Resveratrol Promotes Clearance of Alzheimer's Disease Amyloid-β Peptides
Several epidemiological studies indicate that moderate consumption of wine is associated with a lower incidence of Alzheimer's disease. Wine is enriched in antioxidant compounds with potential neuroprotective activities. However, the exact molecular mechanisms involved in the beneficial effects of wine intake on the neurodegenerative process in Alzheimer's disease brain remain to be clearly defined. Here we show that resveratrol (trans-3,4′,5-trihydroxystilbene), a naturally occurring polyphenol mainly found in grapes and red wine, markedly lowers the levels of secreted and intracellular amyloid-β (Aβ) peptides produced from different cell lines. Resveratrol does not inhibit Aβ production, because it has no effect on the Aβ-producing enzymes β- and γ-secretases, but promotes instead intracellular degradation of Aβ via a mechanism that involves the proteasome. Indeed, the resveratrol-induced decrease of Aβ could be prevented by several selective proteasome inhibitors and by siRNA-directed silencing of the proteasome subunit β5. These findings demonstrate a proteasome-dependent anti-amyloidogenic activity of resveratrol and suggest that this natural compound has a therapeutic potential in Alzheimer's disease. Several epidemiological studies indicate that moderate consumption of wine is associated with a lower incidence of Alzheimer's disease. Wine is enriched in antioxidant compounds with potential neuroprotective activities. However, the exact molecular mechanisms involved in the beneficial effects of wine intake on the neurodegenerative process in Alzheimer's disease brain remain to be clearly defined. Here we show that resveratrol (trans-3,4′,5-trihydroxystilbene), a naturally occurring polyphenol mainly found in grapes and red wine, markedly lowers the levels of secreted and intracellular amyloid-β (Aβ) peptides produced from different cell lines. Resveratrol does not inhibit Aβ production, because it has no effect on the Aβ-producing enzymes β- and γ-secretases, but promotes instead intracellular degradation of Aβ via a mechanism that involves the proteasome. Indeed, the resveratrol-induced decrease of Aβ could be prevented by several selective proteasome inhibitors and by siRNA-directed silencing of the proteasome subunit β5. These findings demonstrate a proteasome-dependent anti-amyloidogenic activity of resveratrol and suggest that this natural compound has a therapeutic potential in Alzheimer's disease. Alzheimer's disease (AD) 2The abbreviations used are: ADAlzheimer's disease; Suc, succinylTMStrans-2,3′,4,5′-tetramethoxystilbeneALLNN-acetyl-LL-norleucinal-CHOELISAenzyme-linked immunosorbent assayECEendothelin-converting enzymeIDEinsulin-degrading enzyme; Aβ, amyloid-βAPPamyloid-β precursor proteinAMCamido-4-methylcoumarin; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineWBWestern blottingIPimmunoprecipitationNEPneprilysinMOPS4-morpholinepropanesulfonic acidERendoplasmic reticulumsiRNAsmall interfering RNATBSTris-buffered saline; sAβ, secreted amyloid-βZbenzyloxycarbonyl is a progressive neurodegenerative disorder leading to the most common form of dementia. Compelling evidence supports the central role of Aβ in the pathogenesis of the disease (1Hardy J. Selkoe D.J. Science. 2002; 297: 353-356Crossref PubMed Scopus (11122) Google Scholar). Aβ is a core component of the senile plaque, a classical lesion found in the neocortex and hippocampus of AD brains, and excessive production of the highly insoluble 42-amino acid-long Aβ42 peptide is almost invariably observed in the presence of mutations in the three genes linked to early onset autosomal dominant familial forms of AD (2Tanzi R.E. Bertram L. Cell. 2005; 120: 545-555Abstract Full Text Full Text PDF PubMed Scopus (1508) Google Scholar). Alzheimer's disease; Suc, succinyl trans-2,3′,4,5′-tetramethoxystilbene N-acetyl-LL-norleucinal-CHO enzyme-linked immunosorbent assay endothelin-converting enzyme insulin-degrading enzyme; Aβ, amyloid-β amyloid-β precursor protein amido-4-methylcoumarin; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine Western blotting immunoprecipitation neprilysin 