ZEBRAFISH MODELS FOR DRAVET SYNDROME RESEARCH AND DISCOVERY

项目摘要/摘要 德拉韦综合征(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、持续的抗药性癫痫和高风险的意外猝死 癫痫。我们最近对具有功能丧失钠通道(SCN1A)的斑马鱼突变体的研究 突变(例如，在约80%的DS患者中发现的基因突变)集中在药物发现和 发育、代谢功能障碍和行为并存。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，成功地鉴定了一种 克霉唑抗癫痫作用的5-羟色胺受体机制及开发的三种新药物 克莱咪唑类似物。利用CRISPR/Cas9基因组编辑技术，我们获得了新的斑马鱼突变系 CHD2、GABRB3、pcdh19和STXBP1(例如，约20%的DS患者出现从头突变)。有趣的是， STXBP1和GABRB3突变体表现出癫痫表型。我们还设计和制造了一种微流控， 斑马鱼仔鱼长期无创电生理多通道电极集成平台 (Hong et al.2016)，并开发了用于研究癫痫宏观和微观的钙成像分析流水线。 活体网络(Liu和Baraban 2019)。拟议的工作将利用这些独特的工具。三个具体的 提出的目标是：(I)解决导致DS斑马鱼幼体癫痫发作的神经网络，(Ii)执行 利用斑马鱼进行高通量药物筛选和(Iii)进一步评价克霉唑及其相关的抗癫痫作用 化合物。这些技术将包括自动运动跟踪，活体斑马鱼电生理学， 药理学，以及使用遗传编码的钙和电压敏感指示器的快速钙成像。 我们的结果承诺同时推进我们的长期目标(I)以更好地理解 遗传性癫痫的病理生理学和(Ii)确定这些顽固性癫痫有希望的新治疗选择 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.