CELL CULTURE MODELS OF MITOCHONDRIAL ENCEPHALOMYOPATHIES

线粒体脑肌病在临床上，形态学上， 和生化多样性的疾病。在过去的几年里， 已经发现特定的线粒体DNA（mtDNA）突变导致 在几种人类疾病中。不幸的是， 线粒体DNA突变的鉴定， 推测的病因学和这些疾病的发病机制。 这项提案的目的是分析病理后果， 几种特定mtDNA突变的分子遗传学原因， 人类疾病这项分析将利用独特的细胞培养 一种允许在中性核中分析mtDNA突变的系统 背景该系统基于人细胞系的分离 完全缺乏mtDNA（p/o细胞系）和重新繁殖能力的细胞， 这些细胞带有外源性线粒体，因此，线粒体DNA。该系统 将被应用于分析疾病MERRF（肌阵挛性癫痫 和破碎红纤维病）。两个点突变，都在tRNA/LYS， 线粒体DNA是导致这种疾病的原因通过检查 生物化学、形态学和遗传学的影响 突变，并比较结果，应该可以确定 致病的精确分子机制以类似的方式， mtDNA编码的tRNA基因的其他点突变与 疾病将被研究。只有在具体的缺陷及其原因是 已知，是否有可能为患者开发合理的治疗方法 患有这些疾病。这种体外系统将允许精确的 呼吸链功能受损细胞的代谢需求 待定 此外，还研究了不同生长条件或处理对 可以检测突变的和野生型的mtDNA。如果一个基因组可以 在其复制中优先受损或抑制，它可能是 有可能为这些目前无法治愈的疾病设计治疗方法， 致命的疾病除了这些正在研究的疾病， 建议，这个模型系统也可以应用到其他的研究 已知或怀疑线粒体参与的疾病， 拟议的分析将为这些未来的 特征化。

The mitochondrial encephalomyopathies are a clinically, morphologically, and biochemically diverse group of disorders. In the past several years, specific mitochondrial DNA (mtDNA) mutations have been found to result in several human diseases. Unfortunately, there has been little or no correlation between the identification of the mtDNA mutations, the presumed etiology, and the pathogenesis of these disorders. The goal of this proposal is to analyze the pathological consequences and the molecular genetic causes of several specific mtDNA mutations causing human disease. This analysis will take advantage of a unique cell culture system that permits the analysis of mtDNA mutations in a neutral nuclear background. This system is based upon the isolation of human cell lines that completely lack mtDNA (p/o cell lines) and the ability to repopulate these cells with exogenous mitochondria, and thus, mtDNA. This system will be applied to the analysis of the disease MERRF (myoclonic epilepsy and ragged-red fiber disease). Two point mutations, both in tRNA/LYS of the mtDNA, are known to result in this disease. By examining the biochemical, morphological and genetic consequences of these two mutations, and comparing the results, it should be possible to determine the precise molecular mechanism of pathogenesis. In a similar fashion, other point mutations of the mtDNA-encoded tRNA genes associated with disease will be studied. Only after specific defects and their causes are known, will it be possible to develop rational therapies for patients suffering from these diseases. This in vitro system will permit the exact metabolic requirements for cells with impaired respiratory chain function to be determined. In addition, the effects of different growth conditions or treatments on the mutated and wild-type mtDNAs can be examined. If one genome can be preferentially damaged or inhibited in its replication, it may be possible to devise treatments for these currently incurable, and often fatal diseases. In addition to those diseases being studied in this proposal, this model system can also be applied to the study of other diseases where mitochondrial involvement is known or suspected, and the proposed analyses will provide a foundation for these future characterizations.