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Keywords:

  • amyloid-beta;
  • donepezil;
  • GSK-3β;
  • nAChRs;
  • neuroprotection;
  • PP2A

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. References
Thumbnail image of graphical abstract

The main purpose of this study was to evaluate whether donepezil, acetylcholinesterase inhibitor, shown to play a protective role through inhibiting glycogen synthesis kinase-3β (GSK-3β) activity, could also exert neuroprotective effects by stimulating protein phosphatase 2A (PP2A) activity in the amyloid-beta (Aβ)42-induced neuronal toxicity model of Alzheimer's disease. In Aβ42-induced toxic conditions, each PP2A and GSK-3β activity measured at different times showed time-dependent reverse pattern toward the direction of accelerating neuronal deaths with the passage of time. In addition, donepezil pre-treatment showed dose-dependent stepwise increase of neuronal viability and stimulation of PP2A activity. However, such effects on them were significantly reduced through the depletion of PP2A activity with either okadaic acid or PP2Ac siRNA. In spite of blocked PP2A activity in this Aβ42 insult, however, donepezil pretreatment showed additional significant recovering effect on neuronal viability when compared to the value without donepezil. Moreover, donepezil partially recovered its dephosphorylating effect on hyperphosphorylated tau induced by Aβ42. This observation led us to assume that additional mechanisms of donepezil, including its inhibitory effect on GSK-3β activity and/or the activation role of nicotinic acetylcholine receptors (nAChRs), might be involved. Taken together, our results suggest that the neuroprotective effects of donepezil against Aβ42-induced neurotoxicity are mediated through activation of PP2A, but its additional mechanisms including regulation of GSK-3β and nAChRs activity would partially contribute to its effects.

We investigated neuroprotective mechanisms of donepezil against Aβ42 toxicity: Donepezil increased neuronal viability with reduced p-tau by enhancing PP2A activity. Despite of blocked PP2A activity, donepezil showed additional recovering effect on neuronal viability, which findings led us to assume that additional mechanisms of donepezil including its inhibitory effect on GSK-3β activity and activating role of nicotinic AChRs might be involved.

Abbreviations used

amyloid-beta

AChE

acetylcholinesterase

AChEIs

acetylcholinesterase inhibitors

AD

Alzheimer's disease

CCK-8

cell counting kit-8

GSK-3β

glycogen synthase kinase-3β

Hcy

homocysteine

IRs

immunoreactivities

LCMT-1

leucine carboxyl methyltransferase-1

MLA

mecamylamine

MTT

methylthiazolyl blue tetrazolium bromide

nAChRs

nicotinic acetylcholine receptors

NBM

neurobasal media

NFTs

neurofibrillary tangles

NMDA

N-methyl-d-aspartate

OA

okadaic acid

OD

optical density

PME-1

phosphatase methyltransferase-1

PP2A

protein phosphatase 2A

SDS-PAGE

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

SD

Sprague-Dawley

Ser

serine

siRNA

small interfering RNA

TBST

tris-buffered saline containing 0.05% Tween-20

Thr

threonine

Alzheimer's disease (AD) is the most common neurodegenerative disease causing dementia, pathologically characterized by extracellular deposits of amyloid-beta (Aβ) protein, intracellular neurofibrillary tangle (NFT) composed of hyperphosphorylated forms of the microtubule-associated Tau protein, neuronal loss and neurotransmitter dysfunction (Selkoe 2004). Numerous attempts at developing novel therapeutics targeting AD based on these pathophysiological findings have failed, but acetylcholinesterase inhibitors (AChEIs; donepezil, galamtamine, and rivastigmine) and N-methyl-d-aspartate (NMDA) antagonist, memantine, are currently the only approved agents for the treatment of AD (Farrimond et al. 2012; Gauthier and Molinuevo 2013). In general, AChEIs have been known to possess no ability to halt the progression of the disease, providing only symptomatic efficacy and marginal therapeutic benefits. However, mounting evidence from pre-clinical and clinical studies points toward neuroprotective roles of AChEIs, especially, donepezil (Leyhe et al. 2009; Shen et al. 2010; Min et al. 2012). Moreover, even in AD patients, neuroprotective roles of AChEIs have been demonstrated through retardation of the progression of brain atrophy, indicating plausible disease modifying effect of donepezil by attenuating neuronal death (Hashimoto et al. 2005). A thorough study of the neuroprotective potential of donepezil, therefore, is appropriate at this juncture.

In our recent study, neuroprotective effects of donepezil against Aβ-induced neuronal toxicity were mediated through inhibition of GSK-3β activity via the activation of Akt in a cholinergic-dependent manner (Noh et al. 2009). One shortcoming, however, was that the effects of donepezil on the protein phosphatase 2A (PP2A) activity were not evaluated.

Glycogen synthase kinase-3β (GSK-3β) is one of the major kinases responsible for Tau hyperphosphorylation (Kosik 1992). Evidence suggesting the presence of mutual regulatory systems between kinases including GSK-3β or Akt and phosphatases such as PP2A (Wang et al. 2007; Chen et al. 2010; Hashiguchi and Hashiguchi 2013) prompted us to evaluate the effects of Aβ on cell viability and regulation of PP2A, GSK-3β activity, and the neuroprotective effects of donepezil on them and the expression level of tau phosphorylation against Aβ-induced neuronal injury. Numerous studies have shown that tau is a downstream target of Aβ, which increases GSK-3β activity resulting in hyperphosphorylation of tau (Koh et al. 2007; Noh et al. 2009; Lahmy et al. 2013). On the other hand, it is reported that Aβ induces tau phosphorylation by decreasing PP2A activity (Park et al. 2012).

