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Evidence That Nonsteroidal Anti-inflammatory Drugs Decrease Amyloid β42 Production by Direct Modulation of γ-Secretase Activity

2003; Elsevier BV; Volume: 278; Issue: 34 Linguagem: Inglês

10.1074/jbc.m303592200

ISSN

1083-351X

Autores

Sascha Weggen, Jason L. Eriksen, Sarah A. Sagi, Claus U. Pietrzik, Victor Ozols, Abdul H. Fauq, Todd E. Golde, Edward H. Koo,

Tópico(s)

Prion Diseases and Protein Misfolding

Resumo

Chronic use of nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with a lower risk of developing Alzheimer's disease. Recent evidence indicates that some NSAIDs specifically inhibit secretion of the amyloidogenic Aβ42 peptide in cultured cells and mouse models of Alzheimer's disease. The reduction of Aβ42 peptides is not mediated by inhibition of cyclooxygenases (COX) but the molecular mechanism underlying this novel activity of NSAIDs has not been further defined. We now demonstrate that NSAIDs efficiently reduce the intracellular pool of Aβ42 in cell-based studies and selectively decrease Aβ42 production in a cell-free assay of γ-secretase activity. Moreover, we find that presenilin-1 (PS1) mutations, which affect γ-secretase activity, differentially modulate the cellular Aβ42 response to NSAID treatment. Overexpression of the PS1-M146L mutation enhances the cellular drug response to Aβ42 lowering NSAIDs as compared with cells expressing wild-type PS1. In contrast, expression of the PS1-ΔExon9 mutation strongly diminishes the Aβ42 response, showing that PS1 mutations can modulate the cellular drug response to NSAID treatment both positively and negatively. Enhancement of the NSAID drug response was also observed with overexpression of the APP V717F mutation but not with Swedish mutant APP, which affects β-secretase cleavage. In sum, these results strongly suggest that NSAIDs represent a founding group of compounds that lower Aβ42 production by direct modulation of γ-secretase activity or its substrate. Chronic use of nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with a lower risk of developing Alzheimer's disease. Recent evidence indicates that some NSAIDs specifically inhibit secretion of the amyloidogenic Aβ42 peptide in cultured cells and mouse models of Alzheimer's disease. The reduction of Aβ42 peptides is not mediated by inhibition of cyclooxygenases (COX) but the molecular mechanism underlying this novel activity of NSAIDs has not been further defined. We now demonstrate that NSAIDs efficiently reduce the intracellular pool of Aβ42 in cell-based studies and selectively decrease Aβ42 production in a cell-free assay of γ-secretase activity. Moreover, we find that presenilin-1 (PS1) mutations, which affect γ-secretase activity, differentially modulate the cellular Aβ42 response to NSAID treatment. Overexpression of the PS1-M146L mutation enhances the cellular drug response to Aβ42 lowering NSAIDs as compared with cells expressing wild-type PS1. In contrast, expression of the PS1-ΔExon9 mutation strongly diminishes the Aβ42 response, showing that PS1 mutations can modulate the cellular drug response to NSAID treatment both positively and negatively. Enhancement of the NSAID drug response was also observed with overexpression of the APP V717F mutation but not with Swedish mutant APP, which affects β-secretase cleavage. In sum, these results strongly suggest that NSAIDs represent a founding group of compounds that lower Aβ42 production by direct modulation of γ-secretase activity or its substrate. Despite considerable advances in the understanding of Alzheimer's disease (AD) 1The abbreviations used are: AD, Alzheimer's disease; NSAID, nonsteroidal anti-inflammatory drug; COX, cyclooxygenase; PS, presenilin; APP, amyloid precursor protein; APPs, soluble APP ectodomain; FAD, familial Alzheimer's disease; ELISA, enzyme-linked immunosorbent assay; WT, wild type; CHO, Chinese hamster ovary; ANOVA, analysis of variance; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; AICD, APP intracellular cytoplasmic domain. pathology, therapeutic interventions that may halt or reverse the underlying disease process are not available (1Wolfe M.S. Nat. Rev. Drug Discov. 