Anthocyanins Protect SK-N-SH Cells Against Acrolein-Induced Toxicity by Preserving the Cellular Redox State

Abstract

In Alzheimer’s disease (AD) and in mild cognitive impairment (MCI) patients, by-products of lipid peroxidation such as acrolein accumulated in vulnerable regions of the brain. We have previously shown that acrolein is a highly reactive and neurotoxic aldehyde and its toxicity involves the alteration of several redox-sensitive pathways. Recently, protein-conjugated acrolein in cerebrospinal fluid has been proposed as a biomarker to distinguish between MCI and AD. With growing evidence of the early involvement of oxidative stress in AD etiology, one would expect that a successful therapy should prevent brain oxidative damage. In this regard, several studies have demonstrated that polyphenol-rich extracts exert beneficial effect on cognitive impairment and oxidative stress. We have recently demonstrated the efficacy of an anthocyanin formulation (MAF14001) against amyloid-β-induced oxidative stress. The aim of this study is to investigate the neuroprotective effect of MAF14001 as a mixture of anthocyanins, a particular class of polyphenols, against acrolein-induced oxidative damage in SK-N-SH neuronal cells. Our results demonstrated that MAF14001, from 5μM, was able to efficiently protect SK-N-SH cells against acrolein-induced cell death. MAF14001 was able to lower reactive oxygen species and protein carbonyl levels induced by acrolein. Moreover, MAF1401 prevented glutathione depletion and positively modulated, in the presence of acrolein, some oxidative stress-sensitive pathways including the transcription factors NF-κB and Nrf2, the proteins γ-GCS and GSK3β, and the protein adaptator p66Shc. Along with its proven protective effect against amyloid-β toxicity, these results demonstrate that MAF14001 could target multiple mechanisms and could be a promising agent for AD prevention.

Keywords

Acrolein Alzheimer’s disease anthocyanins antioxidant glutathione oxidative stress polyphenols

INTRODUCTION

Alzheimer’s disease (AD) is an age-related neurodegenerative disease affecting more than 40 million individuals worldwide with the global cost estimated around US $604 billion in 2010 [1]. The AD brain undergoes progressive dysfunction, degeneration of neurons and synapses in selective areas of the brain leading to the observed cognitive decline. AD is a multifactorial disease characterized by several pathological features including the presence of amyloid-β (Aβ) plaques, neurofibrillary tangles (NFTs), neuritic dystrophy, synaptic loss, and high levels of oxidative damage. These alterations are also observed in various animal models of AD [2]. There is a growing body of evidence supporting the involvement of oxidative stress in AD with consistent decrease in antioxidant levels and activities, increase in protein carbonyls and lipid peroxidation in AD brain (see review by [3]). These oxidative-induced damage are also evidenced in mild cognitive impairment (MCI) patients [4], a transition stage between normal aging and dementia, indicating that oxidative stress is upstream of the pathological features. This notion was also observed in various animal models of AD [2] and is highlighted by the recent demonstration that the production of the Aβ peptide is triggered by lipid peroxidation [5]. These studies indicate that oxidative stress represents an early mechanism in the pathophysiology of AD.

Lipid peroxidation process leads to various toxic by-products such as 4-hydroxynonenal (4-HNE), malondialdehyde (MDA), and, at less extent, the most reactive, acrolein. This latter has been found to be elevated in several brain regions from AD such as hippocampus, amygdala, middle temporal gyrus, frontal and temporal cortices, and cerebellum [6 –8]. Moreover, the production of acrolein was observed earlier and was approximately five times higher than 4-HNE [9, 10]. Levels of acrolein were significantly higher in hippocampus and cerebellum from early AD as compared to MCI and control subjects [9]. In hippocampus from AD, the acrolein/guanosine adducts in nuclear DNA are two-fold higher as compared to age-matched control while it was not significantly different for 4-HNE/guanosine adducts [11, 12]. In cerebrospinal fluid, the ratio Aβ/protein-conjugated acrolein was also higher in MCI as compared to AD and the decrease of this ratio was correlated with cognitive decline and the MMSE score [8]. In plasma, levels of protein-conjugated acrolein were also higher in MCI and AD subjects as it did for stroke patients as compared to control subjects [13, 14]. Moreover, its level was also correlated with the MMSE score [15]. These results indicate that the measurement of acrolein derivatives together with Aβ in biologic fluids is useful to estimate the severity of dementia [16]. These findings also indicated that lipid peroxidation and formation of acrolein are present very early in multiple brain disease. The important contribution of acrolein-associated damage in AD brain and neuronal loss was strengthened by the demonstration that acrolein can induce AD-like pathologies after its administration to rats [17].

