AMP-activated Protein Kinase Signaling Activation by Resveratrol Modulates Amyloid-β Peptide Metabolism*

Alzheimer disease is an age-related neurodegenerative disorder characterized by amyloid-β (Aβ) peptide deposition into cerebral amyloid plaques. The natural polyphenol resveratrol promotes anti-aging pathways via the activation of several metabolic sensors, including the AMP-activated protein kinase (AMPK). Resveratrol also lowers Aβ levels in cell lines; however, the underlying mechanism responsible for this effect is largely unknown. Moreover, the bioavailability of resveratrol in the brain remains uncertain. Here we show that AMPK signaling controls Aβ metabolism and mediates the anti-amyloidogenic effect of resveratrol in non-neuronal and neuronal cells, including in mouse primary neurons. Resveratrol increased cytosolic calcium levels and promoted AMPK activation by the calcium/calmodulin-dependent protein kinase kinase-β. Direct pharmacological and genetic activation of AMPK lowered extracellular Aβ accumulation, whereas AMPK inhibition reduced the effect of resveratrol on Aβ levels. Furthermore, resveratrol inhibited the AMPK target mTOR (mammalian target of rapamycin) to trigger autophagy and lysosomal degradation of Aβ. Finally, orally administered resveratrol in mice was detected in the brain where it activated AMPK and reduced cerebral Aβ levels and deposition in the cortex. These data suggest that resveratrol and pharmacological activation of AMPK have therapeutic potential against Alzheimer disease.

Alzheimer disease is an age-related neurodegenerative disorder characterized by amyloid-␤ (A␤) peptide deposition into cerebral amyloid plaques. The natural polyphenol resveratrol promotes anti-aging pathways via the activation of several metabolic sensors, including the AMP-activated protein kinase (AMPK). Resveratrol also lowers A␤ levels in cell lines; however, the underlying mechanism responsible for this effect is largely unknown. Moreover, the bioavailability of resveratrol in the brain remains uncertain. Here we show that AMPK signaling controls A␤ metabolism and mediates the anti-amyloidogenic effect of resveratrol in non-neuronal and neuronal cells, including in mouse primary neurons. Resveratrol increased cytosolic calcium levels and promoted AMPK activation by the calcium/ calmodulin-dependent protein kinase kinase-␤. Direct pharmacological and genetic activation of AMPK lowered extracellular A␤ accumulation, whereas AMPK inhibition reduced the effect of resveratrol on A␤ levels. Furthermore, resveratrol inhibited the AMPK target mTOR (mammalian target of rapamycin) to trigger autophagy and lysosomal degradation of A␤. Finally, orally administered resveratrol in mice was detected in the brain where it activated AMPK and reduced cerebral A␤ levels and deposition in the cortex. These data suggest that resveratrol and pharmacological activation of AMPK have therapeutic potential against Alzheimer disease.
Alzheimer disease (AD) 2 is a progressive neurodegenerative disorder and the first cause of dementia. Amyloid-␤ (A␤) pep-tides have a central role in the pathogenesis of the disease and represent the core components of the senile plaques, the lesions invariably found in the neocortex and hippocampus of the AD brains (1,2). In the amyloidogenic pathway, the amyloid-␤ precursor protein (APP) is sequentially cleaved by the aspartic protease ␤-secretase/BACE1 and by the ␥-secretase proteolytic complex to produce various A␤ peptides, including the most abundant isoforms A␤1-40 and A␤1-42 (3,4).
Epidemiological data suggest that moderate consumption of red wine is associated with a lower incidence of dementia and AD (5). The naturally occurring polyphenol resveratrol (trans-3,4Ј,5-trihydroxystilbene), which is found in abundance in red wine, has antioxidant and neuroprotective properties in vitro and could explain, in part, the beneficial effects of wine consumption in AD (6,7). Importantly, resveratrol controls A␤ levels by facilitating its proteolytic clearance in cultured cell lines (8). However, the exact molecular mechanism by which resveratrol controls A␤ metabolism is currently unknown. Furthermore, evidence is missing to support the notion that orally administered resveratrol is bioavailable and bioactive in the brain.
A growing body of literature has demonstrated the beneficial effect of resveratrol on age-related metabolic deterioration and its protective role in metabolic diseases, such as type 2 diabetes and obesity. Resveratrol mimics caloric restriction by extending the lifespan of different small organisms, including Saccharomyces cerevisiae and Caenorhabditis elegans (9,10), and by delaying several aging phenotypes in mice (11). Resveratrol also appears to be protective against the deregulation of energy homeostasis observed in mouse models for metabolic syndromes via the activation of key metabolic sensor proteins, such as the AMP-activated protein kinase (AMPK) and the deacetylase from the sirtuin family SIRT1 (12,13).
