InteractiveFly: GeneBrief

Neuroligin-1 & Neuroligin-2 Biological Overview | References


Gene name - Neuroligin-1 & Neuroligin-2

Synonyms -

Cytological map positions - 84D11-84D12 & 27C3-27C4

Functions - Transmembrane receptors

Keywords - synaptic junction, synaptic growth and maturation, postsynaptic component of neuromuscular junction, mesoderm

Symbol - Nlg1 and Nlg2

FlyBase ID: FBgn0051146 and FBgn0031866

Genetic map positions - chr3R:3483971-3520596 & chr2L:6875064-6906896

Classification - catalytically inactive acetylcholinesterase-like domain, C-terminal, intracellular PDZ-domain-binding motif

Cellular location - Surface transmembrane, synaptic junctions



NCBI links for Neuroligin-1: Precomputed BLAST | EntrezGene

NCBI links for Neuroligin-2: Precomputed BLAST | EntrezGene

Recent literature
Qian, J., Tu, R., Yuan, L. and Xie, W. (2016). Intronic miR-932 targets the coding region of its host gene, Drosophila neuroligin2. Exp Cell Res [Epub ahead of print]. PubMed ID: 26844630
Summary:
Despite great progress for two decades in microRNAs (miRNAs), the direct regulation of host gene by intragenic (mostly intronic) miRNA is conceptually plausible but evidence-limited. This study reports that intronic miR-932 could target its host gene via binding with coding sequence (CDS) region rather than regular 3'UTR. The conserved miR-932 is embedded in the fourth intron of Drosophila neuroligin2 (dnlg2), which encodes a synaptic cell adhesion molecule, DNlg2. In silico analysis predicted two putative miR-932 target sites locate in the CDS region of dnlg2 instead of regular 3'-UTR miRNA binding sites. Employing luciferase reporter assay, miR-932 was further proved to regulates expression of its host gene dnlg2 via the binding CDS region of dnlg2. Consistently, miR-932 downregulated expression of dnlg2 in S2 cell, and the repression of dnlg2 by miR-932 at both protein and RNA level. Furthermore, CDS-located site1 is dominant for regulating expression of host dnlg2 by miR-932. In addition to providing thorough examination of one intronic miRNA targeting the CDS region of its host gene, this genome-wide analysis indicated that nearly half of fruitfly and human intronic miRNAs may target their own host gene at coding region. This study would be valuable in elucidating the regulation of intronic miRNA on host gene, and provide new information about the biological context of their genomic arrangements and functions.
Tu, R., Qian, J., Rui, M., Tao, N., Sun, M., Zhuang, Y., Lv, H., Han, J., Li, M. and Xie, W. (2017). Proteolytic cleavage is required for functional Neuroligin 2 maturation and trafficking in Drosophila. J Mol Cell Biol 9(3):231-242. PubMed ID: 28498949
Summary:
Neuroligins are transmembrane cell adhesion molecules playing essential roles in synapse development and function. Genetic mutations in neuroligin genes have been linked with some neurodevelopmental disorders such as autism. These mutated Neuroligins are mostly retained in the endoplasmic reticulum (ER). However, the mechanisms underlying normal Neuroligin maturation and trafficking have remained largely unknown. This study found that Drosophila Neuroligin 2 (DNlg2) undergoes proteolytic cleavage in the ER in a variety of Drosophila tissues throughout developmental stages. A region encompassing Y642-T698 is required for this process. The immature non-cleavable DNlg2 is retained in the ER and is non-functional. The C-terminal fragment (CTF) of DNlg2 instead of the full-length or non-cleavable DNlg2 is able to rescue neuromuscular junction defects and GluRIIB reduction induced by dnlg2 deletion. Intriguingly, the autism-associated R598C mutation in DNlg2 leads to similar marked defects in DNlg2 proteolytic process and ER export, revealing a potential role of the improper Neuroligin cleavage in autism pathogenesis. Collectively, these findings uncover a specific mechanism that controls DNlg2 maturation and trafficking via proteolytic cleavage in the ER, suggesting that the perturbed proteolytic cleavage of Neuroligins likely contributes to autism disorder.
Corthals, K., Heukamp, A. S., Kossen, R., Grosshennig, I., Hahn, N., Gras, H., Gopfert, M. C., Heinrich, R. and Geurten, B. R. H. (2017). Neuroligins Nlg2 and Nlg4 affect social behavior in Drosophila melanogaster. Front Psychiatry 8: 113. PubMed ID: 28740469
Summary:
The genome of Drosophila melanogaster includes homologs to approximately one-third of the currently known human disease genes. Flies and humans share many biological processes, including the principles of information processing by excitable neurons, synaptic transmission, and the chemical signals involved in intercellular communication. Studies on the molecular and behavioral impact of genetic risk factors of human neuro-developmental disorders [autism spectrum disorders (ASDs), schizophrenia, attention deficit hyperactivity disorders, and Tourette syndrome] increasingly use the well-studied social behavior of D. melanogaster, an organism that is amenable to a large variety of genetic manipulations. Neuroligins (Nlgs) are a family of phylogenetically conserved postsynaptic adhesion molecules present (among others) in nematodes, insects, and mammals. Impaired function of Nlgs (particularly of Nlg 3 and 4) has been associated with ASDs in humans and impaired social and communication behavior in mice. Making use of a set of behavioral and social assays, this study analyzed the impact of two Drosophila Nlgs, Dnlg2 and Dnlg4, which are differentially expressed at excitatory and inhibitory central nervous synapses, respectively. Both Nlgs seem to be associated with diurnal activity and social behavior. Even though deficiencies in Dnlg2 and Dnlg4 appeared to have no effects on sensory or motor systems, they differentially impacted on social interactions, suggesting that social behavior is distinctly regulated by these Nlgs.

BIOLOGICAL OVERVIEW

Precise apposition of presynaptic and postsynaptic domains is a fundamental property of all neuronal circuits. Experiments in vitro suggest that Neuroligins and Neurexins function as key regulatory proteins in this process. In a genetic screen, several mutant alleles of Drosophila neuroligin 1 (dnlg1) were uncovered that cause a severe reduction in bouton numbers at neuromuscular junctions (NMJs). In accord with reduced synapse numbers, these NMJs show reduced synaptic transmission. Moreover, lack of postsynaptic DNlg1 leads to deficits in the accumulation of postsynaptic glutamate receptors, scaffold proteins, and subsynaptic membranes, while increased DNlg1 triggers ectopic postsynaptic differentiation via its cytoplasmic domain. DNlg1 forms discrete clusters adjacent to postsynaptic densities. Formation of these clusters depends on presynaptic Drosophila Neurexin (DNrx). However, DNrx binding is not an absolute requirement for DNlg1 function. Instead, other signaling components are likely involved in DNlg1 transsynaptic functions, with essential interactions organized by the DNlg1 extracellular domain but also by the cytoplasmic domain (Banovic, 2010).

Synapses are specialized membrane contacts between presynaptic and postsynaptic cell compartments that are connected by cell-cell adhesion proteins, which regulate the assembly and maturation of synapses. Different classes of synaptic adhesion proteins have been identified, including members of the immunoglobulin superfamily, Eph/Ephrins, Cadherins, and the Neurexin/Neuroligin families. A typical transsynaptic complex is formed by the heterophilic interaction of presynaptic Neurexins (Nrxs) and postsynaptic Neuroligins (Nlgs) (Dean, 2006). Nlgs are encoded by four independent genes in rodents and five genes in humans. Nlgs possess a catalytically inactive acetylcholinesterase-like domain, which interacts with presynaptic Nrxs. Both Nrxs and Nlgs contain C-terminal, intracellular PDZ-domain-binding motifs believed to recruit scaffolding proteins for organization of either the presynaptic release machinery or the postsynaptic neurotransmitter receptors. Therefore, the interaction of Nrxs with Nlgs has the potential to assemble a large transsynaptic complex that mediates the precise apposition of presynaptic and postsynaptic membranes (Banovic, 2010).

Nlgs localize to postsynaptic regions and, when expressed in nonneuronal cells, induce cocultured neurons to form presynaptic specializations onto the nonneuronal cell (Song, 1999; Scheiffele, 2000). In support for a central role in the formation of synaptic contacts, overexpression of Nlgs in cultured neurons increases not only the number and density of synapses, but also synaptic function (Chih, 2005; Levinson, 2005; Sara, 2005 Chubykin, 2007). Conversely, knockdown of Nlgs by RNA interference (RNAi) leads to a reduction of synapse numbers (Chih, 2005), suggesting a role for Nlgs in synapse formation, stability, or both. Mice that are triply deficient in Nlgs 1–3 die immediately after birth due to respiratory failure, likely as a consequence of reduced synaptic transmission in the brainstem centers controlling respiration (Varoqueaux, 2006). Unexpectedly, however, brain cytoarchitecture and synapse density were not visibly altered, indicating that Nlgs are dispensable for the initial formation of synapses in vivo, and rather, control synaptic function. The differentiation and maturation of central synapses in the brain is technically difficult to analyze at the single-synapse level and particularly might be subject to compensatory regulations. It would thus be desirable to also explore the function of Nlgs in synaptic differentiation/maturation and its relation to Nrxs at a genetically accessible and comparatively simple synaptic terminal (Banovic, 2010).

In a large-scale, unbiased mutagenesis screen for genes that regulate synaptic terminal growth in Drosophila, mutations were isolated in a neuroligin homolog (dnlg1) resulting in neuromuscular junctions (NMJs) with strongly reduced numbers of synaptic boutons. NMJ in vivo imaging showed that the structural defects in dnlg1 mutants are due to a deficit in bouton addition, but not to subsequent deficits in bouton stability. DNlg1 is specifically expressed and functionally required at the postsynaptic side of NMJs, forming discrete clusters adjacent to, but not overlapping with, glutamate receptor (GluR) clusters. Lack of DNlg1 provoked severe deficits in postsynaptic differentiation, with individual active zones (AZs) or even entire boutons lacking postsynaptic GluR fields. The phenotypes identified by this analysis might be valuable for the further mechanistic analysis of Nlg-mediated signaling, and might shed light on Nlg-associated diseases such as autism (Banovic, 2010).

Nlgs are generally considered to play an important role in the establishment of fully functional neuronal circuits (Varoqueaux, 2006; Hoon, 2009). Nlgs bind Nrxs, and both proteins are sufficient to induce synapse formation in cultured cells. Major issues, however, concerning the precise role of Nlgs for synapse formation, maturation, and maintenance have therefore remained open and are actively discussed. These aspects include whether Nlgs can execute actual synaptogenic functions or are restricted to synapse maturation, maintenance, or both. To what extent functions of Nlgs can be reduced to retrograde signaling via Nrxs is another question (Banovic, 2010).

In an unbiased EMS mutagenesis screen, this study identified a Drosophila Nlg family protein, DNlg1. Null mutations in Drosophila dnlg1 dramatically reduced the number of synaptic boutons. Consistent with a reduction in terminal size, the number of the remaining synapses per NMJ was similarly reduced. Electrophysiological analysis suggested that the reduction in synapses provoked a similar reduction in the amount of neurotransmitter released per action potential. In contrast to findings in mice, where electrophysiological, but not structural, abnormalities were observed in nlg triple mutants (Varoqueaux, 2006), the functional defects at Drosophila NMJs seem to be largely a consequence of the structural defects (Banovic, 2010).

Notably, DNlg1 is not required for the initial formation of synaptic terminals per se, because NMJs form on all muscles of dnlg1 mutant animals, with an apparently normal timing. In addition, approximately 50% of the synapses are still present and largely functional, also at later stages. DNlg1, however, is required for effective addition of synaptic boutons during NMJ development and growth. Extended in vivo imaging of synaptic terminals was performed at wild-type and mutant NMJs, finding that the dnlg1 phenotype clearly reflects a genuine inability to effectively add new synaptic boutons to a synaptic terminal, but does not arise as a secondary deficit in the stability of previously assembled boutons. Thus, the inability to add new boutons, identified as the hallmark of this complementation group in the unbiased screen, leads to the reduction of NMJ size at the end of larval development. The reduction in bouton numbers also correlated with a reduction in the total number of synapses per NMJ. Establishment of a direct causal relation awaits further genetic dissection of DNlg1 signaling. Clearly, however, DNlg1 is not absolutely essential, because residual boutons still form. Thus, DNlg1 might be regarded more as a regulatory factor than an essential building block of synapses, consistent with its localization adjacent to, but not overlapping with, PSDs labeled by GluRs (Banovic, 2010).

