ZEBRAFISH MODELS FOR DRAVET SYNDROME RESEARCH AND DISCOVERY

项目概要/摘要 Dravet综合征（DS）是一种灾难性的儿童癫痫，与严重的智力残疾有关， 社会发展受损、持续的耐药性癫痫发作和突发意外死亡的高风险， 癫痫我们最近对斑马鱼钠通道功能丧失突变体（scn1a）的研究 突变（例如，在约80%的DS患者中发现了基因突变），专注于药物发现， 发育、代谢功能障碍和行为共病。使用基于表型的高通量 筛选策略和药物化学，我们筛选了近3000种药物，成功地确定了一种 克立咪唑抗癫痫作用5-羟色胺（5-HT）受体机制，并开发了三种新的 克立咪唑类似物。使用CRISPR/Cas9基因组编辑技术，我们产生了新的斑马鱼突变系。 对于CHD 2、GABRB 3、PCDH 19和STXBP1（例如，在约20%的DS患者中观察到新发突变）。有趣的是， STXBP 1和GABRB 3突变体表现出癫痫表型。我们还设计和制造了一个微流体， 一种用于斑马鱼幼鱼长期无创电生理的多通道电极集成平台 (Hong等人，2016年），并开发了一种钙成像分析管道，用于研究癫痫发作的宏观和微观 体内网络（Liu and Baraban 2019）。拟议的工作将利用这些独特的工具。三个具体 提出的目标：（i）解决神经网络负责癫痫发作的幼虫DS斑马鱼，（ii）执行 使用斑马鱼的高通量药物筛选和（iii）进一步评估克立咪唑和相关的抗癫痫 化合物.技术将包括自动运动跟踪，体内斑马鱼电生理学， 药理学和使用遗传编码的钙和电压敏感指示剂的快速钙成像。 我们的结果承诺同时推进我们的长期目标（i）更好地了解 遗传性癫痫的病理生理学和（ii）确定有前途的新的治疗选择，这些难治性癫痫 条件

Project Summary/Abstract Dravet syndrome (DS), a catastrophic childhood epilepsy, is associated with severe intellectual disability, impaired social development, persistent drug-resistant seizures and a high risk of sudden unexpected death in epilepsy. Our recent investigation of zebrafish mutants featuring a loss-of-function sodium channel (scn1a) mutation (e.g., a gene mutation identified in ~80% of DS patients) focused on drug discovery and development, metabolic dysfunction and behavioral comorbidities. Using a high-throughput phenotype-based screening strategy and medicinal chemistry, we screened nearly 3000 drugs, successfully identified a serotonin (5HT) receptor mechanism underlying anti-seizure activity of clemizole and developed three novel clemizole analogs. Using CRISPR/Cas9 genome editing technology we generated new zebrafish mutant lines for chd2, gabrb3, pcdh19 and stxbp1 (e.g., de novo mutations seen in ~20% of DS patients). Interestingly, stxbp1 and gabrb3 mutants exhibit epileptic phenotypes. We also designed and manufactured a microfluidic, multi-channel electrode-integrated platform for long-term non-invasive electrophysiology on larval zebrafish (Hong et al. 2016) and developed a calcium imaging-analysis pipeline for studying seizure macro- and micro- networks in vivo (Liu and Baraban 2019). The proposed work will leverage these unique tools. Three specific aims are proposed: (i) to resolve neural networks responsible for seizures in larval DS zebrafish, (ii) to perform high-throughput drug screening using zebrafish and (iii) to further evaluate clemizole and related anti-seizure compounds. Techniques will include automated locomotion tracking, in vivo zebrafish electrophysiology, pharmacology, and fast calcium imaging using genetically encoded calcium- and voltage-sensitive indicators. Our results promise to simultaneously advance our long-term goals (i) to better understand the pathophysiology of genetic epilepsies and (ii) identify promising new treatment options for these intractable conditions.