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. 2010 Mar 19;285(12):9100-13.
doi: 10.1074/jbc.M109.060061. Epub 2010 Jan 14.

AMP-activated protein kinase signaling activation by resveratrol modulates amyloid-beta peptide metabolism

Valérie Vingtdeux  1 , Luca Giliberto, Haitian Zhao, Pallavi Chandakkar, Qingli Wu, James E Simon, Elsa M Janle, Jessica Lobo, Mario G Ferruzzi, Peter Davies, Philippe Marambaud
Affiliations

AMP-activated protein kinase signaling activation by resveratrol modulates amyloid-beta peptide metabolism

Valérie Vingtdeux et al. J Biol Chem. .

Abstract

Alzheimer disease is an age-related neurodegenerative disorder characterized by amyloid-beta (Abeta) 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 Abeta 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 Abeta 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-beta. Direct pharmacological and genetic activation of AMPK lowered extracellular Abeta accumulation, whereas AMPK inhibition reduced the effect of resveratrol on Abeta levels. Furthermore, resveratrol inhibited the AMPK target mTOR (mammalian target of rapamycin) to trigger autophagy and lysosomal degradation of Abeta. Finally, orally administered resveratrol in mice was detected in the brain where it activated AMPK and reduced cerebral Abeta levels and deposition in the cortex. These data suggest that resveratrol and pharmacological activation of AMPK have therapeutic potential against Alzheimer disease.

