Coordinated pre- and postsynaptic protein dynamics underlie rapid Sema4D-mediated inhibitory synapse assembly

In the mammalian hippocampus, synapses are either excitatory or inhibitory as defined by the presynaptic neurotransmitter (glutamate or GABA, respectively) and the specific ligand-gated ion channel receptors localized to the postsynaptic specialization. While numerous studies explore the formation of excitatory synapses, the process of inhibitory synapse formation is less understood. Using both loss- and gain-of-function approaches, our lab previously identified the class 4 Semaphorin Sema4D as a key regulator of inhibitory synaptogenesis. Here, using recombinant Sema4D protein as a tool to rapidly induce GABAergic synapse formation in cultured hippocampal neurons, we employ two-channel live imaging to identify changes to pre- and postsynaptic protein dynamics during inhibitory synapse formation. We find that Sema4D treatment promotes the mobility of presynaptic GAD65 protein assemblies while having a negligible effect on the behavior of the postsynaptic gephyrin scaffold, leading to increased colocalization of these proteins. In addition, Sema4D treatment promotes the recruitment of GABA_ARγ2 subunits to immature gephyrin scaffolds, suggesting that Sema4D primes these scaffolds for receptor recruitment. Surprisingly, we observe new colocalization events between existing gephyrin and GABA_AR puncta, suggesting that clustering of either the gephyrin scaffold or the GABA_AR is sufficient to nucleate assembly of the postsynaptic specialization. Overall our results support a model in which Sema4D signaling coordinates dynamic changes in both pre- and postsynaptic compartments to assemble inhibitory synapses on rapid timescales.