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

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.

It has been reported that the transient expression of wild-type SIRT1 stimulates the conversion of LC3-I to LC3-II in HCT116 cells [11]. To investigate the effect of cilostazol on LC3-II expression, a model of retinoic acid-induced neuronal differentiation was used. As shown in Fig 2D, LC3-II levels in N2a cells were elevated by 238 ± 11.0% (P < 0.001) in culture media containing 10 μM retinoic acid as compared with the non-retinoic acid control, and interestingly, cilostazol (10 μM) increased LC3-II levels by 491.3 ± 21.9% (P < 0.001). Furthermore, this effect of cilostazol was significantly blocked by 3-methyladenine (2.5 mM; an inhibitor of autophagy), indicating that cilostazol enhanced autophagosome formation.

Immunoprecipitation and Immunofluorescence studies
To examine the effect of SIRT1 on autophagy components, we assessed and compared the deacetylations of LC3 by cilostazol or recombinant SIRT1 (rSIRT1) in the N2aSwe cells. Neuronal cells were exposed to Tetfor 24 h in the absence and presence of 10 μM of cilostazol or 300 nM of rSIRT1, and then one part of each whole cell lysates was immunoblotted for LC3. The other portions were immunoprecipitated with LC-3 antibody and immunoblotted for acetylated LC3 using an anti-acetyl lysine antibody. As shown in Fig 6A and 6B, LC3-II levels were markedly elevated by treatment with cilostazol or rSIRT1. However, after immunoprecipitating LC-3, the immunoblotted band intensity of acetylated LC3-II was markedly reduced (by~30%) in samples treated with cilostazol or rSIRT1. In addition, the effect of cilostazol was further examined with respect to autophagy initiation by assaying LC3 puncta in N2a cells treated with or without cilostazol (10 or 30 μM). Fluorescent puncta were significantly increased by cilostazol at 10 and 30 μM (both P < 0.001), but these increases were significantly blocked by 3-methyladenine (2.5 mM, P < 0.001), bafilomycin A1 (100 nM, P < 0.001), or sirtinol (20 μM, P < 0.001). Overall, these results show that like rSIRT1, cilostazol stimulates the conversion of LC3-I to LC3-II, which is suggestive of increased autophagosome formation.

Cell viability enhancement by cilostazol
The cytotoxic effects of exogenously applied Aβ1-42 in N2a cells and of endogenously released Aβ in N2aSwe cells were assessed using an MTT assay. Exposure of N2a cells to 10 μM of Aβ1-42 for 24 h resulted in a significant decline in cell viability by 51.6 ± 2.7% (P < 0.001). The decreased viability induced by Aβ1-42 was largely recovered by 10 or 30 μM of cilostazol to 81.7 ± 2.6% (P < 0.001) and 88.0 ± 3.7% (P < 0.001), respectively. Furthermore, this effect of cilostazol was significantly blocked by pretreating cells with 3-methyladenine (2.5 mM, a chemical inhibitor of autophagy) (Fig 7A).

