Intracellular Abeta is increased by okadaic acid exposure in transfected neuronal and non-neuronal cell lines.
Intracellular Abeta was examined in both a neuronal cell line (B103) expressing human APP with Swedish mutation and a non-neuronal cell line (Chinese hamster ovary, CHO) expressing wild human APP. Exposure of the APP695sw-transfected B103 cells to okadaic acid for 3 h, Abeta immunostaining was enhanced, as demonstrated by two independent anti-Abeta antibodies. The confocal microscopic study revealed that the immunoreactivity of Abeta was mainly colocalized with a Golgi marker and partially with an ER marker. Quantitative analyses, using Abeta sandwich ELISA, showed significantly increased intracellular Abeta. False positive detection of Abeta by antibody cross-reaction with APP was ruled out by extracting the fraction with formic acid and making it alkaline before subjecting it to ELISA. This procedure resulted in a fraction that contained little APP. Using CHO cells, OA treatment was also shown to be effective in increasing Abeta, as demonstrated by Western blot. The increased full-length APP and decreased APPC99 were also observed. This is the first study to demonstrate that OA treatment significantly increases intracellular Abeta.
Sun
Xiaoyan
X
Laboratory for Alzheimer's Disease, Brain Science Institute of the RIKEN, 2-1 Hirosawa, Wako, 351-0198, Saitama, Japan.
Cole
Gregory M
GM
Chu
Teresa
T
Xia
Weiming
W
Galasko
Douglas
D
Yamaguchi
Haruyasu
H
Tanemura
Kentaro
K
Frautschy
Sally A
SA
Takashima
Akihiko
A
D016229:Amyloid beta-Peptides / Q000096:biosynthesis* / Q000235:genetics; D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007150:Immunohistochemistry; D007202:Indicators and Reagents; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D019319:Okadaic Acid / Q000494:pharmacology*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D011233:Precipitin Tests; D013268:Stimulation, Chemical; D013778:Tetrazolium Salts; D013844:Thiazoles; D014162:Transfection
D016229:Amyloid beta-Peptides; D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D013778:Tetrazolium Salts; D013844:Thiazoles; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases; C022616:thiazolyl blue