PROJECT 1: EMORY PARKINSON'S DISEASE COLLABORATIVE ENVIRONMENTAL RESEARCH CENTER

正如在初步数据中详细说明的那样，我们已经证明，通过 VMAT 2表达的减少导致多巴胺末梢和细胞体的进行性损失， 黑质、突触核蛋白聚集和L-DOPA反应性行为缺陷，使得VMAT 2 缺陷小鼠是PD的独特进行性模型。我们已经证明，几种环境毒物会改变 VMAT 2功能。我们推测，多巴胺储存能力的降低导致了脑组织中的氧化应激。 突触前多巴胺神经元，对细胞成分的损伤和神经元的死亡。因此 多巴胺储存不当后发生的氧化性损伤可能是疾病过程的基础。 我们最近表明，氧化还原状态（还原和氧化组分之间的动态平衡） 可以通过亚细胞区室在空间上分辨神经元，因为动物模型中使用的化合物 帕金森氏病可以优先氧化细胞质，线粒体！，或核氧化还原组分， 如硫氧还蛋白（Trx）或谷胱甘肽（GSH）。因此，多巴胺改变对氧化还原状态的扰动 体内平衡可能是毒性的关键介质。鉴于VMAT 2、α-突触核蛋白和氧化还原的关键作用， 陈述多巴胺系统，本项目的目标是确定这三者之间的相互作用 为了更好地了解介导细胞损伤的潜在致病过程， 帕金森氏病假设：多巴胺的区室化改变破坏了细胞的氧化还原平衡。 导致多巴胺神经元Aim 1的脆弱性增强的神经元。确定asynuclein的作用 调节VMAT 2的定位和功能。我们有初步证据表明 VMAT 2和α-突触核蛋白的调节相互作用。在这个目标中，我们将确定这种分子的性质， 和功能相互作用，使用定点诱变，共聚焦显微镜，和囊泡摄取 测定。目标二。确定a-突触核蛋白表达是否改变多巴胺介导的损伤的脆弱性 这是由于VMAT 2表达减少。通过从具有不同分化的动物中获取中脑多巴胺神经元， VMAT 2和α-突触核蛋白的表达，我们将研究增加细胞质多巴胺的作用， α-突触核蛋白对多巴胺介导的细胞死亡的影响。目标3：为了确定减少或增加的影响 VMAT 2的表达对外源性多巴胺或MPTP的易感性的影响。目标4：扩大我们目前的 多巴胺代谢的动力学-动力学模型，包括环境毒物的目标。我们将 扩展我们的模型来解释线粒体发生的氧化应激！功能障碍与 α-synculein对多巴胺系统关键成分的影响。完成上述具体目标 将提高我们对环境和遗传因素如何破坏适当储存的机械理解 多巴胺和有助于帕金森病的发病机制

As detailed in the preliminary data, we have demonstrated that mishandling of dopamine via reduced VMAT2 expression causes a progressive loss of dopamine terminals and cell bodies in the substantia nigra, synuclein aggregation, and L-DOPA responsive behavioral deficits, making the VMAT2 deficient mice a unique progressive model of PD. WE have shown that several environmental toxicants alter VMAT2 function. We hypothesize that the reduced ability to store dopamine causes oxidative stress in the presynaptic dopamine neuron, damage to cellular constituents, and the death of the neuron. Thus, the sxidative damage that occurs following improper storage of dopamine may underlie the disease process. We have recently shown that the redox state (dynamic balance between reduced and oxidized components) of neurons can be spatially resolved by subcellular compartment in that compounds used in animal models of Parkinson's disease can preferentially oxidize cytoplasmic, mitochondria!, or nuclear redox components, such as thioredoxin (Trx) or glutathione (GSH). Thus, the perturbation of redox state by altered dopamine homeostasis may be a key mediator of toxicity. Given the critical role of VMAT2, a-synuclein, and redox state the dopamine system, the goal of this project is to identify the interactions among these three components in order to better understand the underlying pathogenic processes that mediate cellular damage n Parkinson's disease. Hypothesis: Altered compartmentalization of dopamine disrupts the redox balance of the neuron resulting in enhanced vulnerability of the dopamine neuron Aim 1. To determine the role of asynuclein in regulation of the localization and function of VMAT2. We have preliminary evidence of a direct regulatory interaction of VMAT2 and a-synuclein. In this aim we will determine the nature of this molecular and functional interaction using site-directed mutagenesis, confocal microscopy, and vesicular uptake assays. Aim 2. t To determine if a-synuclein expression alters the vulnerability of dopamine-mediated injury due to reduced VMAT2 expression. By harvesting midbrain dopamine neurons from animals with differential expression of VMAT2 and a-synuclein, we will examine the effects of increased cytoplasmic dopamine and a-synuclein on dopamine-mediated cell death. Aim 3. To determine the effects of reduced or increased expression of VMAT2 on vulnerability to exogenous dopamine or MPTP. Aim 4: To expand our current kinetic-dynamic model of dopamine metabolism to encompass targets of environmental toxicants. We will extend our model to account for the oxidative stress that occurs with mitochondria! dysfunction and the impact of a-synculein on key components of the dopamine system. Completion of the above specific aims will improve our mechanistic understanding of how environmental and genetic factors disrupt proper storage of dopamine and contribute to the pathogenesis of Parkinson's disease