Regulatory role of cytosolic phospholipase A2alpha in NADPH oxidase activity and in inducible nitric oxide synthase induction by aggregated Abeta1-42 in microglia.
In Alzheimer's disease, extracellular deposits of amyloid beta(1-42) (Abeta(1-42)) may induce activation of microglial cells by releasing proinflammatory factors that contribute to the neurodegeneration process. Since the activation of cytosolic phospholipase A(2)alpha (cPLA(2)alpha) has been reported in inflammatory conditions, its role in primary rat microglial cell activated by aggregated Abeta(1-42) was elucidated. The results of the present study show that activation of microglia by 5 microM aggregated Abeta(1-42) (as evident by the amoeboid morphology and increased CD68 immunofluorescence reactivity) caused an immediate activation of cPLA(2)alpha, measured by its phosphorylated form and its specific activity, followed by a gradual elevation of its expression and activity during 24 h. Inhibition of cPLA(2)alpha expression and activity by the presence of 1 microM specific antisense resulted in a significant decrease in NADPH oxidase activity that releases superoxides, PGE(2) formation, iNOS expression, and NO production, indicating a major role for cPLA(2)alpha in the regulation of these inflammatory processes. NADPH oxidase activity, which is under cPLA(2)alpha regulation, was found to upregulate cPLA(2)alpha and COX-2 protein expression through the redox-sensitive NFkappaB activation as evident by its phosphorylation on Ser-536, resulting in increased PGE(2) formation. The secreted PGE(2) induced the synthesis of iNOS and the production of NO through the PKA-CREB pathway. Taken together, our results suggest that the response of cPLA(2)alpha to aggregated Abeta(1-42) is probably a key player in the oxidative stress present in AD, regulating potent oxidative agents: the production of superoxides by NADPH oxidase and NO formation by iNOS.
Szaingurten-Solodkin
I
I
Infectious Diseases and Immunology Laboratory, Department of Clinical Biochemistry, Faculty of Health Sciences, Soroka University Medical Center and Ben-Gurion University of the Negev, Beer Sheva, Israel.
Hadad
N
N
Levy
R
R
D016229:Amyloid beta-Peptides / Q000378:metabolism*; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D015214:Antigens, Differentiation, Myelomonocytic / Q000378:metabolism; D015153:Blotting, Western; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D015232:Dinoprostone / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D054513:Group IV Phospholipases A2 / Q000378:metabolism*; D047468:Immunoprecipitation; D007249:Inflammation / Q000378:metabolism; D017628:Microglia / Q000378:metabolism*; D018613:Microscopy, Confocal; D019255:NADPH Oxidases / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism*; D020319:Oligodeoxyribonucleotides, Antisense; D018384:Oxidative Stress / Q000502:physiology; D010446:Peptide Fragments / Q000378:metabolism*; D010766:Phosphorylation; D011863:Radioimmunoassay; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors; D000097382:CD68 Molecule
D016229:Amyloid beta-Peptides; D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D015232:Dinoprostone; D054513:Group IV Phospholipases A2; D019255:NADPH Oxidases; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; D020319:Oligodeoxyribonucleotides, Antisense; D010446:Peptide Fragments; C067980:CD68 antigen, human; D000097382:CD68 Molecule; C075222:amyloid beta-protein (1-42)