Using Drosophila Models to Understand Patient-Specific Mechanisms of Genetic Seizure Disorders

While thousands of epilepsy-causing mutations have been identified throughout the SCN1A gene encoding voltage-gated sodium channel Nav1, a protein crucial for regulating neuronal excitability, how most of these mutations lead to seizures is unknown. To effectively treat patients, it is important to understand how individual mutations alter cellular and channel properties and affect the brain. My thesis has focused on two different mutations occurring at one position within SCN1A: R1648C associated with the severe disorder Dravet Syndrome, and R1648H, with the milder disorder GEFS+. It is not yet possible to examine cellular activity in the living human brain, so we used genetically modified fruit flies (Drosophila) to explore how these two different mutations at the same amino acid location contribute to different behavioral symptoms and diagnostic outcomes. We used CRISPR-Cas9 gene editing to create Drosophila lines with the R-C or R-H mutation, or R-R control substitution in the fly sodium channel gene para. Animals heterozygous for R-C or R-H mutations displayed reduced lifespans, altered circadian behavior and sleep, and spontaneous and temperature-induced seizures not observed in R-R controls. These behavioral phenotypes, particularly heat induced (“febrile”) seizures, are reminiscent of symptoms observed in patients. To examine the underlying cellular mechanisms, we obtained electrophysiological recordings from neurons in the intact brains of adult flies. In GABAergic inhibitory neurons, the R-C and R-H mutants exhibited sustained neuronal depolarizations and altered firing frequency that were exacerbated at elevated temperature. R-H and R-C inhibitory neurons also displayed similar and constitutively hyperpolarized sodium current deactivation thresholds. In contrast, excitatory cholinergic neurons carrying R-C or R-H did not display sustained depolarizations or other obvious differences from controls, implicating decreased inhibition as the main neuronal impact of R1648 mutations. Further, the similarity of the effect of the R-C and R-H mutations in Drosophila with identical genetic backgrounds suggests that genetic modifiers play a crucial role in human presentations of seizure disorders. Finally, this work with SCN1A mutant Drosophila was conducive to the creation of a classroom module connecting biological principles used in lecture to real-world research, and resulted in high engagement for both undergraduate students and graduate teaching assistant