Project Summary Epilepsy affects up to 1% of the population worldwide, and 3 million in the United States alone. A growing proportion of pediatric epilepsies are tied to causative variants in ion channel genes, including the voltage-gated sodium channel gene SCN2A. The 2020 Epilepsy Research Benchmarks of NINDS prioritize identifying how genetic variants cause epilepsy and related neurodevelopmental disorders. SCN2A variants that manifest with loss-of-function are associated with severe neurodevelopmental disorders and late-onset epilepsy. On the other hand, gain-of-function SCN2A variants predominantly have a phenotype of early-onset epilepsy. The encoded sodium channel (NaV1.2) is highly expressed in excitatory glutamatergic neurons early in development, presenting a unique opportunity to examine how excitatory neuron dysfunction leads to early-onset epilepsy. Animal and human tissue-derived neuron models have brought mechanistic insight to how Dravet syndrome results in interneuron dysfunction and epilepsy. Among SCN2A-related diseases, animal models illuminate how loss-of-function leads to autism spectrum disorder with late-onset epilepsy. Due to lack of readily available disease models, there is sparse mechanistic understanding of how excitatory neuron dysfunction early in development leads to early-onset epilepsy. This proposal will exploit two early-onset epilepsy variants of SCN2A that have a convergent clinical phenotype yet divergent biophysical mechanisms. Patient-derived neuron models and mouse models provide the opportunity to define the point of mechanistic convergence at multiple scales: from single neurons to neural circuits influencing epilepsy phenotype. Aim 1 will determine how two gain- of-function SCN2A variants, encoding missense mutations M1879T and E430A, confer increased excitability by distinct mechanisms. Functional analysis of iPSC-derived neurons in isolation and in elementary circuits will define how the different variants impact excitability and thus converge toward an epileptic phenotype. Aim 2 will define hippocampal higher-level circuit perturbations in epileptic mice designed with genome editing to recapitulate the SCN2A-E430A human epileptic encephalopathy. Ex vivo analysis of changes in excitability, synaptic signaling, and network output in the hippocampus will lead to new understanding of how gain-of-function SCN2A variants affect neuronal networks. EEG and depth electrodes will provide spatiotemporal correlate to the in vivo epilepsy phenotype. This proposal will propel the awardee to independence as a physician-scientist by incorporating new expertise in multi-scale modeling of genetic epilepsy, focused relevant didactics, and a diverse career development team specializing in neurodevelopmental and genetic disorders, all in a highly collaborative environment fostering junior faculty development. This award will provide a platform to 1) define variant-specific contributions to epilepsy phenotype in self-lim...