Selective Overexpression of Collybistin in Mouse Hippocampal Pyramidal Cells Enhances GABAergic Neurotransmission and Protects against PTZ-Induced Seizures

Abstract

Collybistin (CB) is a rho guanine exchange factor found at GABAergic and glycinergic postsynapses that interacts with the inhibitory scaffold protein, gephyrin, and induces accumulation of gephyrin and GABA A receptors (GABA A Rs) to the postsynapse. We have previously reported that the isoform without the src homology 3 (SH3) domain, CBSH3-, is particularly active in enhancing the GABAergic postsynapse in both cultured hippocampal neurons as well as in cortical pyramidal neurons after chronic in vivo expression in in utero electroporated (IUE) rats. Deficiency of CB in knockout mice results in absence of gephyrin and gephyrin-dependent GABA A Rs at postsynaptic sites in several brain regions, including hippocampus. In the present study, we have generated an adeno-associated virus (AAV) that expresses CBSH3-in a cre-dependent manner. Using male and female VGLUT1-IRES-cre or VGAT-IRES-cre mice, we explore the effect of overexpression of CBSH3-in hippocampal pyramidal cells or hippocampal interneurons. The results show that: 1) the accumulation of gephyrin and GABA A Rs at inhibitory postsynapses in hippocampal pyramidal neurons or interneurons can be enhanced by CBSH3-overexpression, 2) overexpression of CBSH3-in hippocampal pyramidal cells can enhance the strength of inhibitory neurotransmission, and 3) these enhanced inhibitory synapses provide protection against PTZ-induced seizures. The results indicate that this AAV vector carrying CBSH3-can be used for in vivo enhancement of GABAergic synaptic transmission in selected target neurons in the brain.Significance statement: Excessive or imbalanced excitation in the hippocampus can result in acute or chronic pathological conditions, such as seizures, epilepsy, and learning impairments. It is therefore important to uncover target genes that can be manipulated to restore or prevent this imbalance. This study uses a novel adeno-associated virus to express collybistin SH3-in select cells of the hippocampus. We have found that overexpression of this protein enhances GABAergic inhibitory synaptic transmission. The results also bring attention to collybistin as a possible target for therapeutic intervention aimed to restore the balance between excitation and inhibition.

Advances in Epilepsy: Mechanisms, Clinical Trials, and Drug Therapies

“…Enhancement of GABAergic neuro-transmission has been shown to inhibit or attenuate seizures. 37 GABA is mainly synthesized at axonal terminals, which is formed by decarboxylate of glutamate precursor under the action of glutamic acid decarboxylase (GAD). Synthetic GABA is released from vesicles into the synaptic cleft via vesicular GABA transporters after neuron depolarization.…”

GABAergic neurons maturation is regulated by a delicate network

“…Studies in CB knockout mice revealed that loss of CB results in severe disruption to the clustering of gephyrin and GABA A receptors in certain brain regions, including the hippocampus (Papadopoulos et al, 2008). Overexpression of CB2SH3 in specific neuron types enhances postsynaptic accumulation of gephyrin, GABA A receptors, and GABAergic synaptic transmission in the developed hippocampus (George et al, 2021).…”

Regulation of Inhibitory Signaling at the Receptor and Cellular Level; Advances in Our Understanding of GABAergic Neurotransmission and the Mechanisms by Which It Is Disrupted in Epilepsy

“…Previously these investigators demonstrated the relevance of α2 subunit interactions with collybistin by showing that disruption of α2/collybistin interactions made mice susceptible to seizures and early death ( Hines et al, 2018 ). In recent years, the importance of collybistin, encoded by Cdc42 Guanine Nucleotide Exchange Factor 9 (ARHGEF9), to functional inhibitory synapses has become clear with the discovery of multiple patients with ARHGEF9 mutations that present with disease syndromes including epilepsy ( Shimojima et al, 2011 ; George et al, 2021 ; Hines et al, 2022 ). Additionally, a recent study in mice demonstrated that increased expression of collybistin results in elevated GABAergic signaling and mitigates the development of seizures in an SE model ( George et al, 2021 ).…”