On the basis of observations suggesting that Aβ induces tau hyperphosphorylation by decreasing PP2A activity or increasing GSK-3β activity, we first tried to evaluate the effects of Aβ42 on cell viability, and the time course of PP2A and GSK-3β activity. And then, to explore whether donepezil in Aβ-induced toxic condition has neuroprotective role by activating PP2A, direct measurement of PP2A activity and quantitative analysis of methyl/demethylated form of PP2A and expression levels of tau phosphorylation were analyzed with/without depleting PP2A activity by a selective antagonist of PP2A, okadaic acid (OA), or PP2A siRNA transfection.

According to recent evidence, the neuroprotective roles of AChEIs are mediated through the stimulation of α7-nAChRs and PI3K/Akt pathways against Aβ toxicity (Takada et al. 2003; Noh et al. 2009) or glutamate-induced excitotoxicity (Akaike et al. 2010; Shen et al. 2010). Therefore, lastly, we evaluated whether donepezil-mediated nAChRs activation also has a role in regulating PP2A activity in the same model.

In this study, we demonstrated that neuroprotective effects of donepezil against Aβ42-induced model of neurotoxicity were mediated through the activation of PP2A, although additional mechanisms including regulation of GSK-3β and nAChRs activities may contribute to its neuroprotective roles.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. References

Materials

Neurobasal media (NBM) and B27 supplement were purchased from GIBCO (Invitrogen Corporation, Grand Island, NY, USA). Aβ42, OA, mecamylamine (MLA), radio immunoprecipitation assay buffer, protein protease inhibitor cocktail, trypan blue solution, insulin, and DNase I were obtained from Sigma-Aldrich (St. Louis, MO, USA). Donepezil hydrochloride {(±)-2-[(1-benzylpiperidin-4-yl) methyl]-5, 6-dimethoxy-indan-1-one monohydrochloride: E2020} was provided by Eisai Co., Ltd. (Tokyo, Japan). Before use, it was dissolved in distilled water and diluted with culture medium to the desired concentrations.

Primary cultures of cortical neurons

All procedures on animals were performed in accordance with the Hanyang University guidelines for the care and use of laboratory animals. Every effort was made to minimize the number of animals used and animal suffering. All animals were used only once.

Primary cultures of cortical neurons were acquired from the cerebral cortices of fetal Sprague-Dawley (SD) rats (at 16 days gestation, Orient Bio Inc., Seoul, Korea) (Noh et al. 2009). Briefly, rat embryos of both sexes were decapitated, and their brains were isolated and put in a Petri dish half-filled with ice-cold Hank's balanced salt solution (HBSS; Gibco BRL). Single cells separated from whole cerebral cortices were seeded on 100-mm Corning dishes (5 × 106 cells/cm2) coated with poly-l-lysine (Sigma-Aldrich) or glass cover slips placed in 6- or 24-well Nunc plates (5 × 105, 2.5 × 106 cells/cm2) and were suspended in 10% fetal bovine serum (FBS)/modified Eagle's medium (MEM). After 24 h, the media were changed to the serum-free media, NBM supplemented with B27. Cultures were kept at 37°C under a humidified 5% CO2 atmosphere. Two days after plating, non-neuronal cells were removed by adding 5 μM cytosine arabinoside for 24 h. Only mature cultures (7 days in vitro) were used for experiments. The cultures consisted of about 85% primary cortical neurons.

Aβ42 oligomer preparations

Aβ42 peptide (Sigma-Aldrich) was dissolved to 1 mM in hexafluoro-2-propanol and stored in dried form at −80°C after evaporation of the solvent. The dried peptide was resuspended in dimethyl sulfoxide to a final concentration of 5 mM, vortexed thoroughly, and sonicated for 10 min. The peptide was then diluted to 100 μM with ice-cooled phenol red-free Ham's F12 medium. Then, the peptide was placed at 4°C overnight to form Aβ42 oligomers. The oligomer solution was centrifuged briefly, and the supernatant was used for the experiments.

MTT assay, cell counting kit-8

Fifty microliter of 2 mg/mL methylthiazolyl blue tetrazolium bromide (MTT) (Sigma-Aldrich) was added to each well following the addition of 200 μL medium. An aliquot (220 μL) was removed from each well, and 150 μL of dimethyl sulfoxide was added. Optical density (OD) at 540 nm was evaluated on an ELISA plate reader after dissolving precipitates with a microplate mixer for 10 min. All results were corrected for the OD of similarly conditioned wells that did not contain cells (Koh et al. 2003). Live cells were counted with a Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan) by combining WST-8 and 1-Methoxy PMS. Briefly, 10 μL of kit reagent was added to cultured cells in 96-well plates and incubated for an additional 3 h. Cell viability was obtained with an ELISA reader at 450 nm.

PP2A activity assay

Phosphatase assays were performed using a PP2A phosphatase assay kit (catalog number 17-131; Upstate Biotechnology, Temecula, CA, USA). Briefly, cells were lysed with lysis buffer (150 mM NaCl, 25 mM Tris-HCl, 2 mM EDTA, 10% glycerol, 1% NP-40, and Sigma protease inhibitor cocktail). The phosphatase activity of the supernatants was assayed. PP2A was immunoprecipitated with monoclonal anti-PP2A antibody and rocked overnight at 4°C. Twenty microliters of protein G (Sigma-Aldrich) was added to immunoprecipitate the PP2A with rocking for 2 h at 4°C. The PP2A-bound beads were washed sequentially with lysis buffer and pNPP assay buffer. The reactions were initiated by adding pNPP assay buffer. In this reaction, phosphate groups are released from a synthetic substrate PP2A, phosphor peptide (K-R-pT-I-R-R, in which pT is phosphothreonine), over a period of 30 min.

