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• W2087254373 abstract "Presenilin 1 (PS1) plays an essential role in intramembranous “γ-secretase” processing of several type I membrane proteins, including the β-amyloid precursor proteins (APP) and Notch1. In this report, we examine the activity of two familial Alzheimer's disease-linked PS1 variants on the production of secreted Aβ peptides and the effects of L-685,458, a potent γ-secretase inhibitor, on inhibition of Aβ peptides from cells expressing these PS1 variants. We now report that PS1 variants enhance the production and secretion of both Aβ1–42 and Aβ1–40 peptides. More surprisingly, whereas the IC50 for inhibition of Aβ1–40 peptide production from cells expressing wild-type PS1 is ∼1.5 μm, cells expressing the PS1ΔE9 mutant PS1 exhibit an IC50 of ∼4 μm. Immunoprecipitation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry reveal that the levels of Aβ1–43 peptides are elevated in medium of PS1ΔE9 cells treated with higher concentrations of inhibitor. The differential effects of wild-type and mutant PS1 on γ-secretase production of Aβ peptides and the disparity in sensitivity of these peptides to a potent γ-secretase suggest that PS may be necessary, but not sufficient, to catalyze hydrolysis at the scissile bonds that generate the termini of Aβ1–40 and Aβ1-42 peptides. Presenilin 1 (PS1) plays an essential role in intramembranous “γ-secretase” processing of several type I membrane proteins, including the β-amyloid precursor proteins (APP) and Notch1. In this report, we examine the activity of two familial Alzheimer's disease-linked PS1 variants on the production of secreted Aβ peptides and the effects of L-685,458, a potent γ-secretase inhibitor, on inhibition of Aβ peptides from cells expressing these PS1 variants. We now report that PS1 variants enhance the production and secretion of both Aβ1–42 and Aβ1–40 peptides. More surprisingly, whereas the IC50 for inhibition of Aβ1–40 peptide production from cells expressing wild-type PS1 is ∼1.5 μm, cells expressing the PS1ΔE9 mutant PS1 exhibit an IC50 of ∼4 μm. Immunoprecipitation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry reveal that the levels of Aβ1–43 peptides are elevated in medium of PS1ΔE9 cells treated with higher concentrations of inhibitor. The differential effects of wild-type and mutant PS1 on γ-secretase production of Aβ peptides and the disparity in sensitivity of these peptides to a potent γ-secretase suggest that PS may be necessary, but not sufficient, to catalyze hydrolysis at the scissile bonds that generate the termini of Aβ1–40 and Aβ1-42 peptides. presenilin familial Alzheimer disease β-amyloid precursor protein β-amyloid Notch extracellular truncation Notch intracellular domain COOH-terminal fragment immunoprecipitation NH2-terminal fragment wild-type N,N-bis(2-hydroxyethyl)glycine matrix-assisted laser desorption/ionization time-of-flight N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine Presenilin 1 and 2 (PS1 and PS2)1 are polytopic membrane proteins that are mutated in the majority of pedigrees with early onset familial Alzheimer's disease (FAD) (1Levy-Lahad E. Wasco W. Poorkaj P. Romano D.M. Oshima J. Pettingell W.H. Yu C.E. Jondro P.D. Schimidt S.D. Wang K. Crowley A.C. Fu Y.-H. Huenette S.Y. Galas D. Nemens E. Wijsman E.M. Bird T.D. Schellenberg G.D. Tanzi R.E. Science. 1995; 269: 973-977Crossref PubMed Scopus (2230) Google Scholar, 2Rogaev E.I. Sherrington R. Rogaev E.A. Levesque G. Ikeda M. Liang Y. Chi H. Lin C. Holman K. Tsuda T. Mar L. Sorbi S. Nacmias B. Piacentini S. Amaducci L. Chumakov I. Cohen D. Lannfelt L. Frase P.E. Rommens J.M. St. George-Hyslop P. Nature. 1995; 376: 775-778Crossref PubMed Scopus (1791) Google Scholar, 3Sherrington R. Rogaev E.I. Liang Y. Rogaeva E.A. Levesque G. Ikeda M. Chi H. Lin C. Li G. Holman K. Tsuda T. Mar L. Foncin J.