Role of Aβ-receptor for advanced glycation endproducts interaction in oxidative stress and cytosolic phospholipase A₂ activation in astrocytes and cerebral endothelial cells.

Blood-brain barrier (BBB) dysfunctions have been implicated in the progression of Alzheimer's disease. Cerebral endothelial cells (CECs) and astrocytes are the main cell components of the BBB. Although amyloid-β oligomers (Aβ₄₂) have been reported to mediate oxidative damage to the CECs and astrocytes and trigger the downstream mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway, the cell surface binding site for Aβ₄₂ and exact sequence of these events have yet to be elucidated. In this study, the receptor for advanced glycation endproducts (RAGE) was postulated to function as a signal transducing cell surface receptor for Aβ₄₂ to induce reactive oxygen species (ROS) generation from NADPH oxidase and trigger downstream pathways for the phosphorylation of extracellular signal-regulated kinases (ERK1/2) and cytosolic phospholipase A₂ (cPLA₂). We found that Aβ₄₂ competed with the anti-RAGE antibody (Ab(RAGE)) to bind to RAGE on the surfaces of CECs and primary astrocytes. In addition, Ab(RAGE) abrogate Aβ₄₂-induced ROS production and the colocalization between the cytosolic (p47-phox) and membrane (gp91-phox) subunits of NADPH oxidase in both cell types. Ab(RAGE) as well as NADPH oxidase inhibitor and ROS scavenger suppressed Aβ₄₂-induced ERK1/2 and cPLA₂ phosphorylation in CECs. At the same time, only Ab(RAGE), but neither NADPH oxidase inhibitor nor ROS scavenger, inhibited the ERK1/2 pathway and cPLA₂ phosphorylation in primary astrocytes. Therefore, this study demonstrates that NADPH oxidase complex assembly and ROS production are not required for Aβ₄₂ binding to RAGE at astrocytic surface leading to sequential phosphorylation of ERK1/2 and cPLA₂, and suggests the presence of two different RAGE-dependent downstream pathways in the CECs and astrocytes.