NeuroToxicology 40 (2014) 23–32

Contents lists available at ScienceDirect

NeuroToxicology

Donepezil inhibits the amyloid-beta oligomer-induced microglial activation in vitro and in vivo Hyo Geun Kim a,1, Minho Moon b,1,2, Jin Gyu Choi a, Gunhyuk Park c, Ae-Jung Kim d, Jinyoung Hur e,3, Kyung-Tae Lee c, Myung Sook Oh a,c,* a Department of Oriental Pharmaceutical Science, College of Pharmacy, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul 130-701, Republic of Korea b Department of Pharmacology, School of Medicine, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul 130-701, Republic of Korea c Department of Life and Nanopharmaceutical Science and Kyung Hee East-West Pharmaceutical Research Institute, Kyung Hee University, 26 Kyungheedaero, Dongdaemun-gu, Seoul 130-701, Republic of Korea d The Graduate School of Alternative Medicine, Kyunggi University, 71 Chungjeong-ro-2-ga, Seodaemun-gu, Seoul 120-837, Republic of Korea e Department of Brain Korea 21 Project Center, School of Medicine, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul 130-701, Republic of Korea

A R T I C L E I N F O

A B S T R A C T

Article history: Received 7 June 2013 Accepted 23 October 2013 Available online 1 November 2013

Recent studies on Alzheimer’s disease (AD) have focused on soluble oligomeric forms of amyloid-beta (Ab oligomer, AbO) that are directly associated with AD-related pathologies, such as cognitive decline, neurodegeneration, and neuroinflammation. Donepezil is a well-known anti-dementia agent that increases acetylcholine levels through inhibition of acetylcholinesterase. However, a growing body of experimental and clinical studies indicates that donepezil may also provide neuroprotective and disease-modifying effects in AD. Additionally, donepezil has recently been demonstrated to have antiinflammatory effects against lipopolysaccharides and tau pathology. However, it remains unknown whether donepezil has anti-inflammatory effects against AbO in cultured microglial cells and the brain in animals. Further, the effects of donepezil against AbO-mediated neuronal death, astrogliosis, and memory impairment have also not yet been investigated. Thus, in the present study, we examined the anti-inflammatory effect of donepezil against AbO and its neuroinflammatory mechanisms. Donepezil significantly attenuated the release of inflammatory mediators (prostaglandin E2, interleukin-1 beta, tumor necrosis factor-a, and nitric oxide) from microglia. Donepezil also decreased AbO-induced upregulation of inducible nitric oxide synthase and cyclooxygenase-2 protein and phosphorylation of p38 mitogen-activated protein kinase as well as translocation of nuclear factor-kappa B. We next showed that donepezil suppresses activated microglia-mediated toxicity in primary hippocampal cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. In intrahippocampal AbOinjected mice, donepezil significantly inhibited microgliosis and astrogliosis. Furthermore, behavioral tests revealed that donepezil (2 mg/kg/day, 5 days, p.o.) significantly ameliorated AbO-induced memory impairment. These results suggest that donepezil directly inhibits microglial activation induced by AbO through blocking MAPK and NF-kB signaling and, in part, contributing to the amelioration of neurodegeneration and memory impairment. ß 2013 Elsevier Inc. All rights reserved.

Keywords: Donepezil Amyloid-beta oligomer Alzheimer’s disaese Microglia Inflammation

1. Introduction

* Corresponding author at: Department of Oriental Pharmaceutical Science, College of Pharmacy, Kyung Hee University, #1 Hoegi-dong, Dongdaemun-gu, Seoul 130-701, Republic of Korea. Tel.: +82 2 961 9436; fax: +82 2 963 9436. E-mail address: [email protected] (M.S. Oh). 1 These authors contributed equally to this work. 2 Present address: Molecular Neurobiology Laboratory, McLean Hospital/Harvard Medical School, Belmont, MA 02478, United States. 3 Present address: Division of Metabolism and Functionality Research, Korea Food Research Institute, Seongnam-si, Gyeonggi-do, Republic of Korea. 0161-813X/$ – see front matter ß 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neuro.2013.10.004

It is known that Alzheimer’s disease (AD), the most common progressive neurodegenerative disorder, is characterized by neuronal loss and cognitive dysfunction (Donev et al., 2009; Terry et al., 2011). Although the exact mechanisms of AD pathogenesis remain unclear, intracellular amyloid beta (Ab) accumulation and extracellular Ab plaques are thought to be major pathological factors in AD (Moon et al., 2012; Selkoe, 1994). Amyloid pathology is selective to certain brain regions, including the hippocampal formation, and seems to be an underlying cause of cognitive

24

H.G. Kim et al. / NeuroToxicology 40 (2014) 23–32

impairment and neuronal loss (Chen et al., 2000; Hyman et al., 1984; Samuel et al., 1994). Recent studies have shown that ADrelated pathologies are caused primarily by oligomeric forms of the Ab peptide (Ab oligomer, AbO) (Lacor et al., 2007; Sakono and Zako, 2010). It is reported that activation of microglia, indicative of neuroinflammation, is another histological characteristic in brains of AD patients (McGeer et al., 1987). The reactive microgliosis has been demonstrated to contribute to neurodegeneration and memory impairment in AD (Block et al., 2007; Holmes et al., 2009; Pickering et al., 2005). Specifically, AbO directly stimulates microglia through the activation of signaling pathways that include nuclear factor-kappaB (NF-kB), mitogen-activated protein kinases (MAPKs), Lyn, and Syk tyrosine kinases (Dewapriya et al., 2013). These pathways lead to the release of neurotoxic mediators that can lead to neuronal cell loss (Pan et al., 2009). Although AbO is capable of directly inducing neurotoxic effects, neuronal and cognitive deficits are accelerated by pro-inflammatory substances, such as cytokines, NO, and prostaglandins, released from activated microglia (Block et al., 2007; Weninger and Yankner, 2001). Donepezil [R, S-1-benzyl-4-[(5, 6-dimethoxy-1-indanon)-2-yl] methylpiperidine hydrochloride] is a drug used clinically for cognitive dysfunction in AD (Giacobini, 2000; Hansen et al., 2008). Although its major mechanism of action is to inhibit cholinesterase activity, which increases acetylcholine (ACh) level and cholinergic transmission (Giacobini, 2000), several clinical studies have suggested that donepezil also has excellent neuroprotective and disease-modifying effects in AD patients (Hashimoto et al., 2005; Krishnan et al., 2003; Rogers et al., 2000; Winblad et al., 2006). Moreover, in vitro and animal studies using donepezil have also shown that donepezil does not function solely at the level of Ach but does influence AD progression through numerous cellular and molecular effects (Jacobson and Sabbagh, 2008). Additionally, a growing body of evidence suggests that donepezil has potent antiinflammatory effects in AD patients, a tauopathy mouse model, and lipopolysaccharide (LPS)-treated animals (Reale et al., 2005; Tyagi et al., 2007, 2010; Yoshiyama et al., 2010). Recently, it was reported the microglia-deactivating action of donepezil may not be dependent on ACh or its receptor (Hwang et al., 2010). Donepezil inhibited pro-inflammatory gene expression directly, resulting in reduced secretion of tumor necrosis factor-alpha (TNF-a), nitric oxide (NO), and interleukin-1 beta (IL-1b) in LPS-treated BV2 cells, a murine microglia cell line (Hwang et al., 2010). In total, there is now a substantial body of evidence about microglial deactivation mediated by donepezil in LPS and tauopathy models. This background prompted us to hypothesize that donepezil could have anti-inflammatory effects against AbO, a major causative agent of AD pathogenesis. Thus, in the present study, we examined the effects of donepezil on AbO-induced microglial activation in vitro and in vivo, the signaling pathways involved in the effects of donepezil in AbO-treated microglia, and the effects of donepezil on inflammation-mediated pathology, such as neuronal and memory loss. 2. Materials and methods 2.1. Materials Dulbeco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and penicillin-streptomycin (P/S) were purchased from Hyclone Laboratories Inc. (Logan, UT, USA). Neurobasal media and B27 were obtained from Gibco Industries Inc. (Auckland, NZ). Rabbit monoclonal anti-gilal fibrillary acid protein (GFAP) and rabbit polyclonal anti-microtubule-associated protein-2 (MAP-2) were purchased from Millipore Bioscience Research (Bedford, MA, USA). Rat monoclonal anti-CD11b (Mac-1) and rabbit monoclonal

