Mechanisms of Protection and Pathogenesis in Amyotrophic Lateral Sclerosis Mice

描述(申请人提供)：十多年来，与肌萎缩侧索硬化症相关的SOD1突变株在体外和体内都与野生型酶进行了比较，试图了解其发病机制(S)。然而，关于药物延长G93A小鼠存活的分子机制知之甚少。我们已经确定了两种化学上不同的(有机锰)化合物，当在疾病开始时(类似于人类治疗开始的时候)给药时，它们显著延长G93A小鼠的存活。根据我们的结果，正在努力用这些化合物在肌萎缩侧索硬化症进行人体临床试验。我们建议使用这些高效的化合物来探索保护机制，同时继续我们对SOD1突变介导的毒性机制的研究。我们将研究伴随着这些化合物保护和最终失去保护的蛋白质表达的变化(目标1)，以更好地了解涉及的机制。这些化合物延长生命的幅度堪比迄今报道的任何化合物的最好水平，即使是那些症状前给药的化合物。然而，可靠的比较需要类似的治疗方案。我们建议测试在起效给药范例(目标2)中症状前给药时显示有效性的化合物，以测试一些人类试验的失败可能与研究设计的差异有关的假设。令人信服的新证据表明，线粒体对突变体的摄取对疾病至关重要，摄取由金属状态(主要是锌)和内部二硫化物的状态决定。在这里，我们提供了从根本上新的证据，证明体内缺锌突变与疾病严重程度直接相关，并且突变SOD1(但不是野生型)是共价交联的。目标3将识别交叉连接的蛋白质，并确定药物治疗是否会改变它。目的4利用从脊髓中提纯的酶作为“体内表达系统”，扩展我们对突变体体内金属状态和硫醇状态的研究。

DESCRIPTION (Provided by applicant): For over a decade, ALS-associated SOD1 mutants have been compared to wild-type enzyme, both in vitro and in vivo in an attempt to understand the mechanism(s) of pathogenesis. However, little is known about the molecular mechanisms which underlie pharmacologically-mediated extension of survival in G93A mice. We have identified two chemically distinct (organomanganic) compounds which markedly extend survival in G93A mice when given at disease onset (similar to when human treatment would begin). Based on our results, efforts are underway to conduct human clinical trials in ALS with these compounds. We propose to use these highly efficacious compounds to probe the mechanisms of protection, while continuing our work on the mechanisms of SOD1 mutant-mediated toxicity. We will examine protein expression changes which accompany protection by these compounds and the ultimate loss of protection (Aim 1) in order to gain a better understanding of the mechanisms involved. The magnitude of life extension with these compounds rivals the best yet reported for any compound, even those administered presymptomatically. However, reliable comparisons require comparable treatment regimens. We propose to test compounds that show efficacy when given presymptomatically in an onset administration paradigm (Aim 2) to test the hypothesis that the failure of some human trials may relate to differences in study design. Compelling new evidence suggests that mitochondrial uptake of mutants is critical to disease and that uptake is determined by metallation state (primarily zinc) and the status of an internal disulfide. Herein we offer fundamentally new evidence that a zinc-deficient mutant in vivo directly correlates with disease severity, and that mutant SOD1 (but not wild-type) is covalently cross linked. Aim 3 will identify the cross linked proteins and determine if drug treatment alters it. Aim 4 will extend our studies of in vivo metallation states and thiol status of mutants using enzyme purified from spinal cord as an "in vivo expression system".