Two Neto isoforms are required for proper synapse assembly at the Drosophila NMJ. Cathy I Ramos, Oghomwen Igiesuorobo, Mihaela Serpe. NICHD, NIH, Bethesda, MD.

   Synaptogenesis requires the recruitment of neurotransmitter receptors, which comprises of their assembly, trafficking and stabilization at specific membrane locations. The Drosophila neuromuscular junction (NMJ) shares common features with mammalian central synapses. Notably, neurotransmission is sustained by ionotropic glutamate receptor (iGluR) at postsynaptic densities (PSDs). Drosophila NMJ iGluRs are heterotetrameric complexes composed of three essential subunits - GluRIIC, GluRIID, GluRIIE - and either GluRIIA or GluRIIB. Our lab has identified and characterized the Drosophila neto (neuropilin and tolloid-like) as an essential gene required for clustering of both iGluRs types at the NMJ. Our previous studies revealed that Neto is an essential component of the iGluR complexes required for iGluR clustering, organization of PSDs, and synapse functionality. Now, we have found alternative splicing in the neto locus encoding for a second Neto isoform, Neto-B. The Neto-A and Neto-B isoforms share similar extracellular and transmembrane domains but contain different intracellular parts. These two isoforms are conserved in Drosophilidae. We have confirmed that both isoforms are expressed in the larval body-wall muscles in D. melanogaster. Furthermore, either isoform could rescue the iGluRs clustering defects and embryonic lethality of neto null mutants. Using isoform specific mutants and rescue constructs, we are investigating the role of the two Neto isoforms in iGluRs clustering. Preliminary morphological analyses suggest that Neto-B specifically influences the synaptic accumulation of the type-A iGluR complexes. Furthermore, electrophysiological recordings in an allelic series of neto-B mutants revealed quantal sizes that correlated with GluRIIA levels. These findings indicate that the two Neto isoforms may have distinct, non-redundant functions in synapse assembly at the Drosophila NMJ.