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Séminaire - Jack Parent"Induced Pluripotent Stem Cell Modeling of a Genetic Epilepsy"

Abstract :

Dravet Syndrome (DS) is a severe childhood epilepsy typically caused by de novo dominant mutations in the SCN1A gene encoding the voltage-gated sodium channel Nav1.1. Heterologous expression of mutant channels suggests haploinsufficiency, raising the quandary of how loss of sodium channels underlying action potentials produces hyperexcitability. Work in DS mouse models supports a role for decreased Nav1.1 function in interneurons leading to disinhibition as an epilepsy mechanism. However, mutant SCN1A effects in human neurons are unknown. We used induced pluripotent stem cell (iPSC) reprogramming of patient fibroblasts to derive DS forebrain-like neurons and explore disease mechanisms. Two subjects with different SCN1A mutations and three human controls were studied. Patient fibroblasts were obtained and reprogrammed to iPSCs by retroviral mediated gene transfer of Oct4, Klf4, Sox2 and c-Myc. Forebrain-like neurons were generated from the iPSCs and were studied using whole-cell patch clamp recordings and immunostaining for voltage-gated sodium channel alpha subunits. We obtained both GABAergic interneurons and glutamatergic pyramidal-like neurons from the iPSCs. Unexpectedly, voltage-clamp recordings of both bipolar- and pyramidal-shaped DS mutant neurons showed markedly increased sodium current densities compared to controls. With current clamp recordings, DS mutant neurons displayed a lower threshold for action potential generation, more repetitive firing and increased firing frequency than control neurons. Also, DS mutant neurons, but not controls, showed spontaneous bursting activity. Immunocyochemistry for sodium channel alpha subunits showed a lack of immunoreactivity for Nav1.6 in controls but positive immunolabeling in DS mutant neurons, suggesting that the increased sodium current density in DS neurons reflected overcompensation by Nav1.6 for the partial loss of Nav1.1.

We next explored potential Sudden Unexpected Death in Epilepsy (SUDEP) mechanisms in DS, which has a high incidence of this most severe epilepsy complication. Because NaV1.1 is also expressed in heart, we hypothesized that altered Na currents in DS cardiac myocytes (CMs) cause arrhythmias and SUDEP. To test this idea, we generated CMs from 3 DS patient and 1 control iPSCs, as well as from a human embryonic stem cell line (H7). We examined CM markers, beating rate and sodium current density in the iPSC-derived CMs. We also investigated sodium currents and action potentials in CMs from DS knock-in mice that express a human SCN1A mutation causing seizures and premature death. CM differentiation of DS and control iPSCs led to beating cells that expressed CM markers α-actinin and cardiac troponin-T, as well as ventricular- and atrial-specific markers. Similar to DS iPSC-neurons, the DS-iPSC CMs from one patient showed increased sodium current density vs. control, as well as an increased beating rate. Ventricular CMs from P17-19 DS mice show increased sodium current density compared to wild-type, as well as pro-arrhythmogenic alterations of action potentials. The mice displayed frank arrhythmias during telemetry recordings. Characteristics of the sodium currents in DS mouse CMs suggest that they result from enhanced NaV1.5 (SCN5A gene) current that overcompensates for 50% NaV1.1 loss. Together, these findings suggest potential epilepsy and cardiac arrhythmogenic SUDEP mechanisms in DS.

Selected publications

Ganglionic eminence graft pre-eminence in epilepsy. Parent JM, Murphy GG. Nat Neurosci. 2013 Jun;16(6):656-8. doi: 10.1038/nn.3406.

Runaway dendrites: blame the older siblings.Patino G, Parent JM. Epilepsy Curr. 2012 Nov;12(6):222-4. doi: 10.5698/1535-7511-12.6.222.


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