Independent Generation of Aβ42 and Aβ38 Peptide Species by γ-Secretase
2008; Elsevier BV; Volume: 283; Issue: 25 Linguagem: Inglês
10.1074/jbc.m802912200
ISSN1083-351X
AutoresEva Czirr, Barbara A. Cottrell, Stefanie Leuchtenberger, Thomas Kukar, Thomas B. Ladd, Hermann Esselmann, Sabine Paul, Robert Schubenel, Justin W. Torpey, Claus U. Pietrzik, Todd E. Golde, Jens Wiltfang, Karlheinz Baumann, Edward H. Koo, Sascha Weggen,
Tópico(s)Cholinesterase and Neurodegenerative Diseases
ResumoProteolytic processing of the amyloid precursor protein by β- and γ-secretase generates the amyloid-β (Aβ) peptides, which are principal drug targets in Alzheimer disease therapeutics. γ-Secretase has imprecise cleavage specificity and generates themostabundant Aβ40 and Aβ42 species together with longer and shorter peptides such as Aβ38. Several mechanisms could explain the production of multiple Aβ peptides by γ-secretase, including sequential processing of longer into shorter Aβ peptides. A novel class of γ-secretase modulators (GSMs) that includes some non-steroidal anti-inflammatory drugs has been shown to selectively lower Aβ42 levels without a change in Aβ40 levels. A signature of GSMs is the concomitant increase in shorter Aβ peptides, such as Aβ38, leading to the suggestion that generation of Aβ42 and Aβ38 peptide species by γ-secretase is coordinately regulated. However, no evidence for or against such a precursor-product relationship has been provided. We have previously shown that stable overexpression of aggressive presenilin-1 (PS1) mutations associated with early-onset familial Alzheimer disease attenuated the cellular response to GSMs, resulting in greatly diminished Aβ42 reductions as compared with wild type PS1. We have now used this model system to investigate whether Aβ38 production would be similarly affected indicating coupled generation of Aβ42 and Aβ38 peptides. Surprisingly, treatment with the GSM sulindac sulfide increased Aβ38 production to similar levels in four different PS1 mutant cell lines as compared with wild type PS1 cells. This was confirmed with the structurally divergent GSMs ibuprofen and indomethacin. Mass spectrometry analysis and high resolution urea gel electrophoresis further demonstrated that sulindac sulfide did not induce detectable compensatory changes in levels of other Aβ peptide species. These data provide evidence that Aβ42 and Aβ38 species can be independently generated by γ-secretase and argue against a precursor-product relationship between these peptides. Proteolytic processing of the amyloid precursor protein by β- and γ-secretase generates the amyloid-β (Aβ) peptides, which are principal drug targets in Alzheimer disease therapeutics. γ-Secretase has imprecise cleavage specificity and generates themostabundant Aβ40 and Aβ42 species together with longer and shorter peptides such as Aβ38. Several mechanisms could explain the production of multiple Aβ peptides by γ-secretase, including sequential processing of longer into shorter Aβ peptides. A novel class of γ-secretase modulators (GSMs) that includes some non-steroidal anti-inflammatory drugs has been shown to selectively lower Aβ42 levels without a change in Aβ40 levels. A signature of GSMs is the concomitant increase in shorter Aβ peptides, such as Aβ38, leading to the suggestion that generation of Aβ42 and Aβ38 peptide species by γ-secretase is coordinately regulated. However, no evidence for or against such a precursor-product relationship has been provided. We have previously shown that stable overexpression of aggressive presenilin-1 (PS1) mutations associated with early-onset familial Alzheimer disease attenuated the cellular response to GSMs, resulting in greatly diminished Aβ42 reductions as compared with wild type PS1. We have now used this model system to investigate whether Aβ38 production would be similarly affected indicating coupled generation of Aβ42 and Aβ38 peptides. Surprisingly, treatment with the GSM sulindac sulfide increased Aβ38 production to similar levels in four different PS1 mutant cell lines as compared with wild type PS1 cells. This was confirmed with the structurally divergent GSMs ibuprofen and indomethacin. Mass spectrometry analysis and high resolution urea gel electrophoresis further demonstrated that sulindac sulfide did not induce detectable compensatory changes in levels of other Aβ peptide species. These data provide evidence that Aβ42 and Aβ38 species can be independently generated by γ-secretase and argue against a precursor-product relationship between these peptides. A variety of therapeutic strategies in clinical development for Alzheimer disease (AD), 2The abbreviations used are: AD, Alzheimer disease; Aβ, amyloid β-peptide; ANOVA, analysis of variance; APP, amyloid precursor protein; FAD, early-onset familial AD; CHO, Chinese hamster ovary; GSM, γ-secretase modulator; SS, sulindac sulfide; PS, presenilin; TMD, transmembrane domain; WT, wild type; MALDI-TOF, matrix-assisted laser desorption/ionization time of flight mass spectrometry; Bicine, N,N-bis(2-hydroxyethyl)glycine. the most common neurodegenerative disorder, target the amyloid-β (Aβ) peptides that are generated through proteolytic processing of the transmembrane amyloid precursor protein (APP) (1Golde T.E. J. Neurochem. 