A model for elimination of defective mitochondrial genomes

摘要 拟议研究的主要目标是揭示消除有缺陷的线粒体的机制 基因组（mtDNA）通过遗传和表观遗传过程。动物细胞中线粒体DNA的异质状态 允许有缺陷的线粒体DNA积累，并具有潜在的复制优势。质量控制系统功能 去除这种有害的线粒体基因组。我们发现大量带有缺失的 线虫中的 mtDNA，uaDf5，通常与野生型 mtDNA 稳定维持在异质状态 （WT-mtDNA）随着成年年龄的增长而增加，并且这种增加的负担会传递给年长母亲的后代。 这种有缺陷的 mtDNA 在缺乏两种质量控制系统之一的突变体中也会升高：线粒体自噬（粉红色- 1(-)) 或种系程序性细胞死亡 (PCD; ced-13(-))。然而出乎意料的是，我们发现，当两者 Pink-1 和 ced-13 功能被消除，相反的情况发生：uaDf5 被快速且完全地消除， 有效治愈动物线粒体疾病。更令人吃惊的是，两者杂交的后代 Pink-1(-) 或 ced-13(-) 单突变母亲的雄性同时缺乏 PINK-1 和 CED-13 也表现出快速 去除删除的线粒体DNA，并且无论后代基因型不同，都会发生这种效应。这些 研究结果使我们推测这些杂交的起始遗传事件（IGE）触发了有效的 跨代表观遗传过程，可区分并有效去除有缺陷的 mtDNA。有了这些 基于基础发现，我们将研究这一引人注目的 mtDNA 质量控制过程的机制。瞄准 1，我们将分析 IGE 触发的 uaDf5 去除对其年龄依赖性积累的影响和 分析其发育时序。我们将测试消除过程是否对其他线粒体DNA具有通用性 删除并评估大小和序列位置对删除过程的影响。我们将调查 线粒体自噬和 PCD 途径中的其他成分是否在激活清除方面表现出协同作用，并将进行测试 增强的线粒体裂变可能允许区分和消除 uaDf5 的假设。瞄准 2、我们将分析mtDNA质量明显表观遗传跨代传递的基础 控制过程。我们将评估我们发现的 WT-mtDNA 的显着扩增这一假设 发生在去除 uaDf5 的世代之前，去除过程需要去除 uaDf5，并且 uaDf5 是 需要触发此放大。我们将研究 IGE 是否拮抗线粒体 UPR， 它可以保护 uaDf5 不被删除。我们将检验 IGE 是由父亲专门触发的假设 PINK-1 和 CED-13 的丢失。最后，我们将评估 IGE 激活的表观遗传过程是否被传递 通过细胞核或细胞质/线粒体。这些研究将有助于我们理解如何 维持健康的线粒体基因组，这是衰老、神经退行性变和衰老的首要问题 线粒体疾病。

SUMMARY The major objective of the proposed research is to unveil mechanisms that eliminate defective mitochondrial genomes (mtDNA) through genetic and epigenetic processes. The heteroplasmic state of mtDNA in animal cells allows accumulation of defective mtDNAs with potential replicative advantage. Quality control systems function to remove such deleterious mitochondrial genomes. We found that the abundance of a large deletion-bearing mtDNA in C. elegans, uaDf5, that is normally stably maintained in a heteroplasmic state with wild-type mtDNA (WT-mtDNA) increases with adult age and that this increased burden is passed onto offspring of older mothers. This defective mtDNA is also elevated in mutants lacking either of two quality control systems: mitophagy (pink- 1(-)) or germline programmed cell death (PCD; ced-13(-)). Unexpectedly, however, we found that when both pink-1 and ced-13 functions are eliminated, the opposite occurs: uaDf5 is rapidly and completely removed, effectively curing the animal of mitochondrial disease. Even more startling, descendants of crosses between pink-1(-) or ced-13(-) single mutant mothers with males lacking both PINK-1 and CED-13 also show rapid removal of the deleted mtDNA, and this effect occurs irrespective of the differing descendant genotypes. These findings lead us to hypothesize that an initiating genetic event (IGE) from these crosses triggers a potent transgenerational epigenetic process that discriminates and effectively removes defective mtDNA. With these foundational findings, we will investigate the mechanisms of this striking mtDNA quality control process. In Aim 1, we will analyze the effect of the IGE-triggered removal of uaDf5 on its age-dependent accumulation and analyze its developmental timing. We will test whether the elimination process is general to other mtDNA deletions and assess the impact of size and sequence location on the removal process. We will investigate whether other components in the mitophagy and PCD pathways show synergy in activating removal and will test the hypothesis that enhanced mitochondrial fission may allow for discrimination and elimination of uaDf5. In Aim 2, we will analyze the basis for the apparently epigenetic transgenerational transmission of the mtDNA quality control process. We will evaluate the hypothesis that the dramatic amplification of WT-mtDNA that we found occurs prior to the generations in which uaDf5 is removed is required for the removal process and that uaDf5 is required to trigger this amplification. We will investigate whether the IGE antagonizes the mitochondrial UPR, which protects uaDf5 from removal. We will test the hypothesis that the IGE is triggered specifically by paternal loss of PINK-1 and CED-13. Finally, we will assess whether the IGE-activated epigenetic process is transmitted through the nucleus or cytoplasm/mitochondria. These studies will contribute to our understanding of how a healthy mitochondrial genome is maintained, a problem of premier importance to aging, neurodegeneration, and mitochondrial disease.