-F. Bruni A.C. Montesi M.P. Sorbi S. Rainero I. Pinessi L. Nee L. Chumakov I. Pollen D. Brookes A. Sanseau P. Polinsky R.J. Wasco W. Da Silva H.A.R. Haine J.L. Pericak-Vance M.A. Tanzi R.E. Roses A.D. Fraser P. Rommens J.M. St. George-Hyslop P. Nature. 1995; 375: 754-760Crossref PubMed Scopus (3585) Google Scholar). It is now established that PS play an essential role in intramembranous “γ-secretase” processing of type I membrane proteins, including the β-amyloid precursor proteins (APP) (4De Strooper B. Saftig P. Craessaerts K. Vanderstichele H. Guhde G. Annaert W. Von Figura K. Van Leuven F. Nature. 1998; 391: 387-390Crossref PubMed Scopus (1552) Google Scholar, 5Naruse S. Thinakaran G. Luo J.J. Kusiak J.W. Tomita T. Iwatsubo T. Qian X. Ginty D.D. Price D.L. Borchelt D.R. Wong P.C. Sisodia S.S. Neuron. 1998; 21: 1213-1221Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar), the developmental signaling receptor, Notch1 (6De Strooper B. Annaert W. Cupers P. Saftig P. Craessaerts K. Mumm J.S. Schroeter E.H. Schrijvers V. Wolfe M.S. Ray W.J. Goate A. Kopan R. Nature. 1999; 398: 518-522Crossref PubMed Scopus (1800) Google Scholar, 7Schroeter E.H. Kisslinger J.A. Kopan R. Nature. 1998; 393: 382-386Crossref PubMed Scopus (1361) Google Scholar, 8Struhl G. Adachi A. Cell. 1998; 93: 649-660Abstract Full Text Full Text PDF PubMed Scopus (635) Google Scholar), the tyrosine kinase receptor ErbB4 (9Ni C.-Y. Murphy M.P. Golde T.E. Carpenter G. Science. 2001; 294: 2179-2181Crossref PubMed Scopus (756) Google Scholar, 10Lee H.-J. Jung K.-M. Huang Y.Z. Bennett L.B. Lee J.S. Kim T.-W. J. Biol. Chem. 2002; 277: 6318-6323Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar), and N- and E-cadherins (11Marambaud P. Shioi J. Serban G. Georgakopoulos A. Sarner S. Nagy V. Biki L. Wen P. Efthimiopoulos S. Shao Z. Wisniewski T. Robakis N.K. EMBO J. 2002; 21: 1948-1956Crossref PubMed Scopus (624) Google Scholar). For APP, γ-secretase catalyzes proteolysis of a set of membrane-tethered APP derivatives, termed APP-CTFs, resulting in the production and secretion of β-amyloid (Aβ) peptides. On the other hand, γ-secretase-mediated processing of the membrane-tethered Notch1 derivative, termed S2/NEXT, releases the intracellular domain (S3/NICD) that subsequently translocates to the nucleus and activates transcription of target genes (7Schroeter E.H. Kisslinger J.A. Kopan R. Nature. 1998; 393: 382-386Crossref PubMed Scopus (1361) Google Scholar, 8Struhl G. Adachi A. Cell. 1998; 93: 649-660Abstract Full Text Full Text PDF PubMed Scopus (635) Google Scholar). The observation that Aβ and S3/NICD production are completely eliminated in cells derived from mouse blastocysts with compound deletions of PS1 andPS2, lends convincing support to the notion that PS are critical for intramembranous cleavage of APP and Notch1 (12Herreman A. Serneels L. Annaert W. Collen D. Schoonjans L. De Strooper B. Nat. Cell Biol. 2000; 2: 461-462Crossref PubMed Scopus (450) Google Scholar, 13Zhang Z. Nadeau P. Song W. Donoviel D. Yuan M. Bernstein A. Yankner B.A. Nat. Cell Biol. 2000; 2: 463-465Crossref PubMed Scopus (359) Google Scholar). Although the mechanism(s) by which PS facilitates γ-secretase processing of APP and Notch1 have not been fully elucidated, the generation of Aβ (14Li 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, 15Li Y.-M. Lai M.-T. Xu M. Huang Q. DiMuzio-Mower J. Sardana M.K. Shi X.-P. Yin K.-C. Shafer J.A. Gerdell S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6138-6143Crossref PubMed Scopus (499) Google Scholar, 16Shearman M.S. Beher D. Clarke E.E. Lewis H.D. Harrison T. Hunt P. Nadin A. Smith A.L. Stevenson G. Castro J. Biochemistry. 2000; 39: 8698-8704Crossref PubMed Scopus (367) Google Scholar) and S3/NICD (17Martys-Zage J.L. Kim S.-H. Berechid B. Bingham S.J. Chu S. Sklar J. Nye J. Sisodia S.S. J. Mol. Neurosci. 