anti-iNOS were purchased from Chemicon International (Temecula, CA, USA). Goat and rabbit polyclonal anti-cyclooxygenase-2 (COX-2) and a rabbit polyclonal NF-kB were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). A rabbit p38 MAPK and a rabbit phospo-p38 MAPK (pp38 MAPK, Thr180/Tyr182) were purchased from Cell Signaling Tech. (Berverly, MA, USA). Biotinylated horse anti-goat antibody, goat anti-rabbit antibody, rabbit anti-rat antibody, normal goat serum, normal horse serum, and avidin– biotin complex were purchased from Vector Laboratories (Burlingame, CA, USA). A mouse and a rat TNF-a ELISA kits and a mouse IL1b ELISA kit were purchased from Invitrogen Corp. (Carlsbad, CA, USA). A mouse prostaglandin E2 (PGE2) ELISA kit and anti-rabbithorseradish peroxidase secondary antibody were purchased from Assay Designs Inc. (Ann Arbor, MI, USA). Zoletil 501 and Rompun1 were purchased from Virbac (Carros, France) and Bayer Korea (Seoul, Korea), respectively. TEMED, protein standards dual color, western view marker, protein assay, tween-20, protein extraction solution, acrylamide, ammonium persulfate, skim milk, and ECL reagent were purchased from Bio-Rad Laboratories (Hercules, CA, USA). Paraformaldehyde, 3,3-diaminobenzidine (DAB), 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT), sodium chloride, sucrose, ethanol, phosphate buffer (PB), phosphate buffered saline (PBS), poly-L lysine, Griess reagent, 1,1,1,3,3,3-hexafluoro-2-propanol, and dimethyl sulfoxide anhydrous (DMSO) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Donepezil hydrochloride was supplied by Eisai Korea Co. Ltd. (AriceptW; Seoul, Korea). Ab1–42 peptide was purchased from American Peptide (Sunnyvale, CA, USA). 2.2. Preparation of AbO1–42 solution Soluble oligomers were generated as described previously with slight modifications (Chromy et al., 2003; Moon et al., 2013) Briefly, Ab1–42 was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol to a final concentration of 1 mg/mL at room temperature for 3 days. The peptide was aliquoted and dried under vacuum for 1 h. The aliquoted peptide was dissolved in DMSO to a final concentration of 1 mM. Protein determination was performed by Bradford assay to calculate molarities of solution. The Ab1–42 stock in DMSO was diluted directly into PBS at 10 mM and it was incubated at 4 8C for 24 h to make AbO1–42. 2.3. BV-2 cell culture Mouse BV-2 microglial cells were maintained in DMEM, supplemented with 10% heat-inactivated FBS, and 1% P/S in a humidified incubator of 5% CO2 at 37 8C. The cells were subcultured every day. All experiments were carried out 12 h after cells had been seeded at a density of 2.0  105 cells/mL. 2.4. Rat primary microglia culture Cell cultures were prepared from the hippocampus of postnatal day 1 Sprague–Dawley rats (Daehan Biolink Co., Ltd., Eumseong, Korea). Meninges-free hippocampus was collected, dissociated on the ice, and trypsinized at 37 8C. Then, the cells were maintained in DMEM, supplemented with 10% heat-inactivated FBS, and 1% P/S in a humidified incubator of 5% CO2 at 37 8C. After 10 days incubation, the cells were shaken at 180 rpm on a rotary shaker overnight. The microglia cells were collected and seeded at a density of 2.0  105 cells/mL. 2.5. Rat primary hippocampal cell culture Cell cultures were prepared from the hippocampus of 18-day embryos of timed pregnant Sprague–Dawley rats (Daehan Biolink

H.G. Kim et al. / NeuroToxicology 40 (2014) 23–32

Co., Ltd.). Hippocampus was dissected, collected, and dissociated on the ice. Then, hippocampal cells were plated in poly-L lysine pre-coated plates at a density of 1  105 cells/mL. Cultures were maintained in a humidified incubator of 5% CO2 at 37 8C in a neurobasal media with 2 mM glutamine, 2% B27, and 1% P/S. After 3 days incubation, the medium was replaced with a new medium. All experiments were carried out on the days in vitro 7. 2.6. Treatment After cells were approximately 75% confluent, they were treated with 750 nM AbO1–42 1 h prior to the addition of various concentrations of donepezil for the indicated times. For the conditioned media (CM) treatment, rat primary hippocampal cells were first treated with CM from BV-2 cells stimulated with AbO1– 42 and post-treated with donepezil for 24 h.

25

or 6 per cage, had free access to water and food, and maintained under a constant temperature (23  1 8C), humidity (60  10%), and a 12 h light/dark cycle. Animal treatment and maintenance were carried out in accordance with the Principle of Laboratory Animal Care (NIH publication No. 85-23, revised 1985) and the Animal Care and Use Guidelines of Kyung Hee University, Seoul, Korea. Mice were immediately anesthetized by mixture of Zoletil 501 and Rompun1 solution (3:1 ratio, 1 mL/kg, i.p.) and mounted in a stereotaxic apparatus (myNeuroLab, St. Louis, MO, USA). Each mouse was unilaterally injected (at rate 0.5 mL/min) with 3 mL of AbO1–42 (10 mM) into the hilus of dentate gyrus (DG) of hippocampus (coordinates with respect to bregma in mm: AP 2.0, ML 1.5, DV 2.0), according to the stereotaxic atlas of mouse brain (Paxinos and Franklin, 2001). The sham-operated mice were injected with the same volume of saline alone. The accuracy of stereotaxic injection to the targeted region was monitored in all animals by examination of the needle tract within brain sections.