AMPK is a Ser/Thr protein kinase formed by a heterotrimeric complex comprising a catalytic ␣ subunit and regulatory ␤ and ␥ subunits. AMPK is activated by different upstream kinases via phosphorylation within its activation loop at Thr-172 (14,15). The main AMPK-activating kinase is LKB1, a protein expressed ubiquitously and recruited for AMPK phosphorylation after an elevation of the AMP/ATP ratio. The calcium/ calmodulin-dependent protein kinase kinase-␤ (CaMKK␤), a kinase with a more restricted expression in neural tissue, also activates AMPK. AMPK phosphorylation at Thr-172 by CaMKK␤ is triggered by an increase in cytosolic calcium levels. AMPK targets several proteins involved in cellular energy balance, including a regulator of fatty acid biosynthesis, acetyl-CoA carboxylase (ACC). The calcium/CaMKK␤/AMPK signaling pathway also controls mechanisms relevant to protein degradation by controlling mTOR (mammalian target of rapamycin) signaling and autophagy (16). Indeed, mTOR is a potent repressor of autophagy and is negatively controlled by AMPK (14,15).
In recent years, several studies have focused on the potential relationship between AD and metabolic diseases. Obesity and diabetes significantly increase cognitive decline and AD risk (17), supporting the notion that molecular mechanisms of cellular energy homeostasis are linked to AD pathogenesis. Here we identify the mechanism involved in the anti-amyloidogenic effect of resveratrol by showing that this polyphenol lowered A␤ accumulation via activation of the metabolic sensor AMPK in different cell lines and in mouse primary neurons. Resveratrol activated AMPK by increasing intracellular calcium levels and by promoting AMPK phosphorylation at Thr-172 by CaMKK␤. Activation of AMPK by resveratrol resulted in mTOR inhibition and initiation of autophagy and lysosomal clearance of A␤. Importantly, we also demonstrate that resveratrol, orally administered in mice, reached the brain where it activated AMPK and significantly reduced A␤ levels and deposition in the cerebral cortex, showing that resveratrol is both bioavailable and bioactive in the brain after oral dosing.
Cell Lines and Drug Treatments-HEK293 (APP-HEK293) and N2a (APP-N2a) cells stably transfected with human APP695 were treated at confluence with the different drugs and for the indicated concentrations and incubation times. Culture medium was changed, and treatments were continued for another 3 h to allow A␤ secretion. Cells were transiently transfected with CA-AMPK and DN-AMPK cDNAs using Lipofectamine 2000 reagent (Invitrogen), as per the manufacturer's instructions. All cell lines were tested negative for mycoplasma contaminants (18).
Transgenic Mice and Brain Extraction-All animal experiments were performed according to procedures approved by the Feinstein Institute for Medical Research Institutional Animal Care and Use Committee. Fifteen-week-old male APP/PS1 transgenic mice (B6C3-Tg(APPswe, PSEN1dE9)85Dbo/J, The Jackson Laboratory) were randomly assigned to resveratrol or control groups. The control groups received a standard AIN-93G diet (Testdiet), and the resveratrol groups received a standard AIN-93G diet supplemented with 0.35% resveratrol. Resveratrol (Sigma-Aldrich) was mixed to homogeneity during manufacturing of the diet (Testdiet) and stored at Ϫ20°C. Diet was replaced every 3 days. Food intake and body weight were monitored weekly throughout the study. At 30 weeks of age, mice were sacrificed. Brains were excised and hemi-dissected, and immediately one hemi-brain was fixed with 4% paraformaldehyde in 0.1 M phosphate-buffered saline, pH 7.6, whereas the other hemi-brain was sequentially extracted, as described (19). Briefly, brains were homogenized and sonicated in Trisbuffered saline containing 2% SDS and 1ϫ Complete protease inhibitor mixture (Roche Applied Science) and centrifuged at 100,000 ϫ g for 1 h at 4°C. The supernatant was then removed, and the resulting pellet was extracted with 70% formic acid in water. For brain A␤ ELISA, 2% SDS extracts were diluted 1:40. Formic acid extracts were neutralized initially by a 1:20 dilution into 1 M Tris phosphate buffer, pH 11, and then diluted as necessary. Brain A␤1-40 and A␤1-42 were quantified by ELISA.
Primary Neuronal Cultures-Primary neurons were prepared as described before (20) from J20 APP transgenic mice (B6.Cg-Tg(PDGFB-APPSwInd)20Lms/2J, The Jackson Laboratory). Briefly, females were sacrificed at 17.5 days of gestation. Fetuses were processed separately to obtain pure transgenic cultures. Genotyping was carried out by isolating tail DNA and as described online in The Jackson Laboratory data base. Forebrains were dissected in ice-cold Hanks' balanced salt solution (Invitrogen) plus 0.5% w/v D-glucose (Sigma) and 25 mM Hepes (Invitrogen) under a dissection microscope. Dissociation was carried out mechanically in ice-cold dissection medium containing 0.01% w/v papain (Worthington), 0.1% w/v dispase (Roche Applied Science), and 0.01% w/v DNase (Worthington) and by incubation at 37°C twice for 15 min. Cells were then spun down at 220 ϫ g for 5 min at 4°C, resuspended in Neurobasal medium with 2% B27, 1 mM sodium pyruvate, 100 units/ml penicillin, 100 g/ml streptomycin, 2 mM Glutamax (Invitrogen), filtered through a 40-m cell strainer (Fisher), counted, and plated on poly-L-ornithine-and laminin-coated plates at a density of about 10 6 cells/well. Culture medium was completely replaced after 16 -20 h, and new medium (30% of starting volume) was added every 3 days until the end of the culture period.
