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Cilostazol Upregulates Autophagy via SIRT1 Activation: Reducing Amyloid-β Peptide and APP-CTFβ Levels in Neuronal Cells.

Lee HR, Shin HK, Park SY, Kim HY, Bae SS, Lee WS, Rhim BY, Hong KW, Kim CD - PLoS ONE (2015)

Bottom Line: We previously found that cilostazol induced SIRT1 expression and its activity in neuronal cells, and thus, we hypothesized that cilostazol might stimulate clearances of Aβ and C-terminal APP fragment β subunit (APP-CTFβ) by up-regulating autophagy.When N2a cells were exposed to soluble Aβ1-42, protein levels of beclin-1, autophagy-related protein5 (Atg5), and SIRT1 decreased significantly.Further, decreased cell viability induced by Aβ was prevented by cilostazol, and this inhibition was reversed by 3-methyladenine, indicating that the protective effect of cilostazol against Aβ induced neurotoxicity is, in part, ascribable to the induction of autophagy.In conclusion, cilostazol modulates autophagy by increasing the activation of SIRT1, and thereby enhances Aβ clearance and increases cell viability.

View Article: PubMed Central - PubMed

Affiliation: Gene & Cell Therapy Research Center for Vessel-associated Diseases, Pusan National University, Yangsan-si, Gyeongsangnam-do, Republic of Korea; Medical Research Center for Ischemic Tissue Regeneration, Pusan National University, Yangsan-si, Gyeongsangnam-do, Republic of Korea.

ABSTRACT
Autophagy is a vital pathway for the removal of β-amyloid peptide (Aβ) and the aggregated proteins that cause Alzheimer's disease (AD). We previously found that cilostazol induced SIRT1 expression and its activity in neuronal cells, and thus, we hypothesized that cilostazol might stimulate clearances of Aβ and C-terminal APP fragment β subunit (APP-CTFβ) by up-regulating autophagy.When N2a cells were exposed to soluble Aβ1-42, protein levels of beclin-1, autophagy-related protein5 (Atg5), and SIRT1 decreased significantly. Pretreatment with cilostazol (10-30 μM) or resveratrol (20 μM) prevented these Aβ1-42 evoked suppressions. LC3-II (a marker of mammalian autophagy) levels were significantly increased by cilostazol, and this increase was reduced by 3-methyladenine. To evoke endogenous Aβ overproduction, N2aSwe cells (N2a cells stably expressing human APP containing the Swedish mutation) were cultured in medium with or without tetracycline (Tet+ for 48 h and then placed in Tet- condition). Aβ and APP-CTFβ expressions were increased after 12~24 h in Tet- condition, and these increased expressions were significantly reduced by pretreating cilostazol. Cilostazol-induced reductions in the expressions of Aβ and APP-CTFβ were blocked by bafilomycin A1 (a blocker of autophagosome to lysosome fusion). After knockdown of the SIRT1 gene (to ~40% in SIRT1 protein), cilostazol failed to elevate the expressions of beclin-1, Atg5, and LC3-II, indicating that cilostazol increases these expressions by up-regulating SIRT1. Further, decreased cell viability induced by Aβ was prevented by cilostazol, and this inhibition was reversed by 3-methyladenine, indicating that the protective effect of cilostazol against Aβ induced neurotoxicity is, in part, ascribable to the induction of autophagy. In conclusion, cilostazol modulates autophagy by increasing the activation of SIRT1, and thereby enhances Aβ clearance and increases cell viability.

No MeSH data available.


Related in: MedlinePlus

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.
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pone.0134486.g003: 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.

Mentions: To investigate endogenous accumulations of full-length APP and Aβ under pathological situations simulating those in the AD brain, we used mouse neuroblastoma cells stably expressing human APP Swedish mutation (N2aSwe cells). These cells were exposed to Tet+ and Tet- conditions, as previously described by Anekonda et al. [21]. Briefly, cells were exposed to medium containing 1 μg/ml of tetracycline (Tet+) for 48 h, and then removed to tetracycline-free (Tet-) conditions for 3, 12, or 24 h to induce endogenous Aβ overproduction. As shown in Fig 3A, in Tet+ condition, cells showed thin density, but in the Tet- condition, N2aSwe cells exhibited time-dependent increases in full-length APP (~100 kDa) at between 3 and 24 h (using anti-Aβ (6E10) antibody). Accordingly, the accumulation of Aβ (4 kDa) increased from 12 ~ 24 h under the Tet- condition by 165.4 ± 15.0% (P < 0.001) (Fig 3B). This increase in Aβ level was significantly reduced by 65.1 ± 9.0% (P < 0.01) under pretreatment with 10 μM of cilostazol, and this reduction by cilostazol was prevented by co-treating cilostazol with bafilomycin A1 (100 nM; a blocker of autophagosome to lysosome fusion) [22] by 95.6 ± 9.1%, P < 0.05 and with TIMP-1 (10 μM, by 91.0 ± 8.7%, P < 0.05), an ADAM10 inhibitor [23] (Fig 3C).


