Mitochondrial Genetics and Physiology

线粒体遗传学和生理学

• 批准号: 8344891
• 负责人: Hong Xu
• 金额: $ 248.43万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8344891
• 关键词: Adult; Age; Aging; Aging-Related Process; Anabolism; Animals; Appearance; Autophagocytosis; Bacterial Toxins; Behavior; Behavioral Genetics; Biological Assay; Biology; Brain; Cell Aging; Cell Cycle; Cell physiology; Cells; Complex; DNA; DNA biosynthesis; Disease; Drosophila genus; Electron Transport; Engineering; Evolution; Excision; Female; Fertility; Free Radicals; Frequencies; Gene Mutation; Generations; Genetic; Genetic Recombination; Goals; Health; Heart; Histones; Homeostasis; Human; Inherited; Lead; Link; Lipids; Liver; Longevity; Lysosomes; Mitochondria; Mitochondrial DNA; Mitochondrial Diseases; Mitochondrial Inheritance; Mitochondrial Proteins; Mitotic; Modeling; Molecular; Monitor; Mus; Muscle; Mutagenesis; Mutation; Neurodegenerative Disorders; Neurologic; Oogenesis; Organelles; Organism; Ovary; Pathogenesis; Pathway interactions; Physiologic pulse; Physiological; Physiology; Play; Population; Premature aging syndrome; Process; Proteins; RNA Interference; Radial; Reporter; Role; Screening procedure; Source; Stem cells; System; Testing; Tissues; Vesicle; Whole Organism; age related; aged; cell age; egg; feeding; fitness; fly; genetic analysis; genome wide association study; genome-wide; human disease; mitochondrial DNA mutation; multicatalytic endopeptidase complex; normal aging; parkin gene/protein; prevent; repaired; respiratory; response; segregation; stem cell differentiation; stem cell division; tissue/cell culture; transmission process

I. A genetic approach for mitochondrial degradation. Marcomolecules in mitochondria are prone to damage because of their close vicinity to the free radicals, the by products of mitochondrial electron transferring. However there is no significant accumulation of dysfunctional mitochondrial in normally aged tissues. It is conceivable that post mitotic cells ought to constantly monitor mitochondrial abundance and fitness and are able to cleanse the defective mitochondria and replenish with new organelles. Indeed, mitochondria undergo constant turnover in post mitotic cells. On the other hand, accumulation of defective mitochondria has been associated with many types of human neurodegenerative diseases, further indicating a crucial involvement of this mitochondrial surveillance system in maintaining cell wellness and tissue integrity. But it remains to be determined on first ground whether defective mitochondria are selectively removed. Little is known regarding the mechanism of mitochondrial degradation, which is essential for maintaining overall fitness of mitochondria population, and likely play a role in mitochondrial inheritance. We have developed a genetic approach to quantitatively assay mitochondrial degradation, and test in principle whether cells can selectively eliminate damaged mitochondria. To delineate the cellular responses against defective mitochondria, we are applying this assay for a genome wide RNAi screening in S2 cells. We expressed a bacterial toxin, PorB at low level in culture cells and tissues. PorB localized to and damaged a subset of cellular mitochondria. As a reference, an endogenous mitochondrial protein, Tom20 was also expressed. Mitochondrion is believed to be turned over by autophagic engulfment of whole organelle. Thus the behavior of reporter proteins will reflect the fate of the particular mitochondriaon that they reside in. We found that PorB was turned over much faster than Tom20 after a pulse of induction. The fast decline of PorB coincided with the appearance of vesicles containing abnormal mitochondria. Also most of PorB harboring mitochondria were associated with lysosomes, suggesting that defective mitochondria were degraded through a lysosome dependent process. Genetic analysis revealed that proteasome and autophagy were required for the removal of damaged