Glycogen synthase kinase-3 activity assay

GSK-3β activity was evaluated using GSK-3 substrate phospho-glycogen synthase peptide-2 (Upstate) as described previously (Noh et al. 2009).

Western blot analysis

Phosphorylated and non-phosphorylated forms of the protein tau were measured by western blotting. After chemical treatment, cells were lysed with ristocetin-induced platelet agglutination buffer supplemented with phosphatase inhibitor. Cell lysates were centrifuged at 18 000 g for 20 min at 4°C. Protein concentrations were determined using the Bradford protein assay kit (Bio-Rad, Hercules, CA, USA). Equal amounts of protein (40 μg) from each sample were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, Buckinghamshire, UK). Membranes were blocked with 1% skim milk and incubated successively with specific antibodies against the PP2A-C subunit (1 : 1000, Upstate Biotechnology, Temecula, CA, USA), PP2Ac (1 : 500, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), methyl-PP2Ac (L309) (1 : 500, Upstate Biotechnology), demethyl-PP2Ac (L309) (1 : 500, Upstate Biotechnology), tau-5 (total tau; 1 : 1000, Invitrogen), and phospho-tau (Ser396) (1 : 1000, Invitrogen). Samples were washed with Tris-buffered saline containing 0.05% Tween-20 (TBST), and processed using an horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibody (Amersham Pharmacia Biotech, Piscataway, NJ, USA) and reactive bands were detected by ECL (Amersham Pharmacia Biotech, Piscataway, NJ, USA) and quantified with an image analyzer (Bio-Rad, Quantity One-4, 2, 0). The same membranes were probed for GAPDH as an internal control. All figures are representative of at least five independent experiments.

Transfection of small interfering RNA (siRNA)

SH-SY5Y cells were transfected with PP2Ac siRNA. Human PP2Ac small interfering RNA (siRNA; sc-43509) was purchased from Santa Cruz Biotechnology, Inc. The non-silencing control siRNA (sc-36869) and transfection of siRNA have previously been described. In brief, six-well plates of SH-SY5Y cells were cultured to 80% confluence, and then each well was resuspended in 2 mL siRNA transfection medium with 100 μL transfection reagent and 1.5 μg of PP2A siRNA or 1.5 μg of control siRNA. PP2A-Cα (N-25, sc-130237) antibody was used for western blots to monitor PP2AC gene expression knockdown 48 h after siRNA transfection.

Statistical analysis

All data are presented as means ± SEM of five or more independent experiments. Statistical comparisons between groups were analyzed by one-way anova followed by Tukey's post hoc comparisons. Two-tailed p-values less than 0.05 were considered statistically significant. All statistical analyses were performed using the SPSS 17.0 software package for Windows (SPSS, Seoul, Korea).

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. References

Determination of optimal toxic dose of Aβ42 for the assessing neuronal viability and PP2A activity

To determine optimal toxic dose of Aβ42 on neuronal viability, rat cortical neurons were treated with different concentrations of Aβ42 for 6 h. In addition, cell viability was measured using MTT and CCK-8 assays. As shown in Fig. 1a, cell viability was gradually reduced in a concentration-dependent manner. Cell viability was 83.7 ± 0.68% at 1 μM, 74.8 ± 1.28% at 5 μM, 66.8 ± 1.56% at 10 μM, 61.5 ± 0.83% at 20 μM, and 56.4 ± 1.85% at 50 μM, respectively, as compared to non-treated control (p < 0.01, in MTT assay). Based on these data, 10 or 20 μM could be selected as candidates for toxic concentrations because usually about 60–70% viability was appropriate in the study of Aβ42 induced neuronal toxicity.

image

Figure 1. Determination of optimal toxic dose of amyloid-beta (Aβ)42 in rat cortical neurons. (a) To determine optimal toxic dose of Aβ42 in neuronal viability. Rat cortical neurons were treated with different concentrations of Aβ42 for 6 h. Cell viability was measured using MTT and cell counting kit-8 (CCK-8) assays. (b) Effect of Aβ42 on protein phosphatase 2A (PP2A) activity. Rat cortical neurons were treated with different concentrations of Aβ42 for 6 h. And direct measurement of PP2A activity with ELISA method. (c) Relationship between PP2A and glycogen synthase kinase-3β (GSK-3β) activity in 20 μM Aβ42-induced neuronal toxicity model. The time course changes of cell viability, PP2A and GSK-3 activity. The data are mean (% of the non-treated group) ± SEM from five independent experiments. GSK-3 activity data are represented as mean (normalized to the non-treated group) ± SEM from five independent experiments. Each treatment group was compared with the other groups by Tukey's test after one-way anova. *p < 0.01 when compared with the non-treated group.

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To investigate the effect of Aβ42 on PP2A activity and what concentration is suitable for evaluating Aβ42 own inhibitory effect on PP2A activity, rat cortical neurons were treated with different concentrations of Aβ42 for 6 h. In addition, direct measurement of PP2A activity with ELISA method, as described in methods, was done in cell lysates. As shown in Fig. 1b, Aβ42 exhibited dose-dependent inhibitory effect on PP2A activity at all selected concentrations, and each PP2A enzyme activity measured at 1, 5, 10, 20, and 50 μM was 83.7 ± 0.64%, 67.1 ± 1.34%, 54.8 ± 2.92%, 26.5 ± 1.11%, and 8.53 ± 2.72%, respectively, when compared with basal level of the untreated control (p < 0.01, respectively).

It was essential procedure in this study to determine what concentration of Aβ42 was suitable for an appropriate evaluation of the excitatory effect of donepezil on PP2A activity, which was to be suppressed by Aβ42 toxic insult. Therefore, among two candidates of Aβ42 concentration based on viability study, we selected 20 μM as an optimal concentration for the next study. Because the suppressing effect of 20 μM Aβ42 on PP2A was about 70%, we thought that potentially recoverable 70% room was more suitable than the 45% one suppressed at 10 μM Aβ42 for evaluating the effect of donepezil on recovering PP2A activity inhibited by Aβ42.

