InteractiveFly: GeneBrief

Neurexin 1: Biological Overview | References

BIOLOGICAL OVERVIEW

Neurexins have been proposed to function as major mediators of the coordinated pre- and postsynaptic apposition. However, key evidence for this role in vivo has been lacking, particularly due to gene redundancy. Null mutations have been obtained in the single Drosophila Neurexin gene. Nrx loss of function prevents the normal proliferation of synaptic boutons at glutamatergic neuromuscular junctions, while Nrx gain of function in neurons has the opposite effect. Nrx mostly localizes to the active zone of presynaptic terminals. Conspicuously, Nrx null mutants display striking defects in synaptic ultrastructure, with the presence of detachments between pre- and postsynaptic membranes, abnormally long active zones, and increased number of T bars. These abnormalities result in corresponding alterations in synaptic transmission with reduced quantal content. Together, these results provide compelling evidence for an in vivo role of neurexins in the modulation of synaptic architecture and adhesive interactions between pre- and postsynaptic compartments (Li, 2007).

Although cell adhesion molecules have long been postulated and in several cases have been shown to be major participants in synapse development and plasticity, the impact of their function and the molecular mechanisms that they activate remain a puzzle. Particularly intriguing is the function of neurexins, which may provide clues to understanding of synapse organization. Null mutants were isolated in the single Drosophila dnrx gene. Nrx mutants were shown to have striking abnormalities in synapse development and function. A recent study reported that Drosophila neurexin is required for synapse formation in the adult CNS (Zeng, 2007). The current study demonstrates a primary role of Nrx in regulating the formation of synapses, and also reveal the crucial role of Nrx in the proper development of active zones and regulating synaptic function in an intact organism, thus providing insights into understanding the function of neurexins in vivo (Li, 2007).

These studies provide compelling evidence that Nrx plays a prime role during the expansion of the NMJ and, in particular, in defining the cytoarchitecture of the active zones within synaptic boutons. (1) In Nrx mutants, synaptic bouton proliferation is severely disrupted, and therefore NMJ expansion is significantly stunted. (2) Nrx gain of function promotes the formation of new boutons in a gene-dosage-dependent manner. (3) The ultrastructural analyses show that presynaptic densities (PRDs) are not apposed normally to PSDs displaying signs of abnormal adhesion to the postsynaptic density (PSD), although every PRD is exactly juxtaposed to the PSD. (4) In Nrx mutants, critical components of the presynaptic compartment, such as synaptic vesicle proteins and active zone components, are ectopically localized within axons. (5) The distribution of GluRs at the PSD is abnormally large, although this phenotype may arise as a consequence of the presynaptic defects observed in Nrx mutants (Li, 2007).

The great majority of abnormal phenotypes in Nrx mutants could be completely rescued by expressing a wild-type Nrx transgene in neurons; although in some instances the rescue was partial. However, even in the later case, expressing Nrx in both muscles and neurons did not further improve the residual abnormalities, suggesting that Nrx functions primarily if not exclusively in the presynaptic compartment (Li, 2007).

The partial rescue of some of the phenotypes, such as the defects in mEJPs and the morphology of active zones, might be due to the high sensitivity of these processes to the right levels and correct temporal expression of Nrx, which is not completely mimicked by the UAS/Gal4 system. This view is supported by the observation that overexpression of Nrx in a wild-type background also decreased quantal content, suggesting that increased Nrx dosage may have detrimental effects on synapse structure and/or function. However, the data strongly support that the abnormal phenotypes arise from the lack of Nrx: (1) all the experiments were carried out in mutants over a deficiency chromosome in an independent genetic background; (2) a precise excision of the P element did not show any of the mutant phenotypes. Together these data establish a specific role for Nrx in proper synaptic development (Li, 2007).

One of the important findings of this study is that Nrx mutants displayed defective active zones with larger PRD, and especially containing regions of detachment from the PSD. These detachment sites implicate Nrx as a mediator of cell adhesion between the pre- and the postsynaptic cell,. While a complete detachment of active zones is not observed, Nrx mutants have a significant decrease in the number of boutons. This raises the possibility that the phenotypes observed are from those boutons that are maintained and that a more drastic consequence is a failure to form synaptic boutons. Nevertheless, the lack of complete detachment of active zones in Nrx null mutants suggests that Nrx, although an important synapse-organization molecule, is not sufficient for trans-synaptic cell adhesion (Li, 2007).

Another notable phenotype in Nrx mutants was the presence of enlarged PRDs and increased number of T bars. A major feature of Drosophila larval NMJ is its ability to compensate for decreased postsynaptic responses by upregulating neurotransmitter release. For instance, a decrease in the number of postsynaptic GluRs results in an increase in neurotransmitter release, thus maintaining the amplitude of evoked responses. It is plausible that the enlarged PRDs and increase in number of T bars in Nrx mutants are a compensatory mechanism to adjust for defective presynaptic cell adhesion and/or reduced neurotransmitter release. In support of this notion, in Nrx mutants there was a 50% decrease in synaptic bouton number, but this was accompanied by a 2-fold increase in the number of T bars, such that the total number of T bars/NMJ remained constant, despite the changes in bouton number. Similarly, defective presynaptic cell adhesion and/or reduced neurotransmitter release could lead to an increase in GluR accumulation. In these studies, it was found that the length of the PSD was enlarged in Nrx mutants as well as the distribution of GluR clusters (Li, 2007).

The above structural abnormalities were accompanied by corresponding functional deficits. In Nrx mutants, the frequency of mEJPs was strikingly increased. Further, although the T bars were rescued by expression of a Nrx transgene, the length of the PRDs was not, and a similar lack of rescue was observed for mEJP frequency. Thus, there appears to be a notable correlation between the size of the PRD and spontaneous miniature excitatory potential (mEJP) frequency, perhaps due to increased probability of synaptic vesicle release with increased synapse size. In addition, a substantial increase was observed in mEJP amplitude. Two factors may contribute to this change; (1) the distribution of GluR clusters was enlarged, while the GluR intensity remained unchanged, suggesting that more GluRs were accumulated at mutant synapses; (2) an additional contributing factor is that mEJP frequency was increased, and instances of summation were observed (Li, 2007).

Overall, despite the increase in PRD size and the maintenance of overall T bar number, evoked events had a decrease in amplitude and quantal content. Recent studies have suggested that a major constituent of the T bars is Brunchpilot [BRP/CAST (Kittel, 2006; Wagh, 2006)]. In brp mutants, T bars fail to form, but PRDs appear unaltered. Further, although EJP amplitude is decreased, mEJP amplitude and frequency are normal. This has led to the model that T bars per se are not required for synaptic transmission but that they regulate the efficiency of transmission. In Nrx mutants, PRDs are disproportionately large, which could result in asynchronous release, leading to an EJP with decreased amplitude. It is also possible that the presynaptic membrane detachments observed in Nrx mutants could contribute to the functional impairment of neurotransmitter release (Li, 2007).

A recent study demonstrated that in Nrx mutant larvae associative learning is impaired in an olfactory choice paradigm (Zeng, 2007). However, in this study, larval locomotion was not assessed. The current study showing that locomotor behavior is impaired in Nrx mutants raises the possibility that the poor performance of mutant larvae in the conditioning assay might also result from the locomotor abnormalities. Zeng also reported that the number of T bars in the calyx of the mushroom bodies, the learning centers of the fly, was reduced in adult flies. In contrast, a significant increase was found in the number of T bar/bouton, and since Nrx mutants have fewer boutons, this translated in the maintenance of T bar number per NMJ. The differing results might be due to different mechanisms regulating T bar formation in the two tissues (Li, 2007).

The presence of a neurexin in Drosophila strengthened the view that neurexins are highly conserved across species. The synaptic Nrx expression pattern and its function show remarkable parallels with mammalian neurexins. Moreover, the proteins that have been shown to interact with mammalian neurexins also have homologs in Drosophila, which further supports the idea that the function of neurexins and underlying signaling mechanism are evolutionarily conserved. Among these, Drosophila neuroligin and/or Dystroglycan (Dg) might be potential Nrx ligands. Drosophila neuroligin transcription exhibits almost an identical temporal and spatial expression pattern as Nrx during embryonic stages. dg is highly expressed in the somatic musculature of embryos. dg mutants are embryonic lethal, and perturbation of Dg function by RNAi as well as genetic interaction studies suggest an involvement of Dg in muscle maintenance and axonal pathfinding in adult flies (Shcherbata, 2007). Future studies on the identification and characterization of Nrx binding partners in Drosophila should provide additional insights into the mechanisms by which neurexins function in synapse development and function (Li, 2007).

Extensive cell culture studies of neurexins and neuroligins and functional studies using α-neurexin knockout mice have established a central role for neurexins as synaptic adhesive and organizing molecules. Studies on Nrx provide evidence in an intact organism that neurexin is required for important aspects of synapse development and function. Gain-of-function analysis of Drosophila Nrx reveals that overexpression is sufficient to promote the formation of synaptic boutons in vivo, in agreement with the findings from cell culture studies suggesting that mammalian neurexin-neuroligin trans-synaptic complexes can induce pre- and postsynaptic differentiation and synapse formation. Moreover, the accumulations of synaptic vesicle and active zone proteins along axons of Nrx null mutants further support the notion that neurexins may recruit or organize synaptic proteins or organelles during presynaptic differentiation. Phenotypic analyses of α-neurexin knockout mice demonstrated that α-neurexin is essential for synaptic transmission in a process that depends on presynaptic voltage-dependent Ca²⁺ channels. However, triple-knockout mice have normal surface expression of Ca²⁺ channels. These findings have led to the hypothesis that neurexins regulate the coupling between Ca²⁺ channels and the neurotransmitter release machinery. Similarly, in Drosophila Nrx null mutants it was found that the Ca²⁺ sensitivity of evoked responses was abnormal, but the distribution or levels of presynaptic Ca²⁺ channel Cac was unchanged, consistent with the above hypothesis. Notably, Syt I, a synaptic vesicle protein that binds Ca²⁺ and has been proposed to function as a Ca²⁺ sensor during synaptic vesicle exocytosis, was partly mislocalized to axons in Drosophila Nrx mutants. Furthermore, the structure of active zones was impaired in these mutants. Therefore, the organization of active zone proteins including the assembly of neurotransmitter release machinery might be affected in Nrx mutants (Li, 2007).

In conclusion, these studies in Drosophila demonstrate that Nrx is required for both synapse development and function and, in particular, for proper formation of active zones. These studies provide compelling evidence for an in vivo role of neurexins in the modulation of synaptic architecture and adhesive interactions between pre- and postsynaptic compartments (Li, 2007).

Neurexin-1 is required for synapse formation and larvae associative learning in Drosophila

Neurexins are highly polymorphic cell-surface adhesive molecules in neurons. In cultured mammalian cell system, they were found to be involved in synaptogenesis. This study reports that Drosophila neurexin is required for synapse formation and associative learning in larvae. Drosophila genome encodes a single functional neurexin (CG7050; Neurexin-1 or Nrx-1), which is a homolog of vertebrate alpha-neurexin. Neurexin-1 is expressed in central nervous system and highly enriched in synaptic regions of the ventral ganglion and brain. Neurexin-1 null mutants are viable and fertile, but have shortened lifespan. The synapse number is decreased in central nervous system in Neurexin-1 null mutants. In addition, Neurexin-1 null mutants exhibit associative learning defect in larvae (Zeng, 2007).

In mouse, mRNAs of the major neurexin isoforms (α and β) were found throughout the central nervous system. This study detected a widespread expression of Neurexin-1 mRNA during early embryogenesis. As the embryos develop, theNeurexin-1 mRNA is enriched in brain and ventral nerve cord (VNC). Although it is difficult to determine if all neurons in the CNS express Neurexin-1 mRNA, many neurons in VNC and brain are stained. It is concluded that Neurexin-1 mRNA is a neuronal-specific marker, which is expressed in many, if not all, CNS neurons. Expression of Neurexin-1 protein from embryo to adult was also examined in this study (Zeng, 2007).

Using the purified antiserum, which recognizes epitopes from within the 1534–1690 residues not deleted in Nrx-1^Δ83 and Nrx-1^Δ174 mutants, no residual full-length or truncated gene products are detected in adult heads of Nrx-1^Δ83 and Nrx-1^Δ174 mutants. In contrast, a ∼200 KD band can be detected in w¹¹¹⁸ controls and to a less degree in P{XP}Nrx-1. No signal is seen in extract from Nrx-1^Δ83 and Nrx-1^Δ174 mutants, thus confirming the specificity of the antibody. Using this antiserum, immunofluorescence stainings were performed on Drosophila embryo, larval brain and adult brain. Neurexin-1 expression in the nervous system is maintained throughout development into adulthood. Within the embryonic VNC, strong staining is accumulated along the longitudinal tracts of the VNC and brain. Weak staining is also seen in the commissures of VNC. In the adult brain, it is expressed at high levels within the medulla, lobula, lobula plate, mushroom body and antennal lobe (Zeng, 2007).

Neurexin-1 co-localizes with synaptic protein syntaxin and synaptotagmin along the ventral longitudinal tracts where synapses are concentrated in embryo , and Neurexin-1 has the same expression pattern as the synaptic protein syntaxin that is concentrated in synaptic regions of the brain lobes and the ventral ganglion in w¹¹¹⁸ larval brain. In addition, Neurexin-1 also co-localizes with syntaxin in adult head. As expected, staining was totally abolished in larval brain from the loss of function mutant Nrx-1^Δ83 and Nrx-1^Δ174, and staining was decreased in P{XP}Nrx-1 compared to w¹¹¹⁸ and Elav-Gal4 rescue larva brain (Zeng, 2007).

To clarify the function of Neurexin-1 in synapse formation, the expression of some synaptic proteins were detected in Neurexin-1 mutants. Neurexin-1 mutants larval brain were found to have normal expression of vesicle associated protein Syntaxin, Synapsin and CSP, as compared to w¹¹¹⁸; However, as compared to w¹¹¹⁸, the expression of Brp is decreased in Neurexin-1 mutants Nrx-1^Δ83 and Nrx-1^Δ174. Immunofluorescence analysis further indicated that expression level and pattern of the vesicle associated protein Syntaxin, Synapsin, CSP in Nrx-1^Δ83 and Nrx-1^Δ174 is the same as w¹¹¹⁸ in larval brain (data not show); however, the expression level of Brp is decreased. Brp, a CAST (CD3E-associated protein) homolog localized to the pre-synaptic specialization, was used as a synaptic marker to count the number of synapse in Drosophila (Zeng, 2007).

Based on the decreased expression of Brp in Neurexin-1 mutants' larval brain, it was deduced that Neurexin-1 may disrupt the synapse formation in Drosophila. In order to further test this hypothesis, electron micrograph of calyx region of adult brain was performed. An electron-dense, pre-synaptic ribbon with surrounding vesicles defined each synapse. It was found that Nrx-1^Δ83 and Neurexin-1 trans-heterozygous null mutant Nrx-1^Δ83/Df(3R)Exel6191 flies (about 4 synapses per 11.4μm²) have fewer synapse number than w¹¹¹⁸ (about 8 synapses per 11.4μm²), and the synapse number of rescue flies Elav-Gal4/Y; UAS-neurexin; Nrx-1^Δ83 is the same as w¹¹¹⁸. Binding of the neuroligin and neurexin complex can trigger pre-synaptic specializations in vitro. However, the α-neurexins triple knock-out mice indicate that α-neurexins are not essential for synapse formation. This maybe due to the functional redundancy of β-neurexin. There is only one neurexin gene and one transcript in Drosophila, the synapse formation is disrupted in Drosophila when Neurexin-1 is knocked out (Zeng, 2007).

In order to determine whether neurexin might have a role in associative learning in Drosophila, w¹¹¹⁸, P{XP}Nrx-1and Nrx-1^Δ83 larvae were tested for their ability to associate odors with a fructose reward in individual-animal assay. Comparing to w¹¹¹⁸ larvae, the learning is reduced in Neurexin-1 hypomorphic mutants P{XP}Nrx-1, and more severely reduced in Neurexin-1 null mutant Nrx-1^Δ83. Comparing to w¹¹¹⁸ larvae, normalcy is restored when Elav-Gal4 and UAS-Nrx-1 are used in Nrx-1^Δ83, which drive Neurexin-1 expressed in whole nervous system in mutant. Unexpectedly, mushroom body specific expressed Neurexin-1 in Nrx-1^Δ83 using 201Y-Gal4 failed to rescue the learning defect of Nrx-1^Δ83, which indicates that the function of Neurexin-1 in appetitive olfactory learning in Drosophila larvae is not only limited in the olfactory associative learning center, mushroom body (Zeng, 2007).

