Mechanisms of organelle deterioration

描述（由申请人提供）：影响美国人的大多数流行非传染性疾病与细胞损伤和细胞器功能障碍有关。然而，这些疾病发生的机制以及为什么它们的发病率随着人类年龄的增长而急剧增加还不清楚。芽殖酵母是研究细胞器生物发生和功能的重要模式系统。此外，其不对称的细胞分裂的性质表明，“母”细胞的活力随着每次细胞分裂而下降，其有限的复制寿命已被用作研究和确定保守的遗传和环境过程的模型，以延长后生动物的寿命。然而，它在研究衰退过程中的应用受到限制，这在很大程度上是由于分离经历了越来越多的细胞分裂的母细胞的困难。我们最近通过开发母亲富集计划（MEP）克服了这一限制，现在可以分离和检查大量同步老化的细胞。我们已经将我们的新技术与S.酿酒酵母研究自发细胞器下降的分子机制。我们发现最早的变化发生在“中年”和巧合：线粒体碎片和线粒体膜融合的损失，空泡酸度的损失和积累的一个亚类的ER-高尔基体囊泡。我们将确定线粒体、液泡和内质网高尔基体在它们的衰退中是否存在因果联系，如果是这样，我们将定义将它们联系起来的机制。这将为我们识别随着重复细胞分裂而变得有缺陷的特定分子、它们如何变得有缺陷以及缺陷的影响的目标奠定基础。由于这些细胞器是保守的，并与人类疾病有关，我们进行这些研究的独特能力很可能为人类年龄相关性衰退提供新的见解。我们还发现了线粒体膜融合的两个关键蛋白（Fzo 1和Ugo 1）的显着变化。这些变化为我们在母细胞分裂时观察到的线粒体融合的丧失提供了可能的解释。我们建议确定的化学性质，负责，在这些蛋白质的改变，采用生物化学和遗传学方法相结合的过程。类似的线粒体断裂和融合缺陷与神经病、糖尿病和肌肉退化有关。因此，我们的研究结果可能为了解这些疾病的发病和治疗提供一个范例。最后，我们将使用细胞生物学，生物化学和遗传学方法的组合，确定导致液泡酸性丧失的分子缺陷。考虑到溶酶体和一些与年龄相关的疾病（包括阿尔茨海默病）之间的联系，我们在这项提案中开发的关于空泡酸化损失的详细知识水平，其中用于解剖这一过程的工具是无与伦比的，可能会提供关于这些疾病发作的新见解。 公共卫生相关性：折磨人类的主要非传染性疾病与细胞损伤和细胞器功能障碍有关。我们已经开发了一个系统来研究在一个简单的生物体，酵母酿酒酵母（Saccharomyces cerevisiae）中这些相同变化的分子机制。这些研究有可能为克服人类神经病、糖尿病和肌肉退化等疾病提供见解和策略。

DESCRIPTION (provided by applicant): Most prevalent non-infectious diseases affecting people in the United States are associated with cellular damage and organelle dysfunction. Yet the mechanism by which these diseases occur and why their onset dramatically increase as humans age is unclear. The budding yeast Saccharomyces cerevisiae is an important model system for studying organelle biogenesis and function. Furthermore, the nature of its asymmetric cell divisions indicates that the vitality of a "mother" cell declines with each cell division, and its limited replicative life span has been used as a model to study and identify conserved genetic and environmental processes that extend life span in metazoans. However, its use in studying the process of decline has been limited, in large part due to the difficulty in isolating mother cells that have gone through an increasing number of cell divisions. We have recently overcome this limitation by development of the Mother Enrichment Program (MEP), and can now isolate and examine large populations of synchronously aged cells. We have applied our novel technology in combination with the full set of resources available in S. cerevisiae to study molecular mechanisms of spontaneous organelle decline. We discovered the earliest changes occurred at "middle-age" and coincidentally: mitochondrial fragmentation and loss of mitochondrial membrane fusion, loss of vacuolar acidity and accumulation of a subclass of ER-Golgi vesicles. We will determine whether the mitochondria, vacuole and ER-Golgi are causally linked in their decline, and if so, we will define the mechanism(s) that links them. This will lay the groundwork for our goal of identifying specific molecules that become defective with repeated cell divisions, how they become defective and the impact of the defect(s). Because these organelles are conserved and implicated in human diseases, our unique ability to conduct these studies may very well provide new insights about age-associated decline in humans. We also discovered dramatic alterations in two critical proteins of mitochondrial membrane fusion (Fzo1 & Ugo1). These changes offer a likely explanation for the loss of mitochondrial fusion we observe as mother cells divide. We propose to identify the chemical nature of, and processes responsible for, the alterations in these proteins, employing a combination of biochemical and genetic approaches. Similar mitochondrial fragmentation and fusion defects have been linked to neuropathies, diabetes and muscle deterioration. Thus our findings may provide a paradigm for understanding the onset and treatment of these diseases. Lastly, we will identify the molecular defects that give rise to the loss of vacuolar acidity with repeated cell divisions, using a combination of cell biological, biochemical and genetic approaches. Given the link between the lysosome and a number of age-associated diseases, including Alzheimer's disease, the detailed level of knowledge about loss of vacuole acidification that we develop in this proposal, where the tools for dissecting this process are unparalleled, may provide new insights about the onset of these diseases. PUBLIC HEALTH RELEVANCE: The major non-infectious diseases that afflict humans are associated with cellular damage and organelle dysfunction. We have developed a system to study the molecular mechanisms that underlie these same changes in a simple organism, the yeast Saccharomyces cerevisiae. These studies have the potential to provide insight into and strategies to overcome diseases such as neuropathies, diabetes and muscle deterioration in humans.