Abstracts

Rationale: Genetic variation in SCN3A, which encodes the voltage-gated sodium (Na+) channel a subunit Nav1.3, is associated with SCN3A neurodevelopmental disorders (SCN3A-NDD), a spectrum that includes epilepsy and malformation of cortical development (MCD). SCN3A is highly expressed in the embryonic forebrain; however, it remains unclear how genetic variation in SCN3A leads to these striking clinical features, in part due to a lack of model systems available for study.


Methods: To investigate the mechanisms of SCN3A-NDD, we modified a control human induced pluripotent stem cell (iPSC) line using CRISPR/Cas9 gene editing to generate a set of isogenic lines, each expressing a heterozygous pathogenic SCN3A variant associated with a characteristic/defined clinical phenotype within the SCN3A spectrum. Using the well-established Ngn2 rapid induction protocol, we generated glutamatergic forebrain-like neurons (iNeurons) in 2D culture and performed whole-cell patch-clamp recording to determine the effect of each variant on cellular excitability. We also differentiated these lines into 3D cortical spheroids, which better recapitulate the cytoarchitecture of the developing cerebral cortex, to assess the impact of each variant on neurodevelopmental endophenotypes relevant to MCD.

Results: We find that iNeurons expressing either the SCN3A-p.Thr1486Ile variant (associated with severe epilepsy and MCD), or the SCN3A-p.Pro1333Leu variant (associated with severe epilepsy without MCD), show altered intrinsic excitability including a hyperpolarized voltage threshold for action potential generation and wider (prolonged) action potentials compared to control. A prominent subset of iNeurons expressing either patient variant displayed irregular firing patterns with paroxysmal bursting and plateau-like potentials with action potential failure. We find that, at both 2.5 and 4 weeks in culture, SCN3A-p.Thr1486Ile expressing cortical spheroids are larger in diameter, whereas SCN3A-p.Pro1333Leu spheroids are smaller, compared to isogenic control.

Conclusions: We find that iNeurons expressing either the SCN3A-p.Thr1486Ile variant (associated with severe epilepsy and MCD), or the SCN3A-p.Pro1333Leu variant (associated with severe epilepsy without MCD), show altered intrinsic excitability including a hyperpolarized voltage threshold for action potential generation and wider (prolonged) action potentials compared to control. A prominent subset of iNeurons expressing either patient variant displayed irregular firing patterns with paroxysmal bursting and plateau-like potentials with action potential failure. We find that, at both 2.5 and 4 weeks in culture, SCN3A-p.Thr1486Ile expressing cortical spheroids are larger in diameter, whereas SCN3A-p.Pro1333Leu spheroids are smaller, compared to isogenic control.

Funding: NIH NINDS F31 NS129377 to J.P.M. and R01 NS119977 to E.M.G.