Akt pathway and prevents

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Neuroscience Letters 432 (2008) 146–150

Caffeine activates the PI3K/Akt pathway and prevents apoptotic cell death in a Parkinson’s disease model of SH-SY5Y cells Kazuhiro Nakaso ∗ , Satoru Ito, Kenji Nakashima Department of Neurology, Institute of Neurological Sciences, Faculty of Medicine, Tottori University, 36-1 Nishicho, Yonago, 683-8504, Japan Received 25 September 2007; received in revised form 22 November 2007; accepted 10 December 2007

Abstract Parkinson’s disease (PD) is one of the most common neurodegenerative diseases. Recent epidemiological studies suggest that caffeine, one of the major components of coffee, has a protective effect against developing PD. However, the detailed mechanisms of how caffeine suppresses neuronal death have not been fully elucidated. We investigated the cytoprotective mechanisms of caffeine using human dopaminergic neuroblastoma SH-SY5Y cells as a PD model. Caffeine prevented the apoptotic cell death induced by serum/retinoic acid (RA) deprivation, MPP+, rotenone, and 6-OHDA in SH-SY5Y cells in a dose dependent manner. Caffeine lowered caspase-3 activity induced by serum/RA deprivation and 6-OHDA administration, and also decreased the number of apoptotic condensed and/or fragmented nuclei. Akt was phosphorylated 60 min after caffeine administration in a dose dependent manner; PI3K inhibitors, wortmannin and LY294002 canceled this cytoprotective effect of caffeine. On the other hand, MAPKs such as Erk1/2, p38, or JNK were not activated by caffeine. These results suggest that caffeine has a cytoprotective effect due to the activation of the PI3K/Akt pathways in SH-SY5Y cells. © 2008 Published by Elsevier Ireland Ltd. Keywords: Caffeine; Coffee; Neuroprotection; Phosphatidyl inositol 3 kinase (PI3K); Akt/PKB

Parkinson’s disease (PD) is a neuropathological disorder involving the degeneration of dopaminergic neurons in the substantia nigra, with the subsequent loss of their terminals in the striatum. The ensuing loss of dopamine causes most of the debilitating motor disturbances associated with PD. Current PD medications treat the symptoms of the disease without halting or retarding the degeneration of dopaminergic neurons. Recently, there has been considerable interest in neuroprotection as a therapeutic strategy for PD [16], and several drugs have been proposed as candidates for neuroprotective agents for PD [7,12,16,23]. The pathogenesis of PD is caused not only by genetic factors but also by environmental factors such as pesticides and heavy metals [4,18]. The vulnerability to developing PD can be affected by life style, food intake, beverages, and so on. Previous reports suggest that smoking, non-estradiol anti-inflamatory drugs, and coffee/caffeine intake are inversely associated with the onset of PD [9,10,14,15]. Large prospective epidemiologic studies have linked the consumption of coffee to a reduced risk of develop-

∗

Corresponding author. Tel.: +81 859 38 6757; fax: +81 859 38 6759. E-mail address: [email protected] (K. Nakaso).

0304-3940/$ – see front matter © 2008 Published by Elsevier Ireland Ltd. doi:10.1016/j.neulet.2007.12.034

ing PD [15]. Caffeine, one of the main components of coffee, has been believed to be the compound responsible for these observations [15]. Caffeine is known as a specific antagonist of the adenosine A2a receptor [13], and has been found to attenuate neurotoxicity in experimental models of neurodegenerative diseases such as PD and Alzheimer’s disease [5,11,17,21,22]. However, other neuroprotective mechanisms of caffeine in PD may exist, and further evidence associated with neuroprotection by caffeine may emerge. The aim of this study was to clarify the molecular mechanism of how caffeine acts as a neuroprotective agent. Caffeine (Wako) was dissolved in hot water and stored at −20 ◦ C until use. PI3K inhibitor LY294002 and wortmannin were purchased from Alomone Labs. Erk MAPK inhibitor PD98059, p38 MAPK inhibitor SB203580, and JNK inhibitor SP600125 were purchased from Alexis. MPP+ was purchased from Sigma and 6-OHDA and rotenone were obtained from ICN. All-trans-retinoic acid (RA) was from Wako. The following antibodies were used in this study: anti-phospho-Erk (E-4), anti-phospho-p38 (Tyr182-R), anti-phospho-JNK (Thr183/Tyr185-R), anti-Akt (Santa Cruz Biotechnology), anti-phospho-Akt (Ser473) (Cell Signaling Technology).

