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

研究体细胞线粒体常见缺失的影响和动态

• 批准号: 10826448
• 负责人: Agnel Sfeir
• 金额: $ 54.29万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-30 至 2028-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10826448
• 关键词: Aging; Base Pairing; Biogenesis; Brain; Cell Aging; Cell physiology; Cells; Chronic progressive external ophthalmoplegia; DNA Repair; Disease; Drosophila genus; Epithelial Cells; Fibroblasts; Functional disorder; Genetic; Genome; Genome Stability; Genomic Instability; Interphase Cell; Kearns-Sayre syndrome; Link; Mammalian Cell; Methodology; Methods; Mitochondria; Mitochondrial DNA; Mitosis; Monitor; Muscle; Muscle Cells; Mutate; Neurons; Nuclear; Output; Oxidative Phosphorylation; Pathology; Pathway interactions; Physiological; Positioning Attribute; Process; Series; Somatic Cell; Specificity; Testing; Tissues; age related; biological adaptation to stress; cell type; dimer; embryonic stem cell; experience; genome integrity; heteroplasmy; insight; mitochondrial DNA mutation; mitochondrial dysfunction; mitochondrial genome; model organism; mutant; novel; particle; postmitotic; prevent; reconstitution; repaired; respiratory; response; tool; transcription activator-like effector nucleases; transcriptomics

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.

项目总结： 最常见的线粒体DNA异常是4977个碱基对的缺失 称为共同缺失(CD)，与线粒体病理有关，广泛存在于 衰老。CD主要表现在大脑和肌肉中当删除的分子超过 占总拷贝数的60%，称为异质性。然而，导致有害的机制 缺失以及为什么神经细胞和肌肉细胞对镉特别脆弱仍不清楚。 研究cd的主要障碍是缺乏操纵mtDNA的工具，以及无法 以受控方式生成CD。在这里，我们开发了一系列方法来 克服这些障碍。具体地说，我们产生了一个可诱导的准二聚体TALEN 在同基因设置和定义的异质状态下产生Cd。有了这个工具，我们将 在胚胎干细胞中建立低、中、高水平的镉异质性，我们将 然后分化为肌肉、神经元和成纤维细胞，并阐明 这种特定细胞类型的有害缺失及其对细胞衰老的影响。此外， 我们将探索CD的特定细胞类型的分布，确定维持 突变的mtdna在有丝分裂后细胞中的繁殖，同时促进其在分裂细胞中的消除。 将新的遗传工具与在基因组稳定性方面的丰富经验相结合将解决 线粒体DNA优先突变体在有丝分裂后细胞中繁殖的长期谜团 对许多线粒体病理和衰老有重要意义。