Neurexin in embryonic Drosophila neuromuscular junctions

Neurexin is a synaptic cell adhesion protein critical for synapse formation and function. Mutations in neurexin and neurexin-interacting proteins have been implicated in several neurological diseases. Previous studies have described Drosophila neurexin mutant phenotypes in third instar larvae and adults. However, the expression and function of Drosophila neurexin early in synapse development, when neurexin function is thought to be most important, has not been described. This study used a variety of techniques, including immunohistochemistry, electron microscopy, in situ hybridization, and electrophysiology, to characterize neurexin expression and phenotypes in embryonic Drosophila neuromuscular junctions (NMJs). The results surprisingly suggest that Neurexin in embryos is present both pre and postsynaptically. Presynaptic Neurexin promotes presynaptic active zone formation and neurotransmitter release, but along with postsynaptic Neurexin, also suppresses formation of ectopic glutamate receptor clusters. Interestingly, this study found that loss of neurexin only affects receptors containing the subunit GluRIIA. This study extends previous results and provides important detail regarding the role of neurexin in Drosophila glutamate receptor abundance. The possibility that neurexin is present postsynaptically raises new hypotheses regarding neurexin function in synapses, and the results provide new insights into the role of neurexin in synapse development (Chen, 2010).

The first complete knockout of neurexin function was achieved in Drosophila, and Zeng (2007) provided the first report of Drosophila neurexin mutants. Using western blots and immunohistochemistry, Zeng (2007) showed that neurexin mutants had reduced brp expression in larval brain, and reduced synapse density in adult brain. Both results are consistent with the idea that neurexin promotes synapse formation or maintenance, as previously argued by many studies in mammalian cells and alpha neurexin mouse mutants. Another study subsequently provided a detailed examination of neurexin mutant larval NMJs, and showed that larval NMJ arborizations were smaller, similar to what is describe in this study in embryos. Smaller NMJs were reported in larval neurexin mutants. However, a large increase in the number of presynaptic densities ('T-bars') and boutons (with no obvious decrease in active zone density per bouton) was reported in neurexin mutant larval NMJs, which contrasts with the idea that neurexin promotes synaptogenesis and the observation that the number of presynaptic densities (measured either by EM or brp staining) is reduced in neurexin mutant embryonic NMJs. The proliferation of active zones observed in neurexin mutant larvae may therefore represent developmental compensation for reduced muscle excitation. Increased active zone proliferation during larval development has previously been observed in other mutants with reduced NMJ transmission (Chen, 2010).

Apparent disruptions were reported in cell adhesion between presynaptic neurons and postsynaptic muscle in larval neurexin mutant NMJs, which appeared as widened synaptic clefts visible by electron microscopy. No such changes were observed s in any of the dozens of sections from 14 separate embryonic NMJs that were examined. The comparison suggests that these ultrastructural changes may occur during larval development rather than initial synapse formation (Chen, 2010).

Mouse alpha neurexin mutants show dramatic defects in calcium-dependent neurotransmitter release. Drosophila neurexin mutants also show reductions in neurotransmitter release, both at the larval stage, and in embryos. In embryos, decreased neurotransmission in neurexin mutant NMJs appears entirely attributable to the decrease in NMJ active zone number that was observed observed in this study. In older stages, and in mammals, some of the decreased neurotransmitter release is attributed to defects in calcium-secretion coupling (Chen, 2010).

Miniature postsynaptic potentials are larger in larval neurexin mutant NMJs. In contrast, no change was observed in embryonic neurexin mutant sEJC amplitude, and no change in the size of individual receptor cluster sizes that typically go along with such changes. Instead, an increase was seen in the number of postsynaptic receptor clusters without any corresponding increase in presynaptic active zones. The data, taken together with larval results, suggests that loss of neurexin initially causes an increase in nonfunctional postsynaptic receptor clusters. During larval development, many of these receptor clusters then become paired with presynaptic active zones, so that there is an increase in synapse number. At this stage, increased receptor expression is manifest as an increase in individual receptor cluster sizes and miniature postsynaptic potentials. Besides the insights into neurexin function, this is interesting because it suggests that glutamate receptors in embryos can semi-autonomously form clusters, but receptors in larvae are preferentially added to pre-existing synapses (Chen, 2010).

Importantly, the data demonstrate that neurexin mutant glutamate receptor phenotypes, at least in embryos, are restricted to A-type receptors. This suggests a mechanism. Drosophila A-type, but not B-type, glutamate receptors depend on postsynaptic F-actin for localization/stabilization, and this process involves a direct interaction between the C-terminus of GluRIIA and the Drosophila 4.1 protein 'coracle'. The intracellular C-terminus of mammalian neurexin has been shown to bind to the PDZ domain protein CASK, and interactions between mammalian neurexin and CASK in combination with 4.1 protein have been shown to nucleate F-actin assembly. CASK knockout mice are lethal but show no dramatic synaptic alterations except increased neuroligin protein levels and higher spontaneous event frequency at glutamatergic synapses, consistent with the cureen data and a subtle role in synaptic protein organization. The Drosophila genome encodes one CASK homolog, which interacts with neurexin to regulate walking behavior and neuromuscular transmission. Drosophila neurexin may therefore work with CASK and coracle (4.1) to regulate A-type glutamate receptor organization via actin rearrangements. Mammalian beta neurexin also appears to regulate AMPA receptor abundance in a subunit-specific manner, and alpha neurexin appears to regulate NMDA but not AMPA receptor function. But in these cases receptors are recruited by the presence of neurexin rather than repressed, as is seen in Drosophila. This added complexity may be due to differing molecular functions of neurexin within pre and postsynaptic cells. Interestingly, complete loss of neurexin in both pre and postsynaptic cells led to the same increase in GluRIIA as loss of either pre or postsynaptic neurexin. The increase in GluRIIA immunoreactivity observed after loss of neurexin on any side of the synapse might therefore represent a physiological limit, or (more likely) either pre or postsynaptic neurexin is sufficient to suppress GluRIIA in WT animals (Chen, 2010).

The most controversial suggestion provided by these data is the possibility that neurexin in Drosophila NMJs might be present in postsynaptic muscle, where it appears to contribute (along with presynaptic neurexin) to formation of proper glutamate receptor clusters in embryos. Previous studies did not report neurexin expression in muscles. However, the embryonic cuticle forms at approximately the same time in development as the body wall muscles, and suppresses the ability of RNA probes to enter tissues, making it difficult to detect expression of muscle genes that are expressed late in embryonic development. Indeed, initially it was found to be difficult to unambiguously detect neurexin expression in body wall muscles of intact embryos by in situ hybridization. Previous studies did not test explicitly whether postsynaptic neurexin affected postsynaptic receptor clustering, but agree with the result that postsynaptic neurexin does not affect presynaptic differentiation or function. It has also been suggested that neurexin might function postsynaptically in mammalian cells. Specifically, it was suggested that postsynaptic neurexin could interact with postsynaptic neuroligin to reduce transsynaptic neurexin-neuroligin interactions. This hypothesis does not quite fit with the curren data, however, since knockdown of presynaptic neurexin and postsynaptic neurexin had the same receptor phenotype. If postsynaptic neurexin counteracted presynaptic neurexin function, one would expect postsynaptic neurexin knockdown to have the opposite effect of presynaptic neurexin knockdown. The simplest hypothesis that allows for all the results is that presynaptic neurexin works via transynaptic interactions (with neuroligin or other proteins), while postsynaptic neurexin works primarily via intracellular C-terminal interactions to regulate receptor clustering. Note that these postsynaptic neurexin intracellular interactions do not exclude any previously demonstrated interactions between the intracellular C-terminus of postsynaptic neuroligin and other proteins, including PSD-95 (Chen, 2010).

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).

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 p70^S6K , and further elucidation of its role as a signal 'integrator' in synapse plasticity should be interesting. The fact that Nrx-1 levels were increased at Spn NMJs and, most importantly, that genetic removal of a single nrx-1 gene copy effectively suppressed the Spn AZ phenotype, indicates an important role of the Spn/Nrx-1 interaction in this context. Affinity of Spn-PDZ for the Nrx-1 C-term was somewhat lower than that of the Syd-1-PDZ, both in ITC and Y2H experiments. Nonetheless, overexpression of Spn was successful in reducing the targeting effect of Syd-1 on overexpressed Nrx-1^GFP. It will be interesting to see whether this interaction can be differentially regulated, for example, by (de)phosphorylation. It is worth noting that apart from Syd-1 and Spn, several other proteins containing PDZ domains, including CASK, Mint1/X11, CIPP and Syntenin, were found to bind to the Nrxs C-termini. CASK was previously shown to interact genetically with Nrx-1, controlling endocytic function at Drosophila NMJs. However, when this study tested for an influence of CASK on Nrx-1^GFP motility using FRAP, genetic ablation of CASK had no effect (Muhammad, 2015).

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

The Neurexin-NSF interaction regulates short-term synaptic depression

Although Neurexins, which are cell adhesion molecules localized predominately to the presynaptic terminals, are known to regulate synapse formation and synaptic transmission, their role in the regulation of synaptic vesicle release during repetitive nerve stimulation is unknown. This study shows that Drosophila neurexin mutant synapses exhibit rapid short-term synaptic depression upon tetanic nerve stimulation. Moreover, the intracellular region of Neurexin was demonstrated to be essential for synaptic vesicle release upon tetanic nerve stimulation. Using a yeast two-hybrid screen, it was found that the intracellular region of Neurexin interacts with N-ethylmaleimide sensitive factor (NSF), an enzyme that mediates soluble NSF attachment protein receptor (SNARE) complex disassembly and plays an important role in synaptic vesicle release. The binding sites of each molecule were mapped, and it was demonstrated that the Neurexin-NSF interaction is critical for both the distribution of NSF at the presynaptic terminals and SNARE complex disassembly. These results reveal a previously unknown role of Neurexin in the regulation of short-term synaptic depression upon tetanic nerve stimulation and provide new mechanistic insights into the role of Neurexin in synaptic vesicle release (Li, 2015).

Previous studies have shown that the α-NRXs functionally couple Ca2+ channels to the presynaptic machinery to mediate synaptic vesicle exocytosis and that defective synaptic vesicle release can be restored with 1 mm Ca2+ (Li, 2007). In the current experiments, 1.8 mm Ca2+ was used to eliminate the effects of abnormal Ca2+ sensitivity in neurotransmitter release. Under this condition, it was shown that nrx mutant synapses exhibit rapid short term synaptic depression and reduced quantal content of nerve-evoked synaptic currents at the steady state during tetanic stimulation. These observations suggest that NRX regulates activity-dependent synaptic plasticity. Similar observations have been reported in the fast-twitch diaphragm muscle of the α-NRX double knock-out mouse, which indicates that presynaptic efficacy but not presynaptic homeostatic plasticity is normal under basal conditions (Li, 2015).

Deficits in both the presynaptic neurotransmitter release machinery and the postsynaptic neurotransmitter receptors may cause rapid short term synaptic depression. As a presynaptic adhesion molecule, NRX interacts with the postsynaptic adhesion molecule, Neuroligin, and bridges the synaptic cleft that aligns the presynaptic neurotransmitter release machinery with the postsynaptic neurotransmitter receptors. It has been suggested that the activity-dependent regulation of the NRX/Neuroligin interaction mediates learning-related synaptic remodeling and long term facilitation. A recent study reveals that the alternative splicing of presynaptic NRX-3 controls postsynaptic AMPA receptor trafficking and long term plasticity in mice (Aoto, 2013). This study showed that the kinetics of the individual synaptic currents were stable during tetanic stimulation in the nrx mutant synapses. Moreover, NRX was shown to mediate synaptic plasticity through the presynaptic machinery, which was supported by rescue experiments (Li, 2015).

The intracellular sequence of NRX, including the C-terminal PDZ-binding motif, is identical across several vertebrate and invertebrate species. Mammalian NRXs only possesses a short cytoplasmic tail (55 amino acids), including a PDZ-binding motif. To identify the potential binding partners, several laboratories have conducted yeast two-hybrid screening by using the cytosolic tail of mammalian NRX as a bait. Two PDZ-containing proteins, CASK and syntenin, have been identified in the previous screening, and further studies show that the binding between CASK and NRX is abolished by deletion of the last three amino acids of the intracellular C-terminal region of NRX. However, it seems that the previous screenings are not saturated, as the NRX-interacted protein Mints was not identified in these screenings (Li, 2015).

Drosophila NRX contains a long cytoplasmic tail (122 amino acids), including a functional PDZ-binding motif. Thus, it is possible that the long cytoplasmic tail may associate with some partners in a PDZ-independent manner. In this study, normalized Drosophila cDNA libraries were used in the screening, which helps to identify the binding partner with low abundance. In the screening, 23 cDNAs clones that encode the fragments of 18 proteins were recovered. Previous study has demonstrated that the cytoplasmic tail of NRX associates with RFABG and facilitates the retinol transport. This study identified the intracellular region of NRX binds with NSF through the amino acid sequence 1788-1813 but not the PDZ-binding motif of NRX. This NRX/NSF interaction was found to be essential for the NSF recruitment to the presynaptic terminals and plays an important role in synaptic vesicle release. In addition, multiple lines of evidence that NRX associates with NSF at the presynaptic terminals. An alignment analysis of the NSF-binding site of NRX revealed that this sequence is conserved across different species. Moreover, this sequence is present across many proteins that serve different functions, which implies that this sequence may be essential for protein interactions (Li, 2015).

Each NSF molecule contains an N-terminal domain that is responsible for the interaction with α-SNAP and the SNARE complex, a low affinity ATP-binding domain (D1 domain) whose hydrolytic activity is associated with NSF-driven SNARE complex disassembly and a C terminal high affinity ATP-binding domain (D2 domain). This study mapped the NRX-binding sites of NSF to the D2 domain, which mediates the ATP-dependent oligomerization of NSF. An alignment analysis showed that both the D2 domain in NSF and the NSF-binding site in NRX are highly conserved. This result suggests that the NRX/NSF interaction may occur in other species, a possibility that needs to be investigated further (Li, 2015).

It has been documented that the D2 domain of NSF is essential for its hexamerization. This study shows that NRX exhibits a declined NSF binding capacity in high Ca2+ concentration. These observations suggest that NSF can be released from NRX under stimulation. Although NRX and NSF do not bind with Ca2+ directly, it is possible that they undergo Ca2+ signaling-dependent modifications or bind with some Ca2+-binding proteins. In the yeast two-hybrid screening, one potential Ca2+-binding protein (CG33978) has been identified. However, the mechanism of how NRX releases NSF under high Ca2+ concentration need to be further investigated (Li, 2015).

Previous studies have shown that the PDZ-binding motif of NRX associates with several molecules involved in the synaptic vesicle exocytosis machinery, including synaptotagmin and the PDZ domain-containing proteins CASK and Mints. Synaptotagmin functions as a Ca2+ sensor and controls synaptic membrane fusion machinery. Adaptor protein Mints regulates presynaptic vesicle release (Ho, 2006), whereas the other adaptor protein CASK is not essential for the Ca2+-triggered presynaptic release. In contrast, CASK interacts with NRX and protein 4.1 to form a trimeric complex and regulates synapse formation. The process of synaptic vesicle release includes several consecutive steps, docking, priming, and fusion. Depending on the stimulation given to synapses, different synaptic vesicle trafficking steps become rate-limiting for synaptic vesicle release. This causes short term changes in synaptic transmission that determine many higher brain functions such as sound localization, sensory adaptation, or even working memory. Thus, the PDZ-binding motif-linked synaptic vesicle exocytotic machinery might regulate the different steps during synaptic vesicle release. Indeed, nrx mutant synapses that express NRXΔ4 exhibit a rapid current decline over the first several dozen stimulations. In contrast, the expression of NRXΔ4 largely restores the reduced steady-state mean EJCs and quantal content in neurexin mutant synapses. Together with the C-terminally truncated NRX rescue experiments, these data suggest that intracellular regions of NRX other than the PDZ-binding motif also regulate short term synaptic depression. The existing literature extensively documents the roles of NSF in SNARE complex disassembly and short term synaptic depression. This study extended these findings to show that the NRX/NSF interaction facilitates the recruitment of NSF to the presynaptic terminals and promotes the subsequent SNARE complex disassembly (Li, 2015).

