InteractiveFly: GeneBrief

Neuropilin and tolloid-like: Biological Overview | References


Gene name - Neuropilin and tolloid-like

Synonyms - CG32635

Cytological map position -

Function - transmembrane receptor

Keywords - auxiliary subunit of ionic Glutamate receptor required for receptor clustering, neuromuscular junction

Symbol - Neto

FlyBase ID: FBgn0265416

Genetic map position - chrX:13339939-13413663

Classification - CUB (for complement C1r/C1s, UEGF, BMP-1) domains followed by an LDLa (low-density lipoprotein receptor domain class A) motif

Cellular location - surface transmembrane



NCBI links: Precomputed BLAST | EntrezGene
Recent literature
Han, T.H., Dharkar, P., Mayer, M.L. and Serpe, M. (2015). Functional reconstitution of Drosophila melanogaster NMJ glutamate receptors. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 25918369
Summary:
The Drosophila larval neuromuscular junction (NMJ), at which glutamate acts as the excitatory neurotransmitter, is a widely used model for genetic analysis of synapse function and development. Despite decades of study, the inability to reconstitute NMJ glutamate receptor function using heterologous expression systems has complicated the analysis of receptor function, such that it is difficult to resolve the molecular basis for compound phenotypes observed in mutant flies. This study finds that Drosophila Neto functions as an essential component required for the function of NMJ glutamate receptors, permitting analysis of glutamate receptor responses in Xenopus oocytes. In combination with a crystallographic analysis of the GluRIIB ligand binding domain, the Serpe lab used this system to characterize the subunit dependence of assembly, channel block, and ligand selectivity for Drosophila NMJ glutamate receptors.

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).


Functions of Neto orthologs in other species

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).


REFERENCES

Search PubMed for articles about Drosophila Neto

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

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

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

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

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

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

Straub, C., et al. (2011a). Distinct functions of kainate receptors in the brain are determined by the auxiliary subunit Neto1. Nat. Neurosci. 14: 866-873. PubMed ID: 21623363

Straub, C., Zhang, W., Howe, J. R. (2011b). Neto2 modulation of kainate receptors with different subunit compositions. J. Neurosci. 31: 8078-8082. PubMed ID: 21632929

Sulkowski, M., Kim, Y. J. and Serpe, M. (2013). Postsynaptic glutamate receptors regulate local BMP signaling at the Drosophila neuromuscular junction. Development 141(2):436-47. PubMed ID: 24353060

Tang, M, et al. (2011). Neto1 is an auxiliary subunit of native synaptic kainate receptors. J. Neurosci. 31: 10009-10018. PubMed ID: 21734292

Tomita, S., et al. (2003). Functional studies and distribution define a family of transmembrane AMPA receptor regulatory proteins. J. Cell Biol. 161: 805-816. PubMed ID: 12771129

von Engelhardt, J., et al. (2010). CKAMP44: A brain-specific protein attenuating short-term syn- aptic plasticity in the dentate gyrus. Science 327: 1518-1522. PubMed ID: 20185686

Walker, C. S., et al. (2006). Conserved SOL-1 proteins regulate iono- tropic glutamate receptor desensitization. Proc. Natl. Acad. Sci. 103: 10787-10792. PubMed ID: 16818875

Zhang, W., et al. (2009). A transmembrane accessory subunit that modulates kainate-type glutamate receptors. Neuron 61: 385-396. PubMed ID: 16818875

Zheng, Y., et al. (2004). SOL-1 is a CUB-domain protein required for GLR-1 gluta- mate receptor function in C. elegans. Nature 427: 451-457. PubMed ID: 14749834


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date revised: 15 June 2015

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