Mitochondrial Dysfunction in Alzheimer's Disease

大量证据表明，阿尔茨海默病（AD）的发病机制可能涉及线粒体功能障碍和氧化损伤。线粒体功能障碍的发生可能是由于原发性基因突变或由于获得性线粒体DNA （mtDNA）突变，这可能与氧化损伤有关。在目前的提案中，我们将研究与正常对照相比，AD患者死后脑组织中mtDNA突变的发生率是否增加。我们将利用直接mtDNA测序和变性凝胶电泳来检测低频突变，我们将关联8-羟基-2-脱氧鸟苷（OH8dG）的水平，这是DNA氧化损伤的标志。我们开发了一种灵敏而准确的OH8dG检测方法，可用于检测体液中的浓度。我们打算利用这种方法来测量阿尔茨海默病患者和对照组尿液、血浆和脑脊液中的OH8dG。我们将利用从特征明确的阿尔茨海默病患者身上获得的血小板制造杂交体。-淀粉样蛋白沉积可引起氧化应激和/或氧化应激可增加-淀粉样蛋白的产生。我们将通过直接测序来检测携带APP V717F突变的转基因小鼠是否增加了mtDNA突变。我们将把这与ELISA测定的b-淀粉样蛋白浓度以及氧化损伤标志物（包括丙二醛、OH8dG和5-硝基- γ -生育酚）联系起来。我们将检测缺乏线粒体自由基清除酶锰超氧化物歧化酶的转基因小鼠系。我们还将利用缺乏谷胱甘肽过氧化物酶的小鼠，这种酶可以解毒线粒体内的过氧化氢。我们将确定这些小鼠是否在mtDNA氧化损伤中出现年龄依赖性增加，mtDNA突变数量增加，以及这是否与可提取的b-淀粉样蛋白水平的增加相关。这些研究旨在帮助进一步阐明线粒体功能障碍和氧化损伤在正常衰老和AD中的作用。

There is substantial evidence that the pathogenesis of Alzheimer's Disease (AD) may involve mitochondrial dysfunction and oxidative damage. Mitochondrial dysfunction could occur as either a consequence of primary genetic mutations or due to acquired mitochondrial DNA (mtDNA) mutations, which may be related to oxidative damage. In the present proposal, we will examine whether there is an increased incidence of mtDNA mutations in postmortem brain tissue from AD patients as compared to normal controls. We will utilize direct mtDNA sequencing as well as denaturing gelelectrophoresis to detect low frequency mutations, and we will correlate levels of 8-hydroxy-2- deoxyguanosine (OH8dG), a marker of oxidative damage to DNA. We have developed a sensitive and accurate assay for OH8dG, which is useful in examining concentrations in body fluids. We intend to utilize this assay to measure OH8dG in urine, plasma and CSF of AD patients and controls. We will make cybrids utilizing platelets obtained from well characterized AD patients. *-amyloid deposition may cause oxidative stress and/or oxidative stress may increase -amyloid production. We will examine whether transgenic mice with the APP V717F mutation have increased mtDNA mutations as assessed by direct sequencing. We will correlate this with concentrations of b-amyloid as measured by ELISA, as well as markers of oxidative damage including malondialdehyde, OH8dG and 5-nitro-gamma-tocopherol. We will examine transgenic mouse lines which are deficient in the mitochondrial free radical scavenging enzyme manganese superoxide dismutase. We will also utilize mice, which are deficient in glutathione peroxidase, which detoxifies hydrogen peroxide within mitochondria. We will determine whether these mice develop age-dependent increases in oxidative damage within mtDNA, increased numbers of mtDNA mutations and whether this correlates with increases in extractable levels of b-amyloid. These studies are designed to help to further elucidate the role of mitochondrial dysfunction and oxidative damage in normal aging and AD.