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

果蝇和人类 iPSC 衍生神经元癫痫突变的功能研究

• 批准号: 8990893
• 负责人: DIANE K O'DOWD
• 金额: $ 52.62万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-02-15 至 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8990893
• 关键词: 5-Hydroxytryptophan; 6 year old; Action Potentials; Address; Adult; Affect; Behavior; Behavioral Assay; Biological Models; Brain; Comorbidity; Complex; Data; Development; Developmental Delay Disorders; Disease; Disease model; Drosophila genus; Electrophysiology (science); Epilepsy; Exhibits; Febrile Convulsions; Fever; Fibroblasts; Fire - disasters; Generations; Genes; Genetic; Genetic Models; Genotype; Health; Heating; Hereditary Disease; Human; Human Genetics; Individual; Interneurons; Ion Channel; Knock-in; Life; Maps; Mutation; Neurons; Patients; Pharmaceutical Preparations; Phenotype; Preclinical Drug Evaluation; Predisposition; Property; Publishing; Recurrence; Reporting; SCN1A protein; Seizures; Serotonergic System; Serotonin; Siblings; Signal Transduction; Sodium; Sodium Channel; Symptoms; Syndrome; Temperature; Testing; Therapeutic Agents; Whole-Cell Recordings; cell type; cost; disease-causing mutation; early childhood; feeding; fly; induced pluripotent stem cell; interest; monoamine; nerve stem cell; nervous system disorder; new therapeutic target; novel; novel therapeutics; relating to nervous system; research study; stem cell biology; voltage

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.

描述（由申请人提供）：编码Nav1.1电压门控钠通道的SCN 1A基因突变导致多种人类癫痫发作疾病。这些包括Dravet综合征（DS）和遗传性癫痫伴热性惊厥+（GEFS+）。DS和GEFS+都是常染色体显性遗传疾病，但对癫痫发作的细胞机制知之甚少。在这里，我们建议使用两种互补的遗传模型系统来评估致病突变对神经元活性的功能后果：具有SCN 1A突变的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，敲入的GEFS+ SCN 1A突变（K1270 T）到果蝇钠通道基因，帕拉，导致半显性温度诱导癫痫发作表型。成年GEFS+果蝇脑中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（S1231 R）敲入引起的癫痫发作表型比GEFS+更严重。在这种遗传疾病模型中，果蝇和人类之间的基因型-表型图谱的一致性为使用敲入果蝇研究这些复杂的人类遗传疾病的机制铺平了道路。前两个目标集中在使用果蝇钠通道敲入系，以进一步探索导致热诱导癫痫发作的潜在细胞机制，并作为低成本，高效率的平台，用于发现抑制癫痫发作表型的遗传修饰剂和药物。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衍生神经元中的神经元活性进行平行研究，这些突变在敲入果蝇中进行了检查。在这两个模型系统中识别共同的细胞机制有可能识别用于开发新疗法以减少或消除癫痫患者癫痫发作的靶点。