Assembly of the postsynaptic apparatus did not take place for a significant fraction of boutons and individual synapses, whereas the accumulation of presynaptic markers was essentially normal. Again, live imaging was used to demonstrate a genuine postsynaptic assembly deficit, because boutons lacking SSR differentiation develop and continuously add presynaptic BRP-positive AZs without signs of presynaptic dedifferentiation. It thus appears that DNlg1 coordinates the formation of the postsynaptic compartment at the larval NMJ, including the proper localization of GluR clusters and the formation of the SSR and PSDs. Previous work has shown that a genetically induced lack of GluR complexes interferes with formation of the SSR (Schmid, 2006). Thus, an inability to target, transport, or maintain GluRs sufficiently (or some combination thereof) might be at the center of the postsynaptic differentiation or maturation deficits (Banovic, 2010).

The links between bouton defects and individual AZ deficits remain to be addressed. Mutations in dnlg1 affected NMJs both at the single-bouton level and at the single-synapse level, but they affected these synaptic structures only partially. However, increased DNlg1 levels were able to trigger molecular aspects of postsynaptic differentiation even at type II boutons, emphasizing the rate-limiting character DNlg1 can play for assembly processes in this system. The partial character of these phenotypes is not due to residual DNlg1 activities in the alleles because a deletion allele with the entire dnlg1 open reading frame removed resulted in the very same phenotypes. Pathways operating in parallel, upstream, or both of DNlg1 and related differentiation processes need to be addressed in future analyses. The electron microscopy analysis showed that planar appositions between presynaptic AZ membranes and postsynaptic membranes, a hallmark of synapse formation, still formed in bouton regions where the postsynaptic assembly largely failed (indicated by a lack of SSR). Thus, consistent with genetic analysis in mammals, at least some fundamental aspects of synapse formation -- likely involving the deposition of specific cell adhesion proteins at both presynaptic and postsynaptic membrane -- continue in dnlg1 mutants (Banovic, 2010).

The prominent in vivo phenotype that this study reports for an Nlg family protein allow a mechanistic analysis of this important gene family at the Drosophila NMJ. All evidence, particularly functional rescue analysis, conclusively demonstrated that DNlg1 operates in the postsynaptic muscle compartment. When overexpressed, DNlg1 lacking the cytoplasmic domain (DNlg1-GFPΔcyto) displayed a drastic dominant-negative phenotype. Because DNlg1-GFPΔcyto was effectively targeted to the NMJ, it appears plausible that it still incorporates into DNlg1 signaling complexes but abrogates their functionality. Thus, apart from ectodomain-mediated interactions to proteins other than DNrx, the cytoplasmic domain seems also essential for the role of DNlg1 complexes in addition to that of presynaptic boutons. The cytoplasmic interactions of DNlg1 most likely consist of physical links to submembrane scaffold proteins. This is true, at least in part, for Nlg-2, which connects to the PSD proteins gephyrin and collybistin at GABAergic and glycinergic synapses (Poulopoulos, 2009). At vertebrate excitatory synapses, interactions similar to postsynaptic scaffolding proteins such as PSD-95 support Nlg function). The fact that DNlg1-GFPΔextra (ectodomain deleted) is still localized to type I NMJ terminals and triggers ectopic clusters of postsynaptic proteins further underlines the role of the cytoplasmic domain in mediating protein-protein interactions. Thus, while future mechanistic analysis should also include expression of similar constructs under physiological expression levels, screening for interactions with the loss- and gain-of-function phenotypes is warranted (Banovic, 2010).

Interaction with presynaptic Nrxs is thought to be of prime importance for Nlg function. However, depending on the assay and context studied, results that conflict with this hypothesis are reported. In preliminary cell aggregation and immoprecipitation experiments, this study was unable to detect direct interaction between DNrx and DNlg1. It thus remains to be shown that DNlg1 interacts with DNrx directly. In principle, DNrx and DNlg1 could be part of larger complexes that might also comprise Drosophila homologs of an alternative postsynaptic Nrx receptor, called LRRTM2 (de Wit, 2009; Ko, 2009). Irrespective of the exact nature of the protein-protein interactions, this study has presented evidence that presynaptic Drosophila Nrx promotes DNlg1 function, but is not an absolute prerequisite for it. First, while some aspects of the dnlg1 phenotype are similar to dnrx mutant terminals (reduction of bouton numbers, ruffles in AZ, irregular receptor fields), they all are quantifiably less pronounced. Second, the most extreme phenotype (entire boutons lacking postsynaptic differentiation) was absent at dnrx terminals. Third, the severity of the dnlg1 phenotype did not increase upon simultaneous elimination of DNrx, consistent with the idea that both proteins regulate a similar biological process or that DNrx functions are fully mediated via DNlg1 (Banovic, 2010).

Endogenous DNlg1 forms discrete clusters close to, but not identical with, PSD regions. In fact, loss of presynaptic DNrx severely reduced the numbers of DNlg1 clusters. DNrx and DNlg1 clusters often appear apposed at corresponding presynaptic and postsynaptic sites, perhaps defining a new synaptic 'compartment.' The DNlg1 ectodomain together with the transmembrane region seems to be sufficient for the assembly of DNlg1 clusters, while active signaling seems to depend on the cytoplasmic domain. Nrx binding might contribute to this ectodomain-mediated integration, because the dominant-negative effect of DNlg1 overexpression could be suppressed by either blocking DNrx binding by a point mutation or expressing it in a dnrx mutant background. Taken together, these data imply that presynaptic Nrx binding promotes accumulation of Nlg clusters at the postsynaptic membrane. Loss of this Nrx-binding activity weakens, but does not eliminate, Nlg signaling (Banovic, 2010).

Drosophila neuroligin 1 regulates synaptic growth and function in response to activity and phosphoinositide-3-kinase

Neuroligins are postsynaptic neural cell adhesion molecules that mediate synaptic maturation and function in vertebrates and invertebrates, but their mechanisms of action and regulation are not well understood. At the Drosophila larval neuromuscular junction (NMJ), previous analysis demonstrated a requirement for Drosophila neuroligin 1 (dnlg1) in synaptic growth and maturation. The goal of the present study was to better understand the effects and mechanisms of loss-of-function and overexpression of dnlg1 on synapse size and function, and to identify signaling pathways that control dnlg1 expression. Consistent with reduced synapse size, evoked excitatory junctional currents (EJCs) were diminished in dnlg1 mutants but displayed normal Ca(2+) sensitivity and short-term plasticity. However, postsynaptic function was also perturbed, in that glutamate receptor staining and the distribution of amplitudes of miniature excitatory junctional currents (mEJCs) were abnormal in mutants. All the above phenotypes were rescued by a genomic transgene. Overexpression of dnlg1 in muscle resulted in synaptic overgrowth, but reduced the amplitudes of EJCs and mEJCs. Overgrowth and reduced EJC amplitude required Drosophila neurexin 1 (dnrx1) function, suggesting that increased DNlg1/DNrx1 signaling attenuates synaptic transmission and regulates growth through a retrograde mechanism. In contrast, reduced mEJC amplitude was independent of dnrx1. Synaptic overgrowth, triggered by neuronal hyperactivity, absence of the E3 ubiquitin ligase highwire, and increased phosphoinositide-3-kinase (PI3K) signaling in motor neurons reduced synaptic DNlg1 levels. Likewise, postsynaptic attenuation of PI3K, which increases synaptic strength, was associated with reduced DNlg1 levels. These observations suggest that activity and PI3K signaling pathways modulate growth and synaptic transmission through dnlg1-dependent mechanisms (Mozer, 2012).

To gain insight into the role of NLGs in synapse maturation and function, this study determined how loss-of-function and overexpression of dnlg1 affects growth and synaptic transmission, and signaling pathways were identified that regulate dnlg1 expression. Based on loss-of-function and overexpression experiments, DNlg1 signals via a retrograde mechanism to regulate synapse size and function in a DNrx1-dependent manner. Loss-of-function reduced the number of boutons with a concomitant reduction in the number of functional release sites. Conversely, overexpression drove synaptic growth, but attenuated evoked glutamate release, signaling to the presynaptic neuron via DNrx1. DNlg1 also regulates GluR responsiveness release through a DNrx1-independent pathway. Therefore, in the proposed model, DNlg1 mediates bi-directional signaling across the synapse with DNrx1 to regulate synapse growth and function (see A Model of the proposed function of dnlg1 in the regulation of synaptic growth and activity in the neuromuscular junction, Figure 6 of Moser, 2012).

dnlg1 expression is modulated by multiple anterograde signals from presynaptic motor neurons and cell autonomous signaling in muscle. Synaptic overgrowth triggered by constitutive PI3K signaling in motor neurons, neuronal hyperactivity, or the absence of highwire (hiw), correlate with reduced DNlg1 levels at the NMJ. Likewise, increased synaptic strength and reduced synapse size associated with attenuation of PI3K signaling in the motor neurons or in muscle also correlate with lowered DNlg1 levels. Thus, downregulation of dnlg1 expression appears to be a common output of signaling pathways implicated in synaptic plasticity and suggests that attenuation of dnlg1 signaling contributes to mechanisms that coordinate growth with synaptic activity. While it is difficult to reconcile these observations in a simple model, these data suggest that reduced signaling by dnlg1 contributes to plasticity, but is unlikely the sole determinant of the polarity of the response. Reduced levels of DNlg1 may be a prerequisite for assembly or disassembly of synapses, but other factors (perhaps additional cell adhesion molecules) likely determine whether these changes result in an increase or a decrease in synaptic strength (Mozer, 2012).

Previous analysis (Banovic, 2010) suggested that dnlg1 did not directly regulate synaptic function, because the reduction of synaptic strength in mutants was a consequence of a defect in synaptic growth. This study confirmed that evoked release in dnlg1 mutants generated smaller synaptic currents and it was found, in addition, that mutant synapses respond normally to changes in extracellular Ca2 + concentration, and have normal release probability. However, the current findings that dnlg1 overexpression attenuates release and that DNlg1 levels determine the postsynaptic response to quantal release suggest a more nuanced view (Mozer, 2012).

In dnlg1 loss-of-function mutants, distribution of mEJCs in the mutant was shifted significantly to the right, indicating an increased number of larger responses. Postsynaptic overexpression of dnlg1 had a reciprocal effect, increasing the number of smaller mEJCs. Although changes in vesicle size or alteration in glutamate transport by vesicular ATPases can affect mEJC distributions, ultrastructural analysis of dnlg1 mutant synapses did not reveal defects in synapse vesicle size (Banovic, 2010). Instead, the altered GluRIII staining in the mutants and the apparent muscle autonomy of the overexpression phenotype indicate that dnlg1 negatively regulates the clustering or turnover, and possibly the sensitivity of GluRs (Mozer, 2012).

Banovic (2010) demonstrated that dnlg1 is necessary for synaptic growth, and the effects of dnlg1 overexpression presented in this study show that dnlg1 signaling is sufficient for the formation of nascent synapses in the NMJ. Thus, dnlg1 has a unique function in the regulation of synapse growth and differs from the mammalian neuroligins, which are not required for synapse formation in vivo. However, the data also show that dnlg1 signaling modulates postsynaptic function and can attenuate neurotransmitter release, suggesting a determinative regulatory role in synapse maturation. Vertebrate neuroligins have been proposed to drive synaptic maturation through the recruitment of scaffolding molecules or other PDZ domain binding proteins to the postsynaptic complex (Sudhof, 2008), and in the case of nlg1, promote synaptic GluR localization (Heine, 2008; Mondin, 2011). The accumulation of synaptic GluRs in dnlg1 mutants and the inhibitory effects of dnlg1 overexpression on synaptic transmission suggest that dnlg1 may inhibit GluR function by antagonizing signals the promote GluR synaptic localization or organization (Mozer, 2012).

