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.
Cell Adhesion Molecules, Neuronal/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster , Nerve Tissue Proteins/metabolism , Neurogenesis , Neuromuscular Junction/metabolism , Presynaptic Terminals/metabolism , Protein Serine-Threonine Kinases/metabolism , Receptors, Cell Surface/metabolism , Animals , Animals, Genetically Modified , Bone Morphogenetic Protein Receptors, Type I/agonists , Bone Morphogenetic Protein Receptors, Type I/metabolism , Bone Morphogenetic Proteins/metabolism , Cell Adhesion Molecules, Neuronal/genetics , Drosophila Proteins/agonists , Drosophila Proteins/genetics , Gene Knockout Techniques , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Microscopy, Electron, Transmission , Microscopy, Fluorescence , Mutation , Nerve Tissue Proteins/genetics , Neuromuscular Junction/enzymology , Neuromuscular Junction/ultrastructure , Presynaptic Terminals/enzymology , Presynaptic Terminals/ultrastructure , Protein Multimerization , Recombinant Fusion Proteins/metabolism , Recombinant Proteins/metabolism , Signal Transduction , Synapses/enzymology , Synapses/metabolism , Synapses/ultrastructure , Synaptic Transmission
Neuroligins (Nlgs) are a family of cell adhesion molecules thought to be important for synapse maturation and function. Mammalian studies have shown that different Nlgs have different roles in synaptic maturation and function. In Drosophila melanogaster, the roles of Drosophila neuroligin1 (DNlg1), neuroligin2, and neuroligin4 have been examined. However, the roles of neuroligin3 (dnlg3) in synaptic development and function have not been determined. In this study, we used the Drosophila neuromuscular junctions (NMJs) as a model system to investigate the in vivo role of dnlg3. We showed that DNlg3 was expressed in both the CNS and NMJs where it was largely restricted to the postsynaptic site. We generated dnlg3 mutants and showed that these mutants exhibited an increased bouton number and reduced bouton size compared with the wild-type (WT) controls. Consistent with alterations in bouton properties, pre- and postsynaptic differentiations were affected in dnlg3 mutants. This included abnormal synaptic vesicle endocytosis, increased postsynaptic density length, and reduced GluRIIA recruitment. In addition to impaired synaptic development and differentiation, we found that synaptic transmission was reduced in dnlg3 mutants. Altogether, our data showed that DNlg3 was required for NMJ development, synaptic differentiation, and function.
Cell Adhesion Molecules, Neuronal/physiology , Drosophila Proteins/physiology , Drosophila melanogaster/growth & development , Gene Expression Regulation, Developmental , Membrane Proteins/physiology , Nerve Tissue Proteins/physiology , Animals , Animals, Genetically Modified , Cell Differentiation , Crosses, Genetic , Drosophila melanogaster/genetics , Endocytosis , Microscopy, Electron , Mutation , Neuromuscular Junction/metabolism , Neuronal Plasticity , Presynaptic Terminals/metabolism , Receptors, Glutamate/metabolism , Synapses/metabolism
