Investigating the impact and dynamic of mitochondrial common deletion in somatic cells

项目摘要： 最常见的线粒体DNA（mtDNA）异常是4977个碱基对的缺失 称为共同缺失（CD），与线粒体病理学相关，广泛分布于 衰老当删除的分子超过时，CD主要表现在大脑和肌肉中 占总拷贝数的60%，称为异质性。然而，造成有害的机制 缺失以及为什么神经元和肌肉细胞特别容易受到CD的影响仍然不清楚。 研究CD的主要障碍是缺乏操纵mtDNA的工具， 以受控的方式生成CD。在这里，我们开发了一系列方法， 克服这些障碍。具体来说，我们产生了一种诱导型准二聚体TALEN， 在同基因设置和定义的异质性状态下产生CD。有了这个工具，我们将 在胚胎干细胞中建立低、中、高水平的CD异质性， 然后分化成肌肉、神经元和成纤维细胞，并阐明 这种有害的缺失以细胞类型特异性的方式及其对细胞衰老的影响。此外，委员会认为， 我们将探索CD的细胞类型特异性分布，确定维持CD的途径， 突变mtDNA在有丝分裂后细胞中增殖，同时促进其在分裂细胞中的消除。 将新的遗传工具与基因组稳定性方面的丰富经验相结合， 有丝分裂后细胞中优先突变mtDNA繁殖的长期谜团， 对许多线粒体病理学和衰老有重要意义。

Project Summary: The most common mitochondrial DNA (mtDNA) abnormality is a deletion of 4977 base pairs called the common deletion (CD), associated with mitochondrial pathologies and widespread in aging. The CD primarily manifests in the brain and muscles when deleted molecules exceed 60% of total copies, known as heteroplasmy. However, the mechanisms that cause harmful deletions and why neuronal and muscle cells are particularly vulnerable to CD remain unclear. Major obstacles to studying the CD is the lack of tools to manipulate mtDNA and the inability to generate the CD in a controlled manner. Here, we developed a series of methodologies to overcome these barriers. Specifically, we generated an inducible quasi-dimeric TALEN that generates the CD in isogenic settings and at defined heteroplasmy states. With this tool, we will establish low, medium, and high levels of CD heteroplasmy in embryonic stem cells that we will then differentiate into muscle, neuronal, and fibroblast cells and elucidate the consequence of this harmful deletion in a cell-type-specific manner and its impact on cellular aging. Furthermore, we will explore the cell-type-specific distribution of CD, Identifying the pathways that sustain mutant mtDNA propagation in post-mitotic cells while promoting its elimination in dividing cells. Combining novel genetic tools with extensive experience in genome stability will resolve the long-standing mystery of preferential mutant mtDNA propagation in post-mitotic cells and have significant implications for numerous mitochondrial pathologies and aging.