BACE1 Cleavage Site Selection Critical for Amyloidogenesis and Alzheimer's Pathogenesis

Abstract

Mutations in amyloid β precursor protein (APP) gene alter APP processing, either causing familial Alzheimer's disease (AD) or protecting against dementia. Under normal conditions, β-site APP cleaving enzyme 1 (BACE1) cleaves APP at minor Asp¹ site to generate C99 for amyloid β protein (Aβ) production, and predominantly at major Glu¹¹ site to generate C89, resulting in truncated Aβ production. We discovered that A673V mutation, the only recessive AD-associated APP mutation, shifted the preferential β-cleavage site of BACE1 in APP from the Glu¹¹ site to the Asp¹ site both in male and female transgenic mice in vivo and in cell lines and primary neuronal culture derived from timed pregnant rats in vitro, resulting in a much higher C99 level and C99/C89 ratio. All other mutations at this site, including the protective Icelandic A673T mutation, reduced C99 generation, and decreased the C99/C89 ratio. Furthermore, A673V mutation caused stronger dimerization between mutant and wild-type APP, enhanced the lysosomal degradation of the mutant APP, and inhibited γ-secretase cleavage of the mutant C99 to generate Aβ, leading to recessively inherited AD. The results demonstrate that APP673 regulates APP processing and the BACE1 cleavage site selection is critical for amyloidogenesis in AD pathogenesis, and implicate a pharmaceutical potential for targeting the APP673 site for AD drug development.SIGNIFICANCE STATEMENT β-site APP cleaving enzyme 1 (BACE1) is essential for amyloid β protein production. We discovered that A673V mutation shifted the BACE1 cleavage site from the Glu¹¹ to the Asp¹ site, resulting in much higher C99 level and C99/C89 ratio. All other mutations at this site of amyloid β precursor protein (APP) reduced C99 generation and decreased the C99/C89 ratio. Furthermore, A673V mutation resulted in stronger dimerization between mutant and wild-type APP, enhanced the lysosomal degradation of the mutant APP, and inhibited γ-secretase cleavage of the mutant C99 to generate amyloid β protein, leading to recessively inherited Alzheimer's disease (AD). The results demonstrate that APP673 regulates APP processing, and the BACE1 cleavage site selection is critical for amyloidogenesis in AD pathogenesis, and implicate a pharmaceutical potential for targeting the APP673 site for AD drug development.

Keywords: Alzheimer's disease; BACE1.

Figure 1.

Effects of APP A673V (rIta) mutation on APP processing and Aβ generation. A, APP_Wt and APP_rIta adeno-associated viruses were transduced into the brains of 3 neonatal mice, and CTFs were analyzed after immunoprecipitation. PBS was injected into 2 mice as control. In the “control IP,” only lysis buffer, but not brain lysate, was added to the C20 antibody immobilized on protein-G beads. cDNAs coding for C99, C89, and C83 with a methionine in the N termini were cloned into pcDNA4, confirmed by DNA sequencing, and expressed in HEK293 cells as CTF markers. B, APP695_WT, APP695r_Ita, and APP695_Swe plasmids were transfected into PC12 cells. APP and its CTFs were detected by C20 antibody. C99 levels in APP variants were normalized to that in APP_WT. C, D, Aβ40 and Aβ42 in the conditioned media of PC12 cells expressing APP_WT, APP_rIta and APP_Swe were quantified by ELISA and expressed as pg/ml. E, 293B2 were transfected with APP695_Wt, APP695r_Ita, or APP695_Swe plasmids, and CTF markers and cell lysates were blotted for APP, BACE1, and CTFs. C99/C89 ratios in APP mutants were normalized to that of APP_Wt. F, In vitro BACE1 cleavage kinetics. APP fragments spanning from 639 to 681 (β-site between M671–D672) with or without the rIta mutation at A673 were inserted between GFP and FLAG-His tags and expressed in bacteria. The recombinant proteins were purified with His tag beads. The 0.35 μg of recombinant proteins in 35 μl buffered solutions were incubated at 37°C with or without 2.1 μl recombinant BACE1 (R&D Systems, 500 μg/ml) at different pH values for the indicated times. The Western blot bands by anti-FLAG antibody were quantified and plotted. G, APP_Wt and APP_Wt carrying M671V (to inhibit Asp-1-cleavage) and/or Y681A (to inhibit Glu-11-cleavage) mutations were expressed in 293B2 cell, and the whole-cell lysates were blotted for CTFs, full-length APP, and BACE1. H, APP_rIta with or without the Y681A mutation and APP_Swe with or without the Y681 mutation were expressed in 293B2 cells, and whole-cell lysates were blotted for CTFs, APP, and BACE1. Data are mean ± SEM; n = 3 technical replicates. Statistical analysis: one-way ANOVA with post hoc Newman–Keuls tests.

Figure 2.

The A673V mutation altered APP processing independently of expression level and enabled BACE1 to cleave nascent APP. To determine whether APP_rIta expression levels affect β-cleavage site selection and Aβ generation, an inducible APP expression system (Li et al., 2006) was introduced to 293B2 cells to control APP expression at different levels. A, pIND-APP_Wt, pIND-APP_rIta, and pIND-APP_Swe were transfected into 293B2 cells. After ponasterone A treatment at different concentrations, cell lysates were blotted for full-length APP, CTFs, and BACE1. Full-length APPs (B), CTFs (C), Aβ40 (D), and Aβ42 (E) were quantified. F, APP_Wt, APP_Swe, and APP_rIta bearing the KKQN ER retention signal were expressed in 293B2 cells. C99s were quantified. Data are mean ± SEM; n = 3 technical replicates. Statistical analysis: one-way ANOVA with post hoc Newman–Keuls tests.