2.7. Measurement of cell viability 2.11. Drug administration Treated cells were incubated with 1 mg/mL of MTT at 37 8C in a CO2 incubator for 3 h. MTT medium was carefully aspirated from the wells, and the formazan dye was eluted using DMSO. Absorbance was measured using a spectrophotometer (Versamax microplate reader; Molecular Device, Sunnyvale, CA, USA) at a wavelength of 570 nm and then expressed as a percentage of the value in the vehicle-treated control culture. 2.8. Determination of extracellular NO, TNF-a, IL-1b, and PGE2 The supernatants from BV-2 and primary microglia cells stimulated with AbO1–42 and post-treated with donepezil for 24 h were transferred to a separate plate. The accumulated level of nitrite in culture supernatants was reacted with 100 mL of Griess reagent in the dark for 10 min at room temperature. Absorbance at 550 nm was measured. For each experiment, freshly prepared NaNO2 that had been serially diluted was used as a standard, in parallel with culture supernatants. The cytokine levels in culture supernatants were detected by TNF-a, IL-1b, and PGE2 ELISA kits according to the manufacturers’ protocols. The standard smooth curves were plotted with fitting software, four parameter algorithm, and data were expressed as standards of TNF-a, IL1b, and PGE2 (pg/mL). 2.9. Western blot analysis The cells were lysed with protein extraction buffer for whole protein. Nuclear extraction was performed with nucleus fraction kit according to the manufacturer’s protocol. Cell lysates were separated on 10% SDS-polyacrylamide gel electrophoresis and transferred to a membrane. The membranes were incubated with 5% skim milk and primary antibodies (1:1000 dilutions); rabbit anti-iNOS, anti-COX-2, anti-p38 MAPK, anti-pp38 MAPK, anti-p65 subunit of NF-kB, anti-b-actin, and anti-laminin B. Then, the horseradish peroxidase-conjugated secondary antibody was incubated for 1 h. Antibody detection was carried out using ECL detection kit and visualized by LAS-4000 mini system (Fujifilm Corporation, Tokyo, Japan). The intensities of the bands were normalized to the b-actin or laminin B band intensity using Multi Gauge software (Fujifilm Corporation). 2.10. Animals and surgery procedure An AD mouse model induced by intrahippocampal injection of AbO was constructed according to a previous method (Choi et al., 2011; Moon et al., 2011). Male ICR mice (8 weeks, 27–30 g) were purchased from the Daehan Biolink Co. Ltd. Animals were housed 5

The mice were randomly divided into 3 groups (n = 6 in each group); (1) Sham group (sham-operated plus intraorally salinetreated group), (2) AbO1–42 group (AbO1–42-lesioned plus intraorally saline-treated group), (3) AbO1–42 + donepezil 2 mg/kg/day group (AbO1–42-lesioned plus intraorally donepezil 2 mg/kg/day treated group). The dose (2 mg/kg/day) of donepezil used in this experiment was referred from previously reported studies about the anti-inflammatory, cognition-enhancing and AD diseasemodifying effects of donepezil in rodent (Dong et al., 2009; Jiang et al., 2013; Narimatsu et al., 2009; Tyagi et al., 2010; Yoshiyama et al., 2010). Donepezil dissolved in saline was administered once per day for 5 consecutive days after stereotaxic injection. 2.12. The step-through passive avoidance test Learning and memory test was performed using a twocompartment step-through passive avoidance apparatus. The box was divided into bright (21 cm  21 cm  21 cm) and dark (21 cm  21 cm  21 cm) compartments by a guillotine door. The bright compartment contained an 50 W electric lamp, and the floor of the dark compartment was composed of 2 mm stainless steel rods spaced 1 cm apart. Mice were treated with donepezil or vehicle 1 h before the acquisition trial and initially placed in the bright compartment for the acquisition trial. The door between the two compartments was opened 10 s later. Then, when the hind legs of the mice entered into the dark chamber, the guillotine door was closed and electrical foot shock (0.6 mA) was delivered through the grid floor for 3 s. The mice were again placed in the bright chamber for the retention trial 24 h after the acquisition trial. The time taken for a mouse to enter the dark chamber after the door opening was defined as the latency time. The latency time was recorded for up to 300 s. Passive avoidance test was conducted at 5 (acquisition) and 6 (retention) days after AbO1–42 injection (Supplemental file 1). Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.neuro.2013. 10.004. 2.13. The Y-maze test The Y-maze test is used as a method of immediate short term spatial memory (Sarter et al., 1988). Short term spatial memory was investigated using Y-maze 4 days after stereotaxic surgery (Supplemental file 1). The Y-maze apparatus is composed of a three-arm with equal angles between all arms. Mice were placed in the one arm, and were to explore freely through the maze. The

26

H.G. Kim et al. / NeuroToxicology 40 (2014) 23–32

spontaneous alternation and total entries were manually measured for each mouse over an 8 min period. An actual alternation was defined as entries into all three arms consecutively (i.e., ABC, CAB, or BAC but not ABA). Mice were treated with donepezil (2 mg/ kg, p.o.) or vehicle 1 h before this test. The percentage of alternations was calculated as shown by the following equation: [(the number of alternations)/(the total number of arm entries 2)]  100. 2.14. Brain tissue preparation At 24 h after examination of memory behavior, mice were perfused transcardially with 0.05 M PBS, and then fixed with cold 4% paraformaldehyde in 0.1 M PB. Brains were removed, post-fixed overnight at 4 8C, and then immersed in a solution containing 30% sucrose in 0.05 M PBS for cryoprotection. Serial 30 mm-thick coronal sections were cut on a freezing microtome (Leica Microsystems Inc., Nussloch, Germany) and stored in cryoprotectant (25% ethylene glycol, 25% glycerol, and 0.05 M PB) at 4 8C until use. 2.15. Immunohistochemistry For immunohistochemical study, the free floating sections and cells on cover slips were briefly rinsed in PBS and treated with 1% hydrogen peroxide for 15 min to remove endogenous peroxidase activity. They were incubated with a rabbit anti-GFAP antibody (1:3000 dilution), a rat anti-Mac-1 (1:1000 dilution), a goat antiCOX-2 (1:500 dilution), or a rabbit anti-MAP-2 (1:1000 dilution) overnight at 4 8C in the presence of 0.3% Triton X-100 and normal goat serum or normal horse serum. Then, they were incubated with biotinylated anti-rabbit, anti-rat, or anti-goat IgG (1:200 dilution) for 90 min, followed by incubation in avidin–biotin complex (1:100 dilution) for 1 h at room temperature. Peroxidase activity was visualized by DAB in 0.05 M Tris–buffered saline (pH 7.6). After every incubation step, they were washed three times with PBS. Finally, the sections were mounted on gelatin-coated slices, dehydrated, and cover-slipped using histomount medium. The cover slips were mounted on glass slides and air dried. The optical densities of GFAP and Mac-1-immunoreactivity in the hilus of DG were analyzed with ImageJ software (Bethesda, MD, USA). The images were photographed at 400 magnification using an optical light microscope (Olympus Microscope System BX51; Olympus, Tokyo, Japan) equipped with a 20 objective lens. Data were presented as percentages of the value in the vehicle treated sham group or control culture. 2.16. Statistical analysis All statistical parameters were calculated using Graphpad Prism 4.0 software (GraphPad Software Inc., San Diego, CA, USA). Values were expressed as the mean  standard error of the mean (SEM). The result of Y-maze test was analyzed by student t-test followed by the Mann–whitney test. The other results were analyzed by one-way analysis of variance followed by the Tukey’s post hoc test. Differences with a p value less than 0.05 were considered to be statistically significant. 3. Results 3.1. Inhibitory effect of donepezil on production of inflammatory mediators in AbO1–42-stimulated microglia cells It has been well known that donepezil significantly inhibits the LPS-mediated neuroinflammation (Hwang et al., 2010; Tyagi et al., 2007, 2010; Yoshiyama et al., 2010). We tested whether donepezil also had anti-inflammatory action on AbO1–42-stimulated micro-

