Fig 1.
Time-dependent decreases in beclin-1 (A), Atg5 (B), and SIRT1 (C) protein expression in N2a cells exposed to exogenous Aβ1–42 (10μ μM).
Means ± SDs are expressed as percentages of zero time values (N = 4). **P < 0.01, ***P < 0.001 vs. Zero time.
Fig 2.
Increases in the expressions of beclin-1 (A), Atg5 (B), and SIRT1 protein (C) by cilostazol (CSZ, 3–30 μM; incubation for 3 h) and resveratrol (RES, 20 μM) in the presence of exogenous Aβ1–42 (10 μM) in N2a cells. D. Enhancement of LC3-II levels in the culture media containing 10 μM retinoic acid by cilostazol (10 μM), and its blockade by 3-methyladenine (3-MA, 2.5 mM).
Means ± SDs are expressed as percentages of DMSO (vehicle) or absence of retinoic acid (RA-) (N = 4). ###P < 0.001, RA−; *P < 0.05, **P < 0.01, ***P < 0.001 vs. DMSO; †††P < 0.001 vs. 10 μM cilostazol.
Fig 3.
A. Time-dependent increases in the expressions of full-length APP (FL-APP) and Aβ in N2aSwe cells determined using anti-Aβ (6E10) antibody. To evoke endogenous Aβ overproduction, cells were exposed to medium containing 1 μg/ml of tetracycline (Tet+) for 48 h and then switched to tetracycline-free medium (Tet-) for 3, 12, and 24 h. B. Aβ accumulation after culturing cells in Tet- condition for 24 h. C. Cilostazol-induced suppression of Aβ expression induced by Tet- condition, and prevention of this inhibition by bafilomycin A1 (BFA, 100 nM) and TIMP-1 (10 μM), respectively. D. Effect of cilostazol on the increased CTFβ (11 kDa) and CTFα (9 kDa) levels cultured in Tet- condition (Western blot using rabbit polyclonal CTFβ (751–770) antibody). The Western blots shown are representative of four independent experiments that yielded similar results. E. Prevention by bafilomycin A1 (BFA, 100 nM) and TIMP-1 (10 μM) of cilostazol-induced decreases in the CTFβ expressions.
Results are the means ± SDs of percentages (N = 4). ###P < 0.001 vs. Tet+; **P < 0.01, ***P < 0.001 vs. DMSO; †P < 0.05 vs. cilostazol (CSZ, 10 μM) alone.
Fig 4.
A—C. Inhibition of Tet- condition-induced reductions in beclin-1 (A), Atg5 (B) and SIRT1 expressions (C) by cilostazol (10 or 30 μM) in N2aSwe cells. D. Inhibition of Tet- condition-induced endogenous increases in Aβ level by cilostazol as determined by Western blotting (D) and of intracellular Aβ accumulation as determined by ELISA (E, F). The inhibitory effects of cilostazol were blocked by KT5720 (1 μM), sirtinol (20 μM) (E), bafilomycin A1 (BAF, 100 ng/ml), or 3-methyladenine (3-MA, 2.5 mM) (F).
Results are presented as means ± SDs (N = 4–5). ##P < 0.01, ###P < 0.001 vs. Tet+ condition (as control), **P < 0.01 ***P < 0.001 vs. DMSO; †P < 0.05, †††P < 0.001 vs. cilostazol alone (CSZ, 10 μM). PBS, phosphate-buffered saline.
Fig 5.
A. Analysis of the effects of SIRT1-knockdown in N2aSwe cells. Proteins (30 μg) from a negative control and SIRT1- knockdown samples were loaded onto 10 ~ 15% SDS-polyacrylamide gels. In N2aSwe cells transfected with 200 nM of SIRT1 siRNA, SIRT1 protein levels were at ~ 40% of the level in negative controls (A). Cilostazol failed to elevate the expressions of SIRT1 (B), beclin1 (C), Atg5 (D), and (E) LC3 in SIRT1-siRNA treated N2a cells, as contrasted to the levels of all four in the negative control cells.
Results are the means ± SDs of 4 experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. DMSO. ††P < 0.01 vs. cilostazol alone (CSZ, 10 μM).
Fig 6.
A and D. Immunoprecipitation analysis. Whole cell lysates were obtained from N2aSwe cell lysates that were cultured in Tet- condition for 24 h with or without cilostazol (10 μM, A) or rSIRT1 (recombinant SIRT1, 300 nM; B). Upper panels (A and B): the effects of cilostazol and rSIRT1 on LC3-II expression were confirmed. Lower panels (A and B): cell lysates were immunoprecipitated with LC-3 antibody and then immunoblotted for acetylated LC3 LC3-1/II-Ac) using an anti-acetyl lysine antibody. The blot shown is representative of three experiments that produced similar results. C. Immunofluorescent assay of LC3 puncta in N2a cells treated with or without cilostazol (10 and 30 μM) after being pretreated with 3-methyladenine (2.5 mM), bafilomycin A1 (100 nM) or sirtinol (20 μM). D. Quantitative analysis was performed by counting numbers of LC3 puncta/cell. Results are the means ± SDs of 4 experiments.
***P < 0.001 vs. DMSO. †††P < 0.001 vs. cilostazol (10 μM).
Fig 7.
Effect of cilostazol on Aβ-induced cytotoxicity.
Decrease in cell viability in response to exogenous Aβ1–42 in N2a cells (A) and to endogenously overproduced Aβ in the N2aSwe cells (B), and the recovery by cilostazol in the absence and presence of 3-methyladenine (3-MA, 2.5 mM). Results are the means ± SDs of three experiments. ###P < 0.001 vs. PBS (A) and Tet- condition (B); ***P < 0.001 vs. DMSO; †P < 0.05, ††P < 0.01, †††P < 0.001 vs. 10 or 30 μM cilostazol alone. PBS, phosphate-buffered saline. C. Hypothetical model: Neuroprotective effect of cilostazol against Aβ-induced neurotoxicity is ascribable to the increased induction of autophagy by increasing the cAMP/PKA coupled SIRT1 activation, thereby enhances Aβ and CTFβclearance and increases cell viability.