2002; 1: 859-866Crossref PubMed Scopus (179) Google Scholar, 2Golde T.E. J. Clin. Invest. 2003; 111: 11-18Crossref PubMed Scopus (156) Google Scholar). Numerous epidemiological studies support the finding that chronic intake of nonsteroidal anti-inflammatory drugs (NSAIDs) can decrease the risk for AD by more than 50% (3Akiyama H. Barger S. Barnum S. Bradt B. Bauer J. Cole G.M. Cooper N.R. Eikelenboom P. Emmerling M. Fiebich B.L. Finch C.E. Frautschy S. Griffin W.S. Hampel H. Hull M. Landreth G. Lue L. Mrak R. Mackenzie I.R. McGeer P.L. O'Banion M.K. Pachter J. Pasinetti G. Plata-Salaman C. Rogers J. Rydel R. Shen Y. Streit W. Strohmeyer R. Tooyoma I. Van Muiswinkel F.L. Veerhuis R. Walker D. Webster S. Wegrzyniak B. Wenk G. Wyss-Coray T. Neurobiol Aging. 2000; 21: 383-421Crossref PubMed Scopus (3705) Google Scholar, 4Stewart W.F. Kawas C. Corrada M. Metter E.J. Neurology. 1997; 48: 626-632Crossref PubMed Scopus (1048) Google Scholar, 5McGeer P.L. Schulzer M. McGeer E.G. Neurology. 1996; 47: 425-432Crossref PubMed Scopus (1294) Google Scholar, 6t' Veld B.A. Ruitenberg A. Hofman A. Launer L.J. van Duijn C.M. Stijnen T. Breteler M.M. Stricker B.H. N. Engl. J. Med. 2001; 345: 1515-1521Crossref PubMed Scopus (1133) Google Scholar). This protective effect of NSAIDs has generally been ascribed to a diminution of deleterious inflammatory processes in the AD brain (3Akiyama H. Barger S. Barnum S. Bradt B. Bauer J. Cole G.M. Cooper N.R. Eikelenboom P. Emmerling M. Fiebich B.L. Finch C.E. Frautschy S. Griffin W.S. Hampel H. Hull M. Landreth G. Lue L. Mrak R. Mackenzie I.R. McGeer P.L. O'Banion M.K. Pachter J. Pasinetti G. Plata-Salaman C. Rogers J. Rydel R. Shen Y. Streit W. Strohmeyer R. Tooyoma I. Van Muiswinkel F.L. Veerhuis R. Walker D. Webster S. Wegrzyniak B. Wenk G. Wyss-Coray T. Neurobiol Aging. 2000; 21: 383-421Crossref PubMed Scopus (3705) Google Scholar). However, recent findings suggest a direct impact of some NSAIDs on the amyloid pathology in AD. Treatment of various cultured cells with the NSAIDs sulindac sulfide, ibuprofen, indomethacin, and flurbiprofen specifically inhibited the release of the amyloidogenic Aβ42 peptide (7Weggen S. Eriksen J.L. Das P. Sagi S.A. Wang R. Pietrzik C.U. Findlay K.A. Smith T.E. Murphy M.P. Bulter T. Kang D.E. Marquez-Sterling N. Golde T.E. Koo E.H. Nature. 2001; 414: 212-216Crossref PubMed Scopus (1333) Google Scholar, 8Morihara T. Chu T. Ubeda O. Beech W. Cole G.M. J. Neurochem. 2002; 83: 1009-1012Crossref PubMed Scopus (155) Google Scholar). Aβ42 is a proteolytic fragment derived from the β-amyloid precursor protein (APP) by β- and subsequent γ-secretase cleavage activities and is believed to play a central role in AD pathology (9Younkin S.G. J. Physiol. (Paris). 1998; 92: 289-292Crossref PubMed Scopus (252) Google Scholar, 10Golde T.E. Eckman C.B. Younkin S.G. Biochim. Biophys. Acta. 2000; 1502: 172-187Crossref PubMed Scopus (259) Google Scholar). Short term administration of ibuprofen to APP transgenic mice lowered brain levels of Aβ42 and chronic high dose ibuprofen treatment significantly reduced amyloid plaque numbers and plaque-associated pathology in aging APP transgenic mice (7Weggen S. Eriksen J.L. Das P. Sagi S.A. Wang R. Pietrzik C.U. Findlay K.A. Smith T.E. Murphy M.P. Bulter T. Kang D.E. Marquez-Sterling N. Golde T.E. Koo E.H. Nature. 