Molecular and Functional Mechanisms Underlying Cortical Activity in CACNA1A Epilepsy

PROJECT SUMMARY/ABSTRACT The CACNA1A gene encodes the α1A subunit of the P/Q-type voltage-gated calcium channel Caᵥ2.1 which facilitates presynaptic neurotransmitter release in neurons. Loss-of-function (LOF) and gain-of-function (GOF) variants in CACNA1A are both associated with developmental and treatment-resistant epileptic encephalopathy in patients. How do variants with divergent effects on Caᵥ2.1-mediated calcium influx converge to produce overlapping epileptic symptoms? Electrophysiology studies from mouse models have revealed LOF variants selectively disrupt inhibitory neuronal function while GOF variants selectively affect excitatory neurotransmission. We propose that cortical excitatory/inhibitory balance is disrupted in both cases, with LOF leading to weakened inhibition and GOF leading to excessive excitation. Selective disruption of inhibitory neurons in LOF and excitatory neurons in GOF have led to the hypothesis that cell types may functionally compensate by upregulating other calcium channels in LOF while downregulating calcium channels in GOF. However, molecular mechanisms underlying this compensation—including specific channels affected and the degree to which they successfully rescue calcium influx and normal neuronal activity—appear species-specific and cell type-specific. No published study has examined consequences of CACNA1A mutations on expression of genes beyond other calcium channels. To bridge these gaps and improve our understanding of how cortical hyperexcitability emerges in human CACNA1A-related epilepsy, I will characterize cell type-specific functional activity and gene expression in CACNA1A mutations with a 3D, human-derived in vitro model of the developing cortex. Utilizing forebrain assembloids containing cortical cell types from engineered LOF, GOF, and unedited isogenic control lines, this proposal compares function and gene expression of excitatory and inhibitory neurons between genotypes. Aim 1 utilizes calcium imaging to assess calcium transient rates and calcium influx of excitatory and inhibitory neurons alongside network-level measures of excitability. Aim 2 employs single-cell RNA sequencing to ask how gene expression of excitatory and inhibitory neurons is differentially affected in LOF and GOF relative to controls. Together, these experiments may identify expression changes in calcium channels and novel pathways in Aim 2 that explain cell type-specific functional abnormalities in LOF versus GOF revealed in Aim 1. The central hypothesis is that excessive activity and insufficient downregulation of calcium channels in excitatory neurons leads to hyperexcitable cortical networks in LOF while reduced activity and insufficient upregulation of calcium channels in inhibitory neurons leads to hyperexcitable cortical networks in GOF. Completion of these aims will provide novel insights into compensatory mechanisms, functional abnormalities, and etiology of epileptic phenotypes in CACNA1A for the first time in a human-derived model system—potentially informing novel therapeutic targets and variant classification criteria.