2006; 99: 689-707Crossref PubMed Scopus (110) Google Scholar). In the Aβ-producing pathway, APP is cleaved by two aspartyl proteases, first by β-secretase within its ectodomain and subsequently by γ-secretase, which cleaves APP within its transmembrane domain (TMD) (2Wolfe M.S. Biochemistry. 2006; 45: 7931-7939Crossref PubMed Scopus (182) Google Scholar). γ-Secretase is a multiprotein complex with the presenilin (PS) proteins at its enzymatic core (2Wolfe M.S. Biochemistry. 2006; 45: 7931-7939Crossref PubMed Scopus (182) Google Scholar). Because of its imprecise cleavage specificity, γ-secretase generates Aβ peptides of variable length at the carboxyl terminus, with the highly amyloidogenic Aβ42 isoform thought to be the key pathogenic species (3Leuchtenberger S. Beher D. Weggen S. Curr. Pharm. Des. 2006; 12: 4337-4355Crossref PubMed Scopus (37) Google Scholar). A central role of Aβ42 developed largely from genetic research demonstrating that mutations in the APP and PS genes associated with early-onset familial AD (FAD) invariably increase the Aβ42/Aβ40 ratio in primary fibroblasts and plasma of affected individuals, in transfected cells, and in transgenic animals (3Leuchtenberger S. Beher D. Weggen S. Curr. Pharm. Des. 2006; 12: 4337-4355Crossref PubMed Scopus (37) Google Scholar). In contrast to pan γ-secretase inhibitors that prevent all γ-secretase-mediated cleavage events in APP and other substrates, γ-secretase modulators (GSMs) have been shown to selectively lower production of the Aβ42 species without affecting other γ-secretase substrates (3Leuchtenberger S. Beher D. Weggen S. Curr. Pharm. Des. 2006; 12: 4337-4355Crossref PubMed Scopus (37) Google Scholar, 4Beher D. Clarke E.E. Wrigley J.D. Martin A.C. Nadin A. Churcher I. Shearman M.S. J. Biol. 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GSMs such as sulindac sulfide and ibuprofen were first discovered in the class of non-steroidal anti-inflammatory drugs (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 (1346) Google Scholar), and recent derivatives have shown promise as therapeutic agents in AD animal models and clinical trials (9Imbimbo B.P. Del Giudice E. Colavito D. D'Arrigo A. Dalle Carbonare M. Villetti G. Facchinetti F. Volta R. Pietrini V. Baroc M.F. Serneels L. De Strooper B. Leon A. J. Pharmacol. Exp. Ther. 2007; 323: 822-830Crossref PubMed Scopus (81) Google Scholar, 10Wilcock G.K. Black S.E. Haworth J. Hendrix S. Laughlin M. Zavitz K. Christensen D. Bass S. Swabb E. Alzheimers Dement. 2006; 2: S81-S82Abstract Full Text Full Text PDF Google Scholar). Several characteristics indicate that GSMs act directly on the γ-secretase complex or its substrate APP, including their activity in cell-free γ-secretase assays (4Beher D. Clarke E.E. Wrigley J.D. Martin A.C. Nadin A. Churcher I. Shearman M.S. J. Biol. Chem. 2004; 279: 43419-43426Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 5Eriksen J.L. Sagi S.A. Smith T.E. Weggen S. Das P. McLendon D.C. Ozols V.V. Jessing K.W. Zavitz K.H. Koo E.H. Golde T.E. J. Clin. Investig. 2003; 112: 440-449Crossref PubMed Scopus (0) Google Scholar, 6Takahashi Y. Hayashi I. Tominari Y. Rikimaru K. Morohashi Y. Kan T. Natsugari H. Fukuyama T. Tomita T. Iwatsubo T. J. Biol. Chem. 2003; 278: 18664-18670Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 11Fraering P.C. Ye W. Strub J.M. Dolios G. LaVoie M.J. Ostaszewski B.L. van Dorsselaer A. Wang R. Selkoe D.J. Wolfe M.S. Biochemistry. 2004; 43: 9774-9789Crossref PubMed Scopus (205) Google Scholar, 12Weggen S. Eriksen J.L. 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In addition, mass spectrometry analysis has shown that Aβ42-lowering GSMs induced a concomitant increase in shorter Aβ species, primarily Aβ38 (5Eriksen J.L. Sagi S.A. Smith T.E. Weggen S. Das P. McLendon D.C. Ozols V.V. Jessing K.W. Zavitz K.H. Koo E.H. Golde T.E. J. Clin. Investig. 2003; 112: 440-449Crossref PubMed Scopus (0) Google Scholar, 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 (1346) Google Scholar). Conversely, inverse GSMs such as fenofibrate selectively increased Aβ42 production with a concomitant decrease in Aβ38 and other shorter Aβ species (15Kukar T. Murphy M.P. Eriksen J.L. Sagi S.A. Weggen S. Smith T.E. Ladd T. Khan M.A. Kache R. Beard J. Dodson M. Merit S. Ozols V.V. Anastasiadis P.Z. Das P. Fauq A. Koo E.H. Golde T.E. Nat. Med. 2005; 11: 545-550Crossref PubMed Scopus (260) Google Scholar). Interestingly, certain pan γ-secretase inhibitors elevated Aβ42 levels at subinhibitory concentrations, and this also correlated with a decrease in Aβ38 levels (16Zhao G. Tan J. Mao G. Cui M.Z. Xu X. J. Neurochem. 