2001; 414: 212-216Crossref PubMed Scopus (1333) Google Scholar, 11Lim G.P. Yang F. Chu T. Chen P. Beech W. Teter B. Tran T. Ubeda O. Ashe K.H. Frautschy S.A. Cole G.M. J. Neurosci. 2000; 20: 5709-5714Crossref PubMed Google Scholar). The reduction in Aβ42 levels was achieved without affecting other APP processing pathways, like secretion of the soluble APP ectodomain (APPs), and is not the result of enhanced degradation or cell-mediated clearance of Aβ42. Importantly, within the concentration range tested, Aβ42-lowering NSAIDs did not inhibit processing of the NOTCH receptor and presumably other substrates of γ-secretase (7Weggen S. Eriksen J.L. Das P. Sagi S.A. Wang R. Pietrzik C.U. Findlay K.A. Smith T.E. Murphy M.P. Bulter T. Kang D.E. Marquez-Sterling N. Golde T.E. Koo E.H. Nature. 2001; 414: 212-216Crossref PubMed Scopus (1333) Google Scholar). NSAIDs are the first small molecules reported to specifically target Aβ42 without apparent overall inhibition of γ-secretase activity, and it will be important to elucidate the molecular mechanism of this activity if more active compounds are to be developed. The NSAID effect on Aβ42 levels is fully retained in cells deficient in cyclooxygenase-1 (COX1) and COX2 enzymatic activity, thereby excluding the primary pharmacological targets of NSAIDs as mediators of the Aβ42 reduction (7Weggen S. Eriksen J.L. Das P. Sagi S.A. Wang R. Pietrzik C.U. Findlay K.A. Smith T.E. Murphy M.P. Bulter T. Kang D.E. Marquez-Sterling N. Golde T.E. Koo E.H. Nature. 2001; 414: 212-216Crossref PubMed Scopus (1333) Google Scholar). COX-independent mechanisms for NSAID activity are well established (12Tegeder I. Pfeilschifter J. Geisslinger G. Faseb. J. 2001; 15: 2057-2072Crossref PubMed Scopus (675) Google Scholar). For example, NSAIDs have emerged as potentially valuable drugs in the chemoprevention of certain cancers, and they affect several molecular pathways regulating cellular proliferation and apoptosis (13Baron J.A. Sandler R.S. Annu. Rev. Med. 2000; 51: 511-523Crossref PubMed Scopus (256) Google Scholar, 14Thun M.J. Henley S.J. Patrono C. J. Natl. Cancer Inst. 2002; 94: 252-266Crossref PubMed Scopus (1284) Google Scholar). Many of these mechanisms seem to be COX independent, including nuclear factor κB activation (15Yamamoto Y. Yin M.J. Lin K.M. Gaynor R.B. J. Biol. Chem. 1999; 274: 27307-27314Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar), inhibition of lipoxygenases (16Shureiqi I. Lippman S.M. Cancer Res. 2001; 61: 6307-6312PubMed Google Scholar) and modulation of peroxisome proliferator-activated receptor signaling (17Lehmann J.M. Lenhard J.M. Oliver B.B. Ringold G.M. Kliewer S.A. J. Biol. Chem. 1997; 272: 3406-3410Abstract Full Text Full Text PDF PubMed Scopus (1059) Google Scholar, 18He T.C. Chan T.A. Vogelstein B. Kinzler K.W. Cell. 1999; 99: 335-345Abstract Full Text Full Text PDF PubMed Scopus (1034) Google Scholar) and potentially any of these signaling pathways could contribute to the observed NSAID effect on Aβ42 secretion. Alternatively, NSAIDs might directly alter γ-secretase function. This possibility is attractive because the reduction in Aβ42 level is accompanied by an increase in shorter Aβ species, particularly Aβ38. This suggests a subtle alteration of γ-secretase cleavage pattern rather than a selective inhibition of Aβ42 generation (7Weggen S. Eriksen J.L. Das P. Sagi S.A. Wang R. Pietrzik C.U. Findlay K.A. Smith T.E. Murphy M.P. Bulter T. Kang D.E. Marquez-Sterling N. Golde T.E. Koo E.H. Nature. 2001; 414: 212-216Crossref PubMed Scopus (1333) Google Scholar, 19De Strooper B. Konig G. Nature. 