Distinctivein vitrophenotypes in iPSC-derived neurons from patients with gain- and loss-of-functionSCN2Adevelopmental and epileptic encephalopathy

Abstract

SCN2Aencodes Nav1.2, an excitatory neuron voltage-gated sodium channel and major monogenic cause of neurodevelopmental disorders, including developmental and epileptic encephalopathies (DEE) and autism. Clinical presentation and pharmocosensitivity vary with nature ofSCN2Avariant dysfunction with gain-of-function (GoF) cases presenting with pre- or peri-natal seizures and loss-of-function (LoF) patients typically having infantile spasms after 6 months of age. Here, we established and assessed patient induced pluripotent stem cell (iPSC) - derived neuronal models for two recurrentSCN2ADEE variants with GoF R1882Q and LoF R853Q associated with early- and late-onset DEE, respectively. Patient-derived iPSC lines were differentiated using aNeurogenin-2overexpression yielding populations of cortical-like glutamatergic neurons. Electrophysiological and transcriptomic profiles were assessed after 2-4 weeks in culture. Increased neuronal activity at both cellular and network level was observed for R1882Q iPSC-derived neurons at three weeks of differentiation. In contrast, R853Q neurons showed only subtle changes in excitability after four weeks in vitro. In alignment with the reported efficacy in some GoFSCN2Apatients, phenytoin (sodium channel blocker) reduced excitability of neurons to the control levels in R1882Q neuronal cultures. Transcriptomic alterations in neurons were detected for each variant and convergent pathways pointed at the shared mechanisms underlyingSCN2ADEE.

Emerging neurodevelopmental mechanisms in patient induced pluripotent stem cells-derived spheroids modellingSCN1ADravet Syndrome

“…Extraction of RNA samples and set up of RT-qPCR reactions were performed as previously described (21). Immunoreactivity.…”

“…Cells were fixed and stained as previously described (21). Immunoreactivity was tested against NANOG (#ab21624, Abcam, 1:100) and TRA-1-60(R) (#ab16288, Abcam, 1:100), The secondary antibodies were Alexa Fluor-conjugated 488 anti-Mouse (#A11001, Life Technologies) and Alexa Fluor-conjugated 594 anti-Rabbit (#A21207, Life Technologies).…”

“…A total of n ≥ 2 biological replicates per timepoint was collected with multiple spheroids pooled together for each replicate. Samples were processed and analysed as described by (21).…”

Slc35a2 mosaic knockout impacts cortical development, dendritic arborisation, and neuronal firing in the developing brain

“…This includes reduced AP firing of dysmorphic neurons from an electroporation-based mouse models of FCD type II that has a robust spontaneous seizure phenotype 14,15 . It is also well established for some 'channelopathies' that loss of function pathogenic variants can cause epilepsy with reduced AP firing in individual neurons occurring concomitantly with increased network-level excitability in model systems [28][29][30] .…”

“…Furthermore, the role of heterochromatin of the inactive X chromosome in female may also slow Cas9-mediated cutting 30,31 , with the additional two weeks before analysis at P21-25 providing opportunity for continued indel introduction in transfected cells.…”