Potential Link between Amyloid β-Protein 42 and C-terminal Fragment γ 49–99 of β-Amyloid Precursor Protein

A novel cleavage of β-amyloid precursor protein (APP), referred to as ϵ-cleavage, occurs downstream of the γ-cleavage and generates predominantly a C-terminal fragment (CTFγ) that begins at Val-50, according to amyloid β-protein (Aβ) numbering. Whether this cleavage occurs independently of, or is coordinated with, γ-cleavage is unknown. Using a cell-free system, we show here that, although Aβ40 and CTFγ 50–99 were the predominant species produced by membranes prepared from cells overexpressing wild-type (wt) APP and wt presenilin (PS) 1 or 2, the production of CTFγ 49–99, which begins at Leu-49, was remarkably enhanced in membranes from cells overexpressing mutant (mt) APP or mtPS1/2 that increases the production of Aβ42. Furthermore, a γ-secretase inhibitor, which suppresses Aβ40 production and paradoxically enhances Aβ42 production at low concentrations, caused the proportion of CTFγ 50–99 to decrease and that of CTFγ 49–99 to increase significantly. These results strongly suggest a link between the production of Aβ42 and CTFγ 49–99 and provide an important insight into the mechanisms of altered γ-cleavage caused by mtAPP and mtPS1/2. A novel cleavage of β-amyloid precursor protein (APP), referred to as ϵ-cleavage, occurs downstream of the γ-cleavage and generates predominantly a C-terminal fragment (CTFγ) that begins at Val-50, according to amyloid β-protein (Aβ) numbering. Whether this cleavage occurs independently of, or is coordinated with, γ-cleavage is unknown. Using a cell-free system, we show here that, although Aβ40 and CTFγ 50–99 were the predominant species produced by membranes prepared from cells overexpressing wild-type (wt) APP and wt presenilin (PS) 1 or 2, the production of CTFγ 49–99, which begins at Leu-49, was remarkably enhanced in membranes from cells overexpressing mutant (mt) APP or mtPS1/2 that increases the production of Aβ42. Furthermore, a γ-secretase inhibitor, which suppresses Aβ40 production and paradoxically enhances Aβ42 production at low concentrations, caused the proportion of CTFγ 50–99 to decrease and that of CTFγ 49–99 to increase significantly. These results strongly suggest a link between the production of Aβ42 and CTFγ 49–99 and provide an important insight into the mechanisms of altered γ-cleavage caused by mtAPP and mtPS1/2. Senile plaques, one of the neuropathological hallmarks of Alzheimer's disease (AD), 1The abbreviations used are: AD, Alzheimer's disease; Aβ, amyloid β-protein; APP, β-amyloid precursor protein; PS, presenilin; CTF, carboxyl-terminal fragment; CHO, Chinese hamster ovary; HEK, human embryonic kidney; wt, wild type; mt, mutant; RP-HPLC, reverse-phase high performance liquid chromatography; Pipes, 1,4-piperazinediethanesulfonic acid. 1The abbreviations used are: AD, Alzheimer's disease; Aβ, amyloid β-protein; APP, β-amyloid precursor protein; PS, presenilin; CTF, carboxyl-terminal fragment; CHO, Chinese hamster ovary; HEK, human embryonic kidney; wt, wild type; mt, mutant; RP-HPLC, reverse-phase high performance liquid chromatography; Pipes, 1,4-piperazinediethanesulfonic acid. are composed primarily of amyloid β-protein (Aβ) (1Selkoe D.J. Physiol. Rev. 2001; 81: 741-766Google Scholar). Two major Aβ species consisting of 40 and 42 residues are generated mainly in neurons and constitutively secreted. A shorter one, Aβ40, is predominant, and a longer one, Aβ42, is a minor species (<10%) among secreted Aβ species. Aβ is produced from β-amyloid precursor protein (APP), through sequential cleavage by proteases referred to as β- and γ-secretases. β-Secretase was identified as a type I membrane aspartic protease β-site APP-cleaving enzyme (BACE) (2Vassar R. Bennett B.D. Babu-Khan S. Kahn S. Mendiaz E.A. Denis P. Teplow D.B. Ross S. Amarante P. Loeloff R. Luo Y. Fisher S. Fuller J. Edenson S. Lile J. Jarosinski M.A. Biere A.L. Curran E. Burgess T. Louis J.C. Collins F. Treanor J. Rogers G. Citron M. Science. 