Using Mitochondrial DNA Mutations to Understand Limitations of Mitochondrial Quality Control

项目摘要 线粒体DNA（mtDNA）突变随着年龄的增长而积累，并在受影响的患者中以病理水平存在。 某些线粒体疾病和退行性疾病的组织（综述见[1，2]）。mtDNA突变是 易受线粒体质量控制，有缺陷的线粒体的选择性自噬过程，如 通过观察证明，刺激线粒体自噬可以降低这些突变的水平， 模式生物[3-6]和细胞培养[7，8]以及线粒体质量控制中的缺陷与 线粒体DNA突变水平增加[1，2，9]。然而，线粒体DNA的质量控制的敏感性差 因为它受到多个复杂过程的影响，包括质量的变化敏感性， 控制mtDNA基因产物中不同类型的缺陷， 细胞器通过分裂和融合，突变对mtDNA复制的影响，以及 线粒体DNA的转换和遗传。 这项工作旨在了解不同mtDNA突变的性质如何决定 无论它们倾向于在小区内增加还是减少频率（该频率被称为 异质性）。为此，我开发了一个简单的细胞培养系统，使实验人员能够进行调整 mtDNA突变率和每个细胞的拷贝数，如初步数据所示。使用这个系统，我 产生细胞文库，每个细胞含有一个或几个存在于中间水平的新mtDNA突变， 细胞内的频率。响应于不同处理的细胞内突变频率的变化， 特别是自噬刺激，然后可以使用DNA测序来跟踪。实验来阐明 介导这些变化的机制利用了大大改进的线粒体 纯化最近在萨巴蒂尼实验室开发。具体目标是：I）阐明患病率和 mtDNA突变的机制，使mtDNA复制偏向。II）为了了解线粒体质量如何 控制作用于不同类型的mtDNA突变。 该方法的关键新奇在于测量了许多不同类型mtDNA的频率变化 高通量的突变。先前的研究集中在与药物相关的一小部分突变上。 耐药标志物或相对常见的线粒体疾病[12，13]。相反，这里的目标是获得 在单个细胞类型中的数千个突变的类似数据，接近饱和水平的突变。 ~16.5kb哺乳动物线粒体基因组。拟议的工作旨在阐明限制效率的因素 线粒体质量控制方面的各种形式的mtDNA编码的线粒体功能障碍。 了解这些限制可能会提示是什么使细胞更容易或更不容易下降， 线粒体在衰老、退行性疾病和线粒体DNA紊乱中的功能。

Project Summary Mitochondrial DNA (mtDNA) mutations accumulate with age and are present at pathological levels in affected tissues of certain mitochondrial disorders and degenerative diseases (reviewed in [1, 2]). mtDNA mutations are susceptible to mitochondrial quality control, the process of selective autophagy of defective mitochondria, as evidenced by observations that stimulation of mitochondrial autophagy can reduce levels of these mutations in model organisms [3-6] and cell culture [7, 8] and that defects in mitochondrial quality control correlate with increased levels of mtDNA mutations [1, 2, 9]. However, the susceptibility of mtDNA to quality control is poorly understood because it is influenced by multiple complex processes, including the varied sensitivity of quality control to different types of defects in mtDNA gene products, the exchange of gene products between organelles through fission and fusion, the impact of mutations on mtDNA replication, and stochasticity of mtDNA turn-over and inheritance. The proposed work seeks to understand how the nature of different mtDNA mutations determines whether they tend to increase or decrease in frequency within the cell (this frequency is known as the level of heteroplasmy). To do this, I have developed a simple cell culture system that enables the experimenter to tune the mtDNA mutation rate and copy number per cell, as shown in preliminary data. Using this system, I am generating a library of cells, each containing one or few new mtDNA mutations present at intermediate frequency within the cell. Changes in intracellular frequencies of mutations in response to different treatments, particularly autophagy stimulation, can then be tracked using DNA sequencing. Experiments to elucidate mechanisms mediating these changes take advantage of greatly improved methods of mitochondrial purification recently developed in the Sabatini Lab. The specific aims are: I) To elucidate the prevalence and mechanisms of mtDNA mutations that bias mtDNA replication. II) To understand how mitochondrial quality control acts on different types of mtDNA mutations. The key novelty of the approach is to measure changes in frequency of many different kinds of mtDNA mutations in high throughput. Prior studies focused on a small set of mutations corresponding to drug resistance markers or relatively-common mitochondrial diseases [12, 13]. In contrast, the goal here is to obtain similar data for thousands of mutations in a single cell type, approaching saturation-level mutagenesis of the ~16.5kb mammalian mitochondrial genome. The proposed work aims to elucidate the factors limiting efficiency of mitochondrial quality control with respect to diverse forms of mtDNA-encoded mitochondrial dysfunction. Understanding these limitations may suggest what renders cells more or less prone to the decline of mitochondrial function in aging, degenerative disease and mtDNA disorders.