CHOLINERGIC MECHANISMS IN AGING AND ALZHEIMER'S DISEASE

该提议涉及脑中的毒蕈碱受体机制， 开发新的治疗脑部疾病的方法， 胆碱能功能的显著变化。 阿尔茨海默病（AD） 皮质和海马的主要M1、M3和M4受体是 广泛失神经支配和新的选择性激动剂的发展， 胆碱能替代疗法似乎是必要的。 帕金森病 (PD)纹状体中的主要M1和M4受体被认为 被过度激活，新的选择性拮抗剂可能会显着 优于现有的非选择性药物。 PD的治疗也可能是 针对在脑实质中异常普遍的M5受体， 黑质和苍白球，这可能有助于控制的活动， 多巴胺能神经 该实验室已经表明，ml受体（结合 1 nM 3 H-哌仑西平的位点）在数量、亲和力、 对于激动剂，与内源性G蛋白偶联，以及促进 磷酸肌醇水解，在AD和/或慢性实验后 胆碱能去神经 因此，选择性激动剂应该在AD中起作用， 只要它们能够被开发出来，只要细胞 受体的位置不太异常。 设计及测试新 激动剂和拮抗剂的研究至少有证据表明， 不同受体亚型上的3个配体结合位点。 本实验室曾 发现相同数量的高（H）和低（L）亲和力位点的激动剂上 ml和m2受体，提示二价或二聚体受体，加上at 变构拮抗剂的至少多一个位点，一些拮抗剂还 似乎与相等数量的高和低亲和力位点结合。 解决 两个网站的物理意义，将试图显示两个 膜和细胞膜中二价受体上3 H-QNB的结合位点 溶液 如果受体出现二聚体，进一步的研究将涉及 单体和G蛋白的交联，以及尺寸的测量 两个单体。 为了解决两个网站的功能意义， m1-m4受体上的H或L位点将在膜中被阻断， 培养的细胞，与拮抗剂，和其他网站将进行表征 对于其激动剂和拮抗剂的结合，功能偶联，其pK 质子化的封锁，以及网站的贡献， 变构现象 H和L位点的可能同一性， 对某些拮抗剂显示出高和低的亲和力， 然后使用ml和m2受体检查两个不同的pK值 有4个点突变的天冬氨酸残基 当每个网站都清楚地 有助于激动剂和拮抗剂的结合和功能， 将开发M1-M5激动剂和拮抗剂的筛选程序， 基于这些方法。 为了解决受体在细胞中的位置， 正常、AD和PD脑，放射自显影将使用新的 毒素显示出明显的ml + m3 > > M2 + m4选择性。 小说 应获得AD和PD的形态学数据，这些数据应 提供良好的 依据 生理 研究毒素 为了理解， 细胞最有可能 可控制， 毒蕈碱配体。

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.