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. 2011 Jan;25(1):219-31.
doi: 10.1096/fj.10-167361. Epub 2010 Sep 17.

Novel synthetic small-molecule activators of AMPK as enhancers of autophagy and amyloid-β peptide degradation

Valérie Vingtdeux  1 , Pallavi Chandakkar, Haitian Zhao, Cristina d'Abramo, Peter Davies, Philippe Marambaud
Affiliations

Novel synthetic small-molecule activators of AMPK as enhancers of autophagy and amyloid-β peptide degradation

Valérie Vingtdeux et al. FASEB J. 2011 Jan.

Abstract

AMP-activated protein kinase (AMPK) is a metabolic sensor involved in intracellular energy metabolism through the control of several homeostatic mechanisms, which include autophagy and protein degradation. Recently, we reported that AMPK activation by resveratrol promotes autophagy-dependent degradation of the amyloid-β (Aβ) peptides, the core components of the cerebral senile plaques in Alzheimer's disease. To identify more potent enhancers of Aβ degradation, we screened a library of synthetic small molecules selected for their structural similarities with resveratrol. Here, we report the identification of a series of structurally related molecules, the RSVA series, which inhibited Aβ accumulation in cell lines nearly 40 times more potently than did resveratrol. Two of these molecules, RSVA314 and RSVA405, were further characterized and were found to facilitate CaMKKβ-dependent activation of AMPK, to inhibit mTOR (mammalian target of rapamycin), and to promote autophagy to increase Aβ degradation by the lysosomal system (apparent EC(50) ∼ 1 μM). This work identifies the RSVA compounds as promising lead molecules for the development of a new class of AMPK activating drugs controlling mTOR signaling, autophagy, and Aβ clearance.