1999; 286: 735-741Google Scholar), but the identity of γ-secretase has remained unknown. γ-Secretase cleaves APP in the middle of the transmembrane domain, releasing Aβ and its counterpart, C-terminal fragment γ of APP (CTFγ). Most recent studies have shown that γ-secretase forms a large complex composed of presenilin (PS) 1 or 2, nicastrin, PEN-2, and APH-1, and the activity of γ-secretase is now known to depend on these proteins (3Steiner H. Haass C. Nat. Rev. Mol. Cell. Biol. 2000; 1: 217-224Google Scholar, 4Yu 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: 48-54Google Scholar, 5Francis R. McGrath G. Zhang J. Ruddy D.A. Sym M. Apfeld J. Nicoll M. Maxwell M. Hai B. Ellis M.C. Parks A.L. Xu W. Li J. Gurney M. Myers R.L. Himes C.S. Hiebsch R. Ruble C. Nye J.S. Curtis D. Dev. Cell. 2002; 3: 85-97Google Scholar, 6Steiner H. Winkler E. Edbauer D. Prokop S. Basset G. Yamasaki A. Kostka M. Haass C. J. Biol. Chem. 2002; 277: 39062-39065Google Scholar, 7Lee S.F. Shah S. Li H. Yu C. Han W. Yu G. J. Biol. Chem. 2002; 277: 45013-45019Google Scholar). One of the Aβ species, Aβ42, has a much higher aggregation potential (8Hilbich C. Kisters-Woike B. Reed J. Masters C.L. Beyreuther K. J. Mol. Biol. 1991; 218: 149-163Google Scholar, 9Burdick D. Soreghan B. Kwon M. Kosmoski J. Knauer M. Henschen A. Yates J. Cotman C. Glabe C. J. Biol. Chem. 1992; 267: 546-554Google Scholar) and is believed to be initially deposited in senile plaques (10Iwatsubo T. Odaka A. Suzuki N. Mizusawa H. Nukina N. Ihara Y. Neuron. 1994; 13: 45-53Google Scholar). It is reasonable to postulate that Aβ42 accumulation in the brain is the very initial event in the development of AD including sporadic AD. Indeed, all mutations of PS1/2 and some mutations of APP that cause familial AD result in increased Aβ42 production (11Sisodia S.S. St. George-Hyslop P.H. Nat. Rev. Neurosci. 2002; 3: 281-290Google Scholar). Recently, we and other groups found that APP is cleaved by PS-dependent γ-secretase, not only in the middle of the transmembrane domain (γ-cleavage) but also near the cytoplasmic membrane boundary (ϵ-cleavage) (12Gu Y. Misonou H. Sato T. Dohmae N. Takio K. Ihara Y. J. Biol. Chem. 2001; 276: 35235-35238Google Scholar, 13Sastre M. Steiner H. Fuchs K. Capell A. Multhaup G. Condron M.M. Teplow D.B. Haass C. EMBO Rep. 2001; 2: 835-841Google Scholar, 14Yu C. Kim S.H. Ikeuchi T. Xu H. Gasparini L. Wang R. Sisodia S.S. J. Biol. Chem. 2001; 276: 43756-43760Google Scholar, 15Weidemann A. Eggert S. Reinhard F.B. Vogel M. Paliga K. Baier G. Masters C.L. Beyreuther K. Evin G. Biochemistry. 2002; 41: 2825-2835Google Scholar). The major product of the latter process is a CTFγ of APP that begins at Val-50. This cleavage site is a few residues inside the membrane from the cytoplasmic/membrane boundary and is similar to site 3 cleavage of Notch (16Schroeter E.H. Kisslinger J.A. Kopan R. Nature. 1998; 393: 382-386Google Scholar). Since production of CTFγ is inhibited by a dominant negative mutant of PS1 (17Wolfe M.S. Xia W. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.J. Nature. 1999; 398: 513-517Google Scholar), ϵ-cleavage is PS-dependent as well as γ-cleavage (13Sastre M. Steiner H. Fuchs K. Capell A. Multhaup G. Condron M.M. Teplow D.B. Haass C. EMBO Rep. 2001; 2: 835-841Google Scholar, 14Yu C. Kim S.H. Ikeuchi T. Xu H. Gasparini L. Wang R. Sisodia S.S. J. Biol. Chem. 2001; 276: 43756-43760Google Scholar, 15Weidemann A. Eggert S. Reinhard F.B. Vogel M. Paliga K. Baier G. Masters C.L. Beyreuther K. Evin G. Biochemistry. 