Discussion
The present study demonstrates both applied Aβ1-42 and endogenously generated Aβ from activated N2aSwe cells decrease the expressions of beclin-1, Atg5, and SIRT1 at the protein level, and that these suppressions are prevented by cilostazol pretreatment. Interestingly, cilostazol-stimulated upregulations of SIRT1-associated beclin-1, Atg5, and LC3-II reduced the accumulations of Aβ and APP-CTF β in N2aSwe cells. Furthermore, the decreased viability induced by Aβ was largely prevented by cilostazol, and this prevention was blocked by 3-methyladenine. These results suggest cilostazol protects against Aβ-induced neurotoxicity by enhancing the induction of autophagy. The present results also reveal that increases in Aβ and CTFβ expressions induced by Tet + and Tetprocedures are attenuated by cilostazol. Previous study [24] also showed activated N2aSwe cells by depletion of FBS (1%) induced large accumulations of full-length APP (100 kDa), which peaked at 12 h post activation. These accumulations were significantly and concentration-dependently diminished by pretreatment with cilostazol (3~30 μM).
Many authors have reported autophagy induces the degradation of aggregated proteins that cause AD, and that dysfunction of the autophagy-lysosome system contributes to Aβaccumulation and to the formation of tau oligomers [25][26][27]. Therefore, pharmacological regulation of autophagy-lysosome protein degradation is emerging as an important strategy for the treatment of AD.
Intriguingly, Lee et al. [11] emphasized SIRT1 deacetylase is an important regulator of autophagy: they showed that transiently augmenting SIRT1 activity is sufficient to activate autophagy, whereas SIRT1 -/mouse embryonic fibroblasts did not fully activate autophagy under starvation conditions. In addition, the level of beclin-1, a protein that plays a key role in autophagy, was reported to be diminished in the affected brain regions of AD patients, although other studies failed to observe this effect [28][29][30].
To explore relationships between the effects of Aβ and the expressions of molecular components, such as, beclin-1, Atg5, and LC3-II, of the machinery of autophagy under pathological situations, we evoked endogenous Aβ overproduction in N2aSwe cells. N2aSwe cells were exposed to Tet + for 48 h, and then placed to Tetcondition instead of using serum-depleted culture medium, because we observed that serum depletion per se stimulates the upregulations of beclin-1 and Atg5 in a manner similar to starvation conditions. When N2aSwe cells were exposed to Tet + /Tet -, they exhibited time-dependent increases in Aβ and APP-CTFβ levels. The present results also showed elevated intracellular Aβ1-42 levels were markedly inhibited by cilostazol and that this inhibition was blocked by KT5720 and sirtinol, indicating that cAMP-dependent protein kinase and SIRT1 underlie the action mechanism of cilostazol, as has been previously reported by Lee et al. [20].
Our observations of increases in beclin-1, Atg5, and SIRT1 levels by cilostazol (10 or 30 μM) are considered to be related to reduced accumulations of Aβ and APP-CTFβ, which were induced by Tetin the presence of cilostazol, because the cilostazol-induced suppressions of APP-CTFβ expression and intracellular Aβlevels were significantly blocked by bafilomycin A1 (a blocker of autophagosome to lysosome fusion) [22] or 3-methyladenine (an inhibitor of autophagy) [31]. In light of the facts that cilostazol-induced decreases in CTFβ and Aβ accumulation were significantly inhibited by bafilomycin A1 or TIMP-1 (an ADAM10 inhibitor) [23], the inhibition of CTFβ and Aβ accumulation by cilostazol is ascribed to increased autophagic clearance and to reduced production of Aβ through ADAM 10 (α-secretase) activation [24].
It was also reported some time ago LC3 is necessary for the formation of autophagosomes and that it localizes to autophagosome membranes [32]. The conversion of LC3-I to LC3-II via proteolytic cleavage is a hallmark of mammalian autophagy: the amount of LC3-II and LC3-II/ LC3-I ratio are closely related to autophagosome formation [33]. Thus, to evaluate the level of autophagy in a more specific way, we assessed levels of autophagy protein LC3, a marker of mammalian autophagy. Cilostazol markedly increased LC3-II levels in N2a cells, and this increase was significantly blocked by 3-methyladenine, indicating that the autophagy pathway is up-regulated by cilostazol. Interestingly, after immunoprecipitating LC-3, the acetylated LC3-II band was markedly decreased (by~30%) in cells pretreated with cilostazol or rSIRT1. Moreover, numbers of immunofluorescent puncta were significantly increased by cilostazol, and this increase was blocked by 3-methyladenine, bafilomycin A1, or sirtinol, respectively. These results strongly suggest that like rSIRT1, cilostazol stimulates the conversion of LC3-I to LC3-II, which is indicative of increased autophagosome formation.
To confirm that the cilostazol-induced elevations of beclin-1 and Atg5 were mediated by the activation of SIRT1, N2aSwe cells were transfected with SIRT1 siRNA. After silencing the SIRT1 gene, the expressions of beclin-1, Atg5, LC3-II, and SIRT1 were not induced by cilostazol, whereas negative control cells (transfected with scrambled siRNA duplex) were obviously responsive to cilostazol. These observations indicate that increases in beclin-1, Atg5, and LC3-II levels were evoked by cilostazol via SIRT1 activation.
These postulations are strongly supported by the previous reports, in that cilostazol rescued HT22 apoptosis induced by Aβ toxicity by downregulating phosphorylated p53 (Ser 15), Bax, and caspase-3 levels and upregulating Bcl-2 levels, and protected against the suppression of neurite elongation by Aβ [34]. Moreover, cilostazol suppressed the accumulations of Aβ by increasing the expression of ADAM10 and α-secretase activity via the upregulation of cAMPdependent protein kinase-linked SIRT1 expression in activated N2aSwe mutant cells [24]. Based on these reports and the present study, it is considered that cilostazol prevents Aβinduced cell viability reduction. A question arises as to how cilostazol clears APPswe metabolites released from N2aSwe cells, since clearance of metabolites generated from APPswe cells may mechanistically differ from soluble β-amyloid as suggested by Haass et al. [35]. Further study is required to define the autophagic assessment of metabolites from APPswe cells by cilostazol.
Taking these results and those regarding the pharmacological inhibition and gene silencing of SIRT1, cilostazol appears to protect neuronal cells from Aβ-induced neurotoxicity by upregulating the autophagy machinery, as demonstrated by the up-regulations of beclin-1, Atg5, and LC3-II, via activating SIRT1 expression and decreasing Aβ peptide production, and thereby improves cell viability (Fig 7C).

