In the central nervous system (CNS), the inhibitory system plays a key role in neuronal network excitability. To identify novel genes and mechanisms involved in the formation and regulation of inhibitory synapses, we use the inhibitory GABAergic neuromuscular junction of the nematode C. elegans as a genetically tractable model. At these synapses, fast neurotransmission is ensured by type A ionotropic GABA receptors (GABAAR), which form post-synaptic clusters in front of GABAergic buttons. We performed an unbiased EMS genetic screen based on the visualization of fluorescently tagged GABAAR in vivo in a knock-in strain. A second EMS genetic screen has been performed on this knock-in strain in a sensitized background. We identified 56 (31 + 25, respectively) mutants with abnormal GABAAR localization among 3,648 (1,728 + 1,920, respectively) mutagenized haploid genomes. For 36 mutants, we used a novel WGS strategy to simultaneously map and identify causative mutations without any prior time-consuming genetic mapping. We found 7 alleles of genes already known to be involved in synaptogenesis, such as genes encoding master regulators of neuronal identity (the transcription factors UNC-30, a Pitx family member, and UNC-3, a COE motif family member), presynaptic organizers (SYD-2/liprin), postsynaptic scaffold proteins (FRM-3, an ERM domain protein, LIN-2/CASK and the netrin receptor UNC-40/DCC). For 10 mutant strains, we are currently completing the validation of candidate genes, which are known to be involved in various cellular processes, such as transcription factors, motor proteins or extracellular matrix components. In addition, we undertook the functional characterization of a novel candidate gene, tentatively named nsp-3, which encodes an evolutionarily conserved transmembrane protein. An early stop in nsp-3 causes a severe reduction of synaptic GABAAR content and the presence of small ectopic punctae in muscle cells. We reproduced this phenotype by generating a complete deletion of this locus using CRISPR technology. We are now investigating NSP-3 expression pattern, subcellular localization and role in GABAAR trafficking and localization. Though a few publications showed that other members of this family were involved in cellular adhesion, phagocytosis and immune response in Drosophila, nothing is known about the function of these proteins in the CNS. Our data point to novel functions of these proteins in the traffic or synaptic localization of neurotransmitter receptors in the nervous system.

Affiliation:
- Institut Neuromyogene, Universite Claude Bernard Lyon 1 - INSERM U1217 - CNRS UMR5310, Villeurbanne CEDEX, Rhone, FR