Department of Biological Engineering, University of Missouri, Columbia, Missouri 65211, USA.
Neuroscience. 2011 Dec 29;199:375-85. doi: 10.1016/j.neuroscience.2011.09.038. Epub 2011 Sep 28.
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.
血脑屏障（BBB）功能障碍与阿尔茨海默病的进展有关。脑内皮细胞（CEC）和星形胶质细胞是 BBB 的主要细胞成分。尽管已经报道了淀粉样β寡聚体（Aβ₄₂）介导对 CEC 和星形胶质细胞的氧化损伤，并触发下游丝裂原活化蛋白激酶/细胞外信号调节激酶（MAPK/ERK）途径，但 Aβ₄₂的细胞表面结合位点和这些事件的确切序列尚未阐明。在这项研究中，晚期糖基化终产物受体（RAGE）被假定为 Aβ₄₂的信号转导细胞表面受体，以诱导 NADPH 氧化酶产生活性氧（ROS）并触发细胞外信号调节激酶（ERK1/2）和细胞质磷脂酶 A₂（cPLA₂）的磷酸化下游途径。我们发现 Aβ₄₂与抗 RAGE 抗体（Ab（RAGE））竞争，与 CEC 和原代星形胶质细胞表面的 RAGE 结合。此外，Ab（RAGE）消除了 Aβ₄₂诱导的 ROS 产生以及两种细胞类型中 NADPH 氧化酶的细胞质（p47-phox）和膜（gp91-phox）亚基之间的共定位。Ab（RAGE）以及 NADPH 氧化酶抑制剂和 ROS 清除剂抑制了 CEC 中 Aβ₄₂诱导的 ERK1/2 和 cPLA₂磷酸化。同时，只有 Ab（RAGE），而不是 NADPH 氧化酶抑制剂或 ROS 清除剂，抑制了原代星形胶质细胞中 ERK1/2 途径和 cPLA₂磷酸化。因此，这项研究表明，NADPH 氧化酶复合物组装和 ROS 产生对于 Aβ₄₂与星形胶质细胞表面的 RAGE 结合以导致 ERK1/2 和 cPLA₂的顺序磷酸化不是必需的，并表明 CEC 和星形胶质细胞中存在两种不同的 RAGE 依赖性下游途径。
