Investigating Molecular Mechanisms of Alternating Hemiplegia of Childhood Using C. elegans Models

PROJECT SUMMARY/ABSTRACT This proposal addresses fundamental questions about Na+, K+ ATPase function both in wild-type animals and in a new C. elegans model for a rare disease called Alternating Hemiplegia of Childhood (AHC). AHC is one of many diseases caused by dominant mutations in ATP1A3, a Na+, K+ ATPase. It is unknown how mutations in ATP1A3 perturb cellular function, leading to disease. I propose a novel hypothesis to explain AHC pathophysiology: defective ATP1A3 upregulates other P-type ATPases that compete for β subunits; β is expressed at limited levels and essential for ATPase function. I rigorously test this hypothesis in vivo using C. elegans models of AHC that I have developed. I created the first C. elegans models of AHC by direct CRISPR/Cas9-based editing of patient missense mutations into eat-6, the C. elegans ortholog of ATP1A3. Heterozygous AHC model animals have dominant neuromuscular junction (NMJ) defects, unlike heterozygous eat-6 null animals. Therefore, AHC defects are caused by a mechanism other than simple loss of ATPase activity. In Aim 1, I use the C. elegans models of AHC to determine where wild-type and AHC model EAT-6 are required for normal and impaired NMJ function. In Aim 2, I determine if AHC patient mutations upregulate other P-type ATPases such as CATP-2 and if this contributes to NMJ defects in AHC model animals. In Aim 3, I determine which β subunits interact with EAT-6 and test if β is a limiting factor. This information is essential for future studies that delineate the cellular mechanisms perturbed in AHC and investigate targets for therapy development. I will complete this fellowship at Brown University in the Molecular Biology, Cell Biology, and Biochemistry Graduate Program. Institutional and departmental resources strongly support my training goals. I will learn new genetic and biochemical techniques such as determining where gene function is required in a live animal using inducible gene expression, analyzing gene expression at the RNA and protein level, and testing protein interactions based on pull down assays. Additionally, I will dedicate time and effort to professional development and networking, teaching, and mentoring to become a strong candidate for a career in academia.