Title: RNA Interference-mediated Silencing of X11α and X11β Attenuates Amyloid β-Protein Levels via Differential Effects on β-Amyloid Precursor Protein Processing
Abstract: Processing of the β-amyloid precursor protein (APP) plays a key role in Alzheimer disease neuropathogenesis. APP is cleaved by β- and α-secretase to produce APP-C99 and APP-C83, which are further cleaved by γ-secretase to produce amyloid β-protein (Aβ) and p3, respectively. APP adaptor proteins with phosphotyrosine-binding domains, including X11α (MINT1, encoded by gene APBA1) and X11β (MINT2, encoded by gene APBA2), can bind to the conserved YENPTY motif in the APP C terminus. Overexpression of X11α and X11β alters APP processing and Aβ production. Here, for the first time, we have described the effects of RNA interference (RNAi) silencing of X11α and X11β expression on APP processing and Aβ production. RNAi silencing of APBA1 in H4 human neuroglioma cells stably transfected to express either full-length APP or APP-C99 increased APP C-terminal fragment levels and lowered Aβ levels in both cell lines by inhibiting γ-secretase cleavage of APP. RNAi silencing of APBA2 also lowered Aβ levels, but apparently not via attenuation of γ-secretase cleavage of APP. The notion of attenuating γ-secretase cleavage of APP via the APP adaptor protein X11α is particularly attractive with regard to therapeutic potential given that side effects of γ-secretase inhibition due to impaired proteolysis of other γ-secretase substrates, e.g. Notch, might be avoided. Processing of the β-amyloid precursor protein (APP) plays a key role in Alzheimer disease neuropathogenesis. APP is cleaved by β- and α-secretase to produce APP-C99 and APP-C83, which are further cleaved by γ-secretase to produce amyloid β-protein (Aβ) and p3, respectively. APP adaptor proteins with phosphotyrosine-binding domains, including X11α (MINT1, encoded by gene APBA1) and X11β (MINT2, encoded by gene APBA2), can bind to the conserved YENPTY motif in the APP C terminus. Overexpression of X11α and X11β alters APP processing and Aβ production. Here, for the first time, we have described the effects of RNA interference (RNAi) silencing of X11α and X11β expression on APP processing and Aβ production. RNAi silencing of APBA1 in H4 human neuroglioma cells stably transfected to express either full-length APP or APP-C99 increased APP C-terminal fragment levels and lowered Aβ levels in both cell lines by inhibiting γ-secretase cleavage of APP. RNAi silencing of APBA2 also lowered Aβ levels, but apparently not via attenuation of γ-secretase cleavage of APP. The notion of attenuating γ-secretase cleavage of APP via the APP adaptor protein X11α is particularly attractive with regard to therapeutic potential given that side effects of γ-secretase inhibition due to impaired proteolysis of other γ-secretase substrates, e.g. Notch, might be avoided. The study of amyloidogenic β-amyloid precursor protein (APP) 1The abbreviations used are: APP, β-amyloid precursor protein; Aβ, amyloid β-protein; APPsα, secreted α-secretase cleavage product of APP; APPs, secreted N-terminal ectodomain of APP; RNAi, RNA interference; APP-FL, full-length APP; DAPT, N-(N-(3,5-difluorophenacetyl)-l-alanyl)-S-phenylglycine t-butyl ester; siRNA, small interfering RNA; APP-CTF, APP C-terminal fragment. processing at the gene, protein, and cellular levels has been a major focus of Alzheimer disease neuropathogenesis research since the isolation of the APP gene in 1987 (see reviews in Refs. 1Tanzi R.E. Bertram L. Neuron. 2001; 32: 181-184Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar, 2Selkoe D.J. Physiol. Rev. 2001; 81: 741-766Crossref PubMed Scopus (5191) Google Scholar, 3Sisodia S.S. St. George-Hyslop P.H. Nat. Rev. Neurosci. 2002; 3: 281-290Crossref PubMed Scopus (487) Google Scholar). Genetic, neuropathological, and biochemical findings indicate that excessive production and/or accumulation of the amyloid β-peptide (Aβ) plays a fundamental role in the pathogenesis of Alzheimer disease (see reviews in Refs. 1Tanzi R.E. Bertram L. Neuron. 