The effect of muscle overexpression of dnlg1 on synaptic growth and neurotransmitter release suggests a retrograde signaling mechanism. Overexpression of wild-type dnlg1 in larval muscle using the BG487-Gal4 driver increased synaptic bouton number but reduced quantal content, and both effects were suppressed by mutation in dnrx1. Thus, increased synaptic growth and inhibition of neurotransmitter release resulting from retrograde DNlg1 signaling is mediated by DNrx1. Consistent with this hypothesis, overexpression of dnrx1 in motor neurons increased synapse bouton number but reduced quantal content\, a phenocopy of dnlg1 overexpression in muscle. Furthermore, dnrx1 mutants suppressed the synaptic growth defects of dnlg1-gfp overexpression driven by mef2-gal4 (Banovic, 2010; Mozer, 2012).

Increased retrograde signaling by DNlg1/DNrx1 promotes growth, but the nascent synapses have reduced function not accounted for by defects in apposition of pre and postsynaptic components or by a reduction in the number of active zones. Overexpression of dnlg1 likely attenuates presynaptic function by increasing synaptic DNrx1 levels in the terminal. Banovic (2010) showed that dnrx1 is required to maintain DNlg1 levels at the synapse, suggesting that trans-synaptic complex formation stabilizes both binding partners. Because NRXs function to recruit molecules implicated in exocytosis, increased signaling may have deleterious effects on presynaptic function, by altering subunit stoichiometry of components of the release machinery. Alternatively, overexpression of DNlg1 may preclude binding of additional DNrx1 ligands important for presynaptic function. Three additional neuroligins encoded by the fly genome are potential DNrx1 binding partners including DNlg2 which binds DNrx1 in vitro and is expressed both in muscle and motor neurons (Sun, 2011), DNlg3 which is expressed in all neurons, and DNlg4 (Mozer, 2012).

Constitutive activation of PI3K signaling in motor neurons, mutations in hiw, a neuronal E3 ubiquitin ligase, and shaggy, which encodes the Drosophila homolog of GSK3‐β, cause an increase in synapse bouton numbers and reduce quantal content. Because of the shared phenotype and site of action in motor neurons PI3K, hiw and shaggy may signal through a common pathway and epistasis experiments suggest that hiw and shaggy act upstream of AP1 to regulate synaptic growth (Franciscovich, 2008). This study shows that dnlg1 overexpression in muscle increased synapse bouton numbers and reduced quantal content. This observation suggests that retrograde signaling by dnlg1 drives growth and attenuates neurotransmitter release by inhibition of hiw or shaggy or the activation of PI3K signaling in motor neurons (Mozer, 2012).

At the Drosophila NMJ, neuronal hyperexcitability increases synaptic growth and function through alterations of cAMP levels and changes of gene expression mediated by transcription factors including AP1 and CREB, the cyclic AMP responsive element binding protein. This study finds that DNlg1 expression is reduced during synapse overgrowth in eag Sh mutants or following neural overexpression of AP1 suggesting that dnlg1 is negatively regulated by activity. Similarly, it has been shown that reduced expression of the homophilic cell adhesion molecular fasciclinII (fasII) is necessary for synaptic overgrowth in eag Sh mutants. Whether down-regulation of dnlg1 is required for synaptic overgrowth or represents a homeostatic response to pro-growth signaling remain unanswered questions. In eag Sh mutants, dnlg1 RNA is substantially reduced, suggesting that the postsynaptic response to overgrowth involves changes in gene expression and not only protein turnover. Future experiments will be needed to determine if transcriptional repression or alterations of mRNA splicing or turnover account for the decreased dnlg1 RNA levels. Furthermore, the fact that dnlg1 expression induces growth under some conditions (the present paper) and inhibits growth in others (Banovic, 2010) suggests that spatial and/or temporal regulation of dnlg1 is likely to be complex (Mozer, 2012).

In vertebrates, PI3K signaling has been implicated in mechanisms that regulate synaptic function and plasticity. PI3K signaling activates mTOR kinase and has been postulated to increase translation of synaptic proteins, but the downstream effectors that mediate synaptic changes have not been identified. In the fly NMJ, presynaptic PI3K signaling regulates synaptic growth and dampens synaptic activity mediated by presynaptic metabotropic glutamate receptors. Alterations of synapse size and function mediated by reduced PI3K signaling in neurons results in changes in adult Drosophila behavior affecting odor perception and sensitivity to alcohol. This study found that increasing PI3K signaling in the presynaptic neuron, or inhibiting it in either the neuron or muscle, caused dramatic reduction of dnlg1 expression at the Drosophila NMJ. Whether reduced levels of DNlg1 are required to mediate PI3K dependent alterations of synaptic size and strength, and the nature of the anterograde signal to the muscle, remain to be determined. These observations suggest that neuroligins are candidate downstream effectors of PI3K signaling pathways and that reduced dnlg1 signaling contributes to PI3K dependent synaptic changes and altered behavior (Mozer, 2012).

A current model of autism proposes that increased translation of synaptic proteins due to elevated PI3K/TOR signaling underlies synaptic dysfunction and the behavioral phenotypes associated with the disease (Stern, 2011). Elevated PI3K signaling has been implicated in autism associated with certain neurodevelopmental syndromes including fragile X. In this context, constitutive activation of presynaptic PI3K signaling in the Drosophila NMJ is a simple model of synaptic function of the autistic brain. Proteins whose levels increase in response to elevated PI3K signaling likely contribute to pathogenic mechanisms of synaptic dysfunction, so the downregulation of dnlg1 observed in this study may not be relevant to disease. However, expression of Nlg1 was reduced in the hippocampus of fmr1 mutant mouse brain suggesting that downregulation of nlg expression in response to increased PI3K signaling occurs in vertebrate synapses in a mouse model of autism. The current study provides evidence of linkage between PI3K/mTOR and nlg signaling at the synapse and warrants further investigation of the role of neuroligins in PI3K-dependent signaling processes relevant to neurodevelopmental diseases (Mozer, 2012).

The postsynaptic t-SNARE Syntaxin 4 controls traffic of Neuroligin 1 and Synaptotagmin 4 to regulate retrograde signaling

Postsynaptic cells can induce synaptic plasticity through the release of activity-dependent retrograde signals. A Ca(2+)-dependent retrograde signaling pathway mediated by postsynaptic Synaptotagmin 4 (Syt4) has been previously described in this context. To identify proteins involved in postsynaptic exocytosis, this study conducted a screen for candidates that disrupt trafficking of a pHluorin-tagged Syt4 at Drosophila neuromuscular junctions (NMJs). The study further characterized one candidate, the postsynaptic t-SNARE Syntaxin 4 (Syx4). Analysis of Syx4 mutants reveals that Syx4 mediates retrograde signaling, modulating the membrane levels of Syt4 and the transsynaptic adhesion protein Neuroligin 1 (Nlg1). Syx4-dependent trafficking regulates synaptic development, including controlling synaptic bouton number and the ability to bud new varicosities in response to acute neuronal stimulation. Genetic interaction experiments demonstrate Syx4, Syt4, and Nlg1 regulate synaptic growth and plasticity through both shared and parallel signaling pathways. These findings suggest a conserved postsynaptic SNARE machinery controls multiple aspects of retrograde signaling and cargo trafficking within the postsynaptic compartment (Harris, 2016).

Synaptic connections form and mature through signaling events in both pre- and postsynaptic cells. The release of signaling molecules into the synaptic cleft depends on SNARE proteins that drive membrane fusion. This machinery is well understood for neurotransmitter release from the presynaptic cell: in response to an action potential, a v-SNARE in the synaptic vesicle membrane (Synpatobrevin/VAMP) engages t-SNARES in the presynaptic membrane (Syx1 and SNAP-25), forming a four-helix structure that brings the membranes into close proximity and initiates fusion. Although SNARE-dependent fusion drives membrane dynamics in all cell types, it is specialized in the presynaptic terminal to be Ca2+-dependent, employing Ca2+ sensors like Synaptotagmin 1 (Syt1) to link synaptic vesicle fusion to Ca2+ influx following an action potential (Harris, 2016).

The postsynaptic cell also exhibits activity-dependent exocytosis. Altering the composition of the postsynaptic membrane, including regulated trafficking of neurotransmitter receptors, is an important plastic response to neural activity (Chater, 2014). The postsynaptic cell also releases retrograde signals into the synaptic cleft to modulate synaptic growth and function. These retrograde messengers include lipid-derived molecules like endocannabinoids, gases like nitric oxide, neurotransmitters, neurotrophins, and other signaling factors like TGF-β and Wnt. Adhesion complexes that provide direct contacts across the synaptic cleft also participate in retrograde signaling (Harris, 2016).

Although retrograde signaling is a key modulator of synaptic function, little is known about how postsynaptic exocytosis is regulated and coordinated. Components of a postsynaptic SNARE complex have been recently identified in mammalian dendrites. The t-SNAREs Syntaxin 3 (Stx3) and SNAP-47 are required for regulated AMPA receptor exocytosis during long term potentiation, while the v-SNARE synaptobrevin-2 regulates both activity-dependent and constitutive AMPAR trafficking (Jurado, 2013). Stx4 has also been implicated in activity-dependent AMPAR exocytosis (Kennedy, 2010). In Drosophila, a Ca2+-dependent retrograde signaling pathway relies on the postsynaptic Ca2+ sensor Syt4. Syt4 vesicles fuse with the postsynaptic membrane in an activity-dependent fashion (Yoshihara, 2005), and loss of Syt4 leads to abnormal development and function of the NMJ. Syt4 null animals have smaller synaptic arbors, indicating a defect in synaptic growth, and also fail to exhibit several forms of synaptic plasticity seen in control animals, including robust enhancement of presynaptic release in response to high frequency stimulation, and rapid budding of synaptic boutons in response to strong neuronal stimulation (Barber, 2009; Korkut, 2013; Piccioli, 2014; Yoshihara, 2005). However, a detailed understanding of how the postsynaptic cell regulates constitutive and activity-dependent signaling of multiple retrograde pathways is lacking. In addition to exocytosis, it is likely that many cellular processes including vesicle trafficking and polarized transport of protein and transcript are specialized to facilitate postsynaptic signaling. Identifying such regulatory mechanisms is crucial for understanding synaptic development and function (Harris, 2016).

This study carried out a candidate-based transgenic RNAi screen to identify regulators of postsynaptic exocytosis at the Drosophila NMJ, a model for studying glutamatergic synapse growth and plasticit. Using a fluorescently tagged form of the postsynaptic Ca2+ sensor Syt4, candidate gene products were screened that disrupted the localization of Syt4 at the postsynaptic membrane. This study describes characterization of one candidate from this screen, Syntaxin 4 (Syx4). Drosophila Syx4 is the sole homolog of the mammalian Stx 3/4 family of plasma membrane t-SNAREs that also includes Syntaxin 1. The mammalian Stx3 and Stx4 homologs regulate activity-dependent AMPA receptor trafficking in mammalian neurons (Jurado, 2013; Kennedy, 2010), while Stx4 also participates in regulated secretory events in several other mammalian cell types, including insulin-stimulated delivery of the glucose transporter to the plasma membrane in adipocytes and glucose-stimulated insulin secretion from pancreatic beta cells (reviewed by Jewell, 2010). The results demonstrate that the Drosophila Syx4 homolog is essential for retrograde signaling, regulating the membrane delivery of both Syt4 and Neuroligin (Nlg1), a transsynaptic adhesion protein that plays important roles in synapse formation and function, and is linked to autism spectrum disorder (ASD). Through genetic interaction experiments, this study defined functions of the Syx4, Syt4, and Nlg1 pathway in regulating multiple aspects of synaptic growth and plasticity within the postsynaptic compartment (Harris, 2016).

To identify regulators of postsynaptic exocytosis, a screen was conducted for gene products regulating Syt4 plasma membrane accumulation, resulting in the identification of the plasma membrane t-SNARE Syx4. Analysis of a Syx4 null mutant indicates that Syx4 is essential for development of the Drosophila NMJ and regulates the membrane delivery of at least two proteins that are important for synaptic growth and plasticity: the postsynaptic Ca2+ sensor Syt4 and the transsynaptic adhesion protein Nlg1 (Harris, 2016).

