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

Abstract

With the wide adoption of whole-exome sequencing in children having seizures, an increasing number of SCN2A variants has been revealed as possible genetic causes of epilepsy. Voltage-gated sodium channel Nav1.2, encoded by gene SCN2A, is strongly expressed in the pyramidal excitatory neurons and supports action potential firing. One recurrent SCN2A variant is L1342P, which was identified in multiple patients with early-onset encephalopathy and intractable seizures. Our biophysical analysis and computational modeling predicted gain-of-function features of this epilepsy-associated Nav1.2 variant. 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, in which L1342P was engineered by CRISPR/Cas9 mediated genome-editing. Using patch-clamping and micro-electrode array (MEA) recording, we found that the cortical neurons derived from hiPSCs carrying heterozygous L1342P variant presented significantly increased intrinsic excitability, higher sodium current density, and enhanced bursting and synchronous network firing, showing clear hyperexcitability phenotypes. Interestingly, the L1342P neuronal culture displayed a degree of resistance to the anti-seizure medication (phenytoin), which likely recapitulated aspects of clinical observation of patients carrying the L1342P variant. In contrast, phrixotoxin-3 (PTx3), a Nav1.2 isoform-specific blocker, was able to potently alleviate spontaneous and chemical-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.

Disruption of the autism-associated gene SCN2A alters synaptic development and neuronal signaling in patient iPSC-glutamatergic neurons

“…The major finding from this line was an enhancement of the action potential waveform, similar to a human neuronal model of the SCN2A-L1342P variant (Que et al, 2021). However, the L1342P variant is gain-of-function from computational modelling and its association with epilepsy, but it has mixed gain-and loss-of-function phenotypes (Begemann et al, 2019;Que et al, 2021). Even with action potential waveform enhancements, we did not observe increases in neuronal excitability, dissimilar to the L1342P variant.…”

“…The G1744* variant is loss-of-function but the individual presents with early-onset seizures, and the variant is past the predicted non-sense mediated decay breakpoint (Nagy and Maquat, 1998;Sanders et al, 2018). The major finding from this line was an enhancement of the action potential waveform, similar to a human neuronal model of the SCN2A-L1342P variant (Que et al, 2021). However, the L1342P variant is gain-of-function from computational modelling and its association with epilepsy, but it has mixed gain-and loss-of-function phenotypes (Begemann et al, 2019;Que et al, 2021).…”

“…In G1744* iNeurons, we speculate the reduction of spikes during early development was compensated by the early onset seizures. This could explain why at later timepoints the only difference was the synchronization of the network with no bursting (Que et al, 2021;Tidball et al, 2020). Additionally, G1744* iNeurons may have compensatory activity early in development and recover, which may be due to a truncated protein being produced that avoids non-sense mediated decay.…”

“…Understanding how variants in SCN2A contribute to neurological disorders is beneficial as it may inform potential therapies or long-term clinical outcomes. Cell line and computational studies have been instrumental in understanding the potential impact of the SCN2A variants (Begemann et al, 2019;Ben-Shalom et al, 2017;Echevarria-Cooper et al, 2021;Que et al, 2021). These studies have indicated that SCN2A gain-of-function variants are likely associated with an enhancement of channel function and epileptic encephalopathy.…”

The core PCP protein Prickle2 regulates axon number and AIS maturation by binding to AnkG and modulating microtubule bundling

“…Recent studies have reported similar hyperexcitable neurons that carry ASD-and epilepsy-related mutations for SCN2A (Sodium channel protein type 2 subunit alpha) (67,68). Both epilepsy and ASD are also reported in patients with PRICKLE2 mutations (26,27), while Prickle2 knockout mice display a lower threshold for seizures (26) and ASD-like behaviors (27).…”