2001; 32: 181-184Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar, 2Selkoe D.J. Physiol. Rev. 2001; 81: 741-766Crossref PubMed Scopus (5191) Google Scholar, 3Sisodia S.S. St. George-Hyslop P.H. Nat. Rev. Neurosci. 2002; 3: 281-290Crossref PubMed Scopus (487) Google Scholar). Aβ is produced from APP through proteolytic processing by two proteases, β- and γ-secretase. Specifically, APP is first hydrolyzed in the extracellular domain, either between Met671 and Asp672 or between residues 682 and 683, by the aspartyl protease β-site APP-cleaving enzyme or β-secretase, a type I transmembrane glycosylated aspartyl protease found in post-Golgi membranes and at the cell surface (4Vassar R. Bennett B.D. Babu-Khan S. Kahn S. 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Like X11α, X11β also stabilizes cellular APP and diminishes the levels of APPs and Aβ (24McLoughlin D.M. Irving N.G. Brownlees J. Brion J.P. Leroy K. Miller C.C. Eur. J. Neurosci. 1999; 11: 1988-1994Crossref PubMed Scopus (75) Google Scholar, 25Biederer T. Cao X. Sudhof T.C. Liu X. J. Neurosci. 2002; 22: 7340-7351Crossref PubMed Google Scholar). X11α and X11β can interact with presenilin-1 via their PDZ domains (26Lau K.F. McLoughlin D.M. Standen C. Miller C.C. Mol. Cell. Neurosci. 2000; 16: 557-565Crossref PubMed Scopus (81) Google Scholar). Recent studies have shown that overexpression of X11α can impair APP trafficking and may inhibit Aβ production (27King G.D. Perez R.G. Steinhilb M.L. Gaut J.R. Turner R.S. Neuroscience. 2003; 120: 143-154Crossref PubMed Scopus (49) Google Scholar). Furthermore, King et al. (28King G.D. Cherian K. Turner R.S. J. Neurochem. 2004; 88: 971-982Crossref PubMed Scopus (32) Google Scholar) suggested that X11α may specifically interfere with γ-secretase (but not β-secretase)-mediated cleavage of APP and Aβ production. To date, the effects of reduced expression of APBA1 and APBA2 or X11α and X11β on APP processing and Aβ production have not been assessed. For this purpose, we established RNA interference (RNAi) for APBA1 and APBA2 in H4 cells overexpressing either full-length APP (APP-FL) or APP-C99 and evaluated the effects of RNAi-mediated silencing of APBA1 and APBA2 on APP processing and Aβ production. Cell Lines—We used H4 naïve human neuroglioma cells and H4 cells stably transfected to express either APP-FL or APP-C99. APP-C99 is the product of β-secretase and therefore contains α- and γ-cleavage (but not β-cleavage) sites. This cell line provides a valid system to assess whether any effects on APP processing are dependent on γ-secretase-mediated APP processing and independent of β-secretase-mediated APP processing. All cell lines were cultured in high glucose Dulbecco's modified Eagle's medium containing 9% heat-inactivated fetal calf serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mml-glutamine. Stably transfected H4 cells were additionally supplemented with 200 μg/ml G418. RNAi and DAPT Treatment—Small interfering RNA (siRNA) duplexes designed against human APBA1, the gene encoding X11α (5′-GGATGCTCAGCTGATTGCA-3′), and APBA2, the gene encoding X11β (5′-GGTGAAGCTCAACATTGTC-3′), were obtained from Dharmacon, Inc. (Lafayette, CO). Scrambled siRNA (5′-AAATGTGTGTACGTCTCCTCC-3′) (29Luo W.J. Wang H. Li H. Kim B.S. Shah S. Lee H.J. Thinakaran G. Kim T.W. Yu G. Xu H. J. Biol. Chem. 2003; 278: 7850-7854Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar) was used as the control siRNA. siRNAs were transfected into cells by electroporation (Amaxa Inc., Gaithersburg, MD). We mixed 1 million cells, 100 μl of amaxa electroporation transfection solution, and 10 μl of 20 μm siRNA together, and we used the C-9 program in the amaxa electroporation device for cell transfection. The transfected cells were placed in a 6-well plate containing 1.5 ml of cell culture medium. The cells were harvested 48 h after siRNA treatment. The γ-secretase inhibitor DAPT (250 nm, 18-h treatment) was employed in the experiments as a positive control. Cell