The screen identified 15 candidate gene products that altered the localization of Syt4-pH. In addition to Syx4, several other candidates motivate interesting hypotheses about regulatory pathways for postsynaptic exocytosis. MyoV is a Ca2+-sensitive unconventional myosin that regulates polarized traffic. Thus, MyoV could play a role linking Ca2+ influx to vesicle delivery or release at the synapse. Indeed, MyoV homologs have been implicated in regulated AMPA trafficking in mammalian dendrites. Two Rab regulators (Gdi and Rabex) suggest that key vesicle trafficking steps en route to the synapse are modulated by Rab activation states. Also, two cell adhesion molecules (Neuroglian and Contactin) indicate potential transsynaptic mechanisms regulating retrograde signaling. Neuroglian has been shown to be required for synaptic stability and it is possible that Syt4-mediated retrograde signaling plays some role in this process (Harris, 2016).

Syt4 has also been shown to be transferred transsynaptically from the presynaptic terminal to the postsynaptic terminal on exosomes (Korkut, 2013). Thus, the approach of expressing Syt4-pH postsynaptically may not reveal components for the biosynthetic synthesis and transport of presynaptic Syt4. Nevertheless, the requirement for Syt4 in the postsynaptic cell for retrograde signaling is clear, and the results of the screen highlight regulators of Syt4 trafficking to and from the postsynaptic membrane where Syt4 vesicles fuse in an activity-dependent manner. The observation that endogenously expressed Syt4-GFP (Syt4GFP-2M) shows a similar distribution to Syt4-pH supports the biological relevance of the screen data for identifying regulators of Syt4 trafficking in the postsynaptic cell (Harris, 2016).

The Syx4 null allele phenocopies the Syx4-RNAi knockdown, reducing the delivery of Syt4-pH to the postsynaptic membrane. Consistent with this finding, loss of Syx4 produces similar phenotypes to loss of Syt4. Both null mutants exhibit a reduction in the total number of boutons at the NMJ, indicating a defect in synaptic growth. Moreover, genetic interaction experiments clearly indicate that Syx4 and Syt4 interact with respect to synaptic growth. A strong genetic interaction between Syx4 and Syt4 is also evident at the level of lethality, as double mutant animals are lethal at a much earlier stage than either single mutant alone. Thus, even though Syx4 affects the localization of Syt4, suggesting they act in the same pathway, the genetic interaction data do not support a simple epistatic relationship. The difference in phenotypic severity, with the Syx4 bouton number defect being significantly stronger than the Syt4 defect, also points to Syt4 not being absolutely required for Syx4 signaling. A similar phenomenon is observed presynaptically where the t-SNARE Syx1 is indispensible for synaptic vesicle fusion, while fusion is only reduced in the absence of the synaptic vesicle Ca2+ sensor Syt1. Taken together, it is hypothesized that (1) Syx4 and Syt4 act together in a single pathway where Syx4 regulates the exocytosis of vesicles containing Syt4, and (2) Syx4 and Syt4 also act in divergent pathways, where Syt4 cooperates with other t-SNARES, and Syx4 mediates the exocytosis of vesicles in a Syt4-independent manner. This model allows for multiple possible postsynaptic SNARE complexes, regulating distinct release events. Dissecting the other components of these fusion machineries, and distinguishing activity-dependent from constitutive release events, will be important to build understanding of how retrograde signaling is regulated (Harris, 2016).

In addition to affecting the localization of Syt4, Syx4 mutants also exhibit a decrease in the amount of Nlg1 at the postsynaptic membrane. Nlg1 has several functions at the synapse, along with its presynaptic binding partner Nrx-1. Together they regulate bouton number as well as the size and spacing of active zones and glutamate receptors, though some aspects of Nlg1 signaling appear to be independent of Nrx-1. Mutations in Nrx and Nlg family genes are also linked to autism spectrum disorder (ASD), highlighting the importance of Nrx-Nlg signaling in neuronal development. Consistent with a reduction of Nlg1 levels at the synapse, strong genetic interactions were observed between Syx4, Nlg1 and Nrx-1 with respect to bouton number. However, the prominent AZ/GluR defects seen in Nlg1 and Nrx-1 mutants were not observed in Syx4 mutants, and heterozygous combinations did not produce these defects. It is likely that Syx4 mutants exhibit a partial loss of function of Nlg1, and that bouton number is sensitive to this loss while AZ/GluR organization can be maintained with low levels of Nlg1 (Harris, 2016).

A dramatic change in distribution of Nlg1Δcyto is observed in the Syx4 mutant background, providing further evidence that Syx4 regulates the localization of Nlg1. The redistribution of Nlg1Δcyto to large accumulations is striking compared to full-length Nlg1, which is simply reduced at the synapse in the Syx4 mutant background. This observation points to complex Syx4-dependent regulation of Nlg1 localization. One model is that trafficking of Nlg1 involves both a Syx4-dependent pathway and a second pathway that depends on an interaction with the Nlg1 C-terminus, which includes a PDZ-domain-binding motif. In this scenario, a severe Nlg1 trafficking defect is revealed only when both pathways are compromised. A second possibility is that in the absence of Syx4, a portion of the Nlg1 content in the cell is degraded, but that this degradation step depends on the presence of the Nlg1 cytoplasmic tail, leading to the observed aggregation of Nlg1Δcyto in Syx4 mutants (Harris, 2016).

Analysis of Nlg1 trafficking in live animals reveals that Nlg1 is strikingly stable, in both control and Syx4 mutant backgrounds. A motivation in performing these experiments was to test possible mechanisms underlying the decrease in Nlg1 levels in Syx4 mutants. It is possible that some Nlg1 mobility would be observed over a longer time course. Mammalian Nlg has been shown to undergo significant turnover at postsynaptic sites under LTP conditions in neuronal cell culture. Also, synaptic activity has been shown to induce cleavage of Nlg and the subsequent destabilization of the Nrx-Nlg complex. Thus, it remains a possibility that Nlg1 would be mobilized in response to activity in the preparation; however, no increased mobility was observed in response to high K+ incubations in preliminary tests. The data are most consistent with Syx4 regulating Nlg1 over a developmental time course. A detailed examination of the relationship between Syx4 and Nlg1 dynamics will be crucial to understand how Syx4 contributes to this important pathway in synaptic development (Harris, 2016).

A strong suppression of acute structural plasticity was observed in null mutants of Syx4, Syt4 and Nlg1. Double heterozygous combinations also indicated strong genetic interactions between all three of these genes with respect to plasticity. GB budding is regulated by both acute and developmental signaling. Because Syt4 postsynaptic vesicles fuse in an activity-dependent manner, it is possible that Syt4-dependent signaling releases an acute instructive cue for GB budding. Thus, one attractive model is that Nlg1 is delivered to the membrane in response to stimulation, depending on the Ca2+ sensitivity of Syt4 and the presence of the t-SNARE Syx4 at the membrane. It is also possible that Syx4-Syt4-Nlg1 signaling is required throughout development to potentiate the synapse to respond to strong neuronal stimulation. In conclusion, Syx4, Syt4, and Nlg1 interact to regulate several aspects of synaptic biology. The data support multiple overlapping signaling pathways regulated by these proteins, reflecting a complex modulation of retrograde signaling to control synaptic growth and plasticity at the Drosophila NMJ (Harris, 2016).

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

Using full length and truncated forms of Neurexin (Dnrx) and Neuroligins (Dnlg) together with cell biological analyses and genetic interactions, this study reports novel functions of dnrx and dnlg1 in clustering of pre- and postsynaptic proteins, coordination of synaptic growth and ultrastructural organization. dnrx and dnlg1 extracellular and intracellular regions are required for proper synaptic growth and localization of dnlg1 and Dnrx, respectively. dnrx and dnlg1 single and double mutants display altered subcellular distribution of Discs large (Dlg), which is the homolog of mammalian post-synaptic density protein, PSD95. dnrx and dnlg1 mutants also display ultrastructural defects ranging from abnormal active zones, misformed pre- and post-synaptic areas with underdeveloped subsynaptic reticulum. Interestingly, dnrx and dnlg1 mutants have reduced levels of the BMP receptor Wishful thinking (Wit), and dnrx and dnlg1 are required for proper localization and stability of Wit. In addition, the synaptic overgrowth phenotype resulting from the overexpression of dnrx fails to manifest in wit mutants. Phenotypic analyses of dnrx/wit and dnlg1/wit mutants indicate that Dnrx/Dnlg1/Wit coordinate synaptic growth and architecture at the NMJ. These findings also demonstrate that loss of dnrx and dnlg1 leads to decreased levels of the BMP co-receptor, Thickveins and the downstream effector phosphorylated Mad at the NMJ synapses indicating that Dnrx/Dnlg1 regulate components of the BMP signaling pathway. Together these findings reveal that Dnrx/Dnlg are at the core of a highly orchestrated process that combines adhesive and signaling mechanisms to ensure proper synaptic organization and growth during NMJ development (Banerjee, 2016).

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

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

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

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

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

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

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

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

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

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

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

Cooperation of Syd-1 with Neurexin synchronizes pre- with postsynaptic assembly

Synapse formation and maturation requires bidirectional communication across the synaptic cleft. The trans-synaptic Neurexin-Neuroligin complex can bridge this cleft, and severe synapse assembly deficits are found in Drosophila melanogaster neuroligin (Nlg1, dnlg1) and neurexin (Nrx-1, dnrx) mutants. This study shows that the presynaptic active zone protein Syd-1 interacts with Nrx-1 to control synapse formation at the Drosophila neuromuscular junction. Mutants in Syd-1 (RhoGAP100F, dsyd-1), Nrx-1 and Nlg1 share active zone cytomatrix defects, which are nonadditive. Syd-1 and Nrx-1 form a complex in vivo, and Syd-1 is important for synaptic clustering and immobilization of Nrx-1. Consequently, postsynaptic clustering of Nlg1 is affected in Syd-1 mutants, and in vivo glutamate receptor incorporation is changed in Syd-1, Nrx-1 and Nlg1 mutants. Stabilization of nascent Syd-1–Liprin-α (Liprin-α) clusters, important to initialize active zone formation, is Nlg1 dependent. Thus, cooperation between Syd-1 and Nrx-1–Nlg1 seems to orchestrate early assembly processes between pre- and postsynaptic membranes, promoting avidity of newly forming synaptic scaffolds (Owald, 2012)

The Nrx and Nlg families include autism susceptibility genes, and their proteins are needed for proper synapse formation during circuit development. It has so far, however, remained largely unclear how they molecularly integrate into the synapse formation process, particularly in regard to the assembly of the presynaptic active zone scaffold. Thus, identifying proteins coupling Nrx-Nlg to the assembly process itself and defining where in the sequence of events Nrx-Nlg acts is critical for a deeper understanding of synapse formation and remodeling (Owald, 2012).

Independent work in model organisms has identified and characterized proteins guiding active zone assembly, with Syd-1 proteins functioning upstream of Syd-2 (Liprin-α). In vivo imaging demonstrated that both Syd-1 and Liprin-α accumulate very early during synapse assembly earlier than postsynaptic GluRs, and much earlier than presynaptic BRP. In vivo FRAP analysis now suggests that Syd-1 increases the dwell time of Nrx-1 near active zones and can actively recruit Nrx-1 in a PDZ-dependent manner. Likewise, Liprin-α cluster mobility is elevated in the Syd-1 mutant background, implying a retention function of Syd-1 for both Nrx-1 and Liprin-α at assembling active zones. This study also suggests that the assembly of initially forming Syd-1 and Liprin-α scaffolds is reversible. The success rate of establishing stable Syd-1 and Liprin-α scaffolds dropped in the absence of Nlg1. As postsynaptic overexpression of Nlg1 increased the expression of presynaptic Nrx-1, interaction of these initial active zone scaffolds is likely to be directly dependent on local Nrx-1 interacting with Syd-1. It is tempting to speculate that the Nrx-1–Syd-1 interaction provides binding sites at newly forming active zones to drive the accumulation of Liprin-α scaffolds past a critical point, to enter an essentially irreversible maturation process (characterized by the onset of GluRIIA incorporation). Such a cooperative scheme might be optimized for the integration of regulatory elements and protect the system from untimely and aberrant assembly. In fact, the active zone component BRP has been shown to be under constitutive phosphorylation to avoid premature assembly (Owald, 2012).

