Autism-associated ARHGEF9 variants impair GABAergic synapses and ultrasonic communication by reducing gephyrin...

Missense and truncating variants of the X-chromosome-linked ARHGEF9 gene encoding collybistin (Cb) cause intellectual disability and epilepsy. Here we report novel rare variants in ARHGEF9 identified via exome sequencing in male individuals with autism spectrum disorders (ASD). Functional analyses revealed that p.R290C promotes abnormal gephyrin clustering in COS-7 cells and reduces inhibitory synapse density in cultured hippocampal neurons. Electrophysiological recordings revealed that p.V374F and p.G485S, in conjunction with p.R290C, induce defective inhibitory synaptic transmission. Furthermore, only the p.G485S markedly decreased phosphatidylinositol 3-phosphate-binding activity. Mice with conditional knockout (cKO) of Cb displayed altered inhibitory synaptic density and transmission in the medial prefrontal cortex (mPFC). Moreover, ultrasonic vocalization was impaired in mPFC-specific Cb-cKO mice. Strikingly, proteomic analyses showed that mPFC-specific Cb-cKO reduced gephyrin phosphorylation levels. Expression of a subset of ASD-associated ARHGEF9 variants failed to rescue impaired GABAergic synaptic transmission and reduced gephyrin phosphorylation levels in mPFC-specific Cb-cKO mice. Our data suggest that perturbation of diverse facets of Cb activity potentially triggers the onset and/or progression of ASDs via inducing altered balance in gephyrin phosphorylation.