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

Area of Science:

• Neuroscience
• Cell Biology
• Synaptic Plasticity

Background:

• Inhibitory synapse formation is less understood than excitatory synapse formation.
• The precise molecular mechanisms coordinating pre- and postsynaptic assembly are unclear.
• Class 4 Semaphorin Sema4D was previously identified as a key regulator of inhibitory synaptogenesis.

Purpose of the Study:

• To investigate the dynamic changes in pre- and postsynaptic protein behavior during Sema4D-induced inhibitory synapse formation.
• To elucidate the spatiotemporal coordination of synaptic proteins during GABAergic synapse assembly.
• To understand the role of Sema4D in recruiting key synaptic components.

Main Methods:

• Utilized recombinant Sema4D protein to induce rapid GABAergic synapse formation in cultured hippocampal neurons.
• Employed two-channel live imaging to track protein dynamics in real-time.
• Analyzed the mobility and colocalization of presynaptic (GAD65) and postsynaptic (gephyrin, GABA_ARγ2) proteins.

Main Results:

• Sema4D treatment increased the mobility of presynaptic GAD65 assemblies.
• Sema4D had a negligible effect on postsynaptic gephyrin scaffold mobility, but increased colocalization.
• Sema4D promoted the recruitment of GABA_ARγ2 subunits to gephyrin scaffolds, suggesting priming for receptor integration.
• Observed de novo colocalization events between gephyrin and GABA_AR, indicating independent clustering can nucleate assembly.

Conclusions:

• Sema4D signaling orchestrates dynamic pre- and postsynaptic protein changes to assemble inhibitory synapses rapidly.
• The findings reveal a spatiotemporal sequence of molecular events in inhibitory synapse assembly.
• This research provides insights into neurodevelopmental disorders associated with disrupted inhibition.