In this study mutants for Nlg1 and Nrx-1 showed aberrant active zone organization reflected in over-grown (star-shaped) T-bar. These phenotypes have been observed in Syd-1 mutants (Owald, 2010). All three mutants (Syd-1, Nrx-1 and Nlg1) assemble fewer active zones per NMJ. Consequently, levels of unused active zone scaffold components, such as BRP, might locally accumulate along their NMJ terminals. This increase in building blocks in turn might result in over-growth of the remaining active zone scaffolds. Additionally, Syd-1, Nrx-1 and Nlg1 might define an assembly sequence, which in turn could be a precondition to properly terminating assembly. One might speculate that an improperly assembled scaffold could retain free valences and that the scaffold could outgrow improperly. As active zone localization of Syd-1 clustering did not strongly depend on either Nrx-1 or Nlg1, it is suspected that a complex of Syd-1 with Nrx-1 might be important for the regulation of BRP incorporation. Potentially, binding to Nrx-1 (in a trans-synaptic complex with Nlg1) might unmask additional domains of Syd-1 for assembly and thereby allow the effective stabilization of Liprin-α scaffolds. Notably, mammalian Nlg1 has also been implicated in induction and maturation of the presynaptic terminal (Owald, 2012)

Nrx-1 and Syd-1 (Owald, 2010) are both expressed throughout the CNS, whereas Nlg1 is not. It is likely that other Drosophila Nlgs substitute for Nlg1 at central synapses. Of note, star-shaped T-bars were found at adult CNS synapses of Syd-1 mutants as well, suggesting that similar mechanisms as described in this study for NMJ synapses apply to CNS synapses (Owald, 2012)

Although Syd-1 remains cytoplasmic and depends on the presence of Nrx-1 to localize to the plasma membrane in non-neural cells (salivary gland epithelial cells, Syd-1 can also localize to active zones in the absence of Nrx-1. Consistently, Syd-1 mutated in its PDZ-domain (Gal4-UAS expressed) still localized to active zones, at least to a fair extent. Thus, nascent active zones seemingly contain additional proteins providing binding sites for Syd-1 (that also may be needed to stabilize a complex of Syd-1 and Nrx-1). Binding sites are still present after deletion of either Liprin-α or BRP -- despite a direct interaction of Syd-1 with BRP (Owald, 2010). Additional proteins representing potential upstream functions, such as the adaptor protein Neurabin that was shown to recruit C. elegans Syd-1 and Syd-2 to F-actin foci (Chia, 2012) are prime candidates for the localization of Syd-1 (Owald, 2012).

Unlike those of endogenous Syd-1, levels of Gal4-UAS–expressed Syd-1 depended on the presence of Nrx-1. Thus, uncomplexed, excessive Syd-1 might be subjected to degradation. Of note, Liprin-α is a downstream effector and possible substrate of the E3 ubiquitin ligase APC/C (Owald, 2012).

Early and rapid GluRIIA-mediated growth of nascent PSDs (younger than 24 h) is selectively impaired in Syd-1, Nrx-1 and Nlg1 mutants, where young PSDs are characterized by a high GluRIIB content. Mutants for GluRIIA, but not for GluRIIB, fail to grow sufficient synapses per terminal when challenged by high-temperature rearing. In addition, terminals of Syd-1, Nrx-1 and Nlg1 all suffer from under-growth of synaptic terminals. Thus, this under-growth might partially be a consequence of reduced initial GluRIIA incorporation. However, this leaves the question of how Nlg1 dictates GluRIIA incorporation. Nlg1 clusters, functionally associated with proteins regulating initial synapse assembly, might selectively promote GluRIIA incorporation directly. Notably, Nrx-Nlg complexes have been associated with GluR subunit–specific recruitment into PSDs in mammals. In that system, overexpression of Nlg1 selectively decreases the surface mobility of GluA2-containing AMPA-type glutamate receptors, in a manner mediated by a PSD95-Nlg1 interaction, while having no effect on GluA1 homomers. Indeed, Nlg1 is able to recruit the PSD95 ortholog Discs large (Dlg) to the Drosophila NMJ. Nlg1 clustering instructed by Syd-1 and Nrx-1 might create a seed for GluRIIA clustering mediated by Dlg and other scaffold proteins. It should be noted, however, that GluRIIA receptors still incorporate at Nlg1-mutant PSDs, although at a later time point of assembly, with PSDs also overshooting in size. Thus, Nlg1 seems particularly important for providing binding sites for GluRIIA complexes during early assembly, and choosing the right temporal sequence also seems important for the proper termination of the assembly process (Owald, 2012).

Trans-synaptic Teneurin signalling in neuromuscular synapse organization and target choice

Synapse assembly requires trans-synaptic signals between the pre- and postsynapse, but understanding of the essential organizational molecules involved in this process remains incomplete. Teneurin proteins are conserved, epidermal growth factor (EGF)-repeat-containing transmembrane proteins with large extracellular domains. This study shows that two Drosophila Teneurins, Ten-m and Ten-a, are required for neuromuscular synapse organization and target selection. Ten-a is presynaptic whereas Ten-m is mostly postsynaptic; neuronal Ten-a and muscle Ten-m form a complex in vivo. Pre- or postsynaptic Teneurin perturbations cause severe synapse loss and impair many facets of organization trans-synaptically and cell autonomously. These include defects in active zone apposition, release sites, membrane and vesicle organization, and synaptic transmission. Moreover, the presynaptic microtubule and postsynaptic spectrin cytoskeletons are severely disrupted, suggesting a mechanism whereby Teneurins organize the cytoskeleton, which in turn affects other aspects of synapse development. Supporting this, Ten-m physically interacts with alpha-Spectrin. Genetic analyses of teneurin and neuroligin reveal that they have differential roles that synergize to promote synapse assembly. Finally, at elevated endogenous levels, Ten-m regulates target selection between specific motor neurons and muscles. This study identifies the Teneurins as a key bi-directional trans-synaptic signal involved in general synapse organization, and demonstrates that proteins such as these can also regulate target selection (Mosca, 2012).

Vertebrate Teneurins are enriched in the developing brain, localized to synapses in culture and pattern visual connections. Both Drosophila Teneurins, Ten-m and Ten-a, function in olfactory synaptic partner matching and were further identified in neuromuscular junction (NMJ) defect screens, with Ten-m also affecting motor axon guidance (Zheng, 2011). This study examined their roles and underlying mechanisms in synapse development (Mosca, 2012).

Both Teneurins are enriched at the larval NMJ. Ten-a was detected at neuronal membranes: this staining was undetectable beyond background in ten-a null mutants and barely detectable following neuronal ten-a RNAi, indicating that Ten-a is predominantly presynaptic. Partial colocalization has been observed between Ten-a and the periactive zone marker Fasciclin II as well as the active zone marker Bruchpilot, suggesting a localization between these regions. Ten-m appeared strongly postsynaptic and surrounded each bouton. Muscle-specific ten-m RNAi eliminated the postsynaptic staining, but uncovered weak presynaptic staining that ubiquitous ten-m RNAi eliminated. Thus, the Ten-m signal was specific and, while partly presynaptic, enriched postsynaptically. Consistently, muscle Ten-m colocalized extensively with Dlg and completely with α-spectrin and is thus, likely coincident with all postsynaptic membranes (Mosca, 2012).

The localization of Ten-a and Ten-m suggested their transsynaptic interaction. To examine this, myc-tagged Ten-a was co-expressed in nerves using the Q system, and HA-tagged Ten-m was expressed in muscles using GAL4. Muscle Ten-m was able to co-immunoprecipitate nerve Ten-a from larval synaptosomes, suggesting that the Teneurins form a heterophilic transsynaptic receptor pair at the NMJ (Mosca, 2012).

To determine Teneurin function at the NMJ, the ten-a null allele and larvae with neuron or muscle RNAi of ten-a and/or ten-m were examined. Following such perturbations, bouton number and size were altered: the quantity was reduced by 55% and the incidence of large boutons markedly increased. Both elements represent impaired synaptic morphogenesis. The reduction in bouton number was likely cumulative through development, as it was visible in first instar ten-a mutants and persisted. In the ten-a mutant, bouton morphogenesis was rescued by restoring Ten-a expression in neurons, but not muscles. Neuronal Ten-m overexpression could not substitute for the lack of Ten-a, revealing their nonequivalence. Neuronal knockdown of Ten-a or Ten-m both showed an impairment, indicating presynaptic function for both, though presynaptic Ten-a plays a more significant role. Moreover, knocking down postsynaptic Ten-m in the ten-a mutant did not enhance the phenotype. Thus, presynaptic Ten-a (and to a lesser extent, Ten-m) and postsynaptic Ten-m are required for synapse development (Mosca, 2012).

Perturbation of teneurins also caused defects in the apposition between presynaptic active zones (release sites) and postsynaptic glutamate receptor clusters: up to 15% of the active zones/receptor clusters lacked their partner compared to 1.8% in controls. Under electron microscopy, active zones are marked by electron dense membranes and single presynaptic specializations called T-bars, which enable synapse assembly, vesicle release and Ca2+ channel clustering. Teneurin disruption caused defects in T-bar ultrastructure, membrane organization and apposition to contractile tissue. Teneurin perturbation also impaired postsynaptic densities while increasing membrane ruffling, further indicating organizational impairment. These phenotypes resemble mutants with adhesion and T-bar biogenesis defects, suggesting a role for Teneurins in synaptic adhesion and stability. Synaptic vesicle populations similarly required Teneurins for clustering at the bouton perimeter and proper density. As these effects are not synonymous with active zone disruption, Teneurins are also required for synaptic vesicle organization (Mosca, 2012).

Synapses lacking teneurin were also functionally impaired. The mean amplitude of evoked excitatory postsynaptic potentials (EPSP) in larvae was decreased by 28% in the ten-a mutant . Spontaneous miniature EPSPs (mEPSPs) showed a 20% decrease in amplitude, a 46% decrease in frequency and an altered amplitude distribution compared with control). These defects resulted in a 20% reduction in quantal content, which could be partly due to fewer boutons and release sites. However, release probability may also be reduced, as suggested by an increased paired pulse ratio in ten-a mutants. The decay kinetics of responses were faster in ten-a mutants, suggesting additional postsynaptic effects on glutamate receptors and/or intrinsic membrane properties. Further, FM1-43 dye loading revealed markedly defective vesicle cycling in ten-a mutants. Consistent with physiological impairment, teneurin-perturbed larvae exhibited profound locomotor defects. In summary, Teneurins are required for multiple aspects of NMJ organization and function (Mosca, 2012).

As a potential mechanism for synaptic disorganization following teneurin perturbation, the pre- and postsynaptic cytoskeletons were examined. In the presynaptic terminal, organized microtubules contain Futsch (a microtubule-binding protein)-positive “loops” while disorganized microtubules possess punctate, “unbundled” Futsch. Each classification normally represented ~10% (often distal) of boutons. Following teneurin perturbation, many more boutons had unbundled Futsch while those with looped microtubules were decreased by 62%–95%. Therefore, proper microtubule organization requires pre- and postsynaptic Teneurins. In contrast to mild active zone/glutamate receptor apposition defects, most boutons displayed microtubule organizational defects (Mosca, 2012).

Removal of teneurins also severely disrupted the postsynaptic spectrin cytoskeleton, with which Ten-m colocalized. Postsynaptic α-spectrin normally surrounds the bouton. Perturbing neuronal or muscle Teneurins markedly reduced postsynaptic α-spectrin without affecting Dlg. Postsynaptic β-spectrin, Adducin and Wsp were similarly affected. In the muscle, α-spectrin is coincident with and essential for the integrity of the membranous subsynaptic reticulum (SSR). Consistent with this, teneurin disruption reduced SSR width up to 70% and increased the frequency of 'ghost' boutons, which are failures of postsynaptic membrane organization). Thus, Teneurins are involved in the organization of the pre- and postsynaptic cytoskeletons and postsynaptic membranes. Further, endogenous α-spectrin co-immunoprecipitates with muscle-expressed, FLAG-tagged Ten-m, suggesting that Ten-m physically links the synaptic membrane to the cytoskeleton (Mosca, 2012).