Relationship between PP2A and GSK-3β activity in 20 μM Aβ42-induced neuronal toxicity model

To ascertain the time course changes of cell viability, PP2A and GSK-3 activity simultaneously, all of each was measured at different time points after 20 μM Aβ42 treatment. As shown in Fig. 1c, time course curves of PP2A and GSK-3 activity showed time-dependent reverse linear-like pattern toward the direction of accelerating neuronal cell death. That is, Aβ42 gradually suppressed PP2A activity with time, which was accompanied by stepwise increment of GSK-3 activity.

The interesting result obtained at 1 h time point was that activity changes of PP2A and GSK-3 preceded neuronal death. Activity changes of both were already detected before the commencement of cell death based on the data showing that cell viability was still 100% at that point. These findings mean that cell viability does not exactly reflect the level of PP2A enzyme activity. In other words, PP2A activity is not always dependent on cell viability.

Determination of optimal concentration of donepezil for evaluating its role in PP2A activity in 20 μM Aβ42-induced neurotoxicity model

To evaluate the effect of donepezil on neuronal viability, cortical neuronal cells were pre-treated with several concentrations of donepezil for 24 h, followed by 20 μΜ Aβ42 treatment for 6 h, and then we measured cell viability. Donepezil treatment gradually increased cell viability in a concentration-dependent manner to the level of 10 μM, but viability did not increase when donepezil concentration was over 10 μM, and decreased at 100 μM (Fig. 2a). Therefore, 10 μM was selected as a candidate concentration of donepezil to be avoided for its own toxic effect on neuronal cell death.

image

Figure 2. Determination of optimal concentration of donepezil in 20 μM amyloid-beta (Aβ)42-induced neuronal toxicity model. (a) Effect of donepezil in 20 μM Aβ42-induced neurotoxicity on neuronal viability. Rat cortical neuronal cells were pre-treated with several concentrations of donepezil (0.1, 1, 10, and 100 μΜ) for 24 h, followed by 20 μΜ Aβ42 treatment for 6 h, and then cell viability was measured using MTT and cell counting kit-8 (CCK-8) assays. (b) Effect of donepezil in 20 μM Aβ42-induced neurotoxicity on protein phosphatase 2A (PP2A) enzyme activity. And direct measurement of PP2A activity with ELISA method. The data are presented as means (% of non-treated group) ± SEM from five independent experiments. Each treatment group was compared with the other groups using Tukey's test after one-way anova. *p < 0.01 when compared with the non-treated group. #p < 0.01 when compared with the group treated with 20 μM Aβ42 only.

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In the next step, we tried to investigate whether 10 μM, candidate concentration of donepezil in cell viability study, was also sufficient for increasing PP2A enzyme activity in the model of 20 μΜ Aβ42-induced neuronal injury. To address this issue, cortical neuronal cells were pre-treated with different concentrations of donepezil (0.1, 1, 10, and 100 μΜ) for 24 h, followed by 20 μΜ Aβ42 for 6 h. And direct measurements of PP2A activity with ELISA method, as described in methods, were done in cell lysates.

Despite Aβ42-induced decrement of PP2A activity, Donepezil pre-treated groups showed gradual increment of PP2A activity in a concentration-dependent pattern (Fig. 2b). An exclusive treatment of 20 μΜ Aβ42 decreased PP2A activity to 28.7 ± 1.71% level of non-treated control (p < 0.01, Fig. 2b), but each PP2A activity in different concentrations of donepezil-treated group was slowly restored by increasing the concentration. Each PP2A activity was 39.6 ± 0.09% at 0.1 μM, 47.3 ± 0.82% at 1 μM, 65.9 ± 3.43% at 10 μM, and 77.1 ± 0.03% at 100 μM, respectively. The fact that 10 μM donepezil had the capacity of recovering PP2A activity up to 65% of basal level allowed us to select 10 μM as an optimal final concentration for subsequent studies. These results suggest that the protective effects of donepezil on rat cortical neurons are mediated at least in part through an increase in PP2A activity

Effect of OA on cell viability, PP2A activity, and tau phosphorylation in rat cortical neurons

To explore the effect of OA, a selective PP2A inhibitor, on neuronal viability, rat cortical neurons were treated with different concentrations of OA for 3 h. As shown in Fig. 3a, no viability changed at 25 and 50 nM, but significantly decreased to 84.2% at the concentration of 100 nM and their viability reached to 74.1% at 200 nM OA, as compared to the control (p < 0.01, in MTT assay). However, each PP2A activity measured at same concentrations was gradually reduced by increasing the dosage of OA. At 25, 50, 100, and 200 nM, each PP2A activity was 82.1 ± 0.26%, 57.6 ± 2.41%, 32.4 ± 0.03%, and 29.7 ± 3.01%, respectively, as compared to control.

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Figure 3. Effect of okadaic acid on cell viability, protein phosphatase 2A (PP2A) activity, and tau phosphorylation in rat cortical neurons. (a) Effect of okadaic acid (OA), a selective PP2A inhibitor, on neuronal viability. Rat cortical neurons were treated with different concentrations of OA (25, 50, 100, and 200 nΜ) for 3 h. Cell viability was measured using MTT and cell counting kit-8 (CCK-8) assays. (b) Effect of OA on PP2A activity. Rat cortical neurons were treated with different concentrations of OA for 3 h. And direct measurement of PP2A activity with ELISA method. The data are presented as means (% of non-treated group) ± SEM from five independent experiments. Each treatment group was compared with the other groups using Tukey's test after one-way anova. (c) Effect of OA on tau phosphorylation at S-396 site by inhibiting PP2A activity. Rat cortical neurons were treated with different concentrations of OA (10, 30, 50, and 70 nΜ) for 3 h. IRs of phosphorylated Tau (S-396) and Tau were assessed by western blotting. Quantitative data were expressed in arbitrary units normalized to the simultaneously assayed non-treated group's value. Each treatment group was compared with the other groups using Tukey's test after one-way anova (n = 5). *p < 0.01 compared with the non-treated group.