Western Blot (WB) Analyses and A␤ ELISA-5-20 g of cell extracts were analyzed by SDS-PAGE using antibodies listed above. Secreted and intracellular total A␤ or A␤1-40 and A␤1-42 were analyzed by WB or ELISA as described before (8) and in the supplemental Materials and Methods.
Human Phosphoprotein Array and Calcium Measurements-APP-HEK293 cells were treated with 40 M resveratrol (Sigma-Aldrich) or vehicle (DMSO) for 24 h. Cell lysates (250 g of total proteins per array) were applied to the phosphoprotein array following the manufacturer's instructions (Proteome Profiler Human Phosphokinase Array kit, R&D Systems). Free cytosolic calcium was measured using the fluorescent calcium indicator Fluo-4 in cells plated on poly-L-lysine-coated 35-mmdiameter culture dishes, as per the manufacturer's recommendations (Fluo-4 NW Calcium Assay kit, Molecular Probes). Calcium add-back assays were performed as described previously (21) and in the supplemental Materials and Methods.
Immunohistochemistry-For A␤ staining, paraformaldehyde-fixed brain hemispheres were paraffin-embedded and processed according to standard protocols. In brief, sections were pretreated with 70% formic acid for 15 min and immersed in 1% H 2 O 2 for 30 min. Sections were then incubated with 5% milk in Tris-buffered saline for 1 h and with 6E10 primary antibody (1:1000 dilution) overnight at 4°C. Incubation with biotin-coupled anti-mouse IgG1 secondary antibodies (1:1000 dilution) was performed before incubation with streptavidin- horseradish peroxidase (Southern Biotech) and diaminobenzidine tetrahydrochloride. For immunofluorescence, brains were immersion-fixed in 4% paraformaldehyde overnight at 4°C. Staining was performed on mouse brain sagittal Vibratome sections (50 m). Sections were blocked in 5% milk in 0.25% Triton X-100 phosphate-buffered saline for 1 h at room temperature. Sections were then incubated in the presence of primary antibodies directed against NeuN, glial fibrillary acidic protein, and pAMPK for 16 h at 4°C. After washing, sections were incubated with appropriate secondary antibody conjugated to Alexa fluorophores (Invitrogen). Finally, sections were incubated with Sudan Black B (0.3%, 10 min), mounted on glass slides using Vectashield (Vector laboratories), and observed using a confocal microscope.

Analysis of Resveratrol Stability and Content in the Supplemented
Diet-Resveratrol-supplemented diet samples were exposed to air under room temperature conditions without direct sun light for different time points. The samples were then analyzed using a developed LC/UV/MS method, as described in the supplemental Materials and Methods.
Resveratrol Pharmacokinetics and Brain Accumulation in Rodents-All animal procedures were approved by the Purdue Animal Care and Use Committee. Sprague-Dawley rats were dosed orally by gavage with resveratrol in a dose escalation schedule and after an oral 400mg/kg dose. Blood sampling was obtained in the Culex TM automated system (Bioanalytical Systems, West Lafayette, IN). Rats were then fasted for 8 h, dosed orally with 400 mg/kg resveratrol, and sacrificed 1 h postdose. The vascular system was flushed with cold saline. The brains were excised and snap-frozen in liquid nitrogen for subsequent MS analysis. In addition to pharmacokinetic assessment in rats, wild type C57BL/6J mice (The Jackson Laboratory) were fed with AIN-93G diet supplemented or not with resveratrol for 2 weeks. Mice were then sacrificed, and brains were excised and hemi-dissected. One hemi-brain was snap-frozen in liquid nitrogen and stored at Ϫ80°C until extraction for WB analysis. The other hemi-brain was placed in a saline solution containing 0.1% ascorbic acid and frozen at Ϫ80°C for subsequent MS analysis. Blood was collected by intracardiac puncture. Resveratrol was extracted from plasma and homogenized brain tissue (22). Resveratrol metabolites were extracted from plasma using solid phase extraction, as described before (23) and in the supplemental Materials and Methods. LC-MS and MS/MS analyses were completed using an Agilent 1100 HPLC system equipped with an MSD-TOF and a model 6400 QQQ. Separations were conducted using a Varian C18-amide column (2.1 ϫ 150 mm) (Varian Inc., Palo Alto, CA) maintained at a temperature of 30°C. Resveratrol and resveratrol-glucuronide were detected at 227 and 403 m/z, respectively, in rat plasma and brain extracts. Multiple reaction monitoring mode using the transition from m/z 227.1 3 143 was used for detection of low levels of resveratrol in mouse brain and plasma extracts. Quantification was performed from a standard response curve prepared from analysis of authentic standards.

