Variant-specific changes in persistent or resurgent Na+ current in SCN8A-EIEE13 iPSC-derived neurons

Missense variants in the voltage-gated sodium channel (VGSC) gene, SCN8A, are linked to early-infantile epileptic encephalopathy type 13 (EIEE13). EIEE13 patients exhibit a wide spectrum of intractable seizure types, severe developmental delay, movement disorders, and elevated risk of sudden unexpected death in epilepsy (SUDEP). The mechanisms by which SCN8A variants lead to epilepsy are poorly understood, although heterologous expression systems and mouse models have demonstrated altered sodium current (INa) properties. To investigate these mechanisms using a patient-specific model system, we generated induced pluripotent stem cells (iPSCs) from three patients with missense variants in SCN8A: p.R1872>L (P1); p.V1592>L (P2); and p.N1759>S (P3). Using small molecule differentiation into excitatory neurons, iPSC-derived neurons from all three patients displayed altered INa. P1 and P2 had elevated persistent INa, while P3 had increased resurgent INa compared to controls. Further analyses focused on one of the patients with increased persistent INa (P1) and the patient with increased resurgent INa (P3). Excitatory cortical neurons from both patients had prolonged action potential (AP) repolarization and shorter axon initial segment lengths compared to controls, the latter analyzed by immunostaining for ankyrin-G. Using doxycycline-inducible expression of the neuronal transcription factors Neurogenin 1 and 2 to synchronize differentiation of induced excitatory cortical-like neurons (iNeurons), we investigated network activity and response to pharmacotherapies. Both patient neurons and iNeurons displayed similar abnormalities in AP repolarization. Patient iNeurons showed increased burstiness that was sensitive to phenytoin, currently a standard treatment for EIEE patients, or riluzole, an FDA-approved drug used in amyotrophic lateral sclerosis and known to block persistent and resurgent INa, at pharmacologically relevant concentrations. Patch-clamp recordings showed that riluzole suppressed spontaneous firing and increased the AP firing threshold of patient-derived neurons to more depolarized potentials. Our results indicate that patient-specific neurons are useful for modeling EIEE13 and demonstrate SCN8A variant-specific mechanisms. Moreover, these findings suggest that patient-specific iPSC neuronal disease modeling offers a useful platform for discovering precision epilepsy therapies.