Synapse development is critical for the formation of neuronal networks while defects in synapse formation lead to neurodevelopmental disorders. Cell adhesion molecule signaling plays a prominent role in synapse formation, and strong evidence has emerged linking dysfunction of the cell adhesion molecule neurexin to autism spectrum disorders and intellectual disability. However, the precise role of this molecule in regulating synapse formation remains elusive. In humans, three neurexin genes are expressed as thousands of splice isoforms, all of which share a conserved intracellular domain. Understanding the intracellular signaling pathway mediated by neurexin may lead to the development of therapeutics for a multitude of neurexin-associated disorders. C. elegans has long been established as a powerful tool for gene discovery. To elucidate which proteins interact with neurexin's intracellular domain (ICD) we are employing a recently developed enzyme-catalyzed proximity labeling method called TurboID. In TurboID, a protein of interest is tagged with a promiscuous labeling enzyme - the E. coli biotin ligase BirA - which biotinylates neighboring proteins located within a few nanometer radius. Biotinylated proteins are pulled down using streptavidin. To determine the appropriate position for insertion of BirA within neurexin's ICD while not interfering with neurexin's function, we engineered transgenes with various BirA locations. Transgenes carrying BirA inserted just before the C-terminal PDZ binding motif fully rescued the synaptic assembly defects observed in neurexin null mutants, so we chose this location for endogenous insertion via CRISPR/Cas9. The endogenously-tagged BirA strain was then validated using our previously developed presynaptic assembly markers and showed no synaptic defects when compared to wild-type animals. Streptavidin pull-downs followed by mass spectrometry were performed using our endogenously-tagged neurexin-BirA strain and 70 potential neurexin ICD interactors were identified when compared to controls (cytosolic BirA and wild type strains). Of those, we are following up on 21 hits that, by gene ontology analysis, include cytoskeletal-related, endo/exocytitc, metabolic and adhesion processes. This approach will reveal the signaling pathway downstream of neurexin with the potential of identifying therapeutic targets widely applicable to various neurexin-associated disorders.

Affiliation:
- Albert Einstein College of Medicine