2000; 15: 189-204Crossref PubMed Scopus (43) Google Scholar) have been show to be inhibited by highly potent and selective aspartyl protease transition state inhibitors that bind specifically to PS1 and PS2 (14Li 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, 18Esler W.P. Kimberly W.T. Ostaszewski B.L. Diehl T.S. Moore C.L. Tsai J.Y. Rahmati T. Xia W. Selkoe D.J. Wolfe M.S. Nat. Cell Biol. 2000; 2: 428-434Crossref PubMed Scopus (506) Google Scholar). These data, taken with the description of a family of signal peptide peptidases with limited homology to PS (19Weihofen A. Binns K. Lemberg M.K. Ashman K. Martoglio B. Science. 2002; 296: 2215-2218Crossref PubMed Scopus (456) Google Scholar), have led to the conclusion that PS are the elusive γ-secretases (20Wolfe M.S. Selkoe D.J. Science. 2002; 296: 2156-2157Crossref PubMed Scopus (56) Google Scholar). While appealing, the “PS is γ-secretase” model has several weaknesses. First, mutagenesis studies have revealed that γ-secretase has relaxed substrate selectivity within the APP transmembrane domain and occurs at heterogeneous sites (21Lichtenthaler 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 (193) Google Scholar, 22Murphy M.P. Hickman L.J. Eckman C.B. Uljon S.N. Wang R. Golde T.E. J. Biol. Chem. 1999; 274: 11914-11923Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar), while γ-secretase cleavage of Notch1 is highly sequence-specific and appears to generate a single S3/NICD species (7Schroeter E.H. Kisslinger J.A. Kopan R. Nature. 1998; 393: 382-386Crossref PubMed Scopus (1361) Google Scholar). Second, whereas endocytosis and recycling of APP-CTFs are required for the generation of Aβ (23Koo E.H. Squazzo S.L. J. Biol. Chem. 1994; 269: 17386-17389Abstract Full Text PDF PubMed Google Scholar), S3/NICD production does not require endocytic trafficking of the Notch derivative, S2/NEXT (24Struhl G. Adachi A. Mol. Cell. 2000; 6: 625-636Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar). Third, the identification of several PS-interacting membrane proteins, including nicastrin (25Yu 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), APH-1 (26Goutte C. Tsunozaki M. Hale V.A. Priess J.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 775-779Crossref PubMed Scopus (371) Google Scholar), and PEN2 (27Francis R. McGrath G. Zhang J. Ruddy D.A. Sym M. Apfred J. Nicoll M. Maxwell M. Hai B. Ellis M.C. Parks A.L. Xu W. Li J. Gurnney 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) that also modulate production of S3/NICD (25Yu 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, 26Goutte C. Tsunozaki M. Hale V.A. Priess J.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 775-779Crossref PubMed Scopus (371) Google Scholar, 27Francis R. McGrath G. Zhang J. Ruddy D.A. Sym M. Apfred J. Nicoll M. Maxwell M. Hai B. Ellis M.C. Parks A.L. Xu W. Li J. Gurnney 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) and Aβ (25Yu 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, 28Chen F. Yu G. Arawaka S. Nishimara M. Kawarai T. Yu H. Tandon A. Supala A. Song Y.Q. Rogaeva E. Milman P. Sato C. Yu C. Jamus C. Lee J. Song L. Zhang L. Fraser P.E. St. George-Hyslop P.H. Nat. Cell Biol. 2001; 3: 751-754Crossref PubMed Scopus (112) Google Scholar) suggests that a protein complex, comprised of PS and other factors are required for intramembranous proteolysis of APP and Notch1. Finally, PS1 harboring a substitution of aspartate 257 with alanine is capable of processing APP to Aβ peptides (29Capell A. Steiner H. Romig H. Keck S. Baader M. Grim M.G. Baumeister R. Haass C. Nat. Cell Biol. 2000; 2: 205-211Crossref PubMed Scopus (139) Google Scholar,30Kim S.