Mechanisms of Protection and Pathogenesis in Amyotrophic Lateral Sclerosis Mice

描述（由申请人提供）：十多年来，als相关的SOD1突变体已经与野生型酶进行了比较，无论是在体外还是在体内，试图了解发病机制。然而，对于药物介导的G93A小鼠生存延长的分子机制知之甚少。我们已经确定了两种化学上不同的（有机和有机）化合物，在疾病发作时（类似于人类治疗开始时）给予G93A小鼠显着延长生存期。基于我们的研究结果，我们正在努力用这些化合物对ALS进行人体临床试验。我们建议利用这些高效化合物来探索其保护机制，同时继续研究SOD1突变介导的毒性机制。为了更好地理解所涉及的机制，我们将研究伴随这些化合物保护和最终保护丧失的蛋白质表达变化（目的1）。这些化合物的寿命延长幅度与迄今为止报道的任何化合物相媲美，即使是那些在症状前施用的化合物。然而，可靠的比较需要比较的治疗方案。我们建议测试在症状前给药模式下显示疗效的化合物（目的2），以验证一些人体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，突变体的线粒体摄取对疾病至关重要，并且摄取是由金属化状态（主要是锌）和内部二硫化物的状态决定的。在此，我们提供了新的证据，证明体内锌缺乏突变体与疾病严重程度直接相关，并且突变体SOD1（但不是野生型）是共价交联的。目的3将鉴定交联蛋白并确定药物治疗是否会改变它。Aim 4将利用从脊髓中纯化的酶作为“体内表达系统”来扩展我们对突变体体内金属化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".