InteractiveFly: GeneBrief

Neuropilin and tolloid-like: Biological Overview | References

BIOLOGICAL OVERVIEW

Neurotransmitter receptor recruitment at postsynaptic specializations is key in synaptogenesis, since this step confers functionality to the nascent synapse. The Drosophila neuromuscular junction (NMJ) is a glutamatergic synapse, similar in composition and function to mammalian central synapses. Various mechanisms regulating the extent of postsynaptic ionotropic glutamate receptor (iGluR) clustering have been described, but none are known to be essential for the initial localization and clustering of iGluRs at postsynaptic densities (PSDs). This study identified and characterized the Drosophila neto (neuropilin and tolloid-like) as an essential gene required for clustering of iGluRs (GluRIIA, GluRIIB, and GluRIIC) at the NMJ. Neto colocalizes with the iGluRs at the PSDs in puncta juxtaposing the active zones. neto loss-of-function phenotypes parallel the loss-of-function defects described for iGluRs. The defects in neto mutants are effectively rescued by muscle-specific expression of neto transgenes. Neto clustering at the Drosophila NMJ coincides with and is dependent on iGluRs. These studies reveal that Drosophila Neto is a novel, essential component of the iGluR complexes and is required for iGluR clustering, organization of PSDs, and synapse functionality (Kim, 2012).

Once neurons reach their correct postsynaptic targets, a cascade of events marks the beginning of synaptogenesis. The pre- and postsynaptic compartments are kept in register by adhesion molecules, while active zone precursor vesicles and synaptic vesicles arrive at the presynaptic specialization. The assembly of the presynaptic active zones appears to involve the delivery of prefabricated transport packets, although sequential arrival of components has been observed at specialized synapses. The postsynaptic assembly, however, seems to largely depend on gradual de novo clustering of component proteins. The formation of the postsynaptic densities (PSDs) culminates with the recruitment of neurotransmitter receptors. Neuronal activity triggers further synthesis and aggregation of receptor complexes and synapse maturation, stabilization, and growth (Kim, 2012 and references therein).

In contrast to the rich understanding of nicotinic acetylcholine receptor (nAChR) clustering at the mammalian neuromuscular junction (NMJ), clustering of the ionotropic glutamate receptors (iGluRs) that form the majority of central synapses remains less understood. Considerable advances have been made toward identifying proteins that interact with the C-terminal tails of iGluRs and regulate their membrane trafficking, anchoring at the synapses, and involvement in intracellular signaling cascades. In the postsynaptic compartment, proteins that contribute to glutamate receptor clustering at the synapses include PDZ domain-containing proteins, cytoskeleton-binding and scaffolding components, and proteins that control endosomal trafficking. Receptor trafficking and assembly signals have also been found in the N-terminal domains of the iGluRs. Moreover, recent studies using reconstituted synapses have identified a number of presynaptic adhesion molecules and secreted factors that participate in receptor clustering through trans-synaptic protein interactions. For example, Narp (neuronal activity-regulated pentraxin) or other pentraxins secreted from the presynaptic neurons (NP1 and NRP) bind to the N-terminal domain of GluA4 and are critical trans-synaptic factors for GluA4 recruitment at the synapses. The direct coupling of the N-terminal domain of GluA2 to N-cadherin promotes enrichment of AMPA receptors at synapses and maturation of spines, although this interaction could occur in cis or in trans, since N-cadherin is present on both pre- and postsynaptic membranes. These trans-synaptic clustering strategies apply to subsets of iGluR subunits, and it is not clear whether they have a central role in the organization of postsynaptic domains in vivo or rather provide modulatory functions (Kim, 2012).

The Drosophila NMJ is a glutamatergic synapse similar in composition and function to the mammalian central AMPA/Kainate synapses. The fly NMJ iGluRs are heterotetrameric complexes composed of three essential subunits—IIC, IID, IIE—and either IIA or IIB. Type A and type B receptor complexes differ in their single-channel properties, synaptic responses and localization, and regulation by second messengers. Previous studies have shown that the nascent synapses are predominantly type A complexes and change their subunit compositions toward more B-type complexes upon maturation that relies at least in part on CaMKII activity (Kim, 2012 and references therein).

How do iGluR complexes traffic to and cluster at the NMJ? In flies, none of the NMJ iGluR subunits have PDZ- binding motifs. Live-imaging studies on growing synapses have shown that iGluRs from diffuse extrasynaptic pools stably integrate into immature PSDs, but Discs large (Dlg), the fly PSD-95 ortholog, and other postsynaptic proteins remain highly mobile. Dlg does not colocalize with the iGluR receptors at the PSDs and instead is adjacent to the PSDs. Moreover, iGluRs are localized and clustered normally at the NMJ of dlg mutants, although the type B receptor is reduced in levels. The only protein shown to bind directly to iGluR subunits is Coracle, a homolog of mammalian brain 4.1 proteins. Coracle appears to stabilize type A but not type B receptors by anchoring them to the postsynaptic spectrin-actin cytoskeleton (Chen, 2005). Several more postsynaptic proteins have been identified that regulate the subunit compositions and the extent of iGluR synaptic localization, but no molecules other than the receptors themselves were shown to be absolutely required for clustering of the receptor complexes (Kim, 2012).

One possible link in understanding the trafficking and clustering of iGluRs at the fly NMJ could be provided by the emerging families of auxiliary subunits. Auxiliary subunits are transmembrane proteins that avidly and selectively bind to mature iGluRs and form stable complexes at the cell surface. They can modulate the functional characteristics of iGluRs and may also mediate surface trafficking and/or targeting to specific subcellular compartments (Jackson, 2011). Auxiliary proteins described so far include stargazin and its relatives (Tomita, 2003; Milstein, 2008), cornichon homolog-2 and homolog-3 (Schwenk, 2009), Cysteine-knot AMPAR-modulating protein (von Engelhardt, 2010), SynDIG1 (Kalashnikova, 2010), neuropillin and tolloid-like proteins Neto1 and Neto2 (Ng, 2009; Zhang, 2009), and Caenorhabditis elegans SOL-1 (Zheng, 2004). Studies in tissue culture and heterologous systems suggested that some of the auxiliary subunits have the potential to contribute to clustering of iGluRs, since they promote the accumulation of receptors at the cell surface (for review, see Jackson, 2011). However, no auxiliary protein has been implicated in the clustering of iGluRs in vivo. In fact, it is unclear whether surface iGluRs must be associated with auxiliary subunits to be functional. For C. elegans, auxiliary subunits are essential for functional receptors, but for vertebrate and Drosophila iGluRs, this remains an open question (Kim, 2012).

Drosophila has several genes reported to encode for auxiliary subunits, including a stargazin-type molecule (Stg1) (Liebl, 2008), two cornichon proteins (cni and cnir), the SOL-1-related protein CG34402 (Walker, 2006), and one Neto-like protein. Among them, neto mRNA was found to be expressed in the Drosophila striated muscle. Similar to vertebrate Neto1 and Neto2, this study found that Drosophila Neto is a multidomain, transmembrane protein with two extracellular CUB (for complement C1r/C1s, UEGF, BMP-1) domains followed by an LDLa (low-density lipoprotein receptor domain class A) motif. Unlike vertebrate Netos, Drosophila neto was found to be an essential locus: neto-null embryos are completely paralyzed and cannot hatch into the larval stages. Flies with suboptimal Neto levels, such as in neto hypomorphs, do not fly and have defective NMJ structure and function. Neto was found to be essential in the striated muscle for the synaptic trafficking and clustering of the iGluRs at the PSDs. Moreover, Neto and iGluR synaptic clustering depend on each other. It is proposed that Neto functions as an essential nonchannel component of the iGluR complexes at the Drosophila NMJ (Kim, 2012).

Drosophila neto is an essential locus that encodes for a protein dynamically expressed throughout development. The neto transcript is maternally loaded, and the protein could be detected by Western analysis at all stages of embryogenesis. In spite of a significant maternal pool, the absence of zygotic neto expression produces 100% embryonic paralysis and lethality, suggesting a crucial role for Neto in the later stages of embryogenesis. Fully penetrant embryonic paralysis has been described only for two types of mutants: with defects in epithelial integrity or with nonfunctional NMJ. In the first class, disruption of the blood-brain barrier allows for the potassium-rich hemolymph to flood the CNS, causing hyperactivity and action potential failure. The second class includes mutants that impair the NMJ function. Muscle expression of Neto rescued the lethality and defects of neto- null mutants, indicating an essential role for Neto at the NMJ. These findings fit best with a model in which Neto and iGluRs are engaged in targeting each other to PSDs via direct interaction. In this model, Neto functions as a nonchannel, essential subunit of the iGluR complexes (Kim, 2012).