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When OA concentration was over 50 nM, neuronal cell deaths began to be detected and PP2A inhibitory effect of OA at that concentration was about 43%, as compared to the untreated control (Fig. 3a and b). Therefore, 50 nM was deemed appropriate concentration for studies to be performed next to evaluate the regulating effects of donepezil on PP2A activity and tau phosphorylation against the 20 μM Aβ42-induced toxicity model. This idea was based on the fact that OA at 50 nM concentration could be avoided for unwanted cell death and about 43% inhibitory effect on PP2A enzyme activity at that concentration would be sufficient for designing conditions of PP2A blockade.

In addition, we tried to evaluate whether OA is able to increase the level of tau phosphorylation at S-396 site by inhibiting PP2A activity. Western blots were performed and dose-dependent effects of OA on tau phosphorylation were evaluated by measuring the ratio of p-tau (S-396) and total tau. To confirm whether 50 nM is the best candidate concentration of OA, each different OA concentrations below 100 nM was used for this study, from 10 to 70 nM. The level of tau phosphorylation at S-396 site was gradually increased by increasing OA concentration, as shown in Fig. 3c. The ratio of p-tau/total tau measured at relatively lower concentrations did not significantly change (0.98 ± 0.06 at 10 nM and 1.12 ± 0.03 at 30 nM) as compared to the control. The ratio measured at higher concentrations, however, significantly increased at concentrations of 50 and 100 nM (1.28 ± 0.04 and 1.30 ± 0.05, p < 0.01, Fig. 3c). From these results, we were able to deduce that 50 nM OA could sufficiently inhibit PP2A activity without altering cell viability, thereby inducing tau phosphorylation at S-396 site. Therefore, the concentration of 50 nM was determined as an optimal one of OA, and this was used as an eliminating agent for donepezil's own enhancing capability of PP2A activity in Aβ- induced cell injury model.

Effects of donepezil on cell viability and assessment of its effect on PP2A activity and the level of tau phosphorylation when PP2A activity is deleted by OA

We also investigated the effect of donepezil on PP2A methylation and PP2A demethylation. We found that the ratio of IRs for methylated PP2AC (Leu309) to that for the PP2AC subunit itself [methylated PP2AC (Leu309)/PP2AC subunit] decreased in 20 μM Aβ42-treated cells (0.71 ± 0.07, p < 0.01), and that this effect was reversed by pre-treatment with 10 μM donepezil (0.95 ± 0.02, p < 0.01) in Fig. 4a. Furthermore, additional treatment with OA (0.66 ± 0.07, p < 0.01), a PP2A inhibitor, or exposure to OA alone resulted in a reduced ratio of IRs for methylated PP2AC (Leu309) to that for the PP2AC subunit itself. The ratio of IRs for demethylated PP2AC (Leu309) showed in a complementary fashion to that for methylated PP2AC (Aβ42 treated; 1.22 ± 0.06, p < 0.01, combined Aβ42 with donepezil pre-treated group; 1.07 ± 0.03, p < 0.01, and additional treatment with OA group; 1.33 ± 0.04, p < 0.01, right blots in Fig. 4a), as might be expected, and the effects of donepezil on PP2A activity paralleled those on PP2AC methylation (Aβ42 treated; 22.6 ± 1.61%, p < 0.01, combined Aβ42 with donepezil pre-treated group; 56.6 ± 2.04%, p < 0.01, and additional treatment with OA group; 28.7 ± 0.95%, p < 0.01 in Fig. 4b). These finding suggest that donepezil plays a role in PP2A methylation/demethylation.

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Figure 4. Effect of donepezil by using okadaic acid (OA) in amyloid-beta (Aβ)42-induced neurotoxicity on protein phosphatase 2A (PP2A) activity and tau phosphorylation. (a) Effect of donepezil by using OA on PP2A methylation and PP2A demethylation. Rat cortical neuronal cells were pre-treated with 10 μM donepezil for 24 h, followed by 20 μΜ Aβ42 treatment for 6 h. Donepezil plus/minus 50 nM OA was added for 3 h before Aβ42. IRs of PP2AC, methylated PP2A (Leu309) and demethylated PP2A (Leu309) were assessed by western blotting. Quantitative data were expressed in arbitrary units normalized to the simultaneously assayed non-treated group's value. (b) Effect of donepezil by using OA on PP2A activity. PP2A activity was measured using ELISA method. The data are represented as means (% of non-treated group) ± SEM from five independent experiments. (c) Effect of donepezil with/without treatment OA on tau phosphorylation (S-396). IRs of phosphorylated Tau (S-396) and Tau were assessed by western blotting. Quantitative data were expressed in arbitrary units normalized to the simultaneously assayed non-treated group's value. Each treatment group was compared with the other groups using Tukey's test after one-way anova (n = 5). *p < 0.01 when compared with the non-treated group. #p < 0.01 when compared with the group treated with 20 μM Aβ42 only. +p < 0.01 when compared with the group treated with 20 μM Aβ42 and 10 μM donepezil.

