CHOLINERGIC MECHANISMS IN AGING AND ALZHEIMER'S DISEASE

这一建议涉及大脑中的毒鼠碱受体机制和 开发治疗脑部疾病的新疗法 胆碱能功能的显著改变。阿尔茨海默病(AD) 大脑皮层和海马区的m1、m3和m4受体主要有 广泛失神经和新的选择性激动剂的开发 胆碱能替代疗法似乎是有必要的。在帕金森氏症中 (PD)纹状体中主要的M1和M4受体被认为是 过度激活，新的选择性拮抗剂可能会显著 比现有的非选择性药物要好。帕金森氏症的治疗也可能是 靶向M5受体，这种受体在脑实质中异常普遍 黑质和苍白球，这可能有助于控制 多巴胺能神经。这个实验室已经证明了m1受体(结合 1 nM~3H-哌仑西平的结合部位)在数量、亲和力 对于激动剂，偶联内源性G蛋白，以及促进 肌醇磷脂在AD和/或慢性实验后的水解性 胆碱能神经丧失。因此，选择性激动剂应该在AD中起作用， 只要它们能被开发出来，如果细胞 受体的位置并不太异常。新产品的设计和测试 激动剂和拮抗剂根据证据是非常复杂的，至少在 3不同受体亚型上的配基结合部位。这个实验室有 发现相同数量的激动剂的高(H)和低(L)亲和力部位 M1和m2受体，提示为二价或二聚体受体，加上at 至少多了一个变构拮抗剂的位置，一些拮抗剂也 似乎与相同数量的高亲和力和低亲和力结合。致信地址 两个站点的物理含义，将尝试展示两个 ~3H-QNB与膜上和膜上二价受体的结合部位 解决方案。如果受体出现二聚体，进一步的研究将涉及 单体与G蛋白的交联性及大小测定 这两个单体。为了解决两个站点的功能含义， M1-M4受体上的H或L位点将在膜和 培养的细胞，带有拮抗剂，另一部位将被表征 对于它与激动剂和拮抗剂的结合，功能偶联，它的PK 因为质子化的阻挡，以及该地点对 变构现象。H、L遗址的可能同一性 对某些拮抗剂表现出高亲和力和低亲和力，以及显示 然后将使用m1和m2受体检查两个不同的pk值。 带有4个点突变的天冬氨酸残基。当清楚地知道每个站点如何 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， 在此基础上，提出了一种新的研究方法。为了解决受体在细胞中的位置 正常、AD和PD大脑，放射自显影将使用一种新的 毒素具有明显的M1+M3&Gt；&Gt；M2+M4选择性。小说 应获得AD和PD的形态数据，这些数据应 为毒素的生理工作提供良好的基础，以及 为了了解哪些细胞最有可能被控制 毒蛾碱类配体。

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.