Role of Aß-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-ß oligomers (Aß42) 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 Aß42 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 Aß42 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 Aß42 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 Aß42-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 Aß42-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 Aß42 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.

Low energy laser light (632.8 nm) suppresses amyloid-ß peptide-induced oxidative and inflammatory responses in astrocytes.

Oxidative stress and inflammation are important processes in the progression of Alzheimer's disease (AD). Recent studies have implicated the role of amyloid ß-peptides (Aß) in mediating these processes. In astrocytes, oligomeric Aß induces the assembly of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complexes resulting in its activation to produce anionic superoxide. Aß also promotes production of pro-inflammatory factors in astrocytes. Since low energy laser has previously been reported to attenuate oxidative stress and inflammation in biological systems, the objective of this study was to examine whether this type of laser light was able to abrogate the oxidative and inflammatory responses induced by Aß. Primary rat astrocytes were exposed to Helium-Neon laser (λ=632.8 nm), followed by the treatment with oligomeric Aß. Primary rat astrocytes were used to measure Aß-induced production of superoxide anions using fluorescence microscopy of dihydroethidium (DHE), assembly of NADPH oxidase subunits by the colocalization between the cytosolic p47(phox) subunit and the membrane gp91(phox) subunit using fluorescent confocal microscopy, phosphorylation of cytosolic phospholipase A(2) cPLA(2) and expressions of pro-inflammatory factors including interleukin-1ß (IL-1ß) and inducible nitric-oxide synthase (iNOS) using Western blot Analysis. Our data showed that laser light at 632.8 nm suppressed Aß-induced superoxide production, colocalization between NADPH oxidase gp91(phox) and p47(phox) subunits, phosphorylation of cPLA(2,) and the expressions of IL-1ß and iNOS in primary astrocytes. We demonstrated for the first time that 632.8 nm laser was capable of suppressing cellular pathways of oxidative stress and inflammatory responses critical in the pathogenesis in AD. This study should prove to provide the groundwork for further investigations for the potential use of laser therapy as a treatment for AD.