2007; 100: 1234-1246Crossref PubMed Scopus (42) Google Scholar). Finally, mutations in a GXXXG motif in the APP TMD decreased Aβ42 generation and increased Aβ38 levels from the mutant substrate (17Munter L.M. Voigt P. Harmeier A. Kaden D. Gottschalk K.E. Weise C. Pipkorn R. Schaefer M. Langosch D. Multhaup G. EMBO J. 2007; 26: 1702-1712Crossref PubMed Scopus (250) Google Scholar). Taken together, these findings strongly indicated coordinated production of Aβ42 and shorter species such as Aβ38 by γ-secretase, but no confirmation for or against inter-dependence between these peptides has been provided so far. Importantly, this issue is of significance not only for the mode of action of GSMs but also to understand the molecular mechanism of Aβ generation by γ-secretase. In this respect, it has been proposed that Aβ peptides are generated by sequential cleavage of longer into shorter peptide species (16Zhao G. Tan J. Mao G. Cui M.Z. Xu X. J. Neurochem. 2007; 100: 1234-1246Crossref PubMed Scopus (42) Google Scholar, 17Munter L.M. Voigt P. Harmeier A. Kaden D. Gottschalk K.E. Weise C. Pipkorn R. Schaefer M. Langosch D. Multhaup G. EMBO J. 2007; 26: 1702-1712Crossref PubMed Scopus (250) Google Scholar, 18De Strooper B. EMBO Rep. 2007; 8: 141-146Crossref PubMed Scopus (295) Google Scholar, 19Qi-Takahara Y. Morishima-Kawashima M. Tanimura Y. Dolios G. Hirotani N. Horikoshi Y. Kametani F. Maeda M. Saido T.C. Wang R. Ihara Y. J. Neurosci. 2005; 25: 436-445Crossref PubMed Scopus (325) Google Scholar, 20Wolfe M.S. EMBO Rep. 2007; 8: 136-140Crossref PubMed Scopus (166) Google Scholar, 21Zhao G. Cui M.Z. Mao G. Dong Y. Tan J. Sun L. Xu X. J. Biol. Chem. 2005; 280: 37689-37697Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Further, FAD PS1 mutations might lower the catalytic activity of γ-secretase, thereby reducing the turnover of Aβ42 into shorter species (18De Strooper B. EMBO Rep. 2007; 8: 141-146Crossref PubMed Scopus (295) Google Scholar, 20Wolfe M.S. EMBO Rep. 2007; 8: 136-140Crossref PubMed Scopus (166) Google Scholar, 22Bentahir M. Nyabi O. Verhamme J. Tolia A. Horre K. Wiltfang J. Esselmann H. De Strooper B. J. Neurochem. 2006; 96: 732-742Crossref PubMed Scopus (367) Google Scholar, 23Walker E.S. Martinez M. Brunkan A.L. Goate A. J. Neurochem. 2005; 92: 294-301Crossref PubMed Scopus (151) Google Scholar). We have recently shown that several FAD PS1 mutations, characterized by their aggressive nature with disease onset in the second to fourth decade in life, rendered cells non-responsive to the ability of GSMs to lower Aβ42 (14Czirr E. Leuchtenberger S. Dorner-Ciossek C. Schneider A. Jucker M. Koo E.H. Pietrzik C.U. Baumann K. Weggen S. J. Biol. Chem. 2007; 282: 24504-24513Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Now, we have used this tissue culture model to investigate a potential precursor-product relationship between Aβ42 and Aβ38 peptide species. Unexpectedly, treatment with structurally divergent GSMs increased Aβ38 production to similar levels in PS1 mutant cell lines as compared with wild type PS1 control cells despite the insensitivity of the mutants to Aβ42 reduction. These data are inconsistent with strictly coordinated cleavages and support independent generation of Aβ42 and the shorter Aβ38 peptides by γ-secretase. Drugs, Antibodies, Cell Lines, and Cell Culture—The non-steroidal anti-inflammatory drugs sulindac sulfide, ibuprofen, and indomethacin were purchased from BIOMOL (Plymouth Meeting, PA). All other chemicals were from Sigma-Aldrich. Monoclonal antibody Ab9 against amino acids 1–16 of human Aβ and the carboxyl terminus-specific Aβ antibodies BAP24, BAP15, and BAP29 have been described (24Narlawar R. Perez Revuelta B.I. Baumann K. Schubenel R. Haass C. Steiner H. Schmidt B. Bioorg. Med. Chem. Lett. 2007; 17: 176-182Crossref PubMed Scopus (26) Google Scholar, 25Levites Y. Das P. Price R.W. Rochette M.J. Kostura L.A. McGowan E.M. Murphy M.P. Golde T.E. J. Clin. Investig. 2006; 116: 193-201Crossref PubMed Scopus (173) Google Scholar). Biotinylated monoclonal antibody 6E10 recognizing amino acids 1–17 of human Aβ was purchased from Signet (Dedham, MA). Generation of Chinese hamster ovary (CHO) cells with stable coexpression of wild type APP751 and wild type PS1 or the PS1 mutations PS1-P117L, PS1-L166P, PS1-G384A, and PS1-ΔExon9 has been described previously, and comparable PS1 and APP expression in all cell lines has been demonstrated (14Czirr E. Leuchtenberger S. Dorner-Ciossek C. Schneider A. Jucker M. Koo E.H. Pietrzik C.U. Baumann K. Weggen S. J. Biol. Chem. 2007; 282: 24504-24513Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). All cell lines were maintained in α-minimum essential medium supplemented with 10% fetal bovine serum, 1 mm sodiumpyruvate, 2 mm l-glutamine, and 