With growing evidence of the implication of acrolein in human diseases such as AD, the development of strategies preventing acrolein-induced damage and/or to scavenge acrolein is of great relevance. In this regard, we have previously demonstrated that curcumin, a standardized bacopa monniera extract, and caffeic acid can protect neuronal cells against acrolein toxicity [18 –23]. Other polyphenols such as the catechin sub-class, theaflavins, cyanomaclurin, phloretin, and phloridzin have also been demonstrated to be efficient scavengers of acrolein [24, 25].

Several studies have demonstrated that a daily consumption of polyphenols, especially anthocyanin-rich extracts, has beneficial effects on cognitive performance and prevents memory deficits. For instance, anthocyanin-rich food supplementation showed a significant cognitive improvement in subjects with early signs of dementia and MCI [26, 27]. In animal models, the administration of anthocyanins can also prevent cognitive and motor behavioral deficits [28, 29].

Anthocyanins, a subfamily of polyphenols, which are abundant in red wine, bilberry, and black currant, have the ability to cross the blood-brain barrier and reach the central nervous system [30 –34]. Anthocyanins possess multifunctional benefits including anti-oxidative and anti-inflammatory properties, cardioprotection, and chemoprevention for cancer. Anthocyanins are secondary plant metabolites responsible for the blue, purple, and red color of many plants and occur primarily as glycosides of their respective anthocyanidin chromophores [35]. Currently, interest in anthocyanin pigments has drastically intensified because of their potential health-promoting benefits. Anthocyanins are potent antioxidants and the protective effect of anthocyanins against oxidative stress-induced damage is promising as shown on in vivo models [36, 37]. For instance, cyanidin-3-O-β-D-glucopyranoside fraction from mulberry fruit reduced the level of 8-hydroxy-2-deoxyguanosine and upregulated superoxide dismutase in streptozotocin-induced diabetic rats [38]. In HepG2 cells and in diabetic db/db mice as well, cyanidin-3-0-glucoside was able to reduce oxidative stress level monitored by various markers through the modulation of the glutathione (GSH) synthesis pathway [39]. Also, cyanidin-3-glucoside reduced ethanol-induced MDA formation in neurons from a mouse model of ethanol exposure [40]. Finally, cyanidin-3-O-glucoside and pelargonidin-3-O-glucoside attenuated mitochondrial oxidative stress and apoptosis induced by Bcl-2 inhibitor in cultured cerebellar granule neurons [41]. However, the effect of anthocyanins on acrolein-induced cell damage has not been investigated. We hypothesized that anthocyanins could be potent agents for protecting neuronal cells against acrolein-induced toxicity. We recently made up a mixed anthocyanin formulation (MAF14001) using four different anthocyanins which showed a protective effect against Aβ-induced SK-N-SH cell toxicity by the prevention of oxidative stress and mitochondrial dysfunction among others [42]. In this study, we found that MAF14001 protects neuronal cells against acrolein through the attenuation of reactive oxygen species (ROS) and carbonyl elevation, the preservation of the GSH level and the modulation of cellular redox pathways e.g. Nrf2, γ-GCS, NF-κB, p66Shc, and GSK3β.

MATERIALS AND METHODS

Materials

Kuromanin chloride, oenin chloride, callistephin chloride, and peonidin chloride were from Extrasynthese (Z.I Lyon Nord, France). Acrolein, hydrogen peroxide (H₂O₂), AmidoBlack and coomassie blue dyes, ochratoxin, cell survival assay Tox-8-Resazurin-based, monochlorobimane (MCB), Minimal Essential Medium Eagle (MEM), fetal bovine serum (FBS), penicillin, streptomycin, sodium pyruvate, 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 2-4 dinitrophenylhydrazine (DNPH), monochlorobimane (MCB), DL-buthionine-(S,R)-sulfoximine (BSO) and bovine serum albumin (BSA) were obtained from Sigma-Aldrich (Oakville, ON, Canada). Prolong gold antifade reagent was from Life Technologies (ON, Canada). Cytotoxicity Detection Kit-LDH (lactate dehydrogenase) was purchased from Roche Diagnostics (Laval, Quebec, Canada). 2^′,7^′-dichlorofluorescein-diacetate (DCF-DA) was from Invitrogen (Burlington, ON, Canada). Nuclear protein extraction kit and BCA protein estimation kit were obtained from Active Motif (California, USA) and from Pierce Biotechnology (Rockford, USA), respectively.

