The Transmembrane Aspartates in Presenilin 1 and 2 Are Obligatory for γ-Secretase Activity and Amyloid β-Protein Generation

The discovery that a deficiency of presenilin 1 (PS1) decreases the production of amyloid β-protein (Aβ) identified the presenilins as important mediators of the γ-secretase cleavage of β-amyloid precursor protein (APP). Recently, we found that two conserved transmembrane (TM) aspartates in PS1 are critical for Aβ production, providing evidence that PS1 either functions as a required diaspartyl cofactor for γ-secretase or is itself γ-secretase. Presenilin 2 (PS2) shares substantial sequence and possibly functional homology with PS1. Here, we show that the two TM aspartates in PS2 are also critical for γ-secretase activity, providing further evidence that PS2 is functionally homologous to PS1. Cells stably co-expressing TM Asp → Ala mutations in both PS1 and PS2 show further accumulation of the APP-derived γ-secretase substrates, C83 and C99. The production of Aβ is reduced to undetectable levels in the conditioned media of these cells. Furthermore, endoproteolysis of the exogenous Asp mutant PS2 is absent, and endogenous PS1 C-terminal fragments are diminished to undetectable levels. Therefore, the co-expression of PS1 and PS2 TM Asp → Ala mutants suppresses the formation of any detectable PS1 or PS2 heterodimeric fragments and essentially abolishes the production of Aβ. These results explain the residual Aβ production seen in PS1-deficient cells and demonstrate the absolute requirement of functional presenilins for Aβ generation. We conclude that presenilins, and their TM aspartates in particular, are attractive targets for lowering Aβ therapeutically to prevent Alzheimer's disease. The discovery that a deficiency of presenilin 1 (PS1) decreases the production of amyloid β-protein (Aβ) identified the presenilins as important mediators of the γ-secretase cleavage of β-amyloid precursor protein (APP). Recently, we found that two conserved transmembrane (TM) aspartates in PS1 are critical for Aβ production, providing evidence that PS1 either functions as a required diaspartyl cofactor for γ-secretase or is itself γ-secretase. Presenilin 2 (PS2) shares substantial sequence and possibly functional homology with PS1. Here, we show that the two TM aspartates in PS2 are also critical for γ-secretase activity, providing further evidence that PS2 is functionally homologous to PS1. Cells stably co-expressing TM Asp → Ala mutations in both PS1 and PS2 show further accumulation of the APP-derived γ-secretase substrates, C83 and C99. The production of Aβ is reduced to undetectable levels in the conditioned media of these cells. Furthermore, endoproteolysis of the exogenous Asp mutant PS2 is absent, and endogenous PS1 C-terminal fragments are diminished to undetectable levels. Therefore, the co-expression of PS1 and PS2 TM Asp → Ala mutants suppresses the formation of any detectable PS1 or PS2 heterodimeric fragments and essentially abolishes the production of Aβ. These results explain the residual Aβ production seen in PS1-deficient cells and demonstrate the absolute requirement of functional presenilins for Aβ generation. We conclude that presenilins, and their TM aspartates in particular, are attractive targets for lowering Aβ therapeutically to prevent Alzheimer's disease. presenilin amyloid β-protein Chinese hamster ovary β-amyloid precursor protein N-terminal fragment C-terminal fragment transmembrane enzyme-linked immunosorbent assay wild type polyacrylamide gel electrophoresis immunoprecipitation N-2-hydroxy-1,1-bis(hydroxymethyl)ethylglycine Mutations in presenilin 1 (PS1)1 and presenilin 2 (PS2) are responsible for the most severe and common form of dominant, familial Alzheimer's disease (1.Selkoe D.J. Trends Cell Biol. 1998; 8: 447-453Abstract Full Text Full Text PDF PubMed Scopus (808) Google Scholar). These missense mutations have been shown to subtly alter the proteolytic processing of the β-amyloid precursor protein (APP). APP is cleaved by α- or β-secretase, and the membrane-retained 83- and 