Cell culture
Mouse neuroblastoma N2a wild-type cells and N2aSwe cells were kindly donated by Dr. Takeshi Iwatsubo (Department of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, The University of Tokyo) [36]. These cells were cultured in media containing 45% Dulbecco's Modified Eagle's Medium (DMEM, Gibco, Carlsbad, CA), 55% Opti-MEM (Gibco), supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), 100 units/ml penicillin, 100 μg/ml streptomycin, 1% glutamine, and 0.09% Hygromysin B (Sigma-Aldrich, St. Louis, MO) in a humidified 5% CO 2 / 95% air atmosphere at 37°C. To evoke endogenous Aβ overproduction, N2a and N2aSwe cells were cultured under the above condition in the presence of 1 μg/ml of tetracycline (Tet + , which was used as a control) for 48 h and then placed in tetracycline-free condition (Tet -) and cultured for 3, 12, and 24 h, respectively. When treatment with either cilostazol or resveratrol was required, cells were pretreated with these drugs for 3 h in Tet + , and then switched to Tetcondition containing the same drugs and cultured for the indicated times.

Measurement of Aβ levels by ELISA
Cell lysates from cilostazol-treated and untreated cells were collected, and Aβ1-42 levels were determined using ELISA kit Aβ1-42 (FIVEphoton Biochemicals, San Diego, CA). Optical densities were read at 450 nm using a plate reader, and Aβ1-42 concentrations were determined using standard curves. All readings taken fell within the linear range of the assay.

Immunofluorescence experiments
For immunofluorescence studies, N2a cells were fixed in 4% (w/v) paraformaldehyde for 30 min at room temperature, permeabilized with PBS containing 3% bovine serum albumin and 0.1% (v/v) Triton X-100 for 30 min, and then incubated for 1 h at room temperature under constant shaking with antibody against LC3 (rabbit monoclonal LC3A/B antibody; dilution 1:1,000; Cell Signaling). After several washes with PBS, cells were incubated for 1 h with secondary antibody conjugated to Alexa Fluor 488 and 594 (Invitrogen, Carlsbad, CA) at room temperature, washed again with PBS, labeled with DAPI, and mounted in Fluoprep (Bio-Merieux, Craponne-Pays, France). All fluorescent images were acquired at ×100 using an Axiovert 200 (Carl Zeiss, Jena, Germany) fluorescence microscope.

Immunoprecipitation
For the immunoprecipitation assay, N2aSwe cells were lysed with lysis buffer containing 50 mM HEPES-OH pH 7.5, 120 mM NaCl, 1 mM EDTA, 10 mM pyrophosphate, 10 mM glycerophosphate, 50 mM NaF, 1 mM PMSF, 1.5 mM Na 3 VO 4 , 0.3% CHAPS, and protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). Precleared lysates containing 200 μg of whole lysate proteins diluted with lysis buffer were then mixed with 2 μg of protein G agarose conjugated acetyl lysine antibody (Cell Signaling) and incubated for 2 h. Samples were washed four times with lysis buffer and subjected to Western blot analysis.

Determination of cell viabilities
The cytotoxicities of exogenous Aβ1-42 and endogenously released Aβwere assessed using a MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium] assay. N2A and N2aSwe cells were seeded onto 12-well plates and cultured for 24 h before beginning the experiment. After one wash with PBS, cells were placed in a medium containing phenol red-free DMEM and 1% serum. Cells were pretreated with cilostazol when necessary for 2 h, and then 10 μM Aβ 1-42 was added and incubated for 24 h. Alternatively, cells were plated in Tet + (1 μg/ml of tetracycline) or Tetcondition containing vehicle or cilostazol with or without 3-methyladenine (3-MA, 2.5 mM). Wells containing medium without cells served as background controls, and cells cultured in Tet + condition served as a positive control. The MTT assay was performed by adding 0.5 mg/ml of MTT and then incubating for 2 h at 37°C. The formazan salt generated by viable cells was dissolved in DMSO and absorbances were measured at 450 nm.

Statistical Analyses
Results are expressed as means ± SDs. One-way analysis of variance followed by Tukey's post hoc multiple comparisons was used to determine the significances of differences between vehicle and cilostazol treatment groups. Student's t-test was used to determine the significance of difference between the mean of untreated cells and those treated with inhibitors. Statistical significance was accepted for P values of < 0.05.