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. 2019 Apr;14(4):658-665.
doi: 10.4103/1673-5374.247469.

Mutations of beta-amyloid precursor protein alter the consequence of Alzheimer's disease pathogenesis

Nuo-Min Li  1 , Ke-Fu Liu  1 , Yun-Jie Qiu  1 , Huan-Huan Zhang  1 , Hiroshi Nakanishi  2 , Hong Qing  1
Affiliations

Mutations of beta-amyloid precursor protein alter the consequence of Alzheimer's disease pathogenesis

Nuo-Min Li et al. Neural Regen Res. 2019 Apr.

Abstract

Alzheimer's disease is pathologically defined by accumulation of extracellular amyloid-β (Aβ). Approximately 25 mutations in β-amyloid precursor protein (APP) are pathogenic and cause autosomal dominant Alzheimer's disease. To date, the mechanism underlying the effect of APP mutation on Aβ generation is unclear. Therefore, investigating the mechanism of APP mutation on Alzheimer's disease may help understanding of disease pathogenesis. Thus, APP mutations (A673T, A673V, E682K, E693G, and E693Q) were transiently co-transfected into human embryonic kidney cells. Western blot assay was used to detect expression levels of APP, beta-secretase 1, and presenilin 1 in cells. Enzyme-linked immunosorbent assay was performed to determine Aβ1-40 and Aβ1-42 levels. Liquid chromatography-tandem mass chromatography was used to examine VVIAT, FLF, ITL, VIV, IAT, VIT, TVI, and VVIA peptide levels. Immunofluorescence staining was performed to measure APP and early endosome antigen 1 immunoreactivity. Our results show that the protective A673T mutation decreases Aβ42/Aβ40 rate by downregulating IAT and upregulating VVIA levels. Pathogenic A673V, E682K, and E693Q mutations promote Aβ42/Aβ40 rate by increasing levels of CTF99, Aβ42, Aβ40, and IAT, and decreasing VVIA levels. Pathogenic E693G mutation shows no significant change in Aβ42/Aβ40 ratio because of inhibition of γ-secretase activity. APP mutations can change location from the cell surface to early endosomes. Our findings confirm that certain APP mutations accelerate Aβ generation by affecting the long Aβ cleavage pathway and increasing Aβ42/40 rate, thereby resulting in Alzheimer's disease.

Keywords: APP mutations; Alzheimer's disease; amyloid β; cellular localization; liquid chromatography-tandem mass chromatography; long Aβ; nerve regeneration; neural regeneration; β-amyloid precursor protein.

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Conflict of interest statement

None

Figures

Figure 1
Figure 1
APP mutations affect the APP cleavage pathway. Expression levels of APP, CTFs, BACE1, and presenilin1 (PS1) in HEK293 cells expressing vehicle, APP-WT, APP mutants A693T, A693V, E682K, E693Q, or E693G plasmids. (A) Western blot assay of cell lysates from HEK293 cell line transfected with APP mutations and BACE1. (B) APP and APP-CTFs in cell lysates. (C–F) Quantification results of BACE1 (C), APP (D), presenilin 1 (E), and CTF99/CTF83 (F). CTFs include CTF99 and CTF83. Data are expressed as the mean ± SD (one-way analysis of variance). *P < 0.05, ***P < 0.01, vs. APP-WT group. APP: β-Amyloid precursor protein; WT: wild-type; HEK: human embryonic kidney; CTFs: C-terminal fragments; BACE1: beta-secretase 1.
Figure 2
Figure 2
Effect of APP mutations on production of Aβ variants (enzyme-linked immunosorbent assay). HEK293 cells were transfected with APP-WT, or APP mutants (A693T, A693V, E682K, E693Q, or E693G) plasmids. (A) Secreted Aβ42 in conditioned medium was quantified by enzyme-linked immunosorbent assay. APP-A673T vs. APP-WT (P = 0.0006), APP-A673V vs. APP-WT (P = 0.0011), APP-E682K vs. APP-WT (P = 0.005), APP-E693G vs. APP-WT (P = 0.07), and APP-E693Q vs. APP-WT (P = 0.028). (B) Secreted Aβ40 in conditioned medium was quantified by enzyme linked immunosorbent assay. APP-A673T vs. APP-WT (P = 0.017), APP-A673V vs. APP-WT (P = 0.002), APP-E682K vs. APP-WT (P = 0.004), APP-E693G vs. APP-WT (P = 0.004), and APP-E693Q vs. APP-WT (P = 0.18). Data are expressed as the mean ± SD (one-way analysis of variance). *P < 0.05, **P < 0.01, ***P < 0.001, vs. APP-WT group. APP: β-Amyloid precursor protein; Aβ: amyloid-beta; WT: wild-type; HEK: human embryonic kidney.
Figure 3
Figure 3
Effect of APP-WT and APP mutations on long Aβ cleavage by LC-MS/MS. HEK293 cells were transfected with BACE1 and APP-WT or APP mutants (A673T, A673V, E682K, E693G, and E693Q). (A) Relative VVIA levels in cell lysates. (B) Relative IAT levels in cell lysates. (C) Relative VIT, TVI, and VVIA levels in cell lysates during Aβ48–38 formation. Results are expressed as a ratio of APP-WT. (D) Relative ITL, VIV, and IAT levels in cell lysates during Aβ49–40 formation. Results are expressed as a ratio of APP-WT. Data are expressed as mean ± SD, and analyzed by one-way analysis of variance. *P < 0.05, **P < 0.01, vs. APP-WT group. APP: β-Amyloid precursor protein; Aβ: amyloid-beta; WT: wild-type; LC-MS/MS: liquid chromatography-tandem mass spectrometer; HEK: human embryonic kidney; BACE1, beta-secretase 1.
Figure 4
Figure 4
Expression of APP in the HEK293 cell line. HEK293 cells transfected with BACE1 and APP-WT or APP mutants (A673T, A673V, E682K, E693G, and E693Q). Double immunofluorescence of APP/APP mutations and EEA1 in HEK293 cells. APP or APP mutants are shown in green (Alexa Fluor 488) (arrows) and EEA1 in red (TRITC). The nucleus was stained with Hoechst 33258. Scale bars: 10 μm. APP: β-Amyloid precursor protein; WT: wild-type; HEK: human embryonic kidney; BACE1: beta-secretase 1; EEA1: early endosome antigen 1.
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