99-residue C-terminal fragments produced by these cleavages (C83 and C99, respectively) serve as the substrates for a second cleavage by a protease designated γ-secretase. The products of the C99 cleavage include the 40- and 42-residue amyloid β-peptides (Aβ), which accumulate to high abundance in myriad amyloid plaques in the brain. The familial Alzheimer's disease mutations in the presenilins appear to selectively increase the production and deposition of Aβ42 in cell culture (2.Scheuner D. Eckman C. Jensen M. Song X. Citron M. Suzuki N. Bird T.D. Hardy J. Hutton M. Kukull W. Larson E. Levy-Lahad E. Viitanen M. Peskind E. Poorkaj P. Schellenberg G. Tanzi R. Wasco W. Lannfelt L. Selkoe D. 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Neuron. 1997; 19: 939-945Abstract Full Text Full Text PDF PubMed Scopus (901) Google Scholar, 7.Citron M. Westaway D. Xia W. Carlson G. Diehl T. Levesque G. Johnson-Wood K. Lee M. Seubert P. Davis A. Kholodenka D. Motter R. Sherrington R. Perry B. Yao H. Strome R. Lieberburg I. Rommens J. Kim S. Schenk D. Fraser P. St. George-Hyslop P. Selkoe D.J. Nat. Med. 1997; 3: 67-72Crossref PubMed Scopus (1169) Google Scholar), and in the brains of familial Alzheimer's disease patients (8.Lemere C.A. Lopera F. Kosik K.S. Lendon C.L. Ossa J. Saido T.C. Yamaguchi H. Ruiz A. Martinez A. Madrigal L. Hincapie L. Arango L.J.C. Anthony D.C. Koo E.H. Goate A.M. Selkoe D.J. Arango V.J.C. Nat. Med. 1996; 2: 1146-1148Crossref PubMed Scopus (436) Google Scholar). Furthermore, in vitroevidence demonstrates that Aβ42 is more likely than Aβ40 to aggregate and polymerize into fibrils (3.Xia 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 (272) Google Scholar, 9.Jarrett J.T. Berger E.P. Lansbury Jr., P.T. Biochemistry. 1993; 32: 4693-4697Crossref PubMed Scopus (1768) Google Scholar). These Aβ42 fibrils appear to act as a seed for subsequent plaque formation (1.Selkoe D.J. Trends Cell Biol. 1998; 8: 447-453Abstract Full Text Full Text PDF PubMed Scopus (808) Google Scholar). The mechanism by which the presenilins cause this selective increase in Aβ42 has remained a mystery until recently. The discovery that a deficiency in PS1 inhibits the normal processing of APP and reduces both Aβ40 and Aβ42 production suggested that PS1 facilitates γ-secretase activity (10.De Strooper B. Saftig P. Craessaerts K. Vanderstichele H. Gundula G. Annaert W. Von Figura K. Van Leuven F. Nature. 1998; 391: 387-390Crossref PubMed Scopus (1560) Google Scholar). The region of function of PS1 in this regard has been further localized to two aspartates within the putative 6th and 7th transmembrane (TM) domains, which are required for Aβ production (11.Wolfe M.S. Xia W. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.J. Nature. 1999; 398: 513-517Crossref PubMed Scopus (1699) Google Scholar). The mutation of either aspartate 257 (TM6) or aspartate 385 (TM7) to alanine interferes with both the endoproteolysis of PS1 and the generation of Aβ. This occurs by a dominant negative mechanism that replaces endogenous PS1 N- and C-terminal fragments (NTFs and CTFs) with the nonfunctional mutant holoprotein. However, in neurons that lack PS1, or in Chinese hamster ovary (CHO) cells that overexpress PS1 Asp → Ala mutant protein, residual Aβ production is still present (10.De Strooper B. Saftig P. Craessaerts K. Vanderstichele H. Gundula G. Annaert W. Von Figura K. Van Leuven F. Nature. 1998; 391: 387-390Crossref PubMed Scopus (1560) Google Scholar, 11.Wolfe M.S. Xia W. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.J. Nature. 