Modeling Ataxia Telangiectasia (A-T) in a Novel Laminar System for Human Stem Cell-Derived Purkinje Cells - PROJECT SUMMARY Ataxia-telangiectasia (A-T) is an autosomal recessive, multi-system disorder caused by mutations in the ataxia- telangiectasia mutated (ATM) gene affecting approximately 1:40,000-1:100,000 births (Chun and Gatti, 2004; Rothblum-Oviatt et al., 2016). Characterized by progressive cerebellar neurodegeneration, there are no effective treatments for A-T. The cause of cerebellar neurodegeneration, chiefly affecting Purkinje cells (PCs), 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. Knowledge Gap: Human A-T model system that captures the cerebellar phenotype needs to be developed to identify physiological and molecular differences between patient and isogenic, control PCs. In these studies, we propose to use a novel laminar microfluidic model system we developed for human induced pluripotent stem cells (iPSCs), differentiated into hPCs by a protocol developed in our lab (Buchholz et al, 2020; Sundberg et al, 2018), to analyze A-T iPSC-derived PCs and isogenic, control iPSC-derived PCs. Results will provide novel insight into the pathophysiological mechanisms of cerebellar degeneration in A-T. Our overall hypothesis is that a human A-T model system will lead to the discovery of basic mechanisms underlying A-T pathophysiology. We will 1) Determine whether loss of function A-T patient mutations cause deficits in hPC differentiation, including dendrite patterning and synapse density and ATM-dependent pathways of the DNA damage response, 2) Assess whether loss of function A-T patient mutations cause deficits in iPSC-PC physiology and calcium dynamics, 3) Test whether the expression of ion channels or synaptic proteins are dysregulated in A-T patient- derived PCs, and 4) Test whether inhibition of ATM kinase, using a potent, highly specific and reversible ATM inhibitor, KU-60019 (Haj et al, 2025), will phenocopy loss of ATM and whether deficits at different stages of hPC development are reversible. Taken together, the results of these four sets of experiments will provide novel insights into the pathophysiological mechanisms of A-T in human cerebellar PCs and provide the field with an important new model system for human A-T cerebellar cells and their isogenic controls. IMPACT: The proposed studies will yield a paradigm shift in understanding A-T biology by identifying molecular mechanisms underlying A-T pathophysiology. While this proposal is focus on A-T, this work will provide a framework for interrogating other genetic disorders of the cerebellum.
