Mechanisms of PhIP-induced dopaminergic neurotoxicity

 描述（由申请人提供）：大多数帕金森病（PD）病例的原因尚不清楚，约90%为“散发性”，约10%归因于遗传。环境因素，包括农药和溶剂，长期以来一直被怀疑，但没有令人信服的有毒物质已被确定。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激酶2（LRRK 2），一种具有未知内源性功能的大型多结构域蛋白。许多LRRK 2突变导致PD。然而，人类的不完全突变和表达突变的动物对多巴胺能（DA）神经元毒物的敏感性增加表明基因-环境相互作用的重要性。来自Cannon实验室和其他实验室的大量数据表明，杂环芳香胺（哈斯）具有神经毒性，并与神经退行性疾病（包括PD）相关。哈斯主要作为实验室动物的致癌物和潜在的人类致癌物进行研究。哈斯是在高温肉类和家禽烹饪过程中形成的。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，而且接触水平更高。本提案检验了以下假设：2-氨基-1-甲基-6-苯基咪唑[4，5-B]吡啶（PhIP）是熟肉和家禽中含量最高的HAA，通过新提出的N-羟基化神经毒性作用机制（与PhIP介导的遗传毒性相同的代谢物）显示出选择性多巴胺能神经毒性。我们的数据表明，PhIP神经毒性的机制研究对于理解PD的潜在作用至关重要。本提案的主要目标是：1）表征PhIP介导的体内神经毒性; 2）确定PhIP暴露是否会增强表达突变（G2019 S）LRRK 2的动物的病理学（PD最常见的遗传原因）; 3）通过检查N-羟基化是否是关键致病事件来鉴定PhIP介导的DA选择性神经毒性的关键机制，以及通过评估N-氧化PhIP代谢物与主要生物分子广泛形成加合物的倾向。还将检查特定PD相关蛋白质的修饰和加合物形成。该项目的成功将带来几项重大进展。1)鉴定可能的致病因素：PhIP是熟肉中产生的常见毒物，可能比罕见的已知DA毒物消耗更高的剂量; 2）利用最常见的PD致病突变和人类广泛暴露的化合物创建新的基因-环境相互作用模型; 3）新的PD机制：机制研究可能会发现新的致病途径，可能是治疗靶点; 4）及时随访流行病学和生物标志物研究。总之，使用体内和体外系统，我们将仔细表征PhIP诱导的神经变性和确定DA神经毒性的机制。如果PhIP暴露被证明在PD中具有致病作用，则可以向公众提供建议，因为PhIP暴露可以通过改变烹饪方法来减轻。

 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.