-H. Leem J.Y. Lah J.J. Slunt H.H. Levey A.I. Thinakaran G. Sisodia S.S. J. Biol. Chem. 2001; 276: 43343-43350Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), but fails to generate S3/NICD from a truncated Notch1 molecule, termed NotchΔE (31Song W. Nadeau P. Yuan M. Yang X. Shen J. Yankner B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6959-6963Crossref PubMed Scopus (313) Google Scholar). Similarly, expression of several FAD-linkedPS1 variants (30Kim S.-H. Leem J.Y. Lah J.J. Slunt H.H. Levey A.I. Thinakaran G. Sisodia S.S. J. Biol. Chem. 2001; 276: 43343-43350Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 31Song W. Nadeau P. Yuan M. Yang X. Shen J. Yankner B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6959-6963Crossref PubMed Scopus (313) Google Scholar, 32Moehlmann T. Winkler E. Xia X. Edbauer D. Murell J. Capell A. Kaether C. Zheng H. Ghetti B. Haass C. Steiner H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8025-8030Crossref PubMed Scopus (240) Google Scholar) or the experimental L286E or L286RPS1 mutants (33Chen F. Gu Y.J. Hasegawa H. Ruan X. Arawaka S. Fraser P. Westaway D. Mount H. St. George-Hyslop P. J. Biol. Chem. 2002; 277: 36521-36526Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar) leads to exaggerated overproduction of highly fibrillogenic Aβ42 peptides, but surprisingly, thesePS1 variants fail to generate S3/NICD from NotchΔE. Intrigued by the apparent discordance between the activities of FAD-linked PS1 mutants on the production of Aβ42 peptides and S3/NICD production, we examined the activity of these FAD-linked PS1 variants on the production of secreted Aβ peptides and the effects of a potent aspartyl protease transition state inhibitor of γ-secretase, termed L-685,458 (15Li Y.-M. Lai M.-T. Xu M. Huang Q. DiMuzio-Mower J. Sardana M.K. Shi X.-P. Yin K.-C. Shafer J.A. Gerdell S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6138-6143Crossref PubMed Scopus (499) Google Scholar, 16Shearman M.S. Beher D. Clarke E.E. Lewis H.D. Harrison T. Hunt P. Nadin A. Smith A.L. Stevenson G. Castro J. Biochemistry. 2000; 39: 8698-8704Crossref PubMed Scopus (367) Google Scholar) on the production of these Aβ species. We now report that while PS1 variants enhance production of Aβ42, as expected, there is an unexpected enhancement in levels of secreted Aβ40 peptides. We also provide the first demonstration that in the conditioned medium of “pools” of stable cell lines that express individual FAD-linked mutant PS1, both Aβ1–40 and 1–42 peptides accumulate to higher levels than the Aβ peptide variants in medium of cell pools that express wild-type PS1. More surprisingly, under conditions at which the γ-secretase inhibitor completely eliminates production of all Aβ-related peptides from cells expressing wild-type PS1, we now report that the inhibitor is not fully effective at lowering production of Aβ variants from cells expressing two independent FAD-linked PS1 mutants. Hence, we argue that production of Aβ peptides are differentially regulated by the expression of wild-type and FAD-linked PS1 variants. Mouse neuroblastoma N2a cells that constitutively expresses human Swedish APP695 (N2a swe.10) (35Chen C. Okayama H. Mol. Cell. Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4821) Google Scholar) were maintained in 50% Dulbecco's modified Eagle's medium and 50% Opti-MEM (Invitrogen) supplemented with 5% fetal bovine serum. To generate stable cell lines expressing wild-type PS1, the FAD-linked ΔE9, or E280A variants, N2a swe.10 cells were cotransfected with 10 μg of PS1 cDNAs (in pAG3Zeo vector) and 100 ng of pIREShygro using the calcium phosphate method (36Thinakaran G. Teplow D.B. Siman R. Greenberg B. Sisodia S.S. J. Biol. Chem. 1996; 271: 9390-9397Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar). Cells expressing transgene were selected with 400 μg/ml hygromycin. Hygromycin-resistant colonies were further screened in medium containing 400 μg/ml zeocin (Invitrogen) to generate a stable pool. Approximately 100–200 zeocin-resistant colonies were pooled and analyzed. For γ-secretase inhibitor assays, cells were incubated for 16 h in medium containing 2 μm (or indicated concentrations) of the γ-secretase inhibitor, L-685,458 (16Shearman M.S. Beher D. Clarke E.E. Lewis H.D. Harrison T. Hunt P. Nadin A. Smith A.L. Stevenson G. Castro J. Biochemistry. 