Molecular Genetics of Manganese Induced Dopamine Neuron Toxicity

描述(申请人提供)：锰(Mn2+)神经毒性类似于多巴胺(DA)神经元退行性疾病帕金森病(PD)的许多方面。Pd和Mn2+毒性的特征是运动障碍和黑质和其他基底节核团的损伤，多巴胺或其代谢产物被认为是导致这种疾病的原因之一。此外，突触前蛋白α-突触核蛋白的表达和氧化应激诱导的蛋白Parkin被认为与这两种疾病的发病机制有关，职业性暴露于Mn2+可能是帕金森病的易感因素。尽管早在150多年前人们就已经对这种疾病有了初步的认识，并在过去几十年中进行了深入的研究，但其发病机制和参与Mn2+神经毒性的分子决定因素仍未完全阐明。脊椎动物大脑的高度复杂性以及缺乏简便的体内遗传模型来确定和探索细胞死亡的机制是解剖Mn2+诱导的神经毒性的分子组成的一个重要障碍。我们已经开发了一种新的药物遗传学模型，使用遗传上易驯化的线虫来解剖和表征参与DA神经元退化的分子成分(参见Nass等人，PNAS，2002；Nass和Blakely，Ann。托西科尔牧师。Pharmacol.，2003)。在分子水平上，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、包装和重摄取的基因在蠕虫中都存在并具有功能。我们已经证明，整个动物暴露于帕金森病诱导的神经毒素6-羟基多巴胺(6-OHDA)可以选择性地损伤线虫DA神经元(见Nass等人，PNAS，2002)2。我们最近还表明，短暂暴露于Mn2+可导致线虫DA神经元死亡，预先暴露于Mn2+可放大6-OHDA诱导的DA神经元变性，而RNA敲除二价金属转运体1(DMT-1)可部分保护线虫免受锰毒性。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方便而有力地检测DA及其代谢物、内源性蛋白质和神经毒素在体内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查，以确定Mn2+诱导毒性的介体和抑制物。

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.