Molecular mechanisms of Alzheimer disease protection by the A673T allele of amyloid precursor protein

Abstract

Pathogenic mutations in the amyloid precursor protein (APP) gene have been described as causing early onset familial Alzheimer disease (AD). We recently identified a rare APP variant encoding an alanine-to-threonine substitution at residue 673 (A673T) that confers protection against development of AD (Jonsson, T., Atwal, J. K., Steinberg, S., Snaedal, J., Jonsson, P. V., Bjornsson, S., Stefansson, H., Sulem, P., Gudbjartsson, D., Maloney, J., Hoyte, K., Gustafson, A., Liu, Y., Lu, Y., Bhangale, T., Graham, R. R., Huttenlocher, J., Bjornsdottir, G., Andreassen, O. A., Jönsson, E. G., Palotie, A., Behrens, T. W., Magnusson, O. T., Kong, A., Thorsteinsdottir, U., Watts, R. J., and Stefansson, K. (2012) Nature 488, 96-99). The Ala-673 residue lies within the β-secretase recognition sequence and is part of the amyloid-β (Aβ) peptide cleavage product (position 2 of Aβ). We previously demonstrated that the A673T substitution makes APP a less favorable substrate for cleavage by BACE1. In follow-up studies, we confirm that A673T APP shows reduced cleavage by BACE1 in transfected mouse primary neurons and in isogenic human induced pluripotent stem cell-derived neurons. Using a biochemical approach, we show that the A673T substitution modulates the catalytic turnover rate (V(max)) of APP by the BACE1 enzyme, without affecting the affinity (K(m)) of the APP substrate for BACE1. We also show a reduced level of Aβ(1-42) aggregation with A2T Aβ peptides, an observation not conserved in Aβ(1-40) peptides. When combined in a ratio of 1:9 Aβ(1-42)/Aβ(1-40) to mimic physiologically relevant mixtures, A2T retains a trend toward slowed aggregation kinetics. Microglial uptake of the mutant Aβ(1-42) peptides correlated with their aggregation level. Cytotoxicity of the mutant Aβ peptides was not dramatically altered. Taken together, our findings demonstrate that A673T, a protective allele of APP, reproducibly reduces amyloidogenic processing of APP and also mildly decreases Aβ aggregation. These effects could together have an additive or even synergistic impact on the risk of developing AD.

Keywords: Alzheimer Disease; Amyloid Precursor Protein (APP); Amyloid-β (AB); Neurobiology; Proteolytic Enzyme; β-Secretase 1 (BACE1).

FIGURE 1.

Decreased cellular production of β-secretase cleavage products from APP A673T. A, representative Western blot analysis of cell lysates from HEK 293 cells transfected with GFP control, WT, A673T, A673V, or K670N/M671L APP. α- and β-CTF fragments were detected using an antibody specific for the C terminus of APP (A8717), and β-CTF fragments were specifically detected using an antibody just distal to the β-secretase cleavage site (6E10). An antibody to actin was used as a loading control for both blots. B, ELISA quantification of β-CTF cleavage product from these same cell lysates. C and D, ELISA quantification of Aβ40 (C) and Aβ42 (D) from supernatants of cultured mouse cortical neurons transfected with WT, A673T, or A673V human APP. E, ratio of sAPPβ/sAPPα as determined by immunoassay analysis of cell supernatants from the same cortical neurons. Values represent mean ± S.D. of three independent experiments, each with technical replicates. Two-tailed t test was compared with WT APP; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

FIGURE 2.

Decreased β-secretase cleavage products from APP A673T in human iPSC-derived neurons. A, representative immunofluorescence images of differentiated cortical control (WT), isogenic APP-A673T, or isogenic APP-A673V iPS cells. Cells were stained with antibodies to MAP2 (red) and APP (green). B and C, ELISA quantification of Aβ40 (B) and Aβ42 (C) from cell supernatants collected from the different cell lines. D, ratio of sAPPβ/sAPPα as determined by immunoassay analysis of cell supernatants from the same iPSC-derived neurons. Values represent mean ± S.D. of three independent experiments, each with technical replicates. Two-tailed t test was compared with WT APP; *, p < 0.05; **, p < 0.01.

FIGURE 3.

BACE1 processing of APP substrates in a reconstituted in vitro enzyme assay. A and B, TR-FRET analysis of cleavage rates for A673T and A673V peptides in a WT context (A) or in combination with the Swedish (K670N/M671L) mutation (B). C, kinetic analysis of FRET cleavage data. A673T and A673V mutations decreased and increased the rate of catalytic cleavage (V_max) of the short APP substrate, respectively, without affecting the K_m value. D, competitive inhibition of the catalytically efficient Swedish peptide substrate by peptide substrates without Swedish mutation, having a slow cleavage rate. WT, A637T and A673V competitive peptides yielded comparable IC₅₀, suggesting similar binding affinity. Values represent mean ± S.D. of three independent experiments, each with technical replicates.

FIGURE 4.

Aggregation kinetics of Aβ peptides measured by ThT fluorescence. A–C, process of Aβ aggregation was monitored over time using synthetic WT, A2T, or A2V Aβ peptides of pure Aβ(1–40) (A), pure Aβ(1–42) (B), or a 1:9 ratio of Aβ(1–42) to Aβ(1–40) (C). Mixtures of WT, A2T, and A2V are one-to-one molar ratios. Three separate experiments are shown for each set of peptides. Traces represent the mean of three replicate measurements, with ± S.D. shown for the final point. a.u., arbitrary units.

FIGURE 5.

Microglial uptake of WT, A2T, or A2V Aβ peptides. A, primary microglial cultures were incubated with WT, A2T, or A2V mutants of either Aβ(1–40) (upper panels) or Aβ(1–42) (lower panels) for 90 min. Microglia were then fixed and stained for Iba1 (red), DAPI (blue), and Aβ (green). Representative confocal images for each condition show that Aβ(1–42) peptides accumulate in microglia to a greater extent compared with Aβ(1–40). Scale bar, 10 μm. B, quantification of internalized Aβ as measured by average immunofluorescence signal intensity. Values represent mean ± S.D. of three independent experiments, each analyzing >10 cells per condition). Two-tailed t test, compared with WT Aβ; *, p < 0.05. a.u., arbitrary units.

FIGURE 6.

Cortical toxicity by WT, A2T, or A2V Aβ peptides. A and B, cultured mouse cortical neurons (E16.5) were treated with synthetic WT, A2T, or A2V Aβ(1–40) peptides (A) or Aβ(1–42) peptides (B) for 3 days at a concentration ranging from 0.1 to 10 μm. Cell viability was assessed by a luminescent assay. Results are normalized to vehicle-treated controls. Values represent mean ± S.D. of three independent experiments, each with technical replicates.