2000; 39: 8698-8704Crossref PubMed Scopus (367) Google Scholar), prepared in dimethyl sulfoxide (Me2SO) or an equivalent concentration of Me2SO as a vehicle control. Conditioned media were collected and immediately adjusted to 0.5 mm phenylmethylsulfonyl fluoride. Cultured cells were lysed in 1× immunoprecipitation (IP) buffer containing 50 mmTris-HCl (pH 7.4), 150 mm NaCl, 5 mm EDTA, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, and protease inhibitor mixture (Sigma). Nuclei and debris were removed by centrifugation and the protein concentration of detergent-soluble proteins in each lysate was determined using a bicinchoninic acid protein assay kit (Pierce). For immunoprecipitations, we used equivalent volumes of conditioned medium based on the calculation of the protein concentration in each plate of cells to avoid experimental bias because of variations in cell density. Normalized conditioned media were immunoprecipitated with 26D6, a monoclonal antibody raised against Aβ1–12 (37Lamb B.T. Bardel K.A. Kulnane L.S. Anderson J.J. Holtz G. Wagner S.L. Sisodia S.S. Hoeger E.J. Nat. Neurosci. 1999; 2: 695-697Crossref PubMed Scopus (92) Google Scholar), for 16 h at 4 °C. The immune complexes were “bridged” by the addition of rabbit anti-mouse IgG (Pierce), collected with protein A-conjugated agarose beads (Pierce), and eluted by boiling for 5 min in Laemmli SDS sample buffer prior to fractionation on SDS-PAGE. Aliquots of detergent lysate were fractionated on high percentage Tris-Tricine SDS-PAGE gels for detection of full-length APP and APP-CTFs, or Tris glycine SDS-PAGE for analysis of PS1. To detect secreted Aβ40/42, immunoprecipitated samples were fractionated on Bicine/urea gels (38Wiltfang J. Smirnov A. Schnierstein B. Kelemen G. Matthies U. Klafki H.W. Staufenbiel M. Huther G. Ruther E. Kornhber J. Electrophoresis. 1997; 18: 527-532Crossref PubMed Scopus (121) Google Scholar). Fractionated proteins were electrophoretically transferred to polyvinylidene difluoride membranes (Bio-Rad), and the membranes were probed with appropriate primary antibodies. Full-length APP and APP-CTF were detected by CT15, an antisera that recognizes the carboxyl-terminal 15 amino acids of APP (39Sisodia S.S. Koo E.H. Hoffman P.N. Perry G. Price D.L. J. Neurosci. 1993; 13: 3136-3142Crossref PubMed Google Scholar). A polyclonal antibody, PS1NT, was used to detect full-length PS1 and PS1 NTF (40Thinakaran G. Getard J.B. Bouton C.M.L. Harris C.L. Price D.L. Borchelt D.R. Sisodia S.S. Neurobiol. Dis. 1998; 4: 438-453Crossref PubMed Scopus (171) Google Scholar). Soluble APPα and Aβ40/42 were detected by 26D6 (37Lamb B.T. Bardel K.A. Kulnane L.S. Anderson J.J. Holtz G. Wagner S.L. Sisodia S.S. Hoeger E.J. Nat. Neurosci. 1999; 2: 695-697Crossref PubMed Scopus (92) Google Scholar). After incubation with horseradish peroxidase-coupled secondary antibodies (Pierce), bound antibodies were visualized using an enhanced chemiluminescence (ECL) detection system (Perkin-Elmer Life Sciences). N2a cells were starved for 30 min in methionine-free Dulbecco's modified Eagle's medium (Invitrogen) and then labeled with 250 μCi/ml [35S]methionine (PerkinElmer Life Sciences) in methionine-free Dulbecco's modified Eagle's medium supplemented with 1% dialyzed fetal bovine serum (Invitrogen) for 10 min (for pulse-labeling) or 2 h. Conditioned medium was collected and cells were lysed in IP buffer. For immunoprecipitations we used a volume of conditioned medium that was normalized to the calculated trichloroacetic acid-precipitable radioactive counts (cpm) in cell lysates. Soluble APPα and Aβ40/42 were immunoprecipitated with monoclonal antibody, 26D6 (37Lamb B.T. Bardel K.A. Kulnane L.S. Anderson J.J. Holtz G. Wagner S.L. Sisodia S.S. Hoeger E.J. Nat. Neurosci. 