Mitochondrial Dysfunction and Oxidative Stress in Ataxia Telangiectasia

描述（由申请人提供）： 线粒体对于正常的能量代谢是至关重要的，因为它们容纳产生细胞的主要能量货币ATP的氧化磷酸化（OXPHOS）系统。它们还执行数百种其他代谢功能，并密切参与钙稳态和细胞凋亡。然而，线粒体也是毒性活性氧（ROS）的主要来源，其损害细胞组分，促进氧化应激，并引起与线粒体功能障碍相关的病理。线粒体含有编码13个OXPHOS亚基的环状线粒体DNA（mtDNA）基因组，其突变或缺失导致复杂疾病和年龄相关病理。哺乳动物细胞含有数千个mtDNA拷贝，每个组织都有一个特征性的拷贝数，以适应其特定的能量需求和专门的功能。ATM检查点信号传导激酶的突变导致多方面和致命的疾病共济失调-毛细血管扩张症（A-T），其关键病理特征是氧化应激。我们的初步研究结果表明，ATM信号转导的中断导致异常的mtDNA拷贝数，增加mtDNA突变，和细胞ROS积累。我们还发现，ATM缺失小鼠的A-T患者细胞和组织中的常见缺陷是核糖核苷酸还原酶（RNR）的R1亚基的显著耗尽，RNR是一种制造DNA复制和修复所需的脱氧核苷酸所需的酶。该提案的总体目标是了解ATM通路在mtDNA调节和稳定性中的作用，并验证线粒体功能障碍有助于A-T氧化应激相关病理学的新假设。该项目的具体目标是：1）使用ATM被RNAi抑制的培养细胞来确定ATM信号转导的丢失如何影响mtDNA稳态并有助于细胞氧化应激，2）定义体内与A-T相关的线粒体病理学，和3）为了确定在转基因野生型中增加mtDNA拷贝数和稳定性的生理和潜在治疗后果，型和ATM裸小鼠通过过度表达RNR亚基R1或线粒体DNA调节因子，mtTFA。这项研究的广泛意义是，我们将了解到大量关于mtDNA在体内如何调节，以及ATMRNR- mtDNA通路如何被用作A-T，线粒体疾病和衰老的治疗途径。

DESCRIPTION (provided by applicant): 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.