Positioning type A GABA receptors (GABAARs) in front of GABA release sites sets the strength of inhibitory synapses and consequently the excitability of neuronal networks. The C. elegans neuromuscular junction (NMJ) provides a genetically tractable model to analyze the segregation of neurotransmitter receptors because muscle cells receive inhibitory innervation from GABAergic neurons and excitatory innervation from cholinergic neurons. We previously identified an original synaptic organizer, Ce-Punctin/MADD-4, that specifies GABAergic versus cholinergic identity of post-synaptic domains at the NMJs (B. Pinan-Lucarré#, H. Tu#, et al., Nature, 2014). Punctin is secreted by the two types of motoneurons and localizes at NMJs. Alternative promoters generate different isoforms with different functions.
We now show that Punctin controls the clustering of GABAARs through the synaptic adhesion molecule neuroligin (NLG-1) and the netrin receptor UNC‑40/DCC (Deleted in Colorectal Cancer). By using imaging and electrophysiology techniques, we show that NLG-1 is a component of the post-synaptic membrane that specifically clusters GABAAR at the inhibitory NMJ. The short Punctin isoform physically interacts with the extracellular domain of NLG-1, as we demonstrated by co-immunoprecipitation in a heterologous system, and ensures the specific localization of NLG-1 at GABAergic NMJs. In parallel, we performed a genetic screen for UNC-49/GABAAR mislocalization and identified a mutant allele of unc-40. Punctin binds UNC-40/DCC and recruits UNC-40/DCC at post-synaptic sites. The expression of a myristoylated fusion of the intracellular domain of UNC-40, which was shown to behave as a constitutively active receptor, fully rescued GABAAR localization defect in unc-40 mutant despite it was ubiquitously distributed at the muscle cell surface. We therefore favor a model in which UNC-40 activates an intracellular signaling pathway that promotes direct or indirect interaction between GABAARs and the NLG-1 positioned by Punctin in front of GABAergic boutons. In conclusion, Punctin constitutes a new ligand of NLG-1/neuroligin and UNC-40/DCC and functions as a central anterograde organizer of inhibitory synapses. Since the mammalian orthologs of these genes are expressed in the central nervous system and their mutations are implicated in neuropsychiatric diseases, this novel molecular pathway might have been evolutionarily conserved.
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