RESULTS
Our previous studies demonstrated that a 24-h incubation with resveratrol significantly reduced extracellular A␤ levels in APP-transfected cells (8). Both secreted A␤1-40 and A␤1-42 were similarly diminished by resveratrol treatments, with a comparable apparent IC 50  showing that resveratrol reduced A␤ levels without affecting APP processing.
In this study we sought to identify the signaling pathway implicated in the control of A␤ levels by resveratrol. Using a kinase screen analyzing 28 major phosphoproteins, we determined that phosphorylation at Thr-172 on AMPK ␣2 subunit was the most robust effect of resveratrol in HEK293 cells (Fig. 1, A-C). Consistent with an activation of AMPK, resveratrol treatment also resulted in a significant elevation of AMPK ␣1 subunit phosphorylation (Fig. 1, A-C). Additional positive hits were identified, including the AMPK target CREB (24,25) and the previously reported target of resveratrol, the checkpoint kinase Chk2 (26) (Fig. 1, A-C). The effect of resveratrol on AMPK and CREB phosphorylation was confirmed by WB analyses. Resveratrol increased the phosphorylation of AMPK and its target ACC in a dose-dependent manner and at concentrations consistent with the effect of this polyphenol on A␤ levels ( Fig. 1, D and E). In the same concentration range, resveratrol also strongly increased dose-dependently the phosphorylation of CREB and ATF1, another CREB/ATF family member (Fig. 1,  D and E). Notably, resveratrol treatment resulted in a robust increase in c-Fos protein levels (Fig. 1D). c-Fos gene promoter contains cyclic AMP response elements, and CREB is a strong regulator of the transcription of this gene (27,28), suggesting that resveratrol not only increased CREB phosphorylation but also activated its transcriptional activity. Together, these results indicate that one of the primary effects of resveratrol is to target AMPK to increase its phosphorylation at Thr-172 and to promote its activation, as demonstrated by the increased phosphorylation of ACC and CREB upon resveratrol treatment.
Like resveratrol, (ϩ)-catechin is a powerful plant-derived antioxidant (29). Although catechin has some structural similarities with resveratrol, it is ineffective at reducing A␤ levels in cultured cells at concentrations as high as 40 M (see Fig. 1, F and G, and Ref. 8). We found that catechin had no effect on AMPK phosphorylation (Fig. 1G), suggesting that the effect on A␤ levels and AMPK activation is relatively specific to resveratrol and independent of its antioxidant properties.
The activating phosphorylation of AMPK at Thr-172 is primarily controlled by two kinases, LKB1 and CaMKK␤. LKB1 activation is tightly controlled by ATP levels via changes in the AMP/ATP ratio (14,15). Because resveratrol was proposed to influence cellular ATP levels (30), we examined whether resveratrol affects ATP levels upon conditions inhibiting A␤ accumulation in HEK293 cells. We found no effect of 20 or 40 M resveratrol on ATP levels ( Fig. 2A). Furthermore, resveratrol treatment was still able to promote AMPK phosphorylation in the LKB1-deficient HeLa cells (Fig. 2B), indicating that LKB1 activation is not required for the effect of resveratrol on AMPK phosphorylation. We then asked whether resveratrol modulates cytosolic calcium levels in treated cells. Measurements of intracellular calcium levels were conducted under resting conditions in the presence of physiological levels of extracellular calcium. To reveal possible changes in the rate of calcium entry in treated cells, measurements were also performed under extracellular calcium add-back conditions, as previously described (21). These conditions are obtained after a transient external calcium depletion to generate a driving force for calcium entry into the cells. Using the calcium fluorescent dye Fluo-4, we found that resveratrol significantly increased cytosolic calcium levels both at resting conditions and after calcium add-back (Fig. 2, C and D). Because cytosolic calcium levels are also controlled by release of the ion from intracellular stores, such as the endoplasmic reticulum (ER), we also evaluated the effect of resveratrol treatment on ER calcium levels. Measurements of ER calcium levels were performed by blocking the sarco/endoplasmic reticulum calcium-ATPase (SERCA), a manipulation that generates a passive leak of calcium from the ER to the cytosol. In Fluo-4-loaded cells, in the absence of extracellular calcium, and in the presence of the SERCA inhibitor thapsigargin, resveratrol dose-dependently reduced the levels of ER calcium (Fig. 2E). Together these results show that resveratrol impaired intracellular calcium homeostasis by significantly increasing cytosolic calcium levels and by facilitating both calcium entry and ER calcium depletion. Because CaMKK␤ is activated by an increase in cytosolic calcium, we then asked whether CaMKK␤ is involved in the activation of AMPK by resveratrol. We found that the CaMKK␤ inhibitor STO-609 effectively reduced the effect of resveratrol on the phosphorylation of both AMPK and ACC (Fig. 2F), indicating that resveratrol increased cytosolic calcium to promote CaMKK␤dependent phosphorylation and activation of AMPK.