2001; 414: 159-160Crossref PubMed Scopus (30) Google Scholar). γ-Secretase is a multiprotein complex consisting of at least four membrane-bound proteins: presenilin (PS), nicastrin, APH-1, and PEN-2. All of these proteins are required for proper maturation and activity of the complex while the enzymatic core of this activity may reside within presenilin itself (20Yu G. Nishimura M. Arawaka S. Levitan D. Zhang L. Tandon A. Song Y.Q. Rogaeva E. Chen F. Kawarai T. Supala A. Levesque L. Yu H. Yang D.S. Holmes E. Milman P. Liang Y. Zhang D.M. Xu D.H. Sato C. Rogaev E. Smith M. Janus C. Zhang Y. Aebersold R. Farrer L.S. Sorbi S. Bruni A. Fraser P. St George-Hyslop P. Nature. 2000; 407: 48-54Crossref PubMed Scopus (824) Google Scholar, 21Francis R. McGrath G. Zhang J. Ruddy D.A. Sym M. Apfeld J. Nicoll M. Maxwell M. Hai B. Ellis M.C. Parks A.L. Xu W. Li J. Gurney M. Myers R.L. Himes C.S. Hiebsch R. Ruble C. Nye J.S. Curtis D. Dev. Cell. 2002; 3: 85-97Abstract Full Text Full Text PDF PubMed Scopus (713) Google Scholar, 22Selkoe D.J. Physiol. Rev. 2001; 81: 741-766Crossref PubMed Scopus (5170) Google Scholar). γ-Secretase activity displays loose sequence specificity and generates Aβ peptides of varying length, but peptides ending after 40 and 42 amino acids are the species most intensely studied. Over 150 mutations in the PS proteins, which cause the majority of familial forms of Alzheimer's disease (FAD), have been described and they all appear to increase production of Aβ42 (10Golde T.E. Eckman C.B. Younkin S.G. Biochim. Biophys. Acta. 2000; 1502: 172-187Crossref PubMed Scopus (259) Google Scholar). It has been hypothesized that these mutations lead to subtle conformational changes of PS proteins in the γ-secretase complex, thereby altering the stochiometry of the generated Aβ peptides (22Selkoe D.J. Physiol. Rev. 2001; 81: 741-766Crossref PubMed Scopus (5170) Google Scholar). In this way, NSAIDs may affect γ-secretase activity in an analogous but opposite fashion to PS mutations (7Weggen S. Eriksen J.L. Das P. Sagi S.A. Wang R. Pietrzik C.U. Findlay K.A. Smith T.E. Murphy M.P. Bulter T. Kang D.E. Marquez-Sterling N. Golde T.E. Koo E.H. Nature. 2001; 414: 212-216Crossref PubMed Scopus (1333) Google Scholar, 19De Strooper B. Konig G. Nature. 2001; 414: 159-160Crossref PubMed Scopus (30) Google Scholar). In this study, we demonstrate that NSAIDs reduced intracellular Aβ42 levels and selectively decreased Aβ42 production in a cell-free assay of γ-secretase activity. Furthermore, we observed that FAD mutations in the PS and APP modulate the cellular Aβ42 response to NSAID treatment. These studies suggest the γ-secretase complex, either the enzymatic machinery or its substrate, as the molecular target of Aβ42-lowering NSAIDs. Drugs and Antibodies—The NSAIDs sulindac sulfide, indomethacin, and ibuprofen were purchased from Biomol, naproxen was from Sigma, γ-secretase inhibitors z-IL-CHO and Compound E were synthesized as described (23McLendon C. Xin T. Ziani-Cherif C. Murphy M.P. Findlay K.A. Lewis P.A. Pinnix I. Sambamurti K. Wang R. Fauq A. Golde T.E. Faseb J. 2000; 14: 2383-2386Crossref PubMed Scopus (105) Google Scholar, 24Murphy M.P. Uljon S.N. Fraser P.E. Fauq A. Lookingbill H.A. Findlay K.A. Smith T.E. Lewis P.A. McLendon D.C. Wang R. Golde T.E. J. Biol. Chem. 2000; 275: 26277-26284Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 25Seiffert D. Bradley J.D. Rominger C.M. Rominger D.H. Yang F. Meredith Jr., J.E. Wang Q. Roach A.H. Thompson L.A. Spitz S.M. Higaki J.N. Prakash S.R. Combs A.P. Copeland R.A. Arneric S.P. Hartig P.R. Robertson D.W. Cordell B. Stern A.M. Olson R.E. Zaczek R. J. Biol. Chem. 2000; 275: 34086-34091Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). All other chemicals were from Sigma except when otherwise indicated. Monoclonal antibody PSN2 generated against a synthetic peptide corresponding to residues 31–56 of PS1 and APP monoclonal antibody 26D6 recognizing