1. Volume 3, Issue 1, Pages 42-51 (January 2013)
γ-Secretase Modulators and Presenilin 1 Mutants Act Differently on Presenilin/γ- Secretase Function to Cleave Aβ42 and Aβ43 
Masayasu Okochi, Shinji Tagami, Kanta Yanagida, Mako Takami, Takashi S. Kodama, Kohji Mori, Taisuke Nakayama, Yasuo Ihara, Masatoshi Takeda 
Cell Reports 
Volume 3, Issue 1, Pages (January 2013)
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2. Cell Reports 2013 3, 42-51DOI: (10.1016/j.celrep.2012.11.028)
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3. Figure 1 Detection of Aβ and Related Small Peptide Species
Aβ42 and βAPP-CTF cleavage assays were performed at 37°C for 30 min.
(A) Immunoprecipitation-MS analysis of products from the Aβ42 cleavage assay.
(B) Small Aβ-derived peptides (Takami et al., 2009) detected in the Aβ42 cleavage assay.
(C) Levels of total Aβ from βAPP-CTF (black line) and Aβ38 generated from Aβ42 (red line) at various CHAPSO concentrations (0%–1.5%).
(D) Immunoprecipitation-immunoblot detection of Aβ. The volume of each reaction for immunoprecipitation was adjusted to contain the same amount of total Aβ.
(E) Relative rate of the VVIA produced in (D), as defined by [VVIA]/[TVI]. The actual individual data from each of the three experiments are plotted.
See also Figures S1, S2, and S4.
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4. Figure 2
Effects of GSMs/iGSMs and Mutant PS1/γ-Secretases on Aβ42 Cleavage
(A) Fold activation of Aβ42 cleavage by GSMs/iGSMs in the presence of 0.25% CHAPSO. We extracted the PS/γ-secretase fraction from HEK cells stably expressing WT PS1 (Figure S2B). A total of 40 μM GSM1, 10 μM Eisai, 10 μM compound-W (CW), 10 μM SS, 50 μM fenofibrate (FF), or 10 μM S2474 was added to the in vitro assay.
(B) Fold activation of Aβ42 cleavage by GSMs/iGSMs in the presence of 0.5% CHAPSO.
(C) Fold changes of the relative VVIA levels in cell lysates treated with GSMs/iGSM. A total of 4 μM GSM1, 1 μM Eisai, or 30 μM S2474 was added to the cultured medium.
(D) The relative ratio of Aβ species in conditioned medium. The levels of Aβ38, Aβ40, and Aβ42 were measured by ELISA.
(E) Immunoblotting of purified PS/γ-secretase fractions. To show that the same level of each mutant PS1/γ-secretase was used in each reaction, we immunoblotted each fraction with antibodies against all four indispensable elements of PS1/γ-secretase: PS1/2, nicastrin, APh-1-a, and PEN-2. We detected almost equal band densities for all four proteins of mutants and WT PS1/γ-secretase fractions. Exogenous PS1 derivatives displaced the endogenous WT PS2 in the PS/γ-secretase complex. Note that a certain mutant contained a higher level of nicastrin, and thus was omitted from the analysis.
(F) Fold activation of Aβ42 cleavage by purified mutant PS1/γ-secretase.
(G) Fold changes of the relative VVIA levels in cell lysates stably expressing PS1 mutants.
(H) The relative ratio of Aβ species in conditioned medium.
The Aβ38 level was measured by ELISA. Asterisks indicate p < 0.05, Welch’s t test. Error bars represent SD. The actual individual data from each of the three experiments are plotted in (A), (B), and (F). See also Figures S1 and S2.
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5. Figure 3
Enzyme Kinetic Analysis of Aβ42 Cleavage and Biacore Analysis of Aβ42 Dissociation from PS1/γ-Secretase
(A) Proposed reaction mechanism for Aβ42 production.
(B) Proposed formulas of stepwise cleavage for Aβ production.
(C) Hanes-Woolf plots of the Aβ42 cleavage in the presence of 0.25% CHAPSO and GSMs (DMSO, black diamonds; GSM1, green squares; Eisai, blue triangles).
(D) Hanes-Woolf plots of Aβ42 cleavage in the presence of 0.5% CHAPSO and iGSM (DMSO, black diamonds; S2474, red squares).
(E) The relative kcat values (n = 4) of Aβ42 cleavage in the presence of GSMs/iGSM.
