Authors :
Presenting Author: Yukun Yuan, MD, MS, PhD – University of Michigan Medical School
Luis Lopez-Santiago, PhD – University of Michigan Medical School
Louis Dang, MD/PhD – University of Michigan
Grace Lin, PhD – University of Michigan Medical School
Preethi Swaminathan, MS – University of Michigan Medical School
Shivanshi Vaid, MS – University of Michigan
Anna Loughman, BS – University of Michigan Medical School
Andrew Tidball, PhD – University of Michigan Medical School
Katalin Štěrbová, MD – Charles University and Motol Hospital, Prague, Czech Republic
Sarah Weckhuysen, MD, PhD – Neurogenetics Group, Center for Molecular Neurology, VIB, Antwerp, Belgium
Peter DeJonghe, MD, PhD – Neurogenetics Group, Center for Molecular Neurology, VIB, Antwerp, Belgium
Jack Parent, MD – University of Michigan Medical School
Lori Isom, PhD – University of Michigan-Ann Arbor
Rationale: Homozygous loss-of-function variants in
SCN1B, the gene encoding the voltage-gated sodium channel β1/β1B subunits, are linked to Dravet syndrome (DS), a severe developmental and epileptic encephalopathy. We previously showed that a homozygous mouse model with a recessive variant,
SCN1B-c.265C >T, predicting
SCN1B-p.R89C, recapitulates some key DS patient phenotypes including higher susceptibility to hyperthermia-induced seizures, spontaneous generalized seizures, and sudden unexpected death in epilepsy (SUDEP). We reported in a previous abstract that
SCN1B p.R89C patient-derived induced pluripotent stem cell (iPSC) excitatory neurons fired spontaneous action potentials (APs) and evoked APs with a higher frequency compared to controls. Here, we further characterized the differences in neuronal excitability, membrane electrical properties and AP kinetics of iPSC-derived
SCN1B p.R89C excitatory neurons generated from patients from different pedigrees and using different differentiation methods.
Methods: Dermal fibroblasts from individuals
with monoallelic (het) or biallelic (homo)
SCN1B- p.R89C variant were reprogrammed into induced iPSCs by episomal reprogramming, transfecting OCT3/4, SOX2, KLF-4, L-MYC, LIN-28 and shRNA against p53. Dual SMAD inhibition10 or induced expression of neurogenin11 were used to generate cortical-like excitatory neurons. Whole-cell patch-clamp recording techniques were used to compare electrophysiological properties of these patient iPSC neurons with iPSC neurons differentiated in parallel from two healthy controls.
Results:
: In excitatory neurons from neurogenin-driven differentiation of iPSCs, whole cell voltage-clamp recordings showed changes in peak sodium currents, but not persistent currents, between homo-patient and control iPSC neurons. Whole cell current-clamp recordings demonstrated that homo-patient R89C iPSC neurons have significantly decreased capacitance, increased membrane input resistance, lower threshold potential for AP initiation, higher firing frequency of evoked APs and increased rate of spontaneous AP firing compared to controls. In addition, het-patient iPSC neurons had higher maximum AP rise and maximum AP decay rates and thus shorter AP half-amplitude durations compared to controls. Consistently, homo-patient R89C neurons generated from dual SMAD inhibition of iPSCs showed similar changes in passive and active membrane electrical properties.
Conclusions: Taken together, these data suggest that alterations of neuronal membrane electrical properties due to the biallelic
SCN1B-p.R89C variant lead to altered neuronal hyperexcitability and may contribute to pathogenic mechanisms in
SCN1B-linked DS.
Funding: Supported by R37 NS076752 (LLI) and 1U54NS117170-01 (LLI and JMP).