100 units/ml penicillin/streptomycin (Invitrogen). Dose-response Experiments and Statistical Analysis—Aβ secretion of individual cell lines after GSM treatments was compared in dose-response experiments as described (14Czirr E. Leuchtenberger S. Dorner-Ciossek C. Schneider A. Jucker M. Koo E.H. Pietrzik C.U. Baumann K. Weggen S. J. Biol. Chem. 2007; 282: 24504-24513Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). All cell lines intended for comparison were cultured and treated in parallel at similar cell densities. Cells were cultured in serum-containing medium and treated for 24 h with indicated concentrations of GSMs or Me2SO vehicle. Aβ40, Aβ42, and Aβ38 levels in conditioned medium were then analyzed by sandwich immunoassay. Duplicate measurements from each drug concentration were averaged and normalized to Me2SO control condition. These experiments were repeated five times, and results were analyzed by one-way analysis of variance (ANOVA) with Dunnett′s post tests using GraphPad Prism software (San Diego, CA). Aβ Liquid Phase Electrochemiluminescence Assay—Aβ levels were analyzed by sandwich immunoassay as described (14Czirr E. Leuchtenberger S. Dorner-Ciossek C. Schneider A. Jucker M. Koo E.H. Pietrzik C.U. Baumann K. Weggen S. J. Biol. Chem. 2007; 282: 24504-24513Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 24Narlawar R. Perez Revuelta B.I. Baumann K. Schubenel R. Haass C. Steiner H. Schmidt B. Bioorg. Med. Chem. Lett. 2007; 17: 176-182Crossref PubMed Scopus (26) Google Scholar). In brief, the biotinylated antibody 6E10 was used as capture antibody, and carboxyl terminus-specific Aβ antibodies BAP24, BAP15, and BAP29 were labeled with TAG electrochemiluminescent label (Bioveris, Gaithersburg, MD) and used for detection. Culture media were collected following conditioning for 24 h, cell debris was removed, and complete protease inhibitor mixture (Roche Diagnostics) was added. For liquid phase electrochemiluminescence assay analysis, conditioned media were incubated for 3 h with M-280 paramagnetic beads (Invitrogen) and antibodies 6E10 and BAP24-TAG (for Aβ40), BAP15-TAG (Aβ42), or BAP29-TAG (Aβ38). Electrochemiluminescence was quantified using an M-Series M8 analyzer (Bioveris). Mass Spectrometry Analysis—Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry of Aβ peptides was performed on a 4800 MALDI-TOF-TOF (Applied Biosystems/MDS-Sceix, Foster City, CA). Aβ peptides were immunoprecipitated from conditioned medium with Ab9 antibody covalently coupled to Seize™ beads (Pierce). Peptides were eluted from the beads with 25% 0.1% trifluoroacetic acid:75% acetonitrile. Samples were mixed 1:1 with α-cyano-4-hydroxycinnamic acid matrix in methanol:acetonitrile:water (36%:56%:8%) (Agilent, Santa Clara, CA) and spotted on the MALDI target. Mass spectra were acquired from m/z 3500–5000 Da in reflector positive mode at 10,000 shots/spectrum using single shot protection and a delayed extraction time of 420 ns. The area of the isotope pattern (isotopic cluster area) was used as a measure of apparent relative abundance and expressed asa%of total. Results from three independent experiments were averaged for statistical analysis. Urea Gel Electrophoresis—Cells were lysed in radioimmune precipitation buffer (1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, 25 mm Tris-HCl, pH 7.6, 150 mm NaCl), and Aβ peptides from 1.25 mg of total protein were immunoprecipitated using 25 μl of Dynabeads (Dynal Biotech, Hamburg, Germany) coated with 1E8 monoclonal antibody directed against the amino terminus of Aβ (Bayer-Schering AG). Immunoprecipitated material or conditioned supernatants were separated on 10% Bicine/Tris gels containing 8 m urea (22Bentahir M. Nyabi O. Verhamme J. Tolia A. Horre K. Wiltfang J. Esselmann H. De Strooper B. J. Neurochem. 2006; 96: 732-742Crossref PubMed Scopus (367) Google Scholar), and Aβ peptide species were revealed by Western blotting using antibody 1E8. Synthetic Aβ peptides of different size were run in parallel in the same gel system and under the same conditions for the identification and quantification of Aβ peptides by densitometry. Immunoreactive band intensities were quantified with the Quantity One v4.1 software (Bio-Rad). All samples were run as duplicates, and each gel carried a five-step dilution series of a synthetic Aβ peptide mix. Bands were quantified relative to this dilution series. The inter- and intra-assay coefficients were below 10%. Mean values were used for subsequent calculations. We previously demonstrated that aggressive PS1 mutations associated with early onset FAD displayed a dramatic increase in the Aβ42/Aβ40 ratio (14Czirr E. Leuchtenberger S. Dorner-Ciossek C. Schneider A. Jucker M. Koo E.H. Pietrzik C.U. Baumann K. Weggen S. J. Biol. Chem. 2007; 282: 24504-24513Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Further, these mutants were partially non-responsive