Indeed, neto loss-of-function phenotypes parallel the loss-of-function defects described for iGluR complexes. First, neto-null mutant embryos lack any body wall peristalsis and hatching movements and have no detectable iGluR clusters at the NMJ. Second, the animals with suboptimal Neto levels have a dramatically reduced number of synaptic iGluR clusters and reduced frequency and amplitude of miniature synaptic potentials. The sparse iGluR clusters in neto¹⁰⁹ always colocalize with Neto clusters, indicating that the complexes must contain Neto and iGluRs in order to be incorporated at the PSDs. Finally, Neto-deprived animals exhibit a deficit in the maintenance of mature PSDs. A similar deficit was reported for NMJ synapses developing in the near absence of iGluRs. During synapse formation, iGluR incorporation into the postsynaptic membrane is critical to enlarge PSDs. By clustering in concert to iGluRs, Neto is essential for functional iGluR complexes and directly controls synapse formation at the Drosophila NMJ. An important difference between neto¹⁰⁹ and glutamate receptor hypomorpic mutants is that quantal content remains unchanged in neto¹⁰⁹ and there is no presynaptic compensation, as seen in receptor mutants. The cause for this difference is not understood, but it is speculated that the lack of presynaptic compensation in neto mutants may reflect a role for Neto in PSD development and maturation and/or in retrograde signaling (Kim, 2012).

Similar to other postsynaptic components, Neto is distributed between junctional and extrajunctional locations on the muscle, as assessed by antibody staining. Outside the NMJ, Neto appears tightly associated with the muscle membrane in a pattern reminiscent of the T tubules. This distribution suggests that Neto could traffic on the muscle surface and perhaps could be mobilized to the junctions as needed. Fully functional iGluR complexes were also detected on the muscle surface at extrajunctional locations (Kim, 2012).

One way in which Neto could control the iGluR clustering is by engaging the receptor complexes on the muscle membrane followed by trafficking to the synaptic junction. This model would be consistent with the Neto/iGluR codependence for clustering at the synapse; i.e., only components engaged in a productive complex could traffic and be stabilized at the NMJ. This model also predicts that, at suboptimal Neto levels, iGluRs will accumulate on the muscle surface at extrajunctional locations. Indeed, this seems to be the case, since in neto hypomorphs, GluRIIA was detected on the muscle surface, accessible by antibodies in the absence of membrane-permeable detergents (Kim, 2012).

In addition, Neto may have a regulatory role in the synaptic targeting of the iGluRs and control the extent of iGluR clustering at the NMJ. Neto may receive and integrate signals about the cellular status and transduce that information into targeting a certain amount of receptors to the synapses. The intracellular domain of Neto is rich in putative phosphorylation sites that may be used to modulate Neto engagement of iGluRs or to connect the complexes with motors and scaffold proteins. Several kinases have been described to control the extent of the iGluR accumulation at the NMJ. Their substrates may include Neto as part of signaling networks that couple cell status to growth of postsynaptic structures (Kim, 2012).

Live-imaging studies have shown that iGluRs from diffuse extrasynaptic pools stably integrate into immature PSDs, while other postsynaptic proteins remain highly mobile. Neto may mediate stable incorporation and stabilization of iGluRs to newly formed PSDs. For example, Neto could promote iGluR aggregation via CUB-mediated self-association and/or extracellular interactions. CUB-containing proteins have been implicated in the formation of acetylcholine receptor aggregates in C. elegans (Gally, 2004). In flies and vertebrates, synaptic aggregation of the neurotransmitter receptors at the NMJ does not occur in the absence of innervating neurons. In vertebrates, neuronally secreted agrin participates in extracellular interactions that enable receptor clustering and synapse stabilization. In Drosophila, the molecular mechanisms that underlie the requirement for innervation to initiate synaptogenesis at the NMJ are not known. A forward genetic screen identified Mind the gap (MTG), a presynaptically secreted protein that appears to organize the extracellular millieu, but it is unclear how MTG could induce postsynaptic differentiation (Rohrbough, 2007). Neto may provide an entry point in understanding these requirements. These data indicate that by controlling the iGluRs clustering, Neto plays a significant role in the organization and maintenance of the PSDs. Although Neto does not have a PDZ-binding motif, it may participate in both intracellular and extracellular interactions that help stabilize the PSDs (Kim, 2012).

Vertebrate Netos bind to and have a profound impact on the properties of selective kainate receptors: They modulate the agonist-binding affinities and the off kinetics, thus determining the characteristically slow rise time and decay kinetics of synaptic kainate receptors (Zhang, 2009; Straub, 2011a; Straub, 2011b). A role for vertebrate Netos in surface expression of kainate receptors or their redistribution between synaptic and extrasynaptic locations is less clear at this time, as it appears to depend on specific kainate receptor subunits, the neurons and tissues analyzed, and/or the genetic background of the knockout mice tested (Ng, 2009; Copits, 2011; Straub, 2011a). Nevertheless, it is possible that Drosophila Neto also modulates the ligand-gated channel properties for iGluRs and shapes the function of synapses at the NMJ (Kim, 2012).

Recent work from vertebrates has changed ideas regarding iGluRs: They are not companionless complexes at the PSDs, but rather dynamic supramolecular signaling complexes that include components that regulate the trafficking, scaffolding, stability, signaling, and turnover of the receptors. The discovery of Neto reveals that Drosophila iGluRs also form multisubunit complexes modulated by auxiliary proteins at the fly NMJ. Neto is the first auxiliary iGluR subunit described in Drosophila. In vertebrates, Neto and other auxiliary subunits impart diversity and richness to iGluR function, but no auxiliary protein was shown to be essential for in vivo clustering of the receptors. Auxiliary subunits in C. elegans are essential for functional receptors but not for clustering. The fly Neto is the first example of an auxiliary subunit required for iGluR clustering (Kim, 2012).

An intriguing question is why the requirements for Neto are so different in various species. Neto1/Neto2 double knockout mice have defects in long-term potentiation, learning, and memory but are viable (Tang, 2011). More importantly, Neto1 and Neto2 are not essential for iGluR clustering. In contrast, Drosophila neto-null mutants are embryonic-lethal, and Neto is absolutely required for iGluR clustering. This difference could be due to variations in the properties of individual domains of Netos, or it could reflect the diversity among synapse types and the nature and composition of multiprotein complexes where various Netos function. Indeed, there are primary sequence differences among Netos that could translate into functional differences. For example, the LDLa motif in Neto2 binds Ca2+ (Zhang, 2009); the fly Neto lacks the conserved residues predicted to chelate Ca2+ ions. The fly Neto has a long insert between the signal peptide and the first CUB motif. In all Neto proteins, the intracellular domain is rich in potential phosphorylations sites, but in flies, this domain is very acidic (pI 3.86), unlike Neto1 (pI 8.28) and Neto2 (pI 6.62). Secreted isoforms have been reported/predicted for vertebrate Netos but not for Drosophila. Instead, a new transmembrane Neto isoform has been recently entered in the fly database (cDNA reference RE42119). This isoform is predicted to share the exons encoding for extracellular and transmembrane parts, but has alternative exons to encode for a basic (pI 9.17) intracellular domain, with no similarity with vertebrate proteins. While the validated fly Neto isoform is sufficient to provide the essential Neto activity at the NMJ, it will be interesting to investigate whether flies use multiple Neto isoforms at the NMJ or alternate them for tissue- or synapse-specific functions (Kim, 2012).

In flies, Neto is also expressed in subsets of neurons in the CNS; thus, Neto may have additional functions at glutamatergic central synapses. As in vertebrates, neuronal Neto is not essential; only the NMJ function of Neto is required for viability. While a role for Neto at central synapses remains to be determined, it is tempting to speculate that Drosophila Netos might have attained tissue- or context-specific roles in modulation of iGluRs. Thus, Netos constitute a family of conserved proteins that influence the function of glutamatergic synapses and have acquired species- and tissue-specific roles during evolution (Kim, 2012).

Postsynaptic glutamate receptors regulate local BMP signaling at the Drosophila neuromuscular junction

Effective communication between pre- and post-synaptic compartments is required for proper synapse development and function. At the Drosophila neuromuscular junction (NMJ), a retrograde BMP signal functions to promote synapse growth, stability and homeostasis and coordinates the growth of synaptic structures. Retrograde BMP signaling triggers accumulation of the pathway effector pMad in motoneuron nuclei and at synaptic termini. Nuclear pMad, in conjunction with transcription factors, modulates the expression of target genes and instructs synaptic growth; a role for synaptic pMad remains to be determined. This study reports that pMad signals are selectively lost at NMJ synapses with reduced postsynaptic sensitivities. Despite this loss of synaptic pMad, nuclear pMad persisted in motoneuron nuclei, and expression of BMP target genes was unaffected, indicating a specific impairment in pMad production/maintenance at synaptic termini. During development, synaptic pMad accumulation followed the arrival and clustering of ionotropic glutamate receptors (iGluRs) at NMJ synapses. Synaptic pMad was lost at NMJ synapses developing at suboptimal levels of iGluRs and Neto, an auxiliary subunit required for functional iGluRs. Genetic manipulations of non-essential iGluR subunits revealed that synaptic pMad signals specifically correlate with the postsynaptic type-A glutamate receptors. Altering type-A receptor activities via protein kinase A (PKA) revealed that synaptic pMad depends on the activity and not the net levels of postsynaptic type-A receptors. Thus, synaptic pMad functions as a local sensor for NMJ synapse activity and has the potential to coordinate synaptic activity with a BMP retrograde signal required for synapse growth and homeostasis (Sulkowski, 2013).