Previous studies have established that the NSF hexamer serves as the only active form for SNARE complex disassembly. In this study, the binding assay showed that purified NRX binds to NSF in a concentration-dependent and saturable manner. NRXs appear to serve as scaffold proteins to recruit NSF, and the NRX/NSF interaction may promote SNARE complex disassembly in vivo. Live image studies have shown that NSF mutant (i.e. comt) synapses exhibit defective NSF re-distribution during tetanic nerve stimulation. In this study, immunocytochemical and sedimentation analyses revealed that a lack of the NRX/NSF interaction results in an altered distribution of NSF and an accumulation of 7S SNARE complexes. These results imply that NRX/NSF interaction serves as a potential mechanism to restrict the mobilization of NSF (Li, 2015).

Electrophysiological recordings revealed that synaptic depression was comparable between nrx mutant synapses and wild-type synapses in response to low frequency stimulations. The long intervals between low frequency stimulations may allow the remaining NSF to disassemble the SNARE complexes and generate enough free t-SNARE for the subsequent synaptic vesicle fusion events. Another possibility that needs to be investigated further is that tetanic stimulation may have other effects on the re-distribution of NSF (Li, 2015).

In summary, this study provides evidence that the NRX/NSF interaction recruits NSF to the presynaptic terminals and promotes SNARE complex disassembly. These findings have revealed a previously unknown role of NRX in the regulation of neurotransmitter release and provide a linkage between the presynaptic adhesion molecules and the presynaptic plasticity machinery (Li, 2015).

The neuronal protein Neurexin directly interacts with the Scribble-Pix complex to stimulate F-actin assembly for synaptic vesicle clustering

Synaptic vesicles (SVs) form distinct pools at synaptic terminals, and this well-regulated separation is necessary for normal neuro-transmission. However, how SV cluster in particular synaptic compartments to maintain normal neurotransmitter release remains a mystery. The presynaptic protein Neurexin (NRX) plays a significant role in synaptic architecture and function, and some evidences suggest that NRX is associated with neurological disorders, including autism spectrum disorders. However, the role of NRX in SV clustering is unclear. Using the neuromuscular junction at the 2-3 instar stages of Drosophila larvae as a model and biochemical, imaging, and electrophysiology techniques, this study demonstrate that Drosophila NRX (DNRX) plays critical roles in regulating synaptic terminal clustering and release of SVs. DNRX controls the terminal clustering and release of SVs by stimulating presynaptic F-actin. Furthermore, the results indicate that DNRX functions through the scaffold protein Scribble and the GEF protein Pixie to activate the small GTPase Rac1. A direct interaction was observed between the C-terminal PDZ-binding motif of DNRX and the PDZ domains of Scribble and that Scribble bridges DNRX to DPix, forming a DNRX/Scribble/DPix complex that activates Rac1 and subsequently stimulates presynaptic F-actin assembly and SV clustering. Taken together, this work provides important insights into the function of DNRX in regulating SV clustering, which could help inform further research into pathological neurexin-mediated mechanisms in neurological disorders such as autism (Rui, 2017).

Neurons are the basic unit of the nervous system, and they communicate with each other through synapses. After being captured by sensory organs, neural signals pass between synapses in the form of neurotransmitters. As the vehicles of neurotransmitters, synaptic vesicles (SV) are essential for neurotransmission. SVs can be divided into three distinct pools according to their localization and function. The SVs adjacent to the active zone and ready to be released are referred to as the ready release pool. The second pool of vesicles is the exo/endo-cycling pool, which is also found close to release sites and supplies the ready release pool. Finally, the pool located away from the active zone, and that contains the majority of SVs, is referred to as the reserve pool and is considered to be a storage pool. Actin is a major part of the cytoskeleton that is required for maintaining the architecture of synapses as well as contributing to their function, and a significant amount of evidence has shown that the localization, translocation, and release of SVs can be altered by disturbing the polymerization of pre-synaptic actin (Rui, 2017).

The synapse is a highly specialized structure, and synaptogenesis is a highly complex process. Previous studies have shown that synaptic adhesive molecules play important roles in synaptogenesis and neurotransmission, and a number of synaptic cell adhesion molecules, including Neuroligins and Neurexins have been identified over the past few decades. Neurexin was first recognized as a receptor for α-latrotoxin, a black widow spider venom component that triggers massive neurotransmitter release. There are three neurexin genes in mammals, each of which has two promoters generating α-Neurexin and β-Neurexins, whereas there is only one neurexin-1 gene in Drosophila (dnrx). Recent studies both in Drosophila and mammals showed that Neurexin plays a significant role in synaptic architecture and function, and there is evidence suggesting that neurexin is associated with autism spectrum disorders (ASDs). The complexity and redundancy of the neurexin genes in mammals motivated the authors to focus on simpler model systems, such as Drosophila, to investigate the in vivo function of DNRX (Rui, 2017).

Neurexin has been shown to bind to several molecules, including the presynaptic scaffolding proteins Mint (Biederer, 2000), CASK (Sun, 2009), and LRRTM2 (de wit, 2009; Ko, 2009). Recently, DNRX also has been demonstrated to interact with the N-ethylmaleimide sensitive factor to regulate short-term synaptic depression and to interact with Spinophilin to maintain active zone architecture (Muhammad, 2015; Li, 2015). A typical trans-synaptic complex is formed by the heterophilic interaction of presynaptic Neurexins and postsynaptic Neuroligins, and these complexes have attracted much attention as scaffolding complexes that not only maintain the normal structure of the synapse but also function in passing signals across the synapses. It is thus clear that Neurexin is a multifunctional molecule. Despite the identification and characterization of these proteins that functionally associated with DNRX, understanding of the pathways including DNRX that control synaptic function are still incomplete, with other partners and mechanisms yet to be uncovered and analyzed (Rui, 2017).

This study has investigated the role of DNRX in the cluster and release of SVs at synaptic terminals. The effect of DNRX is mediated by presynaptic F-actin and there is a direct interaction between the C-terminal PDZ-binding motif of DNRX and the PDZ domains of the tumor suppressor protein Scribble. Furthermore, Scribble bridges DNRX to DPix, forming a DNRX-Scribble-DPix complex to activate Rac1 and affect presynaptic F-actin assembly and SV clustering. Taken together, these studies provide novel insight into the mechanisms underlying the regulation of neurotransmitter release by DNRX (Rui, 2017).

Neurexin is a highly conserved cell adhesion molecule that is predominantly localized at the presynaptic terminal. Previous studies have shown that Neurexin plays a significant role in synaptic architecture and function. In addition, accumulating evidence has implicated Neurexin in Autism Spectrum Disorders (ASDs). ASDs are neurodevelopmental disorders characterized by deficits in communication and social interaction as well as restricted interests and repetitive and stereotypic patterns of behavior. However, the precise function and underlying molecular mechanisms of Neurexin in both normal physiology and ASDs remain unclear. Drosophila Scribble is a cytoplasmic scaffolding protein that was first recognized as a tumor suppressor that regulates epithelial cell adhesion and migration in mammals. Recently, it has been shown that Scribble is also localized in the nervous system both in invertebrate and vertebrate animals, and plays a role in synaptic plasticity and animal behavior, including learning, memory, social behavior, and olfactory behavior. However, how Scribble functions at the synapse remains unknown. In this study, this study revealed that DNRX interacts directly with the Scribble PDZ domains through the very C-terminal PDZ-binding motif to regulate presynaptic F-actin and SVs (Rui, 2017).

F-actin is highly enriched at synaptic terminals and is vital for SV traffic, localization, and release For decades, F-actin emerged as the major cytoskeleton identified in presynaptic nerve terminals. Considering the especial location of F-actin at the internal space of presynaptic nerve terminals, it raised a hypothesis that F-actin regulates synaptic vesicle localization and release. Previous studies from several groups provided consistent evidence that after disrupting the polymerization of actin, pre-synapse affected SV traffic, localization, and neurotransmitter release. Moreover, cortical actin has been identified as a barrier for vesicles during the process of moving to the active zone in the presynaptic terminal. However, whether and how F-actin participates in the regulation of SV at synapse is still controversial. Therefore, further investigation will be necessary to determine the function of F-actin at synapse and especially for advances in understanding of the relationship with SV. By now the function of F-actin at synapse is still poorly understood. To better understand the effect of F-actin on SV this study assessed the cluster and release of SV after ablating the actin-associated genes and found the obvious defects of SV cluster and release. To further test this possibility, additional experiments are needed to elaborate the vital role of F-actin in SV regulation in future (Rui, 2017).

SV distribution and dynamics are essential for normal neural signal transmission and for synaptic plasticity both at peripheral and central synapses. Disruptions in SV function will lead to various forms of neurological disorders. The process of synaptic vesicle priming, docking, and fusion with the presynaptic membrane has been investigated extensively, and numerous molecules involved in this process have been identified. These include synaptotagmins, synapsins, synaptobrevins, and Munc18. However, how SV cluster in particular compartments and their role in regulating neurotransmitter release at the presynaptic terminal are unclear. The results from this study provide compelling evidences that DNRX plays an essential role in the distribution and release of SVs (Rui, 2017).

In this study, the data suggest the amount of F-actin is significantly reduced. Moreover, presynaptic Cortactin or the active form of DPak, key regulators of the actin cytoskeleton, are able to rescue the defects in SV distribution and spontaneous release frequency in the dnrx mutant. These results illustrate the essential role of presynaptic actin in regulating SVs localization and release. The results are consistent with the recent findings showing that Arp2/3 complex-mediated actin regulation is important for presynaptic neurotransmitter release (Rui, 2017).

Previous work has shown that Scribble is a scaffolding, tumor suppressor protein that through its PDZ domains interacts with a number of proteins, including β-catenin, βPix, and NOS1AP. Although NOS1AP can bind directly to the fourth Scribble PDZ domain, βPix has binding affinity to all four PDZ domains of Scribble. The present study used co-immunoprecipitation to show that DNRX can form a complex with Scribble in the Drosophila nervous system in vivo. DNRX and Scribble are co-localized in both central and peripheral nervous system during embryonic, larval, and adult stages. Importantly, these two proteins are highly expressed in the mushroom body of Drosophila, suggesting a key role in learning and memory. DNRX can directly bind to all four Scribble PDZ domains through its C-terminal PDZ-binding motif, similar to what is observed for βPix. These interaction results suggest that there may be competitions and cross-effect between DNRX and βPix. These interactions may also relate to the mutual effect on the protein level of Scribble and DNRX. Because the mRNA level of Scribble is not altered in the dnrx mutant, it is possible that when the DNRX or Scribble was absent, the complex becomes destabilized and subsequently degraded. Further experiments are needed to address this possibility (Rui, 2017).

What is the functional consequence of the Scribble and DNRX interaction? The present study provides evidence that Scribble may act as a bridge between DNRX and DPix to regulate the actin cytoskeleton. βPix is a GEF specific to Rac1/Cdc2, a key mediator of actin reorganization in response to various stimuli. In the mammalian system, Rac1 is locally activated in dendritic spines, and this spatial restricted activation is regulated by Pix (Zhang, 2005). The present study shows that the active form of Rac1 (Rac1-GTP) is reduced in the dnrx mutant, dnrx, and scribble knockdown flies compared with wild-type for the protein level of DPix in these lines were decreased, supporting the idea that the DNRX and Scribble interaction activates Rac1. Recent studies show that Rac1 plays a critical roles in animal behavior, particularly in the process of forgetting (36). In addition, Scribble has recently been reported to activate forgetting through Rac1 in Drosophila (35). Taken together, it is suggested that DNRX may also be involved in forgetting by regulating the Rac1 signaling pathway. Indeed, it has been demonstrated that Rac1 activation is defective in multiple autism-related gene mutations, including the dnrx gene, and that Rac1 has been proposed to be a converging node linked to ASD (36). Thus, the present study demonstrating that DNRX interacts with Scribble and DPix to regulate Rac1 provides direct mechanistic insight into not only the fundamental mechanisms underlying the roles of the neuroligin-neurexin complex in actin-mediated presynaptic regulation, but also the pathological mechanism of ASD (Rui, 2017).

Coordinated regulation of axonal microtubule organization and transport by Drosophila Neurexin and BMP pathway

Neurexins are well known trans-synaptic cell adhesion molecules that are required for proper synaptic development and function across species. Beyond synapse organization and function, little is known about other roles Neurexins might have in the nervous system. This study reports novel phenotypic consequences of mutations in Drosophila neurexin (dnrx), which alters axonal microtubule organization and transport. dnrx mutants display phenotypic similarities with the BMP receptor wishful thinking (wit) and one of the downstream effectors, futsch, which is a known regulator of microtubule organization and stability. dnrx has genetic interactions with wit and futsch. Loss of dnrx also results in reduced levels of other downstream effectors of BMP signaling, phosphorylated-Mad and Trio. Interestingly, postsynaptic overexpression of the BMP ligand, Glass bottom boat, in dnrx mutants partially rescues the axonal transport defects but not the synapse undergrowth at the neuromuscular junctions. These data suggest that dnrx and BMP signaling are involved in many diverse functions and that regulation of axonal MT organization and transport might be distinct from regulation of synaptic growth in dnrx mutants. Together, this work uncovers a novel function of Drosophila Neurexin and may provide insights into functions of Neurexins in vertebrates (Banerjee, 2018).

Neurexins are some of the most extensively studied synaptic cell adhesion molecules for their role in synapse formation and function. In Drosophila, dnrx mutants have reduced synaptic growth. This reduced synaptic growth is similar to what was observe in the BMP signaling mutants: wit, gbb, mad and trio. Recent studies have shown that dnrx regulates components of the BMP signaling pathway and directs synaptic growth and cytoarchitecture in conjunction with dnlg and wit. The data presented in this study shows that dnrx engages in a complex molecular machinery with the BMP receptor Wit, the ligand Gbb and other downstream effectors to allow smooth axonal transport and proper MT organization (Banerjee, 2018).

Both dnrx and wit mutants display phenotypic similarities with axonal MT organization and transport of various cargo along the segmental nerves. It is interesting to note that axonal MT and transport phenotypes resulting from gain of dnrx function are similar to loss of function mutants. This suggests that a fine balance in levels of dnrx is important, and either too little or too much of it is detrimental for MT organization and the transport machinery. Defects in axonal transport resulting from cell type specific reduction in motor neurons using dnrx-RNAi confirmed that the axon transport defect is autonomous to motor neurons. The defects resulting from the dnrx knockdown, however, were milder than dnrx loss-of-function possibly due to penetrance or expressivity factors. The Gal4-only controls (elav-Gal4 and OK6-Gal4) did not display any axonal transport or MT organization defects further confirming the specificity of the dnrx phenotypes. It is also worth noting that gain of wit did not disrupt MT organization or axonal transport (Banerjee, 2018).

The genetic interaction between dnrx and wit suggest that these proteins regulate axonal MT organization and transport. Recent studies have also showed that dnrx and wit coordinate synaptic organization and growth, which suggests that these proteins are needed for multiple processes during nervous system development. The ultrastructural phenotypes in dnrx and wit mutants reveal a structural disorganization in the arrangement of MT filaments along the axons. These datasets showed that there is indeed disorganization in MT morphology in the mutants and these findings are not artifacts from the altered localization of Futsch (Banerjee, 2018).

Both dnrx and wit are presynaptic proteins and their loss shows a multitude of defects at the axonal, cytoskeletal and synaptic levels. It was therefore important to examine the range of behavioral deficits in these mutant larvae. dnrx and wit mutants displayed defects both in peristalsis and locomotor behaviors. These behaviors are attributable, most likely at least in part, to the defects in axon transport, MT organization and possible dysfunction of the NMJ synapses. Behavior in animals could be a composite of defects at many levels of integration at the CNS, PNS, NMJ and circuits responsible for producing rhythmic patterns of movement and general locomotion. Defects in both peristalsis and wandering behavior in cysteine string protein (csp) mutant larvae, for example, are thought to be due to motor defects at the NMJ and at the levels of CNS output generation. The locomotor behaviors observed in dnrx and wit mutants also indicate that these proteins might play a boarder role in organization of motor circuits (Banerjee, 2018).

Recent studies have implicated BMP signaling in the regulation of synaptic strength, axonal MT organization and transport. While the current studies point to the reliance of MT organization and axonal transport on BMP signaling, there are other studies that show disruption of axonal transport perturbing BMP signaling contributes to synaptic abnormalities in neurodegenerative diseases. It is important to note that mutations that fall under the categories of both positive regulators of BMP, like dnrx8 and negative regulators of BMP such as, Spicthyin and Spartin both lead to MT disorganization. It is, therefore, interesting that while Spichthyin and Spartin function as negative regulators of the BMP signaling by regulating BMP receptor traffic and modulate regulation of MT cytoskeleton, loss of these proteins does not show impaired axonal transport. dnrx loss results in disorganized MTs, but unlike Spichthyin and Spartin, loss of dnrx shows transport defects. These findings suggest that MT cytoskeletal organization could be compromised by both positive and negative regulation of BMP signaling (Banerjee, 2018).

