InteractiveFly: GeneBrief

Neuroligin 4: Biological Overview | References

BIOLOGICAL OVERVIEW

The neuroligin (Nlg) family of neural cell adhesion molecules is thought to be required for synapse formation and development, and has been linked to the development of autism spectrum disorders in humans. In Drosophila melanogaster, mutations in the neuroligin 1-3 genes have been reported to induce synapse developmental defects at neuromuscular junctions (NMJs), but the role of neuroligin 4 (dnlg4) in synapse development has not been determined. This study reports that the Drosophila Neuroligin 4 (DNlg4) is different from DNlg1-3 in that it presynaptically regulates NMJ synapse development. Loss of dnlg4 results in reduced growth of NMJs with fewer synaptic boutons. The morphological defects caused by dnlg4 mutant are associated with a corresponding decrease in synaptic transmission efficacy. All of these defects could only be rescued when DNlg4 was expressed in the presynapse of NMJs. To understand the basis of DNlg4 function, genetic interactions were sought, and connections were found with the components of the bone morphogenetic protein (BMP) signaling pathway. Immunostaining and western blot analyses demonstrated that the regulation of NMJ growth by DNlg4 was due to the positive modulation of BMP signaling by DNlg4. Specifically, BMP type I receptor Tkv abundance was reduced in dnlg4 mutants, and immunoprecipitation assays showed that DNlg4 and Tkv physically interacted in vivo. This study demonstrates that DNlg4 presynaptically regulates neuromuscular synaptic growth via the BMP signaling pathway by modulating Tkv (Zhang, 2017).

The formation, development, and plasticity of synapses are critical for the construction of neural circuits, and the Drosophila larval neuromuscular junction (NMJ) is an ideal model system to dissect these processes. In the past few decades, several subcellular events and signaling pathways have been reported to be involved in regulating synaptic growth at Drosophila NMJs, such as local actin assembly, endocytosis, ubiquitin-mediated protein degradation, the Wingless pathway, and the bone morphogenetic protein (BMP) pathway. Among these, BMP signaling is thought to be a major retrograde pathway that promotes the synaptic growth of NMJs (Zhang, 2017).

At the Drosophila NMJ, the BMP homolog Glass bottom boat (Gbb) is released by muscle cells and binds to the presynaptic type II BMP receptor Wishful thinking (Wit). Wit is a constitutively active serine/threonine kinase and, upon binding to Gbb, forms a complex with the type I BMP receptor Thickvein (Tkv) or saxophone (Sax), which results in their activation by phosphorylation. The activated type I receptor subsequently phosphorylates the downstream R-Smad protein Mothers against decapentaplegic (Mad). Phosphorylated Mad (pMad) then binds to the co-Smad Medea (Med). This complex translocates to the nucleus of motoneurons to activate or repress the transcription of target genes required for NMJ growth. Mutation of any component in the BMP signaling pathway results in a striking deficiency of NMJ growth. In addition, many molecules are reported to affect NMJ growth by negatively regulating BMP signaling at different points in the pathway. This study report that Drosophila Neuroligin 4 (DNlg4), a trans-synaptic adhesion protein, acts as a positive regulator of BMP signaling to regulate NMJ growth (Zhang, 2017).

Neuroligins (Nlgs) were initially reported to be the postsynaptic ligands of the presynaptic adhesion proteins neurexins (Nrxs), and loss of function of Nlgs in humans is thought to be associated with several mental disorders, including autism and schizophrenia. Nlgs are an evolutionarily conserved family of proteins encoded by four independent genes in rodents and five independent genes in humans. Nlgs have been reported to induce synapse assembly by co-cultured neurons when expressed in nonneuronal cells, and overexpression of Nlgs in neurons increases synapse density. These in vitro cell culture studies suggest a role of Nlgs in inducing the formation of synaptic contacts. However, an in vivo study showed, despite severe defects in synaptic transmission, that there was no alteration of synapse number in neurons from nlg1-3 triple knock-out mice. Similarly, loss of Nlg1 specifically in the hippocampus or amygdala did not alter the synapse number, suggesting that the role of Nlgs is not to trigger the initial synapse formation. Rather, it is more likely that upon binding to Nrx, Nlg functions in maturation of nascent synapses, including differentiation and stabilization by recruiting scaffolding proteins, postsynaptic receptors, and signaling proteins (Zhang, 2017).