4-morpholinepropanesulfonic acid endoplasmic reticulum small interfering RNA Tris-buffered saline; sAβ, secreted amyloid-β benzyloxycarbonyl In the amyloidogenic pathway, the amyloid-β precursor protein (APP) is cleaved by the aspartic protease β-secretase/BACE1 to yield the membrane-anchored C-terminal fragments C99 and C89. C99 is then endoproteolyzed by the γ-secretase proteolytic complex to produce various Aβ peptides. The major cleavage takes place after Val-40 producing Aβ40. In an alternative nonamyloidogenic pathway, APP is endoproteolyzed within the Aβ region by α-secretase to generate the C-terminal fragment C83 and the soluble N-terminal fragment secreted APPα. Finally, a γ-secretase-mediated ϵ-cleavage of APP allows the intracellular release of the transcriptionally active APP intracellular domain (AID (3Passer B. Pellegrini L. Russo C. Siegel R.M. Lenardo M.J. Schettini G. Bachmann M. Tabaton M. D'Adamio L. J. Alzheimers Dis. 2000; 2: 289-301Crossref PubMed Scopus (203) Google Scholar) or AICD) (4Checler F. J. Neurochem. 1995; 65: 1431-1444Crossref PubMed Scopus (423) Google Scholar, 5Annaert W. De Strooper B. Annu. Rev. Cell Dev. Biol. 2002; 18: 25-51Crossref PubMed Scopus (198) Google Scholar, 6Marambaud P. Robakis N.K. Genes Brain Behav. 2005; 4: 134-146Crossref PubMed Scopus (61) Google Scholar). Epidemiological studies have shown that moderate wine intake reduces the risk of developing AD (7Luchsinger J.A. Tang M.X. Siddiqui M. Shea S. Mayeux R. J. Am. Geriatr. Soc. 2004; 52: 540-546Crossref PubMed Scopus (297) Google Scholar, 8Lindsay J. Laurin D. Verreault R. Hebert R. Helliwell B. Hill G.B. McDowell I. Am. J. Epidemiol. 2002; 156: 445-453Crossref PubMed Scopus (1031) Google Scholar, 9Orgogozo J.M. Dartigues J.F. Lafont S. Letenneur L. Commenges D. Salamon R. Renaud S. Breteler M.B. Rev. Neurol. (Paris). 1997; 153: 185-192PubMed Google Scholar, 10Truelsen T. Thudium D. Gronbaek M. Neurology. 2002; 59: 1313-1319Crossref PubMed Scopus (258) Google Scholar). Resveratrol, a polyphenol that occurs in abundance in grapes and red wine, is suspected to afford antioxidant and neuroprotective properties and therefore to contribute to the beneficial effect of wine consumption on the neurodegenerative process (11Savaskan E. Olivieri G. Meier F. Seifritz E. Wirz-Justice A. Muller-Spahn F. Gerontology. 2003; 49: 380-383Crossref PubMed Scopus (178) Google Scholar, 12Jang J.H. Surh Y.J. Free Radic. Biol. Med. 2003; 34: 1100-1110Crossref PubMed Scopus (367) Google Scholar, 13Han Y.S. Zheng W.H. Bastianetto S. Chabot J.G. Quirion R. Br. J. Pharmacol. 2004; 141: 997-1005Crossref PubMed Scopus (279) Google Scholar). Here we report that resveratrol has a potent anti-amyloidogenic activity by reducing the levels of Aβ produced from different cell lines expressing wild type or Swedish mutant APP695. We show that resveratrol acts by promoting the intracellular degradation of Aβ by a mechanism that implicates the proteasome. Materials and Antibodies—Quercetin, catechin, resveratrol, piceatannol, phosphoramidon, thiorphan, insulin, N-succinyl-LLVY-7-amido-4-methylcoumarin (Suc-LLVY-AMC), and Suc-AAF-AMC were obtained from Sigma. Trimethoxy-resveratrol and TMS (trans-2,3′,4,5′-tetramethoxystilbene) were from Cayman Chemical. L-685,458, lactacystin, Z-GPFL-CHO, YU101, and N-acetyl-LL-norleucinal-CHO (ALLN) were from Calbiochem. Purified human 20 S proteasome was from Biomol. Anti-Aβ-(1–17) (6E10) and anti-Aβ-(17–24) (4G8, pure and biotinylated) antibodies were from Signet. Anti-APP-(66–81) (22C11) antibody was from Chemicon, and anti-APP C-terminal domain (R1) antibody was provided by Dr. P. D. Mehta, Institute for Basic Research in Developmental Disabilities, Staten Island, NY. Polyclonal antibodies specific for Aβ40 (FCA3340) or Aβ42 (FCA3542) (14Barelli H. Lebeau A. Vizzavona J. Delaere P. Chevallier N. Drouot C. Marambaud P. Ancolio K. Buxbaum J.D. Khorkova O. Heroux J. Sahasrabudhe S. Martinez J. Warter J.M. Mohr M. Checler F. Mol. Med. 1997; 3: 695-707Crossref PubMed Google Scholar) were obtained from Dr. F. Checler, IPMC-Centre National de la Recherche Scientifique, Valbonne, France. Anti-N-cadherin (C32) and anti-β-catenin