ZEBRAFISH MODELS FOR DRAVET SYNDROME RESEARCH AND DISCOVERY

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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.