K. Nakaso et al. / Neuroscience Letters 432 (2008) 146–150

The human dopaminergic neuroblastoma cell line, SH-SY5Y, was purchased from ATCC. SH-SY5Y cells were maintained at 37 ◦ C in 5% in CO2 in DMEM/F12 medium, supplemented with 5% fetal bovine serum and penicillin/streptomycin. Culture medium was exchanged twice a week during cell growth. Experiments were carried out using 1 × 106 cells per well in 6well culture plates for immunoblots and the caspase-3 assay, 5 × 103 cells per well in 96-well culture plates for viability assays, and 1 × 103 cells per well 8-well chamber slides for immunohistochemical analyses. The cells were neuronally differentiated by incubating with 5 ␮M RA for 5 days. Caffeine (10 or 100 ␮M) and kinase inhibitors were added to the medium after the change of serum/RA-free medium, and incubated for the duration indicated in the figure legends. About 5 × 103 SH-SY5Y cells in 96-well plates were exposed to serum/RA-free condition (SRF), MPP+ (1 mM), rotenone (10 nM), and 6-OHDA (50 ␮M). Caffeine was added simultaneously with each toxin. Each kinase inhibitor was added 30 min before caffeine treatment. Cells were incubated for 48 h after exposure to toxins, and cell viability was measured using the MTT assay system (Chemicon) according to the manufacturer’s protocol with some modifications. For the caspase-3 assay, cells that were exposed to SRF condition or 6-OHDA were treated with caffeine. Cells were harvested 20 h after caffeine administration, lysed in buffer A (100 mM HEPES/KOH, pH7.5, 10% sucrose, 0.1% CHAPS, 10mM DTT, 1% TritonX-100, 1 mg/ml PMSF and centrifugation at 14,000 × g for 10 min. Ten microlitres of supernatant was used for the measurement of caspase-3 activity as determined by fluorescent density at (ex 360 nm/em 460 nm) using 100 ␮M Ac-DEVD-MCA (Peptide inc) as an enzyme substrate. For immunohistochemical analyses, cells in 8 well-chamber slides were washed twice with PBS, and fixed with 4% paraformaldehyde. After incubating in ice-cold 95% EtOH/1% acetate and washing with PBS, cells were incubated in antiphospho Akt antibody overnight at 4 ◦ C, followed with an incubation in Texas Red-conjugated secondary antibody. To determine the localization of the nucleus, counterstaining was performed using Hoechst 333492. Condensed or fragmented nuclei were characterized as apoptotic nuclei. For immunoblot analysis, cells were lysed in SDS-sample buffer (50 mM Tris–HCl, pH 6.8, 2% SDS, 10% glycerol, 1 mM PMSF, 2 mM EDTA), and denatured by boiling at 100 ◦ C for 5 min. The samples were stored at −20 ◦ C until use. Total protein content was determined using bicinchoninic acid (BCA) protein assay reagent (Pierce) and 20 ␮g was used for SDS-PAGE. Dye and 2-mercaptoethanol were added to the specimens, and the mixture was boiled just before sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). Each protein was separated by size on 10 or 12.5% polyacrylamide gels, transferred onto a polyvinylidene difluoride membrane (Hybond-P, Amersham), and the blots were incubated in PBS containing 0.1% Tween 20 and primary antibodies. The bound primary antibodies were detected using horseradish peroxidase-conjugated secondary antibodies (Amersham) and ECL detection reagents (Amersham).

