Origins of DNA damage driving pathology in human neurodegeneration

Loss or mutations in both alleles of the gene encoding ataxia-telangiectasia mutated (ATM) kinase results in early-onset cerebellar ataxia and progressive neurodegeneration in humans. Mechanistic explanations for this phenotype, as well as for the related A-T like Disorder (ATLD) caused by rare mutations in the MRE11 gene, have been elusive despite years of work on these enzymes. ATM is a master regulator of the DNA damage response, controlling checkpoint responses and survival of DNA damage in all cell types. We have previously characterized the ATM protein kinase in vitro, using purified proteins to determine that ATM is activated at sites of DNA double-strand breaks and can also be activated independently of breaks by oxidative stress. In recent work we found that loss of ATM kinase activity results in the formation of protein aggregates—detergent-resistant insoluble forms of proteins—enriched for polypeptides with intrinsically disordered domains. These aggregates form in response to hyperactivation of poly-ADP-ribose polymerases (PARPs) that are activated at sites of transcriptional stress. Analysis of 21 patient cerebellum tissue samples also showed massive levels of aggregates as well as hyperPARylation in comparison to controls, consistent with these observations. Based on this evidence we propose that protein aggregation may play a causal role in the neurodegeneration that occurs in this disorder, similar to other forms of cerebellar ataxia and to more common late-onset neurodegeneration in the human population. Here we propose to characterize the origin of single-strand DNA breaks that occur in the absence of ATM function to test the hypothesis that the combined effects of oxidative stress and transcription-dependent damage is responsible for the strand breaks and resulting protein aggregates that are observed with loss of ATM in human neurons. We will also characterize the locations and requirements for strand breaks seen in neurons expressing ATLD alleles of MRE11 and test the hypothesis that the Mre11-Rad50-Nbs1 (MRN) complex promotes single-strand break repair using in vitro biochemistry with purified proteins. These experiments will test novel hypotheses about the functions of ATM and MRN in neurons and the origins of DNA damage during cerebellar neurodegeneration.