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.
With nearly 40,000 children suffering from Dravet Syndrome in desperate need of more effective therapies, and families moving to Colorado based almost on a blind hope for a new treatment, our proposal is squarely focused on a timely and significant unmet need in the pediatric epilepsy community. Combining multiple levels of analysis across several zebrafish models of DS, this proposal aims to provide new insights and treatment for this condition.
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