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Fig. 1. Cytoprotective effect of caffeine against serum/RA deprivation (SRF), MPP+, rotenone, and 6-OHDA. Administration of caffeine (0, 10, 100 ␮M) prevents cell death after 48 h. Caffeine was added simultaneously with PDrelated toxin, and cell viability was quantified by MTT assay system.

Quantitative data were assessed by ANOVA using StatView software. The criterion for statistical significance was p < 0.05. In order to clarify the cytoprotective effect of caffeine, we established a cell toxicity model of PD using the human dopaminergic neuroblastoma cell line, SH-SY5Y. Although SRF conditions decreased cell viability to 77.1% 48 h after treatment, 10 or 100 ␮M caffeine administration prevented this cell death significantly (85.9%, 87.3%, respectively) (Fig. 1A). We also investigated the effect of caffeine against cell death induced by the PD-related neurotoxins MPP+ (1 mM), rotenone (10 nM) and 6-OHDA (50 ␮M) (Fig. 1B–D). Caffeine significantly increased cell viability 48 h after toxin treatment in a dosedependent manner. Caffeine (10 and 100 ␮M) increased cell viability from 69.5% to 74.7% and 85.6%, respectively under MPP+ exposed conditions; from 71.1% to 69.3% and 83.7%, respectively, under rotenone exposed conditions; from 38.4% to 50.7% and 59.9%, respectively, under 6-OHDA exposed conditions. However, 500 ␮M or higher concentrations of caffeine was rather toxic in our experimental condition (data not shown). We investigated whether caffeine could decrease apoptotic nuclei induced by SRF, MPP+ (1 mM), rotenone (10 nM), and 6-OHDA (100 ␮M) treatment. Caffeine treatment decreased the number of apoptotic nuclei (Fig. 2A–H). Caffeine (100 ␮M) decreased the percentage of apoptotic cells with condensed and/or fragmented nuclei from 13.6% to 7.2% under SRF conditions; from 13.1% to 9.7% under MPP+ exposed conditions; from 8.9% to 7.1% under rotenone exposed conditions; from 32.7% to 23.0% under 6-OHDA exposed conditions. We also measured caspase-3 activity as an index for apoptotic cell death. Caffeine decreased caspase-3 activity induced by SRF and 6OHDA toxicity in a dose dependent manner (Fig. 2I). Caffeine (10 and 100 ␮M) decreased caspase-3 activity 20.5% and 22.8% under SRF conditions, and also decreased caspase-3 activity

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Fig. 2. Caffeine inhibits apoptotic cell death induced by PD-related toxins. Apoptotic nuclei were detected using Hoehcst staining (A, C, E, G) and 100 ␮M caffeine suppressed these apoptotic changes after 48 h (B, D, F, H). Caffeine decreased caspase-3 activity under the conditions with SRF and SRF plus 50 ␮M 6-OHDA (I).

30.9% and 38.3% under 6-OHDA exposed condition, respectively. In order to elucidate which intracellular signaling pathways could be involved in the observed caffeine-related cytoprotective action, we investigated the degree of activation of the MAPK and PI3K/Akt pathways as candidate signals for cytoprotective action. As in Fig. 3A, Akt was dramatically phosphorylated 60 min after caffeine treatment in a dose dependent manner (Fig. 3A). The levels of pAkt were 0.89-fold under SRF condition, 1.96-fold under SRF/1 ␮M caffeine, 2.96-fold under SRF/10 ␮M caffeine, 3.08-fold under SRF/100 ␮M caffeine versus control, respectively. p-Akt level under the condition treated with caffeine (100 ␮M) alone was 1.53-fold versus control. Immunohistochemical analysis showed increased Akt phosphorylation in caffeine-treated cells, and translocation of Akt from the cytoplasm to the plasma membrane area (Fig. 3A). We also assessed the phosphorylation of Erk1/2, p38, and JNK. However, these MAPKs were not significantly phosphorylated by caffeine treatment (Fig. 3B). Based on the results described above, we examined whether PI3K inhibitors LY294002 and wortmannin could inhibit the cytoprotective effect of caffeine. Although PD98059 (5 ␮M), SB203580 (5 ␮M), and SP600125 (5 ␮M) (inhibitors of Erk1/2, p38, and JNK, respectively) did not inhibit this cytoprotective effect of caffeine, the PI3K inhibitor LY29004 (10 ␮M) and wortmannin (2 ␮M) dramatically inhibited this effect (Fig. 3E). PD is a common neurodegenerative disease caused by both genetic and environmental factors. Although symptomatic therapy is the mainstay of PD medication, there has been con-