Previous work has described Neto as the first nonchannel subunit required for the clustering of iGluRs and formation of functional synapses at the Drosophila NMJ. Neto and iGluR complexes associate in the striated muscle and depend on each other for targeting and clustering at postsynaptic specializations. This study shows that Neto/iGluR synaptic complexes induce accumulation of pMad at synaptic termini in an activity-dependent manner. The effect of Neto/iGluR clusters on BMP signaling is selective, and limited to synaptic pMad; nuclear accumulation of pMad appears largely independent of postsynaptic glutamate receptors. This study demonstrates that synaptic pMad mirrors the activity of postsynaptic type-A receptors. As such, synaptic pMad may function as an acute sensor for postsynaptic sensitivity. Local fluctuations in synaptic pMad may provide a versatile means to relay changes in synapse activity to presynaptic neurons and coordinate synapse activity status with synapse growth and homeostasis (Sulkowski, 2013).

Drosophila NMJs maintain their evoked potentials remarkably constant during development, from late embryo to the third instar larval stages. This coordination between motoneuron and muscle properties requires active trans-synaptic signaling, including a retrograde BMP signal, which promotes synaptic growth and confers synaptic homeostasis. Nuclear pMad accumulates in motoneurons during late embryogenesis. However, embryos mutant for BMP pathway components hatch into the larval stages, indicating that BMP signaling is not required for the initial assembly of NMJ synapses and instead modulates NMJ growth and development. This study demonstrates that synaptic accumulation of pMad follows GluRIIA arrival at nascent NMJs and depends on optimal levels of synaptic Neto and iGluRs. As type-A receptors have been associated with nascent synapses, and type-B receptors mark mature NMJs, accumulation of synaptic pMad appears to correlate with a growing phase at NMJ synapses. Furthermore, synaptic pMad correlates with the activity and not the net levels of postsynaptic type-A receptors. In fact, expression of a GluRIIA variant with a mutation in the putative ion conduction pore triggered reduction of synaptic pMad levels. Thus, synaptic pMad functions as a molecular sensor for synapse activity and may constitute an important element in synapse plasticity (Sulkowski, 2013).

The synaptic pMad pool has been localized primarily to the presynaptic compartment. However, a contribution for postsynaptic pMad to the pool of synaptic pMad is also possible. Postsynaptic pMad accumulates in response to glia-secreted Mav, which regulates gbb expression and indirectly modulates the Gbb-mediated retrograde signaling (Fuentes-Medel, 2012). RNAi experiments revealed that knockdown of mad in muscle induces a decrease in synaptic pMad, albeit much reduced in amplitude compared with knockdown of mad in motoneurons (Fuentes-Medel, 2012). Also, knockdown of wit in motoneurons, but not in muscle, and knockdown of put in muscle, but not in motoneurons, triggers reduction of synaptic pMad (Fuentes-Medel, 2012). Intriguingly, the synaptic pMad is practically abolished in GluRIIA and neto¹⁰⁹ mutants and cannot be further reduced by additional decrease in Mad levels. Whereas loss of postsynaptic pMad could be due to a Mav-dependent feedback mechanism that controls Gbb secretion from the muscle, the absence of presynaptic pMad demonstrates a role for GluRIIA and Neto in modulation of BMP retrograde signaling (Sulkowski, 2013).

As BMP signals are generally short lived, synaptic pMad probably reflects accumulation of active BMP/receptor complexes at synaptic termini. Recent evidence suggests that BMP receptors traffic along the motoneuron axons, with Gbb/receptors complexes moving preferentially in a retrograde direction. By contrast, Mad does not appear to traffic. Thus, Mad is likely to be phosphorylated and maintained locally by a pool of active Gbb/BMP receptor complexes that remain at synaptic termini for the time postsynaptic type-A receptors are active (Sulkowski, 2013).

The activity of type-A glutamate receptors may control synaptic pMad accumulation (1) indirectly via activity-dependent changes that are relayed to both pre- and postsynaptic cells, or (2) directly by influencing the production and signaling of varied Gbb ligand forms or by localizing Gbb activities. For example, inhibition of postsynaptic receptor activity induces trans-synaptic modulation of presynaptic Ca²⁺ influx. Such Ca²⁺ influx changes may trigger events that induce a local change in synaptic pMad accumulation. One possibility is that changes in Ca²⁺ influx may recruit Importin-β11 at presynaptic termini, which in turn mediate synaptic pMad accumulation (Sulkowski, 2013).

At the Drosophila NMJ, Gbb is secreted in the synaptic cleft from both pre- and postsynaptic compartments. The secretion of Gbb is regulated at multiple levels, transcriptionally and post-translationally. Furthermore, the Gbb prodomain could be processed at several cleavage sites to generate Gbb ligands with varying activities. The longer, more active Gbb ligand retains a portion of the prodomain that could influence the formation of Gbb/BMP receptor complexes. Synaptic pMad may result from signaling by selective forms of Gbb. Or type-A receptors could modulate secretion and processing of Gbb in an activity-dependent manner. Understanding the function of different pools and active forms of Gbb within the synaptic cleft will help explain the multiple roles for Gbb at Drosophila NMJs (Sulkowski, 2013).

Alternatively, active postsynaptic type-A receptor complexes may directly engage and stabilize presynaptic Gbb/BMP receptor signaling complexes via trans-synaptic interactions. CUB domains can directly bind BMPs; thus Neto may utilize its extracellular CUB domains to engage Gbb and/or presynaptic BMP receptors. As synaptic pMad mirrors active type-A receptors, such trans-synaptic complexes will depend on Neto in complexes with active type-A receptors. No capture has yet been shown of a direct interaction between Gbb and Neto CUB domains in co-immunoprecipitation experiments. Nonetheless, a trans-synaptic complex that depends on the activity of type-A receptors could offer a versatile means for relaying synapse activity status to the presynaptic neuron via fast assembly and disassembly (Sulkowski, 2013).

Irrespective of the strategy that correlates synaptic pMad pool with the active type-A receptor/Neto complexes, further mechanisms must act to maintain the Gbb/BMP receptor complexes at synapses and protect them from endocytosis and retrograde transport. Such mechanisms must be specific, as general modulators of BMP receptors endocytosis impact both synaptic and nuclear pMad. A candidate for differential control of BMP/receptor complexes is Importin-β11. Loss of synaptic pMad in importin-β11 is rescued by neuronal expression of activated BMP receptors, by blocking retrograde transport, but not by neuronal expression of Mad. As Mad does not appear to traffic, presynaptic Importin-β11 must act upstream of the BMP receptors, perhaps to stabilize active Gbb/BMP receptor complexes at the neuron membrane. By contrast, local pMad cannot be restored at Neto-deprived NMJs by overactivation of presynaptic BMP receptors or by blocking retrograde transport. As neto and gbb interact genetically, it is tempting to speculate that postsynaptic Neto/type-A complexes localize Gbb activities and stabilize Gbb/BMP receptor complexes from the extracellular side. Additional extracellular factors, for example heparan proteoglycans, or intracellular modulators, such as Nemo kinase, may control the distribution of sticky Gbb molecules within the synaptic cleft and their binding to BMP receptors, or may stabilize Gbb/BMP receptor complexes at synaptic termini (Sulkowski, 2013).

Synaptic pMad may act locally and/or in coordination with the transcriptional control of BMP target genes to ensure proper growth and development of the synaptic structures. A presynaptic pool of pMad maintained by Importin-β11 neuronal activities ensures normal NMJ structure and function. Like importin-β11, GluRIIA and Neto-deprived synapses show a significantly reduced number of boutons. Intriguingly, the absence of GluRIIA induces up to 20% reduction in bouton numbers, whereas knockdown of GluRIIB does not appear to affect NMJ growth. Although the amplitude of the growth phenotypes observed in normal culturing conditions (25°C) was modest, this phenomenon may explain the requirement for GluRIIA reported for activity-dependent NMJ development (at 29°C). Furthermore, knockdown of Neto or any iGluR essential subunit affect synaptic pMad and NMJ growth in a dose-dependent manner. Not significant changes were found in nuclear pMad or expression of BMP target genes in GluRIIA or Neto-deprived animals, but the restoration of synaptic pMad by presynaptic constitutively active BMP receptors rescues the morphology and physiology of importin-β11 mutant NMJs. The smaller NMJs observed in the absence of local pMad may reflect a direct contribution of synaptic pMad to retrograde BMP signaling, a pathway that provides an instructive signal for NMJ growth. Thus, BMP signaling may integrate synapse activity status with the control of synapse growth (Sulkowski, 2013).

