Mitochondrial Dysfunction and Oxidative Stress in Ataxia Telangiectasia

描述(由申请人提供)： 线粒体对正常的能量代谢至关重要，因为它们包含氧化磷酸化(OXPHOS)系统，该系统产生细胞的初级能量货币--三磷酸腺苷。它们还执行数百种其他代谢功能，并与钙稳态和细胞凋亡密切相关。然而，线粒体也是有毒活性氧物种(ROS)的主要来源，ROS破坏细胞成分，促进氧化应激，并导致与线粒体功能障碍相关的病理。线粒体包含一个环状线粒体DNA(MtDNA)基因组，编码13个OXPHOS亚基，其突变或枯竭会导致复杂的疾病和与年龄相关的病理。哺乳动物细胞含有数以千计的mtDNA拷贝，每个组织都有一个根据其特定的能量需求和特殊功能量身定做的独特拷贝数。ATM检查点信号通路的突变导致多方面的致命性疾病共济失调-毛细血管扩张症(A-T)，其主要病理特征是氧化应激。我们的初步结果表明，ATM信号的中断会导致线粒体DNA拷贝数异常，线粒体DNA突变增加，细胞内ROS积累。我们还发现，ATM缺失型小鼠A-T患者细胞和组织中的一个常见缺陷是核糖核苷酸还原酶(RNR)的R1亚基显著耗尽，RNR是制造DNA复制和修复所需的脱氧核苷酸所必需的酶。这项提议的总体目标是了解ATM通路在线粒体DNA调节和稳定性中的作用，并检验线粒体功能障碍导致A-T氧化应激相关病理的新假说。该项目的具体目标是：1)利用ATM被药物或RNAi抑制的培养细胞，确定ATM信号的丢失如何影响mtDNA动态平衡和促进细胞氧化应激；2)在体内确定与A-T相关的线粒体病理；以及3)通过过表达RNR亚单位R1或mtDNA调节因子mtTFA，确定在转基因野生型和ATM缺失小鼠中增加mtDNA拷贝数和稳定性的生理和潜在治疗后果。这项研究的广泛意义在于，我们将了解到令人难以置信的大量关于mtDNA在体内是如何调控的，以及ATMRNR-mtDNA途径如何被用作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.