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To evaluate the effect of donepezil (with or without OA) on hyperphosphorylated tau induced by Aβ42, we examined the IRs of phosphorylated tau (S-396) (Fig. 4c). The level of tau phosphorylation at S-396 site increased in exclusively Aβ42-treated group (1.32 ± 0.05, 2nd lanes in 1st and 2nd blot in Fig. 4c), as compared to the control (p < 0.01). However, when donepezil pre-treatment was combined with Aβ42, the ratio of p-tau/total tau decreased (1.08 ± 0.01, middle lane of 1st and 2nd blot), as compared to the Aβ42 only treated group (p < 0.01). However, OA treatment in this condition reversed the ratio of p-tau/total tau similar to that of exclusive Aβ42 group (1.33 ± 0.05, p < 0.01), which implied that OA abolished the PP2A-activating effect of donepezil. These results suggest that donepezil affects the level of tau phosphorylation at S-396 sites by enhancing PP2A activity.

Effects of donepezil on cell viability, assessment of its effect on PP2A activity and the level of tau phosphorylation when PP2A activity is deleted by PP2Ac siRNA

To further explore the possible neuroprotective effects of donepezil on PP2A activation, cell viability and tau phosphorylation, PP2AC siRNA were transiently transfected into SH-SY5Y cells and compared between the Aβ42 combined with donepezil pre-treated group and the Aβ42 only treated one. Approximately, 40% knockdown was achieved for PP2AC expression as shown in Fig. 5a. As shown in left 3 lanes of 2nd blot in Fig. 5b, IRs of total PP2Ac did not change in non-treated PP2Ac siRNA groups, and about 1.6-fold increase for PP2A demethylation (L309) was noted in the Aβ42 only treated group (1.6 ± 0.08, p < 0.01). However, Aβ42 with donepezil pre-treated one showed only about 1.1-fold increase (1.14 ± 0.06, p < 0.01), as compared to the control. This suggests that donepezil has the ability to trigger PP2A activity (i.e., implies increasing methylated level of PP2Ac) in presence of Aβ42 toxicity. And, as was expected, in the conditions of PP2A siRNA being transfected (lanes 4–6 in Fig. 5b), levels of both PP2A demethylation and total PP2AC significantly decreased in all three groups (non-treated; 0.5 ± 0.01, p < 0.01, only Aβ42 treated; 0.5 ± 0.02, p < 0.01, and combined Aβ42 with donepezil pre-treated group; 0.6 ± 0.07, p < 0.01). These findings mean that the ability of donepezil to activate PP2A is nearly abolished with PP2A siRNA.

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Figure 5. Effects of donepezil by protein phosphatase 2A (PP2A) siRNA transfection on cell viability, PP2A activity, and tau phosphorylation in human neuroblastoma cells. (a) Effects of PP2A siRNA transfection into SH-SY5Y cells. Human neuroblastoma SH-SY5Y cells were transiently transfected with control siRNA and siRNA of PP2AC. After transfection with 48 h, IRs of PP2AC was assessed by western blotting. Quantitative data were expressed in arbitrary units normalized to the simultaneously assayed non-treated group's value. (b) Effects of donepezil by PP2A siRNA transfection in SH-SY5Y cells. After transfection with 48 h, SH-SY5Y cells were pre-treated with 10 μM donepezil for 24 h, followed by 20 μΜ Aβ42 treatment for 6 h. IRs of PP2AC and PP2A demethylation (L309) were assessed by western blotting. Quantitative data were expressed in arbitrary units normalized to the simultaneously assayed non-treated group's value. (c) Effects of donepezil by PP2A siRNA transfection on cell viability and PP2A activity in SH-SY5Y cells. Cell viability was measured using MTT assays and PP2A activity was measured using ELISA method. The data are represented as means (% of non-treated group) ± SEM from five independent experiments. (d) Effects of donepezil by PP2A siRNA transfection on phosphorylated tau (S-396) in SH-SY5Y cells. IRs of phosphorylated Tau (Ser396), Tau-5, and PP2AC were assessed by western blotting. Quantitative data were expressed in arbitrary units normalized to the simultaneously assayed non-treated group's value. Each treatment group was compared with the other groups using Tukey's test after one-way anova (n = 5). *p < 0.01 when compared with the non-treated group. #p < 0.01 compared with the group treated with 20 μM Aβ42 only. +p < 0.01 when compared with the group treated with 20 μM Aβ42 by siRNA PP2A.

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Next, for direct measurement of PP2A activity in the same conditions, phosphatase assays were done using PP2A assay kit, as described in methods. In the absence of PP2Ac siRNA (left 3 black bars of Fig. 5c), PP2A enzyme activity in Aβ42 only treated group significantly decreased to 30.3 ± 1.26% level of the control, but this activity measured in the group of combined Aβ42 and donepezil pre-treatment recovered to about 67.4 ± 3.08% level of the control group (p < 0.01). However, in PP2A siRNA transfected conditions (right 3 black bars in Fig. 5c), PP2A activities significantly decreased in all three groups (non-treated; 23.2 ± 1.37%, Aβ42 treated; 13.3 ± 1.55%, and combined Aβ42 with donepezil pre-treated group; 14.4 ± 1.41%, p < 0.01). And the IRs of PP2Ac obtained from western blots as shown in Fig. 5d showed similar results with direct measurement of PP2A activity.

Importantly, even in depleted PP2A activity with PP2A siRNA transfection, the measured cell viability of donepezil combined with Aβ42 treated group showed a significant twofold increase as compared to the Aβ42 only treated group (p < 0.01, 6th and 5th gray bars in Fig. 5c). Moreover, the IRs of phosphorylated tau level (S-396) detected in combined treated group (0.63 ± 0.02, p < 0.01) had also significantly decreased compared to Aβ42 only treated group (0.82 ± 0.01, p < 0.01, 4th and 3rd dark gray bars in Fig. 5d). These observations led us to assume that other neuroprotective mechanisms of donepezil, including the known inhibitory role of GSK-3β activity or the stimulatory effect on nAChRs, as delineated in our previous data (Noh et al. 2009), might be of considerable significance.