Synaptic pMad may also contribute to synapse stability. Mutants in BMP signaling pathway have an increased number of 'synaptic footprints': regions of the NMJ where the terminal nerve once resided and has retracted. It has been proposed that Gbb binding to its receptors activates the Williams Syndrome-associated Kinase LIMK1 to stabilize the NMJ. Synaptic pMad may further contribute to the stabilization of synapse contacts by engaging in interactions that anchor the Gbb/BMP receptor complexes at synaptic termini. During neural tube closure, local pSmad1/5/8 mediates stabilization of BMP signaling complexes at tight junction via binding to apical polarity complexes. Flies may utilize a similar anchor mechanism that relies on pMad-mediated interactions for stabilizing BMP signaling complexes and other components at synaptic junctions. Local active BMP signaling complexes are thought to function in this manner in the maintenance of stemness and in epithelial-to-mesenchymal transition (Sulkowski, 2013).

Separate from its role in synapse growth and stability, BMP signaling is required presynaptically to maintain the competence of motoneurons to express homeostatic plasticity. The requirements for BMP signaling components for the rapid induction of presynaptic response may include a role for synaptic pMad in relaying acute perturbations of postsynaptic receptor function to the presynaptic compartment. At the very least, attenuation of local pMad signals, when postsynaptic type-A receptors are lost or inactive, may release local Gbb/BMP receptor complexes and allow them to traffic to neuron soma and increase the BMP transcriptional response, promoting expression of presynaptic components and neurotransmitter release. In addition, synaptic pMad-dependent complexes may influence the composition and/or activity of postsynaptic glutamate receptors. Although future experiments will be needed to address the nature and function of local pMad-containing complexes, the current findings clearly demonstrate that synaptic pMad constitutes an exquisite monitor of synapse activity status, which has the potential to relay information about synapse activity to both pre- and postsynaptic compartments and contribute to synaptic plasticity. As BMP signaling plays a crucial role in synaptic growth and homeostasis at the Drosophila NMJ, the use of synaptic pMad as a sensor for synapse activity may enable the BMP signaling pathway to monitor synapse activity then function to adjust synaptic growth and stability during development and homeostasis (Sulkowski, 2013).

Neto-mediated intracellular interactions shape postsynaptic composition at the Drosophila neuromuscular junction

The molecular mechanisms controlling the subunit composition of glutamate receptors are crucial for the formation of neural circuits and for the long-term plasticity underlying learning and memory. This study use the Drosophila neuromuscular junction (NMJ) to examine how specific receptor subtypes are recruited and stabilized at synaptic locations. In flies, clustering of ionotropic glutamate receptors (iGluRs) requires Neto (Neuropillin and Tolloid-like), a highly conserved auxiliary subunit that is essential for NMJ assembly and development. Drosophila neto encodes two isoforms, Neto-α and Neto-β, with common extracellular parts and distinct cytoplasmic domains. Mutations that specifically eliminate Netoβ or its intracellular domain were generated. When Neto-β is missing or is truncated, the larval NMJs show profound changes in the subtype composition of iGluRs due to reduced synaptic accumulation of the GluRIIA subunit. Furthermore, neto-β mutant NMJs fail to accumulate p21-activated kinase (PAK), a critical postsynaptic component implicated in the synaptic stabilization of GluRIIA. Muscle expression of either Neto-α or Neto-β rescued the synaptic transmission at neto null NMJs, indicating that Neto conserved domains mediate iGluRs clustering. However, only Neto-β restored PAK synaptic accumulation at neto null NMJs. Thus, Neto engages in intracellular interactions that regulate the iGluR subtype composition by preferentially recruiting and/or stabilizing selective receptor subtypes (Ramos, 2015).

At the Drosophila NMJ, Neto enables iGluRs clustering at synaptic sites and promotes postsynaptic differentiation. This study shows that Neto-β, the major Neto isoform at the fly NMJ, plays a crucial role in controlling the distribution of specific iGluR subtypes at individual synapses. Similar to other glutamatergic synapses, the subunit composition determines the activity and plasticity of the fly NMJ. The data are consistent with a model whereby Neto-β, via its conserved domains, fulfills a significant part of Neto-dependent iGluRs clustering activities during synapse assembly. At the same time, Neto-β engages in intracellular interactions that regulate iGluR subtypes distribution by preferentially recruiting and/or stabilizing type-A receptors. In this model, Neto-β could directly associate with the GluRIIA-containing complexes and/or regulate the synaptic abundance of type-A receptors indirectly, by recruiting PSD components such as PAK. Thus, Neto-β employs multiple strategies to control which flavor of iGluR will be at the synapses and to modulate PSD composition and postsynaptic organization (Ramos, 2015).

Neto proteins have been initially characterized as auxiliary subunits that modulate the function of kainate (KA) and NMDA receptors. In vertebrates, Neto1 and Neto2 directly interact with KAR subunits and increase channel function by modulating gating properties. Since loss of KAR currents in mice lacking Neto1 and/or Neto2 exceed a reduction that could be attributed to alterations of channel gating, an additional role for Neto proteins in synaptic targeting of receptors has been proposed. The role for vertebrate Neto proteins in KAR membrane and/or synaptic targeting remains controversial and appears to be cell type-, receptor subunit-, and Neto isoform-dependent. Furthermore, the C. elegans Neto has a very small intracellular domain (24 amino acids beyond the conserved domains). This implies that 1) Neto without an intracellular domain constitutes the minimal conserved functional moiety, and 2) the divergent intracellular domains of Neto proteins may fulfill tissue and/or synapse specific modulatory functions. Indeed, Neto2 bears a class II PDZ binding motif that binds to the scaffold protein GRIP and appears to mediate KARs stabilization at selective synapses (Ramos, 2015).

In flies, Neto is an essential protein that plays active roles in synapse assembly and in the formation and maintenance of postsynaptic structures at the NMJ. The Drosophila Neto isoforms do not have PDZ binding motifs, but they use at least two different mechanisms to regulate the synaptic accumulation and subunit composition of iGluRs. First, Neto participates in extracellular interactions that enable formation of iGluR/Neto synaptic complexes; formation of stable aggregates is presumably prevented by the inhibitory prodomain of Neto. Second, the two Neto isoforms appear to facilitate the selective recruitment and/or stabilization of specific iGluR subtypes. It is speculated that Neto-β may selectively associate with and recruit type-A receptors, perhaps by engaging the C-terminal domain of GluRIIA, which is critical for the synaptic stabilization of these receptors. Aside from a possible role in the selective recruitment of iGluR subtypes, Neto-β participates in intracellular interactions that facilitate the recruitment of PAK at PSDs; in turn, PAK signals through two independent, genetically separable pathways (a) to modulate the GluRIIA synaptic abundance and (b) to facilitate formation of SSR (Ramos, 2015).

Whether Neto-β recruits PAK directly or via a larger protein complex remains to be determined. Neto-β contains an SH3 domain that may bind to the proline-rich SH3 binding domain of PAK. However, in tissue culture experiments, attempts to detect a direct interaction between PAK and Neto-β (full-length or intracellular domain) failed. PAK synaptic accumulation is completely abolished at NMJ with mutations in dPix, although not all dpix defects are mediated through PAK. Conversely, PAK together with Dreadlocks (Dock) controls the synaptic abundance of GluRIIA, while PAK and dPix regulate the Dlg distribution. The reduction of GluRIIA and Dlg synaptic abundance observed at neto-β mutant NMJs suggests that Neto-β may interact with both dPix and Dock and enable both PAK activities. In addition, Neto-β may stabilize postsynaptic type-A receptors by enhancing their binding to Coracle, which anchors GluRIIA to the postsynaptic actin cytoskeleton (Ramos, 2015).