amino acids 1–12 of the Aβ sequence have been described (26Okochi M. Ishii K. Usami M. Sahara N. Kametani F. Tanaka K. Fraser P.E. Ikeda M. Saunders A.M. Hendriks L. Shoji S.I. Nee L.E. Martin J.J. Van Broeckhoven C. St George-Hyslop P.H. Roses A.D. Mori H. FEBS Lett. 1997; 418: 162-166Crossref PubMed Scopus (32) Google Scholar, 27Lu D.C. Rabizadeh S. Chandra S. Shayya R.F. Ellerby L.M. Ye X. Salvesen G.S. Koo E.H. Bredesen D.E. Nat. Med. 2000; 6: 397-404Crossref PubMed Scopus (346) Google Scholar). Generation of Retroviral Vectors—Retroviral shuttle plasmids encoding wild type human PS1, PS1-M146L and PS1-ΔExon9 have been described previously (28Soriano S. Kang D.E. Fu M. Pestell R. Chevallier N. Zheng H. Koo E.H. J. Cell Biol. 2001; 152: 785-794Crossref PubMed Scopus (186) Google Scholar). For generation of pseudo-typed retroviral vectors, GP-293 packaging cells were transiently co-transfected with retroviral shuttle plasmids and plasmid encoding VSV-G envelope glycoprotein at 1:1 ratio. Retroviral particles were collected 48 h after transfection and stored at –80 °C. Cell Lines and Cell Culture—WT-APP CHO cells, which stably overexpress wild-type human APP751, and WT-APP HS683 neuroglioma cells, which stably overexpress wild-type human APP695, have been previously described (29Perez R.G. Squazzo S.L. Koo E.H. J. Biol. Chem. 1996; 271: 9100-9107Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 30Xia W. Zhang J. Perez R. Koo E.H. Selkoe D.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8208-8213Crossref PubMed Scopus (239) Google Scholar) and are the parental cell lines for all stably transfected CHO and HS683 cell lines used in this study. WT-APP CHO or HS683 cells were infected with retroviral vectors encoding wild-type PS1, PS1-M146L, or PS1-ΔExon9 and were selected with puromycin (5 or 1 μg/ml, respectively). Stable pools were analyzed without further clonal selection. WT-APP CHO cells stably overexpressing wild type human PS1 have been described (31Xia W. Zhang J. Kholodenko D. Citron M. Podlisny M.B. Teplow D.B. Haass C. Seubert P. Koo E.H. Selkoe D.J. J. Biol. Chem. 1997; 272: 7977-7982Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). Comparable PS1 expression in transfected cell lines was verified by Western blotting with monoclonal antibody PSN2. CHO cell lines overexpressing APP751 harboring the V717F "Indiana" mutation or the "Swedish" mutation (K670N/M671L) have been described (29Perez R.G. Squazzo S.L. Koo E.H. J. Biol. Chem. 1996; 271: 9100-9107Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 32Podlisny M.B. Ostaszewski B.L. Squazzo S.L. Koo E.H. Rydell R.E. Teplow D.B. Selkoe D.J. J. Biol. Chem. 1995; 270: 9564-9570Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar). All cell lines were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 100 units ml–1 penicillin/streptomycin (Invitrogen). ELISA—Aβ secretion was analyzed by sandwich enzyme-linked immunosorbent assay (ELISA) as described previously (24Murphy M.P. Uljon S.N. Fraser P.E. Fauq A. Lookingbill H.A. Findlay K.A. Smith T.E. Lewis P.A. McLendon D.C. Wang R. Golde T.E. J. Biol. Chem. 2000; 275: 26277-26284Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Media were collected following conditioning for 24 h and cell debris was removed by centrifugation. Complete protease inhibitor mixture (PI, Roche Applied Science) was added, and Aβ40 and Aβ42 levels were quantified by BAN50/BA27 and BAN50/BC05 ELISAs or 3160/BA27 and 3160/BC05 ELISAs. All measurements were performed in duplicate. Analysis of Intracellular Aβ Production—Intracellular Aβ levels were analyzed by bicine/urea Aβ Western blot analysis as described (7Weggen S. Eriksen J.L. Das P. Sagi S.A. Wang R. Pietrzik C.U. Findlay K.A. Smith T.E. Murphy M.P. Bulter T. Kang D.E. Marquez-Sterling N. Golde T.E. Koo E.H. Nature. 