2002; 41: 2825-2835Google Scholar). Furthermore, ϵ-cleavage is also inhibited with γ-secretase inhibitors, which are known to selectively bind to PS1/2 (12Gu Y. Misonou H. Sato T. Dohmae N. Takio K. Ihara Y. J. Biol. Chem. 2001; 276: 35235-35238Google Scholar, 13Sastre M. Steiner H. Fuchs K. Capell A. Multhaup G. Condron M.M. Teplow D.B. Haass C. EMBO Rep. 2001; 2: 835-841Google Scholar, 14Yu C. Kim S.H. Ikeuchi T. Xu H. Gasparini L. Wang R. Sisodia S.S. J. Biol. Chem. 2001; 276: 43756-43760Google Scholar, 15Weidemann A. Eggert S. Reinhard F.B. Vogel M. Paliga K. Baier G. Masters C.L. Beyreuther K. Evin G. Biochemistry. 2002; 41: 2825-2835Google Scholar). However, it has remained unknown how ϵ-cleavage relates to the generation of distinct Aβ species or whether this step is essential to generate Aβ. We therefore examined whether there is a link between CTFγ and Aβ production using a cell-free system. Taking advantage of familial AD mutations of APP and PS1/2 and a γ-secretase inhibitor, we show here that, when Aβ40 is predominantly produced, CTFγ 50–99 is the major product of ϵ-cleavage, and when a large amount of Aβ42 is produced, CTFγ 49–99 is predominantly produced. Thus, ϵ-cleavage may be linked to the specificity of γ-cleavage. APP- and PS1/2-overexpressing Cells—Chinese hamster ovary (CHO) cells transfected with cDNA encoding wild-type (wt) APP751 (7WD10 cells) and mutant (mt) APP751 (V717F) were described previously (18Podlisny M.B. Ostaszewski B.L. Squazzo S.L. Koo E.H. Rydell R.E. Teplow D.B. Selkoe D.J. J. Biol. Chem. 1995; 270: 9564-9570Google Scholar). 7WD10 cells were further transfected with cDNA encoding wt, N141I, or D366A PS2 or wt, M146L, M233T, or G384A PS1 as described previously (19Qi Y. Morishima-Kawashima M. Sato T. Mitsumori R. Ihara Y. Biochemistry. 2003; 42: 1042-1052Google Scholar). V717F cells were further transfected with cDNA encoding wt, M233T, or G384A PS1. Human embryonic kidney HEK293 cells were transfected with cDNA encoding wt, V717G, or V717F (according to APP770 numbering) APP695 (20Haass C. Schlossmacher M.G. Hung A.Y. Vigo-Pelfrey C. Mellon A. Ostaszewski B.L. Lieberburg I. Koo E.H. Schenk D. Teplow D.B. Selkoe D.J. Nature. 1992; 359: 322-325Google Scholar). Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and penicillin/streptomycin. Stably transfected CHO cells were further added with G418, zeocin, or puromycin (Invitrogen). Cell-free Assay and Inhibitor Treatment—Confluent cells were harvested and homogenized in homogenization buffer (20 mm Pipes, pH 7.0, 140 mm KCl, 0.25 m sucrose, and 5 mm EGTA). Homogenized cells were centrifuged at 800 × g to remove nuclei and cell debris. The supernatant was further ultracentrifuged at 100,000 × g for 1 h. The resultant pellet, total membrane fraction, was suspended in homogenization buffer. The membrane fraction at a protein concentration of 2.5 mg/ml in homogenization buffer containing protease inhibitor mixture was incubated at 37 °C for the indicated time, and the reaction was stopped by placing the reaction mixture on ice. After extraction of lipids twice with chloroform/methanol (2:1) and chloroform/methanol/water (1:2:0.8), the residue was extracted with 70% formic acid, and the extract was dried. The remaining proteins were dissolved with the SDS sample buffer containing 9 m urea and subjected to 16.5% SDS-PAGE, followed by Western blotting using antibodies BA27 (specific for Aβ40), BC05 (specific for Aβ42), 6E10 (raised against Aβ1–16, and appropriate for assessing total Aβ; Senetek PLC, Maryland Heights, MO), and C4 (raised against the 30 C-terminal