Importantly, this study connects the complex regulatory networks that modulate the PSD composition to the Neto/iGluR clusters themselves. The Neto-β cytoplasmic domain is rich in putative protein interaction motifs, and may function as a scaffold platform to mediate multiple protein interactions that act synergistically during synapse development and homeostasis. Loss of Neto-β-mediated intracellular interactions at netoβshort NMJs reduced the GluRIIA synaptic abundance, but did not affect the GluRIIB synaptic signals. It is unlikely that the remaining cytoplasmic part of Neto-β facilitates the GluRIIB synaptic accumulation at these NMJs at the expense of GluRIIA and PAK. Instead, a model is favored whereby the synaptic stabilization of GluRIIA requires a Neto-β-dependent intracellular network. Disruption of this network diminishes GluRIIA and increases GluRIIB synaptic abundance, pending the availability of limiting subunits, GluRIIC-E and Neto. Conversely, the presence of this network ensures that at least some type-A receptors are stabilized at synaptic sites, even at Neto-deprived synapses, such as in netohypo larvae [12]. Assembly of this network does not require GluRIIA since both Neto-β and PAK accumulated normally at GluRIIA mutant NMJs. Furthermore, in the absence of Neto-β the synaptic abundance of GluRIIA can be partly restored by excess Neto-α or a δ-intracellular Neto variant, suggesting that excess iGluRs 'clustering capacity' overrides the cellular signals that shape PSD composition. What intracellular domain(s) of Neto bind to and how they are modified by post-translational modifications will be critical questions to understand how postsynaptic composition is regulated during development and homeostasis (Ramos, 2015).

The discovery of Drosophila Neto isoforms with alternative cytoplasmic domains and isoform specific activities expands the repertoire of Neto-mediated functions at glutamatergic synapses. All Neto proteins share the highly conserved CUB1, -2, LDLa and transmembrane domains that have been implicated in engaging and modulating the receptors, the central function of Neto proteins. In flies this conserved part is both required and sufficient for iGluRs clustering and NMJ development. In C. elegans the entire Neto appears to be reduced to this minimal functional unit. The only exception is a retina-specific CUB1-only Neto1 isoform with unknown function. In contrast to shared domains, the cytoplasmic domains are highly divergent among Neto proteins. This diversity might have evolved to facilitate intracellular, context specific function for Neto proteins, such as the need to couple the iGluR complexes to neuron or muscle specific scaffolds in various phyla. By engaging in different intracellular interactions, via distinct cytoplasmic domains, different Neto isoforms may undergo differential targeting and/or retention at the synapses and thus acquire isoform-specific distributions and functions within the same cell (Ramos, 2015).

Phylogenetic analyses indicate that the intracellular domains of Neto are rapidly evolving in insects. Blocks of high conservations could be clearly found in the genome of short band insect Tribolium castaneum (Coleoptera) or in Apis mellifera (Hymenoptera). However, most insects outside Diptera appear to have only one Neto isoform, more related to Neto-β. In fact, the only Neto-α isoform outside Drosophila was found in Musca domestica (unplaced genomic scaffold NCBI Reference Sequence: XM_005187241.1). Other neto loci, from Hydra to vertebrates, appear to encode Neto proteins with unique and highly divergent intracellular domains. An extreme example is the C. elegans Neto/Sol-2, with a very short cytoplasmic tail, which requires additional auxiliary subunits, Sol-1 and Stargazin, to control the function of glutamate receptors. Neto proteins appear to utilize their intracellular domains to connect to the signaling networks that regulate the distribution and subunit composition for iGluRs. Such cellular signals converge onto and are integrated by the intracellular domains of the receptors and/or by various auxiliary subunits associated with the iGluR complexes (Ramos, 2015).

Neto proteins modulate the gating behavior of KAR but also play crucial roles in the synaptic recruitment of glutamate receptors in vivo. At the fly NMJ, Neto enables iGluRs synaptic clustering and initiates synapse assembly. In addition, the intracellular domain of Neto-β recruits PSD components and triggers a cascade of events that organize postsynaptic structures and shape the composition of postsynaptic fields. The cytoplasmic domains of Neto proteins emerge as versatile signaling hubs and organizing platforms that directly control the iGluRs subunit composition and augment the previously known Neto functions in modulation of glutamatergic synapses (Ramos, 2015).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Kainate receptor modulation by NETO2

Glutamate-gated kainate receptors are ubiquitous in the central nervous system of vertebrates, mediate synaptic transmission at the postsynapse and modulate transmitter release at the presynapse. In the brain, the trafficking, gating kinetics and pharmacology of kainate receptors are tightly regulated by neuropilin and tolloid-like (NETO) proteins. This study reports cryo-electron microscopy structures of homotetrameric GluK2 in complex with NETO2 at inhibited and desensitized states, illustrating variable stoichiometry of GluK2-NETO2 complexes, with one or two NETO2 subunits associating with GluK2. NETO2 was found to access only two broad faces of kainate receptors, intermolecularly crosslinking the lower lobe of ATD(A/C), the upper lobe of LBD(B/D) and the lower lobe of LBD(A/C), illustrating how NETO2 regulates receptor-gating kinetics. The transmembrane helix of NETO2 is positioned proximal to the selectivity filter and competes with the amphiphilic H1 helix after M4 for interaction with an intracellular cap domain formed by the M1-M2 linkers of the receptor, revealing how rectification is regulated by NETO2 (He, 2021).

Kainate receptors (KARs) are a class of ionotropic glutamate receptors, activated by the neurotransmitter glutamate. They are not only located at the postsynapse to mediate excitatory neurotransmission in many brain regions but also appear at the presynapse to modulate transmitter release on both excitatory and inhibitory synapses. NETO proteins are single-pass transmembrane proteins with an extracellular domain containing two C1r/C1s-Uegf-BMP domains (known as CUB1 and CUB2) and a low-density lipoprotein class A domain (LDLa). These proteins have been identified as an auxiliary component of native KARs and significantly affect KAR trafficking, gating and pharmacology. More specifically, NETO2 modulates KAR gating by slowing deactivation and desensitization, accelerating recovery from desensitization and attenuating polyamine block of calcium-permeable KARs. Despite recent progress in the structural study of isolated KARs, the molecular basis of regulatory roles of NETO proteins remains unclear. This study shows the architectures of the GluK2-NETO2 complex in the antagonist-bound closed state and the agonist-bound desensitized state, illustrating interactions and stoichiometry between GluK2 and NETO2, and the modulation mechanism of NETO2 on GluK2 receptor gating and pore properties. Moreover, a more-complete pore domain, including a detailed structure of the selectivity filter, is provided in these structures (He, 2021).

To investigate the structural basis for modulation of GluK2 gating by NETO2, both the full-length GluK2 (with Gln at the Q/R site) and NETO2 were co-expressed in HEK 293T cells and the complex was purified. Cryo-electron microscopy (cryo-EM) studies generated three distinct assemblies of the antagonist-bound GluK2-NETO2 complexes, including GluK2 bound with one NETO2 (GluK2-1xNETO2) or two NETO2 (GluK2-2xNETO2). The third type of complex features a disrupted ligand-binding domain (LBD) dimer on one side and an intact LBD dimer on the other side, the latter of which is also bound with one NETO2 subunit, and thus this complex is denoted as GluK2-1xNETO2asymLBD. The GluK2-1xNETO2, GluK2-2xNETO2 and GluK2-1xNETO2asymLBD complexes were determined at 4.2 Å, 6.4 Å and 4.1 Å resolutions, respectively. The LBD-transmembrane domain (TMD) focused classification and refinement of the GluK2-1xNETO2 complex yield a 3.9 Å map with more density features (He, 2021).

The overall structure of NETO2 is about 140 Å in height, composed of four domains including CUB1, CUB2, LDLa and TM1. There is a horizontal helix, termed α1, right before TM1 of NETO2 (TM1NETO2). The CUB1, CUB2 and LDLa domains are closely packed together. The linker between LDLa and α1 helix was missing, probably due to conformational heterogeneity. On the GluK2 side, the model of both the GluK2-1xNETO2 and the GluK2-2xNETO2 complexes reveals that the membrane-crossing segment M3 gate is closed and the degree of LBD clamshell closure is nearly identical to the isolated GluK2 LBD structure bound with LY466195, confirming that the complex is stabilized at the antagonist-bound inhibited state. In the case of the GluK2-1xNETO2 complex, the distance between the amino-terminal domain (ATD) and LBD layers at the NETO2-bound side is substantially shortened compared with the non-NETO2-bound side, thus breaking from the ideal overall two-fold symmetry of the receptor complex, as evidenced by the ATD-LBD layers from the A and C positions, which are not superimposable with each other. In addition, the overall structure of the GluK2-2xNETO2 complex also breaks two-fold symmetry. In particular, the interaction between CUB1 and CUB2 on one side is more extensive than that on the other side (He, 2021).

The GluK2-1xNETO2asymLBD complex contains one disrupted LBD dimer, showing that the LBD of the B subunit rotates approximate 72° relative to that of two-fold-related LBDs, which is also consistent with observations from GluK3 structures. One extracellular domain of NETO2 connects ATD with LBD. It is speculated that this domain is CUB1 as it has a similar binding mode with GluK2 ATD. Considering that the TM1NETO2 helix remains present in the structure, the absence of CUB2 and LDLa in the EM map is probably due to conformational flexibility (He, 2021).

