Interrogation of non-canonical roles of VGSCs in neurodevelopmental epilepsy

PROJECT SUMMARY Voltage-gated sodium channels (VGSCs) are transmembrane proteins with well-established roles in neuronal physiology, including action potential initiation and propagation. Mutations in VGSCs are associated with severe pediatric epilepsies. Despite the availability of VGSC-selective pharmacotherapies, many patients do not achieve sufficient seizure control and no existing

treatment is effective for improving cognitive and behavioral impairments. Interestingly, VGSCs are expressed early in the development of the nervous system before neurons are capable of generating action potentials and before neuronal circuits have established. Certain variants in the predominant developmental VGSC gene SCN3A are associated with severe cortical

malformations and Developmental and Epileptic Encephalopathy (DEE). Furthermore, studies of VGSC functions in non-excitable cells have identified under-appreciated yet important functions in a variety of cellular processes termed “non-canonical” functions, including regulation of Na+ homeostasis, inflammation, proliferation, and migration. Together these findings suggest that

VGSCs perform different roles in early development than in the mature brain. I hypothesize that a greater understanding of these functions in VGSC-DEE will be important for developing effective treatments. Here, I will use an innovative human induced pluripotent stem cell (hiPSC) derived brain organoids carrying clinically relevant mutations in SCN3A and isogenic controls. Preliminary

studies have found irregularities in the patterning and morphology of SCN3A mutant cerebral organoids and we have found that pharmacologic inhibition of VGSCs disrupts interneuron migration in control hiPSC-derived cerebral organoids. While this data demonstrates recapitulation of key clinical phenotypes in the cellular model, the mechanisms governing these

effects remains unclear. I propose to study non-canonical cellular functions in the following aims: Aim 1 will examine the influence of SCN3A on cellular migration in the developing brain. Aim 2 will identify the molecular mechanisms by which SCN3A influences cellular processes of brain development using single-cell RNA sequencing analysis to identify transcriptional differences

between control, SCN3A, and pharmacologically-treated organoids. Aim 3 will investigate the therapeutic potential of corrective gene engineering. I will design and test a precise gene editing approach to correct pathogenic SCN3A variants in human cerebral organoids. This proposed project highlights the need for a paradigm-shift in thinking about the role of VGSCs in brain

development and new therapeutic strategies targeting diverse cellular functions of VGSCs.