to pan γ-secretase inhibitors in vitro and in vivo, indicating that certain AD tissue culture and animal models harboring aggressive PS1 mutations might not be suitable to assess the potency and efficacy of γ-secretase inhibitors. In the same study, we showed that these PS1 mutations were also insensitive to the Aβ42-lowering activity of GSMs (14Czirr E. Leuchtenberger S. Dorner-Ciossek C. Schneider A. Jucker M. Koo E.H. Pietrzik C.U. Baumann K. Weggen S. J. Biol. Chem. 2007; 282: 24504-24513Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). For these experiments, CHO cells with stable co-expression of APP and wild type PS1 (PS1-WT) or the PS1 mutants PS1-P117L, PS1-L166P, PS1-G384A, and PS1-ΔExon9 were treated with 30–60 μm of the GSM sulindac sulfide (SS), Aβ42 levels in corresponding cell culture supernatants were determined, and Aβ42 secretion of individual cell lines was assessed. All cell lines expressing mutant PS1 were either completely refractory to SS (PS1-ΔExon9, PS1-L166P) with no Aβ42 reductions or showed only minor Aβ42 reductions (10% reduction at 60 μm, PS1-P117L, PS1-G384A), whereas PS1-WT control cells displayed robust dose-dependent reductions in Aβ42 levels (40% at 60 μm) (14Czirr E. Leuchtenberger S. Dorner-Ciossek C. Schneider A. Jucker M. Koo E.H. Pietrzik C.U. Baumann K. Weggen S. J. Biol. Chem. 2007; 282: 24504-24513Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). We now took advantage of this culture system to investigate a potential precursor-product relationship between Aβ42 and Aβ38 peptides by measuring Aβ38 levels in culture supernatants from the same experiments. If Aβ38 production by γ-secretase were strictly, inversely coupled to Aβ42 production or were dependent on Aβ42 turnover, then treatment of PS1 mutant cell lines with GSMs should also result in greatly diminished Aβ38 elevations as compared with cells expressing PS1-WT. However, we found that sulindac sulfide treatment robustly increased Aβ38 levels in a dose-dependent manner from all PS1 mutant cell lines without any significant difference as compared with PS1-WT control cells (Fig. 1 and Table 1). To exclude a compound-specific effect, we repeated these experiments with the structurally divergent GSM ibuprofen, which belongs to the aryl propionic acid class of non-steroidal anti-inflammatory drugs (Fig. 2). Cell lines expressing PS1-WT or mutant PS1 were treated with 250 μm ibuprofen, and Aβ40, Aβ42, and Aβ38 levels in culture supernatants were measured and compared by one-way ANOVA. At this concentration, ibuprofen did not cause toxicity in CHO cells (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 (1346) Google Scholar) and no significant reductions in Aβ40 levels were observed (data not shown). In PS1-WT control cells, ibuprofen induced a strong reduction in Aβ42 levels whereas all cell lines expressing PS1 mutants displayed a significantly attenuated response (Fig. 2A and Table 2). In contrast, comparable with our findings with sulindac sulfide, Aβ38 levels were elevated by ibuprofen treatment in PS1 mutant cell lines indistinguishable to PS1-WT control cells (Fig. 2B and Table 2). This was further confirmed with the GSM indomethacin (data not shown).TABLE 1Aβ38 levels are similarly increased in cell lines expressing wild type or mutant PS1 after treatment with the γ-secretase modulator sulindac sulfideSulindac sulfideCell lineAβ38 levels (% control ± S.E.)30 μm60 μmPS1-WT147.88 ± 3.37190.44 ± 5.65PS1-ΔExon9164.01 ± 7.49186.59 ± 10.81PS1-P117L157.00 ± 3.55215.01 ± 5.55PS1-L166P137.77 ± 5.62175.07 ± 9.19PS1-G384A146.37 ± 4.84209.264 ± 6.56 Open table in a new tab FIGURE 2PS1 mutants are non-responsive to the Aβ42-lowering activity of ibuprofen but display similar increase of Aβ38 levels as compared with wild type PS1. CHO cells with stable co-expression of wild type APP and wild type PS1 (PS1-WT) or FAD PS1 mutants were treated with 250 μm of the structurally divergent γ-secretase modulator ibuprofen or Me2SO vehicle. Aβ42 and Aβ38 levels were quantified in conditioned medium, and statistical analysis was performed as described in Fig. 1. A, cells overexpressing FAD mutants displayed a strongly diminished Aβ42 reduction as compared with PS1-WT control cells. B, on the contrary, Aβ38 levels were increased to the same level in cells expressing PS1 mutants and PS1-WT control cells (Table 2). n = 5; one-way ANOVA. **, p < 0.01, ***, p < 0.001 Dunnett's post tests.