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Litwin-Zucker Research Center for the Study of Alzheimer's Disease, The Feinstein Institute for Medical Research, North Shore-LIJ, Manhasset, New York 11030Department of Pathology, Albert Einstein College of Medicine, Bronx, New York 10461
Litwin-Zucker Research Center for the Study of Alzheimer's Disease, The Feinstein Institute for Medical Research, North Shore-LIJ, Manhasset, New York 11030Department of Pathology, Albert Einstein College of Medicine, Bronx, New York 10461
* This work was supported, in whole or in part, by National Institutes of Health Grant PO1 AT004511 (NCCAM Project 2; to P. M.). This work was also supported in part by the Alzheimer's Association (to P. M.) and the Institute for the Study of Aging (Alzheimer's Drug Discovery Foundation (to P. M.)). The on-line version of this article (available at http://www.jbc.org) contains supplemental “Materials and Methods” and Fig. S1.
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.
calcium/calmodulin-dependent protein kinase kinase-β
high performance liquid chromatography
light chain 3
cAMP-response element-binding protein
enzyme-linked immunosorbent assay
is a progressive neurodegenerative disorder and the first cause of dementia. Amyloid-β (Aβ) peptides 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 (
). In the amyloidogenic pathway, the amyloid-β precursor protein (APP) is sequentially cleaved by the aspartic protease β-secretase/BACE1 and by the γ-secretase proteolytic complex to produce various Aβ peptides, including the most abundant isoforms Aβ1–40 and Aβ1–42 (
). The naturally occurring polyphenol resveratrol (trans-3,4′,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 (
). However, the exact molecular mechanism by which resveratrol controls Aβ 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 (
). 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 (
AMPK is a Ser/Thr protein kinase formed by a heterotrimeric complex comprising a catalytic α subunit and regulatory β and γ subunits. AMPK is activated by different upstream kinases via phosphorylation within its activation loop at Thr-172 (
). 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-β (CaMKKβ), a kinase with a more restricted expression in neural tissue, also activates AMPK. AMPK phosphorylation at Thr-172 by CaMKKβ 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/CaMKKβ/AMPK signaling pathway also controls mechanisms relevant to protein degradation by controlling mTOR (mammalian target of rapamycin) signaling and autophagy (
), 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 Aβ 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 CaMKKβ. Activation of AMPK by resveratrol resulted in mTOR inhibition and initiation of autophagy and lysosomal clearance of Aβ. Importantly, we also demonstrate that resveratrol, orally administered in mice, reached the brain where it activated AMPK and significantly reduced Aβ levels and deposition in the cerebral cortex, showing that resveratrol is both bioavailable and bioactive in the brain after oral dosing.
Our previous studies demonstrated that a 24-h incubation with resveratrol significantly reduced extracellular Aβ levels in APP-transfected cells (
and supplemental Fig. S1), showing that resveratrol reduced Aβ levels without affecting APP processing.
In this study we sought to identify the signaling pathway implicated in the control of Aβ levels by resveratrol. Using a kinase screen analyzing 28 major phosphoproteins, we determined that phosphorylation at Thr-172 on AMPK α2 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 α1 subunit phosphorylation (Fig. 1, A–C). Additional positive hits were identified, including the AMPK target CREB (
) (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 Aβ 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 (
), 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, (+)-catechin is a powerful plant-derived antioxidant (
). We found that catechin had no effect on AMPK phosphorylation (Fig. 1G), suggesting that the effect on Aβ 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 CaMKKβ. LKB1 activation is tightly controlled by ATP levels via changes in the AMP/ATP ratio (
), we examined whether resveratrol affects ATP levels upon conditions inhibiting Aβ accumulation in HEK293 cells. We found no effect of 20 or 40 μm 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 (
). 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 CaMKKβ is activated by an increase in cytosolic calcium, we then asked whether CaMKKβ is involved in the activation of AMPK by resveratrol. We found that the CaMKKβ 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 CaMKKβ-dependent phosphorylation and activation of AMPK.
We then sought to determine whether direct activation of AMPK controls Aβ metabolism. Pharmacological activation of AMPK by the use of the AMP analog AICAR strongly and significantly lowered extracellular Aβ levels in HEK293 and N2a cells (Fig. 3, A–D). AICAR treatments at concentrations reducing Aβ 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 α1 subunit (
) led to a robust increase of ACC phosphorylation and to a significant reduction of Aβ levels (Fig. 3, E–G). Thus, AMPK activation in cell lines lowered extracellular Aβ accumulation, including Aβ1–40 and Aβ1–42. Furthermore, expression of a dominant-negative form of AMPK (
) significantly inhibited the effect of resveratrol on ACC phosphorylation (Fig. 3, H and I) and on the levels of secreted Aβ (Fig. 3H), including Aβ1–40 and Aβ1–42 (Fig. 3J). Together these data show that resveratrol lowered Aβ levels by activating AMPK.
