Understanding the Impact of CACNA1A/unc-2 Mutations on Presynaptic Structure and Function in C. Elegans - Project Summary/Abstract Mutations in the gene CACNA1A, which encodes Cav2.1, the a1 subunit of P/Q type voltage gated calcium channels, lead to severely debilitating channelopathies. Patients are classically diagnosed with syndromes such as episodic ataxia type 2 (EA2) or familial hemiplegic migraine type 1 (FHM1), though some patients also exhibit intellectual disability (ID) or multiple overlapping symptoms. Prior studies have largely focused on studying biophysical properties of these channels in heterologous systems or on mouse models of loss-of-function (LOF) EA2 mutations that have been linked to aberrant cerebellar Purkinje cell pacemaking. FHM1 has been associated with gain-of-function (GOF) effects leading to overexcitation and cortical spreading depression. Recent studies have suggested that in vitro characterization often obscures more complex or neuronal compartment specific effects in vivo. Cav2.1 plays a prominent role at presynaptic terminals to drive vesicular neurotransmitter release, yet the synaptic effects of many mutations remain understudied. Moreover, the molecular mechanisms that underlie patient symptoms beyond FHM1 and EA2, such as cognitive dysfunction are unknown. The overarching purpose of this proposal is to interrogate how two uncharacterized CACNA1A mutations associated with ID in patients alter synaptic transmission and lead to circuit-wide defects, comparing their effects on synaptic function to more well-characterized LOF and GOF mutations. The nematode C. elegans will be used to model patient-derived mutations in vivo, taking advantage of this powerful genetic system. C. elegans have a single CACNA1A orthologue, unc-2, which displays significant homology to the mammalian channel. The Kurshan lab has thus far generated five mutant CACNA1A/unc-2 strains, modeling either EA2, FHM1, or non-syndromic mutations that lead to ID. Aim 1 of this proposal will determine how synaptic function is altered in these mutant strains by measuring synaptic vesicle release using established and novel behavioral assays, performing electrophysiological recordings, and imaging channel expression in different cell types. Aim 2 will use molecular cloning techniques, calcium imaging and optogenetics to dissect the neural circuits impacted by the mutations, as well as a forward genetic screen to identify additional components of the signaling pathways mediating aberrant circuit output. Completion of this study will lead to a better understanding of the molecular perturbations caused by ID-associated CACNA1A/unc-2 mutations and work to identify synaptic targets for therapeutic intervention to treat patients suffering from these neurological disorders. This proposal will greatly enhance the F31 candidate’s scientific and professional development through strong mentorship and fantastic support from the Albert Einstein College of Medicine, while also leading towards their goal of becoming an independent investigator.