(F) Hanes-Woolf plots of Aβ42 cleavage by WT and mutant PS1/γ-secretase (WT PS1, black diamonds; PS1 L286V, red triangles; PS1 L381V, blue squares; PS1 G384A, purple crosses) in the presence of 0.5% CHAPSO.
(G) The relative kcat values of Aβ42 cleavage by WT and mutant PS1/γ-secretase.
(H) Fitted curves of dissociation for DMSO (purple/black), GSM1 (18 μM, green/gray), and S2474 (135 μM, blue/red).
(I) kb values (n = 3 for each) in the presence of GSM/iGSM treatment compared with those obtained in the presence of DMSO treatment.
(J) Fitted curves of dissociation (WT PS1/γ-secretase, blue/black; PS1 L286V/γ-secretase, purple/red; PS1 G384A/γ-secretase, green/gray).
(K) kb values of mutant PS1/γ-secretases relative to those of WT PS1/γ-secretase.
Asterisks indicate p < 0.05, Welch’s t test. Error bars represent SD. See also Figure S3.
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6. Figure 4 Cleavage of Aβ43, Nβ25, and APL1β28 by PS/γ-Secretase
(A) Representative MALDI-TOF MS spectrum from the Aβ43 cleavage assay (0.5% CHAPSO).
(B) Aβ-derived peptides in the Aβ43 cleavage assay. Addition of L685,458 abolished their generation. Note that Aβ43 cleavage produced much smaller levels of IAT and VVIAT than that of VVIA produced by Aβ42 cleavage. This may be due to the fact that the aggregation property of Aβ43 is higher than that of Aβ42 (Saito et al., 2011).
(C) Aβ species by the βAPP-CTF cleavage assay in the presence of 0.25% or 0.5% CHAPSO.
(D) Aβ species in lysates of HEK293 cells in a 10 cm dish stably expressing sw βAPP and WT PS1.
(E) Fold changes of the relative VVIAT levels in cell lysates treated with GSMs/iGSMs.
(F) Fold changes of the relative VVIAT levels in lysates from cells stably expressing PS1 mutants.
(G) Fold changes of the relative IAT levels in cell lysates treated with GSMs/iGSMs.
(H) Fold changes of the relative IAT levels in lysates from cells stably expressing PS1 mutants.
(I) The relative rates of VVIA (blue) and VVIAT (red) in cells treated with GSMs/iGSMs.
(J) The relative rates of VVIA (blue) and VVIAT (red) in cells stably expressing PS1 mutants.
(K) Representative MALDI-TOF MS spectrum of products from the Aβ45 cleavage assay (0.25% CHAPSO).
(L) Representative MALDI-TOF MS spectrum of products from the Aβ46 cleavage assay (0.75% CHAPSO).
(M) Nβ25 cleavage assay (0.5% CHAPSO).
(N) APL1β28 cleavage assay (0.5% CHAPSO). Insets show an enlargement of the part encircled by the dotted line. Note that APL1β28 cleavage was less efficient than Nβ25 cleavage and Aβ42 cleavage.
Asterisks in (A), (K), (L), and (N) indicate nonspecific peaks, and those in (E), (F), (G), (H), (I), and (J) indicate statistical significance. Error bars represent SD. See also Figures S1 and S4.
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7. Figure S1
Proteolysis of Secreted Aβ Species by PS/γ-Secretase In Vitro, Related to Figures 1, 2, and 4
(A) Aβ38 does not undergo further cleavage by PS/γ-secretase, related to Figure 1A. We used the in vitro γ-secretase assay (Li et al., 2000; Osawa et al., 2008) to investigate whether Aβ38 can also be a substrate for PS/γ-secretase using Aβ38 as a putative substrate and in the presence of 0.125% to 1.5% CHAPSO (upper panel). The de novo generation of shorter Aβ species was not detected, indicating that Aβ38 is not a substrate but is one of the final products of PS/γ-secretase. Simultaneously, we confirmed Aβ42 cleavage under the same experimental conditions (lower panel). MW, molecular weight.
(B) Aβ11–42 is a substrate for PS/γ-secretase and is cleaved between G38 and V39 (Aβ numbering), related to Figure 1A. We used the in vitro γ-secretase assay to examine whether Aβ42 cleavage is dependent on the N-terminus of Aβ with Aβ11–42 as the substrate and in the presence of 0.5% CHAPSO. MALDI-TOF MS analysis showed the de novo generation of an MS peak with an average molecular weight of Da, which matches ionized Aβ11–38. Although we could not detect an MS peak corresponding to Aβ11–39, the results indicate that the N-terminus of Aβ42 is not necessarily the first residue in Aβ to be cleaved by PS/γ-secretase. MW, molecular weight.
