Predicting the neurophysiological consequences of pathogenic SCN2A variants - PROJECT SUMMARY/ABSTRACT The SCN2A gene, which encodes a neuronal voltage-gated sodium channel, is a major genetic risk factor for a wide range of neurodevelopmental disorders, including childhood epilepsies, autism spectrum disorder, and intellectual disability. SCN2A channels are primarily expressed in axon initial segments and dendrites of excitatory glutamatergic neurons, where they drive action potential initiation and backpropagation, respectively. Pathogenic SCN2A mutations affecting protein structure and function modulate neuronal responses to synaptic inputs leading to the cognitive and behavioral impairments and seizures observed in SCN2A-related disorders. We discovered that the location of variants within the SCN2A protein is strong predictor of channel dysfunction, accounting for approximately 40% of the variance in channel biophysical properties. However, the net effects of complex and/or opposing channel biophysical properties on excitatory neuron excitability are difficult to predict and the relationship between specific channel biophysical perturbations and resulting changes to neuronal physiology is unclear. In this project, we propose to determine the consequences of 115 disease-associated SCN2A mutations on channel biophysical properties and intrinsic physiological properties of neocortical pyramidal neurons (Aim 1A). To do this, we will leverage in vitro whole-cell voltage-clamp recordings to build in silico hidden Markov models of each SCN2A variant, which we will use to simulate heterozygous channel dysfunction in a benchmarked mathematical model of a neocortical pyramidal neuron. Subsequently, we will use interpretable machine learning algorithms to explain the relationship between functional perturbations and neuronal physiology (Aim 1B). We also propose to validate neuronal simulations for 10 SCN2A mutations using an experimental system called dynamic action potential clamp (Aim 2A). In this system, we will electrotonically couple mammalian cells heterologously expressing SCN2A variants to a benchmarked model of an axon initial segment and determine effects of heterozygous channel dysfunction on simulated neuronal excitability. Finally, we aim to study 2 prototypical gain-of-function SCN2A mutations that heighten channel activity, potentiate pyramidal neuron firing, and lead to seizures, and evaluate the feasibility of using dynamic action potential clamp to test functional correction of abnormal neuronal excitability using specific sodium channel blockers (Aim 2B). The long-term goal of this entire project is to computationally map and experimentally validate the relationships among protein structure, channel dysfunction, neuronal physiology, and clinical phenotypes in SCN2A-related disorders. Overall, our work will generate a granular genotype-phenotype relationship landscape of SCN2A- related disorders revealing underlying molecular and cellular mechanisms of pathophysiology and will advance a new approach methodology for testing precision medicine interventions for treating neurological disorders.
