Effects of oxidative stress on glial cell membranes

描述(申请人提供)：本提案的目的是研究膜因子在淀粉样蛋白b肽(A？)诱导的神经胶质细胞氧化应激和炎症反应中的作用。其中膜因子包括膜的局部膜相特性和膜受体的细胞骨架连接。AB诱导的氧化应激和炎症与阿尔茨海默病(AD)有关。事实上，已经发现抗体通过激活NADPH氧化酶来诱导氧化应激。细胞内过量的ROS会引起氧化损伤，包括脂质过氧化、RNA、DNA和蛋白质的氧化，从而扰乱正常的细胞过程，包括细胞内信号转导和细胞骨架组织。在这方面，我们以前已经报道，氧化应激通过激活p38Mark和胞浆磷脂酶A2(CPLA2)使星形胶质细胞膜变得更像凝胶一样。我们的初步结果还表明，AB42寡聚体可以诱导cPLA2的激活。由于已经报道NADPH氧化酶的激活效率依赖于其局部的膜环境，我们的初步研究表明，NADPH氧化酶的膜亚单位gp91ph0*主要定位于星形胶质细胞的高GP结构域(即更多的凝胶样膜)，这些发现导致我们假设Af！*2通过激活NADPH氧化酶和MAPK途径来诱导cPLA2的激活，使胶质细胞膜变得更像凝胶，这反过来又成为一个正反馈，进一步放大NADPH氧化酶的激活以产生ROS。其他膜因素，如膜受体的细胞骨架连接，也可以是控制细胞功能的基本因素。据报道，AB42与膜受体CD36、ctePI、CD47和清道夫受体A类结合，在小胶质细胞中引起炎症反应，通过抗体阻断AB42与其中一种受体的结合可以抑制这种炎症反应。这些发现使我们假设，AB诱导的小胶质细胞炎症反应需要这些膜受体之间的协同作用，这种协同作用是通过这些膜受体的细胞骨架连接来建立的。由于阿尔茨海默病与氧化应激和炎症反应密切相关，因此研究膜因子在抗体诱导的神经胶质细胞氧化应激和炎症反应中的作用机制，对于加深对阿尔茨海默病发病机制的认识具有重要意义。新的生物物理技术，包括Laurdan的荧光成像变形(FIMD)和荧光显微镜，以及各种生化技术将被应用来完成这一拟议的项目。

DESCRIPTION (provided by applicant): The objective of this proposal is to investigate the roles of membrane factors in amyloid-b peptide (A¿)- induced oxidative stress and inflammatory responses in glial cells. Here membrane factors include local membrane phase properties and cytoskeletal linkage of membrane receptors. Ab-induced oxidative stress and inflammation are implicated in Alzheimer's disease (AD). In fact, Ab has been found to induce oxidative stress through activation of NADPH oxidase. Excess reactive oxygen species (ROS) in cells, in turn, cause oxidative damage including lipid peroxidation, oxidation of RNA, DNA, and proteins, which subsequently perturb normal cellular processes, including intracellular signaling and cytoskeleton organization. In this regard, we have previously reported that oxidative stress causes astrocyte membrane to become more gel- like through activations of p38 MARK and of cytosolic phospholipase A2 (cPLA2). Our preliminary results also show Ab42 oligomers induce activation of cPLA2. Since it has been reported that the efficiency of NADPH oxidase activation is dependent of its local membrane environment and our preliminary studies show that the membrane subunit of NADPH oxidase, gp91ph0*, is predominately localized at the high GP domains (i.e. more gel-like membranes) in astrocytes, these findings lead us to hypothesize that Af!*2 induces cPLA2 activation through activations of NADPH oxidase and MAPK pathways to cause glial membranes to become more gel-like, which, in turn, becomes a positive feedback to further amplify the activation of NADPH oxidase to produce ROS. Other membrane factors, such as cytoskeletal linkages of membrane receptors, can also be a fundamental element governing cell functions. It has been reported that Ab42 binds to membrane receptors, CD36, ctePi, CD47 and scavenger receptor class A, resulting in inflammatory responses in microglial cells, which can be suppressed by blocking the binding of Ab42 to one of these receptors using their antibodies. These findings lead us to hypothesize that cooperativity between these membrane receptors is required for AB-induced inflammatory responses in microglial cells and this cooperativity is established through the cytoskeletal linkages of these membrane receptors. Since oxidative stress and inflammation are implicated in AD, our study on how membrane factors involved in the mechanisms of Ab-induced oxidative stress and inflammatory responses in glial cells will prove critical to deepen our understandings in the pathogenesis of AD. Novel biophysical techniques including fluorescence imaged deformation (FIMD) and fluorescent microscopy of LAURDAN, and various biochemical techniques will be applied to accomplish this proposed project.