In Drosophila, four Nlgs have been identified. Mutations in dnlg1-3 result in defective synapse differentiation that is primarily characterized by abnormal protein levels or the ectopic postsynaptic localization of glutamate receptors in larval NMJs. In addition, loss of dnlg1-3 separately leads to impairment in NMJ synapse development, as indicated by abnormal synaptic bouton number, but the precise underlying mechanism is poorly understood. A recent study showed that flies with a dnlg4 mutation exhibit an autism-related phenotype of behavioral inflexibility, as indicated by impaired reversal learning (Corthals, 2017). DNlg4 also regulates sleep by recruiting the GABA receptor to clock neurons and thus modulating GABA transmission (Li, 2013), which suggests a role of DNlg4 in synapse differentiation. However, potential molecular mechanisms underlying the behavioral defects caused by dnlg4 mutation and the role of DNlg4 in synapse development have not been reported (Zhang, 2017).

This paper reports the generation of an independent null allele of dnlg4 and characterized the role of DNlg4 in neuromuscular synaptic growth. Loss of DNlg4 led to impaired NMJ synapse growth, as indicated by decreased synaptic bouton numbers and increased bouton size. Presynaptic knockdown of DNlg4 mimicked these phenotypes. These morphological abnormalities in dnlg4 mutants led to corresponding impairment in synaptic transmission efficacy. Unexpectedly, all of these defects were only rescued when DNlg4 was expressed in presynaptic, instead of postsynaptic, areas of NMJs.DNlg4 genetically interacted with components of the BMP pathway and that the presynaptic BMP signaling at NMJs was decreased in dnlg4 mutants. A reduction of the BMP type I receptor Tkv was observed in dnlg4 mutants and DNlg4 physically interacted with Tkv in vivo. Altogether, this study revealed that DNlg4 regulated neuromuscular synaptic growth by positively modulating BMP signaling though Tkv (Zhang, 2017).

Sequence analyses showed that there are four nlg genes in the Drosophila genome, and all four DNlgs share significant amino acid sequence homology and protein structures with vertebrate Nlgs. DNlg1 and DNlg2 have a positive effect on synaptic growth of NMJs, as indicated by an obvious reduction in synaptic boutons in the dnlg1 and dnlg2 mutants. Conversely, loss of DNlg3 led to increased numbers of synaptic boutons at NMJs. In the present study, a dnlg4 null mutant was generated by gene targeting, and this mutant exhibited significant defects in NMJ morphology, including fewer synaptic boutons and increased bouton size. However, neuronal overexpression of two copies of the dnlg4 transgene induced a pronounced increase in bouton number. These results demonstrated a positive role of DNlg4 in regulating synapse development. Interestingly, neuronal overexpression of one copy of dnlg4 in the WT background did not induce an increase in bouton number, but it did when expressed in a dnlg4 mutant background. These results suggested a homeostatic adjustment during synapse development in Drosophila, which could somewhat counteract the effect caused by increased DNlg4. As a result, a moderate increase of DNlg4 in WT flies did not lead to increased synaptic growth of NMJs (Zhang, 2017).

In Drosophila, loss of DNlgs in the dnlg1-3 mutants also induced synaptic differentiation defects that were characterized by decreased protein levels or impaired distribution of glutamate receptors and other postsynaptic proteins at NMJs. In contrast to other dnlg mutants, statistical alteration in the distribution or protein level of glutamate receptors at NMJs in was not observed dnlg4 mutants. In addition, the distribution and protein level of some presynaptic proteins, such as BRP, CSP, and SYT, were normal in the dnlg4 mutants. However, the ultrastructural analyses of the NMJs showed that there were still some defects in synaptic ultrastructure in the dnlg4 mutants, including the increased bouton area per active zone, longer single PSD, and reduced postsynaptic SSR regions. One striking ultrastructural defect in the dnlg4 mutants was the partial detachment of presynaptic membranes from postsynaptic membranes within the active zone, which was rarely observed in WT flies, suggesting the adhesion function of DNlg4 during synaptogenesis. It was interesting that this defect also appeared in dnlg1 mutants and dnrx mutants, suggesting that the DNlg4 might affect the synaptic architecture by a mechanism similar to that of DNlg1 and DNrx (Zhang, 2017).