Another surprising finding was that phosphorylated tau (S-396) level found in PP2A siRNA transfected group without any treatment (2nd band of 1st blot in Fig. 5d) did not increase, instead somewhat decreased, in contrast to our initial expectation. In a previous study, Zhou et al. showed that the blocking of PP2Ac with siRNA reduced tau phosphorylation at the S-396 and S-214 sites, although we observed the phenomenon only at the S-396 site. It is suggested that a complicated regulation of tau phosphorylation exists in neuroblastoma cells when PP2A level is altered (Zhou et al. 2009). It has also been reported that PP2A knockdown increases the level of p-GSK-3β (Ser9), which may play another role in reducing tau phosphorylation at S-396 by inhibiting GSK-3β activity (Zhou et al. 2009). And the roles of these mutual regulatory systems in determining kinase/phosphatase activity are further supported by the co-immunoprecipitation data of GSK-3 and PP2A (Lee et al. 2005), in which undulating GSK-3β phosphorylation/dephosphorylation is regulated by PI3K/Akt (phosphorylation) and PP2A (dephosphorylation), respectively, in neuroblastoma cells.

Thus, donepezil shows its neuroprotective effects not merely through increasing PP2A activity resulting in tau de-phosphorylation, but also by exerting inhibitory effects on GSK-3β activity. This led us to investigate whether the PP2A activating effect of donepezil may also be mediated via nAChRs activation.

Effects of donepezil through nicotinic acetylcholine receptors (nAChRs)

Recent evidence suggests that the neuroprotective roles of AChEIs are mediated via stimulation of α7-nAChRs and PI3K/Akt pathway against Aβ (Takada et al. 2003; Noh et al. 2009) or glutamate-induced excitotoxicity (Akaike et al. 2010; Shen et al. 2010). Therefore, we examined whether donepezil-mediated nAChRs activation also has a role in regulating PP2A activity in the same model.

To demonstrate donepezil-mediated regulation of PP2A activity via nAChRs, we pre-treated a non-specific nAChRs antagonist (10 μM MLA) in donepezil-containing medium for 24 h (Fig. 6). Compared with the 10 μM donepezil and 20 μM Aβ42-combine treatment group (68.73 ± 2.41%, p < 0.01), PP2A activity decreased in the 10 μM MLA-pre-treated group (59.9 ± 0.61%, p < 0.01). MLA did not completely abolish donepezil's effect on PP2A activity. This finding indicates that the effect of donepezil on PP2A activity is partially mediated by nAChRs stimulation. Taken together, our results indicate that the neuroprotective effects of donepezil against Aβ42-induced neuronal toxicity are mediated through activation of PP2A, although additional mechanisms including the regulation of GSK-3β and nAChRs activities may also be involved.

image

Figure 6. Effects of donepezil through nicotinic acetylcholine receptors (nAChRs). We pre-treated a non-specific nAChRs antagonist [10 μM mecamylamine (MLA)] in donepezil-containing medium for 24 h. And direct measurement of PP2A activity with ELISA method. The data are represented as means (% of non-treated group) ± SEM from five independent experiments. Each treatment group was compared with the other groups using Tukey's test after one-way anova. *p < 0.01 when compared with the non-treated group. #p < 0.01 compared with the group treated with 20 μM Aβ42 only. +p < 0.01 compared with the group treated with 20 μM Aβ42 and 10 μM donepezil. ¥p < 0.01 compared with the group treated with 20 μM Aβ42 and 10 μM donepezil and 50 nM okadaic acid (OA).

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. References

Donepezil (R,S-1-benzyl-4-[(5,6-dimethoxy-1-indanon)-2-yl] methylpiperidine hydrochloride) (E2020), a potent AChE inhibitor (Sugimoto et al. 1995), has been suggested as an effective symptomatic therapeutic agent for AD (Takada et al. 2003; Arias et al. 2005; Takada-Takatori et al. 2006, 2008). There is evidence that donepezil exerts its neuroprotective effect by activating PI3K-Akt in various cell types related to cognitive function (Arias et al. 2005; Takada-Takatori et al. 2006) by inhibiting GSK-3 (Koh et al. 2007). However, the exact effects of donepezil on PP2A activity in β42-induced neuronal toxicity have yet to be evaluated.

The main purpose of this study was to demonstrate whether the neuroprotective effects of donepezil against Aβ42-induced neurotoxicity could be mediated by enhancing PP2A activity and to evaluate its effect on the level of tau phosphorylation.

In this study, 20 μM Aβ42 treatment sufficiently suppressed PP2A activity in primary cultured cortical neurons, and 10 μM donepezil pre-treatment suitably activated PP2A enzymes for our subsequent studies. And both okadaic acid and PP2A siRNA transfection abolished donepezil's PP2A activating capacity and tau de-phosphorylating effect. And PP2A activity to be enhanced by donepezil was also partially blocked by nAChRs antagonist. These results suggest that the neuroprotective effects of donepezil against Aβ42-induced neurotoxicity are highly dependent on its stimulatory effect on PP2A activity as well as nAChRs.

With the neuroprotective effects of donepezil focused solely on the aspect of PP2A activation, we may overlook important issues raised in this study. First, there is a mutual regulatory system between kinases and phosphatases activity (Zhou et al. 2009). Second, besides its PP2A activating capacity, donepezil has other important functions including the inhibitory effect on GSK-3β activity via the activation of PI3K/Akt pathway, and the modulating effect of nAChRs activity (Noh et al. 2009). Moreover, in given concentrations Aβ42 tended to accelerate neuronal cell death by increasing GSK-3β activity and decreasing PP2A activity as shown in Fig. 1c. However, in conditions such as Aβ42 toxic insult in neuronal cells, normal compensatory or counteracting mutual regulations of these kinases/phosphatases were found to be disturbed (Fig. 1c). If it is a physiological condition, or if knockdown is induced in one part of the kinase/phosphatase, activity in the counter part of kinase/phosphatase would be appropriately regulated toward the direction of cell survival or maintenance of this balancing system (Zhou et al. 2009).

