Redox Sensitive Proteins in Aging and Neurodegeneration

描述(申请人提供)：蛋白质氧化增加与正常衰老和与年龄相关的神经退行性疾病有关，如阿尔茨海默病(AD)和帕金森病(PD)。实验证明，蛋白质氧化状态的变化，特别是在巯基上的变化，既与细胞信号事件有关，也与氧化损伤有关。使用一种新的氧化还原2D-PAGE蛋白质组学技术来分析分子间二硫键，以及一种具有更高灵敏度的新方法来检测蛋白质羰化作用，我们将与年龄匹配的对照组相比，在人类和小鼠中枢神经系统(CNS)以及人类和转基因小鼠的AD脑中识别其氧化状态在正常老化过程中发生变化的蛋白质。为了确定这些氧化修饰的蛋白质中哪些参与神经细胞死亡，哪些参与其他信号通路，我们将使用小鼠海马细胞系，其中细胞可以通过产生内源性活性氧簇(ROS)而被杀死，或者在长期、高水平的内源性ROS存在的情况下保持存活状态。重要的是，我们还将研究氧化应激对HT22细胞以及神经和神经胶质细胞原代培养中细胞外(分泌)蛋白氧化的影响。在这些培养细胞中被氧化修饰的蛋白质将与那些在正常老化和AD中被修饰的蛋白质进行比较。这些实验将使我们能够确定氧化修饰蛋白质的哪一个子集参与了导致神经细胞死亡的细胞事件。反过来，这些蛋白质将被用作识别潜在的治疗性抗氧化剂的靶点，这些抗氧化剂可能有助于防止与年龄和疾病相关的中枢神经系统损伤。

DESCRIPTION (provided by applicant): Increases in protein oxidation are associated with both normal aging and with age-associated neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Changes in the oxidative state of proteins, particularly at sulfhydryl groups, have been experimentally linked to both cell signaling events and to oxidative damage. Using a novel Redox 2D-PAGE proteomics technique to assay intermolecular disulfide bonds, as well as a new method with improved sensitivity to examine protein carbonylation, we will identify proteins whose oxidation state changes during both normal aging in the human and mouse central nervous system (CNS) and in AD brains from humans and transgenic mice as compared with age-matched controls. To determine which of these oxidatively modified proteins are involved in nerve cell death and which are involved in other signaling pathways, we will use a mouse hippocampal cell line wherein the cells can be killed by the generation of endogenous reactive oxygen species (ROS) or maintained in a viable state in the presence of prolonged, high levels of endogenous ROS. Importantly, we will also examine the effects of oxidative stress on the oxidation of extracellular (secreted) proteins in HT22 cells and in primary cultures of nerve and glia. The proteins that are oxidatively modified in these cultured cells will be compared to those that are modified during normal aging and in AD. These experiments will allow us to determine which subset of oxidatively modified proteins is involved in the cellular events that lead to nerve cell death. These proteins, in turn, will be used as targets for the identification of potentially therapeutic antioxidants that could be useful in preventing age-and disease-associated damage to the CNS.