As the most severe defects following teneurin perturbation were cytoskeletal, it is proposed that Teneurins primarily organize the presynaptic microtubule and postsynaptic spectrin-based cytoskeletons. However, such a solitary role cannot fully explain the observed phenotypes. The bouton number defects associated with cytoskeletal disruption are milder than those following teneurin disruption. Also, while active zone dynamics are affected by cytoskeletal perturbation, defects in apposition are not. Moreover, the T-bar structural defects more closely resemble synapse adhesion and active zone formation defects. Thus, Teneurins may regulate release site organization and synaptic adhesion independent of the cytoskeleton (Mosca, 2012).

These data also indicate that Teneurins act bi-directionally across the synaptic cleft. Ten-a acts predominantly neuronally as evidenced by localization, phenotypes caused by neuronal (but not muscle) knockdown, and mutant rescue by neuronal (but not muscle) expression. Yet, in addition to the presynaptic phenotypes, many others were postsynaptic, including reduced muscle spectrin, SSR and membrane apposition. Similarly, although Ten-m is present both pre-and postsynaptically, muscle knockdown resulted in presynaptic defects, including microtubule and vesicle disorganization, reduced active zone apposition, and T-bar defects. Thus, Teneurins function in bi-directional transsynaptic signaling to organize neuromuscular synapses. This may involve downstream pathways or simply establish an organizational framework by the receptors themselves. Moreover, as the single disruptions of neuronal ten-a or muscle ten-m arevsimilarly severe and not enhanced by combination, they likely function in the same pathway. The finding that Ten-a and Ten-m co-immunoprecipitate from different cells in vitro (Hong, 2012) and across the NMJ in vivo further suggests a signal via trans-synaptic complex. Teneurin function, however, may not be solely transsynaptic. In some cases (vesicle density, SSR width), cell-autonomous knockdown showed stronger phenotypes than knocking down in synaptic partners. This suggests additional cell-autonomous roles unrelated to transsynaptic Teneurin signaling (Mosca, 2012).

Signaling involving the transmembrane proteins Neurexin and Neuroligin also mediates synapse development (Craig, 2007). In Drosophila, Neurexin (dnrx) and Neuroligin1 (dnlg1) mutations cause phenotypes similar to teneurin perturbation: reduced boutons, active zone organization, transmission and SSR. dnlg1 and dnrx mutations do not enhance each other, suggesting their function in the same pathway. Consistently, this study found that dnrx and dnlg1 mutants exhibited largely similar phenotypes. To investigate the relationship between the teneurins and dnrx/dnlg1, focus was placed on the dnlg1 null mutant. Both Ten-m and DNlg1-eGFP occupy similar postsynaptic space. teneurin and dnlg1 loss-of-function also displayed similar bouton number reductions, vesicle disorganization and ghost bouton frequencies. Other phenotypes showed notable differences in severity. In dnlg1 mutants, there was a 29% failure of active zone/glutamate receptor apposition, compared to 15% for the strongest teneurin perturbation. For the cytoskeleton, dnlg1 mutants were mildly impaired compared to teneurin perturbations (Mosca, 2012).

To further examine their interplay, ten-a dnlg1 double mutants were analyzed. Both single mutants were viable, despite their synaptic defects. Double mutants, however, were larval lethal. Rare escapers were obtained that displayed a 72% reduction in boutons, compared to a 50%–55% decrease in single mutants. Active zone apposition in double mutants was enhanced synergistically over either single mutant. Cytoskeletal defects in the double mutant resembled the ten-a mutant. These data suggest that teneurins and dnrx/dnlg1 act in partially overlapping pathways, cooperating to properly organize synapses, with Teneurins contributing more to cytoskeletal organization and Neurexin/Neuroligin to active zone apposition (Mosca, 2012).

In the accompanying manuscript (Hong, 2012), it was shown that while the basal Teneurins are broadly expressed in the Drosophila antennal lobe, elevated expression in select glomeruli mediates olfactory neuron partner matching. At the NMJ, this basal level mediates synapse organization. Analogous to the antennal lobe, elevated ten-m expression was found at muscles 3 and 8 using the ten-m-GAL4 enhancer tra. This was confirmed for endogenous ten-m, and it was determined to be contributed by elevated Ten-m expression in both nerves and muscles. Indeed, ten-m-GAL4 is highly expressed in select motoneurons, including MN3-Ib, which innervates muscle 3. This elevated larval expression also varied along the anterior-posterior axis, and was specific for Ten-m as Ten-a expression did not differ within or between segments (Mosca, 2012).

To test whether elevated Ten-m expression in muscle 3 and MN3-Ib affects neuromuscular connectivity, ten-m RNAi was expressed using ten-m-GAL4. Wild-type muscle 3 was almost always innervated. However, following ten-m knockdown, muscle 3 innervation failed in 11% of hemisegments. This required Ten-m on both sides of the synapse, as the targeting phenotype persisted following neuronal or muscle RNAi suppression using tissue-specific GAL80 transgenes. ten-a RNAi did not show this phenotype, consistent with homophilic target selection via Ten-m. The phenotype was specific to muscle 3, as innervation onto the immediately proximal or distal muscle was unchanged. The low penetrance is likely due to redundant target selection mechanisms. Where innervation did occur, the terminal displayed similarly severe phenotypes to other NMJs. Thus, in addition to generally mediating synaptic organization, Ten-m also contributes to correct target selection at a specific NMJ (Mosca, 2012).

To determine whether Ten-m overexpression could alter connectivity, Ten-m was expressed in muscle 6 (but not the adjacent muscle 7), and the motoneurons innervating both muscles using H94-GAL4. Normally, 60% of the boutons at muscles 6/7 are present on muscle 6 with 40% on muscle 7 . Ten-m overexpression caused a shift whereby 81% of boutons synapsed onto muscle 6 and only 19% onto muscle 7. This shift also required both neuronal and muscle Ten-m as neuronal or muscle GAL80 abrogated it. The effect was specific as Ten-a overexpression did not alter this synaptic balance, nor was it secondary to altered bouton number, which is unchanged. Therefore, elevated Ten-m on both sides of the NMJ can bias target choice. This, combined with evidence that Ten-m can engage homophilic interaction in vitro, supports a transsynaptic homophilic attraction model at the NMJ as in the olfactory system (Mosca, 2012).

In summary, this study has identified a two-tier mechanism for Teneurin function in synapse development at the Drosophila NMJ. At the basal level, Teneurins are expressed at all synapses and engage in hetero- and homophilic bi-directional transsynaptic signaling to properly organize synapses. Supporting this, the Teneurins can mediate homo-and heterophilic interactions in vitro and heterophilic interactions in vivo. At the synapse, Teneurins organize the cytoskeleton, interact with α-spectrin, and enable proper adhesion and release site formation. Further, elevated Ten-m expression regulates target selection in specific motoneurons and muscles via homophilic matching and functions with additional molecules to mediate precise neuromuscular connectivity. Teneurin-mediated target selection at the NMJ is analogous to its role in olfactory synaptic partner matching (Hong, 2012). As the Teneurins are expressed broadly throughout the antennal lobe, it remains an attractive possibility that they also regulate central synapse organization (Mosca, 2012).

Neuroligin 2 is required for synapse development and function at the Drosophila neuromuscular junction

Neuroligins belong to a highly conserved family of cell adhesion molecules that have been implicated in synapse formation and function. However, the precise in vivo roles of Neuroligins remain unclear. This study has analyzed the function of Drosophila neuroligin 2 (dnl2) in synaptic development and function. dnl2 is strongly expressed in the embryonic and larval CNS and at the larval neuromuscular junction (NMJ). dnl2 null mutants are viable but display numerous structural defects at the NMJ, including reduced axonal branching and fewer synaptic boutons. dnl2 mutants also show an increase in the number of active zones per bouton but a decrease in the thickness of the subsynaptic reticulum and length of postsynaptic densities. dnl2 mutants also exhibit a decrease in the total glutamate receptor density and a shift in the subunit composition of glutamate receptors in favor of GluRIIA complexes. In addition to the observed defects in synaptic morphology, it was also found that dnl2 mutants show increased transmitter release and altered kinetics of stimulus-evoked transmitter release. Importantly, the defects in presynaptic structure, receptor density, and synaptic transmission can be rescued by postsynaptic expression of dnl2. Finally, this study shows that dnl2 colocalizes and binds to Drosophila Neurexin (dnrx) in vivo. However, whereas homozygous mutants for either dnl2 or dnrx are viable, double mutants are lethal and display more severe defects in synaptic morphology. Altogether, these data show that, although dnl2 is not absolutely required for synaptogenesis, it is required postsynaptically for synapse maturation and function (Sun, 2011).

Analysis of the Drosophila melanogaster genome revealed the presence of four neuroligin-like genes (CG13772, CG34127, CG34139, and CG31146). All four of the putative neuroligin genes encode proteins that share significant amino acid similarity with vertebrate Neuroligin and a similar predicted protein structure; however, based on protein sequence alignments, the four Drosophila neuroligins and mammalian neuroligins evolved from a common ancestor. As such, it is not possible to draw a direct correlation between any one Drosophila neuroligin and the mammalian neuroligins. A full-length cDNA clone has been identified from a Drosophila brain cDNA library that corresponds to the CG13772 gene, which was submitted to flybase as dneuroligin (dnl). A recent unbiased screen for genes affecting NMJ structure, however, identified CG31146 and named that homolog Drosophila neuroligin 1 (Banovic, 2010). The present study examines the role of the CG13772 homolog originally named dneuroligin, which is now term Drosophila neuroligin 2 (dnl2) to avoid confusion. The dnl2 gene is located on the left arm of the second chromosome at cytological position 27C3-4 and is composed of 13 exons and 12 introns. dnl2 encodes a 1248 amino acid long protein with a predicted molecular weight of 137 kDa. Similar to vertebrate neuroligins, Dnl2 is also predicted to comprise three distinct regions: an N-terminal extracellular acetylcholinesterase-like domain, a single transmembrane region, and a C-terminal cytoplasmic region with a conserved PDZ binding motif. The extracellular domain also contains several putative N-glycosylation sites and a serine/ threonine-rich region for potential O-glycosylation between the acetylcholinesterase-like domain and the transmembrane domain that may affect neurexin binding (Sun, 2011).