The regulation of microtubule stability is essential for axonal transport. The microtubule-associated protein, Futsch, recently implicated in the BMP pathway, has a role in the stabilization of MTs. futsch mutants display axonal transport defects consistent with altered MT organization similar to dnrx and wit mutants. A previous report, however, showed that futsch mutants do not show defects in axonal transport of Brp. This study examined futsch mutants for accumulation of Brp puncta along axons. Mild, yet significant, increase in accumulation of Brp was found along the axons in futsch mutants. These studies are not completely in agreement with the previous report. The discrepancies between the studies could be possibly due to: (1) differences in quantification parameters (Brp area/Hrp area as opposed to number of Brp puncta along nerve length); (2) difference in segmental nerves analyzed (the current analysis was restricted to segmental nerves 5-8); and (3) the region of nerve fiber analyzed (proximal or distal). It is also worth noting that light microscopy studies showed Syt and DCSP2 aggregation along mutant axons did not always co-localize with Brp puncta. These observations are consistent with recently published report that showed Syt co-traffic less efficiently with active zone scaffold proteins Brp and Basoon. Collectively, these findings suggest the possibility of the presence of distinct transport mechanisms for different synaptic cargoes. Future studies will be aimed at addressing the dynamics of axonal membrane traffic in mutants of dnrx and BMP pathway, and whether some synaptic proteins are co-transported and others are not, or whether transport mechanisms are able to discriminate synaptic cargo as it is transported along the axons (Banerjee, 2018).

futsch and dnrx interact genetically as double heterozygotes display axonal transport defects consistent with dose-dependent genetic interactions. This data further corroborates the idea that dnrx is not only necessary for trans-synaptic adhesion and apposition of the pre- and post-synaptic compartments, but also for maintaining the stability of the BMP receptor wit for optimal BMP signaling. Future studies will be aimed at addressing the link between dnrx and Futsch. dnrx is a transmembrane protein with a PDZ domain binding motif and Futsch is a MT-binding protein. Both of these proteins are highly expressed along the axons and could potentially exist as a complex. It is also possible that dnrx might interact with MT motor proteins such as Kinesin or Dynein. The Kinesin superfamily protein, KIF 17, was previously shown to interact with PDZ domain of mLin-10 (Mint1/X11) to transport NMDA receptor 2B. mLin-10 is part of a larger protein complex that includes mLin-2 (Cask) and mLin-7 (Veli). It is likely that the intracellular PDZ domain of dnrx is capable of recruiting protein complexes such as these to form a link between axonal membranes to the axonal MT cytoskeleton (Banerjee, 2018).

While it was not a surprise that levels of the downstream effectors of the BMP cascade, pMad and Trio, would be reduced in dnrx mutant BL/VNC given that previous studies showed reduced levels of Wit, Tkv and pMad at the dnrx mutant NMJ synapses. It is interesting that postsynaptic overexpression of the BMP ligand, Gbb, partially rescues the axonal transport phenotypes in dnrx mutant background but not the NMJ bouton undergrowth phenotype. This indicates that distinct regulatory mechanisms might be involved in proper axonal MT organization and transport and growth during NMJ development, which are still not well understood. While this finding may suggest that Wit/Gbb signaling does not require dnrx to regulate axon transport, it is important to note that the rescue of the axonal transport phenotype was partial and did not reach wild type levels, suggesting that dnrx is required to allow optimal levels of axonal transport to occur. This finding also indicates: (1) that possibly it is the retrograde transport that gets somewhat corrected while there might still be defects in anterograde transport of cargoes in dnrx mutants; (2) that BMP signaling solely is not responsible for trafficking of cargoes and overall MT health in dnrx mutants; and (3) that there might be additional proteins involved in forming a cascade to link dnrx from axonal membrane to MT cytoskeleton. Given the findings reported in this study on Drosophila Nrx, it would be of immense interest to investigate whether mammalian Neurexins will have similar functions separate from their role in synapse formation and their functional modulation, and also axonal microtubular organization. Future studies will further address other aspects of Neurexin functions in the nervous system (Banerjee, 2018).

Antagonistic interactions between two Neuroligins coordinate pre- and postsynaptic assembly

As a result of developmental synapse formation, the presynaptic neurotransmitter release machinery becomes accurately matched with postsynaptic neurotransmitter receptors. Trans-synaptic signaling is executed through cell adhesion proteins such as Neurexin::Neuroligin pairs but also through diffusible and cytoplasmic signals. How exactly pre-post coordination is ensured in vivo remains largely enigmatic. This study identified a 'molecular choreography' coordinating pre- with postsynaptic assembly during the developmental formation of Drosophila neuromuscular synapses. Two presynaptic Neurexin-binding scaffold proteins, Syd-1 and Spinophilin (Spn), spatio-temporally coordinated pre-post assembly in conjunction with two postsynaptically operating, antagonistic Neuroligin species: Nlg1 and Nlg2. The Spn/Nlg2 module promoted active zone (AZ) maturation by driving the accumulation of AZ scaffold proteins critical for synaptic vesicle release. Simultaneously, these regulators restricted postsynaptic glutamate receptor incorporation. Both functions of the Spn/Nlg2 module were directly antagonized by Syd-1/Nlg1. Nlg1 and Nlg2 also had divergent effects on Nrx-1 in vivo motility. Concerning diffusible signals, Spn and Syd-1 antagonistically controlled the levels of Munc13-family protein Unc13B at nascent AZs, whose release function facilitated glutamate receptor incorporation at assembling postsynaptic specializations. As a result, this study has provided direct in vivo evidence illustrating how a highly regulative and interleaved communication between cell adhesion protein signaling complexes and diffusible signals allows for a precise coordination of pre- with post-synaptic assembly. It will be interesting to analyze whether this logic also transfers to plasticity processes (Ramesh, 2021).

Synaptic vesicle (SV) release at chemical synapses depends on the formation of active zone (AZ) scaffolds composed of a canonical apparatus of proteins including Unc13, RIM-binding protein (RIM-BP), Liprin-α and CAST/ELKS (called Bruchpilot [BRP] in Drosophila). The size of individual AZ scaffolds scales with SV release probability. Once matured, each AZ apparently forms an integer number of release sites apposed by postsynaptic glutamate receptors (GluRs), likely spatially coordinated through a trans-synaptic micropattern ('nanocolumns'). Importantly, in the course of maturation, AZ size becomes closely matched to the size of the postsynaptic density (PSD) scaffold clustering neurotransmitter (NT) receptors (Ramesh, 2021).

How the in vivo synapse assembly process and associated regulatory steps achieve this precise pre-post matching during developmental assembly is not fully understood. Notably, trans-synaptic cell-adhesion molecules (CAMs) have the capacity to bidirectionally tune synapse assembly, and Neurexin (Nrx) and Neuroligin (Nlg) interactions represent a regulatory principle conserved across vertebrate and invertebrate synapses. Although many synaptic CAMs and cytoplasmic proteins have been studied in isolation, how different CAMs selectively engage with each other and their cytoplasmic partners to ensure pre-post matching during synapse assembly has remained enigmatic, partly because of the high genetic redundancy among mammalian CAMs. Besides CAM signaling, diffusible signals including NT release at nascent AZs might play a regulatory role in postsynaptic assembly (Ramesh, 2021).

This study characterize mechanisms that ensure pre-post matching during the assembly of individual glutamatergic synapses at the Drosophila neuromuscular junction (NMJ). In developing larvae, synapse maturation ultimately establishes a precisely defined pre-post stoichiometry over the course of several hours. To interrogate these mechanisms, the unique advantages were used of the larval NMJ system, which allows for a synergy of reduced genetic redundancy, super-resolution, and dynamic intravital microscopy and electrophysiology. Nlg1 and Nlg2, two Nlg species previously shown to functionally interact with the only Nrx family protein in Drosophila, Nrx-1. The current results reveal that these two Nlgs serve antagonistic roles and operate in conjunction with two antagonistic presynaptic proteins that bind Nrx-1: Syd-1 cooperating with Nlg1 and Spinophilin (Spn) with Nlg2. Whereas the Spn/Nlg2 functional module promoted AZ maturation (BRP/RIM-BP/Unc13A incorporation) but restrained GluRIIA-containing receptor incorporation, Syd-1/Nlg1 initiated AZ assembly and promoted GluRIIA receptor incorporation through Unc13B recruitment and its glutamate release function. Genetic interaction experiments identified a remarkable degree of crosstalk between these modules, exemplifying a regulatory principle obviously evolved to ensure precise pre-post matching, and integrating Unc13B-dependent glutamate release acting as a diffusible signal. Altogether, these data indicate that synaptic matching is not established via a trans-synaptic 'stoichiometric building principle' to continuously accumulate synaptic components, but via a regulatory crosstalk between antagonistic assembly modules (Ramesh, 2021).

Synapses form out of three interdependent molecular assemblies, each precisely crafted to execute fast and precise information transfer between two cells: the presynaptic AZ where SVs fuse at defined release sites, the synaptic cleft through which NT diffuses, and the postsynaptic compartment where the NT binds its receptors. Importantly, these compartments do not form in isolation, but the size of the AZ (and thus the number of presynaptic release sites per AZ) must closely scale with the number of postsynaptic NT receptors. Super-resolution microscopy identified presynaptic AZ protein nanoclusters to align with concentrated postsynaptic receptors and scaffolding proteins, suggesting the existence of trans-synaptic molecular 'nanocolumns'. Indeed, the exact nanometer location of vesicular release in relation to receptors might be a critical determinant of synaptic strength, which might also contribute to synaptic plasticity (Ramesh, 2021).

A central question now pertains to how trans-synaptic signaling is precisely executed in molecular terms to coordinate pre- with post-synaptic assembly. Candidate molecular scenarios include interactions that directly bridge pre- and postsynaptic membranes like trans-synaptic CAMs, which bidirectionally control synapse formation, remodeling, and elimination. This study exploited the unique features of the Drosophila NMJ system: unique accessibility to intravital imaging to accurately analyze the assembly path, a cytoarchitecture ideal for super-resolution analysis, high-resolution electrophysiological measurements, and a low level of genetic redundancy, to address how presynaptic AZs are matched to postsynaptic GluRs. Moreover, the amount of ELKS protein BRP, easily accessible for STED microscopy, directly scales with presynaptic release at AZs, making it an ideal readout to assess both structural and functional assembly (Ramesh, 2021).

In principle, a strategy of continuously accumulating stoichiometric amounts of pre- and postsynaptic material along the assembly trajectory, potentially via a single transcellular bridge connecting to nucleation processes on both sides, might appear the easiest way to establish pre-post matching. Indeed, such an idea has recently been proposed, where the age of AZs determines their size and strength at the Drosophila NMJ. However, such a solution might lack regulatory flexibility and is also not what was find in this work. Instead, this analysis identifies antagonistic regulatory inputs to be executed by two postsynaptically active Nlg species operating synergistically with their respective 'cognate' presynaptic scaffold proteins, Syd-1 and Spn, previously shown to steer synapse assembly via their Nrx-1-binding function. It here appears likely that autonomy over the presynaptic versus the postsynaptic compartment might be particularly relevant during plasticity processes, shown to involve the specific incorporation of BRP at NMJ synapses. This antagonistic operation might serve to embed contextual information while steering the assembly process and could be particularly robust when utilized in such a highly regulative scheme (Ramesh, 2021).

A model is provided for the functional relations analyzed in this study. Syd-1 and Nlg1 form new AZs in the seeding phase, whereas Spn and Nlg2 promote incorporation of BRP to appropriate levels in the maturation phase. Notably, BRP is the rate-limiting building block of the AZ scaffold, determining the size and functional strength of the AZ specialization. Overactivity of Syd-1/Nrx-1/Nlg1 signaling likely is directly responsible for the Spn AZ phenotype, given that it could be suppressed by lowering the dose of any of these molecules. The same is true for the Nlg2 phenotype as well, suggesting that the Nlg2 AZ phenotype similarly reflects Syd-1/Nlg1 axis overactivity. Furthermore, reduction of Spn efficiently suppressed the normally excessive BRP incorporation at the AZs remaining in Syd-1, Nrx-1, and Nlg-1 mutants. Mechanistically, future analysis will have to clarify whether direct physical interactions of Spn with BRP complexes, co-clustering RIM-BP and Unc13A are of relevance here. Alternatively, the Syd-1 and Spn modules might antagonistically control a downstream process such as the status of F-actin (Ramesh, 2021).

Although in the past, Spn was interpreted as functioning after Syd-1 during the AZ development process, the current data now suggest that Syd-1 and Spn in fact continuously antagonize each other throughout assembly to tune final AZ size and function. Still, intravital imaging of nascent AZs showed that the peak of Syd-1 accumulation precedes the peak of BRP accumulation by hour. The fact that the Syd-1 scaffold is favored over the Spn scaffold during the seeding phase might be explained via a 'quasi-epistatic' relation between these regulators: Syd-1 mutants show lower amounts of Spn, whereas Spn mutants show elevated amounts of Syd-1 suggesting that Syd-1 is needed for Spn accumulation at the AZ, potentially allowing Syd-1-mediated AZ seeding to precede Spn-mediated BRP accumulation. Spn and Syd-1 were shown to interact with each other in Drosophila and in C. elegans. It would be interesting to investigate whether the Spn/Syd-1 interaction plays a role in regulating access to Nrx-1, thereby contributing to define the actual 'assembly mode:' seeding or maturation. Obviously, the assembly modules must communicate to ultimately ensure a well-defined assembly product, e.g., via associated kinase and/or phosphatase activities. For example, the phosphorylation status of BRP can control transport. Furthermore, although Spn attenuation did efficiently suppress the Syd-1, Nrx-1, and Nlg1 AZ phenotypes, Nlg2 attenuation did not suppress the Nrx-1 and Nlg1 phenotypes. This suggests that the trans-synaptic signaling through Nrx-Nlgs might ensure that assembly proceeds from seeding toward maturation during development. This also opens up the possibility that Nlg2 attenuates Syd-1/Nrx-1/Nlg1 function by removing Nrx-1 from the seeding module and/or suppressing Nlg1 activity through cis-heteromerization. FRAP data also indicate that the postsynaptic binding partner identity (Nlg1 or Nlg2) has differential effects on Nrx-1 mobility. Lack of Nlg2 likely boosts the Nrx-1::Nlg1 seeding activity, directly explaining the supernumerary AZs typical for Nlg2 mutant (Ramesh, 2021).

Nlg1 promoted but Nlg2 blocked GluRIIA incorporation, which precedes BRP accumulation. Previous analysis showed that Syd-1 seemingly instructs Nrx-1 to interact with Nlg1 and promotes GluRIIA incorporation before BRP incorporation. Genetic interaction analysis showed that Syd-1/Nlg1 and Spn/Nlg2 execute a mutual regulatory counterplay here. This study now extends the understanding of GluRIIA incorporation to involve the release function of Unc13B, enriched at nascent AZs by Syd-1, a process antagonized by Spn. Spn and Nlg2 functionally cooperate to limit the amount of GluRIIA incorporation in the nascent postsynaptic specialization and match receptor amounts to the AZ size. However, although the Spn mutant phenotype was rescued by Syd-1, Nrx-1, and Nlg1 heterozygosity, the Nlg2 mutant phenotype was only rescued by Syd-1 heterozygosity, suggesting that Nlg1 and Nlg2 have an additional function in mediating GluRIIA incorporation independent of Unc13B. Mechanistically, it might well be that Nlg2 at the nascent postsynaptic compartment directly competes with Nlg1 for the binding of a critical effector, e.g., the ectodomain of the GluR complex or other membrane proteins such as Neto (Ramesh, 2021).

In mice, most synapses formed normally in the absence of NT release during development, but the synapses did not persist as they matured. Experiments in mice have shown in the past that massive local glutamate release could induce spine formation at the postsynapse. However, whether vesicular transmitter release tunes the incorporation dynamics of GluRs during developmental synapse assembly remains inconclusive (Ramesh, 2021).

Unc13B arrives early at nascent NMJ AZs. This recruitment of Unc13B is antagonistically controlled by the two complexes, given that Syd-1 mutants showed reduced but Spn mutants strongly increased synaptic Unc13B amounts. Importantly, the excessive GluRIIA incorporation in Spn mutants critically depended on Unc13B. Notably, treatment of cell cultures with BoNT-C and TNT-E previously was shown to prevent effective postsynaptic insertion of glutamatergic receptors in cultivated hippocampal neurons. However, it cannot be exclude that once Unc13A accumulates at the AZ, Unc13B might continue to mediate GluRIIA incorporation into later stages of synapse assembly (Ramesh, 2021).

