PI: Kurshan, Peri T. PROJECT SUMMARY/ABSTRACT Synapses are the fundamental information processing units in the brain and their dysfunction leads to neurodevelopmental and neuropsychiatric disorders. Synapse development and organization is mediated in part by a class of transmembrane proteins called synaptic cell-adhesion molecules (sCAMs), and mutations in sCAM genes is highly associated with diseases such as autism, schizophrenia and intellectual disability, among others. The dominant model for synapse formation suggests that it is initiated by the trans-synaptic binding of sCAMs, however in vivo evidence for that model is lacking. Here we propose to uncover non-trans- synaptic cell-intrinsic molecular programs for presynaptic assembly that mediate the localization, trafficking and subcellular functions of sCAMs and other integral presynaptic proteins. Our preliminary evidence suggests that the intracellular domains of the presynaptic sCAMs neurexin and syg-2/nephrin are cell-autonomously required for presynaptic organization. Moreover, we find that the localization of neurexin to the presynaptic active zone is mediated by intracellular interactions with active zone scaffold proteins and kinesin motors. Finally, we have found that the dependence of sCAMs and other active zone proteins on transport by kinesins depends on the stage of axonal outgrowth. Based on these findings, we hypothesize that presynaptic assembly is mediated in large part by cell-intrinsic mechanisms. We propose to pursue three Specific Aims to characterize the mechanisms that govern (1) the intracellular recruitment of sCAMs, (2) the intracellular organization of proper synapse spacing by sCAM-mediated cytoskeletal rearrangement, and (3) the delivery and reallocation of sCAMs and other active zone proteins by kinesin- dependent and independent mechanisms at different stages of axonal outgrowth (Aim 3). To investigate these hypotheses, we leverage the genetic tractability and stereotyped nervous system of the nematode C. elegans, along with innovative imaging and genetic approaches. C. elegans has a long history of revealing fundamental synaptic biology and our previous published results and preliminary data position us well to take advantage of the power of this system. Collectively our proposed research will reveal how cell-intrinsic mechanisms govern the function of sCAMs in synapse formation. These studies have the potential to uncover the molecular mechanisms underlying neurodevelopmental disorders that result from defects in sCAM function, and thus provide targets for the development of specific therapeutic interventions.