Mitochondrial DNA Mutations and the Aging Process

描述(申请人提供)：为了增加对衰老分子基础的理解，我们建立了一个小鼠模型(PolgD257A)，该模型应该显示线粒体DNA(MtDNA)自发突变率增加。这个动物模型是通过在ES细胞中引入小鼠DNA聚合酶γ(Polg)的核酸外切酶区域的特定突变而产生的。携带线粒体DNA突变表型的小鼠将使研究人员能够阐明衰老研究中尚未解决的核心问题之一，即线粒体突变对衰老过程的贡献。有待检验的中心假设是，mtDNA突变在许多有丝分裂后组织中与衰老有关，在衰老过程中起着因果作用。 具体目的1.线粒体突变小鼠骨骼肌的特征。携带PolgD257A突变的小鼠是存活的，没有明显的发育缺陷。我们建议测定这些动物线粒体DNA的突变谱，以估计体内的突变频率。我们还建议确定电子传递系统(ETS)异常的水平，通过5个月、15个月和30个月的PolgD257A和野生型对照动物单个肌肉纤维上细胞色素C氧化酶(COX)活性的丧失来确定。分离的线粒体还将被鉴定为与年龄相关的生化异常，包括代谢潜力和OS标志物的变化。 特定目的2.骨骼肌基因表达谱。我们建议表征PolgD257A小鼠成年后骨骼肌的基因表达谱，以确定线粒体突变是否在分子水平上加速衰老过程。具体来说，老鼠将在5个月、15个月和30个月大的时候进行研究。这些实验将涉及野生型和PolgD257A小鼠，并利用高密度寡核苷酸阵列，提供超过12,000个基因和EST的数据。 具体目标3.PolgD257A小鼠的存活和疾病模式。我们建议确定PolgD257A小鼠的存活和疾病模式。动物将回交到B6(C57BL/6小鼠)的遗传背景，我们的实验室已经广泛地描述了这种背景下的疾病模式和死亡率。将对PolgD257A小鼠和野生型对照组进行生存模式，包括生存曲线和死亡率的计算。将观察动物的肿瘤、神经和心脏表型的发展，并相应地在组织病理学水平上进行表征。

DESCRIPTION (provided by applicant): In order to increase understanding of the molecular basis of aging, we have generated a mouse model (PolgD257A) that should display increased spontaneous mutation rates in mitochondrial DNA (mtDNA). This animal model was generated by introducing a specific mutation in the exonuclease domain of mouse DNA Polymerase Gamma (POLG) in ES cells. Mice carrying a "mutator" phenotype in mtDNA will allow investigators to elucidate one of the central unresolved issues in aging research, the contribution of mitochondrial mutations to the aging process. The central hypothesis to be tested is that mutations in mtDNA, known to be associated with aging in many postmitotic tissues, play a causal role in the aging process. Specific Aim 1. Characterization of skeletal muscle in mitochondrial mutator mice. Mice carrying the PolgD257A mutation are viable and display no obvious developmental defects. We propose to determine the mutational spectrum in mtDNA of these animals in order to estimate mutational frequency in vivo. We also propose to determine the level of electron transport system (ETS) abnormalities, as determined by loss of cytochrome c oxidase (COX) activity along individual muscle fibers of 5-month, 15-month and 30-month PolgD257A and wild-type control animals. Isolated mitochondria will also be characterized for age-related biochemical abnormalities, including alterations in metabolic potential and OS markers. Specific Aim 2. Gene expression profiling in skeletal muscle. We propose to characterize the gene expression profile of skeletal muscle of PolgD257A mice over the adult lifespan in order to determine if mitochondrial mutations accelerate the aging process at the molecular level. Specifically, mice will be studied at 5 months, 15 months and 30 months of age. These experiments will involve both wild-type and PolgD257A mice and utilize high density oligonucleotide arrays, which provide data on over 12,000 genes and ESTs. Specific Aim 3. Survival and Disease Patterns in PolgD257A Mice. We propose to determine the survival and disease patterns of PolgD257A mice. Animals will be backcrossed to the B6 (C57BL/6 mice) genetic background, which our laboratory has extensively characterized for disease patterns and mortality. Survival patterns, including survival curves and calculation of mortality rates, will be performed for PolgD257A mice and wild-type controls. Animals will be observed for the development of neoplastic, neurologic and cardiac phenotypes and characterized at the histopathology level accordingly.