Concerning the mode of Unc13B action, the data suggest that evoked Unc13B-mediated glutamate release at nascent sites attracts GluRIIA receptors, which are recruited from diffuse pools at the plasma membrane. Notably, proper gating behavior of GluRIIA in response to presynaptic glutamate release previously was shown to be essential for matching pre- with post-assembly. Unc13B-mediated release is coupled more loosely to Ca2+ channel activity compared with release mediated by the functionally dominant isoform, Unc13A. Likely, sensing glutamate at nascent sites renders GluRIIA into an active state, which allows for postsynaptic incorporation, previously shown to be nearly irreversible. Whether the GluRIIA incorporation subsequent to the glutamate sensing is truly stage dependent, e.g., via specific scaffold or cleft proteins, or whether differences in the spatio-temporal detail of glutamate release between Unc13B and Unc13A are more important here remains to be addressed (Ramesh, 2021).

Nrx-1, Nlg1, and Syd-1 mutants all show reduced NMJ area, whereas Spn and Nlg2 mutants showed normal NMJ sizes, and all of them showed reduced evoked potentials. Previous studies have shown that synaptic terminals can compensate for a change in size by adjusting NT output. A recent study showed that spontaneous neurotransmission is needed for the normal structural maturation of Drosophila NMJ synapses exclusive from the role of evoked neurotransmission. Increasing miniature events was sufficient to induce synaptic terminal growth, and this synapse maturation was locally regulated via a Trio guanine nucleotide exchange factor (GEF) and Rac1 GTPase molecular signaling pathway. Interestingly, Syd-1 was found to interact with Trio signaling. Together with the Rac guanine exchange factor (RacGEF) Trio, Syd-1 GAP activity promotes BRP clustering and independent of its GAP activity, Syd-1 recruits Nrx-1 to boutons. Additionally, mammalian Spn forms a complex with Rac1-GEF Kalirin-7 and, along with Rho-GEF Lfc, control dendritic spine morphology and function. Therefore, it will be interesting to study how Syd-1 and Spn antagonism translates into GAP/GEF signaling, which in turn might control the synapse assembly at Drosophila NMJs (Ramesh, 2021).

Astrocytes close a motor circuit critical period

Critical periods (brief intervals during which neural circuits can be modified by activity) are necessary for proper neural circuit assembly. Extended critical periods are associated with neurodevelopmental disorders; however, the mechanisms that ensure timely critical period closure remain poorly understood. This study defined a critical period in a developing Drosophila motor circuit and identified astrocytes as essential for proper critical period termination. During the critical period, changes in activity regulate dendrite length, complexity and connectivity of motor neurons. Astrocytes invaded the neuropil just before critical period closure, and astrocyte ablation prolonged the critical period. Finally, a genetic screen was used to identify astrocyte-motor neuron signalling pathways that close the critical period, including Neuroligin-Neurexin signalling. Reduced signalling destabilized dendritic microtubules, increased dendrite dynamicity and impaired locomotor behaviour, underscoring the importance of critical period closure. Previous work defined astroglia as regulators of plasticity at individual synapses. This study shows that astrocytes also regulate motor circuit critical period closure to ensure proper locomotor behaviour (Ackerman, 2021).

Critical periods are brief windows during which neural circuit activity can modify the morphological properties of neurons, producing permanent changes to circuit structure and function. Critical periods integrate multiple forms of plasticity to modify neural circuits. 'Homeostatic plasticity' encompasses changes to synapse number, structure and function across an entire neuron, as well as changes to long-range connectivity. Whereas homeostatic plasticity can occur in the adult brain, substantial activity-dependent remodelling peaks in early development. Indeed, failure to terminate critical period plasticity is linked to neurodevelopmental disorders such as autism and epilepsy. Although putative critical period disorders present with motor defects, the field has largely focused on sensory circuits. To that end, this study developed a novel critical period model in a developing motor circuit (Ackerman, 2021).

This study focused on two well-characterized Drosophila motor neurons, aCC and RP2, which are segmentally repeated in the central nervous system. These motor neurons are susceptible to activity-induced remodelling, although pioneering studies used chronic activity manipulations and did not define an end point for homeostatic plasticity. This study expressed the anion channelrhodopsin GtACR215 specifically in the aCC-RP2 motor neurons using the Gal4-upstream activation system (UAS) system and delivered acute 1-h windows of silencing, terminating at progressively later times in development. Silencing motor neurons for the last hour of embryogenesis (stage 17) increased aCC-RP2 dendritic volume at 0 h after larval hatching (ALH), whereas silencing for 1 h at later stages showed progressively less of an effect, with no remodelling occurring at 8 h ALH or beyond. By contrast, acute 1-h windows of activation using the channelrhodopsin Chrimson resulted in significant loss of motor neuron dendrites at 0 h ALH; activation at 8 h ALH and beyond had little or no effect. Activity-induced changes to dendrite length for single-cell RP2 clones [using the MultiColor FlpOut (MCFO) system] showed similar results Note that these experiments used far shorter periods of tonic activation than past studies. Although Tonic activity manipulations were primarily used, identical results were observed using 600 ms:400 ms pulses of activation or silencing, as well as thermogenetics to activate (via TrpA1) or silence [using the temperature-sensitive shibire gene (shibire^ts)] motor neurons. Notably, dendrite loss following acute activation could be rescued by a 22-h period of dark rearing, indicating that activity induces dendrite plasticity and not excitotoxicity. Together, these experiments define a critical period for activity-dependent motor dendrite plasticity represent the first analyses of motor circuit critical period closure within the central nervous system (Ackerman, 2021).

In vertebrates, homeostatic plasticity functions on a slow timescale, from hours to days. To determine the timescale for motor neuron dendrite expansion following GtACR2 silencing, aCC-RP2 motor neurons were silenced for 15 min, 1 h or 4 h in stage 17 embryos, terminating silencing at 0 h ALH. Larvae were then immediately dissected and dendritic morphology was assessed in single, well-spaced RP2 neurons using MCFO17. Increased dendritic arbor size and complexity following 1 h and 4 h of silencing were used. These results were confirmed using shibire^ts. By contrast, embryonic Chrimson activation resulted in decreased dendrite length and complexity at 0 h ALH after as little as 15 min of activation. Furthermore, using live imaging, significant dendrite retraction was observed within 12 min of Chrimson activation. The fact that silencing required more time to show an effect is not surprising, as extension requires generation of new membrane. It is concluded that activity-induced remodelling of Drosophila motor neurons occurs within minutes, much more quickly than previously documented for homeostatic plasticity in mammals (Ackerman, 2021).

This study showed that motor neurons scale dendrite length according to activity. An important question is whether these morphological changes are accompanied by changes in excitatory or inhibitory synaptic inputs. The excitatory cholinergic neuron A18b and inhibitory GABAergic neuron A23a were examined that are synaptically coupled to aCC-RP2 dendrites in a larval transmission electron microscopy (TEM) reconstruction. To quantify excitatory and inhibitory synapse number by light microscopy, a functionally inactive pre-synaptic marker, Bruchpilotshort::Cherry (Brp), was expressed in excitatory cholinergic neuron A18b or inhibitory GABAergic neuron A23a using the complementary LexA-LexAop binary expression system. A23a-inhibitory GABAergic synapses onto aCC-RP2 dendrites were examined, quantifying cell-type specific Brp puncta overlapping with aCC-RP2 dendritic membrane (putative synapses) using published standards. All critical period manipulations terminated at 4 h ALH (stage matched to the TEM data). It was found that 1 h of motor neuron silencing reduced the number of inhibitory synapses between A23a and aCC-RP2 dendrites. Silencing for a longer period (4 h) also yielded a significant increase in A18b excitatory synapses. Decreasing motor neuron activity thus leads to a compensatory reduction of inhibitory inputs and a corresponding increase in excitatory inputs to rebalance network activity. A18b excitatory cholinergic synapse numbers onto aCC-RP2 dendrites were quantified after activation or silencing. Motor neuron activation was found to significantly decreased numbers of A18b excitatory synapses onto aCC-RP2 dendrites following 1 h and 4 h manipulations. A significant increase in inhibitory synapse number following extended motor neuron activation was observed, possibly owing to insufficient dendritic membrane after activity-induced dendrite retraction. Increasing motor neuron activity thus leads to a compensatory reduction of excitatory pre-synaptic inputs. Finally, a functionally inactive reporter of excitatory post-synaptic densities (Drep2::GFP or Drep2::mStrawberry) was observed, specifically in aCC-RP2, and scaling of synapses was observed accross the entire dendritic arbor in response to altered activity—reduced excitatory post-synapses followed motor neuron activation, whereas increased excitatory post-synapses followed motor neuron silencing during the critical period. Of note, homeostatic scaling of motor neuron synapses did not occur after critical period closure. In sum, motor neurons scale excitatory and inhibitory inputs relative to their level of activity during the critical period (Ackerman, 2021).

The mechanisms that close critical periods remain poorly defined. Drosophila astrocytes infiltrate the neuropil at late embryogenesis and progressively envelop motor neuron synapses as the critical period closes. To test whether astrocytes promote critical period closure, all astrocytes were genetically ablated and optogenetics was used to assay for extension of critical period plasticity at 8 h ALH. Astrocyte elimination was confirmed by loss of the astrocyte marker Gat3. As expected, controls closed the critical period by 8 h ALH. By contrast, astrocyte ablation extended dendrite plasticity following Chrimson activation or GtACR2 silencing up to 8 h ALH. This effect was not observed at earlier stages, indicating that astrocytes do not constitutively dampen plasticity. Additionally, it was found that control motor dendrites were less dynamic after critical period closure, but that astrocyte ablation extends dendrite filopodial dynamicity. It is concluded that astrocytes are required for the transition from dynamic to stable filopodia and concurrent critical period closure (Ackerman, 2021).

To determine how astrocytes close the critical period, the astrocyte-specific alrm-gal4 was used to perform a targeted UAS RNA-mediated interference (RNAi) knockdown screen. Flies were assayed for critical period extension following 1 h of Chrimson activation from 7-8 h ALH. Four genes were identified that were required in astrocytes for timely critical period closure: gat (regulates excitatory-inhibitory balance), chpf [synthesizes chondroitin sulfate proteoglycans (CSPGs)] and the Neuroligins (Nlg) 4 and 2 (Ackerman, 2021).

Neuroligins are cell-adhesion proteins that are known to regulate astrocyte morphogenesis. In Drosophila, astrocyte-specific knockdown of nlg2 (the mouse orthologue is known as Nlgn1) had no effect on astrocyte volume or tiling, suggesting a more specific defect in astrocyte-motor neuron signalling. Knockdown of the remaining critical period regulators had variable effects on astrocyte morphology but all extended the critical period. Neuroligins bind cell adhesion proteins called Neurexins. RNAi against nrx-1 was used, which is known to bind both Nlg2 and Nlg4, specifically in aCC-RP2 motor neurons, and critical period extension was observed; this is consistent with astrocyte Nlg2 and motor neuron Nrx-1 acting in a common pathway to close the critical period. Motor neuron-specific RNAi knockdown of the CSPG receptor Lar also extended critical period plasticity. Notably, while Nrx-1 is often pre-synaptic, there is evidence for dendritic localization of these receptors. Furthermore, antibody staining for endogenous Nrx-1 and Nlg2 revealed localization of this receptor-ligand pair on motor dendrites and astrocytes, respectively. Finally, cell-type-specific overexpression of Nrx-1 and Nlg2 could induce precocious critical period closure (assayed by Chrimson activation from 3-4 h ALH). It is concluded that Nlg2-Nrx-1 ligand-receptor signalling between astrocytes and motor neurons is required for timely critical period closure (Ackerman, 2021).

How does Nlg2-Nrx-1 signalling close the critical period? The balance of excitatory to inhibitory synapses in neural circuits can instruct critical period timing. Additionally, numbers of excitatory synapses are decreased following astrocyte-specific knockout of neuroligins in mouse. This study observed no significant changes in excitatory-inhibitory balance following knockdown of nlg2 in astrocytes, suggesting that critical period closure is not dependent on Nlg2-mediated excitatory-inhibitory synapse balance (Ackerman, 2021).

Alternatively, Nrx-1 can promote microtubule stability in axons of motor neurons, suggesting a mechanism for critical period closure involving microtubule stabilization. To test this hypothesis, Chrimson::mVenus was used to activate and visualize aCC-RP2 dendrite membranes at 0 h ALH (peak critical period), and Cherry::Zeus to visualize stable microtubules during and after dendritic retraction. In live preparations, dendrites showed a reduction in Cherry::Zeus intensity immediately preceding activity-dependent retraction, suggesting that microtubule collapse in distal branches can induce dendrite retraction. In fixed preparations, this study found that proximal dendrites with the highest levels of stable microtubules were protected from activity-dependent retraction. Of note, overexpression of Nrx-1 was sufficient to increase both stable microtubules and stable dendrites at 4 h ALH. It is proposed that Nlg2 in astrocytes binds Nrx-1 in motor neurons to stabilize dendritic microtubules and close the critical period (Ackerman, 2021).

In mammals, inappropriate critical period extension has long-term effects on nervous system function. Indeed, this study observed persistent changes in motor neuron connectivity at least 24 h following acute motor neuron activation at the end of the critical period, which lead to an assay for long-term effects on behaviour. The critical period was transiently extended until 12 h ALH (4 h beyond control critical period closure), and then behaviour was assayed 1.5 days later. Control larvae showed persistent linear locomotion; by contrast, larvae with extended critical periods due to transient knockdown of motor neuron genes showed excessive turning, leading to abnormal spiralling behaviour. Similar but less severe effects were seen in larvae following knockdown of astrocyte genes. It is concluded that a modest extension of the critical period can, in some cases, lead to long-lasting alteration in locomotor behaviour (Ackerman, 2021).

Astrocytes regulate synaptogenesis, synaptic pruning and synaptic efficacy. Within critical periods, astrocyte signalling can tune neuronal plasticity, but its role in critical period closure was not known. This study identified astrocytes as promoting closure of a motor critical period, and defined a series of astrocyte-motor neuron signalling pathways required to close the critical period. Based on previous literature, it is hypothesized that astrocytes could modify critical period closure through regulation of excitatory-inhibitory balance or extracellular matrix composition. Consistent with mammalian studies, it was found that perturbing excitatory-inhibitory balance through astrocyte-specific RNAi of the sole GABA transporter gat was sufficient to extend critical period plasticity. Furthermore, it was found that decreasing signalling from inhibitory extracellular matrix CSPGs through RNAi knockdown of Chondroitin polymerizing factor (Chpf) in astrocytes extended critical period plasticity. Thus, astrocytes use similar strategies in Drosophila and mammals to regulate critical period timing. Unexpectedly, this study also identified astrocyte-derived Neuroligins and their neuronal partner Nrx-1 as instrumental for critical period closure. In sum, this study have identified a key role of astrocytes in closure of a motor critical period required for locomotor function (Ackerman, 2021).

Drosophila Syd-1, liprin-α, and protein phosphatase 2A B' subunit Wrd function in a linear pathway to prevent ectopic accumulation of synaptic materials in distal axons

During synaptic development, presynaptic differentiation occurs as an intrinsic property of axons to form specialized areas of plasma membrane [active zones (AZs)] that regulate exocytosis and endocytosis of synaptic vesicles. Genetic and biochemical studies in vertebrate and invertebrate model systems have identified a number of proteins involved in AZ assembly. However, elucidating the molecular events of AZ assembly in a spatiotemporal manner remains a challenge. Syd-1 (synapse defective-1 or Rho GTPase activating protein at 100F) and Liprin-α have been identified as two master organizers of AZ assembly. Genetic and imaging analyses in invertebrates show that Syd-1 works upstream of Liprin-α in synaptic assembly through undefined mechanisms. To understand molecular pathways downstream of Liprin-α, a proteomic screen was performed of Liprin-α-interacting proteins in Drosophila brains. Drosophila protein phosphatase 2A (PP2A; see MTS, the PP2A catalytic subunit) regulatory subunit B' [Wrd (Well Rounded) or PP2A-B'] was identified as a Liprin-α-interacting protein, and it was demonstrated that it mediates the interaction of Liprin-α with PP2A holoenzyme and the Liprin-α-dependent synaptic localization of PP2A. Interestingly, loss of function in syd-1, liprin-α, or wrd shares a common defect in which a portion of synaptic vesicles, dense-core vesicles, and presynaptic cytomatrix proteins ectopically accumulate at the distal, but not proximal, region of motoneuron axons. Strong genetic data show that a linear syd-1/liprin-α/wrd pathway in the motoneuron antagonizes glycogen synthase kinase-3β kinase activity to prevent the ectopic accumulation of synaptic materials. Furthermore, data is provided suggesting that the syd-1/liprin-α/wrd pathway stabilizes AZ specification at the nerve terminal and that such a novel function is independent of the roles of syd-1/liprin-α in regulating the morphology of the T-bar structural protein BRP (Bruchpilot) (Li, 2014).