mitochondria, and demonstrated Parkin dependent degradation of damaged mitochondria in muscle. This study present a direct evidence of selective degradation of defective mitochondria in otherwise healthy cells, also provide a genetic handle dissect the molecular players and cellular pathways participating in the process. A genome wide screening is currently underway. Accumulation of defective mitochondria ahs been linked to the pathogenesis of many neurodegenerative diseases. Most mitochondrial DNA diseases are caused by heteroplasmic mtDNA mutations. Uncovering the mechanism of mitochondrial turnover not only will facilitate our understanding of basic biology regarding mitochondrial homeostasis, it may also provide new angle for managing age-related neurological and mtDNA diseases. II. Mitochondrial DNA mutations on stem cell aging Mitochondria are prone to damage. Besides converting energy to ATP and carrying out biosynthesis, mitochondria also generate much of free radials that can damage proteins, lipids and DNA. To make matters worse, mitochondrial DNA has no histone protection and lacks recombination or other significant repair mechanisms. Mutations on the electron transport chains complexes could result in the generation of more free radicals and exacerbate the mitochondrial damage in a feed-forward cycle. Accumulation of mutations on mtDNA during lifetime has been postulated to cause age related decline of biogenetics and tissue homeostasis. Recent studies that engineered mice with elevated rate of mtDNA mutagenesis display premature aging, attest the mtDNA mutation in aging process in principle. However the levels of mtDNA mutations from various tissue of normally aged animals including liver, heart, brain and others are rather too low to possibly elicit any pathological consequences, which argues against a causative role of mtDNA mutations in physiological aging, particularly in the post mitotic tissues. DNA replication is the source of mutations. In the adulthood, most of tissues are consist of post mitotic cells that have no or very limited replication, which might explain the lack of mtDNA mutations in most somatic tissues (please refer to the next segment for more detail on this issue). On the other hand, mtDNA mutations in these actively dividing cells in the adulthood, i.e., stem cells could reach high level along lifespan and compromise stem cells activities. Considering the essential roles of stem cells in tissue homeostasis, age dependent decline of stem cells functions could contribute to the aging of whole organism. In the past year, we have been utilizing the drosophila female germline stem cells, one of the best-characterized and genetically tractable models to study the aging of stem cells. Normal aged female flies completely cease to produce any egg about six-week old, while most of germline stem cells remain in the niches, indicating that some type of cellular abnormality occurred and impede stem cell differentiation. We used mtDNA mutation selection as an indication of mtDNA mutation load in female germline stem cells. We found that the frequency of escapers increased dramatically in old female comparing to young flies, reflecting a higher level of mtDNA mutations in aged germline stem cells. We also found old ovary demonstrated deficiency in mtDNA encoded respiratory complex, indicating that mtDNA mutations level is high enough to compromise the functionality of aged stem cells. In addition, virgin female that have reduced number of stem cell division cycles has higher mitochondrial activity, fecundity comparing to these crossed flies. Our results suggested that continues division of stem cells would lead to the accumulation of mtDNA mutations, which in turn might compromise stem cell activity and eventually trigger the shutdown of oogenesis. We are now trying to genetically alter mtDNA mutations level and test whether mtDNA mutations play a causal role in stem cell aging.