Lysis and Protein Quantification—Cell pellets were detergent-extracted on ice using 10 mm Tris-HCl (pH 7.4), 150 mm NaCl, 2 mm EDTA, and 0.5% Nonidet P-40 plus protease inhibitors (1 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 μg/ml pepstatin A). The lysates were collected, centrifuged at 12,000 rpm for 10 min, and quantified for total proteins using the BCA protein assay kit (Pierce). Western Blot Analysis of APP Processing—Western blot analysis was performed as described by Xie et al. (30Xie Z. Moir R. Romano D.M. Tesco G. Kovacs D. Tanzi R. Neurodegenerative Dis. 2004; 1: 29-37Crossref PubMed Scopus (30) Google Scholar). Briefly, 40 μg of total protein from each sample was subjected to SDS-PAGE using 4–20% gradient Tris/glycine gels (Invitrogen) under reducing conditions. Next, the proteins were transferred to a polyvinylidene difluoride membrane (BioRad) using a semidry electrotransfer system (Amersham Biosciences). Nonspecific proteins were blocked using 5% nonfat dry milk in Tris-buffered saline/Tween for 1.5 h. Blots were then incubated with a primary antibody, followed by a secondary antibody (horseradish peroxidase-conjugated anti-rabbit antibody, 1:10,000 dilution; Pierce). Blots were washed with 1× Tris-buffered saline/Tween for 30 min between steps. Antibody H-265 (1:200 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used to recognize X11α (105 kDa), and antibody N-20 (1:100 dilution; Santa Cruz Biotechnology, Inc.) was used to detect X11β (135 kDa). Antibody A8717 (1:1000 dilution; Sigma) was used to visualize APP-FL (110 kDa), APP-C83 (12 kDa), and APP-C99 (10 kDa) in Western blot analysis. The intensity of signals was analyzed using the NIH Image Program (Version 1.62). We quantified the Western blots as follows. We used the levels of β-actin to normalize the levels of X11α, X11β, APP-FL, and the APP C-terminal fragment (APP-CTF) (i.e. determining the ratio of the X11α amount to the β-actin amount) to control for loading differences in total protein amounts. We present the changes in the protein levels of X11α, X11β, APP-FL, APP-C99, and APP-C83 in the cells treated with APBA1 or APBA2 siRNA as a percentage of those in the cells treated with control siRNA. Quantitation of Aβ Using Sandwich Enzyme-linked Immunosorbent Assay—Following treatment with saline, electroporation, and treatment with siRNA (control, APBA1, or APBA2), the conditioned medium was collected, and secreted Aβ was measured by a sandwich enzyme-linked immunosorbent assay as described by Xie et al. (30Xie Z. Moir R. Romano D.M. Tesco G. Kovacs D. Tanzi R. Neurodegenerative Dis. 2004; 1: 29-37Crossref PubMed Scopus (30) Google Scholar). Briefly, 96-well plates were coated with mouse monoclonal antibody specific to Aβ40 (antibody 266) or Aβ42 (antibody 21F12). Following blocking with bovine serum albumin, the wells were incubated overnight at 4 °C with test samples of the conditioned cell culture medium, and horseradish peroxidase-conjugated anti-Aβ antibody HR1 was added. The plates were then developed with tetramethylbenzidine reagent, and absorbance was measured at 450 nm. Aβ levels in test samples were determined by comparison with the signal from unconditioned medium supplemented with known quantities of Aβ40 and Aβ42. Statistics—Analysis of variance with repeated measurements was employed to compare the difference from the control group. p < 0.05 was considered statistically significant. APBA1 RNAi Increases APP-CTF Levels and Decreases Aβ Levels in APP-FL-overexpressing H4 Cells—We first established conditions under which APBA1 siRNA treatment would successfully reduce X11α protein levels in H4 cells overexpressing APP-FL (H4-APP-FL cells). The cells were harvested 48 h after transfection with either control or APBA1 siRNA and then subjected to Western blot analyses in which antibody H-265 was used to visualize the protein levels of X11α. As shown in Fig. 1A, X11α immunoblotting revealed a visible reduction in the protein levels of X11α following APBA1 siRNA treatment (lanes 