Abstracts

Rationale: The SCN8A gene encodes for the voltage-gated sodium channel (VGSC) Nav1.6, the most abundant Nav channel. De novo mutations in SCN8A have recently been found to cause an early infantile epileptic encephalopathy, EIEE13. More than 100 EIEE13 patients have been identified with disease typically manifesting as intractable seizures, intellectual disability and high rates of SUDEP (Sudden Unexpected Death in Epilepsy). The vast majority of patients have missense, putative gain-of-function mutations. The goals of our work are to generate EIEE13 patient-derived neurons, characterize their electrophysiological alterations caused by SCN8A mutations and develop a novel platform for identifying effective pharmacological agents for this debilitating disease. Methods: We obtained dermal fibroblasts from two EIEE13 patients with SCN8A missense mutations (Arg1872>Leu and Val1592>Leu) and compared them to two unrelated controls. The fibroblasts were reprogrammed into induced pluripotent stem cells (iPSCs) and then differentiated into excitatory cortical-like neurons. We analyzed the electrophysiological attributes of these cells using whole-cell patch-clamp recordings and multi-well multielectrode arrays. Results: We discovered an increase in the percentage of peak transient sodium current density that is persistent or non-inactivating (control, 2.5%; patient 1, 6.2%; patient 2, 5.3%) and decreased peak transient sodium current density (-284 pA/pF in control; -136 pA/pF for patient 1; -197 pA/pF for patient 2) in the patient neurons. The same iPSC-derived neurons were also cultured on multi-well multieclectrode array plates to measure spontaneous action potentials before and after acute exposure to anti-epileptic drugs (AEDs). We used two AEDs known to have efficacy in the patient population (oxcarbazepine and lamotrigine), one AED thought to be ineffective (levetiracetam), and one compound shown to preferentially bind to VGSCs in the inactivated state (riluzole). All of the drugs, except levetiracetam, partially inhibited activity at concentrations found in patient CSF and showed clear dose-dependent inhibition. Interestingly, at low concentrations, riluzole had a significantly greater effect on control cells (72% inhibition) than patient cells (23% inhibition) potentially indicating dysfunctional inactivation in the mutant channels. Although riluzole had a stronger inhibitory effect on overall activity in controls, the most distinct characteristics of the EIEE13 neurons (increased spikes/burst and burst duration compared to controls) were more strongly attenuated by riluzole in patient neurons than in control neurons. Conclusions: Our data suggest that increased persistent sodium current is a pathogenic mechanism in EIEE13. Our MEA data also suggest that dysfunctional channel inactivation is responsible for this alteration. We are continuing to characterize the effects of riluzole and other drugs, and are applying machine-learning algorithms to the MEA data to more easily identify disease phenotypes. Funding: NINDS: NS090364 (JP), NS058585 (JP) AES/WFE Postdoctoral Fellowship (AT)