The most interesting finding was that even in the 20 μM Aβ42-induced toxic state, aligned with the PP2A-depleted condition via OA or PP2A siRNA transfection, donepezil still exhibited significant effect not only on cellular viability, but also on the level of phosphorylated tau at the S-396 site. Thus, effects of donepezil in this condition were presented as additional increment of neuronal viability and tau de-phosphorylation at S-396 site (Figs 4, 5). We therefore assumed that other neuroprotective mechanisms of donepezil, including its inhibitory role in GSK-3β activity or stimulatory effect on nAChRs, might be involved in these processes.

PP2A is one of the major serine/threonine phosphatases that plays important roles in many biological processes (Janssens and Goris 2001; Lechward et al. 2001). It can dephosphorylate tau at multiple sites (Gong et al. 2000; Liu et al. 2005). PP2A activity is regulated by post-translational modification via phosphorylation (Tyr307) and methylation (Leu309) of its catalytic C subunit (Xing et al. 2008; Eichhorn et al. 2009). Methylation of the C subunit is performed by cytoplasmic leucine carboxyl methyltransferase-1 (LCMT-1), and demethylation is made by nuclear phosphatase methyltransferase-1 (PME-1) (Longin et al. 2004). Decreased methylation of PP2A has been proposed as a link between the elevated plasma homocysteine (Hcy) levels and tau hyperphosphorylation observed in AD (Zhang et al. 2008). Importantly, PP2A activity is down-regulated in brain tissue from AD patients (Gong et al. 1994). The specific inhibitor of serine/threonine PP1 and PP2A, OA, can induce tau hyperphosphorylation and neuronal cell death (Wang et al. 2001; Vale and Botana 2008). In particular, a previous study demonstrated that PP2A regulates an amyloid-beta-induced apoptosis (Yin et al. 2006). Another study showed that the caspase cleavage of the amyloid precursor protein induces tau phosphorylation by decreasing PP2A activity (Park et al. 2012). PP2A has also been implicated in the phosphorylation of tau and aggregation involved in aging and AD (Liu et al. 2005; Wang et al. 2007). Previous studies have shown that PP2A site-specifically dephosphorylates tau in vitro (Qian et al. 2010) and that Aβ induced tau phosphorylation (Koh et al. 2007; Noh et al. 2009; Lahmy et al. 2013) by decreasing PP2A activity or increasing GSK-3 activity (Park et al. 2012). The data presented here demonstrate that exposure to Aβ42 decreases PP2A activity. These results are consistent with the observation that Aβ peptides decrease PP2A activity in endothelial cells (Hsu et al. 2007) and cleavage of the amyloid precursor protein (APP) at the Asp664 residue-induced tau phosphorylation by decreasing PP2A activity (Park et al. 2012).

Recently, the role of PP2A in the pathogenic mechanisms of AD has been emphasized (Sontag et al. 2007). PP2A links Hcy metabolism with regulation of tau and amyloid precursor protein (Liu and Wang 2009). We observed that PP2A inhibitor (OA) treatment increased the level of tau phosphorylation at the S-369 site, which is consistent with previous findings (Lim et al. 2010; Akasofu et al. 2003; Plattner et al. 2006; Zhou et al. 2008; Liu and Wang 2009). Our results also confirm that the effects of donepezil through PP2A activation are inhibited by PP2A inhibitor (OA) (Fig. 4b). As shown in our previous report, donepezil decreases phosphorylated tau (Ser396, Thr231, Ser199) in models of Aβ42-induced neurotoxicity (Noh et al. 2009). Also, effects of donepezil were associated with an increase in PP2A activity, correlated with the level of tau phosphorylation. Consequently, we tried to inhibit PP2Ac expression by means of RNA interference. The effects of donepezil through PP2A activation were also inhibited by PP2A-siRNA (Fig. 5c).

On the other hand, nAChRs exerted neuroprotective effects against a variety of toxicants (Picciotto and Zoli 2008; Del Barrio et al. 2011). Nicotinic agonists have been reported to be effective against Aβ-induced toxicity (Arias et al. 2005). Our previous results suggested that activation of nAChRs plays an important role in the protective effects of donepezil against Aβ-induced toxicity with the PI3K/Akt signaling pathway (Noh et al. 2009). Examining whether PP2A activity was regulated by nAChRs stimulation, we found that PP2A activity was regulated via nAChRs activity. However, even in the depleted activity of nAChRs induced by mecamylamine, a non-specific antagonist of nAChRs, donepezil pre-treatment demonstrated to have an additional residual PP2A activity. This finding indicates that the effects of donepezil on PP2A activity might be partially related to nAChRs stimulation, with the main part being mediated via a direct stimulation of PP2A or other mediating pathway including a mutual regulatory system of kinase/phosphatase.

Conclusion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. References

Taken together, our results suggest that the neuroprotective effects of donepezil against Aβ42-induced neurotoxicity are mediated through activation of PP2A, but its additional mechanisms including regulation of GSK-3β and nAChRs activity also partially contribute to its effects.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. References

This study was supported by a grant of the Korea Health technology R&D Project, Ministry of Health & Welfare, Republic of Korea. (A091049) and by the cluster research fund of Hanyang University (HY-2009-C). There are no conflicts of interest in this study.

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  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. References
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