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 2PS1 mutants are non-responsive to the Aβ42-lowering activity of ibuprofen but display similar increase of Aβ38 levels as compared to wild type PS1IbuprofenCell lineAβ42 levels (% control ± S.E.)Aβ38 levels (% control ± S.E.)250 μmPS1-WT62.89 ± 3.31142.50 ± 5.31PS1-ΔExon994.26 ± 4.21ap < 0.001 Dunnett's post tests.127.40 ± 8.52PS1-P117L81.37 ± 3.60bp < 0.01.139.90 ± 2.82PS1-L166P111.40 ± 4.90ap < 0.001 Dunnett's post tests.124.10 ± 4.82PS1-G384A88.40 ± 1.80 ap < 0.001 Dunnett's post tests.134.30 ± 4.44a p < 0.001 Dunnett's post tests.b p < 0.01. Open table in a new tab To examine the possibility that PS1 mutants would cause overall alterations in the pattern of Aβ peptides or that GSM treatment would induce compensatory changes in other species not detectable with our carboxyl terminus-specific Aβ antibodies, we analyzed the full spectrum of Aβ peptides secreted by PS1-WT control cells or cells expressing PS1-L166P or PS1-ΔExon9 by mass spectrometry. Cells were treated with 60 μm SS, and tissue culture supernatants were immunoprecipitated with antibody Ab9 recognizing amino acids 1–16 of the human Aβ sequence. The immunoprecipitated material was then analyzed by MALDI-TOF-TOF. To determine the relative abundance of individual Aβ peptides, the area of the isotopic cluster for each peptide was analyzed (Fig. 3). We observed that this tended to be more sensitive than measuring peak height alone (data not shown). The profiles of Aβ peptides produced by cells expressing PS1 mutants have not been examined in depth previously. Interestingly, although the full range of Aβ peptides from Aβ1–33 to Aβ1–40 and Aβ42 was produced, we observed that Aβ34 levels appeared slightly higher in PS1 mutant cells as compared with PS1-WT control cells (Fig. 3A). Following SS treatment, the MALDI-TOF results also confirmed our sandwich immunoassay data, namely, cells expressing PS1 mutants displayed an attenuated response to SS with diminished Aβ42 reductions as compared with PS1-WT control cells, whereas the levels of Aβ38 were increased in all cell lines. More importantly, no substantial additional changes in the pattern of Aβ peptides were observed after SS treatment in PS1 mutant cells or PS1-WT control cells (Fig. 3). However, it remains plausible that Aβ38 peptides could be generated by trimming of other longer species such as Aβ45/Aβ46 in PS1 mutant cells. In support of this possibility, replacement of the Aβ40 and Aβ42 cleavage sites in the APP TMD by tryptophan mutagenesis abolished Aβ40 and Aβ42 secretion and caused accumulation of longer Aβ peptides in cell lysates but still allowed secretion of Aβ38 (26Sato T. Tanimura Y. Hirotani N. Saido T.C. Morishima-Kawashima M. Ihara Y. FEBS Lett. 2005; 579: 2907-2912Crossref PubMed Scopus (26) Google Scholar). The highly hydrophobic Aβ peptides longer than Aβ42 are inefficiently secreted and cannot be analyzed by mass spectrometry (19Qi-Takahara Y. Morishima-Kawashima M. Tanimura Y. Dolios G. Hirotani N. Horikoshi Y. Kametani F. Maeda M. Saido T.C. Wang R. Ihara Y. J. Neurosci. 2005; 25: 436-445Crossref PubMed Scopus (325) Google Scholar). Consequently, we used high resolution urea gel electrophoresis to examine longer Aβ species in cell lysates. In cell supernatants, we were able to detect Aβ peptides ranging from Aβ37 to Aβ40 and Aβ42 by urea gel electrophoresis as previously shown (22Bentahir M. Nyabi O. Verhamme J. Tolia A. Horre K. Wiltfang J. Esselmann H. De Strooper B. J. Neurochem. 2006; 96: 732-742Crossref PubMed Scopus (367) Google Scholar). Confirming our sandwich immunoassay and mass spectrometry results, treatment with sulindac sulfide reduced Aβ42 and increased Aβ38 levels in PS1-WT cells, whereas in cells expressing PS1 mutants L166P or G384A Aβ38 levels were increased despite unchanged Aβ42 levels (Fig. 4A). In corresponding cell lysates from the same experiments (Fig. 4B), we further detected minute amounts of longer Aβ species such as Aβ44, Aβ45, and Aβ46, but these peptides were not differentially affected by GSM treatment. In fact, careful quantitative analysis of several independent experiments showed that the intracellular pool of all detectable Aβ peptides, including Aβ42 and Aβ38, was not affected by GSM treatment in either PS1-WT or PS1 mutant cells (Fig. 4C). We have reported previously that sulindac sulfide treatment was able to lower intracellular Aβ42 levels in CHO cells with stable co-expression of APP and the PS1 mutant PS1-M146L (12Weggen S. Eriksen J.L. Sagi S.A. Pietrzik C.U. Ozols V. Fauq A. Golde T.E. Koo E.H. J. Biol. Chem. 2003; 278: 31831-31837Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). However, in the same study, we showed that the PS1-M146L mutation strongly enhances the cellular response to Aβ42-lowering GSMs as compared with PS1-WT (12Weggen S. Eriksen J.L. Sagi S.A. Pietrzik C.U. Ozols V. Fauq A. Golde T.E. Koo E.H. J. Biol. Chem. 2003; 278: 31831-31837Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). Furthermore, at the time, our analysis of intracellular Aβ species using less developed protocols for immunoprecipitation and Western blot detection was evidently close to the detection limit. Our new findings now clearly demonstrate that sulindac sulfide treatment exclusively affects the secreted pool of Aβ42 peptides. This observation