Regulation of mAChR Function and Expression

mAChR 功能和表达的调节

• 批准号: 6931437
• 负责人: DARRELL A JACKSON
• 金额: $ 19.3万
• 依托单位: UNIVERSITY OF MONTANA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-03-15 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6931437
• 关键词: SDS polyacrylamide gel electrophoresis; acetylcholine; autoradiography; cell line; gene expression; genetic transcription; hippocampus; hypoxia; immunoprecipitation; laboratory rat; muscarinic receptor; neurons; phosphorylation; receptor expression; tissue /cell culture; western blottings

DESCRIPTION (provided by applicant): It is estimated that in the United States 150,000 stroke-related deaths occur each year, making stroke the country's third-leading cause of death. In nonfatal strokes, ischemic injury to the hippocampus, a brain region known to be selectively vulnerable to ischemia, results in cognitive and memory impairment. After cerebral ischemia, surviving neurons undergo a variety of cellular responses that are not completely understood. The long-term goal of this research is to elucidate mechanisms of responses to cerebral ischemia. The present proposal focuses on one aspect of ischemia, hypoxia, and its effect on the fimctional expression of muscarinic acetylcholine receptors (mAChRs), which are thought to play a role in learning and memory. Our experiments will test the hypothesis that transient hypoxia causes a decrease in functional responsiveness of the m2 mAChRs and that the mechanism involved includes both pre- and post-translational modifications. Experiments are designed to determine the underlying mechanisms that mediate internalization, desensitization, and down-regulation of m2 mAChRs by transient hypoxia. The proposed study will use cultures of enriched primary hippocampal neurons and human embryonic kidney cells and mouse embryonic fibroblasts that stably express wild-type or mutated forms of the m2 mAChR. In Specific Aim 1, experiments will be performed to test the hypothesis that hypoxic insult induces phosphorylation of the m2 mAChR, thereby leading to internalization and decreased responsiveness to muscarinic agonists. In Specific Aim 2, experiments will be conducted to test the hypothesis that desensitization of the m2 mAChR by transient hypoxia is independent of m2 receptor internalization. In Specific Aim 3, experiments will be carried out to test the hypothesis that transient hypoxia decreases the expression of the m2 mAChR mRNA and/or protein. Together, these experiments will determine how hypoxia induces alterations in mAChR signaling and advance understanding of the consequences of stroke and transient ischemic attacks. Results from these studies will be useful in developing therapeutic interventions to reduce the impairment of learning and memory after ischemic insults.

描述（由申请人提供）：据估计，在美国，每年有 150,000 例与中风相关的死亡，使中风成为该国第三大死因。在非致命性中风中，海马体（已知选择性地容易发生缺血的大脑区域）的缺血性损伤会导致认知和记忆障碍。脑缺血后，幸存的神经元会经历各种尚未完全了解的细胞反应。这项研究的长期目标是阐明脑缺血的反应机制。本提案的重点是缺血、缺氧及其对毒蕈碱乙酰胆碱受体（mAChR）功能表达的影响，该受体被认为在学习和记忆中发挥作用。我们的实验将检验这样的假设：短暂缺氧会导致 m2 mAChR 的功能反应性降低，并且所涉及的机制包括翻译前和翻译后修饰。实验旨在确定短暂缺氧介导 m2 mAChR 内化、脱敏和下调的潜在机制。 拟议的研究将使用富集的原代海马神经元和人胚胎肾细胞以及小鼠胚胎成纤维细胞的培养物，这些细胞稳定表达野生型或突变形式的 m2 mAChR。在具体目标 1 中，将进行实验来检验以下假设：缺氧损伤会诱导 m2 mAChR 磷酸化，从而导致内化并降低对毒蕈碱激动剂的反应性。在具体目标 2 中，将进行实验来检验短暂缺氧导致 m2 mAChR 脱敏与 m2 受体内化无关的假设。在具体目标 3 中，将进行实验来检验短暂缺氧会降低 m2 mAChR mRNA 和/或蛋白质表达的假设。总之，这些实验将确定缺氧如何诱导 mAChR 信号传导的改变，并加深对中风和短暂性脑缺血发作后果的了解。这些研究的结果将有助于制定治疗干预措施，以减少缺血性损伤后学习和记忆的损害。