(C) PS/γ-secretase also cleaves Aβ40 but produces Aβ37 at a very low efficiency, related to Figure 4A. When the in vitro γ-secretase assay was performed using Aβ43 as a substrate and in the presence of 0.5% CHAPSO, MALDI-TOF MS detected a very low level of Aβ37 (Figure 4A). The tripeptide GVV was detected by LC/MS/MS (Figure 4B), but the hexapeptide GVVIAT was not detected (data not shown). Considering these results, we suspected that Aβ43 is first cleaved to Aβ40, which then undergoes further cleavage by PS/γ-secretase to Aβ37. To verify this, we performed a modified in vitro γ-secretase assay using Aβ40 as a substrate with 0.75% CHAPSO. MALDI-TOF MS showed a very small MS peak with an average molecular weight of Da, which matches ionized Aβ37. We also detected an MS peak (asterisk) at a molecular weight close to that of Aβ36 ( Da).
(D) We then repeated the assay several times in the presence and absence of L685,458 to determine whether the MS peak was PS/γ-secretase dependent, related to Figure 2E and 2F. Representative MS spectra are shown. De novo generation of Aβ37 was observed consistently and was PS/γ-secretase dependent. However, the molecular weight of the MS peak close to Aβ36 was always Da, and the height of the MS peak was unaffected by L685,458. Thus, the MS peak close to Aβ36 is distinct from Aβ36. However, because the efficiency of the Aβ40 cleavage is extremely low, Aβ40 can be regarded as the final product of PS/γ-secretase. We could detect only one product from Aβ40. Given that two distinct products are generated from Aβ42 and Aβ43, minor products other than Aβ37 may be generated from Aβ40, but we could not detect them at any concentration of CHAPSO probably because of the lower efficiency of the cleavage (data not shown).
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8. Figure S2
Preparation of the Purified PS1/γ-Secretase and Characteristics of HEK293 Cells Overexpressing Each PS1 Mutant, Related to Figures 1 and 2
(A) Separation of Aβ species in the tris-tricine-urea gel electrophoresis, related to Figure 1D. Synthetic Aβ38, Aβ40, Aβ42, and Aβ43 peptides were electrophoresed in the tris-tricine-urea polyacrylamide gel, and immunoblotted with anti-Aβ-N-terminus antibody, 82E1 (left panel). Note that the gel could separate Aβ38, Aβ40, and Aβ42, but not Aβ42 and Aβ43. Similarly, the synthetic Aβ38, Aβ40, and Aβ42 peptides and the products of the βAPP-CTF cleavage assay (0.25% CHAPSO) were electrophoresed and immunoblotted (right panel). Note that electrophoretic mobility of each of Aβ species produced by PS/γ-secretase is the same as that of its respective synthetic Aβ peptide.
(B) PS/γ-secretase was purified from cells expressing WT PS1 or PS1 pathological mutants for use in the enzyme and binding assays in place of immunoprecipitated PS1/γ-secretase, related to Figure 2E and 2F (Winkler et al., 2009). Briefly, the crude membrane fraction (CMF) from the cells was isolated and solubilized with DDM/digitonin, and the soluble fraction was precipitated with 50% ammonium sulfate. The resulting supernatant was precipitated again with 70% ammonium sulfate. The resulting precipitate was solubilized in 55% ammonium sulfate and then purified further with WGA-agarose. The eluate from WGA-agarose was used as the purified enzyme in the in vitro γ-secretase assay. Aliquots of samples collected during each step of the purification were separated by SDS-PAGE and were analyzed by immunoblotting with anti-PS1-NTF antibody.
CMF fraction (lane 1); (B) DDM/digitonin supernatant (sup., lane 2) and precipitate (ppt., lane 3) fractions; (C) 50% ammonium sulfate sup. (lane 4) and ppt. (lane 5) fractions and 70% ammonium sulfate sup. fraction (lane 6); (D) 70% ammonium sulfate ppt. fraction (lane 7); (E) 55% ammonium sulfate sup. (lane 8) and ppt. (lane 9) fractions and flow through from the WGA-agarose column (lane 10); (F) WGA-agarose eluate (lane 11) and fraction remaining bound to WGA-agarose (lane 12). Asterisks indicate the lanes in which the same sample (E: 55% ammonium sulfate sup. fraction) was applied to both gels (lanes 8 and 13).