During presynaptic development, small synaptic vesicle (SV) precursors, dense-core vesicles (DCVs), and synaptic cytomatrix proteins are generated in the soma, transported along the axon, and eventually incorporated into the nerve terminal. Within the nerve terminal, active zones (AZs) are specialized areas of plasma membrane containing a group of evolutionarily conserved proteins, including ELKS (glutamine, leucine, lysine, and serine-rich protein)[also called CAST (cytomatrix at the active zone-associated structural protein), Drosophila homologue is BRP (Bruchpilot)], Munc13 (mammalian uncoordinated homology 13), RIM (Rab3-interacting molecule), Syd-1 (synapse defective-1), and Liprin-α, in which the releasable pool of vesicles dock and are released on stimulation. Despite intensive studies of the proteins localized at the presynaptic density, the assembly and maintenance of AZs remains enigmatic. Studies conducted in invertebrate model organisms suggested that Syd-1, a putative RhoGAP, and Liprin-α are two master organizers of presynaptic differentiation. Genetic analyses in Caenorhabditis elegans demonstrated that Syd-1 works upstream of Liprin-α in synaptic assembly. Studies in Drosophila further confirmed this hierarchy by showing that Syd-1 regulates and retains proper localization of Liprin-α at the AZ. However, studies also found that Syd-1 regulates Liprin-α-independent processes, such as retention of Neurexin at the presynaptic side and glutamate receptor incorporation at the postsynaptic side. The morphology of the AZ is distinctly different in liprin-α and syd-1 mutants. Therefore, it is unclear how Syd-1- and Liprin-α-mediated signaling collaborate to achieve the complex regulation of presynaptic differentiation. Identifying novel Liprin-α-interacting proteins at the synapse holds the key to delineating the regulatory network mediated by these two genes (Li, 2014).

This study identified protein phosphatase 2A (PP2A) as one prominent Liprin-α-interacting protein complex through an in vivo tandem affinity purification (TAP) approach. PP2A is an abundant heterotrimeric serine/threonine phosphatase that regulates a broad range of cellular processes. PP2A is highly enriched in neurons and is implicated in Tau-mediated neurodegeneration, regulation of long-term potentiation, and presynaptic and postsynaptic apposition. The diverse functions of PP2A are attributed primarily to its many interchangeable regulatory subunits (B, B', B'', or B'''), each showing specific spatial and temporal expression patterns. The Liprin-α-interacting PP2A holoenzyme that this study identified in the fly brain contains the B' regulatory subunit [also called Wrd (Well Rounded) in fly]. Wrd is highly expressed in synapses and regulates synaptic terminal growth at the Drosophila neuromuscular junction (NMJ). Interestingly, the Liprin-α-Wrd physical interaction may be evolutionarily conserved because PP2A B56γ, the human homolog of Wrd, can bind Liprin-α1 in HEK 293 cell. However, the function of the Liprin-α-Wrd/PP2A B56γ interaction in the nervous system is unexplored (Li, 2014).

This study shows that Syd-1, Liprin-α, and Wrd work in a linear pathway to restrain the localization of vesicles and presynaptic cytomatrix proteins at the nerve terminal. Disruption of such a pathway results in ectopic accumulation of SVs and presynaptic proteins at the distal, but not proximal, end of axons (Li, 2014).

Much progress toward understanding presynaptic differentiation has been made through unbiased forward genetic screens in invertebrates. These studies have led to the identification of several key factors for AZ formation, including two evolutionarily conserved master organizer proteins of AZ assembly: syd-1 and syd-2/liprin-α. However, how Syd-1/Liprin-α organize presynaptic sites remains unclear. This study identified a new synaptic player, the PP2A B′ regulatory subunit, that is localized to the synapse by Liprin-α and mediates Syd-1/Liprin-α signaling in stabilizing AZs and their associated vesicles at the nerve terminal (Li, 2014).

Liprin-α was first identified as a protein interacting with the LAR (leukocyte antigen-related-like) family of phosphatases. Studies during the past two decades demonstrate that Liprin-α regulates presynaptic and postsynaptic development, as well as neurotransmitter release through protein–protein interactions with a range of molecules, including CAST/ELKS/BRP, RIM, CASK (calcium/calmodulin-dependent serine protein kinase), GIT (G-protein-coupled receptor kinase-interacting ArfGAP), GRIP (glutamate receptor interacting protein), LAR, CaMKII, and Liprin-β. Proteomic data confirmed the interaction between Liprin-α and BRP/RIM in Drosophila. Another important Liprin-α binding partner was identified at the presynaptic sites, the B′ regulatory subunit of PP2A (Wrd), which depends on Liprin-α for it proper synaptic localization (Li, 2014).

Phenotypic analysis of syd-1, liprin-α, and wrd mutants demonstrate that they share a unique trafficking defect, in which SVs, DCVs, presynaptic scaffolding proteins, and voltage-gated Ca²⁺ channels ectopically accumulate at the distal, but not the proximal, region of the axon. Genetic rescue experiments define a linear pathway, from syd-1 to liprin-α to wrd, that works cell autonomously in the presynaptic neuron to ensure proper localization of presynaptic materials to the nerve terminal and prevents ectopic accumulation. Together, these biochemical and genetic data suggest that Wrd mediates a novel Syd-1/Liprin-α function at the presynaptic site. Such a Syd-1/Liprin-α function is likely independent of their well established roles in regulating the T-bar structure protein BRP/ELKS (Li, 2014).

Two lines of evidence suggest that a Wrd-containing PP2A mediates the function of Syd-1/Liprin-α in regulating AZ stability. First, two rounds of in vivo biochemical purification using either Liprin-α or Wrd as the bait copurified Liprin-α with Wrd and the other two core subunits of PP2A, indicating the presence of a Liprin-α/Wrd/PP2A protein complex in neurons. Second, loss of GSK-3β kinase [sgg (shaggy)] function suppresses the syd-1, liprin-α, and wrd mutant distal axon phenotype, suggesting that a Wrd/PP2A-mediated phosphatase activity normally functions to antagonize a GSK-3β kinase activity in neurons to stabilize AZ and clustering of SVs at the nerve terminal (Li, 2014).

What is the primary cause for the unique distal axon phenotype in syd-1/liprin-α/wrd mutant larvae? Liprin-α was shown to interact with KIF1A (kinesin family member 1A)/Unc-104, a neuron-specific kinesin motor known to transport SV precursors containing synaptophysin, Syt, and Rab1A. It was reported that Drosophila Liprin-α regulates the trafficking of SVs through its interaction with Kinesin-1 and that liprin-α mutant peripheral nerves show accumulation of clear-core vesicles similar to kinesin heavy chain (khc) mutants. However, when this study focused on the location of the phenotypes relative to the entire axonal length, liprin-α mutant accumulation of clear-core vesicles was found to be present exclusively in the distal end (the ventrolateral peripheral nerve bundles, as well as axonal regions proximal to NMJs), whereas khc mutant larvae show massive aggregation of SV-associated proteins in the proximal end (segmental nerve bundles), and very few SV precursors reach the distal of axon. The distribution pattern of the vesicle accumulation in syd-1 and wrd mutants is the same as liprin-α mutants. Such a pattern is distinct from that of typical trafficking defects induced by mutations in vesicle-transporting motors or cargos (Li, 2014).

Although a unique vesicle trafficking defect as the primary cause for the syd-1/liprin-α/wrd mutant axonal phenotype cannot be completely excluded, a number of lines of evidence suggest a plausible explanation: AZ materials at the nerve terminal become destabilized when the syd-1/liprin-α/wrd pathway is impaired, and the floating AZ materials diffuse back to the adjacent axonal regions as ectopic docking sites for vesicles. First, Syd-1, Liprin-α, and Wrd show clear synaptic localization, with little or no axonal localization detected, consistent with a collaborative function of the three at the AZs. Second, EM analysis detected floating AZ materials in the synaptic boutons and the connected axonal regions in syd-1 mutants. Some of the floating materials are very close to or touching the bouton plasma membrane, indicating a possible defect in AZ stabilization and subsequent back-diffusion of detached AZ materials to axonal regions. Third, AZ components such as BRP, RIM, and voltage-gated Ca²⁺ channels are identified in the mutant distal axons along with vesicles, including SVs and DCVs, but not vesicles that transport AZ scaffolding proteins, or other synaptically localized organelles, or transport machineries. This is consistent with an ectopic accumulation of vesicles attracted by ectopic floating AZ components. Fourth, live imaging analysis found that anterogradely transported DCVs accumulate at preferred spots at the mutant distal axons, consistent with the existence of static docking sites at these axonal regions. Fifth, ectopically accumulated vesicles do not participate in release or recycling, consistent with the notion that the vesicles do not dock on the axonal plasma membrane (Li, 2014).

The fact that knockdown of a kinase (GSK-3β) rescues the distal axonal defects of syd-1/liprin-α/wrd mutants indirectly suggests that a Wrd-dependent dephosphorylation event is antagonized by a phosphorylation event (mediated by GSK-3β) to regulate AZ stability. However, these data cannot exclude the possibility that PP2A-independent functions of Wrd are involved. One way to seek direct evidence that Wrd-containing PP2A is involved in regulating AZ stability is to study the loss of function of PP2A; however, this approach has its own set of complications. As a ubiquitous heterotrimetric enzyme, the substrate specificity and subcellular localization of PP2A are greatly dependent on its regulatory subunit (such as Wrd). Mutating the catalytic or structural domain blocks overall PP2A action mediated by all regulatory subunits, which precludes analysis of Wrd-specific PP2A action. For example, mutations in MTS (the PP2A catalytic subunit) cause early lethality. Overexpression of a dominant MTS protein causes massive axonal transport defects in the entire axon, as well as defects in AZ development. Therefore, identifying common substrates shared by Wrd/PP2A and GSK-3β and studying how their phosphorylation status regulates AZ stability and/or vesicle trafficking will ultimately unravel the mechanism by which a PP2A-dependent pathway regulates presynaptic development. In this context, this study set up a model to study how synapse scaffolding proteins can regulate localized phosphorylation/dephosphorylation through recruitment of specific phosphatases or kinases (Li, 2014).

A mammalian homolog of Syd-1 was identified recently as an important regulator of presynaptic differentiation at central synapses, at least partially through its interaction with mammalian Liprin-α2. Given that Liprin-α1 interacts with PP2A B56γ (mammalian homolog of Wrd) in HEK 293 cells, it will be of interest to investigate whether the function of Drosophila Liprin-α in mediating the signaling from Syd-1 to the PP2A B′ subunit is also evolutionarily conserved during vertebrate synapse development (Li, 2014).

Postsynaptic actin regulates active zone spacing and glutamate receptor apposition at the Drosophila neuromuscular junction

Synaptic communication requires precise alignment of presynaptic active zones with postsynaptic receptors to enable rapid and efficient neurotransmitter release. How transsynaptic signaling between connected partners organizes this synaptic apparatus is poorly understood. To further define the mechanisms that mediate synapse assembly, a chemical mutagenesis screen was carried out in Drosophila to identify mutants defective in the alignment of active zones with postsynaptic glutamate receptor fields at the larval neuromuscular junction. From this screen a mutation was identified in Actin 57B that disrupted synaptic morphology and presynaptic active zone organization. Actin 57B, one of six actin genes in Drosophila, is expressed within the postsynaptic bodywall musculature. The isolated allele, act^E84K, harbors a point mutation in a highly conserved glutamate residue in subdomain 1 that binds members of the Calponin Homology protein family, including spectrin. Homozygous act^E84K mutants show impaired alignment and spacing of presynaptic active zones, as well as defects in apposition of active zones to postsynaptic glutamate receptor fields. act^E84K mutants have disrupted postsynaptic actin networks surrounding presynaptic boutons, with the formation of aberrant actin swirls previously observed following disruption of postsynaptic spectrin. Consistent with a disruption of the postsynaptic actin cytoskeleton, spectrin, adducin and the PSD-95 homolog Discs-Large are all mislocalized in act^E84K mutants. Genetic interactions between act^E84K and neurexin mutants suggest that the postsynaptic actin cytoskeleton may function together with the Neurexin-Neuroligin transsynaptic signaling complex to mediate normal synapse development and presynaptic active zone organization (Blunk, 2014).

Genetic interaction between Neurexin and CAKI/CMG is important for synaptic function in Drosophila neuromuscular junction

Neurexins are neuron-specific cell surface molecules thought to localize to presynaptic membranes. Recent genetic studies using Drosophila have implicated an essential role for the single Drosophila Neurexin in the proper architecture, development and function of synapses in vivo. However, the precise mechanisms underlying these actions are not fully understood. To elucidate the molecular mechanism of Neurexin in vivo, dnrx and caki mutant flies, combined with various methods, were used to analyze locomotion, synaptic vesicle cycling and neurotransmission of neuromuscular junctions. Dneurexin (DNRX) was found to be important for locomotion through a genetic interaction with the scaffold protein, CAKI/CMG, the Drosophila homolog of vertebrate CASK. Similar to its mammalian counterparts, DNRX is essential for synaptic vesicle cycling, which plays critical roles in neurotransmission at neuromuscular junctions (NMJ). However, this interaction appears not to be required for the synaptic targeting of DNRX, but may instead be needed for proper synaptic function, possibly by regulating the synaptic vesicle cycling process (Sun, 2009).

It is generally accepted that cell adhesion molecules are major players in synapse development and plasticity. In particular, Neurexin and its postsynaptic binding partners, the Neuroligins, have drawn the most attention since gene mutations in these two molecules have been linked to brain disorders, including autism and mental retardation. In recent studies using the Drosophila model, it has been shown that the single gene product DNRX plays important roles in both synapse development and function. Knocking out Neurexin basically results in a fly with defective nervous system. In dnrx mutants, the cytoarchitecture and function of synapses are severely disrupted. First, dnrx mutants have trouble moving around. Second, dnrx mutants display shortened axon branches with fewer boutons. Third, critical components of the presynaptic components, such as synaptotagmin and active zone components, such as BRP are ectopically localized within axons. Fourth, on the ultrastructural level, dnrx mutants exhibit defective active zones with larger PRD (presynaptic densities) and an increased number of T bars. Fifth, in dnrx mutants, structural abnormalities are accompanied by corresponding functional deficits, such as increased amplitude and frequency of mEJP and decreased amplitude of EJP. However, the molecular mechanisms by which DNRX acts remain unknown. Studies on DNRX binding partners should provide additional insights into the mechanisms by which Neurexins function in synapse development and function (Sun, 2009).

One important issue to be addressed for Neurexins is how they are properly targeted to the synaptic regions. Experiments in vitro indicated that synaptic targeting of Neurexins was regulated by their C-terminal sequences (Fairless, 2008). However, this model has not yet been examined in in vivo systems. One clue to the answer to this question comes from the finding that Neurexins interact with CASK, a membrane associated PDZ domain-containing protein, in a sequence-specific manner (Sun, 2009).

This study took advantage of various Drosophila mutants to investigate the in vivo function and underlying mechanisms of DNRX and CAKI. Using immunostaining, it was shown that DNRX and CAKI are partially co-localized in both larval brain and in NMJs. Furthermore, these two proteins interact with each other in vivo in both co-immunoprecipitation and yeast two-hybrid assays. Surprisingly, the interaction between DNRX and CAKI appeared not to be required for the synaptic targeting of either protein, because they were targeted to the synaptic region correctly in respective mutant flies. In addition, the results showed that the intracellular domain of DNRX, even in the absence of the PDZ binding motif, was localized to the synaptic region, suggesting that DNRX alone (or requiring proteins other than CAKI) is sufficient for the synaptic targeting of DNRX. These results are inconsistent with the model (Fairless, 2008) that has been proposed previously (Sun, 2009).

