Abstracts

Rationale: Dravet syndrome (DS) is a severe epileptic encephalopathy that is often caused by haploinsufficiency in the SCN1A gene, which encodes the voltage-gated sodium channel (VGSC) Na_v1.1. One possible pathogenic mechanism is that SCN1A mutations affect inhibitory neurons disproportionately more than excitatory neurons, leading to network disinhibition and seizures. This “interneuron hypothesis” has been supported by several mouse studies and a few studies using human pluripotent stem cell-derived neurons (hPSCNs). However, the findings in mouse studies have been dependent on the age and strain of the mice, and in hPSCNs, there have been conflicting findings of whether inhibitory or excitatory neurons are more implicated in pathogenesis. We hypothesize that excitatory DS hPSCNs derived in 2-dimensional and 3-dimensional cultures are hyperexcitable, possibly from maladaptive compensatory expression of another VGSC. Methods: We generated induced pluripotent stem cell (iPSC) lines from multiple subjects with SCN1A-related Dravet syndrome. We also used CRISPR gene editing to make iPSC lines with SCN1A mutations along with isogenic controls. iPSCs were differentiated into excitatory cortical-like neurons using forced expression of neurogenin 2 (NGN2). Electrophysiological properties of the excitatory neurons were measured using patch-clamp whole cell electrophysiology as well as longitudinal recordings of firing properties using multielectrode arrays (MEAs). In order to study excitatory as well as inhibitory neurons in a developmental context that better recapitulates the in vivo condition, we also generated excitatory and inhibitory cortical spheroids. Results: In NGN2-induced hPSCNs, MEA recordings show that the mean firing rate was similar between DS and control, and the burst frequency was higher in DS cells. However, the burst duration was shorter, and the interspike interval within a burst was longer in the DS cells. Patch clamp recordings of NGN2-induced neurons demonstrated higher sodium current densities in DS hPSCNs as well as increased spontaneous firing compared to controls, implicating hyperexcitability. Finally, we successfully generated dorsal (excitatory) and ventral (inhibitory) cortical spheroids, and electrophysiological recordings demonstrate spontaneous action potential firing. Conclusions: NGN2-induced DS hPSCNs are hyperexcitable, with increased burst frequency on MEA and higher sodium current densities and spontaneous firing with whole-cell patch clamp recordings. With the generation of inhibitory and excitatory human cortical spheroids, we will be able to better define what cell types are responsible for pathogenesis in DS. Funding: Grant support is provided by a Dravet Syndrome Foundation Fellowship and NIH (NICHD) HD028820 (LTD), and NIH (NINDS) NS088571 (JMP and LLI).