We then sought to determine whether direct activation of AMPK controls A␤ metabolism. Pharmacological activation of AMPK by the use of the AMP analog AICAR strongly and significantly lowered extracellular A␤ levels in HEK293 and N2a cells (Fig. 3, A-D). AICAR treatments at concentrations reducing A␤ resulted in a robust increase in the phosphorylation of AMPK and ACC (Fig. 3, A and B), confirming the activation of AMPK in these conditions. In addition, genetic activation of AMPK by transfection of a constitutively active form of AMPK ␣1 subunit (31) led to a robust increase of ACC phosphorylation and to a significant reduction of A␤ levels (Fig. 3, E-G). Thus, AMPK activation in cell lines lowered extracellular A␤ accumulation, including A␤1-40 and A␤1-42. Furthermore, expression of a dominant-negative form of AMPK (31) significantly inhibited the effect of resveratrol on ACC phosphorylation (Fig. 3, H and I) and on the levels of secreted A␤ (Fig. 3H), including A␤1-40 and A␤1-42 (Fig. 3J). Together these data show that resveratrol lowered A␤ levels by activating AMPK.
AMPK is expressed in many cell types and tissues. In the adult brain, all AMPK isoforms predominantly localize in neurons (32,33). A more restricted expression of some AMPK subunits was also found in hippocampal astrocytes (32). Although AMPK activation has been extensively studied in hypothalamic neurons for its role in food intake (34,35), little is known about the constitutive levels of activation of AMPK in neurons or in glial cells in other brain regions, such as the hippocampus and cortex. By immunofluorescence in adult mouse brain, we found that activated AMPK is present in most cortical and hippocampal neurons, as shown by the strong colocalization between phosphorylated AMPK and the neuronal marker NeuN (Fig. 4A, panels a-f). No significant staining for phosphorylated AMPK was found in hippocampal astro- cytes (Fig. 4A, panels g-i), indicating that under constitutive conditions in the brain, AMPK is mostly active in neurons. Using immunocytochemistry, we confirmed that activated AMPK is present in primary neuronal cultures (Fig. 4B).
In this context and to confirm our findings in a more physiologically relevant system, we assessed the effect of resveratrol on AMPK activation and A␤ levels in primary neurons. We found that resveratrol significantly and in a dose-dependent manner reduced secreted A␤ levels in primary neurons isolated from APP transgenic mouse forebrain (Fig. 4D), whereas full-length APP levels remained unchanged (Fig.  4C). At concentrations reducing A␤, resveratrol led to an increase in phosphorylated AMPK and ACC (Fig. 4C), showing that resveratrol can activate AMPK in forebrain neurons. Direct activation of AMPK with AICAR also resulted in a significant decrease in neuronal A␤ levels (Fig. 4, E and F). Importantly, pretreatment with the AMPK inhibitor, compound C, significantly prevented the effect of resveratrol on neuronal A␤ levels (Fig. 4, G and H). Together, these results show that in neurons resveratrol reduced secreted A␤ accumulation by activating AMPK.
To gain insight into the mechanism by which resveratrol promotes a reduction of A␤ levels, we asked whether the polyphenol facilitates intracellular degradation of A␤. Our previous work has shown that resveratrol does not significantly affect APP processing and A␤ production but instead facilitates intracellular A␤ clearance (8). AMPK is a key regulator of autophagy (or macroautophagy), an evolutionary conserved lysosomal pathway involved in protein and organelle turnover via the formation of vacuoles known as autophagosomes (36). Autophagy is deregulated in AD brain and participates in intracellular A␤ degradation in vitro and in vivo (37,38). AMPK activation promotes autophagy by repressing the Ser/ Thr protein kinase mTOR, which represents a key blocker of autophagosome formation (39). Interestingly, resveratrol was found to induce autophagy in cancer cell lines (40). In this context we asked whether resveratrol (i) inhibits mTOR activity, (ii) promotes autophagy, and (iii) leads to intracellular degradation of A␤ by the lysosomal system.
We observed that treatments of HEK293 cells with resveratrol resulted in a dose-dependent inhibition of the phosphorylation of p70-S6 kinase, eIF4B (eukaryotic initiation factor 4B), and S6 ribosomal protein, three proteins downstream from mTOR activation (Fig. 5, A and B). Importantly, resveratrol treatments also led to a strong and dose-dependent conversion of the light chain 3 (LC3) protein from LC3-I to LC3-II, which represents a marker for autophagy induction (Fig. 5, C-E). Furthermore, formation of autophagosomes immunoreactive for LC3 was observed in the presence of resveratrol (Fig. 5F). Finally, treatment with the lysosomotropic drugs bafilomycin A1 and chloroquine, which neutralize lysosomal degradation, resulted in a significant increase of intracellular A␤ accumulation in the presence of resveratrol (Fig. 5, G and H). In summary, these data show that resveratrol inhibited mTOR to induce autophagy and intracellular degradation of A␤ by the lysosomal system.