The PS1 holoprotein (∼45 kDa) undergoes endoproteolysis once it is integrated into PS/γ-secretase. The PS1 holoprotein is unstable because it is susceptible to degradation by the ubiquitin–proteasome pathway. Therefore, only a trace is detected in the endogenous state. By contrast, the endoproteolyzed NTF and CTF integrated in the γ-secretase complex are stable and can be observed in the endogenous state. However, when PS1 is exogenously overexpressed, excess PS1 holoprotein that is not integrated into complexes can be detected in (A) (Thinakaran et al., 1997) (see Figure S2F). Note that as the purification proceeds, the intensity of the PS1-NTF band increases as that of the PS1 holoprotein decreases. This indicates the progressive purification of the PS1/γ-secretase complex containing endoproteolyzed exogenous PS1.
(C) Next, the purity of the purified PS1/γ-secretase fractions was assessed by SDS-PAGE followed by silver staining, related to Figure 2E and 2F. Samples A–F are as described in panel A. Each sample represents 20% of a tissue culture plate (φ = 10 cm). Only a small fraction of the total proteins in the CMF remained in fraction F.
(D) The WT PS1/γ-secretase fraction that we purified contained high levels of each partner protein: Nicastrin (∼110 kDa), PS1-NTF (∼30 kDa), PS1-CTF (∼18 kDa), Aph-1a (∼20 kDa), and PEN-2 (∼13 kDa), related to Figure 2 and 3. The same level of the purified enzyme fraction used in (B) was subjected to electrophoresis and followed by immunoblotting with antibodies to each complex partner protein (left panels); PS1 fragments, Aph-1aL, nicastrin, and PEN-2 were identified. Next, 25 times as much of the purified enzyme fraction was electrophoresed and silver stained (right panel). The existence of silver-stained bands confirmed the position where each PS1 fragment, Aph-1aL, nicastrin, or PEN-2 band was identified by immunoblotting. The band pattern of the silver-stained gel was very similar to that reported previously (Winkler et al., 2009). These data indicate that the purified PS1/γ-secretase fraction in our study contained considerable levels of the PS1/γ-secretase complex.
(E) The ratio of Aβ42 to the sum of Aβ40 and Aβ42 was elevated in media from cultured HEK cells stably expressing mutant PS1s, related to Figure 2E–2H. The levels of Aβ40 and Aβ42 secreted into the media of HEK293 cells stably expressing WT or mutant PS1 were measured by ELISA. The ratio of Aβ42 to the sum of Aβ40 and Aβ42 was elevated in the media from all of the cells expressing PS1 mutants. These results demonstrate that the exogenous mutant PS1 molecules are integrated into PS/γ-secretase complexes and are functional in the cells. Values represent means ± SEM (n = 5). Asterisks indicate p < 0.01 versus WT PS1 by Welch’s t test.
(F) Endogenous PSs in the PS/γ-secretase complex are replaced almost completely by exogenous WT or mutant PS1 in transfected HEK293 cells, related to Figure 2E–2H. HEK293 cells were stably transfected with cDNAs encoding WT or pathological mutant forms of PS1 (PS1 L85P (Ataka et al., 2004), L286V (Sherrington et al., 1995), L381V (Dintchov Traykov et al., 2009), or G384A (Cruts et al., 1995)). Translated PS1 or PS2 forms a protein complex with Aph-1, PEN-2, and nicastrin to acquire γ-secretase activity. Following integration into the complex, PS1/2 undergoes endoproteolysis, which produces ∼30 kDa PS1/2 NTF and ∼20 kDa PS1/2 C-terminal fragments (CTF). As shown in the upper panel, the level of PS1 holoprotein was much higher in transfected than in nontransfected HEK293 cells. The results indicate that excess PS1 holoprotein that was not integrated into the γ-secretase complex overflowed in cells overexpressing PS1 (Thinakaran et al., 1997). The PS2-CTF band was absent in lysates of the transfected cells because little endogenous PS2 could be integrated into the γ-secretase complex because of an excess of exogenously expressed PS1 derivatives. This indicates that exogenously expressed PS1 derivatives represent most of the PS incorporated into the PS/γ-secretase complex.
