Single-Cell Analysis of Somatic Mutation Rates, Mechanisms, and Impacts in Human Ataxia Telangiectasia Cerebellum

PROJECT SUMMARY Genome maintenance is crucial to cellular and organismal health. In conditions where DNA damage repair (DDR) pathways are disrupted, patients develop severe multisystem disorders. While these diseases impact distinct repair pathways, they share some phenotypic overlap, such as premature aging and neurodegeneration. This observation suggests that genome instability promotes aging and neuronal death. One such DDR disease is Ataxia Telangiectasia (A-T), a life-limiting DDR disease characterized by cerebellar degeneration, immunodeficiency, and susceptibility to cancer. A-T is caused by biallelic loss-of-function (LoF) mutations at the A-T mutated (ATM) locus, which encodes ATM kinase. A-T relevant mutations are thought to interfere with one or more of its functions, most prominently ATM’s role orchestrating DDR and sensing reactive oxygen species. Loss of these functions results in increased DNA damage and higher ROS in A-T patient cells. Ataxia in A-T coincides with loss of cerebellar volume while sparing other brain regions. Histology shows that Purkinje cells (PCs), the sole output of the cerebellar cortex, and cerebellar granule neurons die over the course of A-T. Our preliminary data shows that deletion burden is higher in A-T neurons from the pre- frontal cortex, but mutation burden analysis is lacking from disease-affected cell types. How ATM LoF impacts PC genome stability, and consequently PC function and longevity, remains unknown. I propose to characterize how ATM LoF impacts the PC genome and transcriptome at the single-cell level in the human cerebellum. Post-mortem human brain was chosen over animal models because A-T mammalian models fail to phenocopy human cerebellar pathology. This experiment requires a single-cell analysis because PCs are very rare, comprising <1% of the cells of the cerebellum, so a “bulk” analysis would obscure PC- specific patterns. A pure population of PCs is a prerequisite for single-cell somatic mutation analysis. However, a robust protocol to isolate rare PCs for single-cell molecular analysis was lacking before I started this project. To circumvent this hurdle, I adapted a technique to isolate soma from fresh frozen brain. I applied PC soma to a new muti-omic protocol capable of capturing DNA and RNA from the same single cell to human brain for the first time. Aim 1 will compare single cell DNA mutation burden between PC from A-T and matched controls. Aim 2 will synthesize mutation characteristics – context, distribution, size (single base variants, small/large insertion or deletions, structural variants) – into signatures. Signatures will suggest mechanisms. Aim 3 will investigate the connection between DNA damage and transcriptional changes, linking genotype and phenotype. Further, this work will provide the fellow with exemplary training in genomics, neurobiology, statistics, and bioinformatics.