Regulation of mAChR Function and Expression

描述(申请人提供)：据估计，美国每年发生15万例与中风有关的死亡，使中风成为该国第三大死因。在非致命性中风中，海马体的缺血性损伤会导致认知和记忆障碍。海马体是已知的大脑选择性易受缺血影响的区域。脑缺血后，存活的神经元经历了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对脑缺血的反应机制。目前的建议集中在缺血、缺氧的一个方面，以及它对M受体功能表达的影响，M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一种假设，即短暂的低氧导致m2mAChRs的功能反应性降低，所涉及的机制包括翻译前和翻译后的修饰。实验旨在确定短暂低氧导致m2mAChRs内化、脱敏和下调的潜在机制。 这项拟议的研究将使用培养丰富的原代海马神经元、人类胚胎肾脏细胞和稳定表达野生型或突变形式的m2 mAChR的小鼠胚胎成纤维细胞。在特定的目标1中，将进行实验来验证这样的假设，即低氧侮辱诱导m2mAChR的磷酸化，从而导致内化和对M受体激动剂的反应性降低。在特定的目标2中，将进行实验来验证这样的假设，即瞬时低氧对m2mAChR的脱敏不依赖于m2受体的内化。在具体目标3中，将进行实验，以验证短暂低氧降低m2mAChR mRNA和/或蛋白表达的假设。总之，这些实验将确定低氧如何诱导mAChR信号的变化，并促进对中风和短暂性脑缺血发作后果的理解。这些研究的结果将有助于开发治疗干预措施，以减少缺血损伤后的学习和记忆障碍。

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.