Authors :
Presenting Author: Scott Adney, MD, PhD – Northwestern University Feinberg School of Medicine
Sashmita Panda, PhD – Northwestern University Feinberg School of Medicine
Luis Williams, PhD – Quiver Bioscience
Karthiayani Harikrishnan, MS – Quiver Bioscience
Steve Ryan, PhD – Quiver Bioscience
Owen McManus, PhD – Quiver Bioscience
Chichi Obasi, BS – Northwestern University
Erik Eastwood, BS – Northwestern University Feinberg School of Medicine
Graham Dempsey, PhD – Quiver Bioscience
Alfred George, MD – Northwestern University Feinberg School of Medicine
Rationale: Pathogenic variants in SCN2A are well established as a cause of early-onset epilepsy with variable severity. While the functional consequences of many variants are understood at the channel level, how this impacts neuronal function remains poorly understood. Extension of channel biophysical properties generally relies on
in silico prediction models, which cannot fully recapitulate the dynamic neuronal environment and lack validation in relevant experimental systems such as intact neurons.
Methods:
Induced pluripotent stem cell (iPSC) lines were generated from a patient heterozygous for the SCN2A p.M1879T pathogenic variant that exhibits gain-of-function features. Lines were edited with CRISPR-Cas9 to create two isogenic mutation-corrected clones; all iPSC clones were subsequently differentiated into cortical excitatory "NGN2" neurons using overexpression of Neurogenin2 (NGN2). The all-optical electrophysiology platform Optopatch was utilized with neurons transduced with blue light-activated channelrhodopsin (CheRiff) and the voltage reporter QuasAr. Evoked spike properties were extracted for further analysis. Pharmacology experiments with conventional anti-seizure drugs were performed using the same platform and techniques. In separate experiments, iPSC-derived neurons were examined for intrinsic excitability measures using whole-cell patch-clamp recording. Additionally, patient-derived and isogenic neurons were plated on 48-well multielectrode arrays (MEAs) to investigate population properties and evoked firing with electrical stimulation. Finally, we examined neuronal populations with single-neuron resolution by using the genetically encoded voltage imager (GEVI) ASAP3Kv, delivered by lentivirus to cultured neurons, and imaged with a high-speed sCMOS camera.
Results:
Using the Optopatch platform, multiple functional parameters of excitability were extracted from spike shape and timing that could distinguish the patient iPSC-derived neurons expressing the p.M1879T variant from isogenic controls. Higher firing rates occurred with stronger excitation stimuli in mutant neurons compared to the isogenic controls. Similar results were observed using patch-clamp electrophysiology. Pharmacology experiments with sodium channel blocking anti-seizure drugs demonstrated efficacy in correcting the pathogenic phenotype. In MEA studies, the p.M1879T line could be distinguished from isogenic control neurons using supervised K-means clustering and machine learning-based feature extraction. Finally, using lentivirus-delivered GEVI in iPSC-derived neurons under similar conditions, we demonstrated synchronized firing in the neuronal lines and compared features.
Conclusions:
We demonstrate multimodal analysis of patient-derived neurons with an SCN2A pathogenic variant implicated in early-onset epilepsy. Patient-derived neurons showed increased propensity to fire at higher stimuli, validating the predicted gain-of-function effect. Population assays demonstrate functional separation between the two lines. Our study provides new mechanistic insight into how a novel SCN2A gain-of-function variant impacts patient-derived excitatory neurons.
Funding: K08NS121601(SKA), U54NS108874(ALG)