glia cells. To do this, we first measured inflammatory mediator levels. Treatment of AbO1–42 alone for 24 h in BV-2 cells increased to 2.71 and 2.85 folds of control in NO and TNF-a levels, respectively, whereas posttreatment of donepezil reduced them significantly (Fig. 1A and B). Also, IL-1b and PGE2 levels were measured in BV-2 cells. The exposure of AbO1–42 alone resulted in increase of IL-1b and PGE2 production levels to 5.87 and 2.61 folds of control, respectively, whereas the overproductions were inhibited by donepezil posttreatment at doses of 0.1 and 1 mM (Fig. 1C and D). To confirm the inhibitory effects of donepezil on production of inflammatory mediators induced by AbO1–42, we measured NO and TNF-a levels in rat primary microglia cells. Treatment of AbO1–42 alone for 24 h in rat primary microglia cells increased to 3.42 and 109.86 folds of control in NO and TNF-a levels, respectively, whereas posttreatment of donepezil reduced the increase levels of them significantly (Fig. 1E and F). Using MTT assay, we observed that the treatment of donepezil (0.1 and 1 mM) and AbO1–42 (0.2–1 mM) did not show any significant effect on viability of microglial cells (data not shown). Taken together, these proinflammatory mediators produced by AbO1–42 in microglia cells were greatly inhibited by donepezil in a dose-dependent manner. For the first time, we demonstrated that donepezil has an anti-inflammatory action on AbO-stimulated activated microglia. 3.2. Inhibitory effect of donepezil on upregulation of proinflammatory protein expressions in AbO1–42-stimulated microglia cells In order to determine the effects of donepezil on production of iNOS and COX-2 protein, which are executed through NO and PGE2, respectively, we performed western blot analysis. As shown in Fig. 2, treatment of AbO1–42 alone for 24 h in BV-2 cells showed 1.22  0.03 and 1.00  0.06 folds of b-actin where the control groups showed 0.47  0.06 and 0.49  0.06 folds of b-actin in iNOS and COX2 protein levels, respectively. However, posttreatment of donepezil at doses of 0.1 and 1 mM markedly downregulated the expression to 1.06  0.01 and 0.70  0.11 folds of b-actin for iNOS and 0.77  0.04 and 0.67  0.05 folds of b-actin for COX-2 protein, respectively (Fig. 2). These data suggested that donepezil-mediated attenuation of release of inflammatory mediators may result from inhibition of protein expression of proinflammatory molecules. 3.3. Inhibitory effect of donepezil on activation of p38 MAPK and nuclear translocation of NF-kB p65 in AbO1–42-stimulated microglia cells It has been reported that Ab-stimulated transcription of inflammatory genes responsible for cytokines production and COX-2 and iNOS induction is dependent on NF-kB and the MAPK pathways (Dewapriya et al., 2013; Kim et al., 2004; Pan et al., 2009). To identify the signaling pathways involved with anti-inflammatory activity of donepezil in AbO-treated microglia, we observed p38 MAPK and p65 NF-kB expressions using western blot analysis. Fig. 3 showed that donepezil at 1 mM inhibited AbO1–42-induced p38 MAPK activation by 29.69% compared with the AbO1–42-only treated group. Also, donepezil markedly reduced the increase of NFkB p65 translocation to nucleus by 60.71% compared with the AbO1– 42-only treated group (Fig. 3). These findings suggest that donepezil might regulate the inhibition of microglial activation via suppressing p38 MAPK and NF-kB pathways. 3.4. Inhibitory effect of anti-inflammatory donepezil on neuronal death induced by AbO1–42-stimulated microglia in primary hippocampal cell culture It is very well known that Ab-mediated activated microglia produces the neurotoxic factors, which lead to neuronal death

H.G. Kim et al. / NeuroToxicology 40 (2014) 23–32

27

Fig. 1. Inhibitory effect of donepezil on production of NO, TNF-a, IL-1b, and PGE2 by AbO1–42 in microglia cells. After microglia cells were treated with AbO1–42 for 1 h and incubated without or with donepezil for a further 23 h, the supernatants were collected. NO level was measured using Griess reagent in BV-2 cells (A). TNF-a level was measured using an ELISA kit in BV-2 cells (B). IL-1b and PGE2 levels were measured using ELISA kits in BV-2 cells (C and D, respectively). NO and TNF-a levels were measured in rat primary microglia cells (E and F, respectively). Values are expressed as mean  SEM. *** P < 0.001 indicates that mean value was significantly different from the control group. ###P < 0.001, ##P < 0.01, and #P < 0.05 indicate that mean value was significantly different from the AbO1–42-only treated group. DN: donepezil.

(Block et al., 2007). Thus, to examine whether donepezil has indirect neuroprotective effect against microglia-mediated toxicity caused by AbO1–42, rat primary hippocampal cells were incubated with microglial conditioned media and then cell viability was measured using MTT assay. As shown in Fig. 4, cells treated with AbO1–42-CM for 24 h and 48 h exhibited significantly decreased cell viability to 84.60  1.66% and 61.34  2.01%, respectively, compared with the control group. However, posttreatment of donepezil at 0.1 and 1 mM with AbO1–42-CM treatment inhibited the decrease of cell viability by 107.68–142.27% compared with the AbO1–42-CM treated group. These results indicate that donepezil may function as a neuroprotective agent for hippocampal neurons by acting as a microglia-deactivating factor.

3.5. Inhibitory effect of donepezil on AbO1–42-induced gliosis in intrahippocampal AbO-injected mice To confirm the effect of donepezil on microglial activation induced AbO1–42 in an in vivo system, we administered donepezil to mice whose brains were intrahippocampally injected with AbO1–42 and then performed the histological analysis. In the Mac1-stained brains, for the measurement of microgliosis in the DG, the percentage of the Mac-1-positive area exhibited a significant increase in the AbO1–42-injected group to 252.26  19.19% compared with the sham group (p < 0.001). In contrast, the Mac-1stained area of AbO1–42-injected mice treated with donepezil significantly decreased it to 175.92  14.56% compared with the

28

H.G. Kim et al. / NeuroToxicology 40 (2014) 23–32

Fig. 2. Inhibitory effect of donepezil on regulation of iNOS and COX-2 proteins by AbO1–42 in microglia cells. After BV-2 cells were treated with AbO1–42 for 1 h and incubated without or with donepezil for a further 23 h, the cells were collected and lysed using protein assay buffer for whole protein lysate. Protein levels of iNOS (B) and COX-2 (C) were normalized to b-actin. The representative band images of experiments are shown in (A). Values are expressed as mean  SEM. ***P < 0.001 indicates that mean value was significantly different from the control group. ###P < 0.001 and ##P < 0.01 indicate that mean value was significantly different from the AbO1–42-only treated group. DN: donepezil.