in conjunction with the very low abundance of longer Aβ peptides in our cell lines argues against the possibility that the increased Aβ38 levels in PS1 mutant cells after GSM treatment can be explained by enhanced turnover of intracellular Aβ peptides longer than Aβ42. In conclusion, these data demonstrate that Aβ42 and Aβ38 peptides can be generated independently by γ-secretase and that the production of these peptides is not constrained by a stringent precursor-product relationship.FIGURE 4Urea gel electrophoresis analysis of Aβ peptide species after treatment with the γ-secretase modulator sulindac sulfide. A, CHO cells with stable co-expression of wild type APP and wild type PS1 (PS1-WT) or FAD mutants PS1-L166P or PS1-G384A were treated with 60 μm of the γ-secretase modulator sulindac sulfide (SS) or Me2SO vehicle. Cell supernatants were separated on urea SDS-gels, and Aβ peptides were detected by Western blotting with antibody 1E8. The first three lanes (M) show serial dilutions of synthetic marker peptides for the identification of matching Aβ peptide species in the cell supernatants. Peptides ranging from Aβ1–37 to Aβ1–40 and Aβ1–42 were detected. In accordance with the sandwich immunoassay and mass spectrometry results, cells expressing PS1 mutants displayed diminished Aβ42 reductions in cell supernatants after SS treatment as compared with PS1-WT cells, whereas Aβ38 levels were increased in cell supernatants of both PS1-WT and PS1 mutant cells. Other detectable Aβ peptide species, Aβ37 and Aβ39, were unchanged by SS treatment. One of three representative experiments is shown. B, analysis of Aβ peptide species in corresponding cell lysates of the same experiment. Aβ peptides were immunoprecipitated from cell lysates with antibody 1E8, the immunoprecipitated material was separated by urea gel electrophoresis, and Aβ peptides were detected by Western blotting with antibody 1E8. In addition to the Aβ peptide species detected in cell supernatants, cell lysates contained minute amounts of peptides longer than Aβ42, such as Aβ44, Aβ45, and Aβ46. Levels of these peptides were not affected by SS treatment in either PS1-WT or PS1 mutant cells. The lane on the right side shows separation of a mixture of synthetic peptides ranging from Aβ41 to Aβ49. The urea gel system did not allow clear separation of Aβ41, Aβ42, and Aβ43 or peptides Aβ46, Aβ47, Aβ48, and Aβ49. The lower panel shows a longer exposure of the same gel for the area containing peptides >Aβ42. C, densitometric quantification of Aβ peptide species from three independent experiments demonstrated that the intracellular pool of all detectable Aβ peptides, including Aβ42 and Aβ38, was unchanged by SS treatment in either PS1-WT or PS1 mutant cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT) How do our observations conform to the current models of Aβ generation by γ-secretase? After ectodomain shedding of APP by β-secretase, the remaining membrane-bound fragment becomes a substrate for γ-secretase and is cleaved at multiple sites within its TMD (Fig. 5). The abundance of proteolytic products indicates that predominant γ-secretase cleavages occur after Val-40 (generating Aβ40), after Ala-42 (Aβ42), and after Leu-49 (ϵ-cleavage), the latter producing the APP intracellular domain, a cytosolic fragment with potential signaling functions (2Wolfe M.S. Biochemistry. 2006; 45: 7931-7939Crossref PubMed Scopus (182) Google Scholar). Less abundant peptides are generated by cleavage after various other residues and have been identified in cell supernatants (Aβ37, Aβ38, Aβ39) or cell lysates (Aβ43, Aβ45, Aβ46, Aβ48) (19Qi-Takahara Y. Morishima-Kawashima M. Tanimura Y. Dolios G. Hirotani N. Horikoshi Y. Kametani F. Maeda M. Saido T.C. Wang R. Ihara Y. J. Neurosci. 2005; 25: 436-445Crossref PubMed Scopus (325) Google Scholar). Evidence indicates that the APP TMD has α-helical conformation with 3.6 residues forming one complete turn (27Lichtenthaler S.F. Wang R. Grimm H. Uljon S.N. Masters C.L. Beyreuther K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3053-3058Crossref PubMed Scopus (197) Google Scholar, 28Wolfe M.S. Xia W. Moore C.L. Leatherwood D.D. Ostaszewski B. Rahmati T. Donkor I.O. Selkoe D.J. Biochemistry. 