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. 2012 Aug;11(4):559-68.
doi: 10.1111/j.1474-9726.2012.00817.x. Epub 2012 Apr 9.

Oxidative lipid modification of nicastrin enhances amyloidogenic γ-secretase activity in Alzheimer's disease

A-Ryeong Gwon  1 , Jong-Sung Park, Thiruma V Arumugam, Yong-Kook Kwon, Sic L Chan, Seol-Hee Kim, Sang-Ha Baik, Sunghee Yang, Young-Kwang Yun, Yuri Choi, Saerom Kim, Sung-Chun Tang, Dong-Hoon Hyun, Aiwu Cheng, Charles E Dann 3rd, Michel Bernier, Jaewon Lee, William R Markesbery, Mark P Mattson, Dong-Gyu Jo
Affiliations

Oxidative lipid modification of nicastrin enhances amyloidogenic γ-secretase activity in Alzheimer's disease

A-Ryeong Gwon et al. Aging Cell. 2012 Aug.

Abstract

The cause of elevated level of amyloid β-peptide (Aβ42) in common late-onset sporadic [Alzheimer's disease (AD)] has not been established. Here, we show that the membrane lipid peroxidation product 4-hydroxynonenal (HNE) is associated with amyloid and neurodegenerative pathologies in AD and that it enhances γ-secretase activity and Aβ42 production in neurons. The γ-secretase substrate receptor, nicastrin, was found to be modified by HNE in cultured neurons and in brain specimens from patients with AD, in which HNE-nicastrin levels were found to be correlated with increased γ-secretase activity and Aβ plaque burden. Furthermore, HNE modification of nicastrin enhanced its binding to the γ-secretase substrate, amyloid precursor protein (APP) C99. In addition, the stimulation of γ-secretase activity and Aβ42 production by HNE were blocked by an HNE-scavenging histidine analog in a 3xTgAD mouse model of AD. These findings suggest a specific molecular mechanism by which oxidative stress increases Aβ42 production in AD and identify HNE as a novel therapeutic target upstream of the γ-secretase cleavage of APP.

