Determining How CACNA1C Variants Cause Interneuron Pathophysiology

Stanford University, Stanford CA

Investigators

Abstract

PROJECT SUMMARY: Neurodevelopmental disorders impact up to 24% of children. The pathophysiology of most neurodevelopmental disorders is poorly understood preventing therapeutics; however, calcium dysregulation and interneuron dysfunction are implicated in many disorders. CACNA1C encodes a CaV1.2 L- type calcium channel subunit. Variants in CACNA1C cause a spectrum of neuropsychiatric symptoms. A CACNA1C gain of channel function variant impairs interneuron migration and cortical development in human induced pluripotent stem cell (hiPSC) derived neural organoid and assembloid models. We do not know the neuronal pathophysiology of CACNA1C loss of function or channel-neutral variants. Investigating the interneuron phenotypes of clinically severe CACNA1C variants will elucidate downstream pathways regulating interneuron pathology that contribute to a cortical network phenotype, potentially indicating treatments. The applicant, Dr. Rebecca Levy, proposes to employ gene editing to develop novel hiPSC lines and neural assembloids, then leverage calcium indicators, live imaging, and RNA sequencing analysis to determine how CACNA1C variants impact mechanisms directing interneuron migration. In Aim 1, Dr. Levy will learn CRISPR/Cas9 gene editing to generate 3 novel hiPS cell lines of CACNA1C including variants that cause loss of function and no change in calcium flux in non-neuronal cells. She will hone her skills in neural organoid culture to derive subpallial organoids and quantitate calcium activity with Fura-2 and interneuron-specific GCaMP calcium indicators, as well as CaV1.2 channel agonists and antagonists to rescue phenotypes. In Aim 2, Dr. Levy will develop cortical-subpallial assembloids and use live imaging to measure interneuron migration saltation length and frequency, then how cortical network activity is affected within assembloids. In Aim 3, Dr. Levy will confirm mechanisms via Western blot, pharmacologic rescue, and single cell RNA sequencing analysis. This will also generate new hypotheses about pathways affected in both gain and loss of function variants for future grants. Dr. Levy is a neurogeneticist with experience in mouse and neural organoid models. Her career development plan addresses training gaps in gene editing, neural assembloid culture, advanced imaging techniques, expression analysis, and scientific communication. Her mentor, Dr. Sergiu Pasca, is a leader in neural organoid culture techniques. Her advisory mentors are Dr. Porteus (gene editing), Dr. Hall (Cacna1c rodent models), Dr. Huguenard (systems neuroscience), and Dr. Bernstein (rare disease geneticist). Stanford provides a rich environment including core facilities, seminars, courses, and collaborations. With this dedicated mentorship team, outstanding institutional environment, and thoughtful training and career development plans, Dr. Levy has the resources and support to launch her independent physician-scientist career. This proposal will discover interneuron pathophysiology caused by CACNA1C variants with variable impact on channel function, identifying core mechanisms that could become therapeutic targets.