Functionally, dnlg4 mutants showed a mild impairment in transmitter release at NMJs, as characterized by slightly reduced evoked EJP amplitude and quantal contents. This phenotype was consistent with the morphological impairments and the ultrastructural defects of NMJs, suggesting a probable reduction in the number of total synapses or functional synapses at NMJs. The amplitude of mEJP was not changed in the dnlg4 mutants, which was consistent with the normal protein levels of postsynaptic glutamate receptors. Interestingly, the frequency of mEJPs in the dnlg4 mutants was dramatically increased, indicating that DNlg4 affects spontaneous transmitter release. The detailed mechanism underlying this should be addressed in future work (Zhang, 2017).

In mammals, Nlgs are generally considered to function as postsynaptic adhesion molecules and to help form trans-synaptic complexes with presynaptic Nrxs. In Drosophila, DNlg1 and DNlg3 are reported to be located in postsynaptic membranes. However, there are some exceptions to the postsynaptic localization of Nlgs. For example, an Nlg in Caenorhabditis elegans is reported to be present in both presynaptic and postsynaptic regions. DNlg2 is also required both presynaptically and postsynaptically for regulating neuromuscular synaptic growth. These studies support a more complex mechanism of Nlgs in synapse modulation and function. These data add to this complexity by suggesting a presynaptic role of DNlg4 in larval neuromuscular synaptic growth and synaptic functions (Zhang, 2017).

First, in the VNC of third-instar larvae, DNlg4 was concentrated in the neuropil region where the synapses aggregated. In NMJs, although endogenous DNlg4 was not detected by anti-DNlg4 antibodies, the exogenous DNlg4 that was expressed in motoneurons was located in type I synaptic boutons of NMJs, suggesting a reasonable presynaptic location of DNlg4 at NMJs. In another assay, DNlg4 was expressed using a DNlg4-Gal4 driver to mimic the endogenous expression pattern of the dnlg4 gene. The DNlg4 promoted by DNlg4-Gal4 was distributed in type I boutons of NMJs and was located in presynaptic areas of boutons, which also supported the presynaptic location of endogenous DNlg4 at NMJs, although a simultaneous postsynaptic localization of DNlg4 at NMJs could not be excluded. Second, knockdown of DNlg4 presynaptically led to morphological defects in NMJs similar to those observed in the dnlg4 mutants, indicated by decreased bouton numbers and increased bouton size. However, knockdown of DNlg4 postsynaptically did not cause the same phenotypes. These morphological defects of NMJs in the dnlg4 mutants could be completely rescued when DNlg4 was expressed in presynaptic neurons, but not when it was expressed in postsynaptic muscles. Third, dnlg4 mutants had a significant increase in spontaneous transmitter release frequency, which was usually interpreted as a presynaptic defect. In addition, all of the functional defects in the dnlg4 mutants, including decreased amplitude of EJPs, reduced quantal contents, and increased mEJP frequency, could be rescued by presynaptic expression of DNlg4. These results indicated that presynaptic DNlg4 was essential for proper proliferation and function of synapses at NMJs. Finally, the number of synaptic boutons at NMJs was significantly increased when two copies of UAS-dnlg4 were overexpressed in presynaptic neurons, whereas this phenomenon was not observed when two copies of UAS-dnlg4 were overexpressed in muscles, suggesting that presynaptic DNlg4 alone was sufficient to promote synaptic growth. Altogether, these data provided convincing evidence that DNlg4 functions as a presynaptic molecule in regulating synaptic growth and transmitter release at NMJs (Zhang, 2017).

The BMPs are major retrograde trans-synaptic signals that affect presynaptic growth and neurotransmission both in the CNS and at NMJs. This study presents immunohistochemical and genetic data showing that DNlg4 regulates NMJ growth via the BMP signaling pathway (Zhang, 2017).

