Mitochondrial DNA Mutations and the Aging Process

描述（由申请人提供）：为了增加对衰老的分子基础的理解，我们已经产生了一种小鼠模型（PolgD 257 A），该模型应显示线粒体DNA（mtDNA）中的自发突变率增加。通过在ES细胞中的小鼠DNA聚合酶γ（POLG）的核酸外切酶结构域中引入特异性突变来产生该动物模型。在线粒体DNA中携带“突变体”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，已知与许多有丝分裂后组织衰老相关的mtDNA突变在衰老过程中起着因果作用。 具体目标1。线粒体突变小鼠骨骼肌的特征。携带PolgD 257 A突变的小鼠是可行的，并且没有表现出明显的发育缺陷。我们建议确定这些动物的mtDNA突变谱，以估计体内突变频率。我们还建议确定电子传递系统（ETS）异常的水平，如通过细胞色素c氧化酶（考克斯）活性沿着5个月、15个月和30个月PolgD 257 A和野生型对照动物的个体肌纤维的损失所确定的。分离的线粒体还将表征年龄相关的生化异常，包括代谢潜力和OS标志物的改变。 具体目标2。骨骼肌基因表达谱。我们建议表征PolgD 257 A小鼠骨骼肌在成年期的基因表达谱，以确定线粒体突变是否在分子水平上加速衰老过程。具体而言，将在5月龄、15月龄和30月龄时研究小鼠。这些实验将涉及野生型和PolgD 257 A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。PolgD 257 A小鼠的存活和疾病模式。我们建议确定PolgD 257 A小鼠的生存和疾病模式。动物将与B6（C57 BL/6小鼠）遗传背景回交，我们的实验室已广泛表征了疾病模式和死亡率。将对PolgD 257 A小鼠和野生型对照进行存活模式，包括存活曲线和死亡率计算。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.