1999; 2: 695-697Crossref PubMed Scopus (92) Google Scholar). To examine APP synthesis, cells were pulse-labeled with [35S]methionine for 10 min, and APP was immunoprecipitated with 369 antibody, raised against a peptide corresponding amino acids 649–695 of APP695 (41Buxbaum J.D. Gandy S.E. Cicchetti P. Ehrlich M.E. Czernik A.J. Fracasso R.P. Ramabhadran T.V. Unterbeck A.J. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6003-6006Crossref PubMed Scopus (427) Google Scholar). Immunoprecipitates were fractionated by SDS-PAGE, and radioactive bands were visualized and quantified using a PhosphorImager (AmershamBiosciences). Conditioned media from N2a swe.10 cell pools stably expressing wild-type PS1 or the PS1ΔE9 were immunoprecipitated with 4G8 antibody, specific for amino acids 17–24 of Aβ, and collected with Protein A/G-coupled agarose beads prior to analysis by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometric analysis, as described (42Wang R. Sweeney D. Gandy S.E. Sisodia S.S. J. Biol. Chem. 1996; 271: 31894-31902Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). Each mass spectrum was averaged from at least 500 measurements, and bovine insulin was included as an internal mass calibrant. It is now well accepted that expression of FAD-linked PS1 variants elevate the levels of secreted Aβ1–42 peptides and, in so doing, increase the calculated ratio of Aβ40/Aβ42 peptides. However, the absolute levels of Aβ peptide variants have rarely been reported, a reflection, in large part, of the variability in transgene-encoded APP between individual lines. With this caveat, we chose to transfect a neuroblastoma N2a cell line that constitutively expresses a carboxyl-terminal Myc epitope-tagged human APP 695 harboring the FAD-linked “Swedish” mutations (38Wiltfang J. Smirnov A. Schnierstein B. Kelemen G. Matthies U. Klafki H.W. Staufenbiel M. Huther G. Ruther E. Kornhber J. Electrophoresis. 1997; 18: 527-532Crossref PubMed Scopus (121) Google Scholar) with human wild-type PS1 (wtPS1) or the FAD-linked PS1 variants, PS1ΔE9, or E280A to generate stable pools that express human PS1 polypeptides. Western blot analysis of stable cell pools revealed the accumulation of human PS1 NTF and low levels of full-length precursor in cells expressing wild-type PS1 (Fig.1 A, lane 1), uncleaved ∼43-kDa PS1ΔE9, and low levels of endogenous mouse PS1 NTF in cells expressing PS1ΔE9 (Fig. 1 A, lane 2), and mutant human PS1 NTF and low levels of full-length precursor in cells expressing the E280A variant (Fig. 1 A,lane 3). In these cell pools, “replacement” of the bulk of murine PS1 fragments has occurred (45Petit A. Bihel F. Alves da Costa C. Pourquie O. Checler F. Kraus J.L. Nat. Cell Biol. 2001; 3: 507-511Crossref PubMed Scopus (193) Google Scholar), although residual levels of murine NTF (as seen in the PS1ΔE9 cells) are still present. This would be expected in a cell pool in which transgene-derived products are expressed at varying levels in independent clones. We examined the levels of secreted Aβ-related species by immunoprecipitation with antibody 26D6 (39Sisodia S.S. Koo E.H. Hoffman P.N. Perry G. Price D.L. J. Neurosci. 