DELETIONS OF MITOCHONDRIAL DNA IN NEUROMUSCULAR DISEASE

我们最近发现，基本上所有进行性 眼外肌麻痹（PEO），表现为肌肉中的红色纤维不规则 - 或者是不太严重的形式，称为眼肌病（OM），或者是 这种致命的形式，称为Kearns-Sayre综合征（KSS）， 线粒体DNA（mtDNA）在他们的骨骼肌。 这是第一 时间线粒体DNA，它开辟了新的途径，研究这两种疾病的过程 以及线粒体的基本生物学。 我们已经绘制了缺失断点的图谱并进行了测序;研究了 异质性，水平（即细胞和组织之间）和 垂直缺失的mtDNA;通过分析KSS肌肉切片， 免疫荧光、细胞化学和原位杂交;并获得 正常和缺失的mtDNA分离的证据。 我们现在建议在分子水平上进一步分析这些缺失， 目的是了解PEO的病因和发病机制， 明确证明存在正常和缺失的分离 单个线粒体中的mtDNA。 第二，我们将测量相对 正常和缺失的mtDNA的复制率，看看是否 缺失的mtDNA在PEO中的增殖是由于复制优势。 第三，我们将建立一个体外模型的肌病由于PEO，通过采取 一种新的组织培养系统的优点， 通过显微注射或细胞融合将线粒体引入到细胞中。 细胞质的受体人细胞已经耗尽了所有的他们的 线粒体DNA（rhoo细胞）。 最后，我们将探讨 创建一个PEO的小鼠模型。 在体外和小鼠模型中， 我们将尝试选择性地消除含有缺失的线粒体， 线粒体DNA分子，最终目标是制定标准， 合理的治疗，以治疗这些目前无法治愈的，并在照顾 Kearns-Sayne综合征致命疾病

We recently discovered that essentially all patients with progressive external ophthalmoplegia (PEO) presenting with ragged red fibers in muscle - either with the less-severe form, called Ocular Myopathy (OM), or with the fatal form, called Kearns-Sayre Syndrome (KSS) - harbor large deletions of mitochondrial DNA (mtDNA) in their skeletal muscle. This is the first time a mtDNA, and it opens up new avenues for study of both disease process itself and the basic biology of mitochondria. We have mapped and sequenced the deletion breakpoints; studied heteroplasmy, both horizontally (i.e. among cells and tissue) and vertically deleted mtDNAs; analyzed KSS muscle sections by immunofluorescence, cytochemistry, and in-situ hybridization; and obtained evidence of segregation of normal and deleted mtDNAs. We now propose to analyze these deletions further at the molecular level, with an aim of understanding the etiology and pathogenesis of PEO, and of demonstrate definitively that there is segregation of normal and deleted mtDNAs in individual mitochondria. Second, we will measure the relative rates of replication of normal and deleted mtDNAs, to see whether the proliferation of deleted mtDNAs in PEO is due to replicative advantage. Third, we will create an in-vitro model of myopathies due to PEO, by taking advantage of a novel tissue culture system whereby isolated, intact mitochondria are introduced by microinjection or cell fusion into the cytoplasm of recipient human cells that have been depleted of all their mitochondrial DNA (rhoo cells). Finally, we will explore the possibility of creating a mouse model of PEO. In both the in-vitro and mouse models, we will attempt to eliminate selectively mitochondria containing deleted mtDNA molecules, with the ultimate goal of developing criteria for a rational therapy to treat these currently incurable, and in the care of Kearns-Sayne Syndrome, fatal diseases.