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突变体与野生型酶进行了体外和体内比较，试图了解发病机制。然而，很少有人知道的药理学介导的延长G93A小鼠的生存的分子机制。我们已经鉴定了两种化学性质不同的（有机锰）化合物，当在疾病发作时（类似于人类治疗开始时）给药时，它们显著延长了G93 A小鼠的存活期。基于我们的结果，正在努力用这些化合物进行ALS的人体临床试验。我们建议使用这些高效的化合物来探测保护机制，同时继续我们对SOD 1突变体介导的毒性机制的研究。我们将研究蛋白质表达的变化，伴随着这些化合物的保护和保护的最终损失（目的1），以获得更好地了解所涉及的机制。这些化合物的生命延长幅度与任何化合物的最佳报道相媲美，即使是那些症状前给药的化合物。然而，可靠的比较需要可比的治疗方案。我们建议测试化合物，显示疗效时，给予症状前在发病管理模式（目的2），以测试的假设，一些人体试验的失败可能与研究设计的差异。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，并且该摄取由代谢状态（主要是锌）和内部二硫化物的状态决定。在此，我们提供了全新的证据，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SOD 1（而非野生型）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改变它。目的4将使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代谢状态和巯基状态的研究。

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".