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Figures

FIGURE 1.
FIGURE 1.
Resveratrol activates AMPK. A–C, APP-HEK293 cells were treated for 24 h with 40 μm resveratrol or DMSO (Control). Cell extracts were then probed on human phosphoprotein arrays. Results were expressed as a % of the control levels (A). Representative phosphoprotein array analyses are shown in B and C. Boxes 1–3 indicate phospho-Thr-174 AMPKα1, phospho-Thr-172 AMPKα2, and phospho-Ser-133 CREB, respectively. D, APP-HEK293 cells were treated for 24 h with the indicated concentrations of resveratrol (RSV). Two independent sources of polyphenol were tested: synthetic resveratrol (Sigma, >99%, GC) and purified natural resveratrol isolated from Polygonum cuspidatum (Chromadex, 99%, HPLC). Cell extracts were then analyzed by WB for secreted total Aβ, and cellular phospho-AMPK (pAMPK), AMPK, phospho-ACC (pACC), ACC, actin, phospho-CREB (pCREB), CREB, and c-Fos levels. E, shown are densitometric analysis and quantification of the ratios pAMPK/AMPK, pACC/ACC, and pCREB/CREB in three independent experiments as in D. a.u., arbitrary units. F and G, APP-HEK293 cells were treated for 24 h with the indicated concentrations of catechin or with 40 μm RSV. Secreted Aβ1–40 and Aβ1–42 levels were analyzed by ELISA (F). The levels of the indicated proteins were analyzed by WB (G). Histograms in E and F show the means ± S.D. of three independent experiments. CTRL, control.
FIGURE 2.
FIGURE 2.
Resveratrol increases intracellular calcium levels and promotes CaMKKβ-dependent phosphorylation of AMPK. A, shown are intracellular ATP levels in APP-HEK293 cells treated for 1 or 24 h with the indicated concentrations of resveratrol (RSV) or with 2-deoxy-d-glucose + antimycin A (2DG/AM; used as control for ATP production inhibition). B, shown are WB analyses of pAMPK, AMPK, actin, and LKB1 levels in HEK293 (lane 1) and HeLa (lanes 2–6) cells treated with the indicated concentrations of resveratrol. C, shown are cytosolic calcium measurements with Fluo-4 loading and calcium add-back conditions in APP-HEK293 cells treated for 24 h with the indicated concentrations of RSV. Cells were incubated in calcium-free buffer (0 CaCl2) and then challenged with physiological extracellular calcium concentrations (1.4 mm CaCl2) to monitor the progressive restoration of basal cytoplasmic calcium levels. Traces illustrate the mean relative fluorescence units (RFU) ± S.D. (shaded area) of three independent experiments. CTRL, control. D, peak and steady state of cytosolic calcium measurements are as in C, expressed in ΔF/F0. Histograms show the mean ± S.D. of three independent experiments. *, p < 0.001 (Student's t test). E, shown are cytosolic calcium measurements in cells treated with RSV as in C and incubated with 2 μm thapsigargin in calcium-free buffer. F, shown are WB analyses of pAMPK, pACC, and actin levels in APP-HEK293 cells incubated for 24 h with the indicated concentrations of STO-609 and in the absence (−) or presence (+) of 40 μm RSV.
FIGURE 3.
FIGURE 3.
Resveratrol lowers Aβ levels by activating AMPK. A, APP-HEK293 cells were treated for 24 h with the indicated concentrations of AICAR. Secreted Aβ and cellular pAMPK, AMPK, pACC, ACC, and actin levels were analyzed by WB. B, shown are densitometric analysis and quantification of the pACC/ACC ratio in cells treated as in A. a.u., arbitrary units. C and D, shown are ELISA measurements of secreted Aβ1–40 and Aβ1–42 levels from APP-HEK293 (C) or APP-N2a (D) cells treated with AICAR as in A. E, APP-HEK293 cells were transfected for 24 h with control vector (Vector) or with a Myc-tagged constitutively active form of AMPK (CA-AMPK). Myc-AMPK, pACC, ACC, actin, and secreted Aβ levels were analyzed by WB. F, shown are densitometric analysis and quantification of the pACC/ACC ratio in cells treated as in E. G, shown are ELISA measurements of secreted Aβ1–40 and Aβ1–42 levels from cells treated as in E. H, APP-HEK293 cells were transfected with control vector (V) or with a Myc-tagged dominant negative form of AMPK (DN). 24 h post-transfection, cells were treated for 24 h with DMSO (CTRL) or 40 μm resveratrol (RSV). Myc-AMPK, pACC, ACC, actin, and secreted Aβ levels were analyzed by WB. I, shown are densitometric analysis and quantification of the pACC/ACC ratio in cells treated as in H. J, shown are ELISA measurements of secreted Aβ1–40 and Aβ1–42 levels from cells treated as in H. Histograms in B–D, F, G, I, and J show the mean ± S.D. of 3–4 independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (Student's t test).
FIGURE 4.
FIGURE 4.
Resveratrol lowers Aβ levels by activating AMPK in primary neurons. A, shown is pAMPK (panels a, d, and g), NeuN (panels b and e), GFAP (panel h), and merged (panels c, f, and i) staining of sagittal brain sections of an adult mouse. Hippocampus (panels a–c and g–i) and cerebral cortex (panels d–f) are shown. B, shown is phase contrast (PC), 4′,6-diamidino-2-phenylindole (DAPI), NeuN, pAMPK, and merged staining of 15 days in vitro primary neuronal cultures from J20 APP transgenic mice. C–H, WB analyses of pAMPK, AMPK, pACC, ACC, APP, and actin (C, E, and G) and ELISA measurements of secreted Aβ1-x levels (D, F, and H) in 15 days in vitro J20 mouse primary neurons treated for 24 h with the indicated concentrations of resveratrol (RSV, C and D) or AICAR (E and F). Primary neurons in G and H were treated for 24 h with 80 μm resveratrol or its vehicle (DMSO, control (CTRL)) in the absence or presence of compound C (CC, 20 μm). **, p < 0.01; ns, not significant (Student's t test).
FIGURE 5.
FIGURE 5.
Resveratrol induces autophagy and lysosomal degradation of Aβ. A–D, APP-HEK293 cells were treated for 24 h with the indicated concentrations of resveratrol (RSV). Cell extracts were then analyzed by WB for phospho-p70S6K (p-p70S6K), p70S6K, phospho-eIF4B (peIF4B), phospho-S6 (pS6), and actin (A) and for LC3 (C). Densitometric analysis and quantification of the p-p70S6K/p70S6K (B) and LC3-II/LC3-I (D) ratios are shown. a.u., arbitrary units. E, WB analyses of LC3 and actin in APP-HEK293 cells treated for the indicated times with 40 μm RSV. F, immunocytochemistry with anti-APP (red) and anti-LC3 (green) antibodies in APP-HEK293 cells incubated with for 72 h in the absence (CTRL) or presence of 40 μm RSV. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). G and H, shown are ELISA measurements of intracellular Aβ1–40 and Aβ1–42 levels (G) and WB analysis of immunoprecipitated intracellular Aβ (H) from APP-HEK293 cells treated for 24 h with 40 μm RSV or its vehicle (DMSO, CTRL) in the absence or presence of chloroquine (Chloro, 100 μm) or bafilomycin A1 (Baf, 100 nm). Histograms in B, D, and G show the mean ± S.D. of three independent experiments. *, p < 0.05; **, p < 0.01 (Student's t test).
FIGURE 6.
FIGURE 6.
Resveratrol stability, bioavailability, and bioactivity in the brain. A, shown are overlaid UV chromatograms (enlarged) of resveratrol-supplemented diet samples at 7 days (red) and 19 days (blue) under room conditions. cis-RSV, cis-resveratrol; trans-RSV, trans-resveratrol. The inset shows the entire UV (a) and MS (b) chromatograms of resveratrol-supplemented diet samples at the time point of 19 days. B, 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 ± S.E., n = 4 rats. C, shown is LC-MS separation of RSV and resveratrol-glucuronide (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 → 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 ± S.D. of three independent experiments. *, p < 0.05; **, p < 0.01 (Student's t test).
FIGURE 7.
FIGURE 7.
Resveratrol lowers Aβ 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) Aβ1–40 and Aβ1–42 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-Aβ 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 ± S.E., n = 9. *, p ≤ 0.05; **, p < 0.02; ***, p < 0.005 (Student's t test).
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