Mechanisms of organelle deterioration

描述（由申请人提供）：影响美国人的最普遍的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随着人类年龄的增长，它们的发病率会急剧增加，目前还不清楚。酿酒酵母（Saccharomyces cerevisiae）是研究酿酒细胞器发生和功能的重要模型系统。此外，其不对称细胞分裂的性质表明，“母亲”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延长后生动物寿命的保守遗传和环境过程的模型。然而，它在研究衰退过程中的应用受到了限制，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。我们最近通过母体富集程序（MEP）的发展克服了这一限制，现在可以分离和检查大量同步衰老的细胞。我们将我们的新技术与酿酒酵母的全套可用资源相结合，研究了自发细胞器衰退的分子机制。我们发现最早的变化发生在“中年”，巧合的是：线粒体断裂和线粒体膜融合的丧失，空泡酸度的丧失和er -高尔基囊泡亚类的积累。我们将确定线粒体、液泡和er -高尔基体的衰退是否有因果关系，如果有，我们将确定将它们联系起来的机制。这将为我们的目标奠定基础，即识别在重复细胞分裂中出现缺陷的特定分子，它们是如何出现缺陷的，以及缺陷的影响。因为这些细胞器是保守的，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与年龄相关的衰退提供新的见解。我们还发现线粒体膜融合的两个关键蛋白（Fzo1和Ugo1）发生了戏剧性的变化。这些变化为我们观察到的母细胞分裂时线粒体融合的丧失提供了一个可能的解释。我们建议采用生物化学和遗传方法相结合的方法来确定这些蛋白质变化的化学性质和过程。类似的线粒体断裂和融合缺陷与神经病变、糖尿病和肌肉退化有关。因此，我们的发现可能为理解这些疾病的发病和治疗提供一个范例。最后，我们将利用细胞生物学、生物化学和遗传学方法的结合，确定导致液泡酸度损失的分子缺陷。鉴于溶酶体与包括阿尔茨海默病在内的许多与年龄相关的疾病之间的联系，我们在本提案中开发的关于液泡酸化丧失的详细知识水平，其中解剖这一过程的工具是无与伦比的，可能为这些疾病的发病提供新的见解。

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.