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

Abstract

Objective: The mutant γ-aminobutyric acid type A (GABA_A ) 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 GABA_A 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 electroencephalography (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 γ2^HA subunits, and compared GABA_A 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.

Keywords: Dravet syndrome; Epileptic encephalopathy; GABAA receptors; GABRG2(Q390X) mutation; Gene-replacement therapy; Genetic epilepsies.

Figure 1. Exogenous γ2^HA subunits were introduced in Gabrg2^+/Q390X KI mice by crossing them with Tg(hGABRG2^HA) mice, and the total amount of full length γ2 subunits in KI mice was restored by the transgene

A1. The schematic diagram shows the breeding strategy. Heterozygous Gabrg2^+/Q390X KI mice were crossed with heterozygous Tg(hGABRG2^HA) BAC transgenic mice, generating offspring with four different genotypes: wt;0 denotes Gabrg2^+/+ mice; het;0 denotes Gabrg2^+/Q390X mice; wt;Tg denotes Gabrg2^+/+;Tg(hGABRG2^HA) mice; and het;Tg denotes Gabrg2^+/Q390X;Tg(hGABRG2^HA) mice. A2. PCR and gel electrophoresis were used for genotyping, and the gel presents results from littermates with the four different genotypes. Primers amplifying the endogenous Gabrg2 allele generated one 323 bp band for the wild-type allele and one 405 bp band for the mutant allele. Primers amplifying the transgenic hGABRG2^HA allele generated one specific band of 324 bp for the transgene. B. Coronal brain sections from adult wt;0 and het;Tg mice were stained by an anti-HA antibody. C1. Cortex was collected from adult wt;0, het;0, wt;Tg, and het;Tg mice and blotted by anti-ATPase, anti-HA and anti-γ2 subunit antibodies. The anti-γ2 subunit antibody only recognized the full length γ2 subunits (including endogenous γ2 and transgenic γ2^HA subunits). C2. Expression levels of wild-type γ2 subunits in cortex and thalamus from adult wt;0, het;0, wt;Tg, and het;Tg mice were plotted. The band intensity of γ2 subunits was normalized to that of ATPase and was further normalized to that of wt;0 littermates (n = 7 for each genotype for cortical samples and n = 4 for each genotype for thalamic samples, mean ± SEM). Differences among littermates were analyzed by one way ANOVA followed by two-tailed paired t test (*** p< 0.001; ** p < 0.01; * p < 0.05).

Figure 2. The expression levels of partnering α1 and β2/3 subunits were restored in the somatosensory cortex by the hGABRG2^HA transgene

A. Immunostaining of α1 and β2/3 subunits in somatosensory cortex of wt;0, het;0 and het;Tg mice was performed. Brains from 2 month old wt;0, het;0, and het;Tg mouse littermates were fixed and sectioned on a cryostat at 30 µm. The brain cortices were stained with anti-α1 or anti-β2/3 subunit antibody, and cortical layer VI was visualized. The nuclei were stained with TO-PRO-3 (blue). Images were acquired under confocal microscopy. B. Expression levels from 12 sections from 3 different mice for each genotype were plotted. The total raw fluorescence values of the α1 or β2/3 subunits in somatosensory cortex were quantified by ImageJ. (* p < 0.05; **p < 0.01 vs wt, *p< 0.05 vs het), unpaired t test).

Figure 3. Cortical mIPSCs were restored by the hGABRG2^HA transgene

A. Representative mIPSC traces from layer 6 cortical pyramidal neurons were obtained from wt;0, het;0 and het;Tg mice. The top traces represented 50 continuous overlapped sweeps. The lower traces represented a single 5 s trace that has representative mIPSCs time-expanded to show rising and decaying current phases. Scale bars were indicated as labeled. B. The averaged mIPSC amplitudes (left panel) and the averaged mIPSC frequencies (right panel) recorded from slices from wt;0 (black, n = 11 cells), het;0 (red, n = 7 cells) and het;Tg mice (green, n = 11 cells) mice (* p<0.05; t test) were plotted. C. Normalized cumulative curves of mIPSC amplitude (left) and event interval (right) recorded from slices from wt;0 (black), het;0 (red) and het;Tg (green) mice were plotted. The mIPSCs were recorded from the same number of cells (n = 7, randomly chosen) and for the same duration of recording (10 min).

Figure 4. Increased PTZ seizure sensitivity was reversed by the hGABRG2^HA transgene

A, B. Mice were injected i.p. with PTZ (55 mg/kg) to induce seizures. The susceptibility to (A) PTZ-induced GTCS and (B) death were assessed by survival curves. Differences between littermates were analyzed by the Mantel-Cox method. (A. wt;0 vs. het;0: p = 0.0009; het;0 vs. het;Tg: p = 0.0001; wt;0 vs. het;Tg: p = 0.7713. B. wt;0 vs. het;0: p = 0.0034; het;0 vs. het;Tg: p = 0.0004; wt;0 vs. het;Tg: p = 0.5050).

Figure 5. Spontaneous and evoked thalamocortical oscillations were less intense in het;Tg mice than in het;0 mice

A. Representative extracellular multiple unit recordings (A1, spontaneous and A2, evoked) from slices from wt;0, het;0 and het;Tg mice were presented. One burst was expanded to show the multiple spikes in the burst. Scale bars were indicated as labeled. B. Autocorrelograms generated from spontaneous (B) and evoked (not shown) burst activity calculated using Clampfit. C. Summaries of averaged oscillatory index (C1) from both spontaneous and evoked burst activity for wt;0 (n = 8 slices), het;0 (n = 8 slices) and het;Tg mice (n = 11 slices) and averaged duration of oscillatory activity (C2) were presented. (* p < 0.05, t-test).