Alzheimer presenilin-1 mutations dramatically reduce trimming of long amyloid β-peptides (Aβ) by γ-secretase to increase 42-to-40-residue Aβ

Abstract

The presenilin-containing γ-secretase complex produces the amyloid β-peptide (Aβ) through intramembrane proteolysis, and >100 presenilin mutations are associated with familial early-onset Alzheimer disease (AD). The question of whether these mutations result in AD through a gain or a loss of function remains highly controversial. Mutations in presenilins increase ratios of 42- to 40-residue Aβ critical to pathogenesis, but other Aβs of 38-49 residues are also formed by γ-secretase. Evidence in cells suggests the protease first cleaves substrate within the transmembrane domain at the ϵ site to form 48- or 49-residue Aβ. Subsequent cleavage almost every three residues from the C terminus is thought to occur along two pathways toward shorter secreted forms of Aβ: Aβ49 → Aβ46 → Aβ43 → Aβ40 and Aβ48 → Aβ45 → Aβ42 → Aβ38. Here we show that the addition of synthetic long Aβ peptides (Aβ45-49) directly into purified preparations of γ-secretase leads to the formation of Aβ40 and Aβ42 whether the protease complex is detergent-solubilized or reconstituted into lipid vesicles, and the ratios of products Aβ42 to Aβ40 follow a pattern consistent with the dual-pathway hypothesis. Kinetic analysis of five different AD-causing mutations in presenilin-1 revealed that all result in drastic reduction of normal carboxypeptidase function. Altered trimming of long Aβ peptides to Aβ40 and Aβ42 by mutant proteases occurs at multiple levels, independent of the effects on initial endoproteolysis at the ϵ site, all conspiring to increase the critical Aβ42/Aβ40 ratio implicated in AD pathogenesis. Taken together, these results suggest that specific reduction of carboxypeptidase function of γ-secretase leads to the gain of toxic Aβ42/Aβ40.

Keywords: Amyloid-β (AB); Carboxypeptidase; Enzyme Kinetics; Enzyme Mechanism; Intramembrane Proteolysis.

FIGURE 1.

γ-Secretase trims synthetic Aβ49 and Aβ48 to Aβ40 and Aβ42 in vitro. A, APP substrate is thought to be cleaved sequentially by γ-secretase at the ϵ, ζ, and γ sites, indicated by arrowheads. These cleavage events result in Aβ peptides with the indicated C termini. Ihara and co-workers (16, 18) have proposed Aβ40- and Aβ42-generating pathways (top and bottom, respectively), in which ϵ cleavage that produces AICD50–99 primarily leads to Aβ40, whereas ϵ cleavage that produces AICD49–99 mainly produces Aβ42. B and C, Aβ40 and Aβ42 production from synthetic Aβ49 and Aβ48 and CHAPSO-solubilized membranes from CHO cells overexpressing the four γ-secretase components (B) or purified γ-secretase complexes reconstituted into lipid vesicles (C). Aβ40 and Aβ42 were detected using specific ELISAs. Assays were performed in triplicate, with control reactions as follows: + I reactions contain 1.5 μm γ-secretase inhibitor L-685,458; D1 reactions contain γ-secretase with an inactive D257A mutant PS1; boiled reactions contain heat-inactivated γ-secretase; γ reactions have no substrate added to the assay mixtures; Aβ reactions have no enzyme added to the reaction mixtures. For all of the non-control reactions, the level of Aβ40 produced was significantly different from that of Aβ42 (p < 0.01, Student's t test). n = 3; error bars, S.D.

FIGURE 2.

γ-Secretase modulators lower Aβ42 produced from Aβ49 and Aβ48. Both GSM-1 and Rivkin-2 (compound 2 in Ref. 29) have previously been shown to selectively lower Aβ42 levels and concomitantly increase Aβ38 levels (22, 29). A, as expected, both compounds lower Aβ42 and increase Aβ38 in a concentration-dependent manner using APP C100-FLAG as substrate in CHAPSO-solubilized γ-secretase assays, as detected using a Bicine/urea-polyacrylamide gel electrophoresis system. B, both compounds decreased the amount of Aβ42 generated from Aβ48 and Aβ49 without altering Aβ40 levels. n = 3; error bars, S.D. * indicate values that were significantly different from the Aβ42 value with no compound added (p < 0.05). Aβ40 values were not significantly different at any concentration tested. C, cleavage reactions were performed in CHAPSO with the indicated concentrations of substrate. The levels of Aβ42 generated from C100-FLAG (left) or Aβ48 (right) were measured by ELISA. +GSM, reactions contained 25 μm GSM-1; −GSM, reactions with vehicle alone. V_max values for C100 and Aβ48 substrates were significantly reduced in the presence of GSM-1 (p < 0.05, Student's t test). n = 2; error: S.D.

FIGURE 3.

γ-Secretase trims p3/Aβ49 and p3/Aβ48 to p3/Aβ40 and p3/Aβ42 in cells. A, the expression of APPϵ49 and APPϵ48 in CHO cells was confirmed by Western blot of cell lysates using anti-APP antibody 22C11. The truncated APPs run slightly faster than full-length, endogenous APP. B, p3/Aβ40 and p3/Aβ42 secretion by cells expressing APPϵ49, APPϵ48, or full-length APP. Untransfected controls (no txn) show the endogenous background signal from the cells; for + inhibitor transfections, cells were treated with 10 μm DAPT. *, p < 0.05; **, p < 0.01 (Student's t test). n = 3; error bars, S.E.

FIGURE 4.

γ-Secretase trims synthetic Aβ45, Aβ 46, and Aβ47 to Aβ40 and Aβ42 in vitro. A, enzyme assays using CHAPSO-solubilized membranes from CHO cells overexpressing the four γ-secretase components. B, enzyme assays using purified γ-secretase complexes reconstituted into lipid vesicles. Control reactions are the same as in Fig. 1. The level of Aβ40 generated in all non-control reactions was significantly different from that of Aβ42 (p < 0.01 for Aβ45 and Aβ46 substrates, p < 0.05 for Aβ47 substrate, Student's t test). n = 3; error bars, S.D.

FIGURE 5.

Aβ49 and Aβ48 conversions to Aβ40 and Aβ42 by γ-secretase are dramatically reduced by PS1 FAD mutations. A, equal amounts of each enzyme were used in the reactions, normalizing for PS1 NTF levels in each enzyme preparation based on densitometry of PS1 NTF Western blots (or of full-length PS1 for the uncleaved but proteolytically active Δexon9 mutation). B, cleavage reactions with WT (top panels) or the indicated FAD-mutant PS1 (bottom panels) were carried out in CHAPSO with the indicated concentrations of substrate. Left panels, Aβ49 substrate; right panels, Aβ48 substrate. Levels of Aβ40 (solid lines) and Aβ42 (dashed lines) were determined by ELISA, and data were fit using GraphPad prism 4 non-linear regression analysis. n = 3; error bars, S.E. Note the difference in scale for Aβ levels from WT (top panels) and FAD-mutant PS1 (bottom panels).

FIGURE 6.

FAD-mutant PS1·γ-secretase complexes increase Aβ42/40 independent of effects on ϵ-site endoproteolysis. Aβ42/40 ratios generated in in vitro γ-secretase assays with either WT or the indicated FAD-mutant PS1 from a 70:30 mixture of Aβ49/48 (similar to what is observed normally from APP). **, p < 0.01 (one-way ANOVA and Dunnett's post test). n = 3; error bars, S.D.