Role of Mitochondrial DNA Mutations in Aging in Neuronal Cells

描述（由申请人提供）：我们的长期目标是研究线粒体在衰老中的作用。这项拨款申请的总体目标是测试线粒体！衰老理论，特别是研究线粒体DNA （mtDNA）突变在衰老过程中小鼠大脑中的作用。作为最受欢迎的假说之一，线粒体衰老理论预测体细胞线粒体DNA （mtDNA）突变随着时间的推移而积累，这些突变导致的线粒体功能受损是导致各种衰老表型的原因。然而，目前还没有对衰老过程中mtDNA突变的总体积累进行全面的研究，并且与衰老相关的mtDNA突变的生理后果在很大程度上尚不清楚。我们最近建立了一种将mtDNA从小鼠神经末梢（即突触体）转移到细胞系统的方法，并且我们改进了分离和表征mtDNA突变的方法。结合已建立的线粒体分子遗传学和生化技术，我们首次可以通过评估突触体中的单个mtDNA来研究衰老过程中神经元细胞中mtDNA突变的积累。在这个提议中要测试的特殊假设是，线粒体基因组的突变在小鼠神经元细胞的衰老过程中积累；这些mtDNA突变反过来损害线粒体功能，并可能导致包括神经细胞mtDNA在内的各种细胞成分的氧化损伤。我们将通过将突触体mtDNA转移到细胞系系统中，对衰老相关的mtDNA突变进行遗传和功能分析。这将揭示其他方法无法识别的低频mtDNA突变。通过生成携带衰老相关mtDNA突变的神经元细胞模型，我们还将描述突变的生理后果，特别是那些与氧化损伤相关的突变。这个项目的成功完成不仅有助于测试线粒体衰老理论，而且可以为衰老过程的潜在机制提供见解。

DESCRIPTION (provided by applicant): Our long-term goal is to study the role of mitochondria in aging. The overall goal of this grant application is to test the mitochondria! theory of aging and, in particular, to investigate the role of mitochondrial DNA (mtDNA) mutations in mouse brain during aging. As one of the most favored hypotheses, the mitochondrial theory of aging, predicts that somatic mitochondria DNA (mtDNA) mutations accumulate with time, and the compromised mitochondrial function resulting from these mutations is then responsible for various aging phenotypes. However, there has been no comprehensive study of the overall accumulation of mtDNA mutations during aging, and the physiological consequences of aging-related mtDNA mutations are largely unclear. We recently established a method to transfer mtDNA from mouse nerve endings, i.e., synaptosomes to a cell system, and we have improved methods to isolate and characterize mtDNA mutations. Combined with established mitochondrial molecular genetic and biochemical technologies, we can, for the first time, investigate the accumulation of mtDNA mutations in neuronal cells during aging by evaluating individual mtDNA from the synaptosomes. The particular hypothesis to be tested in this proposal is that mutations in the mitochondrial genome accumulate during aging in mouse neuronal cells; these mtDNA mutations in turn compromise mitochondrial function and may result in oxidative damage to various cellular components including mtDNA in neuronal cells. We will perform genetic and functional analyses of aging-related mtDNA mutations by transferring synaptosomal mtDNA to a cell line system. This will reveal low frequency mtDNA mutations that could not be identified by other methods. By generating neuronal cell models that carry aging-related mtDNA mutations, we will also characterize the physiological consequences of the mutations, in particular those related to oxidative damage. The successful completion of this project will not only help to test the mitochondrial theory of aging, but could also provide insights into the underlying mechanisms of the aging process.