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，并在患者的病理水平上存在 某些线粒体疾病和退行性疾病的组织(在[1，2]中综述)。线粒体DNA突变是 易受线粒体质量控制的影响，有缺陷的线粒体选择性自噬的过程，如 观察证明，刺激线粒体自噬可以降低这些突变的水平 模型生物[3-6]和细胞培养[7，8]以及线粒体质量控制缺陷与 线粒体DNA突变水平增加[1，2，9]。然而，mtdna对质量控制的敏感性很差。 理解是因为它受到多个复杂过程的影响，包括质量的不同敏感度 控制mtDNA基因产物中不同类型的缺陷，基因产物之间的交换 细胞器的分裂和融合，突变对mtDNA复制的影响，以及 线粒体DNA翻转和继承。 这项拟议的工作试图了解不同mtdna突变的性质如何决定 它们是否倾向于在细胞内增加或减少频率(此频率称为 异质性)。为此，我开发了一种简单的细胞培养系统，使实验者能够调整 线粒体DNA突变率和每细胞拷贝数，如初步数据所示。使用这个系统，我正在 生成一个细胞库，每个细胞库包含一个或几个存在于中间体的新mtDNA突变 小区内的频率。细胞内突变频率的变化对不同治疗的响应， 尤其是自噬刺激，然后可以使用DNA测序来追踪。用实验来阐明 调节这些变化的机制利用了大大改进的线粒体方法 萨巴蒂尼实验室最近开发出了纯化技术。具体目的是：i)阐明流行率和 线粒体DNA突变偏向线粒体DNA复制的机制。Ii)了解线粒体质量如何 控制作用于不同类型的线粒体DNA突变。 该方法的主要新奇之处在于测量了许多不同种类的线粒体dna的频率变化。 高吞吐量中的突变。以前的研究集中在与药物相对应的一小部分突变上 抗性标记或相对常见的线粒体疾病[12，13]。相比之下，这里的目标是获得 单个细胞类型中数千个突变的相似数据，接近饱和水平的突变 ~16.5kb的哺乳动物线粒体基因组。这项拟议的工作旨在阐明限制效率的因素 针对不同形式的线粒体DNA编码的线粒体功能障碍的线粒体质量控制。 了解这些限制可能会提示是什么使细胞或多或少地倾向于 线粒体在衰老、退行性疾病和线粒体DNA疾病中的功能。

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.