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，以确定毒蕈碱神经传递的临床相关步骤，这些步骤可以由m1或m4受体的特异性激动剂或拮抗剂控制。目的1涉及这些受体的直接研究。（1a）人AD-m1受体将使用生物素化m1毒素和单亲和素树脂分离。该受体蛋白的生化研究可以解释其与G蛋白的偶联缺陷以及与抗m1抗体的不完全免疫沉淀，并可能找到提高酯酶抑制剂在AD中的有效性的方法。（1b） 125I-m1- Toxin和125I-m4-toxin将用于确定大鼠体内m1+m4受体的分布，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）利用放射自显影技术定位人和大鼠脊髓背角的125i -m4毒素，大鼠脊背根切断术显示m4受体是否像阿片受体一样位于传入神经末梢。结果将显示m4激动剂是否可能被证明对疼痛有用。（1d） 125i -毒素将用于研究毒素受体复合物，以及m4-毒素在4℃和37℃下的结合动力学。（1e）将通过125i -毒素放射自显影研究半帕金森大鼠大脑中m1+m4受体的变化。目的2关注单侧纹状体m4阻断对大鼠运动的影响。实现特异性m4-阻断的最佳条件将在右腔内灌注m4-毒素0.2-2小时后用125i -m4-毒素通过放射自显影评估。然后研究+/-半pd大鼠自发性前臂使用的改变。特异性右m4拮抗剂可增加右半pd大鼠的左运动，纠正左运动缺陷。目的3关注单侧纹状体m1阻断（“ml敲低”）对大鼠运动的影响。预计右侧纹状体内m1毒素不会影响自发左前臂使用本身或右半pd，但预计会改变对阿帕啡（多巴胺激动剂），匹洛卡平和xanomeline（毒碱激动剂）的反应。至少，研究结果将显示一种特定的m1拮抗剂是否可以用于控制癫痫发作。目的4涉及突变m1毒素。m1同工毒素的独特氨基酸将通过定点诱变改变为与m2-m5受体结合的毒素特征残基，从而产生具有新的选择性特征的突变毒素，以确定哪些残基赋予m1毒素显著的亲和力和选择性，并开始研究突变m1毒素与突变m1受体之间的分子契合。

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.