MECHANISMS OF DEGENERATIVE CELL DEATH IN C ELEGANS

线虫退行性细胞死亡的机制

• 批准号: 2750909
• 负责人: MONICA A. DRISCOLL
• 金额: $ 26.49万
• 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/2750909
• 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.

在生物体中已经确定了遗传的神经退行性疾病 像线虫和人一样多样化，但对 涉及的基因和突变等位基因诱导的机制 不当细胞死亡。我们研究一个基因家族的成员，称为 Degenerins，可以突变以诱导晚期发作肿胀和坏死 - 像在易于研究的线虫中特定神经元的死亡 秀丽隐杆线虫。 Degenerin基因家族最初是定义的 MEC4和DEG-I基因的异常主导“杀手”等位基因。三 来自大鼠，α-，β-和伽玛肾脏的脱纳尔蛋白相关基因具有 被证明是编码amiloride敏感上皮的亚基 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，令人兴奋的 工作假设是因为真核机械感知通道组件 以前尚未分离，并且这些分子特性 频道未知。我们的实验表明无效的通道 闭合是启动神经变性的关键事件。 我的长期研究计划是用分子详细说明如何破译 离子通道活性（和其他侮辱）的正调诱导坏死 像死亡并定义在 神经变性。该提案中概述的实验旨在 使我们能够描述退化的开始，什么是蛋白质 需要制定死亡，甚至是如何去除细胞尸体。在 此外，我们将定义退化之间的区别/共同特征 细胞死亡和编程的细胞死亡途径（我们现在相信 重叠比我们最初预期的更多）。最后，我们希望 利用MEC4（d）充当杀手基因的能力创造消融 向量并建立一个模型系统，用于研究脱纳格蛋白诱导的死亡 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的野生型和突变通道，2） 遗传抑制剂筛选以鉴定Mec4（D）诱导的基因 神经变性，3）定义程序性细胞死亡基因对 接触细胞变性，4）确定MEC4（d）和可能是否有可能 致命的脱脂蛋白相关基因的致命变异可以诱导变性 异源系统，开发融合向量，取决于 这些基因的毒性，并为 Degenerin诱发的死亡。 拟议的工作对于与荒地相关的问题很重要，因为它将 提前了解退化性细胞死亡机制，可能 激发预防损伤引起的细胞死亡的新型策略。 此外，这些研究将有助于表征 新的离子通道类具有机械转导可能功能的功能 现象有助于触摸，听力和平衡感的感觉 更多的。