First, the dnlg4 mutants shared similar phenotypes with the components of the BMP signaling pathway in synapse development, synapse architecture, and synapse functions of NMJs, including reduced synaptic bouton number, increased presynaptic membrane ruffles, and decreased synaptic transmission efficacy. Second, several genetic crosses followed by neuromuscular bouton number analyses showed a definite dosage-sensitive genetic interaction between dnlg4 and the components of the BMP signaling pathway, such as tkv, wit, mad, and dad, suggesting that DNlg4 is required for BMP signaling in regulating NMJ growth. Third, both immunohistochemical and Western blot analyses showed that the pMad, which serves as an indicator of BMP signaling, is decreased in both the synaptic boutons of NMJs and motoneuronal nuclei in the dnlg4 mutants, whereas it is increased in dnlg4-overexpressing flies. Finally, the expression of the BMP signaling target gene trio, is significantly reduced in the dnlg4 mutants, but it was increased in dnlg4-overexpressing flies. Together, these results supported the hypothesis that DNlg4 promotes synaptic growth by positively regulating BMP signaling (Zhang, 2017).

The critical question to be addressed, therefore, is the mechanism underlying this regulation. Through Western blot analyses of larval brain homogenates, it is found that the protein level of Tkv (as assessed by ectopically expressed Tkv-GFP) in the dnlg4 mutants is significantly decreased, whereas Wit and Mad, the other two components of the BMP pathway, were not altered. The immunohistochemical assay also showed that the Tkv protein in the dnlg4 mutants is reduced in both the VNC and the synaptic boutons of NMJs. These data indicated the specific positive regulation of Tkv by DNlg4. Previous studies reported that tkv mutants had decreased synaptic bouton number, increased presynaptic membrane ruffles, reduced amplitude of EJP, and unchanged mEJP amplitude. In addition, neuronal overexpression of one copy of transgenic tkv did not induce NMJ overgrowth, but it induced such overgrowth when two copies of transgenic tkv were expressed in neurons. These phenotypes were similar to what was observed in the dnlg4 mutants and dnlg4-overexpressing flies. All of these results strongly demonstrated that DNlg4 regulates BMP signaling by modulating Tkv protein levels (Zhang, 2017).

Because the dnlg4 mutants had a level of tkv mRNA comparable with that of the WT controls, DNlg4 did not affect the transcription of tkv. The possibility of Tkv trafficking defects from the cell body to the axon terminal could also be excluded because no retention of Tkv in the soma of motoneurons was observed. Thus, it seems reasonable to speculate that DNlg4 affects the recruitment or stability of Tkvs at the presynapse. In support of this hypothesis, co-localization of Tkv and DNlg4 at presynaptic regions of NMJs was observed by immunostaining. As further confirmation, it was shown that Tkv could be co-immunoprecipitated with an antibody against DNlg4, and a reverse co-immunoprecipitation assay using anti-GFP antibodies also resulted in the precipitation of DNlg4 by Tkv-GFP, suggesting a physical interaction between DNlg4 and Tkv in vivo. Furthermore, using pull-down assay, the acetylcholinesterase-like domain of the N terminus was shown to be essential for DNlg4 interacting with Tkv (Zhang, 2017).

If the recruitment of Tkv to the presynaptic membrane entirely depends on DNlg4, the existence of Tkv in the presynaptic membrane would not be detected, and accumulation of Tkv proteins in the presynaptic areas of NMJs would probably be observed in the dnlg4 mutants. However, minor Tkv protein level was detected at the presynaptic membrane, and no accumulation of Tkv was observed at NMJs in the dnlg4 mutants. Thus, a more reasonable hypothesis is that DNlg4 affected the stability of Tkv at the presynaptic membranes. The protein level of Tkv in the presynapses of NMJs was reported to be regulated by several pathways, including direct proteasomal degradation, ubiquitin-mediated degradation, and endocytosis. The simplest model is that the DNlg4 might stabilize Tkv via inhibiting its degradation. Several protein kinases, such as the ribosomal protein S6 kinase-like protein (S6KL) and the serine/threonine kinase Fused, have been reported to interact physically with Tkv in vitro and to facilitate its proteasomal degradation. In particular, S6KL has been demonstrated to degrade Tkv at NMJs. DNlg4 might stabilize Tkv by inhibiting these protein kinases, but the detailed mechanism of such activity still needs to be addressed (Zhang, 2017).

In summary, this study demonstrated that DNlg4 positively regulated neuromuscular synaptic growth by modulating BMP signaling through the maintenance of Tkv protein levels in the presynapse of NMJs. To accomplish this function, DNlg4 acted as a presynaptic molecule instead of a postsynaptic molecule. This study further suggested a relationship between Nlgs and BMP signaling and provided a new understanding of the exact role of Nlgs during synapse formation and development (Zhang, 2017).

