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.

项目概要： 最常见的线粒体 DNA (mtDNA) 异常是 4977 个碱基对的缺失 称为常见缺失 (CD)，与线粒体病理相关，广泛存在于 老化。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元和肌肉细胞特别容易受到CD的影响仍不清楚。 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论 克服这些障碍。具体来说，我们生成了一种可诱导的准二聚体 TALEN 在等基因设置和定义的异质性状态下生成 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，我们将 然后分化为肌肉、神经元和成纤维细胞并阐明其结果 这种以细胞类型特异性方式有害的缺失及其对细胞衰老的影响。此外， 我们将探索 CD 的细胞类型特异性分布，确定维持 CD 的途径 突变体 mtDNA 在有丝分裂后细胞中繁殖，同时促进其在分裂细胞中的消除。 将新颖的遗传工具与基因组稳定性方面的丰富经验相结合将解决这个问题 有丝分裂后细胞中突变体 mtDNA 优先繁殖的长期谜团 对许多线粒体病理学和衰老具有重大影响。