Assessing Synaptic and Intrinsic Effects of Patient-Derived ID-Associated CACNA1A Mutations Using Multiple Models

Abstract Mutations in the CACNA1A gene, which encodes the pore-forming subunit of the P/Q type calcium channel (Cav2.1), lead to neurological disorders including Episodic Ataxia type 2 (EA2) and Familial Hemiplegic Migraine type 1 (FHM1). Patients have typically been classified as having one of these disorders or the other, but symptoms are often overlapping and the distinction has been called into question. More recently, CACNA1A patients presenting primarily with cognitive defects such as intellectual disability or developmental delay have been described, suggesting that a more salient dichotomy may lie between mutations that lead to severe motor deficits and those that are characterized primarily by cognitive dysfunction. Motor dysfunction such as ataxia has been attributed to disruption of neuronal excitability and pacemaking function of cerebellar Purkinje cells, where these channels are most highly expressed. In contrast, t he underlying mechanisms leading to cognitive dysfunction remain unknown. However, Cav2.1 channels are also expressed throughout the nervous system at presynaptic terminals where they mediate synaptic vesicle release. The varying functional consequences of different CACNA1A mutations underscore the importance of delineating the impact of each CACNA1A mutation on channel expression and function to understand how each causes the associated disease phenotypes. We hypothesize that mutations that effect primarily neuronal excitability result in classical motor phenotypes, while those that effect synaptic properties may give rise to cognitive deficits. To begin to address this, we propose to characterize an array of CACNA1A patient mutations resulting in either primarily motor or primarily cognitive presentations. We have validated and now propose to combine two model systems to characterize the effect of these mutations: a heterologous expression system (HEK293t cells) to assess cell-surface expression and biophysical properties using molecular, imaging, and whole cell electrophysiology techniques, as well as the nematode C. elegans to investigate in vivo presynaptic localization and synaptic function. This work will lay the foundation for elucidating the mechanism by which CACNA1A mutations affect neuronal function and lead to pleiotropic patient outcomes.