residues of APP). Aβ40/42 and CTFγ were quantified by a LAS-1000plus luminescent image analyzer (Fuji Film, Tokyo, Japan) using defined amounts of authentic Aβ (Bachem, Bubendorf, Switzerland) as a standard. DFK-167 was purchased from Enzyme Systems Products (Livermore, CA) and dissolved in Me2SO. Mass Spectrometric and Amino Acid Sequence Analyses of CTFγ— Membrane fractions prepared from 80 dishes of cultured cells were incubated at 37 °C for 30 min and then ultracentrifuged at 100,000 × g for 1 h to separate the soluble and membrane-bound CTFγ. The membrane-bound fraction was suspended and homogenized in 1% Triton X-100, which completely extracted CTFγ (data not shown). Solubilized CTFγ was immunoprecipitated once with 4G8 (epitope: Aβ17–24; Senetek PLC, Maryland Heights, MO) to remove the other C-terminal fragments of APP (CTFα and CTFβ), and then CTFγ was immunoprecipitated with C4, extracted with 70% formic acid, and dried by vacuum centrifugation. The partially purified samples were subjected to gel filtration on two tandemly arrayed TSK-gel Super SW2000 columns (Tosoh, Tokyo, Japan), which were developed with 6 m guanidine hydrochloride in 10 mm phosphate buffer (pH 6.0). Pooled putative CTFγ fractions were further purified by reverse-phase high performance liquid chromatography (RP-HPLC) on CAPCELL PAK Phenyl SG300 (Shiseido, Tokyo, Japan). The fractions corresponding to peaks 1 and 2 were subjected to an Applied Biosystems model 494 cLC or model 492 protein sequencer. Both CTFγ 50–99 and CTFγ 49–99 were quantified from the yields of Val-50 at the first and second cycles, Mass was by on the CTFγ was extracted with 1% acid, of the were in a using as a bovine was used as an standard. of Aβ42 and CTFγ 49–99 in from CHO Cells mtAPP or whether there is between Aβ40/42 and we used a cell-free Aβ or CTFγ production consisting of membranes prepared from membranes prepared from or cells were Aβ40 was a predominant species produced when the incubated membranes were from mtAPP (V717F) and the proportion of Aβ42 was increased and predominantly the membranes also produced amounts of a of Aβ from the cells overexpressing a dominant negative mutant of D366A W.T. Xia W. T. M.S. Selkoe D.J. J. Biol. Chem. 2000; Scholar), produced amounts of Aβ40/42 and a amount of CTFγ A and This mutant large amounts of the for γ-secretase A and Thus, this cell-free Aβ production the Aβ in in of cell (data not shown). Cell-free production of Aβ and CTFγ very similar with min (19Qi Y. Morishima-Kawashima M. Sato T. Mitsumori R. Ihara Y. Biochemistry. 2003; 42: 1042-1052Google Scholar) (data not shown). A γ-secretase inhibitor, DFK-167 M.S. Citron M. Diehl T.S. Xia W. Selkoe D.J. J. Chem. 1998; 41: Scholar), Aβ40 production but paradoxically enhanced Aβ42 production at low in the membranes from as previously M.S. Citron M. Diehl T.S. Xia W. Selkoe D.J. J. Chem. 1998; 41: Scholar, L. Song L. G. Y. B. E. Biochemistry. 2001; Scholar) and We whether production of Aβ40 and Aβ42 is with species of CTFγ. The generated CTFγ was into soluble and membrane-bound A proportion of CTFγ was into soluble fraction and the remaining proportion of CTFγ was membrane-bound a first step to of with C4 was CTFγ immunoprecipitated from the soluble and membrane-bound fractions was subjected to species of CTFγ were identified in of the membranes the results of which were with (12Gu Y. Misonou H. Sato T. Dohmae N. Takio K. Ihara Y. J. Biol. Chem. 2001; 276: 35235-35238Google Scholar). The in and membranes CTFγ CTFγ 49–99 was a minor in and it a major in mtAPP (V717F) and This the that the of Aβ40 production is to that of CTFγ 50–99 and that of Aβ42 production is to that of CTFγ 49–99 of soluble and membrane-bound