1999; 398: 513-517Crossref PubMed Scopus (1699) Google Scholar). This remaining peptide, which is approximately 20–40% of the normal Aβ level, is postulated to arise from the residual PS2 activity in these cells. To test this hypothesis, we generated Asp → Ala mutations at the equivalent residues in PS2, aspartates 263 and 366. We first showed that these PS2 aspartyl mutants, like their PS1 aspartyl mutant counterparts, have a similar effect on APP processing, providing further evidence that PS2 is functionally homologous to PS1. We then stably introduced the PS2 D366A construct into CHO cells overexpressing human PS1 D257A and wild-type human APP. We found that the presence of both PS1 and PS2 aspartyl mutants completely abolishes γ-secretase processing of APP. We observed additional accumulation of the C83 and C99 APP CTFs and a suppression of Aβ production to levels that undetectable by a This of Aβ with of detectable PS1 and PS2 We conclude that functional PS1 or PS2 are required to Aβ and that the TM aspartates in are critical in both wild-type PS2 as the to or D366A by and the into the either the or The of the mutant by introduced into CHO cell in PS1, PS2, and PS1 have been (11.Wolfe M.S. Xia W. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.J. Nature. 1999; 398: 513-517Crossref PubMed Scopus (1699) Google Scholar, Wasco W. Tanzi J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). Chinese hamster ovary (CHO) cells stably overexpressing the human cells E.H. S. J. Biol. Chem. Full Text PDF PubMed Google designated in Cells stably co-expressing human APP with either PS2 (3.Xia 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 (272) Google or D257A PS1 (11.Wolfe M.S. Xia W. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.J. Nature. 1999; 398: 513-517Crossref PubMed Scopus (1699) Google in CHO cells co-expressing PS1, and PS2 in and CHO cells co-expressing D257A PS1, and D366A PS2 and in and to (11.Wolfe M.S. Xia W. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.J. Nature. 1999; 398: 513-517Crossref PubMed Scopus (1699) Google Scholar, M.B. D. Selkoe D.J. J. Google Scholar). and of P. Seubert and D. Schenk and are or and M.B. Citron M. P. Sherrington R. Xia W. Zhang J. Diehl T. Levesque G. Fraser P. Haass C. Koo Seubert P. St. George-Hyslop P. Teplow D.B. Selkoe D.J. 1997; 3: PubMed Scopus Google Scholar). a of T. Iwatsubo T. Maruyama K. Saido T.C. Kume H. Shinozaki K. Tokuhiro S. Capell A. Walter J. Grunberg J. Haass C. Iwatsubo T. Obata K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2025-2030Crossref PubMed Scopus (355) Google and the residues of the PS2 which the PS2 a of C. Haass J. Grunberg J. A. Haass C. Biochemistry. 1998; PubMed Scopus Google Scholar). in which cells for in media and by a in cells in a 2 and a protease 2 and to protein the protein assay with protein at for and then with and protein for 2 for at in the and then in with This by a in in at for by on or or and to to the in which a with a the test Aβ as K. Lee M. Motter R. K. Gordon G. R. K. Gordon M. H. D. Lieberburg I. Schenk D. Seubert P. L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: PubMed Scopus Google Scholar). The Aβ residues The Aβ residues as a of Aβ produced by the cell and The of PS1 in cells decreases the γ-secretase cleavage of APP (10.De Strooper B. Saftig P. Craessaerts K. Vanderstichele H. Gundula G. Annaert W. Von Figura K. Van Leuven F. Nature. 1998; 391: 387-390Crossref PubMed Scopus (1560) Google Scholar). We that mutation of either of the two transmembrane (TM) aspartates of PS1 a similar suppression of Aβ generation in cell culture (11.Wolfe M.S. Xia W. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.J. Nature. 1999; 398: 513-517Crossref PubMed Scopus (1699) Google Scholar). on this we that PS1 is either γ-secretase itself or a cofactor for γ-secretase. PS1 and PS2 homology E. Wasco W. Poorkaj P. J. Yu, C. K. D. E. Bird T.D. Schellenberg Tanzi PubMed Scopus Google Scholar, Sherrington R. Levesque G. M. Y. H. C. K. T. L. S. B. S. L. I. D. Lannfelt L. Fraser Rommens St. George-Hyslop Nature. PubMed Scopus Google Scholar), and has been suggested that with to APP We the equivalent TM aspartate residues in PS2 to in to the effect on the accumulation of C83 and C99, the APP C-terminal substrates for γ-secretase. the for wild-type human PS1, human PS2, PS1, PS2, or D366A PS2 into Chinese hamster ovary (CHO) cells stably the human APP cell Cell and for protein by immunoprecipitation with a C-terminal APP and with a C-terminal The of the PS1 aspartyl mutant or either of the PS2 aspartyl mutants in