AMPK is expressed in many cell types and tissues. In the adult brain, all AMPK isoforms predominantly localize in neurons (
), 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 astrocytes (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 Aβ levels in primary neurons. We found that resveratrol significantly and in a dose-dependent manner reduced secreted Aβ levels in primary neurons isolated from APP transgenic mouse forebrain (Fig. 4D), whereas full-length APP levels remained unchanged (Fig. 4C). At concentrations reducing Aβ, 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 Aβ levels (Fig. 4, E and F). Importantly, pretreatment with the AMPK inhibitor, compound C, significantly prevented the effect of resveratrol on neuronal Aβ levels (Fig. 4, G and H). Together, these results show that in neurons resveratrol reduced secreted Aβ accumulation by activating AMPK.
To gain insight into the mechanism by which resveratrol promotes a reduction of Aβ levels, we asked whether the polyphenol facilitates intracellular degradation of Aβ. Our previous work has shown that resveratrol does not significantly affect APP processing and Aβ production but instead facilitates intracellular Aβ clearance (
). 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 (
). In this context we asked whether resveratrol (i) inhibits mTOR activity, (ii) promotes autophagy, and (iii) leads to intracellular degradation of Aβ 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 Aβ 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 Aβ 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 Aβ accumulation in the brain of APP/PS1 transgenic mice. This mouse model co-expresses the familial AD-linked mutants of APP (Swedish) and presenilin-1 (PSEN1ΔE9) (
). Because our studies show that AMPK is involved in the effect of resveratrol on Aβ levels in cultured cells, we followed the same protocol to evaluate the in vivo efficacy of resveratrol on Aβ 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 ± 0.053% (n = 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-resveratrol 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 (
), 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 ∼350 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 ∼1.7 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 Aβ1–40 and Aβ1–42 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-Aβ antibody (Fig. 7, F–K). We found a significant decrease in both soluble and insoluble Aβ1–40 (30%, p = 0.018 and 25%, p = 0.05, respectively) and of insoluble Aβ1–42 (25%, p = 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 Aβ1–42 was also observed in resveratrol-treated mice (17%, p = 0.36; Fig. 7C), whereas APP and APP-CTF levels remained unchanged between treated and untreated 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 measurements of both plaque numbers (42% decrease, p = 0.005) and amyloid deposition area (34% decrease, p = 0.042; Fig. 7, F–H). Thus, resveratrol administered in the diet reached the brain where it significantly activated AMPK and reduced Aβ levels and amyloid deposition in the cerebral cortex.
Using an unbiased approach, we showed that AMPK signaling activation is a central event in the anti-amyloidogenic 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 Aβ 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 (
). 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 Aβ 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 (
The present work provides strong evidence that resveratrol activated AMPK by increasing cytosolic calcium levels and by activating CaMKKβ-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 store-operated calcium entry (
) 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 Aβ levels by up-regulating BACE1 expression (
). In this report we employed pharmacological and genetic approaches to demonstrate that the direct activation of AMPK led to a robust inhibition of Aβ accumulation. We showed that the AMP analog AICAR lowered Aβ 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 Aβ levels. These results clearly demonstrate that AMPK activation directly correlated with a decrease of Aβ levels (see also Ref.
). Here, we show that resveratrol activated autophagy and intracellular clearance of Aβ 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 Aβ is due to coupling to the lysosomal system (
). We found that neutralization of lysosomal degradation by the use of lysosomotropic drugs resulted in a significant increase of intracellular Aβ accumulation in the presence of resveratrol. To the best of our knowledge, this study is the first to reveal that resveratrol can induce autophagy to facilitate intracellular degradation of Aβ by the lysosomal system.
). 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.
). 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.
). 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 (
In summary, this work shows that resveratrol lowered Aβ accumulation by activating AMPK signaling in cell lines and in primary neuronal cultures. Resveratrol activated AMPK by increasing cytosolic calcium levels and by promoting CaMKKβ-dependent phosphorylation of AMPK. In addition, resveratrol reduced Aβ accumulation by activating autophagy and by facilitating the lysosomal degradation of Aβ. We further report that orally administered resveratrol in mice crossed the blood-brain barrier, activated brain AMPK, and reduced Aβ 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 Aβ accumulation. This work provides a rationale for exploring the therapeutic potential of resveratrol and AMPK activation in AD (
We thank Dr. Gopal Thinakaran (University of Chicago, Chicago, IL) for kindly providing us with APP695-N2a cells, Dr. Luciano D'Adamio (Albert Einstein College of Medicine, Bronx, NY) for APP695-HEK293 cells, Dr. David Carling (MRC Clinical Sciences Centre, Imperial College, London, UK) for T172D-AMPK and T172A-AMPK cDNAs, Dr. Pankaj D. Mehta (Institute for Basic Research in Developmental Disabilities, Staten Island, NY) for R1 antibody, and Dr. Amanda Chan (The Feinstein Institute for Medical Research, Manhasset, NY) for assistance with microscopy studies.