On the extracellular side of both the GluK2-1xNETO2 and the GluK2-2xNETO2 complexes, the N-terminal CUB1 of NETO2 interacts with the lower lobe in the ATD layer of the A/C subunit of GluK2 (R2-lobeA/C). In the LBD layer, the CUB2 and LDLa domains of NETO2 interact with the upper lobe of subunit B/D (D1-lobeB/D) and the lower lobe of subunit A/C (D2-lobeA/C), respectively. This spatial arrangement of GluK2-NETO2 interactions leads to a stoichiometry between GluK2 and NETO2 of either 4:1 or 4:2, which is distinct from the 4:4 stoichiometry between the AMPA receptor and TARPγ2. Taking a close look at the GluK2-NETO2 interface, it was found that some charged residues from GluK2 contact the extracellular domains of NETO2. For instance, K216/R220 on the ATD layer and E723/R727 on the LBD layer potentially form electrostatic interactions with NETO2. Unlike a point-to-point interaction mode, CUB2 forms more extensive interactions with loop 1 (residues 448-453). Although their sequence identity is low, the loop 1 from different isoforms adopt similar 3D conformations. In the GluK2-1xNETO2asymLBD complex, one of the LBD dimers is disrupted, consequently yielding a new interaction between CUB1 and D1-lobeB/D. In particular, residues I780/Q784 of LBDB/D are involved in the interaction with CUB1. On the basis of sequence and structure alignment, these critical interaction sites are fairly conserved in the amino acid sequence and in the 3D structure, except for ATD of GluK4 and GluK5 (ATDGluK4/5). However, considering that NETO1 and NETO2 have different subunit-dependent regulation on GluK1 and GluK2, it is speculated that these interactions determined in the GluK2-NETO2 complex might not fully reflect in different combinations of the GluK-NETO complex. Some changes of binding geometry or even new interactions might occur, which would profoundly alter regulatory effects of NETO proteins on KARs. In the GluK4 and GluK5 subtypes, R220 is substituted to a negatively charged Asp at the equivalent site, which would repel negatively charged residues from NETO2 and thus prevent CUB1 from interacting with ATDGluK4/5. Interestingly, GluK5 is shown to specifically occupy the B/D positions of the channel; therefore, this unfavourable CUB1-ATDGluK4/5 interaction would not prohibit NETO2 from associating with and modulating the heteromeric GluK2-GluK5 complex, which is a major population in the brain. Nevertheless, this study was unable to identify crucial residues of NETO2 involved in these GluK2-NETO2 interactions due to medium resolution of the NETO2 density (He, 2021).

To explore functional roles of these contacts, corresponding residues of GluK2 to Ala were mutated to disrupt the putative interactions with NETO2. The I780A/Q784A double mutation profoundly accelerated desensitization, probably due to the mutation destabilizing the LBD dimer. NETO2 is able to slow desensitization of GluK2I780A/Q784A. The other mutations showed little effect on gating kinetics of GluK2 alone. However, these mutations substantially decreased the effects of NETO2 on the GluK2 gating kinetics to different extents. In particular, NETO2 lost its function of regulating GluK2 upon introduction of the K216A/R220A mutations (GluK2ATD-2A), in line with a previous study that suggested that negatively charged residues of CUB1 have vital roles in modulation of NETO2. In addition, a triple point mutation (K448A/D450A/K451A) of GluK2 (GluK2D1-3A) was designed to disturb contacts between CUB2 and D1-lobeB/D. Compared with the wild-type GluK2-NETO2 complex, the GluK2D1-3A-NETO2 complex displayed faster desensitization kinetics. Loop 1 of GluK2 was then substituted by the equivalent residues from GluK5 (GluK2K5-loop1), and this mutant significantly reduced the effects of NETO2 on desensitization kinetics. However, an E723A/R727A double mutation in the D2-lobe (GluK2D2-2A), which presumably disrupts the LDLa-D2-lobe interaction, showed a moderate decrease in slowing desensitization by NETO2. NETO2 is able to attenuate inward rectification of the mutants discussed above, suggesting that these mutations could not damage the association between GluK2 and NETO2 (He, 2021).

The LBD-TMD focused refinement yielded a 3.9 Å resolution map, which enabled building of the most complete TMD model of the KAR so far. The pore profile reveals that the constriction sites are T652, A656 and T660. This is consistent with observations in previous studies. Most importantly, the selectivity filter, consisting of the pore helix M2 (residues 608-620) and the pore loop (residues 621-625), was determined. Residues on M2 helices form extensive hydrophobic interactions with M1 and M3 helices from the same subunit and M3 helices from adjacent subunits. Furthermore, the construct has Q621 at the Q/R site located at the tip of the pore loop. Its sidechain points upwards to the central vestibule, and is aligned with residues at the Q/R site observed in AMPA receptor structures. Previous investigations have shown that Arg at this site renders GluK2 calcium impermeable and attenuates polyamine block, presumably due to charge-charge repulsion. In addition, inside the selectivity filter, a cation ligated by carbonyl oxygen groups was determined from the four Q621 residues with bond lengths of 3.5-4 Å. Given Gln is at the Q/R site, it is proposed that this cation is either a calcium or sodium ion (He, 2021).

The long loops between M1 and M2 helices were resolved in the GluK2 map in the presence of NETO2. These loops extend into the cytosol and interact with each other, forming an intracellular cap domain (ICD) underneath the selectivity filter. Moreover, the amphiphilic H1 helix (residues 857-870) was built immediately after M4. The C terminus of H1 helix is positioned proximal to the C terminus of M1 and the M1-M2 loop from an adjacent subunit. Moreover, TM1NETO2 forms extensive hydrophobic interaction with TMD of GluK2. In particular, TM1NETO2 and its N-terminal α1 helix directly contact M1 of GluK2. One strip-like shape density was observed in between TM1NETO2 and M4 of GluK2, and is supposed to be the hydrophobic tail of a lipid molecule and important for bridging indirect contacts between TM1NETO2 and M4. On the intracellular side, the C terminus of TM1NETO2 competes with the H1 helix to interact with the C terminus of M1 and the N terminus of M2 (He, 2021).

To investigate the functional roles of the H1 helix and the ICD, the H1 helix (GluK2ΔΗ1) and substituted the M1-M2 linker with GluA2 equivalent residues (GluK2A2ICD). These mutations do not change receptor gating kinetics in the absence of NETO2. However, in the presence of NETO2, the GluK2ΔΗ1 and GluK2A2ICD mutants showed slower and faster desensitization kinetics than wild-type GluK2, respectively. It is speculated that these diametrically opposite effects on receptor desensitization kinetics result from a distinct ratio of the GluK2-NETO2 complex to the total receptors on the cell surface. Considering that the H1 helix blocks the NETO2-binding site but the ICD directly interacts with NETO2, more GluK2ΔΗ1 would be fully occupied by NETO2 after deleting the H1 helix; by contrast, more NETO2 would dissociate from GluK2A2ICD once the M1-M2 loop is changed to the equivalent loop from GluA2 (He, 2021).

In regarding to inward rectification, the GluK2A2ICD mutant is similar to wild-type GluA2, but distinct from wild-type GluK2, indicating that the M1-M2 loop has major roles in determining the inward rectification property of glutamate receptors. The M1-M2 loop of GluA2 was replaced by that from GluK2 (GluA2K2ICD) and resulted in a substantial decrease of the GluA2 rectification, further supporting that the M1-M2 loop is crucial for rectification. In the absence or presence of NETO2, the GluK2ΔΗ1 receptor shows comparable inward rectification as wild-type GluK2. It is hypothesized that NETO2 competes with the H1 helix to stabilize the ICD of the receptor, creating a physical barrier that prohibits polyamine from diffusing close to the selectivity filter, thereby eliminating both polyamine inhibition and inward rectification (He, 2021).

A 3.8 Å map of the desensitized GluK2-NETO2 complex was obtained, featuring GluK2 receptor binding with only one NETO2 subunit. The channel gate is closed, yet the LBD clamshell closure is nearly identical to the isolated LBD structure bound with kainate, indicating that the complex is stabilized at a desensitized state (GluK2-1xNETO2des). Only CUB1 and TM1 of the NETO2 subunit are clearly visualized. Consistent with desensitized KARs alone, the LBD layer of the GluK2-1xNETO2des complex shows a disrupted LBD dimer and undergoes a remarkable rearrangement. Structural comparison of LBD layers from the GluK2-1xNETO2des complex and desensitized GluK2 alone shows that all of the four D2-lobes connecting to the gating helix M3 are superimposable, adopt a pseudo four-fold symmetry and are close to the channel central axis, suggesting that, in both the absence and the presence of NETO2, GluK2 receptors share similar mechanisms to decouple agonist binding from channel opening. The D1-lobes at B/D positions clearly show displacement between these two complexes. In the presence of NETO2, the D1-lobe of the B subunit approaches the ATD of the A subunit due to interactions with CUB1 of NETO2. In this desensitized state, the CUB2 and LDLa domains were not well determined, suggesting that the interactions observed in the inhibited state were disrupted upon desensitization. Together, it is speculated that, in the presence of NETO2, the inter-subunit connections of both ATDA/C-LBDB/D and LBDA/C-LBDB/D, bridged by extracellular domains of NETO2, hinder rearrangement in the LBD layer upon desensitization, and thus slow down the desensitization kinetics. The ATD-CUB1 interaction is constitutively present in both inhibited and desensitized states. It is hypothesized that the ATD-CUB1 interaction renders CUB1 an N-terminal anchor, whereas TM1 acts as a C-terminal anchor, constricting CUB2 and LDLa domains around the LBD layer during the gating cycle and thus modulating channel gating, underlying the essential role of the ATD-CUB1 interaction in the regulatory function of NETO2 (He, 2021).

