Beta-Amyloid Blockade of Hippocampal Nicotinic Alpha7-R*

海马烟碱 Alpha7-R* 的 β-淀粉样蛋白阻断

• 批准号: 6333445
• 负责人: Darwin K BERG
• 金额: $ 7.6万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-03 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6333445
• 关键词: Alzheimer's disease; amyloid proteins; hippocampus; laboratory rat; neurons; nicotinic receptors; receptor sensitivity; tissue /cell culture; voltage /patch clamp

Alzheimer's Disease Drug Discovery. Alzheimer's disease is a physiologically devastating condition that is threatening to reach epidemic proportions as the population ages. Cholinergic deficit is a widely recognized aspect of Alzheimer's disease, and is now thought to include nicotinic signaling as a critical component. One of the most interesting nicotinic receptors is the subtype composed of alpha7 subunits because it is widely expressed in the nervous system and has a high relative permeability to calcium which enables it to control diverse cellular functions. Recently it has been shown that alpha7-containing receptors bind the beta-amyloid peptide (amino acids 1-42) in the pM- nM range. The mammalian hippocampus is an interesting system for studying such interactions because alpha7-containing receptors are relatively abundant there, and the hippocampus is centrally involved in memory formation which is a major target of Alzheimer's disease. In preliminary studies we have found that the beta-amyloid peptide in the nM range reversibly inhibits alpha7-containing nicotinic receptors on hippocampal neurons in culture. Surprisingly, certain peptides applied exogenously can quickly and reversibly potentiate the alpha7- containing receptor response by an order of magnitude. This raises the real possibility that drugs capable of accessing the same site may be able to compensate for beta-amyloid inhibition of the receptors. This pilot proposal will examine the interactive of beta-amyloid peptide with hippocampal alpha7-containing receptors both functionally and physically. In addition, it will test the hypothesis that exogenous application of appropriate regulatory molecules will overcome the inhibition mediated by beta-amyloid peptide. The approach will use whole-cell patch clamp recording (usually in perforated patch mode) from the neurons while applying test compounds in desired sequences from a multi-bore rapid applicator. Competition binding experiments with radiolabeled probes will also be used to assess direct interactions. Functional characterization will include modulation of spontaneous synaptic events in the cultures by alpha7-containing receptors, and determining the impact of beta-amyloid peptide on the modulation in the presence of absence of the candidate potentiators. If this pilot study proves successful (as the preliminary experiments encourage us to think it will), the results will provide the basis for a subsequent full-scale follow-up to analyze beta-amyloid effects on CNS alpha7-containing nicotinic receptors, to analyze Alzheimer's brain tissue for specific involvement of alpha7-receptors in the disease, and to evaluate compounds capable of crossing the blood-brain barrier and potentiating the receptors in situ as a possible therapeutic strategy.

阿尔茨海默病药物发现。 阿尔茨海默氏病是一种生理上的破坏性疾病，随着人口老龄化，它有可能达到流行病的程度。 胆碱能缺陷是阿尔茨海默病的一个被广泛认可的方面，现在被认为包括烟碱信号传导作为一个关键组成部分。 最令人感兴趣的烟碱受体之一是由α 7亚基组成的亚型，因为它在神经系统中广泛表达，并且对钙具有高的相对渗透性，这使得它能够控制多种细胞功能。 最近，已经显示含α 7的受体在pM-nM范围内结合β-淀粉样肽（氨基酸1-42）。 哺乳动物海马是研究这种相互作用的一个有趣的系统，因为含有α 7的受体在那里相对丰富，并且海马集中参与记忆形成，记忆形成是阿尔茨海默病的主要靶点。 在初步研究中，我们发现，在nM范围内的β-淀粉样肽可逆地抑制培养的海马神经元上含有α 7的烟碱受体。 令人惊讶的是，外源性应用的某些肽可以快速且可逆地增强含α 7的受体应答一个数量级。 这增加了真实的可能性，即能够进入相同位点的药物可能能够补偿β-淀粉样蛋白对受体的抑制。 这个试验性的建议将检查β-淀粉样肽与海马α 7受体的相互作用，包括功能和物理。 此外，它将测试的假设，外源性应用适当的调节分子将克服β-淀粉样肽介导的抑制。 该方法将使用来自神经元的全细胞膜片钳记录（通常以穿孔膜片钳模式），同时从多孔快速施用器以所需顺序施用测试化合物。 放射性标记探针的竞争结合实验也将用于评估直接相互作用。功能表征将包括通过含有α 7的受体调节培养物中的自发突触事件，并确定β-淀粉样肽在存在或不存在候选增效剂的情况下对调节的影响。 如果这个试验性研究成功了（正如初步实验鼓励我们认为的那样），结果将为随后的全面随访提供基础，以分析β-淀粉样蛋白对含有CNS α 7的烟碱受体的影响，分析阿尔茨海默氏症脑组织中α 7受体在疾病中的特异性参与，并评价能够穿过血脑屏障并原位增强受体的化合物作为可能的治疗策略。