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Figures

Figure 1.
Figure 1.
Structures and physicochemical parameters of the RSVA compounds. Structures were drawn using Marvin applet. MW, molecular weight (Da); cLogP, partition coefficient log P; tPSA, topological polar surface area; H-don, hydrogen bond donors; H-acc, hydrogen bond acceptors; H2L ID, Hit2Lead ID number.
Figure 2.
Figure 2.
Effect of the RSVA compounds on extracellular Aβ levels in different cell lines. A, B) APP-HEK293, APP-SY5Y, and SwAPP-N2a cells were treated for 24 h with the indicated concentrations of RSVA compounds. Medium was changed, and drug treatments were continued for another 3 h to allow Aβ secretion. Secreted total Aβ was analyzed by WB (A), and secreted Aβ1–40 and Aβ1–42 were analyzed by ELISA (B). C, D) LDH release measurements in APP-HEK293 cells (C) or primary neurons (D) treated for 24 h with the indicated concentrations of RSVA314 or RSVA405. Triton-X100 (1%) was used as a positive control for cytotoxicity (POS). Values are given as percentage toxicity. Histograms in C, D illustrate the mean ± sd values of 3 independent experiments.
Figure 3.
Figure 3.
RSVA314 and RSVA405 are potent activators of AMPK. A–D) APP-HEK293 cells were treated for 24 h with DMSO (control, A), RSVA314 (3 μM, B), or RSVA405 (3 μM, C). Cell extracts were then probed on human phosphoprotein arrays. Representative phospho-protein array analyses are shown in A–C. Boxes 1–3 in A–C indicate phospho-Thr-172 AMPKα2, phospho-Ser-133 CREB, and phospho-Thr-68 Chk2, respectively. Results are expressed as percentage of the control levels (D). E–G) APP-HEK293 (E), APP-SY5Y (F), and wild-type HEK293 cells (G) were treated for 24 h with the indicated concentrations of RSVA314 and RSVA405. Cell extracts were then analyzed by WB for secreted Aβ or cellular phospho-Thr-172 AMPK (pAMPK), AMPK, phospho-Ser-79 ACC (pACC), ACC, phospho-Ser-133 CREB (pCREB), CREB, phospho-Ser-63 c-Jun (pc-Jun), c-Jun, c-Fos, or actin levels.
Figure 4.
Figure 4.
RSVA314 and RSVA405 promote CaMKKβ-dependent phosphorylation of AMPK. A) Effect of RSVA314 and RSVA405 on recombinant human AMPK (α1β1γ1) activity. AMP was used as a positive control for AMPK activation. Data are means ± sd of 3 independent measurements. B) Intracellular ATP levels in APP-HEK293 cells treated for 24 h with the indicated concentrations of RSVA compounds. Mean ± sd values of 3 independent measurements are shown. C, D) WB analyses of pAMPK, AMPK, pACC, ACC, LKB1, and actin levels in HEK293 (lane 1) and HeLa cells (lanes 2–5) treated for 24 h with the indicated concentrations of RSVA314 (C) or RSVA405 (D). E, F) Densitometric analyses and quantification of the ratios pAMPK/AMPK and pACC/ACC from experiments as in C, D. a.u., arbitrary units. G) WB analyses of pAMPK, AMPK, pACC, ACC, and actin levels in APP-HEK293 cells incubated for 24 h with the indicated concentrations of A23187. H) LDH release measurements in APP-HEK293 cells treated for 24 h with the indicated concentrations of STO-609. Values are given as percentage of toxicity. Histogram illustrates the mean ± sd values of 3 independent measurements. I, J) WB analyses of pAMPK, AMPK, pACC, ACC, 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 3 μM RSVA314 (I) or RSVA405 (J).
Figure 5.
Figure 5.
Effect of RSVA314 and RSVA405 on Aβ levels is inhibited in cells expressing inactive AMPK. APP-HEK293 cells were transfected with control vector (V) and Myc-tagged dominant negative T172A-AMPK (T172A, A–F) or Myc-tagged kinase-dead K45R-AMPK (K45R, G–L). At 24 h post-transfection, cells were treated for 24 h with DMSO (CTRL) or 3 μM RSVA314 (A–C, G-I) or 3 μM RSVA405 (D–F, J–L). Myc-AMPK, pACC, ACC, pRaptor, actin, or secreted Aβ levels were analyzed by WB (A, D, G, J). B, E, H, K) Densitometric analyses and quantification of the ratio pACC/ACC from experiments as in A, D, G, J. C, F, I, L) ELISA measurements of secreted Aβ. Histograms show means ± sd of 3 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's t test).
Figure 6.
Figure 6.
Pharmacological inhibition of AMPK interferes with the effect of RSVA314 and RSVA405 on Aβ levels. A) WB analyses of pACC, ACC, and actin levels in APP-HEK293 cells incubated for 24 h with DMSO (CTRL), 3 μM RSVA314, or 3 μM RSVA405 in the absence (−) or presence (+) of compound C (CompC, 40 μM). B) ELISA measurements of secreted Aβ in 3 independent experiments as in A. C) WB analyses of pAMPK, AMPK, pACC, ACC, and actin levels in APP-HEK293 cells incubated for 24 h in the absence or presence of STO-609 (STO, 40 μM) and RSVA405 (3 μM). D) ELISA measurements of secreted Aβ in 3 independent experiments as in C. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's t test).
Figure 7.
Figure 7.
RSVA314 and RSVA405 inhibit mTOR signaling and induce autophagy. A–C) APP-HEK293 cells were treated for 24 h with the indicated concentrations of RSVAs. Cell extracts were then analyzed by WB for phospho-Thr-389 p70S6K (p-p70S6K), p70S6K, phospho-Ser-366 eEF2K (peEF2K), phospho-Ser-235/236 S6 (pS6), phospho-Ser-792 raptor (pRaptor), raptor, actin, and LC3 (A). B, C) Densitometric analyses and quantification of the p-p70S6K/p70S6K (B) and LC3-II/LC3-I (C) ratios. D) Immunocytochemistry analysis with anti-LC3 (green) antibodies in APP-HEK293 cells incubated for 24 h in the presence of 3 μM RSVA405. Nuclei were stained with DAPI (blue). E) WB analyses of LC3 levels in APP-HEK293 cells incubated for 24 h in the absence (−) or presence (+) of RSVA314 (3 μM), RSVA405 (3 μM), or chloroquine (Chloro, 100 μM).
Figure 8.
Figure 8.
RSVA314 and RSVA405 control mTOR and autophagy via AMPK, and facilitate the lysosomal degradation of Aβ. A–H) APP-HEK293 cells were transfected with control vector (V) or Myc-tagged kinase-dead K45R-AMPK (K45R). At 24 h post-transfection, cells were treated for 24 h with DMSO (CTRL) and 3 μM RSVA314 (A–D) or 3 μM RSVA405 (E–H). pRaptor, raptor, LC3, p62, Myc-AMPK, and actin levels were analyzed by WB (A, E). B–D, F–H) Densitometric analyses and quantification of the ratios pRaptor/Raptor and LC3-II/LC3-I, and of p62 levels from experiments in A, E. I) ELISA measurements of intracellular Aβ (iAβ) from APP-HEK293 cells incubated for 24 h in the absence (−) or presence (+) of RSVA314 (3 μM), RSVA405 (3 μM), chloroquine (Chloro, 100 μM), or bafilomycin A1 (Baf, 100 nM). Histograms show the means ± sd of 3 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's t test).
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