MECHANISMS OF DEGENERATIVE CELL DEATH IN C ELEGANS

线虫退行性细胞死亡的机制

• 批准号: 2273669
• 负责人: MONICA A. DRISCOLL
• 金额: $ 15.67万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-01 至 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2273669
• 关键词: Caenorhabditis elegans; Drosophilidae; Xenopus oocyte; amiloride; cell death; genetic promoter element; genetically modified animals; membrane channels; molecular cloning; neural degeneration; neuropharmacology; phosphorylation; site directed mutagenesis; suppressor mutations; tissue /cell culture; transfection /expression vector; vaccinia virus

Inherited neurodegenerative conditions have been identified in organisms as diverse as nematodes and man, but relatively little is understood about the genes involved and the mechanisms whereby mutant alleles induce inappropriate cell death. We study members of a family of genes, called degenerins, which can mutate to induce late onset swelling and necrotic- like death of specific groups of neurons in the easily studied nematode Caenorhabditis elegans. The degenerin gene family was originally defined by unusual dominant "killer" alleles of the mec4 and deg-I genes. Three degenerin-related genes from the rat, alpha-, beta-, and gamma rENaC, have been demonstrated to encode subunits of the amiloride-sensitive epithelial Na+ channel. We have speculated that the C. elegans degenerins mec-4 and mec-10 encode components of a mechanosensory ion channel, an exciting working hypothesis because eukaryotic mechanosensory channel components have not previously been isolated and the molecular properties of these channels are unknown. Our experiments indicate that ineffective channel closing is the critical event in the initiation of neurodegeneration. My long term research plan is to decipher in molecular detail how misregulation of ion channel activity (and other insults) induce necrotic- like death and to define the molecular events that transpire during neurodegeneration. Experiments outlined in this proposal are designed to enable us to describe how degeneration is initiated, what proteins are needed to enact the death, and even how cell corpses are removed. In addition, we will define distinctions/common features between degenerative cell death and the programmed cell death pathway (which we now believe overlap more than we originally anticipated). Finally, we expect to exploit the ability of mec4(d) to act as a killer gene to create ablation vectors and to set up a model system for studying degenerin-induced death in mammalian cell culture. Specifically, we will: l) assay the activities of wild-type and mutant channels in a oocyte expression system, 2) conduct genetic suppressor screens to identify genes essential for mec4(d)-induced neurodegeneration, 3) define effects of programmed cell death genes on touch cell degeneration, and 4) determine whether mec4(d) and potentially lethal variants of degenerin-related genes can induce degeneration in heterologous systems, develop ablation vectors which depend on the toxicity of these genes, and set up a mammalian culture model for degenerin-induced death. The proposed work is important to heath related issues in that it will advance understanding of degenerative cell death mechanisms and may inspire novel strategies for the prevention of injury-induced cell death. In addition, these studies will contribute to the characterization of a new ion channel class with a likely function in mechanotransduction a phenomenon which contributes to senses of touch, hearing and balance and more.

生物体中已发现遗传性神经退行性疾病 与线虫和人类一样多样化，但人们对它们的了解相对较少 涉及的基因以及突变等位基因诱导的机制 不适当的细胞死亡。我们研究基因家族的成员，称为 退化蛋白，它可以突变导致迟发性肿胀和坏死 就像易于研究的线虫中特定神经元组的死亡一样 秀丽隐杆线虫。退化蛋白基因家族最初被定义 由 mec4 和 deg-I 基因的异常显性“杀手”等位基因引起。三 来自大鼠的退化蛋白相关基因，α-、β-和γ rENaC，具有 已被证明编码阿米洛利敏感上皮亚单位 Na+通道。我们推测秀丽隐杆线虫 degenerins mec-4 和 mec-10 编码机械感觉离子通道的组件，这是一个令人兴奋的 工作假设是因为真核机械感觉通道成分 之前尚未分离出这些物质的分子特性 渠道未知。我们的实验表明，无效的渠道 关闭是神经变性启动的关键事件。 我的长期研究计划是从分子细节上解读如何 离子通道活性（和其他损伤）的错误调节会导致坏死 像死亡一样并定义期间发生的分子事件 神经变性。本提案中概述的实验旨在 使我们能够描述退化是如何开始的，蛋白质是什么 实施死亡所需的信息，甚至细胞尸体如何被移除的信息。在 此外，我们将定义退化之间的区别/共同特征 细胞死亡和程序性细胞死亡途径（我们现在相信 重叠程度超出我们最初的预期）。最后，我们期望 利用 mec4(d) 作为杀手基因的能力来产生消融 载体并建立研究退化蛋白诱导死亡的模型系统 在哺乳动物细胞培养中。具体来说，我们将： l) 分析活动 卵母细胞表达系统中野生型和突变型通道的分析，2) 进行 基因抑制筛选以确定 mec4(d) 诱导所必需的基因 神经变性，3）定义程序性细胞死亡基因对 触摸细胞变性，以及 4) 确定 mec4(d) 是否和潜在的 退化蛋白相关基因的致死变异可诱导退化 异源系统，开发取决于 这些基因的毒性，并建立了哺乳动物培养模型 退化蛋白诱导的死亡。 拟议的工作对于健康相关问题很重要，因为它将 促进对退行性细胞死亡机制的理解，并可能 激发预防损伤诱导的细胞死亡的新策略。 此外，这些研究将有助于表征 新的离子通道类别可能在机械传导中发挥作用 有助于触觉、听觉和平衡感的现象 更多的。