antibodies were from BD Transduction Laboratories. Anti-20 S proteasome subunit β5 antibody was from ABR Affinity BioReagents, and the polyclonal antibody directed against the subunits α5, α7, β1, β5, β5i, and β7 of the 20 S proteasome was from Biomol. Anti-β-tubulin antibody was from Santa Cruz Biotechnology. Cell Lines, Transfections, and Drug Treatments—HEK293 cells stably transfected with human APP695 were provided by Dr. L. D'Adamio, Albert Einstein College of Medicine, Bronx, NY. N2a cells were stably transfected with wild type or Swedish mutant human APP695 cDNAs (obtained from Dr. N. K. Robakis, Mount Sinai School of Medicine, New York, NY). APP695-HEK293 transfectants were grown in Dulbecco's modified Eagle's medium plus 10% fetal bovine serum, penicillin and streptomycin, and 5 μg/ml puromycin. APP695-N2a cells were maintained in 1:1 Dulbecco's modified Eagle's medium/Opti-MEM supplemented with 5% fetal bovine serum, penicillin and streptomycin, and 0.2 mg/ml G418. For drug treatments, cells were treated at confluence for the indicated concentrations and incubation times. Medium was then changed, and treatments were continued for another 2 h to allow Aβ secretion. For siRNA-directed silencing, 200 pmol of purified siRNA directed against the proteasome subunit β5 (SMARTpool, Dharmacon) were transfected with 10 μl of Lipofectamine 2000 (Invitrogen) in APP695-HEK293 cells plated in 35-mm dishes. At 48 h post-transfection, cells were incubated in the absence or presence of 40 μm resveratrol for another 24 h. Cells and conditioned medium were harvested and analyzed by Western blotting (WB) and by proteasome activity assays as described below. Western Blotting—Cells were washed with phosphate-buffered saline and solubilized in ice-cold HEPES buffer (25 mm HEPES, pH 7.4, 150 mm NaCl, 1× Complete protease inhibitor mixture, Roche Applied Science) containing 1% SDS. Ten micrograms of extracts were analyzed by SDS-PAGE. For total sAβ WB, conditioned medium was subjected to 0.2-μm filtration. Twenty microliters of medium were then electrophoresed on 16.5% Tris-Tricine gels and transferred onto 0.2-μm nitrocellulose membranes. Membranes were microwaved for 5 min in phosphate-buffered saline, blocked in 5% fat-free milk in TBS, and incubated with 6E10 (1:1000 in Pierce SuperBlock) overnight at 4 °C. A standard ECL detection procedure was then used. Aβ Immunoprecipitations (IPs)—Cells were solubilized in ice-cold RIPA buffer (50 mm Tris-HCl, pH 8, 150 mm NaCl, 0.1% SDS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1× Complete). Five hundred micrograms of cell extracts (for total intracellular Aβ IP) or 1 ml of the corresponding conditioned medium diluted in 4× RIPA buffer (for sAβ40 and sAβ42 IPs) were precleared with protein A- or protein G-Sepharose (Amersham Biosciences) for 2 h at 4°C. Supernatants were then incubated overnight at 4 °C with 3 μl of antibodies 4G8 (total intracellular Aβ IP), FCA3340 (sAβ40 IP), or FCA3542 (sAβ42 IP). Supernatants were then treated for 2 h at 4 °C with protein A-Sepharose (polyclonal antibodies) or with protein G-Sepharose (monoclonal antibodies). IPs were washed with ice-cold RIPA buffer and analyzed by WB using the 6E10 antibody as described above. Aβ Enzyme-linked Immunosorbent Assay (ELISA)— 6E10 (capture antibody) was coated at 2 μg/ml in coating buffer (2.27 g/liter K2HPO4, 3.48 g/liter KH2PO4, pH 7.2, 8 g/liter NaCl, 0.372 g/liter EDTA, 0.1 g/liter NaN3) into 96-well immunoassay plates for 24 h at 4 °C. The plates were washed with 0.05% Tween 20 in TBS (TTBS) and blocked with Pierce TBS starting block buffer for 1 h at room temperature. The samples (conditioned medium or Aβ1–40 standards) and biotinylated 4G8 (reporter antibody, at 0.5 μg/ml in 20% Pierce SuperBlock) were then added to the plates and incubated at room temperature for 2 h. Following washing with TTBS, streptavidin-horseradish peroxidase (Southern Biotech, at 0.25 μg/ml in 20% SuperBlock) was added to the wells for 1 h at room temperature. The fluorogenic substrate Amplex Ultra Red (Molecular Probes) was added to the plates and incubated for 15 min. Reaction products were quantified using a Tecan Genios Pro plate