Monoclonal anti-rabbit GSK3β (≠9315), rabbit monoclonal anti-phospho-GSK3α/β (P-GSK3α/β-Ser21/9,≠9331) and rabbit polyclonal anti-phospho-tau (P-tau-S400-T403-S404,≠11837) antibodies were purchased from Cell Signaling Technology. Mouse polyclonal anti-NF-κB p50 (≠SC-53744) antibody, rabbit polyclonal anti-γ-glutamylcysteine synthase (γ-GCS,≠SC-22755), rabbit polyclonal anti-p66Shc (≠SC288), rabbit polyclonal anti-Nrf2 (≠SC722) antibodies were from Santa Cruz Biotechnology (Texas, USA). Rabbit monoclonal anti-phospho-S36-p66Shc (P-p66Shc,≠Ab54518) antibody was from Abcam (Cambridge, MA, USA). Mouse monoclonal anti-GAPDH (≠MAB374) antibody was from Chemicon (Millipore, Mississauga, ON, Canada). Rabbit Anti-DNP (# D9656) antibody, anti-rabbit (≠A6154), and anti-mouse (≠115-035-003) horseradish peroxidase (HRP)-conjugated secondary antibodies were from Sigma-Aldrich, Inc. Goat anti-rabbit IgG Alexa Fluor 488-conjugated (≠A11008) antibody was from Invitrogen (Burlington, ON, Canada). MilliQ water was used for all the experiments.

Anthocyanin/anthocyanidin formulation

Cyanidin-3-O-glucoside chloride, malvidin-3-O-glucoside chloride, pelargonidin-3-O-glucoside chloride and peonidin chloride were dissolved in PBS-HCl, except for peonidin chloride which is first dissolved in ethanol (final treatment concentrations do not exceed 0.05%) and stored at -20°C until used as previously described [42]. MAF14001 is an equimolar mixture of the three anthocyanins listed above and the anthocyanidin peonidin. For instance, 5μM MAF14001 corresponds to 5μM of each anthocyanin/anthocyanin listed above and corresponds to total anthocyanins of 9.9μg/ml.

Radical scavenging assay (DPPH)

The test is based on the reduction of DPPH radical absorbance. Briefly, the DPPH radical was dissolved in ethanol/distilled water (70/30 v/v). The reaction was carried out at room temperature (RT) in a 96-well plate for 30 min in dark by mixing 100μl of DPPH (0.2 mM) with 100μl ethanol 70% (v/v) with or without different concentrations of MAF14001. Values recorded for the DPPH radical in absence of MAF14001 and trolox was considered as a 100% . Furthermore, in order to assess the long lasting antioxidant effect of MAF14001, a time course (6 h) of DPPH scavenging was performed using a 0.6 mM DPPH solution as described above. The DPPH radical scavenging activity of each sample is determined as the decrease of the DPPH absorbance in presence of MAF14001 or Trolox compared to the DPPH absorbance in absence of these compounds. Trolox was used as the reference standard. Since the anthocyanins also absorb at 517 nm, the DPPH absorbance was recorded at 600 nm to ovoid interferences with the pigments. The absorbance was measured using the Synergy HT multidetection microplate reader at 30 min or every 10 min for DPPH radical scavenging kinetic.

Cell culture

SK-N-SH cells, a human neuroblastoma cell line from ATCC (Manassas, VA, USA), were maintained in MEM, supplemented with 10% (v/v) FBS, 100 U/ml penicillin, 100μg/ml streptomycin and 1% sodium pyruvate (1 mM) in a humidified incubator at 37°C with 5% CO₂ to 80% confluence.

Cytotoxity assays

SK-N-SH cells were plated at a density of 2.0x10⁴ cells/well in a 96-well polystyrene-coated plate and incubated for 24 h at 37°C then, the media was completely removed and cells were kept in MEM without serum for 1 h before any treatment. Cell death and survival were assessed 24 h after the treatments using, the LDH cytotoxicity detection kit and Tox-8 (Resazurin-based) kit following the manufacturer’s instructions, respectively.

Intracellular reactive oxygen/nitrogen species measurement

Intracellular ROS and reactive nitrogen species (RNS) was assessed by 2^′,7^′-dichlorofluorescein-diacetate (H₂-DCF-DA) as previously described [19]. H₂-DCF-DA is a cell permeable dye which upon hydrolyzed by intracellular esterases reacts with ROS/RNS to produce the highly fluorescent compound 2^′,7^′-dichlorofluorescein (DCF). The H₂-DCF-DA was chosen because acrolein itself does not interfere with the fluorescence of DCF [43]. Briefly, SK-N-SH cells (2×10⁴/well) were plated into 96-well plates and allowed 24 h to attach. The medium was removed and cells were first kept in PBS containing Ca^2 +/Mg^2 + and 10μM H₂-DCF-DA for 20 min, then cells were washed with PBS-Ca^2 +/Mg^2 + to remove the H₂-DCF-DA excess and treated with 10μM acrolein or 1 mM H₂O₂ in the presence or absence of different concentrations (5, 10, and 20μM) of MAF14001 for 1 h. Afterwards, cells were washed twice and the DCF fluorescence was measured with the excitation/emission filters at 485/535 nm using the Synergy HT multidetection microplate reader. The fluorescence recorded for the control, non-treated cells was considered as a 100% .

