CHOLINERGIC MECHANISMS IN AGING AND AD

该实验室正在使用M1-毒素和M4-毒素作为高度特异性的配体，以确定毒副作用神经传递中临床上相关的步骤，这些步骤可以由M1或M4受体的特定激动剂或拮抗剂控制。目标1涉及对这些受体的直接研究。(1A)人AD-M1受体将使用生物素标记的M1-毒素和单亲和素亲和树脂分离。对受体蛋白的生化研究应该解释其与G蛋白的偶联缺陷以及与抗M1抗体的不完全免疫沉淀，并可能揭示提高酯酶抑制剂在AD治疗中的有效性的方法。(1B)125I-M1-毒素和125I-M4-毒素将用于确定M1+M4受体在大鼠体内的分布，以将注意力集中在新药可以起作用的部位，无论是有益的还是产生副作用的。(1C)用放射自显影定位~(125)I-M4-毒素在人和大鼠脊髓背角，在大鼠背根切断将显示M4受体是否在传入神经末梢上，如阿片受体。结果将显示M4激动剂是否可能被证明对疼痛有用。(1D)~(125)I-毒素将用于研究毒素-受体复合体，并在4℃和37℃下与M4-毒素结合动力学。(1e)用~(125)I-毒素放射自显影研究半帕金森病(HMI-PD)大鼠脑内M1+M4受体的变化。目的2研究单侧纹状体M4区阻断对大鼠运动的影响。在右侧纹状体内注射M4-毒素0.2-2小时后，用125I-M4-毒素进行放射自显影，以评估获得特异性M4-阻断的最佳条件。然后，将研究大鼠+/-半侧帕金森病改变后的前臂自发使用情况。特异性的右M4拮抗剂可增加右半帕金森病大鼠的左运动，纠正左运动障碍。目的3研究阻断单侧纹状体M1区对大鼠运动的影响。右侧纹状体内M1毒素本身或右半帕金森病不会影响左前臂的自发使用，但有望改变对阿朴吗啡(多巴胺激动剂)、匹罗卡品和克诺马林(毒鼠碱激动剂)的反应。至少，结果将显示是否可以使用特定的M1拮抗剂来控制癫痫发作。目标4涉及突变的M1毒素。M1-等毒素的独特氨基酸将通过定点突变改变为与M2-M5受体结合的毒素的残基特征，以产生具有新的选择性图谱的突变毒素，确定哪些残基赋予M1-毒素显著的亲和力和选择性，并开始研究突变的M1-毒素和突变的M1受体之间的分子匹配。

This laboratory is using m1-toxin and m4-toxin as highly specific ligands to identify clinically relevant steps in muscarinic neurotransmission that can be controlled by specific agonists or antagonists for m1 or m4 receptors. Aim 1 concerns direct studies of these receptors. (1a) Human AD-m1 receptors will be isolated using biotinylated m1-toxin and a monoavidin affinity resin. Biochemical studies of the receptor protein should explain its defective coupling to G protein and, incomplete immunoprecipitation with anti-m1 antibodies, and may show ways to improve the effectiveness of esterase inhibitors in AD. (1b) 125I-m1- Toxin and 125I-m4-toxin will be used to establish the distribution of m1+m4 receptors in the rat, to focus attention on sites where new drugs can act, either beneficially or to produce side effects. (1c) Autoradiography will be used to localize 125I-m4-toxin in the dorsal horns of the human and rat spinal cord, and dorsal root rhizotomy in the rat will indicate whether m4 receptors are on afferent nerve terminals, like opiate receptors. The results will show whether m4 agonists are likely to prove useful for pain. (1d) 125I-Toxins will be used to study toxin-receptor complexes, and the kinetics of binding of m4-toxin at 4 degrees and 37 degrees C. (1e) Changes of m1+m4 receptors in hemi- Parkinson (hemi-PD) rat brains will be studied by autoradiography with 125I-toxins. Aim 2 concerns the effects of unilateral striatal m4-blockade on movement in rats. Optimum conditions for achieving specific m4- blockade will be assessed with 125I-m4-toxin by autoradiography 0.2-2 hours after right intrastriatal infusion of m4-toxin. Then rats +/- hemi-PD will be studied for altered spontaneous forearm use. Specific right m4 antagonism should increase left movement and correct the defective left movement of rats with right hemi-PD. Aim 3 concerns the effects pf established unilateral striatal m1-blockade ("ml knockdown") on movement in rats. Right intrastriatal m1-toxin is not expected to affect spontaneous left forearm use by itself or in right hemi-PD, but is expected to alter responses to apomorphine (dopamine agonist), pilocarpine and xanomeline (muscarinic agonists). At the least, the results will show whether a specific m1 antagonist can be used to control seizures. Aim 4 concerns mutant m1-toxins. The unique amino acids of the m1-isotoxins will be changed by site-directed mutagenesis to residues characteristic of toxins that bind to m2-m5 receptors, to produce mutant toxins with new selectivity profiles, to determine which residues confer the remarkable affinity and selectivity of m1-toxins, and to begin studies of the molecular fit between mutant m1-toxins and mutant m1 receptors.