Mitochondrial Proteins in Parkinson's Disease

描述(申请人提供)：越来越多的证据表明线粒体功能障碍与帕金森病(PD)的发病机制有关：平均而言，PD患者在Complex I活性方面存在适度的系统性缺陷；一个致病基因编码线粒体激酶(Pinki)；另外两个致病基因编码蛋白质(parkin和DJ-I)，这些蛋白可进出线粒体；系统性抑制线粒体的功能可准确再现PD的许多特征。 该项目汇集了4名知名研究人员-Tim Greenamre、Jun Chen、Valerian Kagan和Teresa Hastings-他们各自对PD的发病机制和线粒体在这种疾病中所起的作用感兴趣。此外，该项目的神经病理核心主任朱夏莲也对线粒体和帕金森病有兴趣和记录。 Greenamre(项目1)将研究铁是如何通过转铁蛋白和先前未知的线粒体转铁蛋白受体(TfR2)介导的新途径在帕金森病中积累的，TfR2选择性地定位于黑质多巴胺能神经元。 Kagan(项目2)正在研究α-突触核蛋白通过与线粒体阴离子磷脂-心磷脂结合而与细胞色素c相互作用的机制和结果。这种由α-核蛋白-心磷脂-细胞色素c组成的复合体可能通过一种新的过氧化物酶活性来阻止凋亡体的形成，同时也促进了氧化应激。 陈(项目3)将研究HSP27易位到线粒体的机制和相关性，以及帕金森病中Aski/JNK的凋亡途径。 黑斯廷斯(项目4)将研究线粒体硒蛋白，如谷胱甘肽过氧化物酶4和硫氧还蛋白还原酶2，在帕金森病神经退化中的作用。 这些单独的项目由两个科学核心支持。分子核心(曹国栋)将协助每个项目设计和生产用于基因过度表达或基因沉默的构建体，产生瞬时和稳定的转基因，并生产用于体内基因转移的病毒载体。由于致病机制是在项目1-4的模型系统中定义的，因此将与神经病理学核心(Charleen Chu)合作，在死后人体组织中确认它们的相关性。 通过(I)项目之间的大量科学互动；(Ii)使用一套通用的体外和活体模型系统；以及(Iii)每个项目将使用的科学核心，统一和加强了整个计划。 公共卫生相关性：作为第二种最常见的神经退行性疾病，帕金森病在美国影响着150万人。帕金森病的神经退行性变的原因尚不确定，但线粒体损伤已被强烈地联系在一起。该计划将确定线粒体蛋白在导致神经退化中的作用，每个项目都将测试旨在减缓或阻止疾病进程的新的治疗概念。 项目1 主要调查者：J·蒂莫西·格林阿梅尔 标题：帕金森病铁蓄积的机制和后果 描述(申请人提供)：我们的实验室已经证明，对含有鱼藤酮的线粒体复合体I的系统抑制在大鼠和猴子身上复制了帕金森病的许多特征，包括多巴胺能变性、路易小体和轴突(α-突触核蛋白)的病理、DJ-I的修饰和移位到线粒体以及泛素-蛋白酶体系统的损害。最近，我们发现在鱼藤酮治疗的大鼠和猴子的黑质中有铁沉积，这似乎与帕金森病脑中看到的相同。在这个模型中，转铁蛋白(Tf)被氧化(在Cys26o)，并在黑质多巴胺能神经元中积聚。在与神经病理学核心主任朱夏莲的合作下，Greenamre发现在帕金森氏症患者的尸检标本中，黑质神经元中存在类似的转铁蛋白积累，这是以前从未描述过的。此外，他还发现转铁蛋白受体2(TfR2)：(I)具有线粒体靶向序列；(Ii)部分定位于线粒体；以及(Iii)蛋白质在大鼠黑质多巴胺神经元中选择性表达。 目的1：我们将使用活体模型和器官型中脑片培养来确定鱼藤酮后积累铁的细胞类型的序列，并最终在Braak分期的人死后标本中评估这一点。 目的2：我们将研究(1)鱼藤酮脑中Tf和TfR2的氧化状态和分布，(Ii)Tf氧化的功能后果，以及(Iii)这些蛋白在人体标本中的分布和氧化状态。 目的：我们将通过在基础条件下和鱼藤酮处理下过度表达或沉默TfR2基因来评估TfR2的功能作用。有待测量的结果包括：铁稳态、氧化应激、α-突触核蛋白/细胞色素c相互作用、细胞死亡的激活和保护机制以及细胞活力。 目的4：最后，我们将通过病毒介导的基因转移来操纵调节或螯合铁的蛋白质。我们将观察操纵铁蛋白和Frataxin对鱼藤酮诱导的毒性的影响。 我们相信该项目将阐明(I)铁在帕金森病中蓄积的机制以及(Ii)其在帕金森病发病机制中的作用。

DESCRIPTION (provided by applicant): Increasing evidence implicates mitochondrial dysfunction in the pathogenesis of Parkinson's disease (PD): on average, PD patients have a modest, systemic defect in complex I activity; one causative gene encodes a mitochondrial kinase (PINKi); two other causative genes encode proteins (parkin & DJ-i) that traffic in and out of mitochondria; and systemic inhibition of mitochondria] function accurately reproduces many features of PD. This program brings together 4 established investigators - Tim Greenamyre, Jun Chen, Valerian Kagan and Teresa Hastings - who are each individually interested in the pathogenesis of PD and the roles that mitochondria play in this disorder. Moreover, the director of this program's neuropathology core, Charleen Chu, also has an interest and track record in mitochondria and PD. Greenamyre (Project 1) will study how iron accumulates in PD via a novel pathway mediated by transferrin and a previously unrecognized mitochondrial transferrin receptor (TfR2) that is selectively localized in substantia nigra dopaminergic neurons. Kagan (Project 2) is studying mechanisms and consequences of the interactions of alpha-synuclein with cytochrome c via binding to the mitochondrial anionic phospholipid, cardiolipin. This complex of alphasynuclein-cardiolipin-cytochrome c may prevent apoptosome formation while also promoting oxidative stress via a novel peroxidase activity. Chen (Project 3) will study mechanisms and relevance of HSP27 translocation to mitochondria and the ASKi/JNK apoptotic pathway in PD. Hastings (Project 4) will study the roles of mitochondrial selenoproteins, such as glutathione peroxidase 4 and thioredoxin reductase 2, in neurodegeneration in PD. The individual projects are supported