Effects of oxidative stress on glial cell membranes

描述（由申请人提供）：本提案的目的是研究膜因子在淀粉样蛋白-b肽（A¿）-诱导的胶质细胞氧化应激和炎症反应中的作用。这里的膜因子包括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Ab通过激活NADPH氧化酶来诱导氧化应激。反过来，细胞中过量的活性氧（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，包括细胞内信号传导和细胞骨架组织。在这方面，我们之前报道过氧化应激通过激活p38 MARK和胞质磷脂酶A2 （cPLA2）导致星形胶质细胞膜变得更像凝胶。我们的初步结果还表明，Ab42寡聚物诱导cPLA2活化。由于有报道称NADPH氧化酶的激活效率依赖于其局部膜环境，我们的初步研究表明，NADPH氧化酶的膜亚基gp91ph0*主要定位于星形胶质细胞的高GP结构域（即更多的凝胶状膜），这些发现使我们假设Af！*2通过激活NADPH氧化酶和MAPK途径诱导cPLA2活化，使胶质膜变得更像凝胶，这反过来又成为进一步放大NADPH氧化酶活化产生ROS的正反馈。其他膜因子，如膜受体的细胞骨架连接，也可以是控制细胞功能的基本元素。有报道称，Ab42与膜受体CD36、ctePi、CD47和A类清除率受体结合，在小胶质细胞中引起炎症反应，可通过使用抗体阻断Ab42与这些受体之一的结合来抑制炎症反应。这些发现使我们假设这些膜受体之间的协同作用是小胶质细胞中ab诱导的炎症反应所必需的，这种协同作用是通过这些膜受体的细胞骨架连接建立的。由于氧化应激和炎症与AD有关，我们对膜因子如何参与ab诱导的胶质细胞氧化应激和炎症反应机制的研究将对加深我们对AD发病机制的理解至关重要。新的生物物理技术，包括荧光成像变形（FIMD）和荧光显微镜的LAURDAN，以及各种生化技术将被用于完成这个拟议的项目。

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.