Molecular Genetics of Manganese Induced Dopamine Neuron Toxicity

描述（由申请人提供）：锰（Mn2+）的神经毒性与多巴胺（DA）神经元退行性疾病帕金森病（PD）的许多方面相似。PD和Mn2+毒性均以运动缺陷和黑质及其他基底神经节核损伤为特征，多巴胺或其代谢物被认为与该疾病有关。此外，突触前蛋白α -突触核蛋白（α -synuclein）和氧化应激诱导蛋白帕金（parkin）的表达被认为与这两种疾病的发病机制有关，而职业性Mn2+暴露也被认为是PD易感性的诱因。尽管早在150多年前就对这种疾病进行了初步描述，并且在过去的几十年里进行了深入的研究，但其发病机制的起源和涉及Mn2+神经毒性的分子决定因素尚未得到充分阐明。解剖Mn2+诱导的神经毒性的分子成分的一个重要障碍是脊椎动物大脑的高度复杂性和缺乏简单的体内遗传模型来确定和探索细胞死亡的机制。我们开发了一种新的药物遗传学模型，利用遗传易感的线虫秀丽隐杆线虫来解剖和表征DA神经元退化的分子成分(见Nass等人，PNAS, 2002； Nass和Blakely， Ann。启Toxicol。杂志。, 2003)。在分子水平上，秀丽隐杆线虫的神经系统在遗传和功能上与哺乳动物高度保守，所有负责DA生物合成、包装和再摄取的基因都存在于线虫中并发挥作用。我们已经证明，秀丽隐杆线虫DA神经元可以通过将整个动物暴露于诱导帕金森病的神经毒素6-羟多巴胺（6- ohda）而选择性受损（见Nass等人，PNAS, 2002）2。我们最近也表明，短暂暴露于Mn2+会导致蠕虫DA神经元细胞死亡，先前暴露于Mn2+会放大6- ohda诱导的DA神经退行性变，并且RNA敲低二价金属转运蛋白-1（一种假定的Mn转运蛋白），部分保护蠕虫免受Mn毒性。在我们的模型系统中，绿色荧光蛋白（GFP）在DA神经元中的表达将使我们能够简单而有力地检测DA、其代谢物、内源性蛋白和神经毒素在体内Mn2+诱导的DA神经元变性中所起的作用。这些研究还将包括一种新的全基因组筛选，以鉴定Mn2+诱导毒性的介质和抑制因子。

DESCRIPTION (provided by applicant): Manganese (Mn2+) neurotoxicity resembles a number of aspects of the dopamine (DA) neuron degenerating disorder Parkinson's disease (PD). Both PD and Mn2+ toxicity is characterized by motor deficits and damage to substantia nigra and other basal ganglia nuclei, and dopamine or its metabolites are believed to contribute to the disorder. Furthermore, expression of the pre-synaptic protein alpha-synuclein, and the oxidative stress- induced protein parkin have been proposed to contribute to the pathogenesis of both disorders, and occupational exposure to Mn2+ has been invoked to predispose individuals to PD. Despite the initial characterization of the disorder over 150 years ago, and intensive research within the past several decades, the origin of the pathogenesis and the molecular determinants involved in Mn2+ neurotoxicity have yet to be fully elucidated. A significant hindrance in dissecting the molecular components of Mn2+-induced neurotoxicity is the high complexity of the vertebrate brain and lack of facile in vivo genetic models to determine and explore the mechanisms involved in the cell death. We have developed a novel pharmacogenetic model using the genetically tractable nematode C. elegans to dissect and characterize the molecular components involved in DA neuron degeneration (see Nass et al, PNAS, 2002; Nass and Blakely, Ann. Rev. Toxicol. Pharmacol., 2003). At the molecular level, the C. elegans nervous system is highly conserved both genetically and functionally with mammals, and all the genes responsible for DA biosynthesis, packaging, and reuptake are present and functional in the worm. We have shown that the nematode C. elegans DA neurons can be selectively damaged by exposure of whole animals to the parkinsonian-inducing neurotoxin 6- hydroxydopamine (6-OHDA) (see Nass et al, PNAS, 2002)2. We have also recently shown that a brief exposure to Mn2+ causes DA neuron cell death in the worm, that prior exposure to Mn2+ amplifies the 6- OHDA-induced DA neurodegeneration, and that RNA knockdown of the divalent metal transporter-1 (DMT- 1), a putative Mn transporter, partially protects against Mn toxicity in the worm. In our model system, the expression of the green fluorescent protein (GFP) in DA neurons will allow us a facile and powerful test to examine the role that DA, its metabolites, endogenous proteins, and neurotoxins play in Mn2+ - induced degeneration of DA neurons in vivo. These studies will also include a novel genome-wide screen to identify mediators and suppressors of Mn2+-induced toxicity.