Mechanisms of PhIP-induced dopaminergic neurotoxicity

 描述(申请人提供)：大多数帕金森病(PD)病例的病因不明，~90%是散发性的，~10%归因于遗传。环境因素，包括杀虫剂和溶剂，长期以来一直被怀疑，但没有令人信服的毒物被确定。大多数病例被认为是由基因-环境相互作用引起的。一个具体的例子是富含亮氨酸的重复蛋白2(LRRK2)，这是一种内源性功能未知的大型多域蛋白。许多LRRK2突变导致帕金森病。然而，人类的不完全外显和表达突变的动物对多巴胺(DA)神经元毒物的高度敏感性表明了基因-环境相互作用的重要性。来自Cannon实验室和其他机构的越来越多的数据表明，杂环芳胺(HAAs)具有神经毒性，与包括帕金森病在内的神经退行性疾病有关。HAAs已被初步研究为实验室动物的致癌物质和潜在的人类致癌物质。在高温烹调肉类和家禽的过程中会形成HAAs。因此，与许多环境毒物相比，通过饮食长期接触HAA可能更为常见，发生的水平也更高。2-Amino-1-methyl-6-phenylimidazo[4，5-b]pyridine(PhIP)是熟肉和禽肉中质量最丰富的一种氨基酸，它通过一种新提出的N-羟基化神经毒性作用机制显示出选择性的多巴胺能神经毒性，N-羟基化是PhIP介导的遗传毒性的代谢物。我们的数据表明，对PhIP神经毒性的机制研究对于了解其在帕金森病中的潜在作用至关重要。这项建议的主要目标是：1)在体内表征PhIP介导的神经毒性；2)确定PhIP暴露是否加强表达突变(G2019S)LRRK2(帕金森病最常见的遗传原因)的动物的病理；3)通过检测N-羟基化是否是关键的致病事件，并通过评估N-氧化PhIP代谢物与主要生物分子广泛形成加合物的倾向，确定PhIP介导的DA选择性神经毒性的关键机制(S)。还将研究特定PD相关蛋白的修饰和加合物的形成。这个项目的成功将带来几个重大进展。1)确定可能的致病因素：PhIP是一种在熟肉中产生的常见毒物，可能比罕见的已知DA毒物消耗的剂量更高；2)利用最常见的PD引起突变和人类广泛接触的化合物创建新的基因-环境相互作用模型；3)新的PD机制：机理研究可能确定可能成为治疗靶点的新的致病途径；4)迅速进行后续流行病学和生物标记物研究。综上所述，利用体内和体外系统，我们将仔细研究PhIP诱导的神经变性的特征，并确定DA神经毒性的机制。如果PhIP暴露被证明在帕金森病中起致病作用，那么可以向公众提供建议，因为改变烹饪方法可以减轻PhIP暴露。

 DESCRIPTION (provided by applicant): The causes of most Parkinson's disease (PD) cases are unknown, ~90% are 'sporadic', ~10% are attributed to inheritance. Environmental factors, including pesticides and solvents, have long been suspected, but no toxicant has been convincingly identified. Most cases are thought to arise from gene -environment interactions. A specific example is leucine-rich repeated kinase 2 (LRRK2), a large multi-domain protein with an unknown endogenous function. Numerous LRRK2 mutations cause PD. However, incomplete penetrance in humans and heightened sensitivity to dopaminergic (DA) neuron toxicants in animals expressing mutations suggest the importance of gene-environment interactions. Mounting data from the Cannon laboratory and others suggests that heterocyclic aromatic amines (HAAs) are neurotoxic and associated with neurodegenerative diseases, including PD. HAAs have been primarily investigated as carcinogens in laboratory animals and as potential human carcinogens. HAAs are formed during high temperature meat and poultry cooking. Thus, chronic HAA exposure through diet may be much more common and occur at higher levels than for many environmental toxicants. This proposal tests the hypothesis that: 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most mass abundant HAA in cooked meats and poultry, exhibits selective dopaminergic neurotoxicity by a newly proposed mechanism of neurotoxic action through N-hydroxylation, a metabolite in common with PhIP's mediated genotoxicity. Our data suggests that mechanistic studies on PhIP neurotoxicity are critical to understanding a potential role in PD. The major goals of this proposal are to: 1) Characterize PhIP-mediated neurotoxicity in vivo; 2) Determine if PhIP exposure potentiates pathology in animals expressing mutated (G2019S) LRRK2 (the most common genetic cause of PD); 3) Identify key mechanism(s) of PhIP-mediated DA-selective neurotoxicity through examination of whether N-hydroxylation is a key pathogenic event, and by assessing the propensity of N-oxidized PhIP metabolites to broadly form adducts with major biomolecules. Modifications and adduct formation in specific PD-implicated proteins will also be examined. Success of this project will lead to several major advances. 1) Identification of a possible causative factor: PhIP is a common toxicant produced in cooked meats and may be consumed in higher doses than rarely encountered known DA toxicants; 2) Creation of a new gene-environment interaction model utilizing the most common PD causing-mutation and a compound to which humans are widely exposed; 3) New PD mechanisms: mechanistic studies would likely identify novel pathogenic pathways that may be therapeutic targets; 4) Prompt follow-up epidemiological and biomarker studies. In summary, using both in vivo and in vitro systems, we will carefully characterize PhIP-induced neurodegeneration and identify mechanisms of DA neurotoxicity. If PhIP exposure is proven to have a causative role in PD, then recommendations can be provided to the public because PhIP exposure can be mitigated by changes in cooking methods.