Role of Neuronal NOS & Superoxide in Neurodegeneration

神经元 NOS 的作用

• 批准号: 7201577
• 负责人: BALARAMAN KALYANARAMAN
• 金额: $ 32.89万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-10 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7201577
• 关键词: 1-Methyl-4-phenylpyridinium; Aconitate Hydratase; Address; Affect; Age; Anabolism; Antibodies; Apoptosis; Apoptotic; Binding; Brain; Caspase; Cell Death; Cell Line; Cell model; Cells; Cerebrospinal Fluid; Chelating Agents; Complex; Cytoplasmic Granules; Data; Detection; Development; Dopa; Dopamine; Dopaminergic Cell; Enzymes; Extracellular Domain; Fluorescence; Fluorescent Probes; Funding; Generations; Goals; Grant; Human; Hydrogen Peroxide; Iron; Iron-Sulfur Proteins; Laboratory Animals; Lewy Bodies; Light; Link; Lipid Peroxides; Measures; Mediating; Messenger RNA; Methods; Mitochondria; Mitochondrial Diseases; Modality; Modification; NBL1 gene; NOS1 protein, human; Narcotics; Nerve Degeneration; Neuroblastoma; Neurodegenerative Disorders; Neurons; Neurotoxins; Nitrates; Nitric Oxide; Nitric Oxide Synthase Type I; Nitrites; Nitrogen; Oxidants; Oxidation-Reduction; Oxygen; Parkinson Disease; Parkinsonian Disorders; Pathogenesis; Patients; Peroxonitrite; Play; Population; Process; Protein Overexpression; Proteins; Purpose; Reactive Nitrogen Species; Reactive Oxygen Species; Research Personnel; Role; Signal Transduction; Spin Trapping; Stress; Substantia nigra structure; Superoxides; Symptoms; Techniques; Testing; Toxic effect; Toxin; Transferrin; Transferrin Receptor; Tyrosine 3-Monooxygenase; age related; alpha synuclein; cytotoxicity; dihydropteridine reductase; dopaminergic neuron; enzyme activity; granule cell; holotransferrin; improved; inhibiting antibody; insight; neuron apoptosis; neurotoxicity; nitrate; novel; presynaptic; programs; receptor expression; response; sepiapterin; synuclein; tetrahydrobiopterin; uptake

DESCRIPTION (provided by applicant): Long-term goal: The broad objectives of this renewal are to understand the mechanism(s) by which mitochondrial neurotoxins such as 1-methyl-4-phenylpyridinium (MPP+) selectively destroy dopaminergic neurons in the substantia nigra, leading to the development of Parkinson's disease (PD). Reactive oxygen and nitrogen species (ROS/RNS)-mediated damage has been implicated in age-related neurodegenerative diseases like PD. Hypothesis: (i) MPP+ generates mitochondria superoxide (02*) and hydrogen peroxide (H202), and inactivates mitochondrial iron-sulfur-proteins (e.g., aconitase). This stimulates transferrin receptor (TfR)-mediated uptake of iron. (ii) MPP+-induced H202 and iron transported through TfR cause enhanced degradation of tetrahydrobiopterin (BH4), an essential co-factor for neuronal nitric oxide synthase (nNOS), tyrosine hydroxylase (TH), and dihydropteridine reductase (DHPR) activities. BH, depletion causes "uncoupling" of nNOS to form O2* and inactivation of TH and DHPR leading to dopamine depletion. (iii) MPP+-induced O2*, H2O2, and Tf-iron stimulate aggregation of a-synuclein, a neuronal presynaptic protein leading to apoptosis or programmed cell death. Aims: 1.) Investigate the effect of TfR-dependent iron and mitochondrial ROS in neuronal cell apoptosis in response to MPP+. 2.) Assess the modulatory effect of BH4 depletion on nNOS-generated nitric oxide ('NO)/O2* ratio and on BH4-dependent enzyme controlling dopamine synthesis. 3.) Elucidate the role of ROS, Tf-iron and BH4 depletion on a-synuclein aggregation and apoptosis in neuronal cells treated with MPP+. Methods: We will use both dopaminergic and non-dopaminergic cells (neuroblastoma and cerebellar granule neurons). The following redox-parameters will be measured: GSH and lipid peroxides; aconitase, complex-I, and iron-regulatory activities; TfR expression and 55Fe uptake; a-synuclein expression and aggregation; caspase activation and apoptosis. ROS/RNS will be determined by fluorescence and spin-trapping techniques. Significance: PD affects about 1% of population over the age of 50. Emerging data allude to environmental mitochondrial toxins as a causative factor. Novelty: This proposal sheds new light on the synergistic role for MPP*-induced mitochondrial ROS, iron, BH4-induced nNOS uncoupling, dopamine depletion and alpha-synuclein aggregation in neuronal toxicity of PD and other mitochondrial diseases.

描述（申请人提供）：长期目标：这种更新的广泛目标是了解线粒体神经毒素（如1-甲基-4-苯基吡啶铵（MPP+））的机制，有选择地破坏了paskine nigra的多巴胺能神经元，导致了帕克森病（Parkinson's Diseal）（pds）的发展。活性氧和氮种（ROS/RNS）介导的损伤与年龄相关的神经退行性疾病（如PD）有关。假设：（i）MPP+产生线粒体超氧化物（02*）和过氧化氢（H202），并使线粒体铁硫蛋白（例如刺刺酶）失活。这刺激了铁蛋白受体（TFR）介导的铁的摄取。 （ii）MPP+诱导的H202和铁通过TFR转运的铁导致四氢无生蛋白的降解（BH4），这是神经元氧化物氧化物合酶（NNOS），酪氨酸羟化酶（TH）和二羟基替氏肽（Dihydropteridine reductase（Dhydihydropperidine reductase）（Dhydihydrobioptasise（NNOS）的必不可少的辅助因子）。 BH，耗尽会导致NNO的“解偶联”形成O2*，并使Th和DHPR灭活导致多巴胺消耗。 （iii）MPP+诱导的O2*，H2O2和TF铁刺激A-突触核蛋白的聚集，这是一种神经元突触前蛋白，导致细胞凋亡或程序性细胞死亡。目的：1。）研究响应MPP+的TFR依赖性铁和线粒体ROS对神经元细胞凋亡的影响。 2.）评估BH4耗竭对NNOS生成的一氧化氮（'no）/O2*比率以及控制多巴胺合成的BH4依赖性酶的调节作用。 3.）阐明ROS，TF铁和BH4耗竭在用MPP+处理的神经元细胞中A核蛋白聚集和凋亡中的作用。方法：我们将同时使用多巴胺能和非多巴胺能细胞（神经细胞瘤和小脑颗粒神经元）。将测量以下氧化还原参数：GSH和脂质过氧化物； a刺酶，复合物I和铁调节活性； TFR表达和55FE吸收； A核蛋白的表达和聚集；胱天冬酶激活和凋亡。 ROS/RNS将由荧光和自旋陷阱技术确定。意义：PD在50岁以上影响约1％的人口。新兴数据将环境线粒体毒素称为致病因素。新颖性：该提案为MPP*诱导的线粒体ROS，铁，BH4诱导的NNOS解偶联，多巴胺耗竭和α-链核蛋白聚集在PD和其他线粒体疾病中的神经元毒性中介绍了新的启示。