Molecular Mechanisms of Purkinje Cell Degeneration in Ataxia-Telangiectasia

点击翻译按钮获取中文摘要

PROJECT SUMMARY Ataxia-telangiectasia (A-T) is an autosomal recessive, multi-system, disorder caused by mutations in the universally expressed ataxia-telangiectasia, mutated (ATM) gene affecting approximately 1:40,000-1:100,000 births, for which there is no cure. Characterized by progressive cerebellar neurodegeneration, there are no effective treatments for A-T, with patients succumbing to chronic sinopulmonary infections or A-T related cancer by the third decade of life. Furthermore, the cause of cerebellar neurodegeneration, chiefly affecting Purkinje cells (PCs), the primary output neuron of the cerebellum, has remained elusive since the first descriptions of A- T nearly 80 years ago, largely because mouse models do not recapitulate the human cerebellar phenotype of PC death. Thus, the critical objectives of this proposal are to develop the first human A-T model system that recapitulates the cerebellar phenotype and to use that system to identify molecular differences between patient and unaffected PCs as well as differences between human and mouse PCs. Toward that end, we will use our recently published protocol (Buchholz et al, 2020) to generate an induced pluripotent stem cell (iPSC) model system and use that system to study the effects of A-T patient mutations on developing human PCs. Using our protocol, we have been able to differentiate cerebellar Purkinje cells that match young adult PCs on a transcriptomic level (Buchholz et al., 2020) and fire specific calcium currents in co-culture with their target neurons, granule cells (GCs). Our specific aims in this proposal are therefore to use this protocol to differentiate iPSCs derived from patients with A-T, as well as unaffected control iPSCs derived from family members, into PCs to study A-T PC phenotypes, including defects in survival and synaptic function in co-culture with GCs. To discover molecular changes in Purkinje cells with the A-T mutation, we will study global gene expression and protein phosphorylation compared to controls. Critically, we will then use CRISPR-Cas9 prime editing to correct the ATM mutation and test for rescue, including identifying key changes in rescued versus mutant gene expression and proteomics. Taken together, these studies will identify pathways involved in A-T PC phenotypes and will discover altered pathways that could provide novel targets for therapy.