Total and fractioned protein extraction

SK-N-SH cells from 80% confluence flasks were starved in MEM for 1 h. Then, cells were treated with 10μM acrolein and/or 10μM MAF14001 for 1 h. Total proteins from SK-N-SH cells were extracted with a lysis buffer containing a cocktail of protease inhibitors. Nuclear and cytoplasmic proteins were extracted using the Active Motif kit following the manufacturer’s instructions. The BCA test was used to protein quantification.

Western blot analysis

Equal amount of protein cell lysates (40μg) were separated on 10% SDS-PAGE gels and transferred onto PVDF membranes using a Trans-Blot Turbo System (Bio Rad). Membranes were blocked for 1 h in TBS with 5% skim milk (or 5% BSA for phosphorylated proteins) and incubated with primary antibodies: anti-P-GSK3β/α and anti-GSK3β (1/1000), anti-P-tau-S400-T403-S404, anti-NF-κB p50 (1/200), anti-p66Shc (1/1000), anti-P-p66Shc (1/500), and anti-GAPDH (1/20000). Then, membranes were incubated with the secondary antibody HRP-conjugated anti-rabbit or anti-mouse (1/5000) for 1 h. Detection was realized with Immobilion Western Chemiluminescent-HRP Substrate and the bands were visualized and quantified by densitometric analysis using luminescent imaging system FluorChem. GAPDH was used as equal protein loading control in each well for cytoplasmic and total proteins and AmidoBlack staining was used for nuclear protein loading. Data are expressed as means of normalized proteins over the control group considered as a 100% .

Protein carbonyl measurements

Briefly, 20μg of whole cell extract was derivatized with 5 mM DNPH in 2 N HCl for 15 min at RT. Derivatized protein samples were then loaded into a 10% polyacrylamide gel for electrophoresis, and transferred onto a PVDF membrane using a Trans-Blot Turbo System (Bio Rad). The membranes were exposed to the rabbit anti-DNP antibody (1/2000) overnight followed by the secondary antibody anti-rabbit IgG HRP (1/5000) for 1 h in TBS-5% skim milk. Detection was realized with ImmobilonWestern Chemiluminescent-HRP Substrate (Millipore, USA) and membranes were stained with Coomassie blue dye in order to control the protein loading in each well. Bands were visualized and quantified by densitometric analysis using FluorChem software. Data are expressed as means of normalized proteins over the control group considered as a 100% .

Intracellular glutathione level measurement

The intracellular GSH level was monitored by confocal microscopy using the probe MCB, a GSH-specific dye. Briefly, SK-N-SH cells were cultured on cover slips coated with poly-D-lysine at a density of 1.5×10⁴ cells/well in 24-well plates. Cells were incubated for 24 h at 37°C and then starved and treated with acrolein (10μM) and/or MAF14001 (10μM) for 30 min. Positive control cells were treated with BSO (200μM), a γ-GCS synthesis inhibitor, for 24 h. At the end of the treatments, cells were washed three times with 1 ml of PBS containing Ca^2 +/Mg^2 +, and then were incubated for 15 min at 37°C in PBS-Ca^2 +-Mg^2 + containing 100μM of MCB in a dark room. Thereafter, cells were washed five times with PBS-Ca^2 +-Mg^2 + and finally fixed with cold 100% methanol at –20°C during 15 min before microscopy visualization.

Immunocytochemistry by fluorescence microscopy

SK-N-SH cells were cultured on cover slips coated with poly-D-lysine at a density of 1.5×10⁴ cells/well in 24-well plates. Cells were incubated for 24 h at 37°C then starved for 1 h and treated with acrolein (10μM) and/or MAF14001 (10μM) for 1 h. We used a 24 h treatment with BSO (200μM) or Ochratoxin (5μM), the Nrf2 inhibitor, as positive controls for γ-GCS synthesis and Nrf2 inhibition, respectively. At the end of the treatments, cells were washed three times with 1 ml of PBS-Ca^2 +-Mg^2 + then fixed with cold 100% methanol at –20°C during 15 min. Once the methanol removed and cells washed, they were permeabilized for 10 min at RT with 1 mL of 0.25% triton x-100 in PBS-Ca^2 +-Mg^2 +. Cells were then washed again three times for 5 min each and blocked 1 h with 5% BSA at RT before incubating overnight with the primary antibodies rabbit anti-γ-GCS (1/250) or rabbit anti-Nrf2 (1/200). Thereafter, cells were washed for 5 min three times with 1 mL of 0.1% Tween 20-PBS-Ca^2 +-Mg^2 + to remove the non-fixed antibody followed by an incubation with the anti-rabbit Alexa Fluor^® 488-conjugated secondary antibody (1/200) for 1 h then washed for 5 min three times, stained with 1μg/ml DAPI for 10 min at RT and washed to remove the DAPI excess.