I.线粒体降解的遗传方法。 线粒体中的marcomolecules由于与线粒体电子转移的产物的近距离附近而容易受到破坏。 但是，在正常老化的组织中，线粒体功能障碍尚无明显的积累。可以想象，后有丝分裂细胞应该不断监测线粒体的丰度和健身，并能够清洁有缺陷的线粒体并用新的细胞器补充。实际上，线粒体在后有丝分裂细胞中经历恒定的周转。 另一方面，线粒体有缺陷的积累与许多类型的人类神经退行性疾病有关，进一步表明该线粒体监测系统在维持细胞健康和组织完整性方面至关重要。但是，是否有选择性去除线粒体有缺陷的线粒体仍有待确定。关于线粒体降解的机制知之甚少，这对于维持线粒体种群的整体适应性至关重要，并且可能在线粒体遗传中起作用。我们已经开发了一种遗传方法来定量测定线粒体降解，并原则上检验细胞是否可以选择性地消除受损的线粒体。 为了描绘针对有缺陷的线粒体的细胞反应，我们将此测定应用于S2细胞中的基因组宽RNAi筛选。 我们表达了一种细菌毒素，在培养细胞和组织中低水平的porb。 PORB定位于细胞线粒体的一部分。 作为参考，还表达了内源性线粒体蛋白的内源性线粒体蛋白。据信线粒体被整个细胞器的自噬吞噬而移交。因此，报道蛋白的行为将反映他们所居住的特定线粒体的命运。我们发现，在诱导脉冲后，PORB的移交比TOM20快得多。 PORB的快速下降与含有异常线粒体的囊泡的出现相吻合。同样，大多数携带线粒体的孔都与溶酶体有关，这表明有缺陷的线粒体通过溶酶体依赖的过程降解。遗传分析表明，去除损坏的线粒体需要蛋白酶体和自噬，并证明了parkin依赖性的肌肉中线粒体受损的降解。 这项研究提供了直接证据表明，在其他健康细胞中有缺陷的线粒体有缺陷的降解，还提供了遗传处理方法，可以剖析参与该过程的分子参与者和细胞途径。目前正在进行一项基因组广泛的筛查。线粒体AHS的积累与许多神经退行性疾病的发病机理有关。大多数线粒体DNA疾病是由异质mtDNA突变引起的。揭示线粒体周转的机制不仅可以促进我们对线粒体稳态的基本生物学的理解，还可能为管理与年龄相关的神经系统疾病和mtDNA疾病提供新的角度。 ii。干细胞老化的线粒体DNA突变 线粒体容易损坏。 除了将能量转化为ATP并进行生物合成外，线粒体还产生许多可能损害蛋白质，脂质和DNA的游离径向。 更糟糕的是，线粒体DNA没有组蛋白保护，并且缺乏重组或其他重要的修复机制。电子传输链复合物上的突变可能导致产生更多的自由基，并在进料前循环中加剧线粒体损伤。假定在生命周期中，MTDNA突变的积累会导致生物遗传学和组织稳态相关的下降。最近的研究以升高的mtDNA诱变率设计了小鼠，表明了过早的衰老，这证明了MTDNA突变原则上的衰老过程。然而，包括肝脏，心脏，大脑和其他人（包括肝脏，心脏，大脑和其他人）的各种组织的mtDNA突变水平相当低，无法引起任何病理后果，这反对MTDNA突变在生理衰老中的致病作用，尤其是在后有丝分裂组织中。 DNA复制是突变的来源。在成年期，大多数组织都是由没有或非常有限复制的有丝分裂细胞组成的，这可能解释了大多数体细胞组织中缺乏mtDNA突变（请参阅下一个部分以获取有关此问题的更多详细信息）。另一方面，在这些积极分裂的细胞中，mtDNA突变在成年后，即干细胞可以沿着寿命和损害干细胞活性达到高水平。考虑到干细胞在组织稳态中的基本作用，干细胞功能的年龄依赖性下降可能有助于整个生物体的衰老。在过去的一年中，我们一直在利用果蝇雌性种系干细胞，这是研究干细胞衰老的最佳和遗传学模型之一。 正常老年雌性苍蝇完全停止产生大约六周大的卵，而大多数种系干细胞仍留在壁ni，表明某种类型的细胞异常发生并阻碍干细胞分化。我们使用mtDNA突变选择作为女性种系干细胞中mtDNA突变载荷的指示。我们发现，与年轻的苍蝇相比，逃生剂的频率急剧增加，反映了老年种系干细胞中较高水平的mtDNA突变。我们还发现，旧的卵巢表现出MTDNA编码的呼吸复合物缺乏症，表明mtDNA突变水平足够高以损害老年干细胞的功能。此外，减少干细胞分裂循环数量的处女女性具有较高的线粒体活性，与这些交叉苍蝇相比，繁殖力。 我们的结果表明，继续分裂干细胞将导致mtDNA突变的积累，进而可能损害干细胞活性并最终触发卵子发生的关闭。现在，我们正在尝试从遗传上改变mtDNA突变水平，并测试mtDNA突变在干细胞衰老中是否起因果作用。