Giant Mitochondria in Aging

描述（由申请人提供）：有重要证据表明线粒体与生物衰老有关，最近的一项理论提出，巨大的线粒体参与有丝分裂后组织的衰老。在这些组织中，巨大的线粒体似乎逃避自噬作用，积累并取代正常的线粒体。在正常线粒体存在的情况下或从正常线粒体中分离出来后对巨型线粒体进行表征的技术是非常必要的。这项建议的短期目标是表征和分离巨型线粒体，并确定这些线粒体是否影响肌肉功能。我们的长期目标是利用并提供新的技术方法，在亚细胞和分子水平上揭示人类生物衰老的变化。这项工作利用了成肌细胞模型的线粒体和Fischer 344大鼠的肌肉。我们有四个目标：(1)在细胞模型中基于它们的细胞生物学特征来表征巨线粒体。(2)纯化从这些体内细胞模型中分离的巨型线粒体，并对这些巨型线粒体进行生化表征。(3)比较巨线粒体和正常线粒体的蛋白质组学。(4)老龄骨骼肌中巨大线粒体的特征。第一个目的是使用共聚焦显微镜，或将该技术与单细胞毛细管电泳相结合，研究大小、DMA含量和合成、DNA突变、羰基积累、ROS产生、膜电位、线粒体融合和自噬作用。第二个目标描述了使用自由流动电泳分离巨型和正常线粒体亚群的方法，然后使用适用于分离细胞器的方法对这些分离的线粒体进行表征。第三个目的是在细胞培养中利用稳定同位素标记来比较巨线粒体和正常线粒体的蛋白质组。最后一个目的是研究含有巨大线粒体的肌肉中与年龄相关的变化。

DESCRIPTION (provided by applicant): There is significant evidence linking mitochondria to biological aging, with a recent theory proposing that giant mitochondria are involved in the aging of post-mitotic tissues. In these tissues, giant mitochondria appear to evade autophagocytosis, accumulate, and replace normal mitochondria. Techniques to characterize giant mitochondria in the presence of, or after isolation from, normal mitochondria are greatly needed. The short- term goal of this proposal is to characterize and isolate giant mitochondria and establish whether or not these mitochondria affect muscle function. The long-term goal is to use and make available novel technological approaches for uncovering the changes that, at the subcellular and molecular level, define biological human aging. The proposed work utilizes mitochondria from a myoblast model and muscle from Fischer 344 rats. We have four aims: (1) Characterize giant mitochondria in vivo in cell models based on their cell biology. (2) Purify giant mitochondria isolated from these in vivo cell models and then characterize these giant mitochondria biochemically. (3) Compare the proteomes of giant and normal mitochondria isolated from these models. (4) Characterize giant mitochondria in aging skeletal muscle. The first aim uses confocal microscopy, or this technique combined with single cell capillary electrophoresis, to investigate size, DMA content and synthesis, DNA mutations, carbonyl accumulation, ROS production, membrane potential, mitochondrial fusion, and autophagocytosis. The second aim describes the methods to isolate subpopulations of giant and normal mitochondria using free-flow electrophoresis and then characterize these isolated mitochondria using methods suitable for isolated organelles. The third aim utilizes stable isotope labeling in cell cultures to compare the proteomes of giant and normal mitochondria. The last aim investigates age-related changes in muscle containing giant mitochondria. Significance to Health. Giant mitochondria remain poorly characterized, their proteomic and genomic characteristics have not been defined, and their relationship to muscle function has not been investigated. The characterization of this class of mitochondria is highly relevant to the public health not only because of their possible role in aging tissues, but also because of their appearance as a consequence of alcoholism, human toxicity, and multiple diseases.