4–6) compared with control siRNA treatment (lanes 1–3). There was no significant difference in the amount of β-actin in control siRNA- or APBA1 siRNA-treated cells. We then quantified all Western blots using the NIH Image program. As shown in Fig. 1B, APBA1 siRNA treatment significantly reduced X11α protein levels by 61% (normalized to β-actin) compared with control siRNA treatment. These results indicated that RNAi for APBA1 significantly knocked down the protein levels of X11α. We next assessed the effects of RNAi-mediated silencing of APBA1 on APP processing in H4-APP-FL cells by measuring the protein levels of APP-FL, APP-C99, and APP-C83 following APBA1 siRNA treatment. 48 h after transfection of APBA1 or control siRNA, the cells were harvested and subjected to Western blot analyses in which antibody A8717 was used to detect APP-FL, APP-C99, and APP-C83. The protein levels of APP-C99 and APP-C83 were increased in the cells treated with APBA1 siRNA (Fig. 1A, lanes 4–6) compared with those treated with control siRNA (lanes 1–3). As a positive control, the γ-secretase inhibitor DAPT was employed to induce the accumulation of APP-C99 and APP-C83 (lanes 7–9) compared with the control treatment (lanes 1–3). To confirm bands corresponding to APP-C99 and APP-C83, synthetic forms of APP-C99 and APP-C83 were employed as markers. No significant difference in the protein levels of APP-FL was observed in APBA1 siRNA-, control siRNA-, or DAPT-treated cells. We also assessed the protein levels of APP-FL, APP-C99, and APP-C83 in H4 naïve cells and found that they were less than those in H4-APP-FL cells. There was no significant difference in the amount of β-actin in control siRNA-, APBA1 siRNA-, or DAPT-treated H4-APP-FL cells and H4 naïve cells. Quantification of APP-FL, APP-C99, and APP-C83 (normalized to β-actin) revealed that APBA1 siRNA treatment led to a 270% increase in the ratio of APP-C99 to APP-FL (p < 0.05) (Fig. 1C) and a somewhat smaller increase (205%) in the ratio of APP-C83 to APP-FL (p < 0.05) (Fig. 1D) compared with control siRNA treatment. Used as a control, the γ-secretase inhibitor DAPT led to 310% (p < 0.05) (Fig. 1C) and 250% (p < 0.05) (Fig. 1D) increases in the ratios of APP-C99 and APP-C83 to APP-FL, respectively. We next measured Aβ levels in the conditioned medium 48 h after treatment with control siRNA, APBA1 siRNA, or DAPT. Because Aβ42 was too low to be detected in many samples, we present only the changes in Aβ40 production from these experiments. As shown in Fig. 1E, both APBA1 siRNA (black bar) and DAPT (gray bar) treatment decreased Aβ levels to a similar extent compared with control siRNA treatment (white bar): 75 pg/ml (APBA1 siRNA) and 70 pg/ml (DAPT) versus 116 pg/ml (control siRNA). Collectively, these data indicated that RNAi silencing of APBA1 affected APP processing and Aβ production in a manner similar to that of DAPT. APBA2 RNAi Decreases Aβ Levels but Does Not Alter APP Processing in H4-APP-FL Cells—We next investigated whether the other X11 family protein, X11β (encoded by gene APBA2), can similarly affect APP processing and Aβ production in H4-APP-FL cells. For this purpose, we established APBA2 RNAi in H4-APP-FL cells. 48 h after transfection of H4-APP-FL cells with APBA2 or control siRNA, the cells were harvested and subjected to Western blot analyses in which antibody N-20 was used to detect the protein levels of X11β. As shown in Fig. 2A, X11β immunoblotting revealed a visible decrease in the protein levels of X11β in the cells treated with APBA2 siRNA (lanes 4–6) compared with those treated with control siRNA (lanes 1–3). Quantification of the Western blots (normalized to β-actin) revealed that APBA2 siRNA treatment decreased the protein levels of X11β by 46% (p < 0.05) (Fig. 2B) compared with control siRNA treatment. We then assessed the effects of RNAi-mediated silencing of APBA2 on APP processing in H4-APP-FL cells. 