Figure 3.

Enhanced dimerization suppressed C99 production from APP_rIta and γ-cleavage of C99_rIta. Nontagged APPWt (APPWt-NT) (A) or APPrIta (APPrIta-NT) (B) were coexpressed with GFP, or APPWt-GFP, or APPrIta-GFP in 293B2 cells, and the cell lysates were blotted for CTFs-NT, APPs, and BACE1. The ratios of C99-NT/C89-NT were plotted. C, APP_rIta-NT (nontagged APP_rIta) plasmid was cotransfected into 293B2 cells with increasing amount of either APP_Wt-GFP or APP_rIta-GFP plasmid. C20 antibody was used to detect APPs and CTFs derived from AP_rIta-NT, and 9E10 for BACE1. Protein bands for full-length APP_Wt/rIta-GFP and full-length APP_rIta-NT were quantified, and the ratios of APP_Wt/rIta-GFP/APP_rIta-NT were indicated. The ratios of C99_rIta in APP-GFP expressing cells to C99_rIta in cells without APP-GFP expression were plotted. The ratios of C99_rIta/C89 over the APP_Wt/rIta-GFP dosage courses were plotted as well. D, Flag-APP_Wt plasmid and empty vector were transfected with APP_Wt-GFP or APP_rIta-GFP. The immunoprecipitated Flag-APP_Wt by the FLAG-beads and the coprecipitated APP-GFPs were both detected with C20 antibody. E, Flag-APP_Wt and empty vector were transfected with C99_Wt or C99_A2V. The immunoprecipitated FLAG-APP_Wt by the FLAG-beads and the coprecipitated C99s were detected with C20 antibody. F, pcDNA4–C99_WT or -C99_A2V with a signal peptide were cotransfected with pEGFP plasmid (as an internal control for transfection) into HEK293 and then treated with indicated catabolism inhibitors overnight. DMSO, control solvent; GSI, γ-secretase inhibitor L-685,458; MG132, proteasome inhibitor. GFP was used for transfection efficiency control, and β-actin was used as for protein loading control. C99 level was quantified and compared between C99_WT and C99_A2V. G, Conditioned medium was collected to determine the amount of Aβ₄₀ in HEK293 with C99_WT (WT) and C99_A2V (A673V) overexpression. Data are mean ± SEM; n = 3 technical replicates. Statistical analysis: one-way ANOVA with post hoc Newman–Keuls tests.

Figure 4.

Lysosome-dependent degradation of mature APP_A673V. A, Plasmid pcDNA4-APP_Wt, pcDNA4-APP_rIta, or pcDNA4-APP_Swe were transfected into HEK293 cells. The nascent APP (imAPP) and modified APP (mAPP) in cell lysate were resolved on 8% glycine SDS-PAGE gel and detected by C20 antibody. B, The ratios of mAPP/imAPP in A were quantified and normalized to that in APP_Wt. C, D, APP_Wt-SNAP and APP_rIta-SNAP were overexpressed in HEK293 cells and pulse-labeled. After indicated chasing time, cells lysate was blotted for pulse-labeled APP with streptavidin (green) and total APP with C20 (red). The maturation rate (as indicated by the reduction of pulse-labeled immature APP) of APP_Wt and APP_rIta were plotted after normalization with total APP. E, pcDNA4-APP_Wt and pcDNA4-APP_rIta were introduced into HEK293 cells and then treated with10 μm MG132 or 100 μm CHL for 45 min. F, Mature APP protein levels were quantified. Data are mean ± SEM; n = 3 technical repeats. Statistical analysis: ANOVA with post hoc Newman–Keuls test. G, Viruses expressing human APP_Wt or APPrIta were tranduced into primary rat neurons on DIV 3, and neuronal cells with overnight treatment of CHL orsolvent H₂O (Ctrl) were harvested for blotting of full-length APP on DIV 10. Ratios of mature APP to immature APP were plotted. H, The SDYQRL trans-Golgi targeting motif (Wong and Hong, 1993) was fused to APP_Wt or APP_rIta, and the fusion proteins were expressed in HEK293 cells, full-length APPs were blotted with C20 antibody, and the levels of mature APP variants after normalization with immature APP variants were plotted. I, The NPTY internalization signal (Perez et al., 1999) was deleted from APP_Wt and APP_rIta to retain them on the plasma membrane (PM). The resulting APP variants with a FLAG tag fused to the N termini after signal peptide removal were overexpressed in HEK293 cells. Full-length APPs were blotted with anti-FLAG antibody as the NPTY motif was within the epitope of C20 antibody. Levels of mature APP variants after normalization with immature APP variants were plotted. G–I, Data are mean ± SEM; n = 2 technical repeats. Statistical analysis: two-way ANOVA with Newman–Keuls tests.

Figure 5.

Effects of amino acid substitution of A673 on β-cleavage. A, C99 with the indicated mutations at position 2 (position 673 in full-length APP) displayed altered gel mobility by SDS-PAGE. B, A673 in APP were substituted with V, D, L, P-R, or S, and mutants were expressed in 293B2 cells. C99 levels were quantified. C, APP_Wt and APP_Ice were expressed in 293B2 cells, and CTFs were quantified. D, APP_Wt and APP_Ice were expressed in PC12 cells, and C99s and C89s were quantified. Secreted Aβ40 (E) and Aβ42 (F) were measured by ELISA. Data are mean ± SEM; n = 3 technical repeats. Statistical analysis: one-way ANOVA with post hoc Newman–Keuls tests.