GABRG2突变在癫痫性脑病发生中的机制研究

Epileptic encephalopathies are a devastating group of severe infantile and childhood onset epilepsies, which are characterized by intractable seizures, neurodevelopmental impairment, and poor prognosis. Current antiepileptic drugs are ineffective in treating this syndrome, due to the lack of understanding of its pathophysiology. Recently, we performed next generation sequencing on patients, and for the first time identified a group of six missense GABRG2 mutations that are highly associated with epileptic encephalopathies. Besides, we characterized the effects of these mutations on GABAA receptors in vitro. In the current project, we propose to generate transgenic zebrafish models carrying this group of six GABRG2 mutations using Gateway technology and Tol-2 kit system. We plan to identify their epileptic phenotypes based on seizure-like behavior, electroencephalographic responses, and the expression of epilepsy-associated gene. Moreover, we plan to provide the assessments of their neurobehavioral comorbidities using a set of assays based on diurnal rhythms, anxiety, cognitive abilities and social activities. Based on this data, we will focus on 1 or 2 mutations with the most severe phenotype, and study their effects on GABAA receptor, synapse structure/function, and gene expression in neuron. Taken together, the generation and characterization of transgenic zebrafish models carrying GABRG2 mutations could prove valuable to the understanding of genetic epileptic encephalopathies.

癫痫性脑病是婴幼儿或儿童期发病的严重癫痫综合征，以难治性癫痫、神经发育障碍以及预后不良为特征。抗癫痫药物对此病症治疗效果欠佳，原因在于对其发病机制知之甚少。我们在国际上首次采用遗传学方法从患儿身上发现一组（６个）与癫痫性脑病高度相关的错义型GABRG2突变，并在细胞水平研究了这些突变对GABAA受体的影响。本项目拟在前期工作的基础上，采用Gateway技术和Tol2-kit系统构建这组（6个）突变型GABRG2转基因斑马鱼模型。通过行为学、脑电图、癫痫标志基因表达等方面分析斑马鱼癫痫表型；从昼夜节律、焦虑水平、认知功能和社交行为等角度分析其神经行为学变化。在此基础上，重点分析1-2个表型最显著的GABRG2突变对GABAA通道受体、突触结构和功能、神经元基因表达的影响。本项目将在建立遗传性癫痫性脑病斑马鱼模型的基础上，揭示GABRG2突变导致癫痫性脑病的病理生理机制。

前期研究中我们首次从癫痫患儿体内发现一组（6个）与癫痫性脑病高度相关的错义型GABRG2突变，本项目通过Gateway克隆技术和Tol2-kit系统，成功构建了其中3种GABRG2突变的转基因斑马鱼模型[Tg(hGABRG2I107T)、Tg(hGABRG2P282S)、 Tg(hGABRG2F343L)]。癫痫评分、 神经行为学分析及癫痫标志基因表达等观察结果显示，3种GABRG2突变的斑马鱼均呈现癫痫样表型，而且其脑内GABAA受体亚基的表达发生显著变化。转录组测序结合GO和KEGG聚类分析结果显示， 3种突变的斑马鱼脑组织与野生型相比，差异基因主要存在于GABA 能受体和代谢相关通路、以及蛋白质降解等通路。研究还发现，Tg(hGABRG2 I107T)和Tg(hGABRG2F343L)斑马鱼突触相关蛋白表达发生改变，并伴有突触结构发育异常，从而使得脑内兴奋性/抑制性失衡。本研究利用突变型GABRG2转基因斑马鱼模型，从整体动物水平确定了我们在临床所发现的GARBG2突变的癫痫致病性，并初步探究其病理机制，为进一步采用哺乳类动物揭示其致病机理奠定了基础。