Fluorescence and confocal microscopy

The glass slides were mounted with prolong gold antifade reagent, protected from light and air-dried. Then, observed under fluorescence microscope (Leica ECB, Germany) and images were captured using the SensiCam High Performance camera under the DAPI and the FITC filters, respectively for DAPI and Alexa Fluor^® 488-conjugated antibody detection using both 10x and 40x lenses. For confocal analysis, images were taken using an inverted confocal laser scanning microscope LSM 780 AxioObserver (Zeiss, Germany) at 2048 x 2048 pixels. Images were captured using 10x and 60x oil immersion lenses and optical section was < 1μm. MCB was excited by laser at 390 nm and the data collected at 490 nm while for Alexa Fluor^® 488 it was at 495 and 519 nm, respectively, for excitation and emission using Zen software (Zeiss, Germany).

Fluorescence data analysis

For MCB and γ-GCS, the mean of the whole cell fluorescence intensities in each condition was quantified and reported to the same arbitrary surface unit for normalization reasons. For Nrf2 activation, only the overlapped green (Alexa Fluor^® 488-conjugated antibody) with the blue (DAPI) fluorescence was quantified. Thereby, hundreds of cells from several images, of at least three independent experiments, were used for quantification by the Image-Pro plus 5.0 software (Media Cybernetics, USA). Log2 change over the vehicle-treated cells, used as controls, was calculated. Results are expressed as log2 change±SEM.

Statistical analysis

Statistical analysis was performed using one-way ANOVA analysis followed by, depending on the experimental procedure, the Dunnett multiple comparisons t-test to compare all versus acrolein-treated cells or the Tukey-Kramer when different pairs were compared together. Differences were considered significant when p-values≤0.05. Analyses were performed using INSTAT software.

RESULTS

MAF14001 is a powerful antioxidant

Polyphenols are known to exhibit antioxidant activity. Herein, we have tested the antioxidant activity of MAF14001 using the radical scavenging cell-free assay (DPPH) and demonstrated that MAF14001, from 5μM, was able to reduce the DPPH radical. As compared to the standard antioxidant trolox, the antioxidant capacity of MAF14001 was higher for 5 and 10μM concentrations (p < 0.01) (Fig. 1A). Moreover, the decrease of the DPPH radical absorbance over the time was significantly higher in presence of 10μM of MAF14001 (p < 0.01) and 20μM of MAF14001 (p < 0.001) while it was non-significant in presence of 20μM of Trolox (Fig. 1B). The significant difference of the DPPH radical absorbance curves, in presence and absence of MAF14001, for the first 4 h (240 min) of the experiment suggests a long-term antioxidant effect of MAF14001. Beyond, the effect of MAF was less clear due to the spontaneous loss of the DPPH radical absorbance.

MAF14001 Protects SK-N-SH cells against acrolein-induced cell death

We have previously demonstrated that the neurotoxic effect of acrolein occurs mainly by the disruption of the cellular redox state [44, 45]. We have thus tested the hypothesis that MAF14001 could protect SK-N-SH neuronal cells against the acrolein-induced toxicity. Results presented in Fig. 2 showed that acrolein at 10μM induced more than 80% (p < 0.01) of cell death. Interestingly, a co-treatment with MAF14001, from 5μM, significantly attenuated the toxicity of acrolein (p < 0.01). These results indicate that MAF14001 could be a potent agent to neutralize acrolein toxicity.

MAF14001 restores some oxidative stress markers disturbed by acrolein

Acrolein is a by-product of lipid peroxidation but is also an indirect initiator of free radical formation mainly by lowering cell antioxidant defense systems. Herein, we demonstrated that MAF14001, from 5μM, was able to decrease ROS levels in SK-N-SH cells treated with acrolein (p < 0.01) (Fig. 3A) or with hydrogen peroxide (p < 0.01) (Fig. 3B). Acrolein is a highly reactive electrophile which easily reacts with side chain-amino acids of proteins to form carbonyls. As seen in Fig. 3 C, acrolein significantly increased by two-fold (p < 0.01), protein carbonyl formation which was notably prevented by MAF14001 (p < 0.05).

MAF14001 preserves glutathione level depleted by acrolein

GSH plays a key role in cellular defenses against oxidative stress injury and represents the most important detoxifying agent of acrolein. As previously reported, we confirmed in this study the effect of acrolein on cellular GSH level (Fig. 4A). Indeed, we found a 4-fold (p < 0.001) decrease on the cellular level of GSH only after 30 min of incubation with acrolein. This decrease of GSH was also observed in the presence of 200μM BSO (p < 0.001) for 24 h (Fig. 4B). Interestingly, the cellular GSH level was 2-fold higher (p < 0.01) in the presence of MAF14001 as compared to acrolein-treated cells (Fig. 4B). The decrease of the reduced form of GSH in the presence of acrolein is likely due to the formation of GSH-acrolein adduct. Thus, the preservation of the GSH level in the presence of MAF14001 suggests a possible direct interaction between MAF14001 and acrolein to prevent acrolein-GSH adduct formation.

