Abstract
In the mammalian central nervous system, the balance between excitatory and inhibitory synaptic transmission is critical for circuit function, and disruptions to this balance are implicated in neurological disorders such as epilepsy, autism spectrum disorders, and schizophrenia. While the molecular mechanisms of excitatory synapse formation have been extensively studied, the process by which inhibitory GABAergic synapses are assembled remains relatively poorly understood. Semaphorins have emerged as critical regulators of synapse development, and prior work from our lab identified the class 4 Semaphorin Sema4D as a specific regulator of GABAergic synaptogenesis acting through its high-affinity receptor Plexin-B1. However, the spatiotemporal dynamics by which Sema4D signaling coordinates pre- and postsynaptic protein behavior during inhibitory synapse assembly are unknown. This dissertation addresses this question by combining recombinant Sema4D-induced synaptogenesis with two-channel live imaging in cultured hippocampal neurons. I first review the current understanding of chemical synapse organization and assembly, with emphasis on the specialized protein architecture of inhibitory postsynaptic sites, trafficking and stabilization of GABAA receptors, and the roles of trans-synaptic signaling molecules in coordinating synapse development. I then present original research in which I analyze simultaneous pre- and postsynaptic protein dynamics during Sema4D-induced synaptogenesis. I find that Sema4D promotes the mobility of presynaptic GAD65-containing boutons while having a negligible effect on the postsynaptic gephyrin scaffold, leading to increased colocalization of pre- and postsynaptic markers. Sema4D also promotes recruitment of GABAA receptors containing the γ2 subunit preferentially to immature gephyrin scaffolds, suggesting that Sema4D primes nascent postsynaptic sites for receptor loading. Unexpectedly, new colocalization events arise from the convergence of pre-existing gephyrin and GABAA receptor puncta, suggesting that either the scaffold or the receptor cluster alone is sufficient to nucleate postsynaptic assembly. Finally, I discuss these findings in the context of a model in which Sema4D/Plexin-B1 signaling coordinates rapid inhibitory synapse formation by mobilizing pre-assembled synaptic protein complexes rather than driving de novo protein synthesis, and I consider future directions for dissecting the intracellular signaling mechanisms linking Plexin-B1 receptor engagement to cytoskeletal remodeling and synapse stabilization.