Fig. 3. Inhibitory effect of donepezil on phosphorylation of p38 MAPK and translocation of NF-kB by AbO1–42 in microglia cells. After BV-2 cells were treated with AbO1–42 for 1 h and incubated without or with donepezil for a further 1 h, the cells were collected and lysed. For p38 MPAK detection, phospho form of p38 (pp38) and total form of p38 antibodies were used in whole protein lysate. For NF-kB detection, p65 and laminin B antibodies were used in nucleus fraction. Levels of p-p38 (B) and p65 (C) were normalized to p38 and laminin B, respectively. The representative band images of experiments are shown in (A). Values are expressed as mean  SEM. ***P < 0.001 and **P < 0.01 indicate that mean value was significantly different from the control group. ##P < 0.01 and #P < 0.05 indicate that mean value was significantly different from the AbO1– 42-only treated group. DN: donepezil.

control group (p < 0.01; Fig. 5A). Accordingly, we could confirm the microglial deactivating effect of donepezil in vivo. Since it is known that activated microglial cells actively recruits the astrocytes, which enhance the inflammatory response (Eikelenboom et al., 2006; Heneka and O’Banion, 2007; White et al., 2005). Thus, to test whether

donepezil influences the astrogliosis in the area injected with AbO, GFAP immunostaining was performed to measure the fraction of GFAP-immunoreactive area in the hilus of DG. The quantification data showed a significant increase in the AbO1–42-injected group to 155.46  9.10% compared with the control group (p < 0.001),

Fig. 4. Neuroprotective effect of donepezil on activated microglia-induced neurotoxicity in rat primary hippocampal cells. The conditioned media from BV-2 cells stimulated with AbO1–42 for 1 h with or without donepezil for a further 23 h were transferred to rat primary hippocampal cells and incubated for 24 h (A) and 48 h (B). Then cell viabilities were measured using MTT assay. Values are expressed as mean  SEM. ***P < 0.001 indicates that mean value was significantly different from the control group. ### P < 0.001, ##P < 0.01, and #P < 0.05 indicate that mean value was significantly different from the AbO1–42-only treated group. DN: donepezil; CM: conditioned media.

H.G. Kim et al. / NeuroToxicology 40 (2014) 23–32

29

Fig. 5. Inhibitory effect of donepezil on AbO1–42-induced activation of microglia and astrocyte in an AD mouse model induced by intrahippocampal injection of AbO1–42. Mice were treated with vehicle or donepezil (2 mg/kg/day) for 5 consecutive days and after stereotaxic injection of AbO1–42 (10 mM, 3 mL). Microglia and astrocyte activations were determined using Mac-1 and GFAP staining, respectively. Quantification of the Mac-1 and GFAP- stained cells were performed by measuring the area fraction of Mac-1immunoreactivity (B) and GFAP-immunoreactivity (C) cells/areas in the hilus of DG. The representative images of experiments are shown in (A). Scale bar = 50 mm. Values are expressed as mean  SEM. ***P < 0.001 indicates that mean value was significantly different from the sham group. ##P < 0.01 and #P < 0.05 indicate that mean value was significantly different from the AbO1–42-only treated group. DN: donepezil; DG: dentate gyrus.

whereas donepezil treatment reduced it to 127.95  3.91% compared with the control group (p < 0.05; Fig. 5B). Our data indicate that denepezil inhibits the AbO1–42-induced microgliosis and astrogliosis in vivo. 3.6. Protective effect of anti-inflammatory donepezil on memory deficits induced by intrahippocampal AbO1–42 injection The pro-inflammatory cytokines have been shown to act as inhibitor of the induction of long-term potentiation (LTP) and inflammation has been proposed to contribute to the cognitive decline in AD (Cho et al., 2011; Holmes et al., 2009; Pickering et al.,

2005). Therefore, to investigate if anti-inflammatory donepezil ameliorates cognitive dysfunction in mice with reactive gliosis stimulated by intrahippocaml injection of AbO1–42, a passive avoidance test was employed. The retention time of the AbO1–42injected group was significantly reduced to 120.88  17.91 s, whereas the sham group showed 256.61  21.45 s (p < 0.001). In contrast, mice treated with donepezil at 2 mg/kg/day exhibited a significant recovery in this test showing 228.46  21.47 s (p < 0.001; Fig. 6A). No differences were observed in passive avoidance latencies during the acquisition trial in any other groups. To confirm the effect of donepezil on spatial working memory, a Y-maze test was also performed. The sham group showed 64.78  48.02% of alternation

Fig. 6. Effect of donepezil on AbO1–42-induced memory impairment in a mouse model of AD. Mice were treated with vehicle donepezil at 2 mg/kg/day for 5 consecutive days and after AbO1–42 (10 mM, 3 mL) stereotaxic injection. Passive avoidance test was conducted at 5 (acquisition) and 6 (retention) days after AbO1–42 injection (A). Y-maze test was carried out at 4 days after AbO1–42 injection (B). Values are expressed as mean  SEM. ***P < 0.001 and **P < 0.01 Mean value was significantly different from the sham group. ##P < 0.001 indicates that mean value was significantly different from the AbO1–42-only treated group. DN: donepezil.

30

H.G. Kim et al. / NeuroToxicology 40 (2014) 23–32

whereas the AbO1–42-injected group showed 48.02  2.95% of it (p < 0.01). Even though donepezil treatment group failed to show significant recovery in Y-maze test, it showed tendency of protection (Fig. 6B). During the test session, no significant differences in total entry were found for any of the groups. Taken together, antiinflammatory donepezil ameliorated AbO1–42-induced memory impairment in mice, suggesting that donepezil-mediated recovery of cognitive dysfunction seems to be, in part, associated with inhibition of microglial activation in the AbO-injected brain. 4. Discussion This study demonstrated that donepezil significantly reduces AbO1–42-induced secretion and expression of pro-inflammatory factors in a microglial cell line and primary cultured microglia. Additionally, donepezil suppressed MAPK p38 activation and the nuclear translocation of NF-kB p65 in AbO1–42-stimulated microglia. We also found that donepezil protected primary hippocampal neurons from toxicity induced by activated microglia. Moreover, we confirmed that donepezil has an antiinflammatory effect against AbO1–42 in the brains of mice. The anti-inflammatory donepezil effect could ameliorate cognitive dysfunction mediated by intrahippocampal injection of AbO1–42. Our data suggest that donepezil may inhibit neuronal death and cognitive decline by repressing the AbO-triggered inflammatory pathways in microglia (Fig. 7). NF-kB has been known to be an important regulator of inflammation by acting as an essential transcription factor for the induction of target genes such as iNOS, COX-2, TNF-a, and IL1b (Lawrence and Fong, 2010). In addition, p38 MAPK has been identified to be mainly involved in neuroinflammation by leading to phosphorylation of nuclear transcription factors such as ATF2, MEF2A/C, and SAP1a and protein kinases in the cytoplasm such as MAPK-activated protein kinases 2 and 3 and p38-regulated/ activated kinase for IL-1, IL-6, TNF-a, COX-2, and iNOS expressions (Koistinaho and Koistinaho, 2002). Thus, the inhibition of NF-kB

