Functional studies of epilepsy mutations in Drosophila and human iPSC-derived neu

描述(由申请人提供)：编码Nav1.1电压门控钠通道的SCN1A基因突变导致多种人类癫痫发作障碍。这些疾病包括德雷特综合征(DS)和遗传性癫痫伴发热性惊厥(GEFS+)。DS和GEFS+都是常染色体显性遗传病，但对癫痫发生的细胞机制知之甚少。在这里，我们建议使用两个互补的遗传模型系统来评估致病突变对神经元活动的功能后果：带有SCN1A突变的敲入果蝇和具有相同突变的患者的IPSC衍生神经元。我们的初步数据表明，在果蝇钠通道基因PARA中敲入GEFS+SCN1A突变(K1270T)，会导致温度诱导的半显性癫痫表型。对成年GEFS+果蝇大脑中GABA能中间神经元的电生理学研究揭示了热诱导癫痫发作的一种新的细胞机制。与人类的疾病症状一致，DS突变(S1231R)的敲入引起的癫痫表型比GEFS+更严重。在这个遗传疾病模型中，果蝇和人类之间的基因到表型图的一致性为利用敲入果蝇来研究这些复杂的人类遗传疾病的机制铺平了道路。前两个目标集中在利用果蝇钠通道敲入线来进一步探索导致热诱导癫痫的潜在细胞机制，并作为一个低成本、高效率的平台来发现遗传修饰物和抑制癫痫表型的药物。在具体目标3中，我们将利用我们在干细胞生物学方面的专业知识，对来自具有相同GEFS+突变的患者的IPSC衍生神经元的神经元活动进行平行研究，这些患者在敲入果蝇中被检测到相同的GEFS+突变。识别这两个模型系统中的共同细胞机制有可能确定新疗法的开发靶点，以减少或消除人类癫痫发作。

DESCRIPTION (provided by applicant): Mutations in the SCN1A gene encoding Nav1.1 voltage-gated sodium channels result in a variety of human seizure disorders. These include Dravet Syndrome (DS) and genetic epilepsy with febrile seizures plus (GEFS+). Both DS and GEFS+ are autosomal dominant disorders but relatively little is known about the cellular mechanisms underlying seizure generation. Here we propose to assess the functional consequences of disease causing mutations on neuronal activity using two complementary, genetic model systems: knock-in Drosophila with SCN1A mutations and iPSC-derived neurons from patients with the same mutations. Our preliminary data demonstrate that knock-in of a GEFS+ SCN1A mutation (K1270T) into the Drosophila sodium channel gene, para, causes a semi-dominant temperature-induced seizure phenotype. Electrophysiological studies of GABAergic interneurons in the brains of adult GEFS+ flies reveal a novel cellular mechanism underlying heat-induced seizure. Consistent with disease symptoms in humans, the seizure phenotype caused by knock-in of a DS mutation (S1231R) is more severe than GEFS+. The congruence of the genotype-to-phenotype map between flies and human in this genetic disease model paves the way for use of knock-in Drosophila to study the mechanisms underlying these complex human genetic disorders. The first two aims are focused on use of Drosophila sodium channel knock-in lines to further explore the underlying cellular mechanisms contributing to heat-induced seizures and as a low cost, high efficiency, platform for discovery of genetic modifiers and drugs that suppress the seizure phenotype. In specific Aim 3 we will employ our expertise in stem cell biology to conduct parallel studies of neuronal activity in iPSC-derived neurons from patients with the same GEFS+ mutations examined in knock-in flies. Identification of common cellular mechanisms in these two model systems has the potential to identify targets for development of novel therapies to reduce or eliminate seizures in humans with epilepsy.