2001; 414: 212-216Crossref PubMed Scopus (1333) Google Scholar, 33Wiltfang J. Smirnov A. Schnierstein B. Kelemen G. Matthies U. Klafki H.W. Staufenbiel M. Huther G. Ruther E. Kornhuber J. Electrophoresis. 1997; 18: 527-532Crossref PubMed Scopus (121) Google Scholar). One 15-cm dish of WT-APP PS1-M146L CHO cells was lysed in 1 ml 2% CHAPS, 50 mm Tris, 150 mm NaCl containing 1× PI and Aβ was immunoprecipitated with monoclonal antibody 26D6. Samples were separated on bicine/urea gels, transferred to nitrocellulose membranes, and immunoblotted with 26D6 antibody. Standard Aβ1–40 and Aβ1–42 peptides (Sigma) were separated on the same gel for identification of the corresponding Aβ species. Representative radiograms are shown. γ-Secretase in Vitro Assay—γ-Secretase in vitro assay was performed using purified detergent solubilized membranes from CHO-overexpressing APP695NL+I as described (23McLendon C. Xin T. Ziani-Cherif C. Murphy M.P. Findlay K.A. Lewis P.A. Pinnix I. Sambamurti K. Wang R. Fauq A. Golde T.E. Faseb J. 2000; 14: 2383-2386Crossref PubMed Scopus (105) Google Scholar, 34Wahrle S. Das P. Nyborg A.C. McLendon C. Shoji M. Kawarabayashi T. Younkin L.H. Younkin S.G. Golde T.E. Neurobiol. Dis. 2002; 9: 11-23Crossref PubMed Scopus (362) Google Scholar). Samples were incubated for 2h at 37 °C in the presence of selected compounds or Me2SO vehicle to allow in vitro production of Aβ and the APP intracellular cytoplasmic domain (AICD, CTFγ) and subsequently stored at –80 °C. Aβ40 and Aβ42 levels were then quantified by ELISA. Net activity during the 2-h incubation period was defined by subtracting background values obtained from samples immediately frozen at time 0. Corrected values were then normalized to the Me2SO control condition and expressed as % control. Dose Response Experiments and Statistical Analysis—The Aβ42 response of individual cell lines to NSAID treatment was compared in dose response experiments. All cell lines intended for comparison were cultured and treated in parallel at similar cell densities. Cells were cultured in serum-containing medium and pretreated overnight with increasing concentrations of sulindac sulfide, ibuprofen, indomethacin, or Me2SO vehicle. Medium was changed and treatment was continued for another 24 h. Aβ40 and Aβ42 levels in conditioned medium were then analyzed by ELISA. Duplicate Aβ42 measurements from each drug concentration were averaged and normalized to the Me2SO control condition. These experiments were repeated 6–10 times, and results were analyzed by two-way ANOVA with Bonferroni post-tests using cell line and drug concentration as categorical variables. Calculations were performed with GraphPad Prism software (GraphPad Software). Matrix-assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry—MALDI-TOF was performed on Aβ peptides immunoprecipitated from conditioned medium of CHO cells as described (35Uljon S.N. Mazzarelli L. Chait B.T. Wang R. Methods Mol. Biol. 2000; 146: 439-452PubMed Google Scholar) with the following modifications. Pi, phosphoramidon, and a synthetic Aβ1–22 peptide that served as an internal control were added, and all of the Aβ1-x were immunoprecipitated from conditioned medium by overnight incubation with anti-mouse IgG-agarose beads and 26D6. Extraction from the beads was with formic acid/water/isopropyl alcohol 1:4:4 (v/v/v). Eluted material was mixed 1:1 with α-cyano-4-hydroxycinnamic acid solution prior to spotting for spectrometry. Treatment-induced changes in Aβ species distribution were determined by normalization of peak heights to Aβ40. Sulindac Sulfide Reduces Intracellular Aβ42 Production— Aβ42-lowering NSAIDs have been shown to reduce secretion of Aβ42 peptides from a variety of cell lines, and this effect is not caused by enhanced degradation or clearance of Aβ42 from tissue culture media (7Weggen S. Eriksen J.L. Das P. Sagi S.A. Wang R. Pietrzik C.U. Findlay K.A. Smith T.E. Murphy M.P. Bulter T. Kang D.E. Marquez-Sterling N. Golde T.E. Koo E.H. Nature. 