Based on these in vitro results in cell lines and in primary neurons, we tested the ability of resveratrol to control AMPK activation and A␤ accumulation in the brain of APP/PS1 transgenic mice. This mouse model co-expresses the familial ADlinked mutants of APP (Swedish) and presenilin-1 (PSEN1⌬E9) (41) and exhibits high levels of soluble brain A␤ and develops a robust amyloid pathology in different brain regions of the hippocampus and cerebral cortex by 30 weeks of age (41).
An efficient metabolic effect of resveratrol has recently been achieved in vivo in mice by oral administration of a supplemented diet (42,43). This effect was found to be mediated by SIRT1 and AMPK activation. The authors indicated that a dose of 0.4% resveratrol in the diet was well tolerated by the mice for 15 weeks of treatment (43). Because our studies show that AMPK is involved in the effect of resveratrol on A␤ levels in cultured cells, we followed the same protocol to evaluate the in vivo efficacy of resveratrol on A␤ accumulation and amyloid deposition in APP/PS1 mice. Two independent groups of 10 male mice (15 weeks old) were fed an AIN-93G diet supplemented or not with 0.4% resveratrol for a period of 15 weeks.
Before administration in animals, the content of resveratrol in the diet was verified using LC/UV/MS methods. The results show that the variation of resveratrol content in the samples was 0.347 Ϯ 0.053% (n ϭ 10). We also analyzed the stability of this polyphenol in the supplemented diet under room conditions during different incubation periods. Resveratrol-supplemented diet samples were exposed to air at room temperature and under conditions without direct sunlight and then analyzed using LC/UV/MS. We found that resveratrol in solid status (mixed in the diet) is very stable under room conditions. No decomposed peak was detected in both UV and MS detections in the samples up to 4 days under room conditions, whereas only trace levels of trans-resveratrol conversion into cis-res-veratrol was observed after 7 days (Fig. 6A). These results show that resveratrol is detected on average at 0.35% in the supplemented diet and, in line with previous studies (44), appears to be very stable under ambient typical room conditions for durations comparable with the one required for the animal treatments.
We determined that the mice ingested ϳ350 mg/kg of body weight daily of resveratrol from the supplemented diet during the 15-week period (Fig. 7B). Based on this information, absorption of resveratrol and accumulation by the brain was investigated in rodents treated either by intragastric gavage or by oral administration through the diet. Plasma and brain levels of resveratrol and its primary metabolite, resveratrol-glucuronide, were evaluated by LC-MS in Sprague-Dawley rats dosed by gavage in escalation at 100, 250, and 400 mg/kg for 3 days at each dose before an acute 400 mg/kg dose of resveratrol (see "Experimental Procedures"). Plasma resveratrol profiles indicated a rapid rise of resveratrol and resveratrol-glucuronide content in the plasma at 4 h after administration followed by a slow decline, indicating a lack of complete clearance of the polyphenol during the test period with repeated dosing (Fig.  6B). Importantly, in the same animals resveratrol was detected in perfused brain at a concentration of ϳ1.7 nmol/g wet weight (Fig. 6C). The levels of resveratrol and resveratrol-glucuronide were also analyzed in mice fed for 2 weeks resveratrol-supplemented diet. Using LC-MS/MS in multiple reaction monitoring mode, resveratrol was detected in deconjugated brain extracts of treated mouse brain but not in control animals (Fig.  6D). Strikingly, in these animals administered with a resveratrol-supplemented diet, we observed an increase in the phosphorylation of both AMPK and ACC in the brain as compared with control mice (Fig. 6, E and F).
The APP/PS1 mice fed the diet supplemented with resveratrol were in apparent good health at the end of the 15-week period. The mice gained weight slightly during the treatment period, with no significant difference between the controls and the resveratrol-fed mice (Fig. 7A). By ELISA and WB analyses, the levels of soluble and insoluble A␤1-40 and A␤1-42 from brain homogenates sequentially extracted with SDS and formic acid, respectively, were analyzed in resveratrol-fed and control mice (Fig. 7, C and D). The hippocampal and cortical regions were also analyzed by immunohistochemistry using 6E10 anti-A␤ antibody (Fig. 7, F-K). We found a significant decrease in both soluble and insoluble A␤1-40 (30%, p ϭ 0.018 and 25%, p ϭ 0.05, respectively) and of insoluble A␤1-42 (25%, p ϭ 0.012) in total brain homogenates in resveratrol-fed mice, as compared with control mice (Fig. 7, C and D). A trend of decrease in soluble A␤1-42 was also observed in resveratroltreated mice (17%, p ϭ 0.36; Fig. 7C), whereas APP and APP-CTF levels remained unchanged between treated and