Hyperexcitability and Pharmacological Responsiveness of Cortical Neurons Derived from Human iPSCs Carrying Epilepsy-Associated Sodium Channel Nav1.2-L1342P Genetic Variant

Abstract

With the wide adoption of genomic sequencing in children having seizures, an increasing number of SCN2A genetic variants have been revealed as genetic causes of epilepsy. Voltage-gated sodium channel Nav1.2, encoded by gene SCN2A, is predominantly expressed in the pyramidal excitatory neurons and supports action potential (AP) firing. One recurrent SCN2A genetic variant is L1342P, which was identified in multiple patients with epileptic encephalopathy and intractable seizures. However, the mechanism underlying L1342P-mediated seizures and the pharmacogenetics of this variant in human neurons remain unknown. To understand the core phenotypes of the L1342P variant in human neurons, we took advantage of a reference human-induced pluripotent stem cell (hiPSC) line from a male donor, in which L1342P was introduced by CRISPR/ Cas9-mediated genome editing. Using patch-clamping and microelectrode array (MEA) recordings, we revealed that cortical neurons derived from hiPSCs carrying heterozygous L1342P variant have significantly increased intrinsic excitability, higher sodium current density, and enhanced bursting and synchronous network firing, suggesting hyperexcitability phenotypes. Interestingly, L1342P neuronal culture displayed a degree of resistance to the anticonvulsant medication phenytoin, which recapitulated aspects of clinical observation of patients carrying the L1342P variant. In contrast, phrixotoxin-3 (PTx3), a Nav1.2 isoform-specific blocker, can potently alleviate spontaneous and chemically-induced hyperexcitability of neurons carrying the L1342P variant. Our results reveal a possible pathogenic underpinning of Nav1.2-L1342P mediated epileptic seizures and demonstrate the utility of genome-edited hiPSCs as an in vitro platform to advance personalized phenotyping and drug discovery.

Cellular and behavioral effects of altered NaV1.2 sodium channel ion permeability in Scn2a K1422E mice

“…These effects can be considered GoF or LoF with regard to individual channels, but the effect of a given variant of the intrinsic properties of neurons represents a complex summation of channel-level effects ( 1 , 4 , 38 ). For example, the DEE-associated variants SCN2A -p.L1342P and p.S1336Y were both shown to have mixed effects on channel function, but were considered to be overall GoF with regard to neuronal excitability ( 5 , 38 , 92 ). However, these variants have different effects on channel function than those we observed for K1422E.…”

Early maturation and hyperexcitability is a shared phenotype of cortical neurons derived from different ASD-associated mutations

“…Here we concentrated on the early developmental physiological alterations in 4 different ASD and epilepsy-related genes. The enhanced maturation and excitability in such young neurons may be deleterious to the cells and may later result in synaptic degeneration as was previously described in neurons derived from ASD and epilepsy patients 70,[75][76][77][78][79][80] . Fig.…”

Early maturation and hyperexcitability is a shared phenotype of cortical neurons derived from different ASD-causing mutations

“…Here we concentrated on the early developmental physiological alterations in 4 different ASD and epilepsy-related genes. The enhanced maturation and excitability in such young neurons may be deleterious to the cells and may later result in synaptic degeneration as was previously described in neurons derived from ASD and epilepsy patients 70,[75][76][77][78][79][80] . The mean rate of synaptic events was higher in the dup7-mutant neurons compared to control neurons (p=0.029).…”

“…Our Findings present a shared phenotype of early maturation and hyperexcitability in four ASD-related mutations using patient-derived cortical neurons, indicating that there may be a common neurophysiological phenotype in ASD-related variants, sharing similar behavioral phenotypes but a different genotype. iPSC-derived neurons were previously used as a research tool for investigating physiological and cellular alterations characterizing various disorders including autism 70,76,78 and epilepsy 77,79,80 . Here we concentrated on the early developmental physiological alterations in 4 different ASD and epilepsy-related genes.…”