Fig. 7. Schematic illustration of the hypothesis that donepezil suppresses ADrelated pathology through inhibiting the AbO-induced microglial activation. AbO induces MAPK p38 activation and nuclear translocation of NF-kB p65 and upregulates the inflammatory genes in microglial cells, resulting in increase of neurotoxic molecule production. Donepezil shows the protective effects on neuronal death and cognitive decline by repressing the AbO-triggered inflammatory pathways. + Indicates the activating pathways and indicates the inhibitory pathways.

p65 and p38 signaling may play a most critical role in suppressing activated microglia cells (Heneka et al., 2010). It also has been well known that AbO directly stimulates microglia through NF-kB and the MAPK-signaling pathways required for cytokine production and COX-2 and iNOS expression (Dewapriya et al., 2013; Kim et al., 2004; Pan et al., 2009). Previous research suggested that AbO also rapidly activates the MAPK signaling pathway, including p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK), in microglia (Pan et al., 2009). However, another report showed that AbO activated p38 MAPK, but not ERK or JNK, in microglia (Sondag et al., 2009). In the present study, we confirmed that phosphorylated ERK1/2 and JNK were not activated in AbO1–42-treated microglia (data not shown), whereas p38 MAPK was phosphorylated after AbO treatment. Taken together, these data show that microglia activation by AbO1–42 induces activation of NF-kB and p38 MAPK, which subsequently results in the elevation of NO, TNF-a, IL-1b, and PGE2 and the upregulation of iNOS and COX-2. Therefore, to prove and understand the antiinflammatory effects of donepezil, we choose to monitor the phosphorylation of p38 MAPK and nuclear translocation of p65, a component of the NF-kB pathway, in AbO-treated microglia. We used the MTT assay to examine whether the antiinflammatory actions of donepezil were involved in the neuroprotective effect against inflammation-mediated toxicity in rat primary hippocampal cells. In the present study, conditioned media from AbO1–42 with or without donepezil-treated microglial cells was applied to rat primary hippocampal cells. The donepeziltreated conditioned media group showed reduced degeneration induced by AbO1–42-mediated microglial activation (Fig. 4). We found that donepezil had indirect neuroprotective effects against microglia-mediated toxicity induced by AbO1–42. Neuroinflammatory processes generally contribute to the pathogenesis of AD, and this response is sustained by activated microglial cells, resulting in neuronal damage due to the liberation of toxic factors, such as free radicals and cytokines (Bruce-Keller, 1999; Streit, 2002). It has been reported that neuroprotection can result from inhibition of pro-inflammatory mediators through down-regulation of iNOS or COX-2 protein (Pan et al., 2009). Additionally, it is also known that selective COX-2 inhibitors, celecoxib, and nonsteroidal anti-inflammatory drugs, have neuroprotective effects in mouse inflammatory models induced by Ab (Imbimbo, 2009). Thus, inhibiting the production of toxic factors using microglia may alleviate inflammation-induced degeneration. Based on our results and those of previous studies, we suggest that the wellknown neuroprotective effects of donepezil may be caused, at least partially, by a microglia de-activating effect. It is well-known that donepezil exhibits memory-enhancing effects in preclinical and clinical studies on AD. To confirm the effect of donepezil on cognitive deficits, we administered it to mice whose brains were lesioned by intrahippocampal injection of AbO1–42 and then performed behavioral tests and histological analyses. It has been reported that intrahippocampal injection of AbO1–42 induces cognitive impairment in rodents by inhibiting long-term potentiation-mediated memory formation (Cleary et al., 2005; Dinamarca et al., 2006). In a passive avoidance test, donepezil treatment significantly improved memory impairment induced by AbO1–42 (Fig. 6A). We also investigated spontaneous alternation behavior, which is related to immediate spatial working memory (Sarter et al., 1988), using the Y-maze test. Although donepezil failed to show significant reversal of AbO1–42induced behavior in the Y-maze, a (non-significant) tendency toward recovery was observed (Fig. 6B). Similarly, a previous report showed that intraperitoneal or subcutaneous donepezil treatment recovered spatial working memory against Ab25–35induced damage (Meunier et al., 2006; Tsunekawa et al., 2008). Taken together, these data show that donepezil treatment

H.G. Kim et al. / NeuroToxicology 40 (2014) 23–32

significantly ameliorated AbO1–42-induced memory impairment. Our data suggest that the anti-inflammatory effects of donepezil may reduce cognitive dysfunction in AD. The capacity of donepezil to ameliorate memory deficits in AD patients may be related to inhibiting Ab-induced activation of microglia. Previous research has shown that soluble AbO leads to neuronal dysfunction and memory loss by inducing microglia and astrocyte activation (Heneka and O’Banion, 2007; Rojo et al., 2008). The induction of a glial-driven inflammatory response results in the release of various inflammatory mediators (e.g., NO, TNF-a, IL-1b, and PGE2) secreted by the glial cells surrounding Ab plaques (Parvathy et al., 2009; Watson et al., 2005). Once microglia are activated, astrocytes are actively recruited, enhancing the inflammatory response and leading to exponential neuronal death through apoptosis, synaptic dysfunction, and inhibition of neurogenesis (Eikelenboom et al., 2006; Heneka and O’Banion, 2007; White et al., 2005). Recently, it has been reported that donepezil treatment suppresses astroglial activation in a tauopathy mouse model (Yoshiyama et al., 2010). To date, there has been no report on the effects of donepezil on the AbO-mediated activation of astrocytes. Thus, to evaluate whether donepezil influenced astrogliosis in vivo, we assessed GFAP, a specific marker for astrocytes, by immunostaining in the hippocampal region of mouse brains. AbO1–42-injected hippocampi showed a significant increase in the number of GFAP-positive cells. In donepeziladministered mice, GFAP-positive immunoreactivity was decreased significantly (Fig. 5). These results indicate that cognitive improvement by donepezil treatment is accompanied by prevention of the inflammatory response associated with astrocytes, suggesting that inhibiting the activation of astrocytes may be associated with cognitive improvement. Some studies have shown that the anti-inflammatory action of donepezil is due to acetylcholinesterase inhibition (Tyagi et al., 2007, 2010; Wang et al., 2003). ACh is known to effectively deactivate macrophages and inhibit pro-inflammatory cytokines (De Simone et al., 2005). Thus, the anti-inflammatory effects of donepezil in our in vivo study were likely mediated through augmentation of ACh. However, a recent study clearly demonstrated that treatment with antagonists of nicotinic acetylcholine receptors did not influence the donepezil-mediated inhibition of microglial activation, indicating that donepezil directly inhibits microglial activation independently of acetylcholine and its receptors (Hwang et al., 2010). Thus, it is possible that donepezil may contribute to anti-inflammatory effects via not only nicotinic acetylcholine receptors, by elevating ACh levels, but also by direct effects on microglia. Some disease-modifying drugs are occasionally used without a thorough understanding of the mechanisms or the assumed target and without explaining the entire spectrum of effects. Although donepezil, as a well-known acetylcholinesterase inhibitor, has long been used clinically for patients with AD to improve memory and other cognitive functions (Rogers et al., 2000), the effects with a focus on inflammation pathology in an AD model induced by AbOstimulating microglia had not been directly revealed before this study. As neuroinflammation has been emerging as an attractive target for the treatment of AD (Shi et al., 2013), manipulation of inflammatory processes is desired to find new and suitable therapeutic options for AD treatment. In addition to our data, there are substantial evidences that donepezil affects inflammatory processes (Reale et al., 2005; Tyagi et al., 2007, 2010; Yoshiyama et al., 2010), suggesting that the disease-modifying effects of donepezil in patients with AD might be mediated by antiinflammatory action on AbO-stimulated microglial cells. Furthermore, from the present study, it can be speculated that NF-kB p65 and the p38 MAPK-signaling pathways may be a therapeutic target for suppressing the memory deficits induced by AbO. Further