Neurexins and Neuroligins are highly conserved cell adhesion molecules that form an asymmetric, trans-synaptic complex required for synapse formation (Sudhof, 2008). The present study examined a homolog of neuroligin (dnl2) in Drosophila expressed at NMJ synapses and in the CNS. dnl2 null mutants are viable and exhibit numerous defects in synaptic morphology and function. Presynaptically, a reduction was observed in axonal branching and fewer synaptic boutons, although the number of active zones per bouton was increased. Postsynaptically, dnl2 mutants exhibit a decrease in GluR density and a shift in the ratio of GluRIIA to GluRIIB receptor complexes in favor of GluRIIA complexes. dnl2 mutants also showed a decrease in complexity of the subsynaptic reticulum. Both presynaptic and postsynaptic defects observed in dnl2 mutants could be recapitulated by knockdown of dnl2 in muscle, and, more importantly, the defects can be rescued by postsynaptic expression of a wild-type dnl2 transgene. Functionally, dnl2 mutants showed an increase in transmitter release and a decrease in paired-pulse plasticity indicative of an increase in transmitter release probability. It is also possible that changes in the active (voltage-gated) properties of the postsynaptic membrane may contribute to the increased amplitude of EJPs. Indeed, the changes in the kinetics of EJPs with little or no change in the kinetics of mEJPs may be indicative of altered membrane conductance. Together, these data indicate that, although dnl2 is not absolutely required for synaptogenesis, it does play an important role in the postsynaptic cell in synapse maturation and function (Sun, 2011).

dnl2 mutants showed several defects in postsynaptic architecture. In vertebrates, neuroligins are thought to regulate postsynaptic organization via a direct interaction with PSD-95 (Irie, 1997). In Drosophila, the homolog of PSD-95, dlg, has been shown to be required for several aspects of postsynaptic organization. Furthermore, the defects in postsynaptic architecture in dnl2 mutants are reminiscent of a defect in dlg function. Loss of dnl2 does not appear to prevent or impair clustering of dlg at postsynaptic sites because the overall dlg levels was not different in dnl2 mutants. Rather, dnl2 may be required for proper dlg signaling (Sun, 2011).

dnl2 mutants also showed several defects in presynaptic architecture mediated by trans-synaptic interactions. The most likely candidate for a trans-synaptic signaling partner is neurexin. Both dnl2 and dnrx null flies are viable but display significant reductions in the number of synaptic boutons. Strong colocalization of dnl2 and dnrx is observed in the CNS and the NMJ, and a complex was detected between the two proteins in vivo. dnl2;dnrx double mutants, however, are lethal and display more severe phenotypes than those observed in single mutants, implying that dnl2 and dnrx may interact with additional partners during synaptic development. For example, dnrx can also interact with other neuroligins in Drosophila to mediate synaptic development. Banovic (2010) found that loss of dnrx did not enhance the morphological defects in dnl1 mutants, suggesting that both genes function within a common pathway. Furthermore, a point mutation in dnl1 that is predicted to abolish binding to dnrx suppressed the phenotype associated with overexpression of dnl1 (Banovic, 2010). There are also two other predicted homologs of neuroligin in flies, but the function of these genes remains to be determined. Neuroligins and neurexins may also form additional complexes with other proteins involved in synaptogenesis. A recent study found that neuroligin was able to induce increases in synaptic density independently of neurexin binding. Similarly, two other recent studies showed that leucine-rich repeat transmembrane proteins can induce presynaptic differentiation when bound to neurexin, providing a novel trans-synaptic neurexin-dependent mechanism for development of presynaptic specializations (Sun, 2011).

If the presynaptic defects observed in dnl2 mutants are not mediated via an interaction with neurexin, the question remains, how does dnl2 affect presynaptic morphology? One possibility is that these changes occur indirectly. The level of postsynaptic GluRIIA expression is correlated with changes in the number of presynaptic T-bars. It is possible that GluRIIA expression is regulated in part by dnl2, and increased GluRIIA expression in dnl2 mutants is responsible for the increased density of T-bars. Normally, the density of T-bars in individual boutons is held constant via homeostatic changes in the expression of the cell adhesion molecule Fas II. In the present study, however, an increase was observed in the number of T-bars per bouton. Moreover, a decrease was seen in the total number of boutons rather than an increase as might be expected based on previous studies. Because the addition of synaptic boutons during NMJ growth requires the downregulation of Fas II expression, the uncoupling between T-bar numbers and bouton expansion in dnl2 mutants may suggest that dnl2 is involved in the GluRIIA-mediated downregulation of Fas II (Sun, 2011).

The Drosophila genome is predicted to have four neuroligin homologs and a single neurexin homolog. Whether all four Drosophila neuroligins are required for synapse development is presently unknown. To date, the only other neuroligin that has been studied in Drosophila is dnl1 (Banovic, 2010). Neither dnl1 nor dnl2 are required for synaptogenesis, yet both genes play a role in the development/maturation of the NMJ, suggesting that the two genes may be functionally redundant. Both proteins are expressed at the NMJ in wild-type animals, and null mutations in either gene lead to significantly reduced bouton numbers, defects in GluR organization, and alterations in the complexity of the subsynaptic reticulum (Sun, 2011).

Despite these similarities, however, there are also a number of differences between dnl1 and dnl2 null mutants. First, dnl2 is expressed in both the CNS and muscles, whereas dnl1 is only expressed in muscle. Second, dnl1 mutants showed a complete loss of GluR expression in ~10% of boutons (Banovic, 2010), whereas dnl2 showed a uniform decrease in total GluR expression in all boutons and an increased abundance of GluRIIA receptor complexes at the expense of GluRIIB complexes. Third, dnl1 mutants showed a decrease in transmitter release (Banovic, 2010), whereas dnl2 mutants showed an increase in transmitter release. Although both mutants showed a decrease in bouton number, dnl2 mutants also showed a significant increase in the number of active zones. As such, the differences in the amplitude of transmitter release observed in dnl1 and dnl2 mutants likely reflect the different presynaptic morphologies of the two mutants (Sun, 2011). dnrx). dnl1 shows very little colocalization with dnrx and none outside the NMJ (Banovic, 2010). In contrast, dnl2 showed a much stronger colocalization at the NMJ and also shows strong colocalization within the CNS. Furthermore, dnl2 forms a complex with dnrx in vivo, although it remains to be shown whether the same is true for dnl1 (Banovic, 2010). dnl2;dnrx double mutants were lethal and showed more severe defects in bouton morphology than either dnl2 or dnrx mutants alone, whereas dnl1;dnrx mutants were viable and did not show any exacerbation of the morphological defects (Banovic, 2010). Together, these results suggest that the interactions between dnl1 or dnl2 and dnrx serve different functions at the NMJ (Sun, 2011)

Because mutations in dnrx or either of the two dnl genes studied thus far all give rise to a reduction in the number of synaptic boutons, it seems likely that bouton number is regulated via an interaction between dnrx and dnl1 and/or dnl2. It is also apparent, however, that these three genes perform functions independently of each other, such that single mutants have similar yet distinct synaptic phenotypes. Banovic (2010) concluded that dnrx promotes but is not necessary for dnl1 function. The results of the present study, however, showing exaggerated synaptic phenotypes and lethality in dnl2;dnrx double mutants may suggest some redundancy between the functions of dnrx and dnl2. Consistent with this model, a recent publication showed that dnrx is expressed both presynaptically and postsynaptically in embryonic NMJs (Chen, 2010). Furthermore, postsynaptic dnrx appears to specifically promote GluRIIA receptor complexes (Chen, 2010). This raises an interesting possibility of a cis-interaction between dnl2 and dnrx in addition to trans-synaptic interactions, although additional work will be required to assess whether cis-interactions occur, and if so, what role they play (Sun, 2011).

Together, the results of the present study combined with the results of Banovic (2010) suggest that neither dnl1 nor dnl2 are absolutely required for synaptogenesis, but both genes play an essential role in synaptic development. Additional studies will be required to determine the function of other neuroligin genes in Drosophila and to determine whether these genes have functionally redundant roles in synapse development and function (Sun, 2011).

Presynaptic Spinophilin tunes Neurexin signalling to control active zone architecture and function

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

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

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

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

The C. elegans Spn homologue NAB-1 (NeurABin1) was previously shown to bind Syd-1 in cell culture recruitment assays. This study found consistent evidence for Syd-1/Nrx-1/Spn tripartite complexes in salivary gland experiments. Moreover, the PDZ domain containing regions of Spn and Syd-1 interacted in Y2H experiments. It would be interesting to dissect whether the interaction of Spn/Syd-1 plays a role in controlling the access of Nrx-1 to one or both factors. For C. elegans HSN synapses, a previous study showed that loss of NAB-1 results in a deficit of synaptic markers, such as Syd-1 and Syd-2/Liprin-α, while NAB-1 binding to F-actin was also found to be important for synapse assembly. Though at first glance rather contradictory to the results described in this study, differences might result from Chia (2012) studying synapse assembly executed over a short time window, when partner cells meet for the first time. In contrast, this study used a model (Drosophila larval NMJs) where an already functional neuronal terminal adds novel AZs. Despite the efforts of this study, no role of F-actin in the assembly of AZs of late larval Drosophila NMJs was demonstrated. F-actin patches might be particularly important to establish the first synaptic contacts between partner cells. Both the study by Chia et al. and this study, however, point clearly towards important regulatory roles of Spn family members in the presynaptic control of synapse assembly. Further, this study described a novel interaction between the Spn-PDZ domain and the intracellular C-term of Nrx-1 at the atomic level. Interestingly, it was found that all functions of Spn reported in this study, structural as well as functional, were strictly dependent on the ligand-binding integrity of this PDZ domain. It is noteworthy that the Spn-PDZ domain binds other ligands as well, for example, Kalirin-7 and p70S6K , and further elucidation of its role as a signal 'integrator' in synapse plasticity should be interesting. The fact that Nrx-1 levels were increased at Spn NMJs and, most importantly, that genetic removal of a single nrx-1 gene copy effectively suppressed the Spn AZ phenotype, indicates an important role of the Spn/Nrx-1 interaction in this context. Affinity of Spn-PDZ for the Nrx-1 C-term was somewhat lower than that of the Syd-1-PDZ, both in ITC and Y2H experiments. Nonetheless, overexpression of Spn was successful in reducing the targeting effect of Syd-1 on overexpressed Nrx-1GFP. It will be interesting to see whether this interaction can be differentially regulated, for example, by (de)phosphorylation. It is worth noting that apart from Syd-1 and Spn, several other proteins containing PDZ domains, including CASK, Mint1/X11, CIPP and Syntenin, were found to bind to the Nrxs C-termini. CASK was previously shown to interact genetically with Nrx-1, controlling endocytic function at Drosophila NMJs. However, when this study tested for an influence of CASK on Nrx-1GFP motility using FRAP, genetic ablation of CASK had no effect (Muhammad, 2015).

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

Monogenic heritable autism gene neuroligin impacts Drosophila social behaviour

Autism spectrum disorders (ASDs) are characterized by deficits in social interactions, language development and repetitive behaviours. Multiple genes involved in the formation, specification and maintenance of synapses have been identified as risk factors for ASDs development. Among these are the neuroligin genes which code for postsynaptic cell adhesion molecules that induce the formation of presynapses, promote their maturation and modulate synaptic functions in both vertebrates and invertebrates. Neuroligin-deficient mice display abnormal social and vocal behaviours that resemble ASDs symptoms. This study shows that in the fly Drosophila melanogaster, deletion of the dnl2 gene, coding for one of four Neuroligin isoforms, impairs social interactions, alters acoustic communication signals, and affects the transition between different behaviours. dnl2-Deficient flies maintain larger distances to conspecifics and males perform less female-directed courtship and male-directed aggressive behaviours while the patterns of these behaviours and general locomotor activity are not different from wild type controls. Since tests for olfactory, visual and auditory perception reveal no sensory impairments of dnl2-deficient mutants, reduced social interactions seem to result from altered excitability in central nervous neuropils that initiate social behaviours. These results demonstrate that Neuroligins are phylogenetically conserved not only regarding their structure and direct function at the synapse but also concerning a shared implication in the regulation of social behaviours that dates back to common ancestors of humans and flies. In addition to previously described mouse models, Drosophila can thus be used to study the contribution of Neuroligins to synaptic function, social interactions and their implication in ASDs (Hahn, 2013).

Postsynaptic Neuroligins contribute to the formation of bidirectional trans-synaptic signalling complexes that promote pre- and postsynaptic differentiation and regulate synaptic functions. It is believed that disturbance of Neuroligin/Neurexin signalling promotes ASDs phenotypes through impairment of synaptic development, synaptic transmission and imbalance of excitatory and inhibitory synapses in brain circuits implicated in the regulation of social behaviour. Both Neuroligins and Neurexins are evolutionary conserved and orthologs of various proteins that associate with them to form functional pre- and postsynaptic complexes in the mammalian nervous system have been identified in invertebrate species. Neuroligin/Neurexin trans-synaptic signalling has been studied in rats and mice, the nematode C. elegans, the mollusc A. californica, the honeybee A. mellifera and at the neuromuscular junction of D. melanogaster. Results of these studies suggest similar functions of Neuroligin/Neurexin signalling in the initiation, maturation, transmission and plasticity of vertebrate and invertebrate synapses (Hahn, 2013).

