Gluthathione Depletion /Mitochondrial Complex I Dysfunct

谷胱甘肽耗竭是帕金森病黑质(SN)最早可检测到的生化事件。 发生在与疾病相关的线粒体复合体I(CI)活性选择性丧失之前。我们有 大量研究表明，细胞内总谷胱甘肽(GSH+GSSG)水平的下调 多巴胺能细胞系导致线粒体功能下降，与CI的选择性下降有关 活动。急性谷胱甘肽耗竭后CI活性丧失似乎是由于 组成该复合体的蛋白质亚基的可逆亚硝化。长期慢性病的影响 然而，谷胱甘肽耗竭对多巴胺能细胞CI活性的影响尚不清楚，但可能与 另外，不可逆转的氧化事件。我们项目的一个主要目标是评估CI抑制在我们的 多巴胺能细胞模型在不同时间和水平的谷胱甘肽耗竭，以确定极限 功能与功能障碍，包括可逆性与不可逆性抑制的阈值以及 涉及的氧化剂种类和蛋白质靶标。一旦确定了这些目标，我们将评估 在新构建的DOX诱导的抗GSH转基因小鼠模型中存在类似的变化 在体内滴定黑质多巴胺能神经元内谷胱甘肽水平作为分子模型 与帕金森氏症相关的事件。 在我们的细胞模型中，谷胱甘肽的急剧减少也会导致GSSG还原酶的抑制 谷胱甘肽还原酶(Glud)和谷氨酰转肽酶(GGT)的上调 将细胞外的谷胱甘肽分解成底物，底物再转运回细胞内生成谷胱甘肽 综合。前者可能参与谷胱甘肽耗竭对线粒体的有害影响 而后者似乎是一种补偿事件。我们还将探索分子 谷胱甘肽库滴定后这些分子变化的机制包括 所涉及的氧化剂和酶靶标以及这些变化是否/如何改变线粒体功能。我们 将评估转基因GSSG RD和GGT表达对CI活性和线粒体的影响 在我们的多巴胺能细胞模型中发挥作用，看看它们是否能减弱任何有害的线粒体效应。

Glutathione depletion is the earliest detectable biochemical event in the Parkinsonian substantia nigra (SN), occurring prior to selective loss of mitochondrial complex I (CI) activity associated with the disease. We have Dreviously demonstrated that down-regulation of total glutathione (GSH + GSSG) levels in cultured dopaminergic cell lines results in decreased mitochondrial function linked to a selective decrease in CI activity. Loss of CI activity following acute glutathione depletion appears to be due to reversible nitrosylation of protein subunits comprising this complex. The effects of prolonged chronic glutathione depletion on CI activity in dopaminergic cells, however, are unknown but may involve additional, irreversible oxidative events. A major goal of our project is to assess CI inhibition in our dopaminergic cell model at various times and levels of glutathione depletion in order to identify the limits of function vs. dysfunction including the thresholds for reversible vs. irreversible inhibition as well as the oxidant species and protein targets involved. Once such targets have been identified, we will assess the presence of similar alterations in a newly constructed dox-inducible antiGSH transgenic mouse model following titration of glutathione levels within SN dopaminergic neurons in vivo as a model for molecular events associated with Parkinson's disease. Acute reduction in glutathione in our cell model also results in inhibition of the GSSG reducing enzyme glutathione reductase (GluRd) and up-regulation of the enzyme g-glutamyl transpeptidase (GGT) which breaks down extracellular GSH to substrates which are transported back into the cell for glutathione synthesis. The former may contribute to the detrimental effects of glutathione depletion on mitochondrial function while the latter appears to be a compensatory event. We will also explore the molecular mechanisms involved in these molecular alterations following titration of glutathione pools including the oxidants and the enzyme targets involved and if/how these changes modify mitochondrial function. We will assess the effects of both transgenic GSSG Rd and GGT expression on CI activity and mitochondrial function in our dopaminergic cell model to see if they act to attenuate any detrimental mitochondrial effects.