MAF14001 modulates glutathione synthesis pathway

We have previously demonstrated by western blot that acrolein stimulated the expression of the γ-GCS, a rate-limiting enzyme involved in GSH synthesis, after 24 h of treatment [44]. This effect was confirmed in the Fig. 5A and 5B by fluorescence microscopy

showing a two-fold increase (p < 0.01) in the γ-GCS

expression only after 1 h of treatment with acrolein.

The specificity of the fluorescence was confirmed

by the decrease of the fluorescence observed with

BSO treatment (p < 0.01). Interestingly, the addition of

MAF14001 restores the γ-GCS expression to the control level (p < 0.01) reflecting a balanced cellular redox state (Fig. 5B). The γ-GCS is one of the multiple phase II enzymes activated by the nuclear transcription factor Nrf2. The elevation of γ-GCS is concomitant with Nrf2 activation by acrolein (p < 0.01). Interestingly, thislatter was also significantly inhibited by the presence of MAF14001 (p < 0.05) or Ochratoxin (p < 0.01), used as Nrf2 inhibitor, as compared to acrolein-treated cells (Fig. 5C, D).

MAF14001 modulates the NF-κB and the p66Shc pathways

We have previously reported that the activity of the redox sensitive transcription factor NF-κB wasregulated by acrolein. Thus, the effect of MAF14001 on the nuclear translocation of the NF-κB subunit p50 was analyzed. Our results showed that neither acrolein nor MAF14001 modulates the expression of p50 after 1 h treatment (Fig. 6A). However, we showed that MAF14001 can prevent the translocation to the nucleus of the subunit p50 of NF-κB induced by acrolein (p < 0.05) (Fig. 6B). Furthermore, as we previously reported, acrolein has the ability to activate p66Shc, the mitochondrial adaptor and redox sensitive protein [44]. Our results showed also no effect on total p66Shc in presence of acrolein or MAF14001 (Fig. 6C). Interestingly, MAF14001 prevented p66Shc phosphorylation at serine 36 (p < 0.01) which was significantly increased by acrolein (p < 0.05) (Fig. 6D).

MAF14001 modulates GSK3β activity

We have previously reported that acrolein inhibits Akt signaling pathway and GSK3β, a downstream effector kinase of Akt [19, 23]. In this study, we observed no effect of acrolein or MAF14001 on the total level of GSK3β (Fig. 7A) but an increase (p < 0.05) on the phosphorylated form of GSK3β after 1 h of treatment with acrolein, reflecting an inhibitory effect of acrolein on GSK3β activity. Interestingly, in the presence of MAF14001, the inhibition of GSK3β was higher as compared to acrolein-treated group (p < 0.05) (Fig. 7B). Since we have reported the beneficial effect of MAF14001 on tau phosphorylation induced by Aβ on S202 site [42], we investigated the effect of MAF14001 on tau phosphorylation at S404, one of the important phosphorylated sites of tau by GSK3β affected in AD brain [46]. However, our results clearly showed no effect of acrolein or MAF14001 on tau phosphorylation using the anti-P-tau S400-T403-S404 antibody (Fig. 7C).

DISCUSSION

Acrolein production is elevated in different regions of the brain from MCI and AD patients [6 –8], and it is considered as a biomarker of brain infarction. Acrolein is a by-product of lipid peroxidation but could also be produced from polyamines, especially from spermine by spermine oxidase. The release of spermine from RNA could reach mM range during tissue damage [47 –49]. Acrolein was found to be conjugated with proteins that were detected in NFTs and dystrophic neuritis surrounding senile plaques in AD brain [50].

Acrolein is a highly electrophilic, unsaturated carbonyl derivate due to the aldehyde group, and the carbon–carbon double bond that strongly facilitates its reactivity with nucleophilic groups. Indeed, as a strongest electrophile among the unsaturated aldehydes, acrolein displays strong reactivity with nucleophilic compounds such as free sulfhydryl groups of cysteine residues in proteins [51, 52]. Accordingly, due to its high reactivity, acrolein is not only a marker of lipid peroxidation but also an inducer of oxidative stress and thereby can trigger tissue damage. Acrolein can then disturb the cellular redox potential by lowering GSH level, the cellular preferential mechanism of acrolein detoxification. Moreover, acrolein-protein adducts has also been associated to numerous intracellular regulations including cell signaling, inhibition of enzymatic activities, and mitochondrial dysfunction. For instance, we have previously reported that acrolein could trigger tyrosine phosphorylation mediated signaling events leading to the activation of MAPK, NF-κB, Nrf2, Sirt-1, p66Shc, ERK1/2, and heme-oxygenase-1 [7 , 53–55]. Therefore, protecting neuronal cells against acrolein-induced toxicity could alleviate or prevent brain damage. Thus, protecting neurons from acrolein toxicity could be a promising strategy to delay neurodegeneration as observed in AD.

