Redox Sensitive Proteins in Aging and Neurodegeneration

描述（由申请人提供）：蛋白质氧化的增加与正常衰老和年龄相关的神经退行性疾病如阿尔茨海默病（AD）和帕金森病（PD）相关。蛋白质氧化状态的变化，特别是巯基的变化，已经被实验证明与细胞信号传导事件和氧化损伤有关。使用一种新的氧化还原2D-PAGE蛋白质组学技术来测定分子间二硫键，以及一种新的方法，提高了灵敏度，以检查蛋白质羰基化，我们将确定蛋白质的氧化态变化在正常老化过程中在人类和小鼠中枢神经系统（CNS）和AD大脑从人类和转基因小鼠相比，与年龄匹配的控制。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，哪些参与其他信号传导途径，我们将使用小鼠海马细胞系，其中细胞可以通过内源性活性氧（ROS）的产生而被杀死，或者在长时间的高水平内源性ROS存在下维持在存活状态。重要的是，我们还将研究氧化应激对HT 22细胞和神经和神经胶质原代培养物中细胞外（分泌）蛋白氧化的影响。这些培养细胞中被氧化修饰的蛋白质将与正常衰老和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个子集参与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鉴定潜在治疗性抗氧化剂的靶点，这些抗氧化剂可用于预防年龄和疾病相关的CNS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.