untreated  shown is the plasma pharmacokinetic response of RSV and its major metabolite, resveratrol-glucuronide (RSV-gluc), after oral gavage of 400 mg/kg body weight of resveratrol after pretreatment with dose escalation of resveratrol, as described under "Experimental Procedures." Data represent the mean Ϯ S.E., n ϭ 4 rats. C, shown is LC-MS separation of RSV and resveratrolglucuronide (RSV-gluc) from an extract of plasma and brain from rats administered resveratrol at 400 mg/kg body weight by oral gavage. Extracted ion chromatograms at 227 and 403 m/z are shown for RSV and RSV-gluc, respectively. D, detection of RSV in extracts of plasma and brain from mice fed the diet supplemented with 0.35% resveratrol for 2 weeks is shown. Extracted ion chromatograms collected in multiple reaction monitoring mode (transitions 227 3 143; m/z) are shown in mouse plasma (Treated plasma) or brain extracts (Treated brain) from resveratrol-treated mice or in brain extracts from non-treated control mice (Control brain). E and F, brain extracts from mice fed for 2 weeks a diet supplemented (RSV) or not (CTRL) with 0.35% resveratrol were analyzed by WB for pAMPK, AMPK, pACC, and ACC. a.u., arbitrary units. F, shown are densitometric analysis and quantification of the pAMPK/AMPK and pACC/ACC ratios in brain extracts from mice treated as in E. Histograms show the mean Ϯ S.D. of three independent experiments. *, p Ͻ 0.05; **, p Ͻ 0.01 (Student's t test). mice (Fig. 7E). Although no significant effect on amyloid deposition in the hippocampus was found (Fig. 7, I-K), a significant reduction of amyloid deposition in the cerebral cortex was observed in resveratrol-fed mice, as determined by measure-ments of both plaque numbers (42% decrease, p ϭ 0.005) and amyloid deposition area (34% decrease, p ϭ 0.042; Fig. 7, F-H). Thus, resveratrol administered in the diet reached the brain where it significantly activated AMPK and reduced A␤ levels and amyloid deposition in the cerebral cortex.

DISCUSSION
Using an unbiased approach, we showed that AMPK signaling activation is a central event in the antiamyloidogenic effect of resveratrol in vitro and in vivo. Specifically, we demonstrated that resveratrol turned on AMPK activity both in cell cultures and in vivo in mouse brain after oral dosing and that AMPK inhibition significantly impaired the effect of resveratrol on A␤ accumulation in cell lines and in primary neurons. The observation that resveratrol activates AMPK is in line with previous studies using cell line cultures and animal models (42,(45)(46)(47)(48)(49). For instance, it was shown that resveratrol administered orally through the diet can activate AMPK at the periphery in mouse liver (42). Resveratrol was also found to activate AMPK in the brain when administered by intraperitoneal injections (46). Using MS separation, we demonstrate in this study that resveratrol administered orally by gavage or by chronic supplementation of the diet led to the accumulation of this polyphenol in the brain. Consequently, we observed a robust activation of cerebral AMPK and a significant reduction of A␤ levels and deposition in the mouse cortex. These results not only revealed that resveratrol is bioavailable and bioactive in the brain after oral dosing but also demonstrated the anti-amyloidogenic potential of this polyphenol in vivo.
The direct target of resveratrol in vitro and in vivo and the exact mechanism by which AMPK is activated is not firmly established. Resveratrol binds in vitro to SIRT1 and activates the deacetylase activity of this enzyme (50,51). SIRT1 may also represent a main target of resveratrol metabolic functions in muscle tissue (43). It was proposed that SIRT1 activation by resveratrol can FIGURE 7. Resveratrol lowers A␤ accumulation and deposition in vivo in mice. A-K, from 15 to 30 weeks of age, male APP/PS1 mice were fed a diet supplemented (RSV) or not (CTRL) with 0.35% resveratrol. Mouse weight (A) and resveratrol (RSV) intake (B) were monitored weekly. C and D, shown are ELISA measurements of soluble (SDS Fraction) and insoluble (formic acid, FA Fraction) A␤1-40 and A␤1-42 levels in total mouse brain. E, brain extracts were analyzed by WB for APP, APP-CTFs, and actin. F-K, shown are amyloid deposition assessments in the cortex (F-H) and hippocampus (I-K) of control (CTRL) and RSV-fed mice by immunohistochemistry staining using 6E10 anti-A␤ antibody. Graphs show the number of plaques per section (F and I) and the percent area occupied with positive staining (G and J). Graphs indicate the mean Ϯ S.E., n ϭ 9. *, p Յ 0.05; **, p Ͻ 0.02; ***, p Ͻ 0.005 (Student's t test). lead to LKB1 and AMPK activation in HepG2 hepatocytes and HEK293T cells (52,53). However, recent studies showed that resveratrol can also activate AMPK independently of SIRT1. Indeed, resveratrol can activate AMPK in SIRT1-deficient mouse embryonic fibroblasts (13) or in the presence of SIRT1 inhibitors in neurons (46).