Numerous epidemiological studies have associated a high consumption of polyphenols from fruits and vegetables to a reduced incidence of late-life cognitive disorders including AD [56 –61]. Moreover, panoply of evidences suggests that a variety of polyphenols including anthocyanins, are able through multiple mechanisms, to improve cognitive decline and to decrease neuropathological hallmarks in animal models of AD [62 –64]. Polyphenols are well recognized for their antioxidant and anti-inflammatory properties. Some of them are inhibitors of Aβ-induced toxicity, of tau hyperphosphorylation and aggregation, and of lipid and protein oxidation by-product formation [61].

Currently, interest in anthocyanin pigments has drastically intensified due to their potent antioxidants and in vivo protective effects against oxidative stress-induced damage [36, 37]. Moreover, anthocyanins are among the rare polyphenols to display the ability to cross the blood brain barrier. We have previously demonstrated that MAF14001, an anthocyanin-rich formulation, could protect neuronal cells against Aβ-induced toxicity [42]. To go further insight into the neuroprotective property of MAF14001, we have investigated its potential to protect SK-N-SH neuronal cells against acrolein-induced toxicity. Our results highlight the relevance of the use of low concentrations of MAF14001, as low as 5μM for each anthocyanin (corresponding to 9.9μg/ml of total anthocyanins) to achieve a beneficial effect against acrolein. These efficient concentrations of MAF14001 are lower than those from other polyphenols to obtain a protection of neuronal cell against acrolein. For instance, the neuroprotective effect of pycnogenol (PYC), a combination of flavonoids (containing mainly monomeric and oligomeric units of catechin, epicathechin and taxifolin) extracted from the bark of French maritime pine (Pinus maritima) against 10μM of acrolein in human neuroblastoma SH-SY5Y cells was obtained with 50μg/ml [65]. Furthermore, the effect of the standardized extracts of bacopa monniera containing as polyphenols, apigenin and luteolin, against 15μM acrolein in human neuroblastoma SK-N-SH, was obtained with 40μg/ml [22]. The effect of MAF14001 is mediated by its greater antioxidant ability as compared to trolox in a cell free system and at the cellular level, by alleviating ROS formation induced by both acrolein and hydrogen peroxide.

We have previously elucidated some mechanisms leading to acrolein toxicity in both SK-N-SH cell line and in rat primary astrocytes [44, 53]. We showed that acrolein was able to quickly deplete the intracellular GSH level and to up-regulate glutathione S-transferase activity. Acrolein reacts rapidly with GSH to form the irreversible acrolein-GSH adduct such as S-(2aldehydo-ethyl)-glutathione and to reduce the cellular GSH reserve. The maintenance of the level of GSH in the presence of MAF14001 suggest a direct interaction between MAF14001 and acrolein as it was demonstrated for the flavan-3-ols and theaflavins sub-classes [24, 25].

The presence of acrolein also induced the expression of γ-GCS to de novo synthetize GSH. This induction was confirmed by the activation of Nrf2. Therefore, the presence of MAF14001 preserved a balanced cellular redox state which could explain the lower protein carbonyl amounts and the non-activation of Nrf2. These properties are of great interest considering the alteration of the balance oxidant/antioxidant and the elevation of acrolein and protein carbonyl observed in AD brain [4 , 67].

Besides the Nrf2 pathway, we have previously showed the ability of acrolein to modulate some other redox sensitive pathways relevant to AD neuropathogenesis [44, 53], including the transcriptional factor NF-κB [68, 69], the protein adaptator p66Shc, and the survival pathway Akt/GSK3 [22 , 53]. Herein, we confirmed the activation of NF-κB by the translocation of its sub-unit p50 to the nucleus. This translocation was abolished by a co-treatment with MAF14001. Despite the complexity of the NF-κB pathway, the activation of this latter has been associated to inflammation, Aβ formation, oxidative stress, tau phosphorylation, and so forth [70 –73]. Thus, the inhibition of this transcriptional factor by MAF14001 could play an interesting role on cell survival.

