PEROXYNITRITE IN NEURODEGENERATIVE DISEASES OF AGING

描述(摘自申请者摘要)：酪氨酸的活性 羟基酶(TH)是生产儿茶酚胺所必需的。在.期间 帕金森病(PD)进展过程中TH活性和 浓缩性已被描述。多巴胺水平的下降没有任何损失 已经描述了TH免疫反应或多巴胺能神经元的 疾病的早期阶段。疾病的中期是 特点是多巴胺和免疫活性TH的丧失而不是 多巴胺能神经元。多巴胺、TH和多巴胺能神经元的丢失 描述疾病的晚期特征。帕金森病中的这些不同事件是 在1-甲基-4-苯基-1，2，3，6-四氢吡啶中忠实地复制 (MPTP)帕金森病小鼠模型。然而，解释这种变化的生化基础 在TH中，多巴胺能神经元死亡前的活性和含量不是 清楚地明白了。我们发布的数据是在过去两年中生成的 资金为这些变化提供了合理的生化解释 在MPTP神经毒性的早期阶段。数据显示，这是一种 硝化的选择性靶标。酪氨酸残基的硝化代表一种 翻译后的蛋白质修饰，其结果是 含蛋白质的硝化剂。过氧亚硝酸盐等硝化剂 在氧化应激过程中形成。氧化应激已被认为与 帕金森病的发病机制及甲氨蝶呤的神经毒性。公布的数据显示 在注射MPTP后的前6小时内，单个酪氨酸的硝化 这会导致该酶失活。停用 与小鼠纹状体中多巴胺水平的下降平行，而 TH蛋白水平保持不变。然而，在最后一次MPTP后12小时 注射，初步数据表明一种明显的非蛋白水解物， 酶处理修复了硝化TH，这反映在增加 蛋白质的催化活性和大脑中的多巴胺水平。在 与此同时，TH的蛋白质水平已经下降到近50% 控制力。蛋白质的丢失似乎是由泛素蛋白酶体介导的。 路径。基于这些初步数据，我们制定了以下工作 假设：蛋白质硝化(特别是TH)代表一种病理生理学 刺激由两个过程管理；非蛋白水解性修复 独特的脱氮酶和/或蛋白质降解。这项工作的关键方面 假说将通过：1)确定修复和修复的动力学 TH在小鼠MPTP和PC12细胞模型中的降解，2)纯化 和表征大脑的反硝酸酶活性，以及3)研究 TH蛋白降解的分子机制。此应用程序是一个 这是我们之前阐明生物化学的工作的自然延伸 酪氨酸羟化酶早期失活机制的研究 MPTP毒性。拟议中的实验将阐明生化、细胞 和TH在MPTP毒性和帕金森病中期的分子变化 结合我们在蛋白质硝化化学和生物方面的经验 反应物种的化学，用霍维茨博士的PC12细胞模型和Dr。 Przedborski的MPTP小鼠模型。三个不同的国家之间的合作 实验室一直很有成效。了解基本的生物化学和 MPTP和PD进展过程中TH的分子变化将促进 纠正多巴胺功能缺陷的方法研究进展 在PD的生产。

DESCRIPTION (Adapted from the Applicant's Abstract): The activity of Tyrosine Hydroxylase (TH) is essential for the production of catecholamines. During the progression of Parkinson's disease (PD) distinct changes in TH activity and concentration have been described. A decrease in dopamine levels without a loss of either TH immunoreactivity or dopaminergic neurons has been described during the early phase of the disease. The middle stage of the disease is characterized by a loss in dopamine and immunoreactive TH without a loss of dopaminergic neurons. Loss of dopamine, TH and dopaminergic neurons characterize late phase of the disease. These distinct events in PD are faithfully reproduced in the 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) mouse model of PD. However, the biochemical basis to explain the changes in TH, activity and content prior to the death of dopaminergic neurons are not clearly understood. Our published data generated during the last two years of funding has provided a reasonable biochemical explanation for the changes in TH during the early phase of MPTP neurotoxicity. The data revealed that TH is a selective target for nitration. Nitration of tyrosine residues represents a post-translational protein modification that results from the reaction of nitrating agents with proteins. Nitrating agents such as peroxynitrite are formed during oxidative stress. Oxidative stress has been implicated in the pathogenesis of PD and in the MTPT neurotoxicity. The published data showed that for the first 6 hours post MPTP injection, nitration of a single tyrosine in TH results in the inactivation of the enzyme. The inactivation of TH paralleled the decline in dopamine levels in the mouse striatum whereas the levels of TH protein remain unchanged. However, 12 hours after the last MPTP injection, preliminary data indicated that an apparent non proteolytic, enzymatic process has repaired nitrated TH and this is reflected by an increase in the catalytic activity of the protein and in brain dopamine levels. At the same time the protein levels of TH have declined to nearly 50 percent of control. The loss of protein appears to be mediated by the ubiquitin-proteosome pathway. Based on these preliminary data we formulated the following working hypothesis: Protein nitration (specifically TH) represents a pathophysiology stimulus that is managed by two processes; non-proteolytic repair involving a unique denitrase, and/or protein degradation. Critical aspects of this working hypothesis will be examined by: 1) determining the kinetics of repair and degradation of TH in the mouse MPTP and in the PC12 cell models, 2) purifying and characterizing the brain denitrase activity, and 3) investigating the molecular mechanisms for the proteolytic degration of TH. This application is a natural extension of our previous work that elucidated the biochemical mechanism for the inactivation of tyrosine hydroxylase during the early stages of MPTP toxicity. The proposed experiments will elucidate biochemical, cellular and molecular changes in TH during the middle stages of MPTP toxicity and PD by integrating our experiences with protein nitration chemistry and biological chemistry of reactive species, with Dr. Horwitz's PC12 cell model and Dr. Przedborski's MPTP mouse model. The collaboration between the three different laboratories has been productive. Understanding the basic biochemical and molecular changes in TH during the progression of MPTP and PD will facilitate the development of approaches to correct the functional deficit in dopamine production in PD.