The A673T mutation in the amyloid precursor protein reduces the production of β-amyloid protein from its β-carboxyl terminal fragment in cells
- Asuka Kokawa†1,
- Seiko Ishihara†1,
- Hitomi Fujiwara1,
- Mika Nobuhara1,
- Minori Iwata2,
- Yasuo Ihara2 and
- Satoru Funamoto1Email author
© Kokawa et al. 2015
Received: 16 September 2015
Accepted: 22 October 2015
Published: 4 November 2015
The A673T mutation in the amyloid precursor protein (APP) protects against Alzheimer’s disease by reducing β-amyloid protein (Aβ) production. This mutation reduced the release of the soluble APP fragment (sAPPβ), which is processed by β-secretase, suggesting a concomitant decrease in the β-carboxyl fragment of APP (C99), which is a direct substrate of γ-secretase for Aβ production. However, it remains controversial whether the level of C99 is significantly reduced in cells expressing APP that carry A673T as the cause of reduced Aβ production. Here, we investigated the effect of the A673T mutation in C99 on γ-cleavage in cells.
We found that the level of C99 in cells expressing APP A673T was indistinctive of that observed in cells expressing wild-type APP, although the release of sAPPβ was significantly reduced in the APP A673T cells. In addition, our reconstituted β-secretase assay demonstrated no significant difference in β-cleavage on an APP fragment carrying the A673T mutation compared with the wild-type fragment. Importantly, cells expressing C99 containing the A673T mutation (C99 A2T; in accordance with the Aβ numbering) produced roughly half the level of Aβ compared with the wild-type C99, suggesting that the C99 A2T is an insufficient substrate of γ-secretase in cells. A cell-free γ-secretase assay revealed that Aβ production from the microsomal fraction of cells expressing C99 A2T was diminished. A sucrose gradient centrifugation analysis indicated that the levels of the C99 A2T that was codistributed with γ-secretase components in the raft fractions were reduced significantly.
Our data indicate that the A673T mutation in APP alters the release of sAPPβ, but not the C99 level, and that the C99 A2T is an inefficient substrate for γ-secretase in cell-based assay.
KeywordsAlzheimer’s disease Aβ APP γ-secretase C99 Lipid raft
Senile plaques are one of neuropathological hallmarks of Alzheimer’s disease (AD) and composed of β-amyloid protein (Aβ). Aβ is a small protein consisting of 38–43 residues that is produced from the amyloid precursor protein (APP) via sequential cleavage by β- and γ-secretases [1–5]. Longer forms of Aβ, such as Aβ42 and Aβ43, are prone to aggregation and are initially deposited in the brain . Familial AD (FAD) mutations in APP, presenilin 1, and presenilin 2 increase the ratio of Aβ42/Aβ40. We found previously that FAD mutations preferentially produced a longer form of the APP intracellular domain (AICD49–99), rather than AICD50–99, and that the expression of Aβ48, as a counterpart of AICD49–99, resulted in an increase in Aβ42/Aβ40 ratio, as found for FAD mutations [7, 8].
Genetic analyses in AD provide not only insights into the molecular mechanisms underlying the pathogenesis of AD, but also a perspective regarding the prevention and cure of the disease. Recently, the A673T mutation in APP was reported as a novel protective mutation against AD that acts by reducing Aβ production . Although this mutation seems to be restricted to Iceland and Nordic countries, this finding, together with FAD mutations, strongly supports the amyloid hypothesis [10–17]. The A673T mutation causes a substantial decrease in the level of the soluble APP fragment processed by β-secretase (sAPPβ) [9, 18]. It is reported that the level of the β-carboxyl fragment of APP (C99, also known as βCTF and CTFβ), which is a direct substrate of γ-secretase for Aβ production, in cells expressing APP A673T was one fourth of that detected in cells expressing APP wild-type (WT), which suggests the concomitant decrease of C99 as a counterpart of sAPPβ . Conversely, Benilova and colleagues reported that cells expressing APP A673T exhibited no significant difference in the levels of C99 compared with cells expressing WT APP, in spite of a nearly two hundredth reduction in sAPPβ levels . Thus, it is unclear whether a decrease in sAPPβ leads to a concomitant decrease in C99 as a cause of reduced Aβ production in cells expressing APP A673T.
Recently, we found that γ-secretase preferentially cleaved substrates with a short ectodomain, which supports the idea that γ-secretase recognizes the amino terminus of the substrate [20, 21]. The A673T mutation (i.e., A2T; in accordance with the Aβ numbering) lies within the amino terminus of the C99 substrate. It remains unknown whether the A2T substitution in C99 affects γ-secretase-dependent cleavage in cells. In this study, we examined the direct effect of A2T substitution in C99 on γ-secretase-dependent cleavage.
Materials and methods
Cell lysates and conditioned media were subjected to western blotting using the following antibodies: 6E10 (total Aβ; Covance), 82E1 (total Aβ; IBL), E50 (total Aβ), BA27 (Aβ40), BC05 (Aβ42), and anti-GFP (Santa Cruz) [22, 23].