Rapid homeostatic modulation of transsynaptic nanocolumn rings

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

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

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

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

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

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

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

Amino-terminal domains of kainate receptors determine the differential dependence on Neto auxiliary subunits for trafficking

The kainate receptor (KAR), a subtype of glutamate receptor, mediates excitatory synaptic responses at a subset of glutamatergic synapses. However, the molecular mechanisms underlying the trafficking of its different subunits are poorly understood. This study used the CA1 hippocampal pyramidal cell, which lacks KAR-mediated synaptic currents, as a null background to determine the minimal requirements for the extrasynaptic and synaptic expression of the GluK2 subunit. GluK2 (Drosophila homolog: KaiR1D) receptor itself, in contrast to GluK1, traffics to the neuronal surface and synapse efficiently and the auxiliary subunits Neto1 (see Drosophila Neto) and Neto2 caused no further enhancement of these two trafficking processes. However, the regulation of GluK2 biophysical properties by Neto proteins is the same as that of GluK1. It was further determined that it is the amino-terminal domains (ATDs) of GluK1 (see Drosophila Ekar) and GluK2 that control the strikingly different trafficking properties between these two receptors. Moreover, the ATDs are critical for synaptic expression of heteromeric receptors at mossy fiber-CA3 synapses and also mediate the differential dependence on Neto proteins for surface and synaptic trafficking of GluK1 and GluK2. These results highlight the fundamental differences between the two major KAR subunits and their interplay with Neto auxiliary proteins (Sheng, 2017).

This study first used the hippocampal CA1 pyramidal neuron as a null background system to study the mechanism regulating the trafficking of KARs because the Schaffer collateral-CA1 synapses lack KAR expression. Unlike GluK1, which requires the auxiliary subunits Neto1 and Neto2 for surface and synaptic trafficking, GluK2 extrasynaptic and synaptic trafficking is independent of Neto proteins. However, its proper synaptic targeting requires the extracellular ATD region. At the mossy fiber-CA3 synapses, which do express KARs, the GluK2 ATD is also critical for the synaptic targeting of GluK2/GluK5 heteromeric receptor complexes. Furthermore, it was determined that it is the ATDs of GluK1 and GluK2 contributing their differential dependence on Neto proteins for surface and synaptic trafficking. The results demonstrate the important role of the interplay of the KAR ATDs and the Neto auxiliary subunits in controlling the surface expression and synaptic incorporation of kainate receptors (Sheng, 2017).

Consistent with a previous study, expressing GluK1 in CA1 pyramidal neurons results in very few KAR synaptic and surface currents. This finding is interpreted as a defect in the forward trafficking of GluK1, although it is formally possible that the removal of surface receptors is greatly enhanced. By contrast, the expression of GluK2 generates huge responses from outside-out patches from the cell body membrane as well as the evoked EPSCs. These differences cannot be explained by the biophysical properties between GluK1 and GluK2 receptors because the rates of their desensitization or deactivation kinetics are very similar. Thus, the trafficking properties of GluK1 and GluK2 are fundamentally different. This finding is consistent with a previous study showing that the surface staining intensity of GluK2 is much higher than GluK1 when expressed in COS-7 or primary cultured hippocampal neurons. This study also found that when coexpressing GluK5 with GluK1 or GluK2 in CA3 cells, the GluK2/GluK5 but not the GluK1/GluK5 heteromeric receptors can target to the mossy fiber-CA3 synapses, suggesting that the low-affinity KARs may also be critical for the expression and localization of the heteromeric receptors. The different trafficking abilities of KARs are presumably due to their different amino acid sequences. Several studies have already shown the importance of the CTDs of KARs for surface trafficking, and thus the effects on synaptic expression were examined by swapping the CTDs between GluK1 and GluK2. Surprisingly, the enhancement of synaptic responses mediated by the GluK2(CTDK1) or GluK2(ΔCTD) mutated receptor is similar to that seen with the wild-type GluK2 receptor. There are several possible reasons underlying the difference between this observation and previous studies. First, many of the previous experiments were carried out in heterologous expression systems in which the regulation of GluK2 trafficking may differ from that in neurons. Consistent with this proposal, it has been reported that in COS-7 cells GluK2a, the splicing isoform with a long CTD and used in the current studies, traffics to the cell membrane more effectively than the shorter isoform GluK2b. However, the same study reported that the surface expression of the two isoforms is similar in primary cultured neurons. Second, another study used a GluK2 mutant containing a GFP tag in the ATD. Given the critical role found for the ATD of GluK2 in trafficking, it is possible that the tag may have masked the role of the ATD of GluK2, allowing the role of the CTD to be dissected. Consistent with this hypothesis, it was previously found that the synaptic expression of N-terminal HA or Myc tagged-GluK1 is impaired even in the presence of Neto proteins, although they could traffic to the surface successfully. Knockin studies have suggested that the GluK2 CTD stabilizes the receptor at synapses and in agreement this study also found that the GluK2 CTD endows GluK1 with the ability to express at synapses. Given that the CTD is not critical for GluK2 synaptic trafficking, focus was placed on the extracellular domains of GluK2. Surprisingly, swapping the entire extracellular domains or just ATDs between GluK1 and GluK2 fully switches their surface and synaptic trafficking abilities. Furthermore, this study showed that the ATDs of GluK1 and GluK2 determine their differential dependence on auxiliary Neto proteins for trafficking. All these results indicate that the ATDs of GluK1 and GluK2 receptors are the major determinant for their trafficking. Recently two studies have revealed that C1q-like proteins interact with the ATD of GluK2 or GluK4 to organize the KARs at mossy fiber-CA3 synapses, and in accordance, this study also found that the ATD of GluK2 is critical for the synaptic expression of GluK2/GluK5 heteromeric receptors at mossy fiber-CA3 synapses (Sheng, 2017).

In contrast to GluK1, GluK2 surface and synaptic expression are independent on the auxiliary subunits Neto1 and Neto2. A similar conclusion was reached in previous studies showing that Neto2 has no effect on GluK2 trafficking in oocytes but does promote GluK1 surface expression in HEK cells and primary cultured neurons. However, the underlying molecular mechanism remains unknown. This differential dependence on auxiliary subunits for trafficking could not be explained by any specific interaction between GluK1 and Neto proteins because it was found that the biophysical effects of Neto proteins on GluK1 and GluK2 are the same. Moreover, the decay of GluK2 mEPSCs is increased by coexpressed Neto2. All these results indicate that GluK2 and Neto proteins indeed interact at both the surface and the synapse. It has been reported that it is the extracellular CUB domains of Neto1 and Neto2 that mediate their interaction with GluK2 , but it is unknown how they bind to GluK1 or which domains of GluK1 and GluK2 mediate their interactions with Neto proteins. Additionally, it has been reported that the extracellular LDLa domain of the Neto proteins is critical for their effects on GluK2 desensitization, but the intracellular C-terminal domain is critical for their regulation of GluK2 rectification, indicating that Neto proteins can regulate KAR function through different domains. This study reports that the ATDs of GluK1 and GluK2 mediate the differential dependence on Neto proteins for trafficking. It will be of interest to identify the detailed structural and molecular basis for the difference. Importantly, in the central nervous system, most native KARs are heteromeric complexes and the presence of high-affinity subunits could affect the function of Neto proteins. Such a scenario might explain the finding that Neto2 but not Neto1 slows homomeric GluK2 mEPSC decay time, whereas previous studies indicate that Neto1 but not Neto2 is critical for the decay of the slow EPSC at mossy fiber-CA3 synapses. It would be of interest to study further the effects of Neto proteins on the trafficking and biophysical properties of specific heteromeric KAR complexes (Sheng, 2017).