1999; 38: 4720-4727Crossref PubMed Scopus (309) Google Scholar), which would align the cleavage sites for Aβ40, Aβ43, Aβ46, and ϵ-cleavage on one surface of the helix and cleavage sites for Aβ38, Aβ42, Aβ45, and Aβ48 on the opposite surface (Fig. 5). It remains to be clarified whether these cleavages occur simultaneously and independently of each other or whether Aβ peptides are generated by sequential trimming of longer into shorter species (18De Strooper B. EMBO Rep. 2007; 8: 141-146Crossref PubMed Scopus (295) Google Scholar, 20Wolfe M.S. EMBO Rep. 2007; 8: 136-140Crossref PubMed Scopus (166) Google Scholar). If the cleavages occur independently of each other, then FAD-associated mutations in PS or APP or GSM treatment might simply result in subtle changes in substrate presentation to the active site of γ-secretase (3Leuchtenberger S. Beher D. Weggen S. Curr. Pharm. Des. 2006; 12: 4337-4355Crossref PubMed Scopus (37) Google Scholar). In case of a γ-secretase complex containing PS1-WT, GSM treatment might favor exposure of the peptide bond at the Aβ38 cleavage site at the expense of production of the longer Aβ42 peptide. In contrast, aggressive PS1 mutations that are non-responsive to GSMs might confer drastic conformational changes on the γ-secretase that are only partially reversible by GSMs, resulting in increased Aβ38 production in the absence of corresponding Aβ42 reductions. On the other hand, several groups have provided strong evidence that Aβ peptides might be generated by sequential proteolytic cleavage, with ϵ-cleavage occurring first and subsequent cleavages taking place at every 3–4 residues along the α-helical surface (16Zhao G. Tan J. Mao G. Cui M.Z. Xu X. J. Neurochem. 2007; 100: 1234-1246Crossref PubMed Scopus (42) Google Scholar, 17Munter L.M. Voigt P. Harmeier A. Kaden D. Gottschalk K.E. Weise C. Pipkorn R. Schaefer M. Langosch D. Multhaup G. EMBO J. 2007; 26: 1702-1712Crossref PubMed Scopus (250) Google Scholar, 19Qi-Takahara Y. Morishima-Kawashima M. Tanimura Y. Dolios G. Hirotani N. Horikoshi Y. Kametani F. Maeda M. Saido T.C. Wang R. Ihara Y. J. Neurosci. 2005; 25: 436-445Crossref PubMed Scopus (325) Google Scholar, 21Zhao G. Cui M.Z. Mao G. Dong Y. Tan J. Sun L. Xu X. J. Biol. Chem. 2005; 280: 37689-37697Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 26Sato T. Tanimura Y. Hirotani N. Saido T.C. Morishima-Kawashima M. Ihara Y. FEBS Lett. 2005; 579: 2907-2912Crossref PubMed Scopus (26) Google Scholar). In this model, and in accordance with findings that many FAD PS mutations seemed to reduce the catalytic activity of γ-secretase (22Bentahir M. Nyabi O. Verhamme J. Tolia A. Horre K. Wiltfang J. Esselmann H. De Strooper B. J. Neurochem. 2006; 96: 732-742Crossref PubMed Scopus (367) Google Scholar, 23Walker E.S. Martinez M. Brunkan A.L. Goate A. J. Neurochem. 2005; 92: 294-301Crossref PubMed Scopus (151) Google Scholar), γ-secretase complexes containing mutant PS might be more prone to release Aβ42 from the active site before further trimming to Aβ38 (18De Strooper B. EMBO Rep. 2007; 8: 141-146Crossref PubMed Scopus (295) Google Scholar, 20Wolfe M.S. EMBO Rep. 2007; 8: 136-140Crossref PubMed Scopus (166) Google Scholar). Conversely, GSMs may strengthen the substrate-enzyme interaction, thereby enhancing the turnover from Aβ42 to Aβ38. Our findings that production of Aβ42 and Aβ38 peptides can be uncoupled favors the first model of independent cleavages by γ-secretase. However, our results do not refute the concept of Aβ generation by sequential γ-secretase cleavage. Our tissue culture model using PS1 mutants and GSM treatment did only allow examining the relationship between Aβ42 and Aβ38 species. Furthermore, we cannot exclude that the presence of mutant PS1 in the γ-secretase complex somehow uncouples Aβ42 and Aβ38 production. During preparation of this report, we learned that another laboratory had also observed uncoupling of Aβ42 and Aβ38 generation after GSM treatment in cell lines expressing mutant PS1 and in an AD transgenic mouse model expressing mutant PS2 (29Page R.M. Baumann K. Tomioka M. Perez-Revuelta B.I. Fukumori A. Jacobsen H. Flohr A. Luebbers T. Ozmen L. Steiner H. Haass C. J. Biol. Chem. 2008; 283: 677-683Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Taken together, these and our findings suggest that a pure sequential cleavage model could be an oversimplification and that the mechanism of Aβ generation by γ-secretase might be even more complex than previously assumed. Moreover, it raises the possibility that selective modulators of Aβ42 production might exist that do not cause induction of shorter Aβ peptides. This is also supported by the observation that the inverse GSM celecoxib did not affect Aβ38 levels (15Kukar T. Murphy M.P. Eriksen J.L. Sagi S.A. Weggen S. Smith T.E. Ladd T. Khan M.A. Kache R. Beard J. Dodson M. Merit S. Ozols V.V. Anastasiadis P.Z. Das P. Fauq A. Koo E.H. Golde T.E. Nat. Med. 2005; 11: 545-550Crossref PubMed Scopus (260) Google Scholar). As a consequence, reliance on Aβ38 elevation as more easily detectable, surrogate readout could lead to false negative results in drug screenings for Aβ42-lowering GSMs. We thank Dr. Manfred Brockhaus (F. Hoffmann-La Roche Ltd.) for providing carboxyl terminus-specific Aβ antibodies, Drs. Dirk Beher (Amgen), Bart de Strooper, and Lutgarde Serneels (KU Leuven, Belgium) for scientific input, Elisabeth Stein for help with the figures, and Dr. Guido Reifenberger (University of Düsseldorf, Germany) for encouragement and support.
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