Wrd-specific PP2A substrates that regulate AZ stability Project Summary / Abstract Neuronal communication depends on the precisely orchestrated release of neurotransmitters at specialized membrane compartments at the axonal terminal called active zones (AZs). Understanding how AZs are assembled and stabilized during development and activity-driven structural plasticity is key to understanding how neural circuitries are established and modified in healthy brains, and how dysfunction in synaptic formation contributes to pathogenesis of neurodevelopment disorders such as autism. A group of evolutionarily conserved presynaptic scaffolding proteins at the cytomatrix of AZ are thought to organize AZ assembly, however, the precise molecular mechanisms remain enigmatic. We recently showed that two master AZ organizers, Syd-1 and Liprin-?, regulate a specific PP2A activity through a AZ-localized PP2A B' regulatory subunit Well-rounded (Wrd) in a linear pathway to stabilize AZ materials and synaptic vesicles at the nerve terminal, and that this pathway antagonizes GSK-3? activity to prevent ectopic accumulation of synaptic materials at the distal axon region. Based on these findings, we hypothesize that Syd-1 and Liprin-? control PP2A/Wrd-mediated dephosphorylation events at the nerve terminal, which antagonize GSK-3?-mediated phosphorylation events to stabilize AZs at nerve terminal. Our preliminary data suggest that Futsch, a microtubule associated protein, is a potential substrate of both Wrd and GSK-3?. We will test the hypothesis that the syd-1/liprin-?/wrd phosphatase pathway and GSK-3? kinase pathway target Futsch to balance AZ retention at the nerve terminal (Aim1). We will also perform unbiased proteomic screens for downstream substrates of Syd-1-Liprin-?-PP2A/Wrd and GSK-3?, and test whether their phosphorylation and dephosphorylation can dynamically regulate AZ stability (Aim2).