Inhibition of BACE1 affected both its Aβ producing and degrading activities and increased Aβ42 and Aβ40 levels at high-level BACE1 expression

Abstract

The beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) is the predominant β-secretase, cleaving the amyloid precursor protein (APP) via the amyloidogenic pathway. In addition, BACE1 as an amyloid degrading enzyme (ADE), cleaves Aβ to produce the C-terminally truncated non-toxic Aβ fragment Aβ34 which is an indicator of amyloid clearance. Here, we analyzed the effects of BACE1 inhibitors on its opposing enzymatic functions, i.e., amyloidogenic (Aβ producing) and amyloidolytic (Aβ degrading) activities, using cell culture models with varying BACE1/APP ratios. Under high-level BACE1 expression, low-dose inhibition unexpectedly yielded a two-fold increase in Aβ42 and Aβ40 levels. The concomitant decrease in Aβ34 and secreted APPβ levels suggested that the elevated Aβ42 and Aβ40 levels were due to the attenuated Aβ degrading activity of BACE1. Notably, the amyloidolytic activity of BACE1 was impeded at lower BACE1 inhibitor concentrations compared to its amyloidogenic activity, thereby suggesting that the Aβ degrading activity of BACE1 was more sensitive to inhibition than its Aβ producing activity. Under endogenous BACE1 and APP levels, "low-dose" BACE1 inhibition affected both the Aβ producing and degrading activities of BACE1, i.e., significantly increased Aβ42/Aβ40 ratio and decreased Aβ34 levels, respectively. Further, we incubated recombinant BACE1 with synthetic Aβ peptides and found that BACE1 has a higher affinity for Aβ substrates over APP. In summary, our results suggest that stimulating BACE1's ADE activity and halting Aβ production without decreasing Aβ clearance could still be a promising therapeutic approach with new, yet to be developed, BACE1 modulators.

Keywords: Alzheimer’s disease; BACE1 amyloidolytic activity; amyloid clearance; amyloid production; secretase inhibitors.

Figure 1

Effects of VBA2092464 (R3) BACE1 inhibitor on human SH-SY5Y cells. Wild-type SH-SY5Y cells were treated with vehicle (V) or varying concentrations of the VBA2092464 (R3) BACE1 inhibitor. A, Western blotting (representative of 4 independent experiments) of cell lysates for the detection of APP (upper band, mature; lower band, immature), BACE1, and actin (control), or cell media for secreted sAPPβ. B–E, conversion of WB data to relative amounts of BACE1, APP, sAPPβ, or mature APP as a function of BACE1 inhibitor concentration. F, quantification of Aβ34, Aβ40, and Aβ42 levels in cell media as a function of BACE1 inhibitor concentration as determined by MSD immunoassay. G, conversion of MSD data (pg/ml) to the percentage change of Aβ levels (relative to the vehicle condition). H–J, conversion of MSD data (pg/ml) to specified Aβ ratios. Statistics: Bars and error bars indicate mean ± s.e.m. Dunnet’s post hoc tests were performed for pairwise comparisons; selected comparisons are highlighted ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05. B, BACE1, 1-WAY ANOVA, F(5,18) = 1.590, p = 0.2133, (C) APP, 1-WAY ANOVA, F(5,18) = 0.8343, p = 0.5423, (D) sAPPβ, 1-WAY ANOVA, F(5,18) = 15.68, p < 0.0001, (E) mature APP, 1-WAY ANOVA, F(5,18) = 3.315, p < 0.05, (F) Absolute Aβ, 2-WAY ANOVA, interaction F(10,54) = 41.65, p < 0.0001, row factor F(5,54) = 74.57, p < 0.0001, column factor F(2,54) = 549.2, p < 0.0001, (H) Aβ42/Aβ40, 1-WAY ANOVA, F(5,18) = 33.71, p < 0.0001, (I) Aβ40/Aβ34, 1-WAY ANOVA, F(5,18) = 56.02, p < 0.0001, (J) Aβ42/Aβ34, 1-WAY ANOVA, F(5,18) = 58.38, p < 0.0001.