31

detailed investigation is required to (1) determine the molecular mechanisms underlying the direct deactivation of microglia by donepezil (i.e., the identification of receptor for donepezil in microglia) and (2) address the direct effects of donepezil on astroglial activation and the mechanisms by which they operate. In summary, we demonstrated that donepezil exhibits inhibitory effects in AbO-induced inflammation in vitro and in vivo. Furthermore, donepezil protects against neuronal death by inhibiting AbO-induced microglial activation and shows a protective effect against memory impairment induced by AbO in vivo (Fig. 7). These results suggest that donepezil could be a disease-modifying drug, suppressing microglia-mediated inflammation in AD.

Conflicts of interest statement The authors declare that there are no conflicts of interest. Acknowledgments This work was carried out with the support of ‘‘IPET(11210502-1-HD040)’’ Korea institute of planning & evaluation for technology in food, agriculture, forestry and fisheries, Republic of Korea. The authors thank Soohyun Kwak (Newton South High School, MA, USA) for drawing the illustration. References Block ML, Zecca L, Hong JS. Microglia-mediated neurotoxicity: uncovering the molecular mechanisms. Nat Rev Neurosci 2007;8:57–69. Bruce-Keller AJ. Microglial-neuronal interactions in synaptic damage and recovery. J Neurosci Res 1999;58:191–201. Chen G, Chen KS, Knox J, Inglis J, Bernard A, Martin SJ, et al. A learning deficit related to age and beta-amyloid plaques in a mouse model of Alzheimer’s disease. Nature 2000;408:975–9. Cho SH, Sun B, Zhou Y, Kauppinen TM, Halabisky B, Wes P, et al. CX3CR1 protein signaling modulates microglial activation and protects against plaque-independent cognitive deficits in a mouse model of Alzheimer disease. J Biol Chem 2011;286:32713–22. Choi JG, Moon M, Kim HG, Mook-Jung I, Chung SY, Kang TH, et al. Gami-Chunghyuldan ameliorates memory impairment and neurodegeneration induced by intrahippocampal Ab1–42 oligomer injection. Neurobiol Learn Mem 2011;96:306–14. Chromy BA, Nowak RJ, Lambert MP, Viola KL, Chang L, Velasco PT, et al. Self-assembly of Abeta(1–42) into globular neurotoxins. Biochemistry 2003;42:12749–60. Cleary JP, Walsh DM, Hofmeister JJ, Shankar GM, Kuskowski MA, Selkoe DJ, et al. Natural oligomers of the amyloid-beta protein specifically disrupt cognitive function. Nat Neurosci 2005;8:79–84. De Simone R, Ajmone-Cat MA, Carnevale D, Minghetti L. Activation of alpha7 nicotinic acetylcholine receptor by nicotine selectively up-regulates cyclooxygenase-2 and prostaglandin E2 in rat microglial cultures. J Neuroinflammation 2005;2:4. Dewapriya P, Li YX, Himaya SW, Pangestuti R, Kim SK. Neoechinulin A suppresses amyloid-beta oligomer-induced microglia activation and thereby protects PC-12 cells from inflammation-mediated toxicity. Neurotoxicology 2013;35:30–40. Dinamarca MC, Cerpa W, Garrido J, Hancke JL, Inestrosa NC. Hyperforin prevents betaamyloid neurotoxicity and spatial memory impairments by disaggregation of Alzheimer’s amyloid-beta-deposits. Mol Psychiatr 2006;11:1032–48. Donev R, Kolev M, Millet B, Thome J. Neuronal death in Alzheimer’s disease and therapeutic opportunities. J Cell Mol Med 2009;13:4329–48. Dong H, Yuede CM, Coughlan CA, Murphy KM, Csernansky JG. Effects of donepezil on amyloid-beta and synapse density in the Tg2576 mouse model of Alzheimer’s disease. Brain Res 2009;1303:169–78. Eikelenboom P, Veerhuis R, Scheper W, Rozemuller AJ, van Gool WA, Hoozemans JJ. The significance of neuroinflammation in understanding Alzheimer’s disease. J Neural Transm 2006;113:1685–95. Giacobini E. Cholinesterase inhibitors stabilize Alzheimer’s disease. Ann N Y Acad Sci 2000;920:321–7. Hansen RA, Gartlehner G, Webb AP, Morgan LC, Moore CG, Jonas DE. Efficacy and safety of donepezil, galantamine, and rivastigmine for the treatment of Alzheimer’s disease: a systematic review and meta-analysis. Clin Interv Aging 2008;3:211–25. Hashimoto M, Kazui H, Matsumoto K, Nakano Y, Yasuda M, Mori E. Does donepezil treatment slow the progression of hippocampal atrophy in patients with Alzheimer’s disease. Am J Psychiatr 2005;162:676–82. Heneka MT, O’Banion MK. Inflammatory processes in Alzheimer’s disease. J Neuroimmunol 2007;184:69–91. Heneka MT, O’Banion MK, Terwel D, Kummer MP. Neuroinflammatory processes in Alzheimer’s disease. J Neural Transm 2010;117:919–47.

