PROENKEPHALIN GENE REGULATION IN HYPOTHALAMIC NEURONS

下丘脑神经元中脑啡肽原基因的调控

• 批准号: 2377410
• 负责人: DAVID BORSOOK
• 金额: $ 11.49万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-03-15 至 2000-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2377410
• 关键词: antisense nucleic acid; beta galactosidase; cAMP response element binding protein; enkephalins; gene expression; genetic enhancer element; genetic regulation; genetically modified animals; hypothalamus; immunocytochemistry; laboratory mouse; neurogenetics; opiate alkaloid; peptide hormone biosynthesis; physiologic stressor; stress; tissue /cell culture; transcription factor

The proenkephalin gene is involved in a number of physiological processes including embryonic development, stress, pain, opioid addiction and opioid tolerance. The present research proposal outlines a strategy to study stress-induced regulation of the proenkephalin gene in hypothalamic neurons in vivo in a transgenic mouse model in which the proenkephalin promoter is fused to the reporter gene, E. coli beta-galactosidase and in vitro organotypic transgenic cultures. The stress response is a major contributor to morbidity in mood and anxiety disorders, acute and chronic pain, surgery, and drug withdrawal. While the regulation of catecholamines, ACTH, and glucocorticoids by stress have been extensively studied, endogenous opioids have-only recently been implicated in the neurochemical mechanisms underlying the stress response. This proposal focuses on the gene encoding proenkephalin, one of the endogenous opioid precursors, because it is highly regulated in the hypothalamus by stress and because its promoter is understood in great detail. The transgenic model provides unique capabilities to combine molecular and integrative studies because the histochemical detection of beta-galactosidase is simple and is easily combined with both immunohistochemistry (e.g. for defining transcription factors such as the immediate early gene c-fos) or culture (organotypic and primary) methods. This proposal aims to further our understanding of opioid regulation of the proenkephalin gene using the stress model. Initially, we will determine whether hypertonic stress and opioid modulation (acute and chronic) of stress induces expression and post-translational modification of positively (e.g. CREB) and negatively acting (e.g. CREM) transcription factors that interact with the transgene. We also wish to demonstrate that CREB is a significant positive regulator of the transgene in conditions of acute stress and acute opioid modulation of stress and that opioid-inducible negative regulators can be identified. Using-isolated hypothalamic organotypic cultures we will map to the proenkephalin second messenger- inducible enhancer and the role of specific transcription factors in regulation of proenkephalin gene expression in vivo can be analyzed by the use of antisense oligonucleotides. The use of transgenic mice for in vivo experiments and as a source for organotyPic cultures will allow a mechanistic analysis of the regulation of an Important model gene in a highly restricted population of identified neurons involved in the well- established biology of the stress response. These studies should contribute significantly to understanding how exogenous opioids regulate proenkephalin gene expression in specific hypothalamic neurons in response to acute stress and the regulation of long-term plasticity of proenkephalin gene expression with chronic stress.

脑啡肽原基因参与了许多生理过程。 包括胚胎发育、压力、疼痛、阿片成瘾和 阿片耐受性。本研究提案概述了一项战略，以 应激诱导下丘脑前脑啡肽原基因调控的研究 原脑啡肽在体内转基因小鼠模型中的神经元 启动子与报告基因大肠杆菌β-半乳糖苷酶和 体外器官型转基因培养。压力反应是一种主要的 急性和慢性情绪和焦虑症发病率的推动者 疼痛、手术和戒毒。在监管的同时 儿茶酚胺、促肾上腺皮质激素和糖皮质激素在应激状态下广泛存在 研究表明，内源性阿片类药物直到最近才被认为与 应激反应背后的神经化学机制。这项建议 重点研究编码内源性阿片类药物之一的前脑啡肽的基因 前体，因为它在下丘脑中受到压力的高度调节 因为人们对它的发起人有非常详细的了解。转基因生物 Model提供了独特的功能，可将分子和整合 研究是因为β-半乳糖苷酶的组织化学检测是 简单且易于与免疫组织化学结合(例如，用于 定义转录因子，如即刻早期基因c-fos) 或培养(器官型和原生型)方法。这项建议旨在 对前脑啡肽基因阿片类调控的进一步认识 使用应力模型。最初，我们将确定高渗性 应激和阿片类药物调节(急性和慢性)应激诱导 积极的表达和翻译后修饰(如CREB) 和负作用(例如CREM)相互作用的转录因子 利用转基因技术。我们还希望证明CREB是一种 急性转化条件下转基因的显著正向调节因子 应激和急性阿片类药物应激调节以及阿片类药物诱导 消极的监管者是可以识别的。使用隔离的下丘脑 器官类型培养我们将映射到前脑啡肽第二信使- 诱导型增强子与特定转录因子在细胞周期调控中的作用 原脑啡肽原基因在体内表达的调节可以用以下方法分析 反义寡核苷酸的使用。转基因小鼠在体内的应用 活体实验和作为器官类型培养的来源将允许 一种重要模式基因调控的机制分析 与井有关的已鉴定神经元的高度受限群体- 建立了应激反应的生物学基础。这些研究应该 有助于理解外源性阿片类药物是如何调节的 脑啡肽原基因在大鼠下丘脑特异性神经元中的表达 对急性应激的反应和对长期可塑性的调节 脑啡肽原基因的表达与慢性应激。