CTFγ 49–99 and in a of Aβ42 and CTFγ 49–99 in from CHO Cells further increased production of CTFγ 49–99, we examined membrane fractions prepared from wt or M233T, and for membranes produced Aβ40 a that is similar to that of and membranes 2, A and the other membranes produced amounts of Aβ42, although the of Aβ42 to Aβ40 among mutations produced CTFγ was immunoprecipitated and subjected to the the was CTFγ in the the peaks for CTFγ 49–99 were found to be and G384A membranes in which Aβ42 was produced predominantly the peaks for CTFγ 49–99 were much higher in the for CTFγ 49–99 was at the as that in and the Aβ42 production was only increased from HEK293 Cells APP an of Aβ42 and CTFγ 49–99 whether these were to an of CHO we examined the membrane fractions from wt, V717G, and V717F HEK293 shown in A and Aβ40 was predominantly produced in the membranes from the proportion of Aβ42 production was increased in membranes from and V717F The CTFγ immunoprecipitated from membranes of HEK293 cells was by The major species was CTFγ 50–99 in the membrane of cells the other the major peaks in the membranes from and HEK293 cells were CTFγ 49–99, which is similar to CHO cells all cell of HEK293 cells to amounts of CTFγ as with CHO an that with C. Kim S.H. Ikeuchi T. Xu H. Gasparini L. Wang R. Sisodia S.S. J. Biol. Chem. 2001; 276: 43756-43760Google Scholar, F. Y. H. Arawaka S. Fraser P. D. H. St. George-Hyslop P. J. Biol. Chem. 2002; 277: Scholar). these it is that, although ϵ-cleavage may show some among cell CTFγ 50–99 is a predominant species in and membranes in which Aβ40 was predominantly produced, and CTFγ 49–99 production is increased in mtAPP and mtPS1/2 in which Aβ42 production was of CTFγ Using an Amino Acid for CTFγ species on may not be Thus, we to species of CTFγ produced using an sequencer. The CTFγ species produced in membranes from cell as shown in 1 were CTFγ was by gel followed by The fractions corresponding to peaks 1 and 2 were subjected to species of CTFγ were identified 1 species and 50–99 and a amount of 2 two species 49–99 and CTFγ 50–99 and 49–99 for of CTFγ in cell and the remaining species and were at but species CTFγ were by The proportion of CTFγ for cell is in I. The of CTFγ 49–99 were and for and I and Both membranes produced Aβ40 predominantly the of CTFγ 49–99 were increased to and for the membranes of mtAPP (V717F) and of which produced increased amounts of Aβ42 These were with the results of that the by is very in the of CTFγ 50–99 at a of further that the increase in the proportion of CTFγ 49–99 is of the cell we examined the of an inhibitor that at low inhibited the production of Aβ40 and paradoxically increased Aβ42 production on the proportion of CTFγ species produced by the membranes The of CTFγ 50–99 and CTFγ 49–99 in the soluble fraction of 10 membrane were to from and to from with the proportion of Aβ40 produced to from and Aβ42 produced to from and These results strongly suggest that Aβ40 and Aβ42 production is to that of CTFγ 50–99 and CTFγ 49–99, of mtAPP and on Aβ and CTFγ increase in CTFγ 49–99 production a decrease in CTFγ 50–99 production was in and whether the of mtAPP and has an on the produced CTFγ species, we mtAPP and wt or or The of Aβ40/42 produced in membranes was the as that in membranes A and the other the proportion of Aβ42 production was increased in membranes and as with This of mtAPP and on Aβ42 production was with the M. Diehl T.S. C. Ostaszewski B.L. Xia W. Levesque G. St. P. Selkoe D.J. 1998; Scholar). the membranes from cells produced much amounts of Aβ and CTFγ and A and It is that this γ-secretase has a cleavage for V717F We CTFγ species produced in these membranes by The major species was CTFγ 49–99 in the