Neuroligins Nlg2 and Nlg4 affect social behavior in Drosophila melanogaster

The genome of Drosophila melanogaster includes homologs to approximately one-third of the currently known human disease genes. Flies and humans share many biological processes, including the principles of information processing by excitable neurons, synaptic transmission, and the chemical signals involved in intercellular communication. Studies on the molecular and behavioral impact of genetic risk factors of human neuro-developmental disorders [autism spectrum disorders (ASDs), schizophrenia, attention deficit hyperactivity disorders, and Tourette syndrome] increasingly use the well-studied social behavior of D. melanogaster, an organism that is amenable to a large variety of genetic manipulations. Neuroligins (Nlgs) are a family of phylogenetically conserved postsynaptic adhesion molecules present (among others) in nematodes, insects, and mammals. Impaired function of Nlgs (particularly of Nlg 3 and 4) has been associated with ASDs in humans and impaired social and communication behavior in mice. Making use of a set of behavioral and social assays, this study analyzed the impact of two Drosophila Nlgs, Dnlg2 and Dnlg4, which are differentially expressed at excitatory and inhibitory central nervous synapses, respectively. Both Nlgs seem to be associated with diurnal activity and social behavior. Even though deficiencies in Dnlg2 and Dnlg4 appeared to have no effects on sensory or motor systems, they differentially impacted on social interactions, suggesting that social behavior is distinctly regulated by these Nlgs (Corthals, 2017).

Absence of Dnlg2 and Dnlg4 altered social interactions of Drosophila males, without causing obvious impairments of sensory functions and execution of movements. Deficiency of Dnlg2 and Dnlg4, which seem to be differentially expressed at excitatory and inhibitory synapses, induced opposing deviations from wild-type behaviors in some behavioral paradigms, such as sleep rhythm, male chaining, group size, and interindividual distance. Other behavioral paradigms, such as center avoidance and stimulation with wt male courtship songs (Cs), revealed equally altered behavior in a non-opposing fashion. Thus, Dnlg2 and Dnlg4 may play different roles in the regulation of synaptic transmission within brain neuropils implicated in the social behavior of D. melanogaster (Corthals, 2017).

Drosophila neuroligin 4 regulates sleep through modulating GABA transmission

Sleep is an essential and evolutionarily conserved behavior that is closely related to synaptic function. However, whether neuroligins (Nlgs), which are cell adhesion molecules involved in synapse formation and synaptic transmission, are involved in sleep is not clear. This study shows that Drosophila Nlg4 (DNlg4) is highly expressed in large ventral lateral clock neurons (l-LNvs) and that l-LNv-derived DNlg4 is essential for sleep regulation. GABA transmission is impaired in mutant l-LNv, and sleep defects in dnlg4 mutant flies can be rescued by genetic manipulation of GABA transmission. Furthermore, dnlg4 mutant flies exhibit a severe reduction in GABAA receptor RDL clustering, and DNlg4 associates with RDLs in vivo. These results demonstrate that DNlg4 regulates sleep through modulating GABA transmission in l-LNvs, which provides the first known link between a synaptic adhesion molecule and sleep in Drosophila (Li, 2013).

Search PubMed for articles about Drosophila Nlg4

Corthals, K., Heukamp, A. S., Kossen, R., Grosshennig, I., Hahn, N., Gras, H., Gopfert, M. C., Heinrich, R. and Geurten, B. R. H. (2017). Neuroligins Nlg2 and Nlg4 Affect Social Behavior in Drosophila melanogaster. Front Psychiatry 8: 113. PubMed ID: 28740469

Li, Y., Zhou, Z., Zhang, X., Tong, H., Li, P., Zhang, Z. C., Jia, Z., Xie, W. and Han, J. (2013). Drosophila neuroligin 4 regulates sleep through modulating GABA transmission. J Neurosci 33(39): 15545-15554. PubMed ID: 24068821

Zhang, X., Rui, M., Gan, G., Huang, C., Yi, J., Lv, H. and Xie, W. (2017). Neuroligin 4 regulates synaptic growth via the Bone morphogenetic protein (BMP) signaling pathway at the Drosophila neuromuscular junction. J Biol Chem [Epub ahead of print]. PubMed ID: 28912273

date revised: 11 June 2022

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