CHOLINERGIC MECHANISMS IN AGING AND ALZHEIMERS DISEASE

衰老和阿尔茨海默病中的胆碱能机制

• 批准号: 2049476
• 负责人: Lincoln T. Potter
• 金额: $ 26.76万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-05-01 至 1996-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2049476
• 关键词: Alzheimer's disease; G protein; Parkinson's disease; aging; aspartate; autoradiography; brain stem; cell membrane; cerebral cortex; chemical binding; crosslink; hippocampus; human tissue; inhibitor /antagonist; laboratory rabbit; laboratory rat; molecular site; muscarinic receptor; neurons; neuropharmacology; neurotoxins; protein structure; protein structure function; receptor binding; scopolamine; site directed mutagenesis; stimulant /agonist; substantia nigra; tissue /cell culture

This proposal concerns muscarinic receptor mechanisms in the brain and the development of new therapies for brain diseases in which there is a significant change in cholinergic functions. In Alzheimer's disease (AD) the predominant ml, m3 and m4 receptors of the cortex and hippocampus are extensively denervated and the development of new selective agonists for cholinergic replacement therapy appears warranted. In Parkinson's disease (PD) the predominant ml and m4 receptors in the striatum are believed to be overactivated, and new selective antagonists could be significantly better than existing, non selective drugs. Therapy for PD might also be directed at m5 receptors which are unusually prevalent in the substantia nigra and globus pallidus and which may help control the activity of dopaminergic nerves. This laboratory has shown that ml receptors (binding sites for 1 nM 3H-pirenzepine) remain nearly normal in numbers, affinity for agonists, coupling to endogenous G protein, and ability to promote phosphoinositide hydrolysis, in AD and/or after chronic experimental cholinergic denervation. Hence selective agonists should work in AD, provided that they can be developed, and provided that the cellular location of receptors is not too abnormal. The design and testing of new agonists and antagonists is greatly complicated by evidence for at least 3 ligand binding sites on various receptor subtypes. This laboratory has found equal numbers of high (H) and low (L) affinity sites for agonists on ml and m2 receptors, suggesting bivalent or dimeric receptors, plus at least one more site for allosteric antagonists, Some antagonists also appear to bind to equal numbers of high and low affinity sites. To address the physical meaning of two sites, attempts will be made to show two binding sites for 3H-QNB on bivalent receptors in membranes and in solution. If receptors appear dimeric, further studies will involve crosslinking of the monomers and G protein, and measurements of the sizes of the two monomers. To address the functional meaning of two sites, either H or L sites on ml-m4 receptors will be blocked in membranes and in cultured cells, with antagonists, and the other site will be characterized for its binding of agonists and antagonists, functional coupling, its pK for blockade by protonation, and for the contribution of the site to allosteric phenomena. The probable identity of H and L sites, sites showing high and low affinity for certain antagonists, and sites showing two different pK values will then be examined, using ml and m2 receptors with 4 point-mutated aspartate residues. When it is clear how each site contributes to the binding and function of agonists and antagonists, new screening procedures for ml-m5 agonists and antagonists will be developed, based on these methods. To address the cellular location of receptors in normal, AD and PD brains, autoradiography will be carried out using a new toxin which shows marked ml + m3 > > M2 + m4 selectivity. Novel morphological data should be obtained for AD and PD, and these data should provide a good basis for physiological work with the toxin, and for understanding which cells are most likely to be controllable with muscarinic ligands.

该提案涉及大脑中的毒蕈碱受体机制 开发新的脑部疾病疗法 胆碱能功能的显着变化。 在阿尔茨海默氏病（AD） 皮质和海马的主要ML，M3和M4受体是 广泛的剥夺和开发新的选择性激动剂 胆碱能替代疗法似乎有必要。 在帕金森氏病中 （PD）纹状体中的主要ML和M4受体被认为是 过度活化，新的选择性拮抗剂可能会显着 比现有的非选择性药物更好。 PD治疗也可能是 针对M5受体，该受体在底座中普遍存在 Nigra和Globus Pallidus，这可能有助于控制 多巴胺能神经。 该实验室表明ML受体（结合 1 nm 3h pirenzepine的站点的数量几乎正常，亲和力 对于激动剂，耦合到内源G蛋白，以及促进的能力 在AD和/或慢性实验之后，磷酸肌醇水解，/或 胆碱能神经。 因此，选择性激动剂应该在广告中工作， 只要可以开发它们，并且只要细胞 受体的位置不太异常。 新的设计和测试 激动剂和拮抗剂至少被证据而变得非常复杂 各种受体亚型上的3个配体结合位点。 这个实验室有 发现相等数量的高（h）和低（l）亲和力位点 ML和M2受体，提示二价或二聚体受体，加上 至少还有一个用于变构拮抗剂的位置，一些拮抗剂也 似乎结合了相等数量的高亲和力位点。 解决 两个站点的物理含义将尝试显示两个站点 3H-QNB的结合位点在膜中的二价受体上和 解决方案。 如果受体出现二聚体，将进一步研究涉及 单体和G蛋白的交联，大小的测量值 在两个单体中。 要解决两个站点的功能含义， ML-M4受体上的H或L位点将在膜和 具有拮抗剂的培养细胞，另一个位置将被表征 为了结合激动剂和拮抗剂，功能耦合，其PK 用于通过质子化的封锁，以及该地点的贡献 变构现象。 H和L位点的可能身份，地点 显示出对某些拮抗剂的高和低亲和力，并且位置显示 然后，将使用ML和M2受体检查两个不同的PK值 有4点熔化的天冬氨酸残基。 当清楚每个网站如何 有助于激动剂和拮抗剂的结合和功能，新的 将开发ML-M5激动剂和拮抗剂的筛选程序， 基于这些方法。 解决受体在 正常，AD和PD脑，放射自显影将使用新的 毒素显示标记为ML + M3 >> M2 + M4选择性。 小说 应获得AD和PD的形态数据，这些数据应 为毒素提供了良好的生理研究基础， 以了解哪些单元最有可能通过 毒蕈碱配体。