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，以确定可以通过m1或m4受体的特异性激动剂或拮抗剂控制的毒蕈碱神经传递中的临床相关步骤。目的1涉及这些受体的直接研究。(1a)将使用生物素化m1毒素和单抗生物素蛋白亲和树脂分离人AD-m1受体。受体蛋白的生化研究应解释其缺陷耦合G蛋白和，不完全免疫沉淀与抗M1抗体，并可能显示如何提高AD的酯酶抑制剂的有效性。(1b)125 I-m1-毒素和125 I-m4-毒素将用于确定m1+m4受体在大鼠中的分布，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)放射自显影术将被用来本地化125 I-m4-毒素在人类和大鼠脊髓的背角，背根切断术在大鼠将表明是否m4受体的传入神经末梢，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-受体复合物，以及在4 ℃和37 ℃下m4-毒素结合的动力学。(1e)本文用~（125）I-毒素放射自显影法研究了偏侧帕金森（hemi-PD）大鼠脑内m_1 + m_4受体的变化。目的2：单侧纹状体m4-阻滞对大鼠运动的影响。在右侧纹状体内输注m4-毒素后0.2-2小时，通过放射自显影术用125 I-m4-毒素评估实现特异性m4-阻断的最佳条件。然后研究大鼠+/-半-PD的自发前臂使用改变。特异性右侧m4拮抗剂应增加左侧运动，纠正右侧偏侧PD大鼠的左侧运动缺陷。目的3是关于建立单侧纹状体m1-阻断（“ml敲低”）对大鼠运动的影响。预计右纹状体内m1毒素本身或右半侧PD不会影响自发性左前臂使用，但预计会改变对阿扑吗啡（多巴胺激动剂）、毛果芸香碱和xanomeline（毒蕈碱激动剂）的反应。至少，研究结果将表明是否可以使用特定的m1拮抗剂来控制癫痫发作。目的4涉及突变m1毒素。通过定点诱变将m1-同种毒素的独特氨基酸改变为与m2-m5受体结合的毒素的特征残基，以产生具有新的选择性特征的突变体毒素，以确定哪些残基赋予m1-毒素的显著亲和力和选择性，并开始研究突变体m1-毒素和突变体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.