Overexpressing wild‐type γ2 subunits rescued the seizure phenotype in Gabrg2^+/Q390X Dravet syndrome mice

Abstract

Summary Objective The mutant γ-aminobutyric acid type A (GABAA) receptor γ2(Q390X) subunit, (Q351X in the mature peptide) has been associated with the epileptic encephalopathy, Dravet syndrome, and the epilepsy syndrome genetic epilepsy with febrile seizures plus (GEFS+). The mutation generates a premature stop codon that results in translation of a stable truncated and misfolded γ2 subunit that accumulates in neurons, forms intracellular aggregates, disrupts incorporation of γ2 subunits into GABAA receptors and affects trafficking of partnering α and β subunits. Heterozygous Gabrg2+/Q390X knock-in (KI) mice had reduced cortical inhibition, spike wave discharges on EEG, a lower seizure threshold to the convulsant drug pentylenetetrazol (PTZ) and spontaneous generalized tonic-clonic seizures. In this proof of principal study, we attempted to rescue these deficits in KI mice using a γ2 subunit gene (GABRG2) replacement therapy. Methods We introduced the GABRG2 allele by crossing Gabrg2+/Q390X KI mice with bacterial artificial chromosome (BAC) transgenic mice overexpressing HA (hemagglutinin) tagged human γ2HA subunits and compared GABAA receptor subunit expression by western blot and immunohistochemical staining, seizure threshold by monitoring mouse behavior after PTZ-injection, and thalamocortical inhibition and network oscillation by slice recording. Results Compared to KI mice, adult mice carrying both mutant allele and transgene had increased wild-type γ2 and partnering α1 and β2/3 subunits, increased miniature inhibitory postsynaptic current (mIPSC) amplitudes recorded from layer VI cortical neurons, reduced thalamocortical network oscillations, and higher PTZ seizure threshold. Significance Based on these results we suggest that seizures in a genetic epilepsy syndrome caused by epilepsy mutant γ2(Q390X) subunits with dominant negative effects could be rescued potentially by overexpression of wild-type γ2 subunits.

A novel GABAergic dysfunction in human Dravet syndrome

“…All these mutations produce altered channels that are probably the primary cause of epileptic phenotype and intellectual disabilities in DS . Nonetheless, mutations in the γ‐aminobutyric acid type A receptor (GABA A R) subunit genes cause DS‐like phenotypes in both animal models and humans …”

Molecular basis for and chemogenetic modulation of comorbidities in GABRG2‐deficient epilepsies

“…In Gabrg2-deficient mice, anxiety was caused by reduction of functional γ2 subunit-containing receptors, whereas enhancing synaptic GABA A receptor function rescued anxiety, similar to epilepsy. 33 Findings from this study suggest that the pathophysiology from the mutation itself may directly contribute to anxiety ( Figure S2). test, the total distance traveled (E) and the ratio of time in center versus periphery (F) were measured.…”

“…We propose that functional impairment in other neuroanatomical substrates could give rise to corresponding comorbid phenotypes via mechanisms similar to those for epilepsy, depending on specific neurocircuits involved. In Gabrg2 deficient mice, anxiety was caused by reduction of functional γ2 subunit-containing receptors while enhancing synaptic GABA A receptor function rescued anxiety, similar to epilepsy 32 . Findings from this study suggest that the pathophysiology from the mutation itself may directly contribute to anxiety (Supplementary Figure 2).…”

The K328M substitution in the human GABAA receptor gamma2 subunit causes GEFS+ and premature sudden death in knock-in mice

“…Brain slices were prepared according to previously published methods ( Zhou et al, 2011 ; Huang et al, 2017 ). Young adult mice (~1-month-old, either gender) were deeply anesthetized with isoflurane and transcardially perfused with ice-cooled dissection solution (4 °C) (mM: 2.5 KCl, 0.5 CaCl 2 , 10 MgSO 4 , 1.25 NaH 2 PO 4 , 24 NaHCO 3 , 11 Glucose, 214 Sucrose).…”