Drosophila CAKI, a homolog of human CASK, has been shown, by means of electrophysiological methods, to be essential for the regulation of neurotransmitter vesicle release. The current study found that CAKI loss-of-function leads to increased synaptic bouton number at larval NMJs, which indicates that CAKI regulates bouton number at the neuromuscular junction negatively. However, most of the boutons seem smaller and unmatured. In contrast, the current results show that the dnrx mutant displays a reduced bouton number at larval NMJs. These results suggest that DNRX and CAKI regulate synaptic bouton development through different pathways. Nevertheless, the interaction between DNRX and CAKI appears to be important in the regulation of behavioral responses and synaptic vesicle cycling because the double heterozygous mutant for both genes showed a more severe deficit than the single heterozygous mutant. These results are consistent with those obtained from the electrophysiological analyses, which show that the EJP amplitude was decreased more severely in flies heterozygous for mutations in both genes than that of the single heterozygous mutants and suggest that the deficit in the synaptic vesicle cycling may underlie the changes in the EJP amplitude in the mutant flies. The functional significance of the genetic interaction between DNRX and CAKI is also evident in that the double heterozygous mutant for both dnrx and caki showed more dramatic changes in both mEJP frequency and amplitude compared to the single heterozygotes. The reason for the altered mEJP amplitude is unknown but may be related to the glutamate receptors at the postsynaptic site. More studies will be needed to elucidate the underlying mechanisms (Sun, 2009).

Since cell adhesion molecules play critical roles in synaptogenesis, synapse maintenance and synaptic vesicle release, it will be important to further investigate the molecular processes that are responsible for the synaptic targeting and functional regulation of these proteins. The Drosophila mutants that this study has generated will provide a unique tool in these studies (Sun, 2009).

Drosophila neuroligin 1 promotes growth and postsynaptic differentiation at glutamatergic neuromuscular junctions

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). 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. 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. Conversely, knockdown of Nlgs by RNA interference (RNAi) leads to a reduction of synapse numbers, 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. 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. 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, 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. 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-GFP^Δcyto) displayed a drastic dominant-negative phenotype. Because DNlg1-GFP^Δcyto 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. At vertebrate excitatory synapses, interactions similar to postsynaptic scaffolding proteins such as PSD-95 support Nlg function. The fact that DNlg1-GFP^Δextra (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. 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).

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).

Finally, there were differences in the interaction between the two neuroligin homologs and neurexin (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).

Neurexin regulates visual function via mediating retinoid transport to promote rhodopsin maturation

Neurexins are cell adhesion molecules involved in synapse formation and synaptic regulation. Mutations in the neurexin genes are linked to a number of neurodevelopmental disorders such as autism. This study shows that the Drosophila homolog of alpha-Neurexin is critical for fly visual function. Lack of Neurexin leads to significantly impaired visual function due to reduced rhodopsin levels. The decreased chromophore levels cause deficits in rhodopsin maturation, and Neurexin is required for retinoid transport. Using yeast two-hybrid screening, it was determined that Neurexin interacts with apolipoprotein I (ApoL I), a product generated by cleavage of retinoid- and fatty acid-binding glycoprotein (RFABG) that functions in retinoid transport. Finally, it was demonstrated that Neurexin is essential for the apolipoproteins level. These results reveal a role for Neurexin in mediating retinoid transport and subsequent rhodopsin maturation and suggest that Neurexin regulates lipoprotein function (Tian, 2013).

This study shows that Neurexin mediates Rh1 maturation through regulating retinoid transport, which is essential for rhodoposin maturation. It was further demonstrated that the intracellular region of Neurexin interacts with ApoL I and is required for the stability of ApoL I and II, key proteins that function in transporting retinoids in the retina. The results reveal a role for Neurexin in mediating retinoid transport and subsequent rhodopsin maturation and suggest that Neurexin regulates lipoprotein function (Tian, 2013).

Membrane receptors are responsible for translating extracellular stimuli into intracellular responses. The successful intracellular transport of rhodopsin to light sensory organelles is essential for photoreceptor function and survival, as defects in rhodopsin transport lead to severe retinal degeneration. Several proteins play a role in Rh1 maturation, and defects in a number of steps in the biosynthetic pathway may affect Rh1 production. The present study shows that Drosophila Neurexin is required for Rh1 maturation. Loss of Neurexin leads to reduced Rh1 levels and impaired visual function. Eye-specific expression of Neurexin rescues the impaired Rh1 level and visual function in the mutant. This study provides the compelling evidence that the cell adhesion molecule is required for rhodopsin maturation and function (Tian, 2013).

Previous studies have shown that Neurexin-1α is expressed in both embryonic chick retina and embryonic mice retina. In this work shows that Drosophila Neurexin is localized in the rhabdomeres in photoreceptors and photoreceptor-specific expression of Neurexin is able to rescue the impaired Rh1 level in the mutant. These results reveal that photoreceptor-derived Neurexin is essential for Rh1 maturation. The canonical binding partners of Neurexins, Neuroligins, are thought to be important for establishing the asymmetry of the synapse. However, unlike with Neurexin, loss of Neuroligin did not alter Rh1 levels. Taken together, these results suggest that Neurexin is probably activating via a Neuroligin and synapse-independent manner to regulated Rh1 maturation in the fly eye (Tian, 2013).

Drosophila is a good model system for genetic and molecular studies of vitamin A metabolism, because vitamin A is not required for fly viability but is critical for the generation of chromophores and for the synthesis of visual pigments. Several mutants affecting vitamin A production have been identified by prolonged depolarization afterpotential (PDA) screening. Using HPLC analysis, this study has shown that the chromophore levels are dramatically decreased in nrx^Δ83 mutants. This finding represents the evidence that Neurexin is linked to retinoid transport and subsequent rhodopsin maturation (Tian, 2013).

In carotenoid-deprived mutants of Drosophila, defective chromophore production observed outside the retina can be rescued by supplying vitamin A in food. However, we are unsuccessful in restoring Rh1 levels in nrx^Δ83 mutants by supplying all-trans retinal in food. In contrast, it was possible to restore Rh1 levels by expressing Neurexin or RFABG in the photoreceptors. These results further support the conclusion that Neurexin functions inside the retina to facilitate chromophore generation or transport (Tian, 2013).

Neurexins are single-pass transmembrane proteins and the intracellular domain of Neurexin interacts with a number of exocytotic proteins, such as Velis, Munc18, and CASK (Biederer, 2000; Butz, 1998; Mukherjee, 2008). This study reveals that expression of the intracellular region of Neurexin is sufficient to restore the Rh1 level. In a yeast two-hybrid screen, two overlapping cDNAs were isolated of ApoL I binding with the intracellular domains of Neurexin. It was further shown that the ApoL protein levels are reduced in nrx^Δ83 mutant retina and overexpression of Neurexin is able to restore ApoL protein levels in the mutant eye. It has been reported that IRBP undergoes rapid turnover (half-life, 10.7hr) in the Xenopus interphotoreceptor matrix. The current results provide evidence that the intracellular region of Neurexin plays an important role in stabilizing ApoL proteins (Tian, 2013).

Drosophila RFABG is thought to be the functional homolog of vertebrate IRBP, and lack of IRBP causes delayed transfer of newly synthesized chromophores from the RPE to photoreceptors in mice (Jin, 2009). This phenotype resembles that of nrx^Δ83 mutant flies with gradual increase in the Rh1 level after eclosion. Drosophila lipophorins have been shown to play an important role in the transport of lipid-linked morphogens and glycophosphatidylinositol-linked proteins. This study has shown that ApoL protein levels are reduced in nrx^Δ83 mutant retina and Rh1 levels are restored upon overexpression of RFABG in the mutant eye. Sustained overexpression of RFABG might compensate the reduced stability of ApoL proteins in the mutant eye. These observations are consistent with the Neurexin rescue experiments, which show expression of Neurexin in photoreceptors is sufficient for restoring Rh1 level in the mutant. This study reveals the linker between and Neurexin and retinoid transport (Tian, 2013).

Nutritional and environmental factors play important roles in ASD, and fatty acid metabolism and abnormal membrane fatty acid composition may contribute to this disorder. It has been reported that apolipoproteins, especially Apo B-100, are reduced in children with AS. Drosophila RFABG show high similarity in its domain structure with vertebrate Apo B-100. This study shows that the region aa 1,390-1,480 of RFABG is sufficient for the interaction with Neurexin. The sequence aa 1,390-1,480 is lysine enriched (13 out of 90 residues). These highly charged residues could be important in mediating the Neurexin/ApoL interaction. In addition, this region is conserved between Drosophila RFABG and vertebrate IRBP and Apo B-100 (20% identity, data not shown), implying that the Neurexin/ApoL I interaction may be conserved among various species. This revealed interaction and function correlation between Neurexin and lipoproteins have put a step forward in the understanding of pathological relations of Neurexin mutations and perturbed fatty acid metabolism in ASD patients (Tian, 2013).

Drosophila Syncrip modulates the expression of mRNAs encoding key synaptic proteins required for morphology at the neuromuscular junction

Localized mRNA translation is thought to play a key role in synaptic plasticity, but the identity of the transcripts and the molecular mechanism underlying their function are still poorly understood. This study shows that Syncrip, a regulator of localized translation in the Drosophila oocyte and a component of mammalian neuronal mRNA granules, is also expressed in the Drosophila larval neuromuscular junction, where it regulates synaptic growth. RNA-immunoprecipitation followed by high-throughput sequencing and qRT-PCR were used to show that Syncrip associates with a number of mRNAs encoding proteins with key synaptic functions, including msp-300, syd-1 (RhoGAP100F), neurexin-1, futsch, highwire, discs large, and alpha-spectrin. The protein levels of MSP-300, Discs large, and a number of others are significantly affected in syncrip null mutants. Furthermore, syncrip mutants show a reduction in MSP-300 protein levels and defects in muscle nuclear distribution characteristic of msp-300 mutants. These results highlight a number of potential new players in localized translation during synaptic plasticity in the neuromuscular junction. It is proposed that Syncrip acts as a modulator of synaptic plasticity by regulating the translation of these key mRNAs encoding synaptic scaffolding proteins and other important components involved in synaptic growth and function (McDermott, 2014).

Localized translation is a widespread and evolutionarily ancient strategy used to temporally and spatially restrict specific proteins to their site of function and has been extensively studied during early development and in polarized cells in a variety of model systems. It is thought to be of particular importance in the regulation of neuronal development and in the plastic changes at neuronal synapses that underlie memory and learning, allowing rapid local changes in gene expression to occur independently of new transcriptional programs. The Drosophila neuromuscular junction (NMJ) is an excellent model system for studying the general molecular principles of the regulation of synaptic development and plasticity. Genetic or activity-based manipulations of synaptic translation at the NMJ has previously been shown to affect the morphological and electrophysiological plasticity of NMJ synapses. However, neither the mRNA targets nor the molecular mechanism by which such translational regulation occurs are fully understood (McDermott, 2014).

Previously work identified CG17838, the fly homolog of the mammalian RNA binding protein SYNCRIP/hnRNPQ, which was named Syncrip (Syp). Mammalian SYNCRIP/hnRNPQ is a component of neuronal RNA transport granules that contain CamKIIα, Arc, and IP3R1 mRNAs and is thought to regulate translation via an interaction with the noncoding RNA BC200/BC1, itself a translational repressor. Moreover, SYNCRIP/hnRNPQ competes with poly(A) binding proteins to inhibit translation in vitro and regulates dendritic morphology (Chen, 2012) via association with, and localization of, mRNAs encoding components of the Cdc-42/N-WASP/Arp2/3 actin nucleation-promoting complex. Drosophila Syp has a domain structure similar to its mammalian homolog, containing RRM RNA binding domains and nuclear localization signal(s), as well as encoding a number of protein isoforms. It was previously shown that Syp binds specifically to the gurken (grk) mRNA localization signal together with a number of factors previously shown to be required for grk mRNA localization and translational regulation (McDermott, 2012). Furthermore, syp loss-of-function alleles lead to patterning defects indicating that syp is required for grk and oskar (osk) mRNA localization and translational regulation in the Drosophila oocyte (McDermott, 2014).

This study shows that Syp is detected in the Drosophila third instar larval muscle nuclei and also postsynaptically at the NMJ. Syp is required for proper synaptic morphology at the NMJ, as syp loss-of-function mutants show a synaptic overgrowth phenotype, while overexpression of Syp in the muscle can suppress NMJ growth. Syp protein associates with a number of mRNAs encoding proteins with key roles in synaptic growth and function including, msp-300, syd-1, neurexin-1 (nrx-1), futsch, highwire (hiw), discs large 1 (dlg1), and α-spectrin (α-spec). The protein levels of a number of these mRNA targets, including msp-300 and dlg1, are significantly affected in syp null mutants. Furthermore, in addition to regulating MSP-300 protein levels, Syp is required for correct MSP-300 protein localization, and syp null mutants have defects in myonuclear distribution and morphology that resemble those observed in msp-300 mutants. It is proposed that Syp coordinates the protein levels from a number of transcripts with key roles in synaptic growth and is a mediator of synaptic morphology and growth at the Drosophila NMJ (McDermott, 2014).

The results demonstrate that Syp is required for the appropriate branching of the motoneurons and the number of synapses they make at the muscle. These observations are potentially explained by the finding that Syp is also required for the correct level of expression of msp-300, dlg1 and other mRNA targets. Given that it was previously shown that Syp regulates mRNA localization and localized translation in the Drosophila oocyte, and studies by others have shown that mammalian SYNCRIP/hnRNPQ inhibits translation initiation by competitively binding poly(A) sequences (Svitkin, 2013), these functions of Syp as occurring at the level of translational regulation of the mRNAs to which Syp binds. Our data are also consistent with other work in mammals showing that SYNCRIP/hnRNPQ is a component of neuronal RNA transport granulesthat can regulate dendritic morphology via the localized expression of mRNAs encoding components of the Cdc-42/N-WASP/Arp2/3 actin nucleation-promoting complex (McDermott, 2014 and references therein).

Translation at the Drosophila NMJ is thought to provide a mechanism for the rapid assembly of synaptic components and synaptic growth during larval development, in response to rapid increases in the surface area of body wall muscles or in response to changes in larval locomotion. The phenotypes observed in this study resemble, and are comparable to, those seen when subsynaptic translation is altered genetically or by increased locomotor activity. In syp null mutants, NMJ synaptic terminals are overgrown, containing more branches and synaptic boutons. Similarly, bouton numbers are increased by knocking down Syp in the muscle using RNAi. In contrast, overexpression of Syp in the muscle has the opposite phenotype, resulting in an inhibition of synaptic growth and branching. Furthermore, expressing RNAi against syp in motoneurons alone does not result in a change in NMJ morphology, indicating that Syp acts postsynaptically in muscle, but not presynaptically at the NMJ to regulate morphology. Interestingly, pan-neuronal syp knockdown or overexpression using Elav-GAL4 also results in NMJ growth defects, revealing that some of the defects observed in the syp null mutant may be attributed to Syp function in neuronal cell types other than the motoneurons, such as glial cells, which are known to influence NMJ morphology. Finally, while Syp is not required in the motoneuron to regulate synapse growth and is not detected in the motoneuron, the possibility cannot be excluded that Syp is present at low levels in the presynapse and regulates processes independent of synapse morphology. A further detailed characterization of the cell types and developmental stages in which Syp is expressed and functions is required to better understand the complex phenotypes that were observe (McDermott, 2014).

RNA binding proteins have emerged as critical regulators of both neuronal morphology and synaptic transmision, suggesting that protein production modulates synapse efficacy. Consistent with this, it has been shown in a parallel study that Syp is required for proper synaptic transmission and vesicle release and regulates the presynapse through expression of retrograde Bone Morphogenesis Protein (BMP) signals in the postsynapse. In this role, Syp may coordinate postsynaptic translation with presynaptic neurotransmitter release. These observations provide a good explanation for how Syp influences the presynapse despite being only detectable in the postsynapse. This study has shown that Syp associates with a large number of mRNAs within third instar larvae, many of which encode proteins with key roles in synaptic growth and function. Syp mRNA targets include msp-300, syd-1, nrx-1, futsch, hiw, dlg1, and α-spec. Syp negatively regulates Syd-1, Hiw, and DLG protein levels in the larval body wall but positively regulates MSP-300 and Nrx-1 protein levels. Dysregulation of these multiple mRNA targets likely accounts for the phenotypes that were observed. Postsynaptically expressed targets with key synaptic roles that could explain the synaptic phenotypes that were observed in syp alleles include MSP-300, α-Spec, and DLG. For example, mutants in dlg1 and mutants where postsynaptic DLG is destabilized or delocalized have NMJ morphology phenotypes similar to those observed upon overexpression of Syp in the muscle. Presynaptically expressed targets include syd-1, nrx-1, and hiw. However, this study has shown that syp knockdown in presynaptic motoneurons does not result in any defects in NMJ morphology. The RIP-Seq experiments were carried out using whole larvae and will, therefore, identify Syp targets in a range of different tissues and cells, the regulation of which may or may not contribute to the phenotype that were observed in syp mutants. It is, therefore, possible that Syp associates with these presynaptic targets in other neuronal cell types such as the DA neurons of the larval peripheral nervous system. It is also possible that Nrx-1 or Hiw are expressed and required postsynaptically in the muscle, but this has not been definitively determined. syp alleles may provide useful tools to examine where key synaptic genes are expressed and how they are regulated (McDermott, 2014).

