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. ? ?
