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.

项目摘要： 最常见的线粒体DNA（mtDNA）异常是缺失4977碱基对 称为公共缺失（CD），与线粒体病理相关，并普遍存在 老化。当删除的分子超过时，CD主要表现在大脑中，肌肉 总副本的60％，称为异质。但是，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。 研究CD的主要障碍是缺乏操纵mtDNA的工具，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。具体而言，我们生成了一个诱导的准二级塔伦 在等基因环境和定义的异质状态下生成CD。使用此工具，我们将 在胚胎干细胞中建立低，中和高水平的Cd杂质者，我们将 然后分化为肌肉，神经元和成纤维细胞，并阐明 这种有害的缺失以细胞类型的特异性方式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，以识别维持的途径 突变的mtDNA在有丝分裂细胞中的传播，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丰富的经验相结合将解决 长期存在的有丝分裂细胞中优先突变mtDNA传播的奥秘，并具有 对众多线粒体病理和衰老的重要意义。