by 2 scientific cores. The Molecular Core (Guodong Cao) will assist each project with design and production of constructs for gene overexpression or gene silencing, generation of transient and stable transfections, and production of viral vectors for in vivo gene transfer. As pathogenic mechanisms are defined in model systems in Projects 1-4, their relevance will be confirmed in postmortem human tissue in collaboration with the Neuropathology Core (Charleen Chu). The overall Program is unified and strengthened by (i) numerous scientific interactions between the projects; (ii) the use of a common set of in vitro and in vivo model systems; and (iii) the scientific cores, which will be used by each project. PUBLIC HEALTH RELEVANCE: As the second most common neurodegenerative disorder, PD affects 1.5 million individuals in the United States. The cause of neurodegeneration in PD is uncertain, but mitochondrial impairment has been strongly implicated. This Program will determine the roles of mitochondrial proteins in causing neurodegeneration, and each project will test novel therapeutic concepts designed to slow or stop the disease process. PROJECT 1 Principal Investigator: J. Timothy Greenamyre Title: Mechanisms & Consequences of Iron Accumulation in PD Description (provided by applicant): Our laboratory has shown that systemic inhibition of mitochondrial complex I with rotenone reproduces in rats and monkeys many features of PD, including dopaminergic degeneration, Lewy body and neurite (alpha-synuclein) pathology, DJ-i modification and translocation to mitochondria, and impairment of the ubiquitin-proteasome system. Most recently, we have found that there is iron deposition in substantia nigra of rotenone treated rats and monkeys, which appears identical to that seen in PD brains. In this model, transferrin (Tf) becomes oxidized (at Cys26o) and accumulates in nigral dopaminergic neurons. In collaboration with Charleen Chu, director of the Neuropathology Core, Greenamyre has found a similar - previously undescribed - accumulation of transferrin in nigral neurons in human postmortem specimens from PD cases. Additionally, he has found that transferrin receptor 2 (TfR2): (i) has a mitochondrial targeting sequence; (ii) is localized, in part, in mitochondria; and (iii) protein is selectively expressed in nigral dopamine neurons in rats. Aim 1: We will determine the sequence of cell types that accumulate iron after rotenone using the in vivo model and organotypic midbrain slice cultures, and we vAW ultimately assess this in human postmortem specimens across Braak staging. Aim 2: We will characterize (i) the oxidation state and distributions of Tf and TfR2 in the rotenone brain, (ii) the functional consequences of Tf oxidation, and (iii) ultimately, the distributions and oxidation states of these proteins in human specimens. Aim 3: We will assess the functional role of TfR2 by overexpressing or silencing the TfR2 gene under basal conditions and with rotenone treatment. Outcomes to be measured include: aspects of iron homeostasis, oxidative stress, a-synuclein/cytochrome c interactions, activation of cell death and protective mechanisms and cell viability. Aim 4: Lastly, we will manipulate - via viral-mediated gene transfer - proteins that regulate or chelate iron. We will look at the effects of manipulating ferritin and frataxin on rotenone-induced toxicity. We believe this project will elucidate (i) the mechanisms by which iron accumulates in PD and (ii) its role in PD pathogenesis.