MECHANISMS OF GLUTAMATE-MEDIATED C-FOS EXPRESSION

谷氨酸介导的 C-FOS 表达机制

• 批准号: 2260015
• 负责人: STEVEN M FINKBEINER
• 金额: $ 7.72万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-15 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2260015
• 关键词: NMDA receptors; PC12 cells; calcium flux; calcium indicator; excitatory aminoacid; gene expression; gene induction /repression; genetic transcription; immunocytochemistry; inhibitor /antagonist; laboratory rat; molecular site; neural plasticity; neurons; northern blottings; phosphoprotein phosphatase; phosphorylation; protein kinase; protooncogene; second messengers; site directed mutagenesis; transfection /expression vector; video microscopy; voltage /patch clamp; western blottings

Neuronal connectivity and function undergo activity-dependent changes that are central to learning, memory and development. The long-term goal of the proposed research is to understand how activity changes synapses. Recent work suggests gene transcription may make activity-dependent synaptic changes permanent. At many synapses, glutamate receptors initiate plasticity through ion fluxes, second messengers and gene transcription. The proposed research tests the hypothesis that glutamate induces gene expression through a unique pathway important for synaptic plasticity. We will use the immediate early gene, c-fos, to probe pathways of glutamate- induced gene expression. Others found that glutamate induces c-fos partly through the N-methyl-D-aspartate (NMDA)-receptor subtype, however, the role of other glutamate receptor subtypes is unclear. It is also unclear whether Ca2+ influx through the NMDA-receptor is sufficient to induce c- fos or whether the channel itself serves some signaling function. Certain NMDA subunits undergo tyrosine phosphorylation and contain peptide domains that may transmit signals to other cytoplasmic proteins. Specific aims and methods are: 1) We propose to test the role of glutamate receptor subtypes in endogenous hippocampal c-fos expression with agonists and antagonists of specific glutamate receptors, second messengers, protein kinases and phosphatases as measured by Northern blotting. Parallel correlative experiments will test the role of cytoplasmic Ca2+ rises for c-fos expression using the Ca2+ -sensitive dye fura-2 and video microscopy. 2) We will test the sufficiency of NMDA receptors for c-fos induction by expressing functional NMDA receptors in PC 12 cells; demonstrating the channels using patch clam,p and Ca2+ imaging methods; and testing for glutamate-induced c-fos expression with Northern immunoblotting. We will deduce gene elements that mediate NMDA-induced c- fos by cotransfecting vectors with intact or mutated c-fos promoters. We will immunocytochemically test for active upstream signaling factors using antiphosphoprotein antibodies. 3) We will test the idea that the NMDA channel itself transmits signals by measuring glutamate-induced NMDA receptor phosphorylation in transfected PC12 cells; identifying specific tyrosine phosphorylation sites by tryptic peptide and phosphoamino analysis and by testing their importance using site-directed mutagenesis and transfections. Other candidate cytoplasmic domains of the NMDA receptor will be tested similarly. The proposed research should improve understanding of glutamate receptor signaling, NMDA receptor structural biology, NMDA gene expression and mechanisms of glutamate-mediated plasticity. Clinically, glutamate kills neurons at high concentrations and may play a role in the pathogenesis of neurodegenerative diseases, epilepsy and stroke. Elucidating mechanisms of glutamate induced gene transcription and plasticity may lead to new treatments for diseases associated with cellular hyperexcitability and glutamate toxicity.

神经元连通性和功能经历活动依赖性变化， 是学习，记忆和发展的核心。 长期目标 拟议的研究是了解活动如何改变突触。 最近的工作表明基因转录可能使活动依赖性 突触变化永久性。在许多突触中，谷氨酸受体启动 通过离子通量，第二信使和基因转录的塑性。 提出的研究检验了谷氨酸诱导基因的假设 通过独特的途径表达对突触可塑性很重要的途径。我们 将使用直接的早期基因c-fos探测谷氨酸 - 诱导基因表达。 其他人发现谷氨酸会诱导C-FOS部分 然而，通过N-甲基-D-天冬氨酸（NMDA） - 受体亚型 其他谷氨酸受体亚型的作用尚不清楚。还不清楚 Ca2+通过NMDA受体涌入是否足以诱导C- FOS或通道本身是否具有某些信号函数。肯定 NMDA亚基经历酪氨酸磷酸化并含有肽结构域 这可能会将信号传输到其他细胞质蛋白。 具体目的和方法是：1）我们建议测试谷氨酸的作用 内源性海马C-FOS表达的受体亚型与激动剂表达 和特定谷氨酸受体的拮抗剂，第二使者， 通过北印迹测量的蛋白激酶和磷酸酶。 平行相关实验将测试细胞质CA2+的作用 使用Ca2+敏感的染料Fura-2和视频来表达C-FOS表达 显微镜。 2）我们将测试NMDA受体的充分性C-FOS 通过在PC 12细胞中表达功能性NMDA受体来诱导； 使用斑块，P和Ca2+成像方法演示通道； 并测试谷氨酸诱导的C-FOS表达 免疫印迹。我们将推断出介导NMDA诱导的C-的基因元素 通过与完整或突变的C-FOS启动子共转移载体的FOS。 我们 会使用免疫细胞化学测试使用的主动信号传导因子 抗磷蛋白抗体。 3）我们将测试NMDA的想法 通道本身通过测量谷氨酸诱导的NMDA传输信号 转染的PC12细胞中的受体磷酸化；确定具体 胰蛋白酶肽和磷酸氨基氨基通过酪氨酸磷酸化位点 分析并通过使用位置定向诱变来测试其重要性 和转染。 NMDA的其他候选细胞质结构域 受体将进行类似的测试。 拟议的研究应提高对谷氨酸受体的理解 信号传导，NMDA受体结构生物学，NMDA基因表达和 谷氨酸介导的可塑性的机理。 临床上，谷氨酸杀死 高浓度的神经元可能在 神经退行性疾病，癫痫和中风。阐明的机制 谷氨酸诱导的基因转录和可塑性可能导致新的 与细胞过度兴奋性相关的疾病治疗 谷氨酸毒性。