stable structure. A fluid condensate would be capable of incorporating and evenly mixing binding partners; by contrast, such binding partners might interact only with the outer shell of a static structure. The components of the active zone form a complex web of interactions; perhaps in the LLPS milieu these interactions attach and align over time, gradually solidifying the structure. It is also conceivable that the observed solidification represents a conversion to a gel-like state, as seen in vitro in this study and with other LLPS motifs. The mixing enabled by an LLPS might be important for macromolecular structures that are built by large numbers of proteins with complex interactions with each other (McDonald, 2020).

It has been reported that RIM/UNC-10 and RIM-BP-two other components of the active zone-also phase separate when mixed in vitro. This study has shown here that the recruitment of UNC-10/RIM is downstream of the LLPS of SYD-2. The functional importance of RIM/UNC-10 and RIM-BP LLPS remains to be tested in vivo, but in vitro it is seen that SYD-2, ELKS-1 and UNC-10 can assemble into the same condensate. Distinct from the active-zone structure, but nearby in the presynapse, the synapsin protein also phase separates in order to cluster synaptic vesicles. Whether and how each of these condensates interacts during the formation and function of synapses requires further study. In sum, phase separation by scaffolding proteins may be a central assembly mechanism that drives the formation of synapses (McDonald, 2020).

Search PubMed for articles about Drosophila Bruchpilot

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date revised: 22 August 2023

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