Cilostazol Upregulates Autophagy via SIRT1 Activation: Reducing Amyloid-β Peptide and APP-CTFβ Levels in Neuronal Cells.

Lee HR, Shin HK, Park SY, Kim HY, Bae SS, Lee WS, Rhim BY, Hong KW, Kim CD - PLoS ONE (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4526537&req=5

pone.0134486.g003: 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.
Mentions: To investigate endogenous accumulations of full-length APP and Aβ under pathological situations simulating those in the AD brain, we used mouse neuroblastoma cells stably expressing human APP Swedish mutation (N2aSwe cells). These cells were exposed to Tet+ and Tet- conditions, as previously described by Anekonda et al. [21]. Briefly, cells were exposed to medium containing 1 μg/ml of tetracycline (Tet+) for 48 h, and then removed to tetracycline-free (Tet-) conditions for 3, 12, or 24 h to induce endogenous Aβ overproduction. As shown in Fig 3A, in Tet+ condition, cells showed thin density, but in the Tet- condition, N2aSwe cells exhibited time-dependent increases in full-length APP (~100 kDa) at between 3 and 24 h (using anti-Aβ (6E10) antibody). Accordingly, the accumulation of Aβ (4 kDa) increased from 12 ~ 24 h under the Tet- condition by 165.4 ± 15.0% (P < 0.001) (Fig 3B). This increase in Aβ level was significantly reduced by 65.1 ± 9.0% (P < 0.01) under pretreatment with 10 μM of cilostazol, and this reduction by cilostazol was prevented by co-treating cilostazol with bafilomycin A1 (100 nM; a blocker of autophagosome to lysosome fusion) [22] by 95.6 ± 9.1%, P < 0.05 and with TIMP-1 (10 μM, by 91.0 ± 8.7%, P < 0.05), an ADAM10 inhibitor [23] (Fig 3C).

Bottom Line: We previously found that cilostazol induced SIRT1 expression and its activity in neuronal cells, and thus, we hypothesized that cilostazol might stimulate clearances of Aβ and C-terminal APP fragment β subunit (APP-CTFβ) by up-regulating autophagy.When N2a cells were exposed to soluble Aβ1-42, protein levels of beclin-1, autophagy-related protein5 (Atg5), and SIRT1 decreased significantly.Further, decreased cell viability induced by Aβ was prevented by cilostazol, and this inhibition was reversed by 3-methyladenine, indicating that the protective effect of cilostazol against Aβ induced neurotoxicity is, in part, ascribable to the induction of autophagy.In conclusion, cilostazol modulates autophagy by increasing the activation of SIRT1, and thereby enhances Aβ clearance and increases cell viability.

View Article: PubMed Central - PubMed

Affiliation: Gene & Cell Therapy Research Center for Vessel-associated Diseases, Pusan National University, Yangsan-si, Gyeongsangnam-do, Republic of Korea; Medical Research Center for Ischemic Tissue Regeneration, Pusan National University, Yangsan-si, Gyeongsangnam-do, Republic of Korea.

ABSTRACT
Autophagy is a vital pathway for the removal of β-amyloid peptide (Aβ) and the aggregated proteins that cause Alzheimer's disease (AD). We previously found that cilostazol induced SIRT1 expression and its activity in neuronal cells, and thus, we hypothesized that cilostazol might stimulate clearances of Aβ and C-terminal APP fragment β subunit (APP-CTFβ) by up-regulating autophagy.When N2a cells were exposed to soluble Aβ1-42, protein levels of beclin-1, autophagy-related protein5 (Atg5), and SIRT1 decreased significantly. Pretreatment with cilostazol (10-30 μM) or resveratrol (20 μM) prevented these Aβ1-42 evoked suppressions. LC3-II (a marker of mammalian autophagy) levels were significantly increased by cilostazol, and this increase was reduced by 3-methyladenine. To evoke endogenous Aβ overproduction, N2aSwe cells (N2a cells stably expressing human APP containing the Swedish mutation) were cultured in medium with or without tetracycline (Tet+ for 48 h and then placed in Tet- condition). Aβ and APP-CTFβ expressions were increased after 12~24 h in Tet- condition, and these increased expressions were significantly reduced by pretreating cilostazol. Cilostazol-induced reductions in the expressions of Aβ and APP-CTFβ were blocked by bafilomycin A1 (a blocker of autophagosome to lysosome fusion). After knockdown of the SIRT1 gene (to ~40% in SIRT1 protein), cilostazol failed to elevate the expressions of beclin-1, Atg5, and LC3-II, indicating that cilostazol increases these expressions by up-regulating SIRT1. Further, decreased cell viability induced by Aβ was prevented by cilostazol, and this inhibition was reversed by 3-methyladenine, indicating that the protective effect of cilostazol against Aβ induced neurotoxicity is, in part, ascribable to the induction of autophagy. In conclusion, cilostazol modulates autophagy by increasing the activation of SIRT1, and thereby enhances Aβ clearance and increases cell viability.

No MeSH data available.


Related in: MedlinePlus