reader at 535 nm excitation and 590 nm emission. Enzymatic Activity Assays—For neprilysin (NEP) activity assays, intact cells were incubated at 37 °C for 2 h in Opti-MEM containing 50 μm Suc-AAF-AMC in the absence or presence of 20 μm thiorphan. Cells were then homogenized, and protein concentrations were determined using a Bradford assay (Bio-Rad). Chymotrypsin-like activity of the endogenous proteasome was determined by solubilizing the cells in activity assay buffer (25 mm HEPES, pH 7.5, 0.5 mm EDTA, 0.05% Nonidet P-40, and 0.001% SDS). Cell extracts (40 μg) were incubated at 37°C for 2 h in 100 μl of activity assay buffer containing 50 μm Suc-LLVY-AMC in the absence or presence of 10 μm of ALLN. For purified proteasome activity assays, 12.5 μg/ml purified human 20 S proteasome were incubated at 37 °C for 2 h in 100 μl of activity assay buffer containing 50 μm Suc-LLVY-AMC. Release of AMC was measured by fluorescence spectrophotometry using wavelengths of 340 nm excitation and 535 nm emission (Tecan Genios Pro). Enzymatic activities were expressed as nmol AMC/min/mg protein. In Vitro γ-Secretase Assays—In vitro assays were performed as described previously (15Marambaud P. Wen P.H. Dutt A. Shioi J. Takashima A. Siman R. Robakis N.K. Cell. 2003; 114: 635-645Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar). Briefly, cells were resuspended in 0.5 ml/35-mm dish of hypotonic buffer (10 mm MOPS, pH 7.0, 10 mm KCl) and homogenized on ice. A postnuclear supernatant was prepared by centrifugation at for 15 min at 4 °C. were from the postnuclear supernatant by centrifugation at for 40 min at 4 °C. The were then resuspended in assay buffer mm sodium pH 1× and incubated at 37 °C for 4 h in the absence or presence of the indicated were then analyzed by evidence is that moderate wine intake reduces the risk of developing AD (7Luchsinger J.A. Tang M.X. Siddiqui M. Shea S. Mayeux R. J. Am. Geriatr. Soc. 2004; 52: 540-546Crossref PubMed Scopus (297) Google Scholar, 8Lindsay J. Laurin D. Verreault R. Hebert R. Helliwell B. Hill G.B. McDowell I. Am. J. Epidemiol. 2002; 156: 445-453Crossref PubMed Scopus (1031) Google Scholar, 9Orgogozo J.M. Dartigues J.F. Lafont S. Letenneur L. Commenges D. Salamon R. Renaud S. Breteler M.B. Rev. Neurol. (Paris). 1997; 153: 185-192PubMed Google Scholar, 10Truelsen T. Thudium D. Gronbaek M. Neurology. 2002; 59: 1313-1319Crossref PubMed Scopus (258) Google we to three antioxidant found in red wine, resveratrol and Aβ this we treated cells with concentrations of the different and analyzed Aβ levels by and secreted Aβ A and secreted Aβ40 and Aβ42 were markedly by μm resveratrol after 24 h of and were at concentrations A and At the resveratrol total intracellular Aβ resveratrol not to intracellular of Aβ we that Aβ was not the effect of resveratrol is APP695-HEK293 cells were then treated for different of with 10 or 20 μm resveratrol not Aβ levels after h of effect on Aβ levels is after incubation of 48 and of resveratrol, and on Aβ levels in APP695-HEK293 A and cells were treated for 24 h with the indicated concentrations of in Medium was changed, and drug treatments were continued for another 2 h to allow Aβ secretion. sAβ was analyzed by and WB sAβ40 sAβ42 and total intracellular Aβ total were analyzed by and WB APP was analyzed by cells were treated for different of with the indicated concentrations of sAβ was then analyzed as in B. The of was to in the of The Western shown of at three a cell we treated N2a cells with that resveratrol total secreted Aβ at the in another cell Aβ produced by N2a cells APP695 the familial AD Swedish was by treatments resveratrol APP we then by WB the levels of APP and proteolytic At the resveratrol APP levels and secreted In resveratrol not the levels of APP C-terminal fragments and C83 and and APP intracellular domain indicate that resveratrol no effect on the or γ-secretase-mediated of APP or on the of APP or C-terminal resveratrol not APP and Aβ that resveratrol not inhibit γ-secretase resveratrol was used in a assay to produce Aβ in In this in vitro from APP695-HEK293 cells were incubated at 37 °C for 4 h in the absence or presence of concentrations of of Aβ was not