Inhibition of MAPKs Signaling Pathways Prevents Acrolein-Induced Neurotoxicity in HT22 Mouse Hippocampal Cells

Abstract

Activation of mitogen-activated protein kinases (MAPKs) in neurons may underlie the pathogenesis of Alzheimer’s disease (AD). Acrolein, a ubiquitous pollutant, has been reported to implicate in the etiology of AD. Our previous data showed that acrolein changed the levels of key AD-associated proteins, including advanced glycation end products (RAGE), A-disintegrin and metalloprotease (ADAM-10), and beta-site amyloid-beta peptide cleaving enzyme 1 (BACE-1). In this study, we investigated whether acrolein-induced alterations of AD-associated proteins are relevant to MAPKs activation, and strategies to inhibit MAPKs activation yield benefits to acrolein-induced neurotoxicity in HT22 mouse hippocampal cells. We found that acrolein activated MAPKs signaling pathways to mediate cells apoptosis, and then altered the levels of AD-associated proteins ADAM-10, BACE-1 and RAGE. Inhibitors of MAPKs signaling pathways attenuated the cells death and restored the proteins levels of ADAM-10, BACE-1 and RAGE in varying degrees induced by acrolein. Taken together, activated MAPKs signaling pathways should be underlying the pathology of acrolein-induced neuronal disorders. Inhibitors of MAPKs pathways might be promising agents for acrolein-related diseases, such as AD.

INTRODUCTION

Alzheimer’s disease (AD) is an age-related neurodegenerative malady characterized by cognitive impairment, memory loss and dementia.¹⁾ Though AD has been studied for more than 100 years since 1906, the exact cause(s) and pathogenic mechanism(s) remain to be clarified. Also, the efficient treatment for AD are unavailable.²⁾ Acrolein (CH₂=CH–CHO), a ubiquitous pollutant, is an α, β-unsaturated aldehyde, showing highly electrophile and reactivity with nucleophiles.³⁾ It is relatively well-known that the toxicity of acrolein is mediated by oxidative stress-induced damage.⁴⁾ Very recently, several lines of evidence indicated acrolein-induced oxidative damage is associated with neurodegenerative disease, including AD.⁵⁾ Acrolein induced hyperphosphorylation of microtubule-associated protein tau^6,7) and promoted amyloid beta peptide (Aβ) aggregation in senile plaque.⁸⁾ Our previous study have also shown that chronic administration of acrolein to rats induced AD-like pathologies such as mild cognitive impairment, hippocampal atrophy, and an upregulation of beta-site amyloid-beta peptide cleaving enzyme 1 (BACE-1).⁹⁾

Acrolein-induced oxidative damage has been reported to activate mitogen-activated protein kinases (MAPKs, namely p38, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK)), and Akt/glycogen synthase kinase 3β (GSK-3β) signaling pathways to mediate cells’ death or self-protection in A549 cells, Chinese hamster ovary cells and SK-N-SH cells.^10–13) Interestingly, MAPKs activation in neurons may trigger tau hypephosphorylation and self-assembly, which underlie the pathogenesis of AD.^7,14) Our previous data showed that acrolein changed the levels of some key AD-associated proteins, such as advanced glycation end products (RAGE), A-disintegrin and metalloprotease (ADAM-10), and BACE-1 in HT22 mouse hippocampal cells, but it still unknown that whether these proteins alterations are relevant to MAPKs activation, and whether strategies to inhibit MAPKs activation yield benefits to acrolein-induced neurodegenerative diseases such as AD.

Here, we investigated the toxicity of acrolein on MAPKs signaling pathway and further explored the effects of MAPKs activation or inhibition on AD-associated proteins RAGE, ADAM-10, and BACE-1 in HT22 mouse hippocampal cells.