2001; 414: 212-216Crossref PubMed Scopus (1333) Google Scholar, 8Morihara T. Chu T. Ubeda O. Beech W. Cole G.M. J. Neurochem. 2002; 83: 1009-1012Crossref PubMed Scopus (155) Google Scholar). These changes in the secreted pool of Aβ42 should be reflected by similar changes in the intracellular pool of Aβ42 if NSAIDs affect Aβ42 production by direct modulation of γ-secretase activity. We therefore analyzed intracellular Aβ42 levels after treatment of CHO cells stably transfected with both APP751 (wild-type APP, WT-APP) and the PS1 mutant M146L (PS1-M146L) with the Aβ42-lowering NSAID sulindac sulfide. Aβ was immunoprecipitated from cell lysates and separated on a gel system that permits resolution of individual Aβ species. Treatment of cells with 60 μm sulindac sulfide consistently reduced intracellular Aβ42 levels and strongly decreased the Aβ42/Aβ40 ratio with minimal effects on Aβ40 levels (Fig. 1). Aβ42-lowering NSAIDs Inhibit Aβ42 Production in a Cell-free Assay of γ-Secretase Activity—We next examined the effect of several NSAIDs on Aβ production in a cell-free assay of γ-secretase activity. If Aβ42-lowering NSAIDs act through direct modulation of γ-secretase activity, they would be expected to inhibit Aβ42 production in cell-free assays, as demonstrated previously for well characterized γ-secretase inhibitors (23McLendon C. Xin T. Ziani-Cherif C. Murphy M.P. Findlay K.A. Lewis P.A. Pinnix I. Sambamurti K. Wang R. Fauq A. Golde T.E. Faseb J. 2000; 14: 2383-2386Crossref PubMed Scopus (105) Google Scholar, 36Li Y.M. Xu M. Lai M.T. Huang Q. Castro J.L. DiMuzio-Mower J. Harrison T. Lellis C. Nadin A. Neduvelil J.G. Register R.B. Sardana M.K. Shearman M.S. Smith A.L. Shi X.P. Yin K.C. Shafer J.A. Gardell S.J. Nature. 2000; 405: 689-694Crossref PubMed Scopus (865) Google Scholar). As predicted, the γ-secretase inhibitors z-IL-CHO and Compound E both inhibited γ-secretase activity, with significant reductions in both Aβ40 and Aβ42 production. NSAIDs, such as indomethacin and sulindac sulfide, that have been shown to affect Aβ42 production in cell-based assays (7Weggen S. Eriksen J.L. Das P. Sagi S.A. Wang R. Pietrzik C.U. Findlay K.A. Smith T.E. Murphy M.P. Bulter T. Kang D.E. Marquez-Sterling N. Golde T.E. Koo E.H. Nature. 2001; 414: 212-216Crossref PubMed Scopus (1333) Google Scholar) displayed very similar activities in the cell-free γ-secretase assay (Fig. 2). At these doses, even when Aβ42 production is virtually abolished as seen with sulindac sulfide and indomethacin, no measurable effect was observed on AICD/CTFγ production (data not shown). Naproxen, an NSAID without Aβ42-lowering properties (7Weggen S. Eriksen J.L. Das P. Sagi S.A. Wang R. Pietrzik C.U. Findlay K.A. Smith T.E. Murphy M.P. Bulter T. Kang D.E. Marquez-Sterling N. Golde T.E. Koo E.H. Nature. 2001; 414: 212-216Crossref PubMed Scopus (1333) Google Scholar), had no effect on Aβ42 production (Fig. 2). The PS1-M146L Mutation Enhances the Cellular Aβ42 Response to NSAID Treatment—In our previous study, the NSAID effect on Aβ42 was observed across multiple cell lines and cell types, including those expressing various PS mutations. In view of the in vitro γ-secretase assay results, we now asked whether different PS1 mutants might subtly influence the cellular Aβ42 response to NSAID treatment. We choose to first analyze the PS1-M146L mutation as preliminary observations suggested that the level of Aβ42 reduction was different from wild-type PS1 cells. For these