Impaired formation of high-order gephyrin oligomers underlies gephyrin dysfunction-associated pathologies

Gephyrin is critical for the structure, function, and plasticity of inhibitory synapses. Gephyrin mutations have been linked to various neurological disorders; however, systematic analyses of the functional consequences of these mutations are lacking. Here, we performed molecular dynamics simulations of gephyrin to predict how six reported point mutations might change the structural stability and/or function of gephyrin. Additional in silico analyses revealed that the A91T and G375D mutations reduce the binding free energy of gephyrin oligomer formation. Gephyrin A91T and G375D displayed altered clustering patterns in COS-7 cells and nullified the inhibitory synapse-promoting effect of gephyrin in cultured neurons. However, only the G375D mutation reduced gephyrin interaction with GABAA receptors and neuroligin-2 in mouse brain; it also failed to normalize deficits in GABAergic synapse maintenance and neuronal hyperactivity observed in hippocampal dentate gyrus-specific gephyrin-deficient mice. Our results provide insights into biochemical, cell-biological, and network-activity effects of the pathogenic G375D mutation. [Display omitted] •Gephyrin G375D reduces the formation of E- domain-mediated gephyrin oligomers•Gephyrin G375D impairs GABAergic synapse maintenance in vivo•Gephyrin G375D increases susceptibility to kainic acid-induced seizures in vivo•Gephyrin G375D displays reduced interactions with GABAA receptors and Nlgn2 in vivo Molecular Biology; Neuroscience; Structural Biology