increase of C83 and C99 with cells PS1 or PS2 mutation of the transmembrane aspartates in both PS1 and PS2 similar on APP we the of both mutant We a PS2 D366A into CHO cells that overexpress APP and PS1 D257A to that stably human We for of the mutant PS2 by which human hamster PS2 with high 2 a high of mutant PS2 as by a of at 2 that with the PS2 in the CHO cell 2 2 and 3.Xia 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 (272) Google Scholar). These present in CHO cells human PS2 2 1 and To the levels of we on cell to APP and to the levels of human APP to the cell 2 APP in is similar to cell 2 and To that the cells similar of APP production, we cells for 1 and then APP with 2 demonstrates equivalent levels of APP and APP in cell We also the of PS1 holoprotein. demonstrates equivalent levels of of human PS1 a cell and cell and and This is with and which endogenous PS1 1 and Therefore, has levels of human APP and PS1 D257A to cell and also high levels of mutant PS2 holoprotein. To the of the aspartyl mutants on APP we of the C83 and C99 APP To the levels of these we also the APP from the by the of the with To for the of in a we the C83 and C99 levels APP and PS2 and PS1, and PS2 The or of these in CHO cells in approximately similar levels of C83 and C99 Therefore, the of the effect of the required to have any effect on APP proteolytic that these we the of APP to a more in protein a and APP we found in the of of APP at endogenous levels in in W. T. and D. J. Therefore, the of appear to have any effect on APP of the of the of cells that stably the aspartate mutant we observed a accumulation of C83 and C99. which high levels of PS1 increase as (11.Wolfe M.S. Xia W. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.J. Nature. 1999; 398: 513-517Crossref PubMed Scopus (1699) Google Scholar). has further levels a that is the cell are and then to and this the C83 is in the aspartate mutant cell To these mutations with α- or processing of we at the of and found that in the aspartate mutant cell with M.S. Xia W. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.J. Nature. 1999; 398: 513-517Crossref PubMed Scopus (1699) Google and the of PS1 D257A and PS2 D366A further accumulation of the C83 and C99 as with PS1 D257A We also the of two additional that PS1 and PS2 aspartate and We to the of the C83 fragments in these as as in and We a of C83 to within cell and that to the for The PS1 and PS2 aspartate mutant and and their than To this we also the C83 to in the cell (11.Wolfe M.S. Xia W. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.J. Nature. 1999; 398: 513-517Crossref PubMed Scopus (1699) Google Scholar). This cell inhibits Aβ to a similar as and in C83 accumulation the of this accumulation of the C83 APP in PS1 PS2 aspartate mutant cell → Ala mutant increase in APP C83 from cell C83 to within cell The for a of and are to that Cell (11.Wolfe M.S. Xia W. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.J. Nature. 1999; 398: 513-517Crossref PubMed Scopus (1699) Google as a that from in C83 The or from in a The C83 to within cell The for a of and are to that Cell (11.Wolfe M.S. Xia W. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.J. Nature. 1999; 398: 513-517Crossref PubMed Scopus (1699) Google as a that from in C83 The or To Aβ production, conditioned media and by a and the for the cell and and a of The Aβ levels from cell then as a of that showed a in Aβ to cells presenilin similar to on this (11.Wolfe M.S. Xia W. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.J. Nature. 1999; 398: 513-517Crossref PubMed Scopus (1699) Google Scholar). showed detectable Aβ production which to a media of Aβ We Aβ42 levels the of Aβ of the Furthermore, the in Aβ levels in conditioned media is a of Aβ is undetectable in in to cells (11.Wolfe M.S. Xia W. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.J. Nature. 1999; 398: 513-517Crossref PubMed Scopus (1699) Google Scholar). from cells Asp mutant are to B. and D. J. Scholar). Therefore, the in Aβ in the media of aspartate mutant cell the of The of the PS1 aspartyl mutants inhibits the cleavage of the exogenous protein and also suppresses the of endogenous hamster PS1 fragments in a (11.Wolfe M.S. Xia W. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.J. Nature. 1999; 398: 513-517Crossref PubMed Scopus (1699) Google Scholar). of the PS1 aspartyl mutants mutant holoprotein. We the accumulation of the PS2 in cell to a similar Cell by and PS2 by with the the and cell present 2 and This in either the APP or the cell which overexpress human PS2 1 human PS2 present in the protein at levels than the human cells detectable co-expression of PS1 and PS2 the formation of the PS2 PS2 is as high in the cells as in This that the lack of fragments in is to the mutation of the aspartate We then to human PS2 with a of for immunoprecipitation and for we in and cells 2 and we to any PS2 CTFs in cell Therefore, the transmembrane aspartates of PS2 are critical for with the effect of the TM aspartates in PS1. The of PS1 or PS2 fragments the formation of the G. C.L. Ratovitski T. F. Price D.L. D.R. Sisodia S.S. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). of PS1 the formation of endogenous fragments of PS2 and on these that PS1 and PS2 for which in endoproteolysis and of the fragments G. C.L. Ratovitski T. F. Price D.L. D.R. Sisodia S.S. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). We the effect of of PS1 and PS2 aspartyl mutants on the endogenous and exogenous PS1 of CHO cells showed the substantial of endogenous hamster with the human fragment 1 and similar of in endogenous PS1 the production of a human observed in Cells overexpressing both PS1 and PS2 showed similar of endogenous fragments as PS1 that PS2 in the presence of PS1 the formation of PS1 is of detectable human or hamster PS1 CTFs Therefore, the high of PS1 and PS2 aspartyl mutants suppresses the formation of endogenous fragments with exogenous human This that detectable presenilin which with lack of Aβ this we evidence that PS1 and PS2 the transmembrane aspartates for γ-secretase cleavage of APP. both these residues are required for the of the presenilin and of the APP C-terminal mutation of these aspartates the function of the We that PS1 either is a for γ-secretase or is itself γ-secretase (11.Wolfe M.S. Xia W. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.J. Nature. 1999; 398: 513-517Crossref PubMed Scopus (1699) Google Scholar). of evidence this from PS1-deficient reduced production of Aβ (10.De Strooper B. Saftig P. Craessaerts K. Vanderstichele H. Gundula G. Annaert W. Von Figura K. Van Leuven F. Nature. 1998; 391: 387-390Crossref PubMed Scopus (1560) Google Scholar). these cells are at the fragment of type transmembrane protein, Strooper B. Annaert W. P. Saftig P. Craessaerts K. E.H. V. M.S. Goate A. R. Nature. 1999; 398: PubMed Scopus Google Scholar). is to the of fragment to the within the transmembrane region E.H. R. Nature. 1998; PubMed Scopus Google Scholar). mutations in presenilin a that the of the presenilin abolishes G. I. Nature. 1999; 398: PubMed Scopus Google Scholar, Y. N. Nature. 1999; 398: PubMed Scopus Google Scholar). Therefore, APP and which both cleavage for presenilin activity for proteolytic on these is that γ-secretase either cleavage or substrates to the cleavage is that the presenilins two transmembrane aspartates that as the functional of aspartyl protease two of that the cleavage in APP γ-secretase activity and that γ-secretase is aspartyl protease M.S. Xia W. C. D. Ostaszewski B. T. I. Selkoe D.J. Biochemistry. 1999; PubMed Scopus Google Scholar). Here, we show that mutation of either aspartate in PS1 or PS2 results in similar on the APP substrates C83 and C99. Furthermore, of the PS2 aspartyl mutant results in of PS2 N- and C-terminal a that at PS2 aspartate mutants in cell type similar H. K. Capell A. H. S. Hardy Yu, X. M. K. Citron M. R. B. S. M. T. Iwatsubo T. R. Haass C. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). These additional evidence that the transmembrane aspartates in both presenilins are critical to their is that are aspartyl of in the of that γ-secretase activity Aβ generation in the presence of presenilin and C99 a that is this we also the dominant negative effect of the presenilin aspartate mutants to more the of of presenilins in Aβ We that the co-expression of Asp → Ala PS1 and PS2 in CHO cells C83 and C99 also Aβ generation to levels detectable to a These cells to any human with that PS1 aspartyl mutants to endoproteolysis (11.Wolfe M.S. Xia W. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.J. Nature. 1999; 398: 513-517Crossref PubMed Scopus (1699) Google Scholar). we to exogenous or endogenous CTFs for PS1, that these cells lack any of presenilin have suggested that the heterodimeric fragments are the functional presenilin the endoproteolysis of protein and the of the fragments is G. C.L. Ratovitski T. F. Price D.L. D.R. Sisodia S.S. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, G. D.R. Lee M.K. L. Kim G. T. F. C. M. Hardy J. Jenkins N.A. Copeland N.G. Price D.L. Sisodia S.S. Neuron. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). are the form present in and human M.B. Citron M. P. Sherrington R. Xia W. Zhang J. Diehl T. Levesque G. Fraser P. Haass C. Koo Seubert P. St. George-Hyslop P. Teplow D.B. Selkoe D.J. 1997; 3: PubMed Scopus Google Scholar, G. D.R. Lee M.K. L. Kim G. T. F. C. M. Hardy J. Jenkins N.A. Copeland N.G. Price D.L. Sisodia S.S. Neuron. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). of PS1 or PS2 has effect on the or of and are H. Capell A. B. Citron M. Selkoe D.J. H. K. Haass C. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). Here, we additional evidence that the heterodimeric fragments are the functional We that in cells that lack detectable is detectable Aβ also further the PS1 and PS2 by G. C.L. Ratovitski T. F. Price D.L. D.R. Sisodia S.S. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google that PS1 the formation of endogenous PS2 The occurs with PS2 that both PS1 and PS2 with for a postulated that the formation of heterodimeric We show that the of both PS1 and PS2 the endogenous protein for the of that the presenilin fragments These cells normal APP processing and Aβ production that is to that endogenous hamster is we found that levels of human PS2 with the cell PS2 fragments a and 2 the co-expression of human PS1 in these cells the formation of the human PS2 in with the observed in G. C.L. Ratovitski T. F. Price D.L. D.R. Sisodia S.S. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). Therefore, the of the of PS1 and PS2 which on levels which cleaved and cell that both PS1 and PS2 aspartate mutants, we observed a similar these mutant their high co-expression replaces endogenous fragments with mutant are We that the of the of Aβ is the of of exogenous aspartate mutant high of either mutant PS1 or PS2 to the activity of endogenous PS1 and PS2 H. K. Capell A. H. S. Hardy Yu, X. M. K. Citron M. R. B. S. M. T. Iwatsubo T. R. Haass C. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). results have for the mechanism of Aβ has been that Aβ generated in on the that Aβ42 is to B. D. J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The of C99, for in Aβ J. J. L. C. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google that in the presence of proteolytic This the Aβ that then J. J. L. C. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). This mechanism for the residual Aβ observed in PS1-deficient neurons (10.De Strooper B. Saftig P. Craessaerts K. Vanderstichele H. Gundula G. Annaert W. Von Figura K. Van Leuven F. Nature. 1998; 391: 387-390Crossref PubMed Scopus (1560) Google and in PS1 aspartate mutant (11.Wolfe M.S. Xia W. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.J. Nature. 1999; 398: 513-517Crossref PubMed Scopus (1699) Google Scholar). However, are with this we that Aβ production is the Therefore, we conclude that the residual Aβ seen in PS1-deficient cells is a of the functional PS2 heterodimeric that in these cells. that is to and at either of the presenilins in the activity of their TM to Aβ We that levels of Aβ and the formation of Aβ and in Alzheimer's disease.