48 h after transfection with either APBA2 or control siRNA, the cells were harvested and subjected to Western blot analyses. As shown in Fig. 2A, immunoblotting with antibody A8717 revealed no significant difference in the protein levels of APP-FL in APBA2 siRNA-treated (lanes 4–6), control siRNA-treated (lanes 1–3), or DAPT-treated (lanes 7–9) cells. In addition, APBA2 siRNA treatment (lanes 4–6) did not alter the protein levels of APP-C99 or APP-C83, whereas DAPT treatment (lanes 7–9), used as a positive control in the experiment, increased the protein levels of both APP-C99 and APP-C83 compared with control siRNA treatment (lanes 1–3). We also assessed the protein levels of APP-FL, APP-C99, and APP-C83 in H4 naïve cells and found that they were less than those in H4-APP-FL cells. Quantification of the protein levels of APP-FL, APP-C99, and APP-C83 in the Western blot revealed that DAPT treatment (gray bars) led to 270% (p < 0.05) (Fig. 2C) and 245% (p < 0.05) (Fig. 2D) increases in the ratios of APP-C99 and APP-C83 to APP-FL, respectively, compared with control treatment (white bars). However, APBA2 siRNA treatment (Fig. 2C, Fig. 2D, black bar) did not alter the ratios of APP-C99 and APP-C83 to APP-FL compared with control siRNA treatment. We next measured Aβ levels in the conditioned medium 48 h following treatment with control siRNA, APBA2 siRNA, or DAPT. As shown in Fig. 2E, both APBA2 siRNA (black bar) and DAPT (gray bar) treatment significantly decreased Aβ production compared with control siRNA treatment (white bar): 68 pg/ml (APBA2 siRNA) and 48 pg/ml (DAPT) versus 109 pg/ml (control siRNA) (p < 0.05). Taken together, these findings indicated that, in contrast to APBA1 siRNA treatment, APBA2 siRNA treatment did not alter APP processing. However, APBA2 siRNA treatment also decreased Aβ production in H4-APP-FL cells. As discussed above, β-secretase cleaves APP-FL to produce APP-C99, and then γ-secretase cleaves APP-C99 to produce Aβ. Therefore, the changes in APP processing and Aβ production following treatment with APBA1 and APBA2 siRNAs could conceivably be due to alterations in either β-secretase and/or γ-secretase activity. In the following experiments, we set out to determine whether the changes in APP processing and Aβ production following treatment with APBA1 or APBA2 siRNA were independent of β-secretase-mediated APP processing and dependent on γ-secretase-mediated APP processing, employing H4 cells overexpressing APP-C99 (H4-APP-C99 cells). APBA1 RNAi Increases APP-CTF Levels and Decreases Aβ Levels in H4-APP-C99 Cells—To eliminate increased β-secretase cleavage of APP-FL as a possible explanation for increased APP-C99, we employed H4-APP-C99 cells. APP-C99 is the product of β-secretase and harbors α- and γ-cleavage (but not β-cleavage) sites. 48 h after transfection of H4-APP-C99 cells with APBA1 or control siRNA, the cells were harvested and subjected to Western blot analyses in which antibody H-265 was used to detect X11α. As shown in Fig. 3A, X11α immunoblotting revealed a visible reduction in the protein levels of X11α (encoded by gene APBA1) in the cells treated with APBA1 siRNA (lanes 5–7) compared with those treated with control siRNA (lanes 2–4). There was no significant difference in the amount of β-actin in control siRNA- or APBA1 siRNA-treated cells. Quantification of X11α in the Western blot (normalized to β-actin) showed that APBA1 siRNA treatment decreased the protein levels of X11α by 50% (p < 0.05) (Fig. 3B). We next assessed the effects of RNAi-mediated silencing of APBA1 on processing of APP-C99. 