This study addressed the question whether impairment of Neuroligin/Neurexin trans-synaptic signalling impacts Drosophila's social behaviour and whether parallels to ASDs-like phenotypes reported in humans and mice are present. Neurexin-deficient males display severe locomotor defects and are not able to produce courtship songs, though unilateral wing extension is occasionally observed. A detailed analysis of neurexin-deficient flies was therefore not performed. In contrast, mutant lines deficient of the central nervously expressed dnl2 display no obvious motor impairments and are able to produce both types of courtship song patterns with the same accuracy as wild-type Drosophila males, suggesting that the central pattern generators for both pulse and sine song seem to function properly. Comparison of acoustic communication patterns of dnl2-deficient and wild-type flies reveals two differences, a reduced intensity of sine songs and shorter duration of inter pulse intervals. The reduced sine song intensity of dnl2KO17 mutants likely results from a weaker synaptic transmission at the neuromuscular junction causing reduced muscle activation and lower amplitudes of wing vibrations. The reduced inter pulse interval must result from altered synaptic properties in thoracic pulse song pattern generating circuits and/or differences in the intensity of their activation by descending brain neurons. The inter pulse interval is the critical parameter for species recognition and song attractiveness and deviation from a species-typical range should reduce Drosophila's courtship success and reproduction. Altered ultrasound vocalization has also been reported from mouse models for autism. While mice with impaired Neuroligin/Neurexin signalling display generally reduced calling rates, other mouse strains with ASDs-like phenotypes display abnormal spectral and temporal song patterns. Similar to the reduced rates of acoustic communication observed in mice with impaired Neuroligin/Neurexin trans-synaptic signalling, reduced courtship singing was also observed in this study with dnl2-deficient Drosophila (Hahn, 2013).

Distances between individuals of D. melanogaster have been studied in different behavioural settings which distinguish and emphasize different aspects including dispersal/exploration, intrinsic social space or group formation. It has been demonstrated that inter individual space may depend on the balance of attractive and repulsive sensory signals, previous social experience like isolation or mating and also on the type of arena and the number of flies used for the assay. The assay performed in this study excludes exploration/dispersal during the first minutes after introduction into the arena, reveals that wild type flies initially establish shorter distances to conspecifics that steadily increase between 5 and 18 min after being placed in the arena, suggesting a gradually decreasing tendency to engage in short range interactions with other individuals. In contrast, dnl2-deficient flies display this low tendency, reflected in large inter individual distances, already after 5 min in the arena without showing consistent changes with progressing time in the arena (Hahn, 2013).

Since dnl2-deficient flies are equally active as wild type flies, display no sensory and motoric impairments, and are able to produce the typical components of courtship and agonistic behaviours, their reduced social interactions and impaired transition between different behaviours (e.g. from courtship singing to subsequent behaviour; between walking and turning) appear to result from altered information processing in central nervous circuits responsible for the initiation and coordination of behaviour. Especially the mushroom bodies and the central complex, that express Dnl2, have been implicated in these functions in insects. Studies in the honeybee Apis mellifera have revealed expression of various neuroligins and neurexin in the mushroom bodies and regulation of brain neuroligin and neurexin expression by social interactions (comparison of isolated versus hive bees) during early adulthood. This suggests that Neuroligin/Neurexin signalling may also be involved in behavioural plasticity resulting from social experience, which modulates the age-related division of labour in honeybee colonies. A similar relevance of activity-dependent neuroligin- and/or neurexin-mediated synaptic plasticity in the mature brain has also been documented in mice and has been implicated in the aetiology of ASDs (Hahn, 2013).

Phenotypes very similar to those described in this study for dnl2-deficient Drosophila have also been reported in various mouse models for ASDs including neuroligin-deficient mice. Behavioural and cognitive impairments of these mice, as well as ASDs-symptoms in humans, have been linked to altered balance of excitation and inhibition in critical brain circuits that normally results from specific functions of different Neuroligin isoforms at excitatory and inhibitory synapses and altered functions of Neuroligin/Neurexin trans-synaptic signalling complexes. Similarly, differential effects on synaptic properties are also mediated by dnl1 and dnl2 at the Drosophila neuromuscular junction. Since dnl1 is expressed in muscle but not in the nervous system, it will be interesting to see, whether dnl3 or dnl4 act as “balancing counterparts” to dnl2 within the nervous system to establish proper excitation/inhibition ratios. Circuits that regulate the initiation and intensity of social behaviours including acoustic communication may be especially sensitive to disturbances of neuroligin-mediated synaptic fine tuning and this sensitivity seems to be shared by humans, mice and Drosophila and probably other social insects like honeybees. Thus, phylogenetical conservation of Neuroligins from flies to humans extends beyond their molecular structure and their direct function at the synapse and also includes their implication in the regulation of social behaviours (Hahn, 2013).


Functions of Neuroligin orthologs in other species

Neuroligins/LRRTMs prevent activity- and Ca2+/calmodulin-dependent synapse elimination in cultured neurons

Neuroligins (NLs) and leucine-rich repeat transmembrane proteins (LRRTMs) are postsynaptic cell adhesion molecules that bind to presynaptic neurexins. This paper shows that short hairpin ribonucleic acid-mediated knockdowns (KDs) of LRRTM1, LRRTM2, and/or NL-3, alone or together as double or triple KDs (TKDs) in cultured hippocampal neurons, did not decrease synapse numbers. In neurons cultured from NL-1 knockout mice, however, TKD of LRRTMs and NL-3 induced an approximately 40% loss of excitatory but not inhibitory synapses. Strikingly, synapse loss triggered by the LRRTM/NL deficiency was abrogated by chronic blockade of synaptic activity as well as by chronic inhibition of Ca(2+) influx or Ca(2+)/calmodulin (CaM) kinases. Furthermore, postsynaptic KD of CaM prevented synapse loss in a cell-autonomous manner, an effect that was reversed by CaM rescue. These results suggest that two neurexin ligands, LRRTMs and NLs, act redundantly to maintain excitatory synapses and that synapse elimination caused by the absence of NLs and LRRTMs is promoted by synaptic activity and mediated by a postsynaptic Ca(2+)/CaM-dependent signaling pathway (Ko, 2011).

Neurexin and neuroligin mediate retrograde synaptic inhibition in C. elegans

The synaptic adhesion molecules neurexin and neuroligin alter the development and function of synapses and are linked to autism in humans. This study found that C. elegans neurexin (NRX-1) and neuroligin (NLG-1) mediated a retrograde synaptic signal that inhibited neurotransmitter release at neuromuscular junctions. Retrograde signaling was induced in mutants lacking a muscle microRNA (miR-1) and was blocked in mutants lacking NLG-1 or NRX-1. Release was rapid and abbreviated when the retrograde signal was on, whereas release was slow and prolonged when retrograde signaling was blocked. The retrograde signal adjusted release kinetics by inhibiting exocytosis of synaptic vesicles (SVs) that are distal to the site of calcium entry. Inhibition of release was mediated by increased presynaptic levels of tomosyn, an inhibitor of SV fusion (Hu, 2012).

Drosophila Neuroligin3 regulates neuromuscular junction development and synaptic differentiation

Neuroligins (Nlgs) are a family of cell adhesion molecules thought to be important for synapse maturation and function. Studies in mammals have shown that different Nlgs have different roles in synaptic maturation and function. The functions of Drosophila Neuroligin1 (DNlg1), DNlg2, and DNlg4 have also been examined. This study reports the role of DNlg3 in synaptic development and function by using Drosophila neuromuscular junctions (NMJs) as a model system. DNlg3 was found to be expressed in both CNS and NMJs where it was largely restricted to the postsynaptic site. By generating and examining dnlg3 mutants, the mutants mutants were found to exhibit an increased bouton number and reduced bouton size compared to the wild-type. Consistent with alterations in bouton properties, pre- and postsynaptic differentiations were also affected including abnormal synaptic vesicle endocytosis, increased PSD length and reduced GluRIIA recruitment. Additionally, synaptic transmission was reduced. Altogether, this study shows that DNlg3 is required for NMJ development, synaptic differentiation and function (Xing, 2014).

Epigenetic suppression of neuroligin 1 underlies amyloid-induced memory deficiency

Amyloid-induced microglial activation and neuroinflammation impair central synapses and memory function, although the mechanism remains unclear. Neuroligin 1 (NLGN1), a postsynaptic protein found in central excitatory synapses, governs excitatory synaptic efficacy and plasticity in the brain. This study found, in rodents, that amyloid fibril-induced neuroinflammation enhances the interaction between histone deacetylase 2 and methyl-CpG-binding protein 2, leading to suppressed histone H3 acetylation and enhanced cytosine methylation in the Nlgn1 promoter region and decreased NLGN1 expression, underlying amyloid-induced memory deficiency. Manipulation of microglia-associated neuroinflammation modulates the epigenetic modification of the Nlgn1 promoter, hippocampal glutamatergic transmission and memory function. These findings link neuroinflammation, synaptic efficacy and memory, thus providing insight into the pathogenesis of amyloid-associated diseases (Bie, 2014).

CaMKII phosphorylation of neuroligin-1 regulates excitatory synapses

Neuroligins are postsynaptic cell adhesion molecules that are important for synaptic function through their trans-synaptic interaction with neurexins (NRXNs). The localization and synaptic effects of neuroligin-1 (NL-1, also called NLGN1) are specific to excitatory synapses with the capacity to enhance excitatory synapses dependent on synaptic activity or Ca(2+)/calmodulin kinase II (CaMKII). This study reports that CaMKII robustly phosphorylates the intracellular domain of NL-1. T739 was shown to be the dominant CaMKII site on NL-1, and it is phosphorylated in response to synaptic activity in cultured rodent neurons and sensory experience in vivo. Furthermore, a phosphodeficient mutant (NL-1 T739A) reduces the basal and activity-driven surface expression of NL-1, leading to a reduction in neuroligin-mediated excitatory synaptic potentiation. These results are the first to demonstrate a direct functional interaction between CaMKII and NL-1, two primary components of excitatory synapses (Bemben, 2014).

Proteomic analysis of unbounded cellular compartments: Synaptic clefts

Cellular compartments that cannot be biochemically isolated are challenging to characterize. This study demonstrates the proteomic characterization of the synaptic clefts that exist at both excitatory and inhibitory synapses. Normal brain function relies on the careful balance of these opposing neural connections, and understanding how this balance is achieved relies on knowledge of their protein compositions. Using a spatially restricted enzymatic tagging strategy, the proteomes of two of the most common excitatory and inhibitory synaptic clefts were mapped in living neurons. These proteomes reveal dozens of synaptic candidates and assign numerous known synaptic proteins to a specific cleft type. The molecular differentiation of each cleft allowed identification of Mdga2 as a potential specificity factor influencing Neuroligin-2 (see Drosophila Nlg2) recruitment of presynaptic neurotransmitters at inhibitory synapses (Loh, 2016).

gamma-Protocadherins Interact with Neuroligin-1 and Negatively Regulate Dendritic Spine Morphogenesis

The 22 γ-Protocadherin (γ-Pcdh) cell adhesion molecules are critical for the elaboration of complex dendritic arbors in the cerebral cortex. This study provides evidence that the γ-Pcdhs negatively regulate synapse development by inhibiting the postsynaptic cell adhesion molecule, neuroligin-1 (Nlg1; see Drosophila Neuroligin). Mice lacking all γ-Pcdhs in the forebrain exhibit significantly increased dendritic spine density in vivo, while spine density is significantly decreased in mice overexpressing one of the 22 γ-Pcdh isoforms. Co-expression of γ-Pcdhs inhibits the ability of Nlg1 to increase spine density and to induce presynaptic differentiation in hippocampal neurons in vitro. The γ-Pcdhs physically interact in cis with Nlg1 both in vitro and in vivo, and evidence is presented that this disrupts Nlg1 binding to its presynaptic partner neurexin1β (see Drosophila Neurexin-1). Together with prior work, these data identify a mechanism through which γ-Pcdhs could coordinate dendrite arbor growth and complexity with spine maturation in the developing brain (Molumby, 2017).


REFERENCES

Search PubMed for articles about Drosophila Neuroligin

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Harris, K.P., Zhang, Y.V., Piccioli, Z.D., Perrimon, N. and Littleton, J.T. (2016). The postsynaptic t-SNARE Syntaxin 4 controls traffic of Neuroligin 1 and Synaptotagmin 4 to regulate retrograde signaling. Elife 5. PubMed ID: 27223326

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date revised: 20 May 2017

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