Environmental Toxicants and Neurodegeneration

描述(申请人提供)：我们的长期目标是研究环境神经毒物引起的神经变性的机制。这项建议是为了研究星形胶质细胞在调节环境神经毒性阳离子水平中的积极作用，从而在调节神经退行性变中发挥作用。根据我们的初步数据，我们假设MPP+(1-甲基-4-苯基吡啶)和百草枯(PQ)等阳离子通过有机阳离子转运蛋白3(Oct3)双向转运穿过星形细胞质膜，并通过这一机制调节神经毒性。因此，Oct3的组织和细胞分布在确定对阳离子神经毒素的不同区域敏感性方面应该是至关重要的。将使用代表两种不同类别的环境神经毒物的阳离子，这些毒物的毒代动力学不同。1-甲基-4-苯基-1，2，3，6-四氢吡啶(MPTP)是一种脂类化合物，将用于在星形胶质细胞内产生MPP+。目的是评估MPP+从星形胶质细胞(v/aOct3)释放到细胞外间隙是如何导致黑质多巴胺能神经元选择性死亡的。PQ是一种广泛使用的阳离子除草剂，与帕金森症有关，将被用来评估星形胶质细胞如何通过(通过Oct3)吸收并从而从细胞外空间移除有毒阳离子来影响神经退行性变。值得注意的是，MPP+和PQ也增加了内源性阳离子多巴胺(DA)的外流，DA在氧化时是神经毒性的。为了检验我们的假设，将使用Oct3缺陷突变小鼠和Oct3抑制剂。在第一个具体目标中，我们将通过细胞培养和动物模型来确定Oct3对MPP+、PQ和DA的摄取和反向运输动力学，以评估Oct3如何调节MPP+、PQ和DA的水平。在第二个具体目标中，我们将评估Oct3如何通过其MPP+和PQ的双向转运来调节神经毒性。我们假设Oct3消融通过隔离星形胶质细胞中的MPP+，减轻MPTP治疗后多巴胺能神经元的死亡。相反，Oct3消融通过阻止MPP+、PQ和DA进入星形胶质细胞，增加MPP+和PQ处理后的多巴胺能神经元的死亡。因此，我们的计划是评估Oct3突变小鼠的多巴胺能神经毒性的程度，以及用MPTP、MPP+或PQ处理的星形胶质细胞和多巴胺能神经元的共培养模型。我们还将评估Oct3在缺乏该转运蛋白的星形胶质细胞中的重新表达是否会逆转神经毒性效应。这项拟议的研究有可能揭开一条仍不为人所知的途径，即大脑中不同类型的细胞相互作用，以调节环境毒物引起的神经退化。此外，这些研究可能为一种新的机制提供重要的见解，这种机制有助于细胞死亡的模式，如散发性帕金森病等神经退行性疾病。

DESCRIPTION (provided by applicant): Our long term goal is to study the mechanism of neurodegeneration induced by environmental neurotoxicants. This proposal is submitted to investigate the active role of astrocytes in regulating the levels of environmental neurotoxic cations and hence, in modulating neurodegeneration. Based on our preliminary data we hypothesize that cations such as MPP+ (1-methyl-4-phenylpyridinium) and paraquat (PQ) are bi- directionally transported across the astrocytic plasma membrane by the organic cation transporter 3 (OCT3) and, through this mechanism, OCT3 modulates neurotoxicity. Thus, the tissue and cellular distribution of OCT3 should be critical in defining the differential regional susceptibility to cationic neurotoxins. Cations representing two different categories of environmental neurotoxicants with different toxicokinetics will be used. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a lipophillic compound that will be used to generate MPP+ inside of astrocytes. The goal is to assess how the release of MPP+ from astrocytes (v/a OCT3) into the extracellular space would subsequently induce selective death in the nigral dopaminergic neurons. PQ, a widely used cationic herbicide that has been linked to parkinsonism, will be used to assess how astrocytes affect neurodegeneration by taking up (via OCT3) and thus removing toxic cations from the extracellular space. Of note, both MPP+ and PQ also increase the outflow of the endogenous cation dopamine (DA), which is neurotoxic upon oxidation To test our hypotheses, mutant mice deficient in OCT3 and an OCT3 inhibitor will be used. In the first specific aim, we will assess how OCT3 regulates the levels of MPP+, PQ and DA by determining its uptake and reverse transport kinetics for these cations using both cell culture and animal models. In the second specific aim, we will evaluate how OCT3 modulates neurotoxicity through its bi-directional transport of MPP+ and PQ. We hypothesize that OCT3 ablation, by sequestrating MPP+ in astrocytes, attenuates dopaminergic neuronal death after MPTP treatment. Conversely, OCT3 ablation, by preventing the uptake of MPP+, PQ, and DA into astrocytes, enhances dopaminergic neuronal death after MPP+ and PQ treatments. Thus, our plan is to assess the magnitude of dopaminergic neurotoxicity in OCT3 mutant mice as well as co-culture models of astrocytes and dopaminergic neurons, treated with MPTP, MPP+ or PQ. We will also assess whether re-expression of OCT3 in astrocytes deficient in this transporter would reverse the neurotoxic effects. The proposed studies have potential to unravel a still unrecognized pathway by which different cell types in the brain interact with each other to modulate neurodegeneration induced by environmental toxicants. In addition, these studies may provide significant insights into a novel mechanism that contributes to the pattern of cell death as seen in neurodegenerative disorders such as sporadic Parkinson's disease.