ROLE OF NEURONAL NOS AND SUPEROXIDE IN NEURODEGENERATION

这项拟议研究的长期目标是了解 ROS和RNS在年龄相关性神经退行性疾病发病中的作用。的 待检验的一般假设是nNOS可以产生超氧化物和 一氧化氮的比例由黄素辅因子调节，氧化还原活性 化合物，L-精氨酸和四氢生物蝶呤（BH 4）。具体的假设是 nNOS在MPP+的神经毒性中起着至关重要的作用， 一种线粒体神经毒素，能引起帕金森症状。具体目标：1） 超氧化物歧化酶和NO的动力学形成从纯化的nNOS在存在 并且将确定MPP+的不存在。2)nNOS在细胞毒作用中的作用 并确定MPP+在神经元细胞中的凋亡作用。类似 实验将在BH 4缺陷的神经元细胞中确定， 在这些系统中，将测量ROS和RNS形成。3)的 MPP+对nNOS神经元细胞毒性和凋亡的影响 将确定敲除小鼠。方法：细胞外生成 超氧化物将通过自旋捕获和细胞内形成 通过监测线粒体顺乌头酸酶活性来测定超氧化物。 用免疫化学法测定酶的羰基化和硝化蛋白质 方法.这项研究的重要结果应该提供新的机制 从nNOS了解ROS和RNS形成。神经元一氧化氮合酶 在一些年龄相关的神经退行性疾病的发病中， 帕金森氏症，亨廷顿氏症，卢伽雷氏症。抑制nNOS和 在动物模型中，活性氧清除剂显示出神经保护作用 模仿这些病理。新的治疗策略， 年龄相关的神经退行性疾病可能从这些研究中出现。 新奇：该提案的新颖之处在于能够同时测量 来自纯化的nNOS的NO和超氧化物响应MPP+和其他 辅因子超氧阴离子介导的乌头酸酶中4Fe-4S簇合物的氧化 使酶失活并增加氧化应激。的可用性 新的超氧化物歧化酶模拟物使得有可能证明更多 彻底关于活性氧的性质，造成失活的乌头酸酶， 细胞内环境

The long-term goal of this proposed research is to understand the role of ROS and RNS in the onset of age-related neurodegenerative diseases. The general hypothesis to be tested is that nNOS can generate both superoxide and nitric oxide in a ratio that is regulated by flavin cofactors, redox active compounds, L-arginine and tetrahydrobiopterin (BH4). The specific hypothesis to be tested is that nNOS plays a crucial role in the neurotoxicity of MPP+, a mitochondrial neurotoxin that induces Parkinsonian symptoms. Specific aims: 1) the kinetic of superoxide and NO formation from purified nNOS in the presence and absence of MPP+ will be determined. 2) The role of nNOS in the cytotoxic and apoptotic effects of MPP+ in neuronal cells will be determined. Similar experiments will be determined in BH4-deficient neuronal cells that do not synthesize NO. In these systems, ROS and RNS formation will be measured. 3) The effect of MPP+ toxicity and apoptosis in neuronal cells isolated from nNOS knockout mice will be determined. Methods: Extracellular generation of superoxide will be measured by spin-trapping and intracellular formation of superoxide will be determined by monitoring mitochondrial aconitase activity. Aconitase carbonyls and nitrated protein will be determined by immunochemical methods. significance results from this study should provide new mechanistic insight into ROS and RNS formation from nNOS. Neuronal NOS has been implicated in the onset of several age-related neurodegeneration disorder including Parkinson's, Huntington's, and Lou Gehrig's diseases. Inhibition of nNOS and scavenger of ROS have been shown to exert neuroprotection in animal model mimicking these pathologies. New therapeutic strategies for treating age-associated neurodegenerative diseases may emerge from these studies. Novelty: Novel aspect of this proposal is the ability to measure simultaneously both NO and superoxide from purified nNOS in response to MPP+ and other co-factors. The superoxide-mediated oxidation of 4Fe-4S clusters in aconitase inactivates the enzyme and increases oxidative stress. The availability of novel superoxide dismutase mimetics has made it possible to prove more thoroughly regarding the nature of ROS causing the inactivation of aconitase in the intracellular milieu.