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9. Figure S3
Enzyme Kinetics of Aβ42 Cleavage by WT PS1/γ-Secretase, Related to Figure 3
Enzyme kinetic analysis of PS/γ-secretase is usually performed using C-terminally elongated and membrane-bound forms of Aβ or βAPP-CTF as substrates. To calculate the Michaelis–Menten kinetic constants, a single proteolysis reaction is required. However, whether Aβ42 is generated from βAPP-CTF by PS/γ-secretase via single cleavage is controversial (Qi-Takahara et al., 2005), and it is unclear whether secreted Aβ species have a precursor–product relationship. Because we found that PS/γ-secretase mediates the proteolysis of Aβ42, producing Aβ38 in a single step (see Figure 1), we analyzed the kinetics of Aβ38 production by PS/γ-secretase.
(A) Aβ42 cleavage assay using purified PS1/γ-secretase in the presence of 0.5% CHAPSO, related to Figure 3C–3G.
(B) Representative Lineweaver–Burk plot from three independent experiments, related to Figures 3C–3G. The linear regression line for the plot was y = 1700x + 4.3, R2 = 0.99.
(C) Eadie–Hofstee replot, related to Figure 3C–3G. The linear regression line for the plot was y = –380x + 0.23, R2 = 0.97.
(D) Hanes–Woolf replot, related to Figure 3C–3G. The linear regression line for the plot was y = 4.5x + 1700, R2 = According to these results, the Km was 370 ± 40 nM (mean ± standard deviation; n = 5). These results demonstrate that Aβ42 cleavage by PS1/γ-secretase conforms to Michaelis–Menten kinetics. V, velocity; S, substrate concentration.
(E) Fitted curves of dissociation for DMSO with (gray) or without (red) L685,458 (10 μM). The data were used for the fitted curve labeled with DMSO in Figure 3H.
(F) Fitted curves of dissociation for GSM1 with (gray) or without (red) L685,458 in Figure 3H.
(G) Fitted curves of dissociation for S2474 with (gray) or without (red) L685,458 in Figure 3H.
(H) Fitted curves of dissociation for WT PS1/γ-secretase with (gray) or without (red) L685,458 from Aβ42. The data were used for the fitted curve labeled with WT in Figure 3J.
(I) Fitted curves of dissociation for PS1 L286V/γ-secretase with (gray) or without (red) L685,458 from Aβ42 in Figure 3J.
(J) Fitted curves of dissociation for PS1 G384A/γ-secretase with (gray) or without (red) L685,458 from Aβ42 in Figure 3J.
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10. Figure S4
Levels and Relative Ratios of Small Peptides Associated with Aβ Generation, Related to Figures 1 and 4
(A) Amounts of tri-, tetra-, and pentapeptides produced during the stepwise processing of βAPP in living CHO cells, related to Figure 4D. The relative level of each Aβ-generation-related peptide (i.e., ITL, VIT, VIV, TVI, IAT, VVIA, and VVIAT) in lysates of CHO cells (7WD10) (Koo and Squazzo, 1994) stably expressing sw βAPP was very similar to that observed in HEK cells.
(B) GSMs drastically increased the relative levels of both VVIA and VVIAT, whereas an iGSM decreased the relative levels of the two peptides, related to Figure 4I. The levels of VVIA and VVIAT were compared with the levels of the peptides produced by major cleavage in the previous step (i.e., TVI for VVIA and VIV for VVIAT) in living cells. Note that a very similar pattern of changes in the relative VVIA and VVAIT ratio in Figure 4I was observed.
(C) The PS1 mutants decreased the relative levels of VVIA and VVIAT; however, the degree of the changes was smaller than that observed for GSM, related to Figure 4J. The levels of VVIA and VVIAT were normalized to the level of each major cleavage in the previous step. Note that a very similar pattern of changes in the relative VVIA and VVAIT ratio in Figure 4J was observed.
(D) CHAPSO increased the relative levels of both VVIA and VVIAT in a dose-dependent manner, related to Figure 1D. The levels of VVIA and VVIAT were normalized to the sum of peptides associated with Aβ generation.
(E) CHAPSO increased the relative levels of both VVIA and VVIAT in a dose-dependent manner, related to Figure 1D. The levels of VVIA and VVIAT were normalized to the level of each cleavage in the previous step.
Asterisks indicate p < 0.05, Welch’s t test.
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