Mechanisms of Neuronal Death in Parkinson's Disease

描述(由申请人提供)：氧化应激是帕金森氏病(PD)的主要因素。多巴胺(DA)本身很容易被氧化成苯醌衍生物和活性氧物种(ROS)，这会损害能量代谢，并与蛋白质形成加合物，如突触核蛋白。由于动物模型中DA的药理耗竭被非特异性的外周和中枢神经系统效应所混淆，因此DA氧化在黑质细胞死亡中的作用以前一直是不可能解决的。因此，这一领域的一个关键假设是DA氧化是帕金森病患者多巴胺能神经元死亡的主要原因。拟议的研究涉及这一假说的几个方面，包括已知环境因素在触发DA氧化中的相互作用。具体地说，帕金森诱导药物MPP+的DA释放潜力是由于其与DA交换和/或降低细胞内pH梯度的能力，这一假设将通过表达来自多巴胺能位点的增强型绿色荧光蛋白(TH+/EGFP)的新小鼠来解决。来自这些动物的原代培养物以及来自多巴胺能终端区的纯化突触体和囊泡制剂将与荧光和放射性探针结合使用，以确定毒素处理后DA释放、细胞膜变化、ROS形成、ATP丢失等时间方面的变化。此外，DA氧化导致多巴胺能细胞死亡的假设将在活体中直接测试，使用的是通过基因工程产生不同水平DA的动物。行为、氧化和免疫细胞化学标准将用于确定DA在急性和慢性MPTP帕金森病模型中的作用。为了测试DA耗竭是否能预防ROS，新的原位检测ROS的方法将与一系列针对硝基酪氨酸、硝化α-突触核蛋白等的抗体一起使用，以暂时评估在急性或慢性MPTP注射后DA缺乏和野生动物的ROS形成。综上所述，拟议的研究将确定DA氧化是否在DA合成细胞的死亡中发挥核心作用，并提供从标准动物模型中无法获得的见解。了解DA诱导的自由基形成的来源和级联反应将有助于回答围绕使用多巴胺替代疗法的风险和益处的争议，并促进PD发病机制中新药和/或治疗策略的开发。

DESCRIPTION (provided by applicant): Oxidative stress is a major factor in Parkinson's Disease (PD). Dopamine (DA) itself is easily oxidized to quinone derivatives and reactive oxygen species (ROS) that impair energy metabolism and form adducts with proteins such as upsilon-synuclein. Because pharmacological depletion of DA in animal models is confounded by non-specific peripheral and central nervous system effects, the role of DA oxidation in nigral cell death has been previously impossible to address. Thus a key unanswered hypothesis in this field is that DA oxidation is a major contributor to the death of dopaminergic neurons in PD. The proposed studies address several aspects of this hypothesis including the interaction of known environmental factors in triggering DA oxidation. Specifically, the hypothesis that the DA-releasing potential of the parkinsonism-inducing drug, MPP+, is due to its ability to exchange with DA and/or to reduce intracellular pH gradients will be addressed using newly derived mice expressing enhanced green fluorescent protein from a dopaminergic locus (TH+/eGFP). Primary cultures derived from these animals as well purified synaptosomal and vesicular preparations from dopaminergic terminal fields will be used in combination with fluorescent and radioactive probes to determine the temporal aspects of DA release, intracellular membrane changes, ROS formation, ATP loss, etc in response to toxin treatment. In addition, the hypothesis that DA oxidation contributes to the death of dopaminergic cells will be directly tested in vivo using animals genetically engineered to have different levels of DA production. Behavioral, oxidative and immunocytochemical criteria will be used to establish the role of DA in both the acute and chronic MPTP model of PD. To test whether DA depletion prevents ROS, new methodologies to detect in situ ROS will be used with a battery of antibodies directed against nitrotyrosine, nitrated alpha-synuclein, etc. to temporally evaluate ROS formation following acute or chronic MPTP administration in DA deficient and wild type animals. Taken together, the proposed studies will determine whether DA oxidation plays a central role in the death of DA synthesizing cells and provide insights impossible to obtain from standard animal models. Knowledge of the source and cascade of events surrounding DA-induced free radical formation will help answer risk-benefit controversies surrounding the use of dopamine replacement therapies as well as facilitate the development of new drugs and/or treatment strategies in the pathogenesis of PD.