Abstracts

Rationale: In recent years, hundreds of novel epilepsy-associated genes have been identified thanks to advancements in next-generation sequencing and human genetics research. This many genes require functional analysis to determine which ones are truly responsible for epilepsy and through which mechanisms defects in these genes lead to the circuit dysfunction that defines epilepsy.

Methods: We developed a high-throughput screening pipeline using zebrafish to identify which of the many genes associated with monogenic human epilepsies are amenable to functional investigation in this system. We amassed a large list of epilepsy candidate genes from the literature. We modeled genes that do not tolerate variation in humans (gnomAD pLI > 0.9, missense Z-score > 3) and that have at least 60% homology between zebrafish and human. We injected constructs designed using a computational guide design algorithm (https://chopchop.cbu.uib.no/) into fertilized zebrafish eggs at the 1-cell stage and bred genetic lines to at least the F2 generation. We assayed larvae of 81 mutant lines at 5 days post fertilization (dpf) for seizure-like activity and hyperexcitability via the following methods: (a) high-throughput automated behavioral assessment for seizure-like swimming patterns; (b) whole brain calcium imaging for increased fluorescence; (c) local field potential recording for direct electrophysiological evidence of hyperexcitability; (d) quantification of interneurons using a transgenic line that labels developing inhibitory interneurons.

Results: Analysis of Crispant vs. WT zebrafish larval behavior in 81 genetic mutant lines revealed 7 promising, functionally heterogenous, candidate genes, which were further characterized by whole brain calcium imaging and single electrode field-recording in larval tectum. Most strikingly abnormal was the potassium channel modulator Kcnv1, which, in addition to displaying seizure-like swim patterns, showed abnormal electrophysiology on LFP recordings, increased neuronal activity by calcium imaging, and a decrease in inhibitory neurons.

Conclusions: We demonstrate a high-throughput and cost-effective method for initial functional assessment of epilepsy-associated genes. Using high-throughput assessment for seizure-like swim patterns, 7 of 81 genetic models displayed abnormalities that were additionally associated with other measures of hyperexcitability. In conclusion, the zebrafish system represents a robust platform to rapidly identify genetic models of epilepsy. These models can then be used to dissect underlying mechanisms of epileptogenesis as well as to screen for potential therapeutic compounds that might rectify the tendency to seizures.

Funding: Please list any funding that was received in support of this abstract.: BCH Translational Research Program.