Superoxide Dismutase, Peroxynitrite and ALS

描述(申请人提供)：描述ALS是一种致命的进行性瘫痪，每年影响34,000名美国人，并导致8,000人死亡。1993年发现的与2-7%的肌萎缩侧索硬化症病例有关的铜，锌-超氧化物歧化酶(铜，锌-超氧化物歧化酶)显性突变的发现，使人们对基于抗氧化剂的肌萎缩侧索硬化症的新治疗方法有望问世。虽然人们普遍认为ALS突变的超氧化物歧化酶损害了稳定性，但与这些突变相关的特定毒性功能获得仍然存在激烈的争论。两种主要的解释是聚集性假说和缺锌假说，前者认为突变超氧化物歧化酶的聚集是疾病的直接原因，后者则认为疾病是由于超氧化物歧化酶氧化还原活性增加所致，超氧化物歧化酶失去了锌，但铜保留在其活性部位。我们提出，基于大量的初步证据，这两个假说与聚集密切相关，聚集通过去除缺锌的SOD具有矛盾的保护作用。锌缺乏假说也提出了一个有趣的可能性，即超氧化物歧化酶可能导致散发性肌萎缩侧索硬化症。我们开发了创新的质谱学方法来定量测定整个脊髓中SOD的金属含量，这使得我们能够批判性地评估缺锌和聚集假说。目的1研究肌萎缩侧索硬化症相关突变如何影响超氧化物歧化酶的二聚体界面和分子内二硫键，从而影响锌和铜的结合以及超氧化物歧化酶的聚集倾向。这一目标将探索金属丢失和聚集的物理基础，以了解突变SOD如何更容易丢失锌，以及为什么突变SOD在遗传中占主导地位。目的探讨转基因动物体内缺锌超氧化物歧化酶(SOD)的铜伴侣蛋白(CCS)和野生型铜锌超氧化物歧化酶如何调节体内锌缺乏的浓度，以及如何从药理上最有效地降低体内锌缺乏超氧化物歧化酶的含量。目的3绘制散发性ALS患者病变区与非病变区不同金属状态的解剖分布图，验证锌缺乏是否为散发性与家族性超氧化物歧化酶的共同联系。新的初步数据表明，FTICR在灵敏度方面有了巨大的改善，使目标3可行。拟议中的实验的完成将关键地测试SOD突变如何导致ALS的发展，以及散发性ALS中野生型SOD是否会导致锌的丢失。与公共卫生相关：只有一小部分卢·格里克病(又称ALS)患者携带一种名为SOD的常见抗氧化剂防御酶的突变。拟议中的实验的完成将有助于解释绝大多数没有SOD突变的散发性ALS患者如何参与其中，这将为治疗这种疾病指明新的方法。我们专门测试了这样一个假设，即当这种酶失去一个锌原子时，它就会中毒，并开发了新的质谱学方法，直接测量脊髓中锌的损失。

DESCRIPTION (provided by applicant): Description ALS is a fatal progressive paralysis affecting 34,000 Americans and killing 8,000 per year. The discovery in 1993 of dominant mutations to Cu,Zn-superoxide dismutase (Cu,Zn-SOD) linked to 2-7% of ALS cases led to hopes that new treatments for ALS based on antioxidants might be forthcoming. Whereas there is broad agreement that ALS mutant SODs have impaired stability, the specific toxic gain-of-function associated with these mutations is still hotly debated. The two leading explanations are the aggregation hypothesis that aggregation of mutant SODs is a direct cause of disease and the zinc-deficient hypothesis that disease is due to increased redox activity of SOD that has lost zinc but retains copper in its active site. We propose, based on extensive preliminary evidence, that the two hypotheses are intimately interconnected with aggregation being paradoxically protective by removing zinc-deficient SOD. The zinc-deficient hypothesis also raises the intriguing possibility that SOD could contribute to sporadic ALS. We have developed innovative mass spectrometric methods to quantitively the metal content of SOD across the spinal cord, which allow us to critically evaluate the zinc-deficient and aggregation hypotheses. Aim 1 will characterize how the dimer interface and the intramolecular disulfide of SOD are affected by ALS-associated mutations to affect zinc and copper binding as well as propensity for aggregation of SOD. This aim will explore the physical basis underlying metal loss and aggregation to understand how mutant SODs are more prone to losing zinc and why mutant SODs are dominant in inheritance. Aim 2 will assess how the concentrations of zinc-deficient SOD are modulated by CCS (the copper chaperone for SOD) and wild-type Cu,Zn SOD in transgenic animals both of which accelerate disease in vivo -- and how to most effectively pharmacologically decrease zinc-deficient SOD in vivo. Aim 3 will map the anatomical distribution of the different metal states of SOD in disease-affected versus unaffected regions in human sporadic ALS patients, testing whether zinc-deficient SOD may be the common connection between sporadic and familial SOD. New preliminary data demonstrate a vast improvement in sensitivity with the FTICR that makes Aim 3 feasible. Completion of the proposed experiments will critically test how SOD mutations lead to the development of ALS and whether loss of zinc occurs from wild-type SOD in sporadic ALS. PUBLIC HEALTH RELEVANCE: Only a small percentage of patients with Lou Gehrig's disease (also known as ALS) carry mutations to a common antioxidant defense enzyme called SOD. Completion of the proposed experiments will help explain how SOD can be involved in the vast majority of sporadic ALS patients who do not have SOD mutations, which will point to new ways to treat the disease. We specifically are testing the hypothesis that this enzyme becomes toxic when it loses one zinc atom and have developed new mass spectrometric methods that directly measure the loss of zinc directly from the spinal cord.