1993; 13: 3136-3142Crossref PubMed Google Scholar), specific for Aβ residues 1–12, fractionation of immune complexes on Bicine/urea gels, and analysis of immunoprecipitated Aβ peptides using 26D6 antibody and enhanced chemiluminescence detection. For these studies, we calculated the protein concentration in each plate of cells, and used normalized volumes of medium so that there would be no experimental bias because of differences in cell density. In Fig. 1, we show that constitutive expression of wild-type PS1 leads to robust secretion of Aβ1–40 peptides, limited levels of secreted Aβ1–37, Aβ1–38, and Aβ1–39 peptides and nearly undetectable levels of Aβ1–42 peptides (Fig. 1 B, lane 1). This level of secreted Aβ peptides is no higher than parental APPswe.10 cells (data not shown). On the other hand, we consistently observed that the levels of accumulated Aβ1–40 and Aβ1–42 peptides were elevated in medium of cells expressing either the PS1ΔE9 or A280E variants (Fig.1 B, lanes 3 and 5, respectively). Even more surprising was the observation that under conditions in which treatment of wtPS1 cells with a potent γ-secretase inhibitor, L-685,458 (17Martys-Zage J.L. Kim S.-H. Berechid B. Bingham S.J. Chu S. Sklar J. Nye J. Sisodia S.S. J. Mol. Neurosci. 2000; 15: 189-204Crossref PubMed Scopus (43) Google Scholar) (2 μm for 16 h), resulted in nearly complete inhibition of secreted Aβ peptides (Fig. 1 B,lane 2), low levels of Aβ1–40 and Aβ1–42 peptides still remained in the medium of cells expressing either PS1 mutant (Fig. 1 B, lanes 4 and 6). Our finding that two FAD-linked PS1 variants enhance secretion of the principal Aβ variant, Aβ1–40, is novel and we felt it important to fully validate this finding. We chose to focus on the PS1ΔE9 pool. First, to establish that the differences in accumulated Aβ peptides between wtPS1 and PS1ΔE9 cell pools was not simply a reflection of differences in synthetic levels of transgene-encoded APPswe, we pulse-labeled cells with [35S]methionine for 10 min and analyzed newly synthesized APP in cell lysates by subjecting equivalent detergent-soluble, trichloroacetic acid-precipitable, radioactivity (cpm) to immunoprecipitation with antibody 369, raised against a peptide corresponding to amino acids 649–695 of APP (43Thinakaran G. Harris C.L. Ratovitski T. Davenport F. Slunt H.H. Price D.L. Borchelt D.R. Sisodia S.S. J. Biol. Chem. 1997; 272: 28415-28422Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar), fractionation of immune complexes on SDS-PAGE, and phosphorimaging. In Fig. 1 C, we show that the synthesis of full-length APP is indistinguishable between the cell pools that express either human wtPS1 or PS1ΔE9. To further quantify the absolute increase in both Aβ1–40 and Aβ1–42 peptides in medium of mutant PS1-expressing cells relative to cells expressing wtPS1, we incubated cell pools with [35S]methionine for 2 h and quantified the levels of secreted Aβ peptides in medium by immunoprecipitation with antibody 26D6, fractionation of immune complexes on Tris-Tricine gels, and phosphorimaging. For these analyses, we quantified total counts/min in detergent-solubilized cell lysates and used normalized volumes of radiolabeled conditioned medium for immunoprecipitations. As we have shown by Western blot analysis (Fig. 1 B), quantitative phosphorimaging analysis revealed an elevation in total Aβ peptides in medium of cells expressing the PS1ΔE9 mutant (Fig. 1 D,lane 3) by 2.8-fold relative to Aβ peptides secreted from cells expressing wtPS1 (Fig. 1 D, lane 1). Notably, the 26D6 antibody, specific for Aβ residues 1–12, also detects soluble derivatives generated by α-secretase, termed APPsa, quantitative phosphorimaging revealed a 1.5-fold increase in levels of APPsa in medium of PS1ΔE9 cells relative to cells expressing wild-type PS1 (Fig. 1 D, comparelanes 3 and 1, respectively), this despite identical synthetic rates of the APPswe precursor between cell pools (Fig. 1 C). These findings offer the suggestion that expression of the PS1ΔE9 variant leads to enhanced trafficking (or processing) of full-length APPswe to cellular compartments in which α-secretase is active, but further studies will be necessary to address this issue. In any event, quantitative phosphorimaging revealed that while treatment of wtPS1 cells with 2 μm