Potentiating NaV1.1 in Dravet Syndrome patient iPSC-derived GABAergic neurons increases neuronal firing frequency and decreases network synchrony

Dravet syndrome is a developmental and epileptic encephalopathy characterized by seizures, behavioral abnormalities, developmental deficits, and elevated risk of sudden unexpected death in epilepsy (SUDEP). Most patient cases are caused by de novo loss-of-function mutations in the gene SCN1A, causing a haploinsufficiency of the alpha subunit of the voltage-gated sodium channel NaV1.1. Within the brain, NaV1.1 is primarily localized to the axons of inhibitory neurons, and decreased NaV1.1 function is hypothesized to reduce GABAergic inhibitory neurotransmission within the brain, driving neuronal network hyperexcitability and subsequent pathology. We have developed a human in vitro model of Dravet syndrome using differentiated neurons derived from patient iPSC and enriched for GABA expressing neurons. Neurons were plated on high definition multielectrode arrays (HD-MEAs), permitting recordings from the same cultures over the 7-weeks duration of study at the network, single cell, and subcellular resolution. Using this capability, we characterized the features of axonal morphology and physiology. Neurons developed increased spiking activity and synchronous network bursting. Recordings were processed through a spike sorting pipeline for curation of single unit activity and to assess the effects of pharmacological treatments. At 7-weeks, the application of the GABAAR receptor agonist muscimol eliminated network bursting, indicating the presence of GABAergic neurotransmission. To identify the role of NaV1.1 on neuronal and network activity, cultures were treated with a dose-response of the NaV1.1 potentiator d-theraphotoxin-Hm1a. This resulted in a strong increase in firing rates of putative GABAergic neurons, an increase in the intraburst firing rate, and eliminated network bursting. These results validate that potentiation of NaV1.1 in Dravet patient iPSC-derived neurons results in decreased firing synchrony in neuronal networks through increased GABAergic neuron activity and support the use of human neurons and HD-MEAs as viable high-throughput electrophysiological platform to enable therapeutic discovery. Overall design: A single cell RNA sequencing experiment was performed to understand the cell identity of Dravet patient induced pluripotent stem cell (iPSC) derived neurons. Neurons were thawed following the manufacturer's protocol and plated in 24-well plate format at 300,000 live cells/well. Base media contained DMEM/F-12 media (0.5x, Gibco), Neurobasal media (0.5x, Gibco), B-27 supplement (1x, Gibco), N-2 supplement (1x, Gibco), GlutaMax (0.5 mM, Gibco), brain-derived neurotrophic factor (BDNF, 10 ng/mL, PeproTech), glial-derived neurotrophic factor (GDNF, 10 ng/mL, PeproTech), and TGF-ß1 (1 ng/mL, PeproTech), for a total of 500 mL/well. For seeding media at DIV 0, GABA neuron seeding supplement (1x, BrainXell) was added. At DIV 4, base media plus Day 4 Supplement (1x, BrainXell) and Supplement K (1x, BrainXell) were added at 500 mL/well. Starting at DIV 7, a 50% media change 2x per week was performed using base media. Cells were disassociated at DIV 21 following protocol for mature neuronal culture outlined (Julie Jerber, 2020). Cells in suspension were 89% viable (Countess II) following disassociation. Initial steps of sample QC and cDNA synthesis were conducted at GENEWIZ, LLC./Azenta US, Inc (Waltham, MA, USA). Library preparations, sequencing reactions, and data analysis were conducted at GENEWIZ, LLC./Azenta US, Inc (South Plainfield, NJ, USA).