Mitochondrial DNA Damage: Cellular Responses, Aging and Disease

描述（由申请人提供）： 项目摘要/摘要知识差距：由于损伤线粒体DNA的物质也会损伤核DNA，因此很难研究线粒体DNA损伤所产生的影响。由于细胞核DNA约占细胞DNA的98%，因此DNA损伤的影响可能是由于细胞核DNA损伤。细胞核和线粒体DNA损伤的同时发生，给我们理解细胞如何对线粒体DNA损伤做出特异性反应造成了关键障碍。已经为该项目开发了小鼠模型，其在不存在并发的核DNA损伤的情况下促进线粒体DNA损伤的实验调节。这些模型将被用来描绘的影响，特别是从线粒体DNA损伤。将检验的总体假设是线粒体DNA损伤导致线粒体、细胞和组织功能障碍。具体目标是：1）确定在小鼠寿命期间对急性线粒体DNA损伤（例如可能在创伤期间发生）的细胞反应，2）确定慢性线粒体DNA损伤（例如可能在糖尿病或帕金森病中发生）在小鼠中的影响，和3）确定线粒体DNA损伤的后果是否可以在小鼠寿命期间逆转。研究设计：已经产生了转基因小鼠模型，其通过基因调控的四环素系统在实验调控下表达限制性内切酶EcoRI。表达的调节可以通过在食物中提供四环素类似物多西环素来关闭基因并提供正常食物（无多西环素）来实现EcoRI的表达来实现。与EcoRI编码序列框内融合的线粒体易位前序列确保蛋白质易位到线粒体基质而不是细胞核。一旦进入线粒体，EcoRI切割线粒体DNA，以双链断裂的形式造成损伤。对于目标1，EcoRI将在青年、中年或老年时开启，以确定年龄如何影响急性线粒体DNA损伤的影响。将检查许多线粒体功能以确定对线粒体功能的影响。对于目标2，EcoRI将在年轻的成年人中开启，并在小鼠的生命周期中保持开启。将在生命周期的指定时间点检查对线粒体功能的影响，以确定慢性线粒体DNA损伤如何影响线粒体功能。对于目标3，将在寿命的规定时间点检查逆转线粒体DNA损伤后果的能力。拟议的研究旨在解决定义线粒体DNA损伤产生的特定影响的目标。 公共卫生相关性： 线粒体DNA损伤威胁着线粒体在ATP产生中发挥作用的能力。大部分细胞的ATP是由线粒体通过氧化磷酸化过程产生的。由于线粒体DNA编码氧化磷酸化所需的13个亚基，因此线粒体DNA的完整性对能量产生很重要。线粒体与中风、严重脓毒症、严重烧伤、衰老和神经退行性疾病等有关。美国退伍军人容易受到这些健康问题的影响。因此，了解线粒体DNA损伤的影响对于了解线粒体如何影响衰老，神经退行性疾病，中风，感染，烧伤和其他与退伍军人相关的临床疾病等过程至关重要。

DESCRIPTION (provided by applicant): Project Summary/Abstract Knowledge gap: The effects that emanate specifically from mitochondrial DNA damage have been difficult to study because agents that damage mitochondrial DNA also damage nuclear DNA. Because nuclear DNA represents approximately 98% of the DNA in a cell, the demonstrated effects of DNA damage have likely been due to nuclear DNA damage. The simultaneous occurrence of nuclear and mitochondrial DNA damage has resulted in a critical barrier to our understanding of how cells respond specifically to mitochondrial DNA damage. Mouse models have been developed for this project that facilitate experimental regulation of mitochondrial DNA damage in the absence of concurrent nuclear DNA damage. These models will be used to delineate the effects specifically emanating from mitochondrial DNA damage. The overall hypothesis that will be tested is that mitochondrial DNA damage results in mitochondrial, cellular and tissue dysfunction. The specific aims are: 1) to determine the cellular responses to acute mitochondrial DNA damage (such as might occur during trauma) across the mouse lifespan, 2) to determine the effects of chronic mitochondrial DNA damage (such as might occur with diabetes or Parkinson's disease) in the mouse, and 3) to determine if the consequences of mitochondrial DNA damage can be reversed throughout the mouse lifespan. Research design: Transgenic mouse models have been produced that express the restriction endonuclease EcoRI under experimental regulation via the tetracycline system of gene regulation. Regulation of expression can be achieved by supplying the tetracycline analogue, doxycycline, in food to turn off the gene and providing normal chow (no doxycycline) to achieve expression of EcoRI. A mitochondrial translocation presequence fused in frame with the EcoRI coding sequences assures that the protein is translocated to the mitochondrial matrix and not to the nucleus. Once in the mitochondria, EcoRI cleaves the mitochondrial DNA creating damage in the form of double-strand breaks. For aim 1, EcoRI will be turned on at young adult, middle-age or old age to determine how age impacts the effects of acute mitochondrial DNA damage. A number of mitochondrial functions will be examined to determine the effects on mitochondrial function. For aim 2, EcoRI will be turned on in young adults and left on for the lifespan of the mice. Effects on mitochondrial function will be examined at defined timepoints in the lifespan to determine how chronic mitochondrial DNA damage affects mitochondrial function. For aim 3, the ability to reverse the consequences of mitochondrial DNA damage will be examined at defined timepoints in the lifespan. The proposed studies are intended to address the goal of defining effects specifically emanating from mitochondrial DNA damage. PUBLIC HEALTH RELEVANCE: Project Narrative Mitochondrial DNA damage threatens the ability of mitochondria to fulfill their role in ATP production. A large portion of the cells's ATP is produced by mitochondria through the process of oxidative phosphorylation. Because mitochondrial DNA encodes 13 subunits required for oxidative phosphorylation, integrity of mitochondrial DNA is important for energy production. Mitochondria have been implicated in stroke, severe sepsis, severe burns, aging and neurodegenerative disorders among others. U.S. veterans are susceptible to these health issues. Therefore, understanding the effects of mitochondrial DNA damage is essential to understanding how mitochondria affect processes such as aging, neurodegenerative disease, stroke, infection, burns and other clinical conditions relevant to veterans.