32

H.G. Kim et al. / NeuroToxicology 40 (2014) 23–32

Holmes C, Cunningham C, Zotova E, Woolford J, Dean C, Kerr S, et al. Systemic inflammation and disease progression in Alzheimer disease. Neurology 2009;73:768–74. Hwang J, Hwang H, Lee HW, Suk K. Microglia signaling as a target of donepezil. Neuropharmacology 2010;58:1122–9. Hyman BT, Van Hoesen GW, Damasio AR, Barnes CL. Alzheimer’s disease: cell-specific pathology isolates the hippocampal formation. Science 1984;225:1168–70. Imbimbo BP. An update on the efficacy of non-steroidal anti-inflammatory drugs in Alzheimer’s disease. Expert Opin Investig Drugs 2009;18:1147–68. Jacobson SA, Sabbagh MN. Donepezil: potential neuroprotective and disease-modifying effects. Expert Opin Drug Metab Toxicol 2008;4:1363–9. Jiang Y, Zou Y, Chen S, Zhu C, Wu A, Liu Y, et al. The anti-inflammatory effect of donepezil on experimental autoimmune encephalomyelitis in C57 BL/6 mice. Neuropharmacology 2013;73:415–24. Kim SH, Smith CJ, Van Eldik LJ. Importance of MAPK pathways for microglial proinflammatory cytokine IL-1 beta production. Neurobiol Aging 2004;25:431–9. Koistinaho M, Koistinaho J. Role of p38 and p44/42 mitogen-activated protein kinases in microglia. Glia 2002;40:175–83. Krishnan KR, Charles HC, Doraiswamy PM, Mintzer J, Weisler R, Yu X, et al. Randomized, placebo-controlled trial of the effects of donepezil on neuronal markers and hippocampal volumes in Alzheimer’s disease. Am J Psychiatr 2003;160:2003–11. Lacor PN, Buniel MC, Furlow PW, Clemente AS, Velasco PT, Wood M, et al. Abeta oligomer-induced aberrations in synapse composition, shape, and density provide a molecular basis for loss of connectivity in Alzheimer’s disease. J Neurosci 2007;27:796–807. Lawrence T, Fong C. The resolution of inflammation: anti-inflammatory roles for NFkappaB. Int J Biochem Cell Biol 2010;42:519–23. McGeer PL, Itagaki S, Tago H, McGeer EG. Reactive microglia in patients with senile dementia of the Alzheimer type are positive for the histocompatibility glycoprotein HLA-DR. Neurosci Lett 1987;79:195–200. Meunier J, Ieni J, Maurice T. The anti-amnesic and neuroprotective effects of donepezil against amyloid beta25-35 peptide-induced toxicity in mice involve an interaction with the sigma1 receptor. Br J Pharmacol 2006;149:998–1012. Moon M, Choi JG, Nam DW, Hong HS, Choi YJ, Oh MS, et al. Ghrelin ameliorates cognitive dysfunction and neurodegeneration in intrahippocampal amyloid-beta 1–42 oligomer-injected mice. J Alzheimers Dis 2011;23:147–59. Moon M, Hong HS, Nam DW, Baik SH, Song H, Kook SY, et al. Intracellular amyloid-beta accumulation in calcium-binding protein-deficient neurons leads to amyloid-beta plaque formation in animal model of Alzheimer’s disease. J Alzheimers Dis 2012;29:615–28. Moon M, Song H, Hong HJ, Nam DW, Cha MY, Oh MS, et al. Vitamin D-binding protein interacts with Abeta and suppresses Abeta-mediated pathology. Cell Death Differ 2013;20:630–8. Narimatsu N, Harada N, Kurihara H, Nakagata N, Sobue K, Okajima K. Donepezil improves cognitive function in mice by increasing the production of insulin-like growth factor-I in the hippocampus. J Pharmacol Exp Ther 2009;330:2–12. Pan XD, Chen XC, Zhu YG, Chen LM, Zhang J, Huang TW, et al. Tripchlorolide protects neuronal cells from microglia-mediated beta-amyloid neurotoxicity through inhibiting NF-kappaB and JNK signaling. Glia 2009;57:1227–38. Parvathy S, Rajadas J, Ryan H, Vaziri S, Anderson L, Murphy GM Jr. Abeta peptide conformation determines uptake and interleukin-1alpha expression by primary microglial cells. Neurobiol Aging 2009;30:1792–804. Paxinos G, Franklin KBJ. The mouse brain in stereotaxic coordinates. 2nd ed. San Diego: Academic Press; 2001.

Pickering M, Cumiskey D, O’Connor JJ. Actions of TNF-alpha on glutamatergic synaptic transmission in the central nervous system. Exp Physiol 2005;90:663–70. Reale M, Iarlori C, Gambi F, Lucci I, Salvatore M, Gambi D. Acetylcholinesterase inhibitors effects on oncostatin-M, interleukin-1 beta and interleukin-6 release from lymphocytes of Alzheimer’s disease patients. Exp Gerontol 2005;40:165–71. Rogers SL, Doody RS, Pratt RD, Ieni JR. Long-term efficacy and safety of donepezil in the treatment of Alzheimer’s disease: final analysis of a US multicentre open-label study. Eur Neuropsychopharmacol 2000;10:195–203. Rojo LE, Fernandez JA, Maccioni AA, Jimenez JM, Maccioni RB. Neuroinflammation: implications for the pathogenesis and molecular diagnosis of Alzheimer’s disease. Arch Med Res 2008;39:1–16. Sakono M, Zako T. Amyloid oligomers: formation and toxicity of Abeta oligomers. FEBS J 2010;277:1348–58. Samuel W, Masliah E, Hill LR, Butters N, Terry R. Hippocampal connectivity and Alzheimer’s dementia: effects of synapse loss and tangle frequency in a twocomponent model. Neurology 1994;44:2081–8. Sarter M, Bodewitz G, Stephens DN. Attenuation of scopolamine-induced impairment of spontaneous alteration behaviour by antagonist but not inverse agonist and agonist beta-carbolines. Psychopharmacology (Berl) 1988;94:491–5. Selkoe DJ. Alzheimer’s disease: a central role for amyloid. J Neuropathol Exp Neurol 1994;53:438–47. Shi S, Wang Z, Qiao Z. The multifunctional anti-inflammatory drugs used in the therapy of Alzheimer’s disease. Curr Med Chem 2013;20:2583–8. Sondag CM, Dhawan G, Combs CK. Beta amyloid oligomers and fibrils stimulate differential activation of primary microglia. J Neuroinflammation 2009;6:1. Streit WJ. Microglia as neuroprotective, immunocompetent cells of the CNS. Glia 2002;40:133–9. Terry AV Jr, Callahan PM, Hall B, Webster SJ. Alzheimer’s disease and age-related memory decline (preclinical). Pharmacol Biochem Behav 2011;99:190–210. Tsunekawa H, Noda Y, Mouri A, Yoneda F, Nabeshima T. Synergistic effects of selegiline and donepezil on cognitive impairment induced by amyloid beta (25–35). Behav Brain Res 2008;190:224–32. Tyagi E, Agrawal R, Nath C, Shukla R. Cholinergic protection via alpha7 nicotinic acetylcholine receptors and PI3K-Akt pathway in LPS-induced neuroinflammation. Neurochem Int 2010;56:135–42. Tyagi E, Agrawal R, Nath C, Shukla R. Effect of anti-dementia drugs on LPS induced neuroinflammation in mice. Life Sci 2007;80:1977–83. Wang H, Yu M, Ochani M, Amella CA, Tanovic M, Susarla S, et al. Nicotinic acetylcholine receptor alpha7 subunit is an essential regulator of inflammation. Nature 2003;421:384–8. Watson D, Castano E, Kokjohn TA, Kuo YM, Lyubchenko Y, Pinsky D, et al. Physicochemical characteristics of soluble oligomeric Abeta and their pathologic role in Alzheimer’s disease. Neurol Res 2005;27:869–81. Weninger SC, Yankner BA. Inflammation and Alzheimer disease: the good, the bad, and the ugly. Nat Med 2001;7:527–8. White JA, Manelli AM, Holmberg KH, Van Eldik LJ, Ladu MJ. Differential effects of oligomeric and fibrillar amyloid-beta 1–42 on astrocyte-mediated inflammation. Neurobiol Dis 2005;18:459–65. Winblad B, Wimo A, Engedal K, Soininen H, Verhey F, Waldemar G, et al. 3-Year study of donepezil therapy in Alzheimer’s disease: effects of early and continuous therapy. Dement Geriatr Cogn Disord 2006;21:353–63. Yoshiyama Y, Kojima A, Ishikawa C, Arai K. Anti-inflammatory action of donepezil ameliorates tau pathology, synaptic loss, and neurodegeneration in a tauopathy mouse model. J Alzheimers Dis 2010;22:295–306.