Huang Xuan, Zhou Chengwen, Tian Mengnan, Kang Jing-Qiong, Shen Wangzhen, Verdier Kelienne, Pimenta Aurea, MacDonald Robert L
The Graduate Program of Neuroscience, Vanderbilt University Medical Center, Nashville, Tennessee, U.S.A.
Department of Neurology, Vanderbilt University Medical Center, Nashville, Tennessee, U.S.A.
Epilepsia. 2017 Aug;58(8):1451-1461. doi: 10.1111/epi.13810. Epub 2017 Jun 6.
The mutant γ-aminobutyric acid type A (GABA ) 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 receptors, and affects trafficking of partnering α and β subunits. Heterozygous Gabrg2 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.
We introduced the GABRG2 allele by crossing Gabrg2 KI mice with bacterial artificial chromosome (BAC) transgenic mice overexpressing HA (hemagglutinin)-tagged human γ2 subunits, and compared GABA 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.
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.
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.
突变的γ-氨基丁酸A型（GABA）受体γ2（Q390X）亚基（成熟肽中为Q351X）与癫痫性脑病、德雷维特综合征以及伴有热性惊厥附加症的癫痫综合征（GEFS +）相关。该突变产生一个过早的终止密码子，导致翻译出一个稳定的截短且错误折叠的γ2亚基，其在神经元中积累，形成细胞内聚集体，破坏γ2亚基并入GABA受体，并影响配对的α和β亚基的运输。杂合的Gabrg2基因敲入（KI）小鼠皮质抑制作用减弱，脑电图（EEG）出现棘波放电，对惊厥药物戊四氮（PTZ）的惊厥阈值降低，并且出现自发性全身性强直阵挛发作。在这项原理验证研究中，我们尝试使用γ2亚基基因（GABRG2）替代疗法来挽救KI小鼠的这些缺陷。
我们通过将Gabrg2 KI小鼠与过表达HA（血凝素）标记的人γ2亚基的细菌人工染色体（BAC）转基因小鼠杂交来引入GABRG2等位基因，并通过蛋白质免疫印迹和免疫组织化学染色比较GABA受体亚基表达，通过监测PTZ注射后小鼠行为来比较惊厥阈值，通过脑片记录来比较丘脑皮质抑制和网络振荡。
与KI小鼠相比，携带突变等位基因和转基因的成年小鼠野生型γ2以及配对的α1和β2/3亚基增加，从VI层皮质神经元记录到的微小抑制性突触后电流（mIPSC）幅度增加，丘脑皮质网络振荡减少，并且PTZ惊厥阈值更高。
基于这些结果，我们认为由具有显性负效应的癫痫突变γ2（Q390X）亚基引起的遗传癫痫综合征中的癫痫发作可能通过野生型γ2亚基的过表达得到挽救。
