Abstract
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. Here, we report 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, we looked for genetic interactions and found connections 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 thickvein (Tkv) abundance was reduced in dnlg4 mutants, and immunoprecipitation assays showed that DNlg4 and Tkv physically interacted in vivo. Our study demonstrates that DNlg4 presynaptically regulates neuromuscular synaptic growth via the BMP signaling pathway by modulating Tkv.
Highlights
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 third-instar larvae, Drosophila neuroligin 4 (DNlg4) accumulated in the brain and ventral nerve cord (VNC), as indicated by co-staining with antibodies against DNlg4 and Bruchpilot (BRP), and it was especially concentrated at the neuropil, where the synapses of neurons aggregate (Fig. 1, B–BЉ)
In the lines in which DNlg4 was overexpressed in muscles, DNlg4 immunoreactivity accumulated at neuromuscular junctions (NMJs), but with a non-uniform distribution at distinct type I synaptic boutons (Fig. 1, C–CЉ)
Summary
DNlg is predominantly expressed in central neurons and is concentrated at glutamatergic NMJs. We expressed one or two copies of UASdnlg in muscle cells with the C57-Gal driver, but no increase in the number of synaptic boutons was observed (supplemental Fig. S4) These results indicated that overexpression of DNlg presynaptically was sufficient to promote the proliferation of synaptic boutons at NMJs and further demonstrated that DNlg functions presynaptically, but not postsynaptically, in regulating NMJ growth. Western blot analyses of larval brain homogenates with anti-Trio antibodies showed that Trio was significantly decreased in the dnlg mutants but increased in the dnlg4-overexpressing flies (Fig. 5, I and J) Taken together, these results demonstrated that DNlg positively regulated BMP signaling, which, in turn, affected the synaptic growth of NMJs. A key question is the mechanism underlying the down-regulation of pMad in dnlg mutants. Our results showed that loss of DNlg led to synaptic transmission defects at NMJs along with reduced larval locomotor activity, and all of the defects were derived from loss of DNlg presynaptically
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