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Figures

Figure 1
Figure 1
The lipid peroxidation product HNE enhances γ-secretase activity. Cultured rat cortical neurons (A and B), hippocampal neurons (C and D), and SH-SY5Y cells (E and F) were pre-incubated with 500 µM glutathione-ethyl ester (GSH) or 2 µM γ-secretase inhibitor (GSI) for 1 h, or with vitamin E (50 ng/ml) for 24 h before being treated with HNE. Cells were collected after 3 h of incubation with HNE or FeSO4. Lysates of primary cultured cortical (A and B) and hippocampal (C and D) neurons were tested for γ-secretase activity. Values are the mean ± S.D. of at least 3 independent experiments. *p < 0.01, **p < 0.05. (E and F) SH-SY5Y cells were transfected with the constructs of C99-GVP with UAS-luciferase reporter gene. Cells were collected after 3h of incubation with HNE or FeSO4. Values are the mean ± S.D. of at least 3 independent experiments. *p < 0.01, **p < 0.05 vs. controls. ##p < 0.05 vs. HNE or FeSO4 treated samples. (G) Total extracts of 2×108 SH-SY5Y cells were immunoprecipitated with anti-PS1 antibody. Immunoprecipitants were incubated with HNE at 37 °C for 30 min and analyzed for γ-secretase activity in vitro. Values are the mean ± S.D. of at least 3 independent experiments. *p < 0.01 vs. non-treated control.
Figure 2
Figure 2
The membrane lipid peroxidation product HNE increases Aβ42/Aβ40 ratio and AICD production. (A and B) HNE treating increased the amounts of secreted Aβ40, Aβ42 and Aβ42/Aβ40. After treating SH-SY5Y cells stably expressing mutant (Swedish) APP with HNE for 24 h, media were harvested and analyzed by sandwich ELISA for secreted Aβ40 (dark bars) and Aβ42 (light bars). In control cultures, the concentrations of Aβ40 and Aβ42 were 2067 ± 134 pg/ml protein and 848 ± 56 pg/ml, respectively (mean ± SD; n=3). *p < 0.01 vs. controls, #p < 0.01 vs. HNE-treated samples. (C) C99/SH-SY5Y cells (left panel) and BACE1KO MEF (right panel) were treated for 3 h with HNE (10 µM) in the presence or absence of the γ-secretase inhibitor DAPT (GSI; 1 µM). C99 and C83 levels were then analyzed using anti-APP-CTF antibody. (D and E) C100-Flag was incubated with the CHAPSO (D) or dodecyl-maltoside (E) solubilized lysate of SH-SY5Y cells at 37 °C; and the reaction was terminated at the indicated times by placing reaction tubes on ice. Reaction mixtures were separated in a 16% Tricine gel and subjected to immunoblotting for AICD using APP-CTF antibody.
Figure 3
Figure 3
Nicastrin is modified by HNE. (A) Left) SH-SY5Y cells were pre-incubated with GSH (500 µM) for 1 h before being treated with HNE (10 µM) or vitamin E (50 ng/ml), both for 24 h, before HNE treatment (3 h). The cell lysates (25 µg/lane) obtained were then immunoblotted using antibodies for the indicated-γ-secretase subunits. Right) Cell lysates (800 µg) in SDS-IP buffer were immunoprecipitated with an anti-HNE antibody and then subjected to immunoblot analysis using antibodies against nicastrin (Nct), PS1, Aph-1, or Pen-2 (* indicates the band corresponding to the light chain of anti-HNE antibody). (B) Cell lysates in SDS-IP buffer were immunoprecipitated with anti-nicastrin antibody and HNE-nicastrin conjugates were then detected using an antibody against HNE protein adducts. (C) HNE-modified nicastrin remained in the γ-secretase complex. Cell lysates in CHAPSO-IP buffer were co-immunoprecipitated with anti-PS1 antibody and then immunoblotted using antibodies against HNE-protein adducts or nicastrin. (D) Nicastrin was modified by HNE in vitro. Total extracts of 2×108 SH-SY5Y cells were immunoprecipitated with anti-nicastrin antibody. The immunopurified nicastrin obtained was incubated with increasing concentrations of HNE for 30 min at 37 °C. After incubation, samples were separated by SDS-PAGE, blotted, and lipid-protein conjugates and immunopurified nicastrin were detected using antibodies against HNE protein adducts or nicastrin. (E) Immunoprecipitants in (D) were incubated with HNE for 30 min at 37 °C and analyzed for γ-secretase activity. Values are the mean and ± SD of at least 3 independent experiments. *p < 0.01, **p < 0.05 vs. the controls. (F) The binding affinity of HNE-modified nicastrin for C100-Flag. To estimate the relative strength of binding between C100-Flag and non-modified and HNE-modified nicastrins, purified nicastrin ectodomain (Nct(ECD)) was preincubated with HNE at molar ratios of 1:0, 1:0.5 or 1:1 and then incubated with purified C100-Flag. After 5 washes, HNE-modified Nct(ECD) co-precipitated with C100-Flag. The addition of Flag peptide to this mixture prevented the precipitation.
Figure 4
Figure 4
HNE-modified nicastrin is associated with increased γ-secretase and BACE1 enzymatic activities in sporadic AD brain tissue samples. (A) Immunoblots showing relative levels of each protein in γ-secretase complex and BACE in samples from the inferior parietal lobule of AD and control subjects. Loaded protein levels were normalized versus β-actin. Experiments were performed at least three times. (B and C) BACE1 (B) and γ-secretase (C) enzymatic activities were evaluated using AD-enzymatic crude extracts incubated with fluorescent-labeled peptides bearing the β-site or γ-site of APP. BACE1 and γ-secretase enzymatic activities in AD brain extracts were normalized versus mean values in controls. BACE1 and γ-secretase activities were significantly greater in AD subjects (p < 0.0001). (D) C100-Flag was incubated with CHAPSO-solubilized membrane lysates of inferior parietal lobule specimens from the brains of AD patients (A1-6) and neurologically normal subjects (N1-6) at 37 °C; reactions were terminated after 1 h by placing reaction tubes on ice. Reaction mixtures were separated in 16% Tricine gel and immunoblotted for AICD. (E) Nicastrin was modified by HNE in the AD brain. Proteins in brain tissue samples from AD and control subjects were immunoprecipitated using antibodies against nicastrin, and then immunoblotted. (F) Linear regression analysis of γ-secretase activity versus HNE-nicastrin levels in inferior parietal lobes from clinically diagnosed and neuropathologically confirmed AD patients, and non-demented control subjects. (G) Linear regression analysis of HNE-nicastrin levels versus neuritic Aβ plaque numbers in AD and control brains. (H) Linear regression analysis of γ-secretase activities versus numbers of neuritic Aβ plaques.
Figure 5
Figure 5
Nicastrin is redistributed to an APP-enriched lipid raft fraction in AD brains and in response to direct exposure of neural cells to HNE. (A and B) Control and HNE-treated (10 µM for 3 h) SH-SY5Y cells (A), and AD and control inferior parietal lobes (B) were lysed in sodium carbonate buffer and subjected to flotation sucrose gradient centrifugation to isolate lipid rafts. Equal volumes of fractions were immunoblotted with antibodies against PS1 CTF, nicastrin, APP, APP CTF, flotillin-1 (a lipid raft marker), and γ-adaptin (a marker of clathrin-coated, non-raft membranes). (C) Lipid raft fractions (fractions 4 and 5) were immunoprecipitated using an antibody against HNE-modified proteins, and precipitated proteins were immunoblotted using anti-nicastrin antibody. (D and E) The signal intensities of PS1 CTF and nicastrin were quantified and fraction 4 versus fraction 5 (F4/F5) ratios were calculated for these proteins. The values shown are mean and S.D. (n=5; #p<0.01, *p<0.05 vs. controls). (F and G) γ-secretase activities in fractions 4 and 5, are reported as F4/F5 ratios. Values are the mean and S.D (n=5; *p<0.05 compared to control).
Figure 6
Figure 6
The histidine analogue histidyl hydrazide (AG/01) reduces the ratio of Aβ42/Aβ40, γ-secretase activity and HNE-modified Nct in the brains of AD mice. Seven month-old male 3xTg-AD mice were treated with AG/01 (20 mg/kg) or vehicle (PBS) intraperitoneally every other day for one month. (A–C) Aβ40 and Aβ42 levels, and the ratio of Aβ42/Aβ40 in the hippocampal and neocortical tissues of 3xTg-AD mice treated with PBS or AG/01. Values are the mean and S.D. (6 mice/group). **p < 0.01 vs. vehicle-treated mice. (D) Hippocampal and neocortical tissues were homogenized and γ-secretase activity was measured using fluorogenic substrates. The values shown are the means and S.D. (6 mice/group). *p < 0.05, **p < 0.01 vs. vehicle-treated mice. (E and F) Hippocampal and neocortical lysates were immunoprecipitated with anti-nicastrin antibody, and HNE-nicastrin conjugates were detected using an antibody against HNE-protein adducts. Signal intensities of HNE-Nct and total amounts of immunoprecipitated nicastrin were quantified and HNE-Nct/total Nct ratios were calculated. Values (panel F) are the mean and S.D. *p < 0.05 vs. vehicle-treated mice.
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