Molecular Genetics of Manganese Induced Dopamine Neuron Toxicity

描述（由申请人提供）：锰（Mn2+）神经毒性在许多方面类似于多巴胺（DA）神经元变性疾病帕金森病（PD）。PD和Mn 2+毒性的特征都是运动缺陷和黑质和其他基底神经节核的损伤，多巴胺或其代谢产物被认为是导致这种疾病的原因。此外，已经提出突触前蛋白α-突触核蛋白和氧化应激诱导的蛋白帕金的表达有助于这两种疾病的发病机制，并且职业暴露于Mn 2+已经被认为使个体易患PD。尽管在150多年前对该疾病进行了初步表征，并且在过去几十年中进行了深入研究，但尚未完全阐明发病机制的起源和涉及Mn 2+神经毒性的分子决定因素。一个显着的障碍，在解剖Mn 2+诱导的神经毒性的分子组成部分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，以确定和探索参与细胞死亡的机制。我们已经开发了一种新的药物遗传学模型，使用遗传易处理的线虫C。elegans来解剖和表征参与DA神经元变性的分子组分（参见Nass等人，PNAS，2002; Nass和Blakely，Ann.Rev.Toxicol.药理学，2003年）。在分子水平上，C.线虫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功能。我们已经证明，线虫C。通过将整个动物暴露于帕金森病诱导性神经毒素6-羟基多巴胺（6-OHDA），可以选择性地损伤线虫DA神经元（参见Nass等，PNAS，2002）。我们最近还表明，短暂暴露于Mn2+导致DA神经元细胞死亡的蠕虫，预先暴露于Mn2+放大6-OHDA诱导的DA神经变性，RNA敲低的二价金属转运蛋白-1（DMT-1），一个假定的锰转运蛋白，部分保护免受锰毒性的蠕虫。在我们的模型系统中，DA神经元中绿色荧光蛋白（GFP）的表达将使我们能够方便和强大的测试，以检查DA，其代谢产物，内源性蛋白质和神经毒素在体内Mn 2+诱导的DA神经元变性中发挥的作用。这些研究还将包括一种新的全基因组筛选，以确定Mn2+诱导毒性的介质和抑制剂。

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.