48 h after transfection with APBA1 or control siRNA, the cells were harvested and subjected to Western blot analyses with antibody A8717. APBA1 siRNA treatment (Fig. 3A, lanes 5–7) did not alter the protein levels of APP-FL compared with control siRNA treatment (lanes 2–4). APP-CTF immunoblotting revealed visible increases in the protein levels of both APP-C99 and APP-C83 in the H4-APP-C99 cells treated with APBA1 siRNA (lanes 5–7) compared with those treated with control siRNA (lanes 2–4). We also assessed the protein levels of APP-C99 and APP-C83 in H4 naïve cells and found that they were less than those in H4-APP-C99 cells. There was no significant difference in the amount of β-actin in control siRNA- or APBA1 siRNA-treated H4-APP-C99 cells and H4 naïve cells. Quantification of APP-FL, APP-C99, and APP-C83 revealed that APBA1 siRNA treatment led to a 224% increase in the ratio of APP-C99 to APP-FL (p < 0.05) (Fig. 3C) and a 273% increase in the ratio of APP-C83 to APP-FL (p < 0.05) (Fig. 3D) compared with control siRNA treatment. We then measured Aβ levels in the conditioned medium from H4-APP-C99 cells 48 h after treatment with either control or APBA1 siRNA. As shown in Fig. 3E, APBA1 siRNA decreased Aβ production compared with control siRNA treatment: 110 pg/ml (APBA1 siRNA) versus 164 pg/ml (control siRNA) (p < 0.05). These findings suggest that APBA1 RNAi inhibits γ-secretase (but not β-secretase)-mediated cleavage of APP. APBA2 RNAi Does Not Alter APP Processing or Aβ Levels in H4-APP-C99 Cells—Finally, we assessed the effects of APBA2 RNAi on APP processing and Aβ production in H4-APP-C99 cells. 48 h after transfection with APBA2 or control siRNA, the cells were harvested and subjected to Western blot analyses in which antibody N-20 was used to detect the protein levels of X11β. X11β immunoblotting revealed a visible reduction in the protein levels of X11β (encoded by gene APBA2) in the cells treated with APBA2 siRNA (Fig. 4A, lanes 6–8) compared with those treated with control siRNA (lanes 3–5). There was no significant difference in the amount of β-actin in control siRNA- or APBA2 siRNA-treated cells. Quantification of X11β (normalized to β-actin) revealed that APBA2 siRNA treatment decreased the protein levels of X11β by 86% (p < 0.05) (Fig. 4B) compared with control siRNA treatment. Immunoblotting with anti-APP antibody A8717 revealed no detectable difference in the protein levels of APP-FL, APP-C99, and APP-C83 in the cells treated with APBA2 siRNA (Fig. 4A, lanes 6–8) compared with those treated with control siRNA (lanes 3–5). We also assessed the protein levels of APP-C99 and APP-C83 in H4 naïve cells and found that they were less than those in H4-APP-C99 cells. There was no significant difference in the amount of β-actin in control siRNA- or APBA2 siRNA-treated H4-APP-C99 cells and H4 naïve cells. Quantification of APP-FL, APP-C99, and APP-C83 revealed that APBA2 siRNA treatment did not significantly alter the ratios of APP-C99 (Fig. 4C) and APP-C83 (Fig. 4D) to APP-FL. We next measured Aβ levels in the conditioned medium from H4-APP-C99 cells 48 h after treatment with control or APBA2 siRNA. As shown in Fig. 4E, APBA2 siRNA treatment did not significantly alter Aβ production compared with control siRNA treatment: 173 pg/ml (control siRNA) versus 167 pg/ml (APBA2 siRNA). Collectively, these results suggest that, in contrast to APBA1 RNAi, APBA2 RNAi does not alter APP processing or Aβ production in H4-APP-C99 cells. Control siRNA or Electroporation Does Not Affect APP Processing or Aβ Levels in H4-APP-FL and H4-APP-C99 Cells—In a control experiment, we also assessed the effects of control siRNA or electroporation on APP processing and Aβ levels in H4-APP-FL and H4-APP-C99 cells. We found that control siRNA or electroporation did not affect APP processing or alter Aβ levels compared with saline treatment in H4-APP-FL and H4-APP-C99 cells (data not shown). These results confirmed that the effects of APBA1 or APBA2 RNAi on APP processing and Aβ levels in our experiments were not due to electroporation or control siRNA (scrambled siRNA), but to the reduction in the protein levels of X11α and X11β. Aβ, the key component in senile plaques, is derived from APP via cleavage by two proteases, β- and γ-secretase (4Vassar 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: 735-741Crossref PubMed Scopus (3327) Google Scholar, 5Hussain I. Powell D. Howlett D.R. Tew D.G. Meek T.D. Chapman C. Gloger I.S. Murphy K.E. Southan C.D. Ryan D.M. Smith T.S. Simmons D.L. Walsh F.S. Dingwall C. Christie G. Mol. Cell. Neurosci. 1999; 14: 419-427Crossr