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bioRxiv · 10.64898/2026.01.21.700908

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

Pranske, Z. J.·Paradis, S.
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Abstract

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 GABAAR{gamma}2 subunits to immature gephyrin scaffolds, suggesting that Sema4D primes these scaffolds for receptor recruitment. Surprisingly, we observe new colocalization events between existing gephyrin and GABAAR puncta, suggesting that clustering of either the gephyrin scaffold or the GABAAR 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. Significance StatementThe assembly of new synaptic contacts requires precise coordination of specialized proteins in pre- and postsynaptic neurons. Inhibitory synapses, which suppress neuronal activity and are essential for circuit stability, contain distinct molecular components, yet the mechanisms governing their assembly remain poorly understood. We used Sema4D, a protein that rapidly induces inhibitory synapse formation, as a molecular tool to dissect how synaptic proteins on either side of the synaptic cleft are coordinated in space and time. Using live imaging we show that Sema4D acts on both pre- and postsynaptic compartments to recruit synaptic proteins with spatiotemporal precision. Together, these findings define the sequence of molecular events underlying inhibitory synapse assembly and have implications for neurodevelopmental disorders in which inhibition is disrupted.

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Pranske, Z. J., Paradis, S.. 2026-01-21. Coordinated pre- and postsynaptic protein dynamics underlie rapid Sema4D-mediated inhibitory synapse assembly. https://doi.org/10.64898/2026.01.21.700908

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