In summary, this study has characterized the critical role of the ATD for KAR trafficking in hippocampal neurons as well as their interplay with auxiliary subunit Neto proteins in this process. The Schaffer collateral-CA1 synapse that normally does not express KARs, was selected to determine the minimal requirements that govern the insertion of KARs into excitatory synapses, and the findings were further confirmed at mossy fiber-CA3 synapses that express heteromeric KARs. These results demonstrate the critical role of the extracellular ATD for KAR surface and synaptic expression as well as their contribution to KARs' differential dependence on Neto proteins for trafficking (Sheng, 2017).

Neto auxiliary proteins control both the trafficking and biophysical properties of the kainate receptor GluK1

Kainate receptors (KARs) are a subfamily of glutamate receptors mediating excitatory synaptic transmission and Neto proteins (see Drosophila Neto) are recently identified auxiliary subunits for KARs. However, the roles of Neto proteins in the synaptic trafficking of KAR GluK1 are poorly understood. Using the hippocampal CA1 pyramidal neuron as a null background system this study found that surface expression of GluK1 receptor itself is very limited and is not targeted to excitatory synapses. Both Neto1 and Neto2 profoundly increase GluK1 surface expression and also drive GluK1 to synapses. However, the regulation GluK1 synaptic targeting by Neto proteins is independent of their role in promoting surface trafficking. Interestingly, GluK1 is excluded from synapses expressing AMPA receptors and is selectively incorporated into silent synapses. Neto2, but not Neto1, slows GluK1 deactivation, whereas Neto1 speeds GluK1 desensitization and Neto2 slows desensitization. These results establish critical roles for Neto auxiliary subunits controlling KARs properties and synaptic incorporation (Sheng, 2015).

Search PubMed for articles about Drosophila Neto

Banovic, D., Khorramshahi, O., Owald, D., Wichmann, C., Riedt, T., Fouquet, W., Tian, R., Sigrist, S. J. and Aberle, H. (2010). Drosophila neuroligin 1 promotes growth and postsynaptic differentiation at glutamatergic neuromuscular junctions. Neuron 66(5): 724-738. PubMed ID: 20547130

Chen, K., Merino, C., Sigrist, S. J., and Featherstone, D. E. (2005). The 4.1 protein coracle mediates subunit-selective anchoring of Drosophila glutamate receptors to the postsynaptic actin cytoskeleton. J. Neurosci. 25: 6667-6675. PubMed ID: 16014728

Copits, B. A., Robbins, J. S., Frausto, S. and Swanson, G. T. (2011). Synaptic targeting and functional modulation of GluK1 kainate receptors by the auxiliary neuropilin and tolloid-like (NETO) proteins. J. Neurosci. 31: 7334-7340. PubMed ID: 21593317

Fuentes-Medel, Y., Ashley, J., Barria, R., Maloney, R., Freeman, M. and Budnik, V. (2012). Integration of a retrograde signal during synapse formation by glia-secreted TGF-beta ligand. Curr Biol 22: 1831-1838. PubMed ID: 22959350

Gally, C., Eimer, S., Richmond, J. E. and Bessereau, J. L. (2004). A transmembrane protein required for acetylcholine receptor clustering in Caenorhabditis elegans. Nature 431: 578-582. PubMed ID: 15457263

Han, T. H., Vicidomini, R., Ramos, C. I., Wang, Q., Nguyen, P., Jarnik, M., Lee, C. H., Stawarski, M., Hernandez, R. X., Macleod, G. T. and Serpe, M. (2020). Neto-alpha controls synapse organization and homeostasis at the Drosophila neuromuscular junction. Cell Rep 32(1): 107866. PubMed ID: 32640231

He, L., Sun, J., Gao, Y., Li, B., Wang, Y., Dong, Y., An, W., Li, H., Yang, B., Ge, Y., Zhang, X. C., Shi, Y. S. and Zhao, Y. (2021). Kainate receptor modulation by NETO2. Nature. PubMed ID: 34552241

Jackson, A. C. and Nicoll, R. A. (2011). The expanding social network of ionotropic glutamate receptors: TARPs and other transmembrane auxiliary subunits. Neuron 70: 178-199. PubMed ID: 21521608

Kalashnikova, E., et al. (2010). SynDIG1: An activity-regulated, AMPA- receptor-interacting transmembrane protein that regulates excitatory synapse development. Neuron 65: 80-93. PubMed ID: 20152115

Kim, Y. J., Bao, H., Bonanno, L., Zhang, B. and Serpe, M. (2012). Drosophila Neto is essential for clustering glutamate receptors at the neuromuscular junction. Genes Dev. 26(9): 974-87. PubMed ID: 22499592

Kiragasi, B., Wondolowski, J., Li, Y. and Dickman, D. K. (2017). A presynaptic glutamate receptor subunit confers robustness to neurotransmission and homeostatic potentiation. Cell Rep 19(13): 2694-2706. PubMed ID: 28658618

Li, Y., Dharkar, P., Han, T. H., Serpe, M., Lee, C. H. and Mayer, M. L. (2016). Novel functional properties of Drosophila CNS glutamate receptors. Neuron 92(5): 1036-1048. PubMed ID: 27889096

Liebl, F. L. and Featherstone, D. E. (2008). Identification and investigation of Drosophila postsynaptic density homologs. Bioinform. Biol. Insights 2: 375-387. PubMed ID: 19812789

Lomash, R. M., Sheng, N., Li, Y., Nicoll, R. A. and Roche, K. W. (2017). Phosphorylation of the kainate receptor (KAR) auxiliary subunit Neto2 at serine 409 regulates synaptic targeting of the KAR subunit GluK1. J Biol Chem 292(37): 15369-15377. PubMed ID: 28717010

Milstein, A. D. and Nicoll, R. A. (2008). Regulation of AMPA receptor gating and pharmacology by TARP auxiliary subunits. Trends Pharmacol Sci 29: 333-339. PubMed ID: 18514334

Kim, Y. J., Bao, H., Bonanno, L., Zhang, B. and Serpe, M. (2012). Drosophila Neto is essential for clustering glutamate receptors at the neuromuscular junction. Genes Dev 26(9): 974-987. PubMed ID: 22499592

Mosca, T. J., Hong, W., Dani, V. S., Favaloro, V. and Luo, L. (2012). Trans-synaptic Teneurin signalling in neuromuscular synapse organization and target choice. Nature 484(7393): 237-241. PubMed ID: 22426000

Kim, Y. J., Bao, H., Bonanno, L., Zhang, B. and Serpe, M. (2012). Drosophila Neto is essential for clustering glutamate receptors at the neuromuscular junction. Genes Dev 26(9): 974-987. PubMed ID: 22499592

Muttathukunnel, P., Frei, P., Perry, S., Dickman, D. and Muller, M. (2022). Rapid homeostatic modulation of transsynaptic nanocolumn rings. Proc Natl Acad Sci U S A 119(45): e2119044119. PubMed ID: 36322725

Ng, D., et al. (2009). Neto1 is a novel CUB-domain NMDA receptor-interacting protein required for synaptic plasticity and learning. PLoS Biol 7(2): e41. PubMed ID: 19243221

Orr, B. O., Gorczyca, D., Younger, M. A., Jan, L. Y., Jan, Y. N. and Davis, G. W. (2017). Composition and control of a Deg/ENaC channel during presynaptic homeostatic plasticity. Cell Rep 20(8): 1855-1866. PubMed ID: 28834749

Petzoldt, A. G., Lee, Y. H., Khorramshahi, O., Reynolds, E., Plested, A. J., Herzel, H. and Sigrist, S. J. (2014). Gating characteristics control glutamate receptor distribution and trafficking in vivo. Curr Biol 24(17): 2059-2065. PubMed ID: 25131677

Pinheiro, P. S., Perrais, D., Coussen, F., Barhanin, J., Bettler, B., Mann, J. R., Malva, J. O., Heinemann, S. F. and Mulle, C. (2007). GluR7 is an essential subunit of presynaptic kainate autoreceptors at hippocampal mossy fiber synapses. Proc Natl Acad Sci U S A 104(29): 12181-12186. PubMed ID: 17620617

Ramos, C. I., Igiesuorobo, O., Wang, Q. and Serpe, M. (2015). Neto-mediated intracellular interactions shape postsynaptic composition at the Drosophila neuromuscular junction. PLoS Genet 11: e1005191. PubMed ID: 25905467

Rohrbough, J., et al. (2007). Presynaptic establishment of the synaptic cleft extracellular matrix is required for post-synaptic differentiation. Genes Dev. 21: 2607-2628. PubMed ID: 17901219

Schwenk, J., et al. (2009). Neto is essential in synaptogenesis Functional proteomics identify cornichon proteins as auxiliary subunits of AMPA receptors. Science 323: 1313-1319. PubMed ID: 19265014

Sheng, N., Shi, Y. S., Lomash, R. M., Roche, K. W. and Nicoll, R. A. (2015). Neto auxiliary proteins control both the trafficking and biophysical properties of the kainate receptor GluK1. Elife 4. PubMed ID: 26720915

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date revised: 2 January 2023

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