The identity of localized mRNAs and the mechanism of localized translation at the NMJ are major outstanding questions in the field. To date, studies have shown that GluRIIA mRNA aggregates are distributed throughout the muscle. The Syp targets identified in this study, such as msp-300, hiw, nrx-1, α-spec, and dlg1, are now excellent candidates for localized expression at the NMJ. Ultimately, conclusive demonstration of localized translation will involve the visualization of new protein synthesis of targets during activity-dependent synaptic plasticity. Biochemical experiments will also be required to establish the precise mode of binding of Syp to its downstream mRNA targets, the basis for interaction specificity, and the molecular mechanism by which Syp differentially regulates the protein levels of its mRNA targets at the Drosophila NMJ. Despite the fact that mammalian SYNCRIP is known to associate with poly(A) tails, this study and other published work have revealed that Syp can associate with specific transcripts. How Syp associates with specific mRNAs is unknown, and future studies are needed to uncover whether the interaction of Syp with specific transcripts is dictated by direct binding of the three Syp RRM RNA binding domains or by binding to other specific mRNA binding proteins. It is also possible that specific mRNA stem–loops, similar to the gurken localization signal, are required for Syp to bind to its mRNA targets (McDermott, 2014).

This study shows that msp-300 is the most significant mRNA target of Syp. MSP-300 is the Drosophila ortholog of human Nesprin proteins. These proteins have been genetically implicated in various human myopathies. For example, Nesprin/Syne-1 or Nesprin/Syne-2 is associated with Emery-Dreifuss muscular dystrophy (EDMD) as well as severe cardiomyopathies. Moreover, Syp itself is increasingly linked with factors and targets that can cause human neurodegenerative disorders. Recent work has revealed that SYNCRIP/hnRNPQ and Fragile X mental retardation protein (FMRP) are present in the same mRNP granule, and loss of expression of FMRP or the ability of FMRP to interact with mRNA and polysomes can cause cases of Fragile X syndrome. Separate studies have also shown that SYNCRIP interacts with wild-type survival of motor neuron (SMN) protein but not the truncated or mutant forms found to cause spinal muscular atrophy, and Syp genetically interacts with Smn mutations in vivo. Understanding Syp function in the regulation of such diverse and complex targets may, therefore, provide new avenues for understanding the molecular basis of complex disease phenotypes and potentially lead to future therapeutic approaches (McDermott, 2014).

alpha-Neurexins are required for efficient transmitter release and synaptic homeostasis at the mouse neuromuscular junction

Neurotransmission at chemical synapses of the brain involves alpha-neurexins, neuron-specific cell-surface molecules that are encoded by three genes in mammals. Deletion of alpha-neurexins in mice previously demonstrated an essential function, leading to early postnatal death of many double-knockout mice and all triple mutants. Neurotransmitter release at central synapses of newborn knockouts was severely reduced, a function of alpha-neurexins that requires their extracellular sequences. This study investigated the role of alpha-neurexins at neuromuscular junctions, presynaptic terminals that lack a neuronal postsynaptic partner, addressing an important question because the function of neurexins was hypothesized to involve cell-adhesion complexes between neurons. Using systems physiology, morphological analyses and electrophysiological recordings, this study shows that quantal content, i.e. the number of acetylcholine quanta released per nerve impulse from motor nerve terminals, and frequency of spontaneous miniature endplate potentials at the slow-twitch soleus muscle are reduced in adult alpha-neurexin double-knockouts, consistent with earlier data on central synapses. However, the same parameters at diaphragm muscle neuromuscular junctions showed no difference in basal neurotransmission. To reconcile these observations, this study tested the capability of control and alpha-neurexin-deficient diaphragm neuromuscular junctions to compensate for an experimental reduction of postsynaptic acetylcholine receptors by a compensatory increase of presynaptic release: Knockout neuromuscular junctions produced significantly less upregulation of quantal content than synapses from control mice. These data suggest that alpha-neurexins are required for efficient neurotransmitter release at neuromuscular junctions, and that they may perform a role in the molecular mechanism of synaptic homeostasis at these peripheral synapses (Sons, 2006).

The structure of neurexin 1alpha reveals features promoting a role as synaptic organizer

alpha-neurexins are essential synaptic adhesion molecules implicated in autism spectrum disorder and schizophrenia. The alpha-neurexin extracellular domain consists of six LNS domains interspersed by three EGF-like repeats and interacts with many different proteins in the synaptic cleft. To understand how alpha-neurexins might function as synaptic organizers, the structure of the neurexin 1alpha extracellular domain (n1alpha) was solved to 2.65 A. The L-shaped molecule can be divided into a flexible repeat I (LNS1-EGF-A-LNS2), a rigid horseshoe-shaped repeat II (LNS3-EGF-B-LNS4) with structural similarity to so-called reelin repeats, and an extended repeat III (LNS5-EGF-B-LNS6) with controlled flexibility. A 2.95 A structure of n1alpha carrying splice insert SS#3 in LNS4 reveals that SS#3 protrudes as a loop and does not alter the rigid arrangement of repeat II. The global architecture imposed by conserved structural features enables alpha-neurexins to recruit and organize proteins in distinct and variable ways, influenced by splicing, thereby promoting synaptic function (Chen, 2011)

SAM68 regulates neuronal activity-dependent alternative splicing of neurexin-1

The assembly of synapses and neuronal circuits relies on an array of molecular recognition events and their modification by neuronal activity. Neurexins are a highly polymorphic family of synaptic receptors diversified by extensive alternative splicing. Neurexin variants exhibit distinct isoform-specific biochemical interactions and synapse assembly functions, but the mechanisms governing splice isoform choice are not understood. This study demonstrates that Nrxn1 alternative splicing is temporally and spatially controlled in the mouse brain. Neuronal activity triggers a shift in Nrxn1 splice isoform choice via calcium/calmodulin-dependent kinase IV signaling. Activity-dependent alternative splicing of Nrxn1 requires the KH-domain RNA-binding protein SAM68 that associates with RNA response elements in the Nrxn1 pre-mRNA. These findings uncover SAM68 as a key regulator of dynamic control of Nrxn1 molecular diversity and activity-dependent alternative splicing in the central nervous system (Iijima, 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).

Membrane-tethered monomeric neurexin LNS-domain triggers synapse formation

Neurexins are presynaptic cell-adhesion molecules that bind to postsynaptic cell-adhesion molecules such as neuroligins and leucine-rich repeat transmembrane proteins (LRRTMs). When neuroligins or LRRTMs are expressed in a nonneuronal cell, cocultured neurons avidly form heterologous synapses onto that cell. This study shows that knockdown of all neurexins in cultured hippocampal mouse neurons did not impair synapse formation between neurons, but blocked heterologous synapse formation induced by neuroligin-1 or LRRTM2. Rescue experiments demonstrated that all neurexins tested restored heterologous synapse formation in neurexin-deficient neurons. Neurexin-deficient neurons exhibited a decrease in the levels of the PDZ-domain protein CASK (a calcium/calmodulin-activated serine/threonine kinase), which binds to neurexins, and mutation of the PDZ-domain binding sequence of neurexin-3β blocked its transport to the neuronal surface and impaired heterologous synapse formation. However, replacement of the C-terminal neurexin sequence with an unrelated PDZ-domain binding sequence that does not bind to CASK fully restored surface transport and heterologous synapse formation in neurexin-deficient neurons, suggesting that no particular PDZ-domain protein is essential for neurexin surface transport or heterologous synapse formation. Further mutagenesis revealed, moreover, that the entire neurexin cytoplasmic tail was dispensable for heterologous synapse formation in neurexin-deficient neurons, as long as the neurexin protein was transported to the neuronal cell surface. Furthermore, the single LNS-domain (for laminin/neurexin/sex hormone-binding globulin-domain) of neurexin-1beta or neurexin-3beta, when tethered to the presynaptic plasma membrane by a glycosylinositolphosphate anchor, was sufficient for rescuing heterologous synapse formation in neurexin-deficient neurons. These data suggest that neurexins mediate heterologous synapse formation via an extracellular interaction with presynaptic and postsynaptic ligands without the need for signal transduction by the neurexin cytoplasmic tail (Gokce, 2013).

Amyloid-beta Oligomers Interact with Neurexin and Diminish Neurexin-mediated Excitatory Presynaptic Organization

Alzheimer's disease (AD) is characterized by excessive production and deposition of amyloid-beta (Abeta) proteins (see Drosophila Appl) as well as synapse dysfunction and loss. While soluble Abeta oligomers (AbetaOs) have deleterious effects on synapse function and reduce synapse number, the underlying molecular mechanisms are not well understood. This study screened synaptic organizer proteins for cell-surface interaction with AbetaOs and identified a novel interaction between neurexins (NRXs; see Drosophila Neurexin) and AbetaOs. AbetaOs bind to NRXs via the N-terminal histidine-rich domain (HRD) of beta-NRX1/2/3 and alternatively-spliced inserts at splicing site 4 of NRX1/2. In artificial synapse-formation assays, AbetaOs diminish excitatory presynaptic differentiation induced by NRX-interacting proteins including neuroligin1/2 (NLG1/2; see Drosophila Neuroligin) and the leucine-rich repeat transmembrane protein LRRTM2. Although AbetaOs do not interfere with the binding of NRX1beta to NLG1 or LRRTM2, time-lapse imaging revealed that AbetaO treatment reduces surface expression of NRX1beta on axons and that this reduction depends on the NRX1beta HRD. In transgenic mice expressing mutated human amyloid precursor protein, synaptic expression of beta-NRXs, but not alpha-NRXs, decreases. Thus these data indicate that AbetaOs interact with NRXs and that this interaction inhibits NRX-mediated presynaptic differentiation by reducing surface expression of axonal beta-NRXs, providing molecular and mechanistic insights into how AbetaOs lead to synaptic pathology in AD (Naito, 2017).

Deletion of LRRTM1 and LRRTM2 in adult mice impairs basal AMPA receptor transmission and LTP in hippocampal CA1 pyramidal neurons

Leucine-rich repeat transmembrane (LRRTM; see Drosophila Toll-6 & Toll-7 and Capricious) proteins are synaptic cell adhesion molecules that influence synapse formation and function. This study took advantage of the generation of a LRRTM1 and LRRTM2 double conditional knockout mouse (LRRTM1,2 cKO) to examine the role of LRRTM1,2 at mature excitatory synapses in hippocampal CA1 pyramidal neurons. Genetic deletion of LRRTM1,2 in vivo in CA1 neurons dramatically impaired long-term potentiation (LTP), an impairment that was rescued by simultaneous expression of LRRTM2, but not LRRTM4. Mutation or deletion of the intracellular tail of LRRTM2 did not affect its ability to rescue LTP, while point mutations designed to impair its binding to presynaptic neurexins (see Drosophila Neurexin) prevented rescue of LTP. These proteins at mature synapses also caused a decrease in AMPA (see Drosophila GluR1A) receptor-mediated, but not NMDA receptor-mediated, synaptic transmission and had no detectable effect on presynaptic function. Imaging of recombinant photoactivatable AMPA receptor subunit GluA1 in the dendritic spines of cultured neurons revealed that it was less stable in the absence of LRRTM1,2. These results illustrate the advantages of conditional genetic deletion experiments for elucidating the function of endogenous synaptic proteins and suggest that LRRTM1,2 proteins help stabilize synaptic AMPA receptors at mature spines during basal synaptic transmission and LTP (Bhouri, 2018).

LRRTMs Organize Synapses through Differential Engagement of Neurexin and PTPsigma

Presynaptic neurexins (Nrxs) and type IIa receptor-type protein tyrosine phosphatases (RPTPs) organize synapses through a network of postsynaptic ligands. This study shows that leucine-rich-repeat transmembrane neuronal proteins (LRRTMs; see Drosophila Capricious) differentially engage the protein domains of Nrx (see Drosophila (see Drosophila Neurexin)) but require its heparan sulfate (HS) modification to induce presynaptic differentiation. Binding to the HS of Nrx is sufficient for LRRTM3 and LRRTM4 to induce synaptogenesis. Mammalian Nrx1gamma was identified as a potent synapse organizer, and LRRTM4 is revealed as its postsynaptic ligand. Mice expressing a mutant form of LRRTM4 that cannot bind to HS show structural and functional deficits at dentate gyrus excitatory synapses. Through the HS of Nrx, LRRTMs also recruit PTPsigma to induce presynaptic differentiation but function to varying degrees in its absence. PTPsigma forms a robust complex with Nrx, revealing an unexpected interaction between the two presynaptic hubs. These findings underscore the complex interplay of synapse organizers in specifying the molecular logic of a neural circuit (Roppongi, 2020).

Structural insights into modulation and selectivity of transsynaptic neurexin-LRRTM interaction

Leucine-rich repeat transmembrane neuronal proteins (LRRTMs) function as postsynaptic organizers that induce excitatory synapses. Neurexins (Nrxns) and heparan sulfate proteoglycans have been identified as presynaptic ligands for LRRTMs. Specifically, LRRTM1 and LRRTM2 bind to the Nrxn splice variant lacking an insert at the splice site 4 (S4). This study reports the crystal structure of the Nrxn1beta-LRRTM2 complex at 3.4 A resolution. The Nrxn1β-LRRTM2 interface involves Ca(2+)-mediated interactions and overlaps with the Nrxn-neuroligin interface. Together with structure-based mutational analyses at the molecular and cellular levels, the present structural analysis unveils the mechanism of selective binding between Nrxn and LRRTM1/2 and its modulation by the S4 insertion of Nrxn (Yamagata, 2018).

Alternative Splicing of Presynaptic Neurexins Differentially Controls Postsynaptic NMDA and AMPA Receptor Responses

AMPA- and NMDA-type glutamate receptors mediate distinct postsynaptic signals that differ characteristically among synapses. How postsynaptic AMPA- and NMDA-receptor levels are regulated, however, remains unclear. Using newly generated conditional knockin mice that enable genetic control of neurexin alternative splicing (see Drosophila Neurexin), this study shows that in hippocampal synapses, alternative splicing of presynaptic neurexin-1 at splice site 4 (SS4) dramatically enhanced postsynaptic NMDA-receptor-mediated, but not AMPA-receptor-mediated, synaptic responses without altering synapse density. In contrast, alternative splicing of neurexin-3 at SS4 suppressed AMPA-receptor-mediated, but not NMDA-receptor-mediated, synaptic responses, while alternative splicing of neurexin-2 at SS4 had no effect on NMDA- or AMPA-receptor-mediated responses. Presynaptic overexpression of the neurexin-1beta and neurexin-3beta SS4+ splice variants, but not of their SS4- splice variants, replicated the respective SS4+ knockin phenotypes. Thus, different neurexins perform distinct nonoverlapping functions at hippocampal synapses that are independently regulated by alternative splicing. These functions transsynaptically control NMDA and AMPA receptors, thereby mediating presynaptic control of postsynaptic responses (Dai, 2019).

Deorphanizing FAM19A proteins as pan-neurexin ligands with an unusual biosynthetic binding mechanism

Neurexins (see Drosophila Neurexin) are presynaptic adhesion molecules that organize synapses by binding to diverse trans-synaptic ligands, but how neurexins are regulated is incompletely understood. This study identified FAM19A/TAFA proteins, "orphan" cytokines, as neurexin regulators that interact with all neurexins, except for neurexin-1gamma, via an unusual mechanism. Specifically, this study showed that FAM19A1-A4 bind to the cysteine-loop domain of neurexins by forming intermolecular disulfide bonds during transport through the secretory pathway. FAM19A-binding required both the cysteines of the cysteine-loop domain and an adjacent sequence of neurexins. Genetic deletion of neurexins suppressed FAM19A1 expression, demonstrating that FAM19As physiologically interact with neurexins. In hippocampal cultures, expression of exogenous FAM19A1 decreased neurexin O-glycosylation and suppressed its heparan sulfate modification, suggesting that FAM19As regulate the post-translational modification of neurexins. Given the selective expression of FAM19As in specific subtypes of neurons and their activity-dependent regulation, these results suggest that FAM19As serve as cell type-specific regulators of neurexin modifications (Khalaj, 2020).

Search PubMed for articles about Drosophila Neurexin 1

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date revised: 11 June 2024

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