experiments, CHO cell lines were treated with three increasing concentrations of sulindac sulfide (20–60 μm) and Aβ40 and Aβ42 levels in culture media were measured by ELISA. The Aβ42 response of individual cell lines was then compared by two-way ANOVA (see "Experimental Procedures" for details). Total Aβ levels (Aβ40+Aβ42) were not significantly affected at these concentrations of sulindac sulfide and treatment did not cause toxicity as reported previously (7Weggen S. Eriksen J.L. Das P. Sagi S.A. Wang R. Pietrzik C.U. Findlay K.A. Smith T.E. Murphy M.P. Bulter T. Kang D.E. Marquez-Sterling N. Golde T.E. Koo E.H. Nature. 2001; 414: 212-216Crossref PubMed Scopus (1333) Google Scholar). ANOVA demonstrated that overexpression of wild type, human PS1 resulted only in minimal, non-significant changes in the Aβ42 response as compared with parental cells expressing endogenous, hamster PS1 (Fig. 3 and Table I). However, overexpression of the PS1-M146L mutation strongly enhanced the cellular Aβ42 response to sulindac sulfide treatment. ANOVA showed a highly significant statistical difference in the Aβ42 reduction as compared with either parental WT-APP CHO cells expressing endogenous PS1 or WT-APP WT-PS1 CHO cells overexpressing wild-type, human PS1 (p < 0.001, Fig. 3 and Table I). At all three concentrations tested, the reduction in Aβ42 levels was almost 20% greater in PS1-M146L cells than that seen in the other two cell lines. Similar trends toward enhanced Aβ42 reduction with the PS1-M146L mutant as compared with WT-PS1 were also observed with indomethacin and ibuprofen and, importantly, in HS683 neuroglioma cells expressing the same PS constructs (data not shown). These results indicated that the enhanced Aβ42 reduction is distinctively associated with the PS1-M146L mutant, and is not limited to the NSAID sulindac sulfide or to a specific cell type.Table IDose-response experiments demonstrate enhanced Aβ42 reduction in cells overexpressing PS1-M1461Sulindac sulfideAβ42CHO WT-APPCHO WT-APP WT-PS1CHO WT-APP PS1-M146L% control ± S.E.20 μm79.28 ± 1.6378.77 ± 2.0163.13 ± 1.82***40 μm70.56 ± 2.4268.31 ± 1.5250.76 ± 2.53***60 μm56.36 ± 1.4951.13 ± 2.9632.80 ± 1.87*** Open table in a new tab The PS1-ΔExon9 Mutation Diminishes the Cellular Aβ42 Response to NSAID Treatment—We next examined CHO cells overexpressing the PS1-ΔExon9 mutation to assess whether another PS1 mutation would similarly alter the cellular drug response to NSAID treatment. We chose this mutation because it results in an exon deletion rather than a missense substitution as seen in virtually all other FAD PS1 mutations and because the clinical manifestation of this mutation is varied (37Brooks W.S. Kwok J.B. Kril J.J. Broe G.A. Blumbergs P.C. Tannenberg A.E. Lamont P.J. Hedges P. Schofield P.R. Brain. 2003; 126: 783-791Crossref PubMed Scopus (49) Google Scholar). Surprisingly, we found that the PS1-ΔExon9 mutation strongly diminished the Aβ42 response to sulindac sulfide treatment. Two-way ANOVA analysis of 10 independent dose-response experiments showed a highly significant attenuation of the Aβ42 response at all three concentrations tested as compared with parental WT-APP CHO cells expressing endogenous PS1 or WT-APP WT-PS1 CHO cells overexpressing wild-type, human PS1 (p < 0.001, Fig. 4, panel A, and Table II). This result was confirmed with HS683 neuroglioma cells overexpressing the PS1-ΔExon9 mutation (Fig. 4, panel B, and Table II). In sum, these findings strongly indicated that PS1 mutations that alter Aβ42 generation are able to modulate the cellular Aβ42 response to NSAIDs treatment.Table IIDose response experiments demonstrate diminished Aβ42 reduction in cells overexpressing PS1-ΔExon9Sulindac sulfideA

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