Figure 2

Effects of VBA2092464 (R3) BACE1 inhibitor on stably BACE1 overexpressing SH-SY5Y cells. BACE1-SH-SY5Y cells were treated with vehicle (V) or varying concentrations of the VBA2092464 (R3) BACE1 inhibitor. A, Western blotting (representative of three independent experiments) of cell lysates for the detection of APP (upper band, mature; lower band, immature), BACE1, and actin (control), or cell media for secreted sAPPβ. B–E, conversion of WB data to relative amounts of BACE1, APP, sAPPβ, or mature APP as a function of BACE1 inhibitor concentration. F, quantification of Aβ34, Aβ40 and Aβ42 levels in cell media as a function of BACE1 inhibitor concentration as determined by ELISA and MSD (Aβ34). G, conversion of data from Figure 2F (pg/ml) to percentage change of Aβ levels (relative to the vehicle condition). H–J, conversion of data from Figure 2F (pg/ml) to specified Aβ ratios. Statistics: Bars and error bars indicate mean ± s.e.m. Dunnet’s post hoc tests were performed for pairwise comparisons; selected comparisons are highlighted ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05. B, BACE1, 1-WAY ANOVA, F(5,12) = 0.06869, p = 0.9959, (C) APP, 1-WAY ANOVA, F(5,12) = 3.730, p < 0.05, (D) sAPPβ, 1-WAY ANOVA, F(5,12) = 25.71, p < 0.0001, (E) mature APP, 1-WAY ANOVA, F(5,12) = 15.25, p < 0.0001, (F) Absolute Aβ, 2-WAY ANOVA, interaction F(10,36) = 39.26, p < 0.0001, row factor F(5,36) = 146.4, p < 0.0001, column factor F(2,36) = 247.4, p < 0.0001, (H) Aβ42/Aβ40, 1-WAY ANOVA, F(5,12) = 2.714, p = 0.0727, (I) Aβ40/Aβ34, 1-WAY ANOVA, F(5,12) = 78.10, p < 0.0001, (J) Aβ42/Aβ34, 1-WAY ANOVA, F(5,12) = 17.73, p < 0.0001.

Figure 3

Effect of inhibitors with different pKa values on stably BACE1 overexpressing SH-SY5Y cells. BACE1-SH-SY5Y cells were treated with vehicle (V) or varying concentrations of the N6, N8, N10 or N11 BACE1 inhibitor. Three independent experiments were performed. Due to the different toxicities of inhibitors, the concentrations covered are different. The concentration of 10⁻⁷ M for all inhibitors is marked with a box in the figure to allow an easier comparison. A, C, E, and G, quantification of Aβ34, Aβ40 and Aβ42 levels in cell media as a function of BACE1 inhibitor concentration as determined by ELISA and MSD (Aβ34). B, D, F, and H, conversion of data from absolute amounts (pg/ml) to the percentage change of Aβ levels (relative to the vehicle condition). Statistics: Bars and error bars indicate mean ± s.e.m. Dunnet’s post hoc tests were performed for pairwise comparisons; selected comparisons are highlighted ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05. A, Absolute Aβ, 2-WAY ANOVA, interaction F(8,30) = 26.14, p < 0.0001, row factor F(4,30) = 69.42, p < 0.0001, column factor F(2,30) = 44.53, p < 0.0001, (C) Absolute Aβ, 2-WAY ANOVA, interaction F(10,36) = 8.300, p < 0.0001, row factor F(5,36) = 16.76, p < 0.0001, column factor F(2,36) = 7.373, p < 0.001, (E) Absolute Aβ, 2-WAY ANOVA, interaction F(6,24) = 7.742, p < 0.001, row factor F(3,24) = 6.049, p < 0.01, column factor F(2,24) = 9.651, p < 0.001, (G) Absolute Aβ, 2-WAY ANOVA, interaction F(8,30) = 11.01, p < 0.0001, row factor F(4,30) = 13.70, p < 0.0001, column factor F(2,30) = 21.08, p < 0.0001.

Figure 4

Effect of BACE1 inhibitor VBA2092464 (R3) on stably APP-C99 overexpressing SH-SY5Y cells. APP-C99-SH-SY5Y cells were treated with vehicle (V) or varying concentrations of the VBA2092464 (R3) BACE1 inhibitor. A, Western blotting (representative of three independent experiments) of cell lysates for the detection of APP (upper band, mature; lower band, immature), BACE1, APP-C99 and actin (control), or cell media for secreted sAPPβ. B–E, conversion of WB data to relative amounts of BACE1, APP, sAPPβ, or mature APP as a function of BACE1 inhibitor concentration. F, quantification of Aβ34, Aβ40, and Aβ42 levels in cell media as a function of BACE1 inhibitor concentration as determined by ELISA. G, conversion of ELISA data (pg/ml) to the percentage change of Aβ levels (relative to the vehicle condition). H–J, conversion of ELISA data (pg/ml) to specified Aβ ratios. Statistics: Bars and error bars indicate mean ± s.e.m. Dunnet’s post hoc tests were performed for pairwise comparisons; selected comparisons are highlighted ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05. B, BACE1, 1-WAY ANOVA, F(5,12) = 1.280, p = 0.3344, (C) APP, 1-WAY ANOVA, F(5,12) = 1.419, p = 0.2860, (D) sAPPβ, 1-WAY ANOVA, F(5,12) = 4.904, p < 0.05, (E) mature APP, 1-WAY ANOVA, F(5,12) = 1.965, p = 0.1568, (F) Absolute Aβ, 2-WAY ANOVA, interaction F(10,36) = 1.006, p = 0.4573, row factor F(5,36) = 2.045, p = 0.0955, column factor F(2,36) = 451.5, p < 0.0001, (H) Aβ42/Aβ40, 1-WAY ANOVA, F(5,12) = 1.807, p = 0.1860, (I) Aβ40/Aβ34, 1-WAY ANOVA, F(5,12) = 15.79, p < 0.0001, (J) Aβ42/Aβ34, 1-WAY ANOVA, F(5,12) = 31.73, p < 0.0001.