The C99-FLAG tag-coding region was fused to the APP signal peptide. Additional Asp and Ala residues were inserted between the APP signal peptide and C99-FLAG, which allowed precise cleavage at the β-cleavage site, generating C99 and Aβ species that start from Asp-1 [8, 24]. This C99-FLAG coding region or full-length APP region was inserted prior to the internal ribosome entry site (IRES) of pMXIG. This construct allows cells to express GFP as an internal standard. For pulse-chase analysis, the full-length APP-FLAG tag-coding region was inserted into pcDNA4/TO (Invitrogen) and transfected in T-Rex CHO cells (Invitrogen).
Pulse-chase analysis of APP processing
T-Rex CHO cells were cultured in 12-well plates and transfected with pcDNA4/TO carrying full-length APP (WT or A673T) . Twenty-four hours later, cells were treated with tetracycline at a concentration of 1 μg/mL for 4 h, to express APP. Cells were washed twice in tetracycline-free medium and incubated in the medium for 24 h. Cells and media were collected every 4 h and subjected to western blotting, to visualize and quantify the levels of APP, sAPPβ, sAPPα, C99, C83, and Aβ.
A γ-secretase assay and coimmunoprecipitation analyses were performed as described previously [21, 26]. Briefly, the C99-FLAG substrate was expressed in sf9 cells and purified using anti-FLAG M2 agarose beads. The C99-FLAG substrate was incubated with a γ-secretase fraction for 4 h and subjected to western blotting. For coimmunoprecipitation of C99 with γ-secretase components, the C99-FLAG substrate was immobilized on anti-FLAG M2 magnetic beads and incubated with a γ-secretase fraction. For β-secretase assay, the human APP fragment 633–685 (numbering from APP751) fused with N-terminal Myc and C-terminal FLAG tags (referred to as APP633–685-FLAG) was expressed in Escherichia coli BL21 cells and affinity purified using ANTI-FLAG M2 beads . The purified APP633–685-FLAG (500 nM) was incubated with β-secretase (Sigma) for 4 h, according to the manufacturer’s instructions. β-Cleaved C-terminal fragments (Aβ33-FLAG) from APP633–685-FLAG were visualized and quantified using the E50 antibody.
Cell-free γ-secretase assay
Cells were cultured in Dulbecco’s modified Eagle’s medium (Sigma) supplemented with 10 % FBS (Invitrogen) and penicillin/streptomycin (Invitrogen). Harvested cells were homogenized in Buffer A (20 mM PIPES, pH 7.0, 140 mM KCl, 0.25 M sucrose, and 5 mM EGTA) with a glass/Teflon homogenizer. Postnuclear supernatants were subjected to ultracentrifugation at 100,000 g for 1 h. The pellets were resuspended in Buffer A at a protein density of 2.5 mg/mL and defined as microsomal fractions . Microsomal fractions from C99 WT and C99 A2T cells were incubated at 37 °C and lipids were extracted with chloroform/methanol before western blotting.
Isolation of CHAPSO-insoluble fractions
The CHO homogenate was adjusted to 40 % sucrose and centrifuged on a discontinuous sucrose gradient for 20 h at 4 °C using an SW 41 Ti rotor (Beckman) [28, 29]. After centrifugation, the homogenate was fractionated into 12 fractions and subjected to western blotting using the following antibodies: N1660 (Nicastrin; 1/3000 in TBS containing 0.1 % Tween; Sigma), anti-Aph-1a C-term antibody (Aph-1; 1/1000 in TOYOBO Can Get Signal; Covance), anti-PS1–CTF antiserum (Presenilin 1 CTF; 1/3000 in TBS containing 0.1 % Tween; gifts from Drs. T. Iwatsubo and T. Tomita, The University of Tokyo), anti-Pen-2 antibody (Pen-2; 1/3000 in TBS containing 0.1 % Tween; a gift from Dr. A. Takashima, National Center for Geriatrics and Gerontology), anti-caveolin antibody (caveolin-1; 1/1000 in TBS containing 0.1 % Tween; Santa Cruz), and anti-flotillin antibody (flotillin-1; 1/1000 in TBS containing 0.1 % Tween; BD).
