Variant-specific changes in persistent or resurgent sodium current in SCN8A-related epilepsy patient-derived neurons

Abstract

Missense variants in the SCN8A voltage-gated sodium channel gene are linked to early-infantile epileptic encephalopathy type 13, also known as SCN8A-related epilepsy. These patients exhibit a wide spectrum of intractable seizure types, severe developmental delay, movement disorders, and elevated risk of sudden unexpected death in epilepsy. The mechanisms by which SCN8A variants lead to epilepsy are poorly understood, although heterologous expression systems and mouse models have demonstrated altered sodium current properties. To investigate these mechanisms using a patient-specific model, we generated induced pluripotent stem cells from three patients with missense variants in SCN8A: p.R1872>L (Patient 1); p.V1592>L (Patient 2); and p.N1759>S (Patient 3). Using small molecule differentiation into excitatory neurons, induced pluripotent stem cell-derived neurons from all three patients displayed altered sodium currents. Patients 1 and 2 had elevated persistent current, while Patient 3 had increased resurgent current compared to controls. Neurons from all three patients displayed shorter axon initial segment lengths compared to controls. Further analyses focused on one of the patients with increased persistent sodium current (Patient 1) and the patient with increased resurgent current (Patient 3). Excitatory cortical neurons from both patients had prolonged action potential repolarization. Using doxycycline-inducible expression of the neuronal transcription factors neurogenin 1 and 2 to synchronize differentiation of induced excitatory cortical-like neurons, we investigated network activity and response to pharmacotherapies. Both small molecule differentiated and induced patient neurons displayed similar abnormalities in action potential repolarization. Patient induced neurons showed increased burstiness that was sensitive to phenytoin, currently a standard treatment for SCN8A-related epilepsy patients, or riluzole, an FDA-approved drug used in amyotrophic lateral sclerosis and known to block persistent and resurgent sodium currents, at pharmacologically relevant concentrations. Patch-clamp recordings showed that riluzole suppressed spontaneous firing and increased the action potential firing threshold of patient-derived neurons to more depolarized potentials. Two of the patients in this study were prescribed riluzole off-label. Patient 1 had a 50% reduction in seizure frequency. Patient 3 experienced an immediate and dramatic seizure reduction with months of seizure freedom. An additional patient with a SCN8A variant in domain IV of Nav1.6 (p.V1757>I) had a dramatic reduction in seizure frequency for several months after starting riluzole treatment, but then seizures recurred. Our results indicate that patient-specific neurons are useful for modelling SCN8A-related epilepsy and demonstrate SCN8A variant-specific mechanisms. Moreover, these findings suggest that patient-specific neuronal disease modelling offers a useful platform for discovering precision epilepsy therapies.

Sodium channel Nav1.2-L1342P variant displaying complex biophysical properties renders hyperexcitability of cortical neurons derived from human iPSCs

“…Studies found that enhanced excitability of hiPSC-derived excitatory neurons is the primary mechanism underlying the gain-of-function Nav1.6 variant related seizure. 64 Loss-of-function variants of Nav1.6 are also found in patients, but these patients did not develop epileptic phenotypes. 65 In contrast, the association between variants in Nav1.2 and seizure are more complicated.…”

“…Epilepsyassociated Nav1.6 variants are suggested to be gain-of-function. Study found that these gain-of-function Nav1.6 mutations led to enhanced excitability of excitatory neurons derived from hiPSCs, which are suggested as the main mechanism underlying the Nav1.6 variant related seizure (Tidball et al, 2020). Loss-of-function variants of Nav1.6 are also found in patients, but these patients do not have develop epileptic phenotypes (Gardella and Moller, 2019).…”

“…Pairing with MEA, a highthroughput, extracellular recording assay of neuronal activity (Odawara et al, 2016) (Odawara et al, 2018Tukker et al, 2020b), study has shown a successful application of patient-specific disease modeling to discover epilepsy therapies (Tidball et al, 2020). Using the hiPSC-derived neurons, it was found that riluzole and phenytoin can target specific Nav1.6 variants to attenuate bursting phenotypes in vitro, and suppress patient seizure events in the clinical study (Tidball et al, 2020). Here we showed that hiPSCderived neurons carrying the drug-resistant Nav1.2-L1342P variant are not sensitive to phenytoin, but have equal or even stronger sensitivity towards an experimental isoformspecific Nav1.2 blocker.…”

“…Pairing with MEA, a high-throughput, extracellular recording assay of neuronal activity, 57,74,75 such studies have been attempted with neurons carrying Nav1.6 variants. 64 Using the hiPSC-derived neurons, it was recently found that riluzole and phenytoin can target specific Nav1.6 variants to attenuate bursting phenotypes in vitro. Importantly, some effective compounds identified in the hiPSC model successfully suppress seizure events of patients in the clinical study, highlighting the translational value of the in vitro hiPSC-based platform.…”

“…Importantly, some effective compounds identified in the hiPSC model successfully suppress seizure events of patients in the clinical study, highlighting the translational value of the in vitro hiPSC-based platform. 64 While the use of patient-derived hiPSC line is desirable and could directly model subject-specific phenotypes and interindividual differences, 47 WT and L13432P neuron culture after KA-stimulation. (E) PTx3 significantly inhibited the bursting frequency in both WT and L13432P neuron culture after KA-stimulation.…”