Mechanisms of PhIP-induced dopaminergic neurotoxicity

多巴胺（DA）能神经变性是帕金森病（PD）的病理学标志， 主要的运动功能文献中的主要空白仍然是关于常见的饮食暴露是否以及如何可能 有助于发病机制。该提案旨在通过对以下方面的高度机械性研究来解决这些差距： 来自被称为杂环芳香胺（哈斯）的饮食毒物的神经毒性。第一周期 R 01 ES 025750中，我们取得了重大进展，证明哈斯可产生选择性DA能神经毒性， 细胞、线虫和啮齿动物模型系统。我们还鉴定了HAA诱导的氧化损伤、蛋白质 聚集、自噬破坏和DNA加合物形成是关键的生化和分子结果 对帕金森病至关重要在这个数据集中，我们已经取得了总体的机械进步， 为注重行动机制的更新奠定基础。首先，神经黑色素（NM）对HAA至关重要 细胞内蓄积和神经毒性。这一发现指向选择性，因为NM是在 人的儿茶酚胺能神经元，以及NM细胞和动物模型中的关键翻译需求。 哈斯研究（大多数PD模型中缺乏NM）。第二，哈斯选择性地靶向线粒体， 指出可能的选择性，因为DA能神经元对线粒体毒性特别敏感。基于 根据这些数据和文献，我们将检验以下机制假设： 神经毒性是通过NM和线粒体功能障碍之间的生物化学相互作用介导的， 产生神经毒性级联反应我们将通过三个目标来检验这一假设。在目标1中，我们将确定 NM形成大鼠表现出较高的HAA诱导DA能神经毒性。在新的NM形成大鼠中，我们将 评估HAA蓄积、HAA脑代谢和神经毒性，以确定PD相关性。在目标2中， 将确定介导HAA诱导的神经毒性的线粒体靶点。我们会发现 通过定量在线粒体DNA加合物中形成的加合物，研究哈斯的线粒体DNA加合物介导的神经毒性 线粒体和基因组DNA的对比此外，我们将确定HAA生物活化途径，导致 线粒体和基因组DNA加合物形成。最后，我们将确定特定的线粒体基因， 由DNA损伤引起的蛋白质损伤。在目标3中，我们将展示NM， 线粒体功能障碍和蛋白质聚集。使用无细胞，细胞和动物模型系统，我们将 确定NM对HAA介导的线粒体功能、自噬（特别是 线粒体自噬），以及使用生物化学和组织学方法的PD相关蛋白聚集的传播 技术.总体而言，NM、线粒体自噬/自噬和蛋白质聚集之间的相互作用的阐明， 对HAA神经毒性作用机制至关重要，预计将大大促进对HAA的理解， 诱导的神经毒性和更广泛的环境诱导的DA能神经毒性。这些研究 预期将显著推进对PD发病机制的理解。

Dopamine (DA)-ergic neurodegeneration is a pathological hallmark of Parkinson’s disease (PD) that produces the cardinal motor features. Major gaps in the literature remain on if and how common dietary exposures may contribute to pathogenesis. This proposal aims to address these gaps through highly mechanistic studies of neurotoxicity from dietary toxicants known as heterocyclic aromatic amines (HAAs). In the first cycle of R01ES025750, we made major advances demonstrating that HAAs produce selective DAergic neurotoxicity in cellular, nematode, and rodent model systems. We also identified HAA-induced oxidative damage, protein aggregation, autophagy disruption, and DNA adduct formation as key biochemical and molecular outcomes that are of critical importance to PD. Within this dataset, we have made overarching mechanistic advances that set the stage for a mechanism-of-action-focused renewal. First, neuromelanin (NM) is critical to HAA intracellular accumulation and neurotoxicity. This finding points to selectivity because NM is formed in catecholaminergic neurons in humans, and a critical translational need for NM cell and animal models in the study of HAAs (NM is lacking in most PD models). Second, HAAs selectively target mitochondria, again pointing to possible selectivity because DAergic neurons are especially sensitive to mitochondrial toxicity. Based on these data and the literature, we will test the following mechanistic hypothesis: HAA-induced DAergic neurotoxicity is mediated through biochemical interactions between NM and mitochondrial dysfunction that produce a neurotoxic cascade. We will test this hypothesis through three aims. In Aim 1, we will determine if NM-forming rats exhibit heightened HAA-induced DAergic neurotoxicity. In novel, NM-forming rats, we will assess HAA accumulation, HAA brain metabolism, and neurotoxicity to establish PD relevance. In Aim 2, we will identify mitochondrial targets that mediate HAA-induced neurotoxicity. We will discover the role of mitochondrial DNA adducts of HAAs in mediating neurotoxicity by quantifying adducts formed in mitochondrial versus genomic DNA. Further, we will identify HAA bioactivation pathways that lead to mitochondrial and genomic DNA adduct formation. Finally, we will identify specific mitochondrial gene and protein impairments resulting from DNA damage. In Aim 3, we will demonstrate connections between NM, mitochondrial dysfunction, and protein aggregation. Using cell-free, cellular and animal model systems, we will determine the effects of NM on HAA-mediated perturbations of mitochondrial function, autophagy (especially mitophagy), and the propagation of PD-relevant protein aggregation using biochemical and histological techniques. Overall, elucidation of interactions between NM, mitophagy/autophagy, and protein aggregation as critical to HAA neurotoxic mechanism of action is expected to significantly advance understanding of HAA- induced neurotoxicity and, more broadly, environmentally induced DAergic neurotoxicity. These studies are expected to significantly advance understanding of PD etiopathogenesis.