by the presence of resveratrol in vitro However, the levels of Aβ produced in vitro by from cells treated in with the polyphenol were that resveratrol Aβ β- and γ-Secretase several type the and (15Marambaud P. Wen P.H. Dutt A. Shioi J. Takashima A. Siman R. Robakis N.K. Cell. 2003; 114: 635-645Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar, P. Shioi J. G. A. S. L. Wen P. S. T. Robakis N.K. J. 2002; PubMed Scopus Google Scholar). of by γ-secretase the transcriptionally active intracellular fragment from the proteolytic cleavage of the C-terminal fragment (15Marambaud P. Wen P.H. Dutt A. Shioi J. Takashima A. Siman R. Robakis N.K. Cell. 2003; 114: 635-645Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar). a assay we determined that production of was not by resveratrol the absence of effect of resveratrol on γ-secretase with the that resveratrol not the levels of APP and C-terminal proteolytic indicate that resveratrol not an Aβ-producing activity but Aβ on we to resveratrol promotes Aβ Aβ peptides in by at endothelin-converting and and and insulin-degrading enzyme L. N. 2004; PubMed Scopus Google Scholar). evidence that with resveratrol promotes activity in cells F. 2002; Google Scholar). assays on intact we that activity was resveratrol However, of with or in APP695-HEK293 cells could not the decrease of Aβ levels by resveratrol and we that activities were not involved in the decrease of with insulin, acts as a inhibitor of not Aβ levels resveratrol resveratrol not Aβ degradation by and or in evidence that resveratrol promotes the degradation of a specific of H. M. D. 2002; Google the E. M.J. J. 2004; PubMed Scopus Google or the J. C. 2004; PubMed Scopus Google Scholar). the proteasome has shown to Aβ levels 2004; PubMed Scopus Google we proteasome Aβ levels resveratrol that treatments with the selective proteasome lactacystin, Z-GPFL-CHO, or YU101, prevented the resveratrol-induced decrease of the of the proteasome in the anti-amyloidogenic effect of resveratrol, we to proteasome activity by siRNA-directed The proteasome is a protease complex by different subunits by several genes M. S. 2000; PubMed Scopus Google Scholar). is highly selective for the activity of the proteasome M. J. R. Biol. Full Text PDF PubMed Scopus Google Scholar) and because the subunit β5 is for this proteolytic activity M. S. 2000; PubMed Scopus Google we siRNA-directed silencing of the proteasome subunit β5 the resveratrol-induced decrease of of directed against the subunit β5 β5 protein and of the activity of the proteasome we observed a of the resveratrol-induced Aβ decrease We then resveratrol proteasome In the activity of purified proteasome was in vitro incubation with The activity was from endogenous proteasome in cell extracts after with the polyphenol in cell effect of resveratrol on the activity of purified or endogenous proteasome was observed in and resveratrol Aβ levels after 24 h of we resveratrol proteasome subunit We found that resveratrol does not markedly the levels of several proteasome subunits in cells and demonstrate that resveratrol promotes a proteasome-dependent intracellular degradation of Aβ via a mechanism that does not total proteasome with this levels of a protein by the proteasome H. A. J. A. R. J. 1997; PubMed Scopus Google not by resveratrol in cells We then the anti-amyloidogenic effect of several resveratrol were an at and and the three were to decrease Aβ levels However, with resveratrol and were TMS a in reducing the of Aβ show that resveratrol reduces Aβ produced by different cell lines expressing wild type or Swedish mutant APP695. Resveratrol acts by promoting the intracellular degradation of the peptide by a mechanism that implicates the proteasome. studies show that of the previously and or involved in this Finally, we demonstrate the anti-amyloidogenic activity of of resveratrol, and that of resveratrol be in the of and and therefore therapeutic studies be to the role of the proteasome in this mechanism of intracellular of A of of the proteasome in the of Aβ have to the complex 2004; PubMed Scopus Google Scholar). 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D. for with and cell lines.