siderable interest recently in neuroprotection as a therapeutic strategy for PD [16]. A recent meta-analysis of eight case–control studies and five cohort studies revealed a 30% risk reduction of PD in coffee drinkers [15]. Although some negative results for the cytoprotective effect by caffeine and/or coffee have been reported [19,20], a large number of reports have demonstrated that caffeine and/or coffee intake have a preventive effect against PD-related neuronal death or PD onset itself [2,3,6,8]. However, the cytoprotective effect of coffee drinking in PD is less well examined in detail. Various authors have alluded to a number of putative mechanisms for this protective effect, but it has not been well characterized. Since caffeine is an antagonist of adenosine A2a receptor, it might favorably alter the course as well as the symptoms of PD. Furthermore, caffeine may be expected to have other beneficial and adverse effects elsewhere in the central nervous system such as neuroprotection. In order to clarify this point, we examined the neuroprotective effects of caffeine in the present study. In our experimental conditions, caffeine prevented apoptotic cell death induced by SRF and PD-related toxins (Fig. 1). On the other hand, it has been reported that caffeine has the cytoprotective effect against MPP+-induced apoptosis but not against serum withdrawal in cerebeller granule neurons (CGNs) [1]. The cytoprotective mechanism of caffeine in SH-SY5Y cells may be different in CGNs. Although the cytoprotective effect of caffeine was not very powerful, caffeine prevented the activation of caspase-3 in a dose-dependent manner, suggesting that the cytoprotective effect of caffeine is due to the suppression of apoptotic signals or the upregulation of cytoprotective


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gate the suppressive effect of several foods rich in caffeine against PD. Acknowledgements This study was supported by the grant from All Japan Coffee Association, and by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan. References

Fig. 3. The cytoprotective effect depends on PI3K/Akt signaling. Caffeine upregulated phosphorelated Akt (p-Akt) 60 min after treatment in a dosedependent manner (A). Low expression of p-Akt before caffeine treatment (B) and high expression of p-Akt 60 min after treatment of 100 ␮M caffeine (C). Lower panels in (B, C) show distribution and quantification of p-Akt. MAPKs (Erk, p38, JNK) were not phosphorylated by caffeine (D). Cytoprotective effect of caffeine was inhibited by the PI3K inhibitors LY294002 and wortmannin (E). PD: PD98059 (5 ␮M), SB: SB203580 (5 ␮M), SP: SP600125 (5 ␮M), LY: LY294002 (10 ␮M), WO: wortmannin (2 ␮M).

signals. Therefore, we investigated the signal transduction pathways in SH-SY5Y cells following caffeine administration. In our experimental conditions the PI3K/Akt system, one of the major neuroprotective signaling pathways, was activated after caffeine treatment (Fig. 3A–C). On the other hand, MAPKs were not activated to the same level by the administration of caffeine (Fig. 3D). Moreover, the inhibition of the PI3K/Akt system abolished the cytoprotective effect of caffeine (Fig. 3E), suggesting that the cytoprotective effect of caffeine is associated with PI3K/Akt activation. Caffeine is a constituent of coffee, tea, and several other foods such as cacao. It might be beneficial to investi-

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