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P4–090: Lovastatin inhibits amyloid–precursor protein (APP) beta–cleavage through reduction of APP level in caveolae–like lipid rafts: Possible involvement of APP endocytosis
Author(s) -
Won Je-Seong,
Singh Avtar K.,
Contreras Miguel A.,
Inderjit Singh
Publication year - 2006
Publication title -
alzheimer's and dementia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 6.713
H-Index - 118
eISSN - 1552-5279
pISSN - 1552-5260
DOI - 10.1016/j.jalz.2006.05.1828
Subject(s) - endocytosis , lovastatin , lipid raft , caveolae , chemistry , endosome , amyloid precursor protein , microbiology and biotechnology , biochemistry , cholesterol , biology , signal transduction , medicine , alzheimer's disease , receptor , disease
with human Swedish APP695 (N2aAPPswe). Objective(s): To evaluate anti-amyloidogenic efficacy of aged garlic extract in N2aAPPswe cells. Methods: Swedish APP695 (N2aAPPswe) cells, N2a cells transfected with wild type human APP695 (N2aAPPwt) and non-transfected N2a cells (N2a) were propagated and maintained in 45% DMEM, 45% Opti-MEM and 10% FBS, containing 200 g/ml G418 and 1% Penn/Strep at 37C. G418 was omitted for N2a. Cells were seeded at a density of 10 cells/well, and cell viability determined with Trypan Blue dye exclusion. 24h prior to AGE treatment, cells were incubated with lipoprotein-free serum, and then treated with different concentrations of AGE (1/2/4/8/16mg/ml) in a serumfree medium for 24h. After treatment, conditioned media were harvested, reacted with protease inhibitor cocktail, centrifuged at 10,000g at 4C for 10 min, supernatant protein-concentration determined (BCA, Pierce). 100 l aliquots containing equal amount of protein were subjected to sandwich ELISA for A 40/42 (Signet), values expressed as pg/100 l. Results: [1] AGE treatment between 1-8mg/ml concentration range showed cell viability above 95%, while at the highest AGE concentration (16mg/ml) cell viability declined to 75%; [2] Both N2aAPPwt and N2aAPPswe cells showed release of A 40/42 peptides in conditioned media with the predominance of A 40, which were decreased after AGE treatment, but more robust reductions were observed in N2aAPPswe cells; [3] AGE treatment produced dose-dependent reduction in the secreted levels of A 40 by 12.9% (1mg), 23.1% (2mg), 40.8% (4mg), and 40.3% (8 and 16 mg), and reduction in the secreted levels of A 42 by 7.5% (1mg), 16.1% (2mg), 21.5% (4mg), 21.5% (8mg), and 20.4% (16 mg) in N2aAPPswe cells. Conclusions: AGE treatment reduced secreted levels of A peptides in a dose-dependent manner peaking at 4mg/ml optimum concentration in N2aAPPswe cells while highest concentration of AGE is cytotoxic. Although preliminary, this is the first report showing amyloidlowering effects of AGE on N2aAPPswe cells.