Mitochondria are critical for normal energy metabolism because they house the oxidative phosphorylation (OXPHOS) system that produces the cell's primary energy currency, ATP. They also perform hundreds of other metabolic functions and are intimately involved in calcium homeostasis and apoptosis. However, mitochondria are also a primary source of toxic reactive oxygen species (ROS) that damage cellular components, promote oxidative stress, and cause pathology associated with mitochondrial dysfunction. Mitochondria contain a circular mitochondrial DNA (mtDNA) genome that encodes thirteen OXPHOS subunits, mutation or depletion of which causes complex diseases and age-related pathology. Mammalian cells contain thousands of copies of mtDNA, with each tissue having a characteristic copy number tailored to its particular energy demands and specialized functions. Mutations in the ATM checkpoint signaling kinase cause the multi-faceted and fatal disease Ataxia-Telangiectasia (A-T), a key pathologic feature of which is oxidative stress. Our preliminary results show that disruption of ATM signaling causes aberrant mtDNA copy number, increased mtDNA mutagenesis, and cellular ROS accumulation. We have also discovered that a common defect in A-T patient cells and tissues of ATM null mice is significant depletion of the R1 subunit of ribonucleotide reductase (RNR), an enzyme required to make deoxynucleotides needed for DNA replication and repair. The overall goal of this proposal is to understand the role of the ATM pathway in mtDNA regulation and stability and to test the novel hypothesis that mitochondrial dysfunction contributes to the oxidative stress-associated pathology of A-T.
The specific aims of the proposed project are 1) To determine how loss of ATM signaling impacts mtDNA homeostasis and contributes to cellular oxidative stress using cultured cells in which ATM is inhibited pharmacologically or by RNAi, 2) To define the mitochondrial pathology associated with A-T in vivo, and 3) To determine the physiological and potentially therapeutic consequences of increasing mtDNA copy number and stability in transgenic wild-type and ATM null mice via overexpression of RNR subunit R1 or the mtDNA-regulatory factor, mtTFA. The broad implications of this study are that we will learn an incredible amount about how mtDNA is regulated in vivo, and how the ATMRNR- mtDNA pathway can be exploited as a therapeutic avenue for A-T, mitochondrial diseases, and aging.
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