Figure 5

Effects of γ-secretase inhibitor VBA1697787 (R1) and γ-secretase modulator VBA2092479 (R2) on stably APP-C99 overexpressing SH-SY5Y cells. APP-C99-SH-SY5Y cells were treated with vehicle (V) or varying concentrations of the VBA1697787 (GSI R1) (A–D) or VBA2092479 (GSM R2) (E−H) compounds. Three independent experiments were performed. A and E, quantification of Aβ34, Aβ40 and Aβ42 levels in cell media as a function of γ-secretase inhibitor/modulator concentrations as determined by ELISA. b and f. Conversion of ELISA data (pg/ml) to the percentage change of Aβ levels (relative to the vehicle condition). C, D, G, and H. conversion of ELISA data (pg/ml) to specified Aβ ratios. Statistics: Bars and error bars indicate mean ± s.e.m. Dunnet’s post hoc tests were performed for pairwise comparisons; selected comparisons are highlighted ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05. A, Absolute Aβ, 2-WAY ANOVA, interaction F(10,36) = 34.14, p < 0.0001, row factor F(5,36) = 96.84, p < 0.0001, column factor F(2,36) = 218.1, p < 0.0001, (C) Aβ40/Aβ34, 1-WAY ANOVA, F(4,10) = 167.1, p < 0.0001, (D) Aβ42/Aβ34, 1-WAY ANOVA, F(3,8) = 4.049, p = 0.05, (E) Absolute Aβ, 2-WAY ANOVA, interaction F(10,36) = 36.03, p < 0.0001, row factor F(5,36) = 90.07, p < 0.0001, column factor F(2,36) = 236.3, p < 0.0001, (F) Aβ40/Aβ34, 1-WAY ANOVA, F(4,10) = 164.8, p < 0.0001, (G) Aβ42/Aβ34, 1-WAY ANOVA, F(4,10) = 58.65, p < 0.0001.

Figure 6

MALDI-TOF analysis of BACE1-cleaved peptides. In vitro digestion of (A) Aβ40 or (B) Aβ42 by BACE1. Synthetic peptides (50 μg/ml of Aβ40 (equals 11.6 μM) and Aβ42 (equals 11.1 μM)) were incubated with 10 μg/ml recombinant BACE1 (0.14 μM) at 37 °C for varying time points as indicated. A, representative MALDI-TOF spectra (top to bottom: time = 0, 10 min, 30 min, and 2 h) to monitor the cleavage of Aβ40 substrate (4327 m/z) to Aβ34 product (3785 m/z) by BACE1 enzyme. B, representative MALDI-TOF spectra (top to bottom: time = 0, 10 min, 30 min, and 48 h) to monitor the cleavage of Aβ42 substrate (4512 m/z) to Aβ34 product (3786 m/z) by BACE1 enzyme. Modest differences between the observed peaks and theoretical monoisotopic masses (Aβ34: 3784.9 + 1H+ = 3785.9 Da; Aβ40: 4327.1 + 1H+ = 4378.1 Da; Aβ42: 4511.3 + 1H+ = 4512.3 Da) were due to the linear detection method used to maximize MALDI-TOF assay sensitivity.

Figure 7

Kinetics of BACE1 cleavage of Aβ40 and Aβ42 and the effect of pH. Michaelis-Menten kinetics calculated for BACE1-mediated cleavage of freshly solubilized Aβ42 and kinetic curves of Aβ42 incubated for times as indicated (A), the Lineweaver-Burk plot for freshly solubilized Aβ42 (B), Michaelis-Menten kinetics for Aβ40 (C) at different pHs (E, G and I) with Vmax and Km values indicated on respective graphs. Lineweaver-Burk plots for Aβ40 are shown in (D, F, H, and J). Synthetic Aβ peptides were diluted to indicated concentrations and incubated with a constant concentration of BACE1. The initial reaction velocities (v0) were determined by measuring the product (Aβ34) concentrations over time by ELISA. Initial velocities were plotted against the used substrate concentrations and fitted using the Michaelis-Menten equation. Mean of three independent experiments is shown. Bars and error bars indicate mean ± SD.

Figure 8

Schematic overview of effects of BACE1 inhibition in three cellular models tested. Inhibition of BACE1 affected Aβ42, Aβ40 and Aβ34 levels depending on the substrate to enzyme (Aβ:BACE1) ratio. In WT cells (model#1) with endogenous levels of substrate and enzyme, BACE1 inhibition decreased levels of all three Aβ peptides tested. At high levels of substrates, i.e., Aβ42 and Aβ40 (model#3: APP-C99 overexpression bypasses the amyloidogenic activity of BACE1), Aβ34 levels are reduced while Aβ42 and Aβ40 levels remain unaffected. BACE1 inhibition under BACE1 overexpression condition (model#2) results in reduced Aβ34 levels and a significant concomitant increase of Aβ40 and Aβ42 levels indicating that amyloidolytic activity of BACE1 is more susceptible to inhibition over a certain concentration range than its amyloidogenic activity.