The present work provides strong evidence that resveratrol activated AMPK by increasing cytosolic calcium levels and by activating CaMKK␤-dependent phosphorylation of AMPK. Our data indicate that resveratrol promoted both extracellular calcium entry into the cells and ER calcium depletion. These consequences of resveratrol treatments could both contribute to the observed elevation of cytosolic calcium levels. ER calcium release, which leads to a transient depletion in ER calcium levels, is coupled to the mechanism of calcium entry called storeoperated calcium entry (54). Interestingly, a similar effect of resveratrol on ER calcium release (55,56) and store-operated calcium entry (55) has also been reported in vascular myocyte cultures and breast cancer cells. Additional studies will be required to determine whether resveratrol increased cytosolic calcium by directly triggering ER calcium release to facilitate the resulting calcium influx via store-operated calcium entry.
Recently, it was shown that the anti-diabetic drug metformin increases A␤ levels by up-regulating BACE1 expression (57). The authors proposed that the effect of the drug on A␤ levels is dependent on AMPK activation (57). The exact mechanism of action of metformin in vivo is not known. The drug activates AMPK in several cell lines and requires the AMPK-activating kinase LKB1 to control glucose homeostasis in mice (58). However, AMPK-independent molecular targets of metformin were also identified in vivo (59). In this report we employed pharmacological and genetic approaches to demonstrate that the direct activation of AMPK led to a robust inhibition of A␤ accumulation. We showed that the AMP analog AICAR lowered A␤ levels at concentrations promoting AMPK activation. We also found that expression of a constitutively active form of AMPK resulted in a marked reduction of extracellular A␤ levels. These results clearly demonstrate that AMPK activation directly correlated with a decrease of A␤ levels (see also Ref. 74). Therefore, it is likely that the effect of metformin on A␤ levels and BACE1 expression is controlled in part by AMPK-independent pathways.
Strong evidence indicates that autophagy is deregulated in AD brain and participates in intracellular A␤ degradation in cell lines and in vivo in animal models (37,38). Our previous work proposed that resveratrol did not affect APP processing but instead promoted A␤ degradation (8). Here, we show that resveratrol activated autophagy and intracellular clearance of A␤ by the lysosomal pathway. Indeed, resveratrol was found to potently inhibit the activity of mTOR, an AMPK target critically involved in autophagy repression. This inhibition of mTOR by resveratrol resulted in LC3 conversion and in the formation of LC3 positive vesicles, two key markers of autophagy induction. Autophagy is coupled to lysosomal degradation, and evidence is increasing that the effect of autophagy on A␤ is due to coupling to the lysosomal system (38,60). We found that neutralization of lysosomal degradation by the use of lysosomotropic drugs resulted in a significant increase of intracellular A␤ accumulation in the presence of resveratrol. To the best of our knowledge, this study is the first to reveal that res-veratrol can induce autophagy to facilitate intracellular degradation of A␤ by the lysosomal system.
The anti-amyloidogenic effect of resveratrol (8) was just confirmed in vivo in mice (61). In this study, which occurred as our study was completed, the mice received an identical diet as the one used in our report (AIN-93G) supplemented with 0.2% resveratrol. Although in our study the diet was supplemented with a higher amount of resveratrol (0.35%), the estimated daily dosage was comparable between the two studies, between 300 and 350 mg/kg (see Fig. 7B and Ref. 61). In agreement with our data, the authors found that resveratrol intake lowered both plaque numbers and plaque area in different brain regions, with the exception of the hippocampal region where no statistically significant changes were noted in both studies (see Fig. 7 and Ref. 61).
Concordant epidemiological data suggest that moderate consumption of red wine is associated with a lower incidence of dementia and AD (62)(63)(64)(65). Studies in AD mouse models have also shown that red wine intake attenuates cerebral amyloid deposition and A␤-associated cognitive deterioration (66,67). Resveratrol is found in abundance in grape skin and red wine and has potential antioxidant, anti-amyloidogenic, and neuroprotective properties (6,7,9). Indeed, resveratrol delays A␤-induced toxicity in different neuronal cell culture models (68 -70) and exerts anti-aggregation and anti-fibrillogenic effects on A␤ in vitro (71,72). Resveratrol, delivered by intracerebroventricular injections, was also found to reduce neurodegeneration in p25 transgenic mice, a model for AD and tauopathies (73). Together with the present work demonstrating the anti-amyloidogenic effect of resveratrol in vivo, these data indicate that resveratrol could explain in part the beneficial effects of wine consumption in AD (7,8).
In summary, this work shows that resveratrol lowered A␤ accumulation by activating AMPK signaling in cell lines and in primary neuronal cultures. Resveratrol activated AMPK by increasing cytosolic calcium levels and by promoting CaMKK␤dependent phosphorylation of AMPK. In addition, resveratrol reduced A␤ accumulation by activating autophagy and by facilitating the lysosomal degradation of A␤. We further report that orally administered resveratrol in mice crossed the blood-brain barrier, activated brain AMPK, and reduced A␤ levels and deposition in the cerebral cortex. Evidence is emerging to support the potential of resveratrol against neurodegenerative disorders. However, no clear neuroprotective mechanism has been proposed so far. This study identifies AMPK as a key neuroprotective kinase against A␤ accumulation. This work provides a rationale for exploring the therapeutic potential of resveratrol and AMPK activation in AD (7).