to V717F membranes the other the peaks for CTFγ 50–99 were found to be in and these the peaks of CTFγ 50–99 were also remarkably as with membranes and are not we not whether or how much CTFγ 49–99 in these membranes was increased as with that in However, these results strongly suggest that mtAPP and have an on CTFγ species produced. It has that mutations of APP to the of Aβ the cleavage specificity of γ-secretase, to increased Aβ42 we that APP mutations γ-cleavage on ϵ-cleavage that occurs near the cytoplasmic membrane However, as shown in and mtAPP (V717F) a on the ϵ-cleavage and generated a large amount of CTFγ 49–99 This large increase in the production of CTFγ was increased proportion of Aβ42 produced for V717F in CHO cells) is the the of Aβ42 production is not to the of CTFγ generated Aβ42 predominantly the proportion of CTFγ 49–99 was and similar results be among other mutations of PS1 Furthermore, the of 10 DFK-167 increased production of Aβ42 by an that of CTFγ 49–99 Thus, the proportion of Aβ40 and Aβ42 not that of CTFγ 50–99 and to CHO HEK293 cell membranes produced a amount of CTFγ CTFγ was increased in mtAPP in CHO CTFγ to be very increased in the membranes from mtAPP and mtPS1/2 cells and Thus, the production of CTFγ may also be to that of Aβ42, although further is to this A of recent have for a between γ-cleavage and Notch site 3 PS1 mutations that increase the production of Aβ42 were found to Notch site 3 cleavage F. Y. H. Arawaka S. Fraser P. D. H. St. George-Hyslop P. J. Biol. Chem. 2002; 277: Scholar, D. D. G. Sisodia S.S. I. S. A. Scholar, D.M. Levitan D. Yu G. Nishimura M. Chen F. Tandon A. Kawarai T. Arawaka S. Supala A. Song Y.Q. Rogaeva E. Liang Y. Holmes E. Milman P. Sato C. Zhang L. St. George-Hyslop P. 2000; Scholar, L. J. Multhaup G. Teplow D.B. R. H. Capell A. Steiner H. Haass C. S. A. 2000; Scholar, T. Winkler E. Xia Edbauer D. J. Capell A. C. H. B. Haass C. Steiner H. S. A. 2002; Scholar) and production of CTFγ as well F. Y. H. Arawaka S. Fraser P. D. H. St. George-Hyslop P. J. Biol. Chem. 2002; 277: Scholar, T. Winkler E. Xia Edbauer D. J. Capell A. C. H. B. Haass C. Steiner H. S. A. 2002; Scholar). These results are with the on that the total amounts of CTFγ produced in the membranes are to of of membranes Thus, mtPS1/2 not only a in the of ϵ-cleavage but also an in the proportion of CTFγ species an increase of This with which not the of ϵ-cleavage F. Y. H. Arawaka S. Fraser P. D. H. St. George-Hyslop P. J. Biol. Chem. 2002; 277: Scholar) but an large amount of CTFγ 49–99 and It is therefore reasonable to that this the mechanisms for increased Aβ42 production by mtAPP and mtPS1/2 The of mtPS1/2 and mtAPP further the between and identified of γ-secretase are cleaved at or near the cytoplasmic membrane and some are also in the middle of the transmembrane domain (16Schroeter E.H. Kisslinger J.A. Kopan R. Nature. 1998; 393: 382-386Google Scholar, I. Y. D. T. N. T. H. J. Biol. 2001; Scholar, P. J. G. A. S. L. P. S. T. EMBO J. 2002; Scholar, S. M. M. C. Capell A. Edbauer D. J. Steiner H. Haass C. J. Biol. Chem. 2002; 277: Scholar, M. Steiner H. A. H. T. T. T. T. M. Haass C. EMBO J. 2002; Scholar). This that γ-cleavage and ϵ-cleavage are in a of type I membrane a potential link between Aβ42 and CTFγ 49–99 further or ϵ-cleavage, and how one cleavage the We to a CTFγ longer CTFγ by or One is that is first cleaved at the and cleaved and and Aβ40/42 are secreted. studies the of Aβ by J. 2000; Scholar, A. A. E.A. L.S. Nature. 2001; Scholar). However, in longer Aβ species were although we the that the of in the are the It is also that and ϵ-cleavage or the a membrane that be to and of for or ϵ-cleavage to a and then to a of the between two of