GROWTH ASSOCIATED PROTEINS IN CNS REGENERATION

中枢神经系统再生中的生长相关蛋白

• 批准号: 2020075
• 负责人: J H PATE SKENE
• 金额: $ 22.09万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-12-08 至 1998-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2020075
• 关键词: antisense nucleic acid; axon; brain injury; gel electrophoresis; gene expression; growth cones; immunoprecipitation; laboratory mouse; laboratory rabbit; laboratory rat; membrane proteins; microinjections; monoclonal antibody; nervous system regeneration; neural plasticity; neurogenetics; neurotrophic factors; nonblood lipoprotein; posttranslational modifications; protein biosynthesis; receptor mediated endocytosis; spinal cord injury; tissue /cell culture

Permanent paralysis after human spinal cord injuries or other CNS trauma results from the failure of most long CNS axons to regenerate in adults. This project examines the hypothesis that regeneration in the mammalian CNS is substantially limited by the failure of most injured neurons to induce expression of a small set of genes whose protein products are necessary for axon elongation. Previous work has shown that successful axon regeneration is strongly correlated with elevated synthesis of certain axonal proteins, including one of the most abundant protein components of growth cone membranes, GAP-43. Expression of these "growth-associated proteins" is low in the majority of adult CNS neurons and remains low after the most common types of CNS injury. The present proposal is to determine whether failure to induce GAP expression after axotomy actually limits the ability of adult mammalian neurons to regenerate their axons. Taking GAP-43 as an accessible example of this class of proteins, the proposed studies ask whether neurons that ordinarily would extend long axons show a reduced proclivity for axon growth after experimental disruption of GAP expression. Expression of GAP-43 will be interrupted in axotomized adult neurons in tissue culture by microinjecting antibodies targeted against specific sites on the GAP-43 molecules necessary for its processing and transport to growth cone membranes, or by injecting oligonucleotides complementary to GAP-43 mRNA. The cells then will be monitored for their ability to extend axons on a series of cellular and extracellular matrix substrata previously shown to support long axonal outgrowth. If GAP-43 proves to be necessary for some steps in axon growth, then effective regeneration of injured axons will require not only induction of GAP-43 synthesis in neuron cell bodies, but also delivery of the protein to axonal growth cones and appropriate regulation of the protein's activity in response to specific cues in the extracellular environment. Intracellular routing of GAP-43 and its interaction with growth cone membranes appears to be regulated physiologically by post-translational addition and removal of covalently linked fatty acid chains to the protein. Preliminary pharmacological evidence suggests that inhibition of the acylating enzyme leads to a rapid disruption of growth cone activity and retraction of neurites in vitro. This application therefore proposes to continue investigating post-translational modification of GAP-43 in response to specific cues in the PNS and CNS environments, with particular emphasis on the biochemical regulation of the protein-acylating reaction in CNS growth cones and its functional role in axon regeneration. Finally, to continue the search for additional growth-associated proteins, a recently isolated monoclonal antibody will be used to characterize a new protein antigen expressed on growth cones, but not on mature axon terminals. Metabolic labeling, immunoprecipitation, and protein microsequencing will be used to determine whether the strongly developmentally regulated appearance of this antigen represents growth=associated expression of a new GAP gene.

人脊髓损伤或其他中枢神经系统创伤后的永久瘫痪 大多数长中枢神经系统轴突在成年人中都无法再生的结果。 该项目研究了哺乳动物中枢神经系统再生的假设 大多数受伤神经元失败的限制很大程度上受到诱导 表达一小部分基因，其蛋白质产物是必需的 轴突伸长。 以前的工作表明成功的轴突再生 与某些轴突蛋白的合成升高密切相关， 包括生长锥最丰富的蛋白质成分之一 膜，GAP-43。 这些“生长相关蛋白”的表达很低 在大多数成年中枢神经系统神经元中，在最常见之后保持较低 中枢神经系统损伤的类型。 目前的建议是确定失败是否 诱导轴切开后的间隙表达实际上限制了成人的能力 哺乳动物神经元再生其轴突。 将GAP-43作为 拟议的研究提出了这类蛋白质的可访问示例 通常会延伸长轴突的神经元是否显示出降低 间隙表达实验破坏后，轴突生长的倾向。 GAP-43的表达将在轴突化的成年神经元中中断 通过针对特定位点的微注射抗体进行的组织培养 在GAP-43分子上处理和运输所需的分子 生长锥膜，或通过注射寡核苷酸 GAP-43 mRNA。 然后，将监控细胞的扩展能力 先前的一系列细胞和细胞外基质基质上的轴突 显示以支持长轴突生长。 如果证明GAP-43对于轴突生长的某些步骤是必需的，那么 有效的受伤轴突的再生将不仅需要诱导 神经元细胞体中的GAP-43合成，但也递送蛋白质 轴突生长锥和蛋白质活性的适当调节 响应细胞外环境中的特定提示。 GAP-43的细胞内路由及其与生长锥的相互作用 膜似乎受到翻译后的生理调节 将共价连接的脂肪酸连接到蛋白质的添加和去除。 初步药理证据表明，抑制 酰化酶会导致生长锥活性的快速中断和 在体外神经突的缩回。 因此，本申请建议 继续研究GAP-43的翻译后修改 对PNS和CNS环境中特定提示的响应， 强调蛋白质酰化反应的生化调节 在中枢神经系统生长锥中及其在轴突再生中的功能作用。 最后，继续寻找其他与增长相关的 蛋白质，最近分离的单克隆抗体将用于 表征在生长锥上表达的新蛋白质抗原，但不在 成熟的轴突末端。 代谢标签，免疫沉淀和 蛋白质微链测序将是 用于确定是否受到强烈发展的监管 该抗原的外观代表生长=新的表达 间隙基因。