The mitochondrial adaptor protein p66Shc has been associated with mitochondrial oxidative stress and longevity. Mice lacking p66Shc gene have less ROS generation, live 30% longer, are resistant to oxidative stress, and are also protected from diabetes [74, 75]. Our results showed an increase of p66Shc phosphorylation in the presence of acrolein and its prevention by MAF14001. Under oxidative stress condition, the phosphorylation of p66Shc on Ser36 is mediated by ERK1/2 [76], suggesting an effect of MAF14001 on these critical kinases that are also activated by acrolein [22]. As the phosphorylation of p66Shc is critical for Aβ mediated toxicity [77], the inhibition of p66Shc by MAF14001 could be beneficial in the protection against Aβ toxicity.

The activation of the kinase GSK3 is well recognized to play a pivotal role in the pathophysiology of AD including tau phosphorylation and NFTs, Aβ plaque formation, inflammation, oxidative stress, memory impairment, and neuronal plasticity and loss (for review see [78]). Herein, we showed that acrolein inhibits GSK3β with higher effect being observed in the presence of MAF14001. These results are consistent with our previous studies showing the inhibition of Akt and GSK3 by acrolein in SK-N-SH and in HT22 cells, respectively [19, 23]. Similar to our findings, in postnatal hippocampal neurons, anthocyanins extracted from Korean black soybeans induced the inhibition of GSK3 and the reduction of oxidative stress-induced neurodegeneration [79]. The inhibition of GSK3β by MAF14001 might attenuate tau phosphorylation at S202 site as we have previously reported for Aβ-induced tau phosphorylation [42]. Moreover, our results are consistent with the inhibitory effect of tau phosphorylation and filament formation by the anthocyanins delphinidin [80] and cyaninidin [81], respectively. Furthermore, the reduction of tau phosphorylation may also result on a reduction of its aggregated form. Hence, as previously reported for other polyphenolic compounds such as oleuropein [82, 83] and epigallocatechin gallate [84], MAF14001 could also have a benefit effect on tau aggregation. These mechanisms could underlie the attenuation of cognitive impairment in rats receiving cyanidin-3-O-glucoside [85]. Therefore, inhibiting tau phosphorylation by MAF14001 could be interesting in the treatment of cognitive impairment.

Previous studies have shown that polyphenols are brain permeable. For instance, a total of 92 and 192 ng of anthocyanins/g tissue (which correspond approximately to 1 and 2 ng/mg protein, respectively) were detected in the brain from rats orally fed with 2% of blueberry-supplemented diet for 8–10 weeks or following a single dose of 8 mg/kg of body weight of a pure anthocyanin mixture, respectively [31 , 86]. In our experimental conditions, the amounts of anthocyanins used (5 or 10μM) would correspond to 100 and 200 ng/mg protein, which are in accordance with the in vivo brain availability. More interestingly, there is a brain region-specific accumulation of anthocyanins with high concentrations being found in hippocampus and other regions involved in cognitive functions [31, 32] as well as the production of acrolein in AD brain [9, 10]. Levels of extractable acrolein measured in hippocampus and amygdala from AD brain were 5±1.6 nmole/mg protein and 2.5±0.9 nmole/mg protein, respectively [9, 10]. Accordingly, considering our experimental conditions, the amount of acrolein used (200 nmole/mg protein) is certainly higher than those found in AD brain. However a chronic exposition to acrolein even at low concentrations, likely occurs in AD brain, and its accumulation could have harmfuleffect to neurons by triggering the alterations of cellular mechanisms as observed in our experimental conditions. Interestingly, the ratio between total anthocyanin (1 and 2 ng/mg protein) and acrolein (2.5-5 nmole/mg) concentrations in vivo and that used in our study for MAF14001 (100 and 200 ng/mg protein) and acrolein (200 nmole/mg protein) are roughly the same. Thus, our results could easily be extrapolated to the in vivo conditions. However, our experiments were achieved in non-differentiated neuronal cell line in culture media, which do not reflect a whole in vivo system. Although anthocyanins were found in their intact forms in the brain after oral administration to animals, due to their low concentrations, a direct antioxidant effect of anthocyanins in human remains to be clarified.

Finally, taken together these results demonstrate for the first time, a preventive effect of an original anthocyanin formulation (MAF14001) against acrolein-induced toxicity. Thereby, highlight the idea of MAF14001 as a potent antioxidant. Jointly with our previous results on Aβ-induced SK-N-SH toxicity, MAF14001 performs at various levels including Aβ fibrillation and toxicity, tau hyperphosphorylation, restoration of the cellular redox balance disrupted by acrolein through GSH saving, decreasing ROS level and modulation of cellular pathways. Since all these hallmarks are involved in AD neuropathogenesis, MAF14001 provide a promising approach to prevent or slow AD progression.

Footnotes

ACKNOWLEDGMENTS

Financial support was obtained from NSERC (CR) and Chaire Louise & André Charron on Alzheimer’s disease.

Author’s disclosures available online ().

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