inhibitor reduced production of newly synthesized Aβ peptides to ∼0.4% of untreated controls (Fig. 1 D,lane 2), the inhibitor diminished the levels of total Aβ peptide species in medium of PS1ΔE9 cells to ∼12% of the untreated control (Fig. 1 D, lane 4). Further examination of the complexity of radiolabeled Aβ peptide variants by Bicine/urea gels (Fig. 1 E) revealed that the absolute levels of both Aβ40 and Aβ42 variants were elevated in medium of PS1ΔE9 cell medium (Fig. 1 E, lane 3; quantified in Fig.1 F, left panel) relative to the levels in medium of cells expressing wild-type PS1 (Fig. 1 E, lane 1; quantified in Fig. 1 F, left panel). Furthermore, under conditions where the γ-secretase inhibitor almost completely eliminated Aβ1–40 species in medium of cells expressing wild-type PS1, this compound diminished Aβ1–40 peptide levels to ∼13% in medium of cells expressing PS1ΔE9 (Fig. 1 E; quantified in Fig. 1 F, left panel). Similarly, the γ-secretase inhibitor fully eliminates secreted Aβ1–42 peptides from cells expressing wild-type PS1, but L-685,458 treatment only reduced the level of Aβ1–42 peptides to ∼58% in medium of cells that expres" @default.
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• W2087254373 date "2003-02-01" @default.
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• W2087254373 title "Familial Alzheimer Disease-linked Presenilin 1 Variants Enhance Production of Both Aβ1–40 and Aβ1–42 Peptides That Are Only Partially Sensitive to a Potent Aspartyl Protease Transition State Inhibitor of “γ-Secretase”" @default.
• W2087254373 cites W1480987708 @default.
• W2087254373 cites W1485366536 @default.
• W2087254373 cites W1493502680 @default.
• W2087254373 cites W1502820594 @default.
• W2087254373 cites W1516399319 @default.
• W2087254373 cites W1518012529 @default.
• W2087254373 cites W1524261859 @default.
• W2087254373 cites W1554824558 @default.
• W2087254373 cites W1626267121 @default.
• W2087254373 cites W1655404681 @default.
• W2087254373 cites W1660631781 @default.
• W2087254373 cites W1675105744 @default.
• W2087254373 cites W1783111952 @default.
• W2087254373 cites W1967566501 @default.
• W2087254373 cites W1979240613 @default.
• W2087254373 cites W1982825693 @default.
• W2087254373 cites W1991549727 @default.
• W2087254373 cites W1994453752 @default.
• W2087254373 cites W2000550532 @default.
• W2087254373 cites W2001914338 @default.
• W2087254373 cites W2006257247 @default.
• W2087254373 cites W2019917942 @default.
• W2087254373 cites W2030082602 @default.
• W2087254373 cites W2030394610 @default.
• W2087254373 cites W2033796514 @default.
• W2087254373 cites W2036755286 @default.
• W2087254373 cites W2045153164 @default.
• W2087254373 cites W2046209489 @default.
• W2087254373 cites W2049130898 @default.
• W2087254373 cites W2049665829 @default.
• W2087254373 cites W2055247763 @default.
• W2087254373 cites W2057713102 @default.
• W2087254373 cites W2063733034 @default.
• W2087254373 cites W2076444547 @default.
• W2087254373 cites W2077674018 @default.
• W2087254373 cites W2079302243 @default.
• W2087254373 cites W2083633251 @default.
• W2087254373 cites W2087932565 @default.
• W2087254373 cites W2089824266 @default.
• W2087254373 cites W2094555107 @default.
• W2087254373 cites W2094842946 @default.
• W2087254373 cites W2101381508 @default.
• W2087254373 cites W2120304489 @default.
• W2087254373 cites W2128084592 @default.
• W2087254373 cites W2134365974 @default.
• W2087254373 cites W2138447190 @default.
• W2087254373 cites W2146955477 @default.
• W2087254373 cites W2153723509 @default.
• W2087254373 cites W2164742714 @default.
• W2087254373 cites W2167959613 @default.
• W2087254373 cites W2775777157 @default.
• W2087254373 cites W2833026885 @default.
• W2087254373 doi "https://doi.org/10.1074/jbc.m209252200" @default.
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