Effect of the A673T mutation on C99 levels in cells
Effect of the A673T mutation on the β-cleavage of the APP fragment
Effect of the A2T substitution on C99 regarding Aβ production in cells
No effect of the A2T substitution in C99 on its γ-cleavage in a membrane-soluble state
Effect of the A2T substitution on Aβ production in microsomal fractions
Altered distribution of C99 A2T in lipid rafts
The A673T mutation in APP has been recognized as a protective variant of late-onset of AD and has been related to longevity in an Icelandic population . Although this variant is extremely rare in non-Nordic countries, it is important to explore the mechanism of reduced Aβ production . Biochemical and cell-based assays demonstrated reductions in the β-cleavage on an APP A673T fragment and in sAPPβ release from APP A673T cells, which suggests that reduced C99 production results in a reduction of Aβ production [9, 18]. In fact, β-cleavage has been a promising drug target for anti-AD therapeutics, to reduce C99 and Aβ [2, 33]. However, Benilova and colleagues observed no significant difference in the levels of C99 between APP WT and A673T cells, despite a substantial reduction in sAPPβ level . This discrepancy prompted us to revisit this issue. In the present study, we showed that the level of C99 in APP A673T cells was comparable to that detected in APP WT cells, despite a significant reduction in sAPPβ. Currently, we do not have clear interpretations of the discrepancy between the amount of sAPPβ and C99 levels in APP A673T cells. However, it is interesting to note that the generation rate of C83 was distinct from that of C99, although the generation curves of sAPPα and sAPPβ had some resemblance to each other (see Fig. 2b and c). This suggests that the levels of those soluble APP fragments do not reflect the levels of their stubs in cells precisely.
Our reconstituted β-secretase assay demonstrated that the APP633–685 fragment carrying A673T was cleaved by β-secretase, as was the WT APP638–685 fragment. This result was consistent with our quantitative analyses of C99 levels on A673T cells; however, it was inconsistent with the result of the cell-based assay of sAPPβ as reported previously [9, 18]. One possible interpretation for the inconsistent results between the reconstituted β-secretase assay and the cell-based assay is that APP A673T is preferentially processed by an unknown enzyme, leading to a reduced level of sAPPβ from APP A673T cells. Recently, Willem and colleagues reported η-secretase as a novel APP-processing enzyme that produces a high-molecular-weight carboxyl terminal fragment of APP (CTFη), which can be processed into C83 and C99 by α- and β-secretases, respectively . One can safely say that η-secretase cleaves APP A673T preferentially. If so, APP A673T cells produce a lesser amount of sAPPβ, but an equal level of C99, compared with APP WT cells. Alternatively, η-secretase may preferentially cleave sAPPβ produced from APP A673T cells.
We have shown that C99 A2T cells also release a lower amount of Aβ compared with C99 WT cells. This is direct evidence that the A2T substitution on C99 alters Aβ production. This was also observed for the other cell lines. Our western blotting assay using 6E10 and E50 revealed that Aβ A2T and C99 A2T were transferred onto nitrocellulose, as were Aβ WT and C99 WT. Our data demonstrated that 82E1 failed to recognize Aβ A2T and C99 A2T on western blot and to capture the C99 A2T substrate on immunoprecipitation. This indicates that our evaluation by western blotting is reliable and that C99 A2T is an inefficient substrate for Aβ production in cells. Importantly, the amount of the p3 peptide in the media of C99 WT and C99 A2T cells was indistinctive, which suggests that the A2T substitution affects the γ-cleavage of C99, but not that of C83. We also found that the A2T substitution in C99 caused no accumulation of intracellular Aβ; rather, it reduced the level of intracellular Aβ. Our data demonstrated that reduced Aβ levels in the medium of C99 A2T cells were caused by impaired γ-cleavage of C99 A2T in cells.
We expected that C99 A2T would be an inefficient substrate for γ-secretase even in the CHAPSO-solubilized γ-secretase assay. However, we observed no significant reduction in Aβ production from the recombinant C99 A2T substrate, and an interaction with γ-secretase components in the CHAPSO-solubilized state. This implies that membrane solubilization disrupts the intact distribution of C99 A2T, which allows free collision between the enzyme and the substrate in the solution. To mimic the cell-based assay in a biochemical analysis, we chose a cell-free assay that used the microsomal fraction of cells. This approach provides an assessment of Aβ production in intact membranes. As expected, the cell-free assay reproduced the altered Aβ production in a membrane fraction of C99 A2T cells. This suggests that the subcellular distribution of C99 A2T is altered, and that this redistribution reduces the interaction between C99 A2T and γ-secretase.
This report indicates that the C99 level in APP A673T cells is comparable to that in APP WT cells despite a significant reduction in released sAPPβ level, suggesting vulnerable correlation between levels of sAPPβ and C99 in cells. Our data demonstrate that the A673T mutation in C99 impairs γ-cleavage the in cell-based assay. Assessment of observed results in vivo may be crucial to elucidate the protective mechanism of A673T mutation against AD.
Availability of supporting data
The data set supporting the results of this article are included within the article and its additional files.
APP intracellular domain
amyloid precursor protein
β-carboxyl fragment of APP
internal ribosome entry site
soluble APP fragment processed by β-secretase
We wish to thank Dr. A. Asami, Takeda Chemical Industries, for BA27 and BC05; Drs. T. Tomita and T. Iwatsubo, The University of Tokyo, for the anti-PS1-CTF anti-serum; Dr. A. Takashima, National Center for Geriatrics and Gerontology, for anti-Pen-2 antibody and Dr. F. Kametani, Tokyo Institute of Psychiatry, for E50. This work was supported in part by The Takeda Science Foundation (to S.F.), Izumi Science Technology Foundation (to S.F.) and The Naito Foundation (to S.F.).
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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