Superoxide Dismutase, Peroxynitrite and ALS

描述（由申请人提供）：ALS是一种致命的进行性瘫痪，每年影响34000名美国人，造成8000人死亡。1993年发现的Cu， zn -超氧化物歧化酶（Cu,Zn-SOD）显性突变与2-7%的ALS病例有关，这使人们希望基于抗氧化剂的ALS新疗法可能即将到来。尽管人们普遍认为ALS突变sod的稳定性受损，但与这些突变相关的特异性毒性功能获得仍存在激烈争论。两种主要的解释是聚集假说，突变SOD的聚集是疾病的直接原因；缺锌假说，疾病是由于SOD氧化还原活性增加，失去锌，但在其活性位点保留了铜。我们提出，基于广泛的初步证据，这两种假设是密切相关的聚集是矛盾的保护通过去除缺锌SOD。锌缺乏假说也提出了一种有趣的可能性，即SOD可能导致散发性ALS。我们开发了创新的质谱方法来定量测定脊髓中SOD的金属含量，这使我们能够批判性地评估锌缺乏和聚集假说。目的1将描述SOD的二聚体界面和分子内二硫化物如何受到als相关突变的影响，从而影响锌和铜的结合以及SOD的聚集倾向。本研究旨在探索金属丢失和聚集的物理基础，以了解突变型sod如何更容易丢失锌，以及为什么突变型sod在遗传中占主导地位。目的2将评估CCS （SOD的铜伴侣）和野生型Cu，Zn SOD在转基因动物中的浓度是如何被调节的，这两种情况都会加速体内疾病，以及如何最有效地从药理学上减少体内缺锌SOD。Aim 3将绘制人类散发性ALS患者患病区与未患病区SOD不同金属状态的解剖分布，检测缺锌SOD是否可能是散发性和家族性SOD之间的共同联系。新的初步数据表明，FTICR在灵敏度上有了巨大的提高，这使得Aim 3成为可能。上述实验的完成将严格测试SOD突变如何导致ALS的发展，以及散发性ALS中野生型SOD是否会发生锌的损失。公共卫生相关性：只有一小部分患有卢伽雷氏病（也被称为ALS）的患者携带一种常见的抗氧化防御酶SOD的突变。完成所提出的实验将有助于解释SOD如何参与绝大多数没有SOD突变的散发性ALS患者，这将指出治疗该疾病的新方法。我们正在测试这种酶在失去一个锌原子时就会有毒的假设，并开发了新的质谱方法，可以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.