MATERIALS AND METHODS

Materials

Acrolein was purchased from Gelei Xiya Chemical Co. (Chengdu, China). Dullbecco’s modified Eagle’s medium (DMEM), and fetal bovine serum (FBS) were purchased from Gibico-BRL (Grand Island, NY, U.S.A.). 3-(3,4-Dimehylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.) unless stated otherwise. The primary and secondary antibodies used in our experiments are summarized in Table 1. Inhibitors of MAPKs and phosphatidylinositol 3-kinase (PI3K) used in this study were obtained from Cell Signaling Technology Inc. (Beverly, MA, U.S.A.), and inhibitor of GSK3 was purchased from Sigma-Aldrich.

Table 1. Summary of Antibodies and Working Conditions Used in the Experiments

Antibody	Species	Source	Dilution
Primary antibodies
Anti-β-actin	Mouse, polyclonal	Sigma-Aldrich, St. Louis, MO, U.S.A.	1 : 10000
Anti-phospho ERK	Rabbit, polyclonal	Cell Signaling Technology, Beverly, MA, U.S.A.	1 : 1000
Anti-total ERK	Rabbit, polyclonal	Cell Signaling Technology, Beverly, MA, U.S.A.	1 : 1000
Anti-phospho JNK	Rabbit, polyclonal	Cell Signaling Technology, Beverly, MA, U.S.A.	1 : 1000
Anti-total JNK	Rabbit, polyclonal	Cell Signaling Technology, Beverly, MA, U.S.A.	1 : 1000
Anti-phospho p38	Rabbit, polyclonal	Cell Signaling Technology, Beverly, MA, U.S.A.	1 : 1000
Anti-total p38	Rabbit, polyclonal	Cell Signaling Technology, Beverly, MA, U.S.A.	1 : 1000
Anti-phospho Akt (Ser 473)	Rabbit, polyclonal	Cell Signaling Technology, Beverly, MA, U.S.A.	1 : 1000
Anti-total Akt	Rabbit, polyclonal	Cell Signaling Technology, Beverly, MA, U.S.A.	1 : 1000
Anti-phospho GSK-3β	Rabbit, polyclonal	Santa Cruz Biotechnology Inc. CA, U.S.A.	1 : 500
Anti-total GSK-3β	Rabbit, polyclonal	Cell Signaling Technology, Beverly, MA, U.S.A.	1 : 1000
Anti-ADAM 10	Rabbit, polyclonal	Santa Cruz Biotechnology Inc., CA, U.S.A.	1 : 500
Anti-BACE 1	Rabbit, polyclonal	Santa Cruz Biotechnology Inc., CA, U.S.A.	1 : 500
Anti-RAGE	Rabbit, polyclonal	Santa Cruz Biotechnology Inc., CA, U.S.A.	1 : 500
Secondary antibodies
Anti-rabbit IgG (HP)	Goat	Santa Cruz Biotechnology Inc., CA, U.S.A.	1 : 5000
Anti-mouse (HP)	Rabbit	Promega, Medison, WI, U.S.A.	1 : 10000

Cell Culture and Cell Treatment

HT22 murine hippocampal neuronal cells (a gift from Prof. Jun Liu, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, China)¹⁵⁾ were maintained in DMEM supplemented with 10% (v/v) FBS and incubated at 37°C under 5% CO₂. Cells were sub-cultured every 3 d. The fresh solution of acrolein (1 M in distill water) was diluted in DMEM supplemented with 0.5% (v/v) FBS immediately before addition to each well at the desired final concentrations. To test the effect of the inhibitors of MAPKs, PI3K and GSK-3β, HT22 cells were seeded in 96-well plates at a density of 4000 cells per well. Cells were pre-treated with each of the inhibitors for 1 h before acrolein exposure. Cells in the control group were treated with vehicle alone.

MTT Assay

Cell viability was determined as we described previously⁹⁾ based on conversion of MTT to MTT-formazan by mitochondrial enzymes. Briefly, cells were seeded into a 96-well plate at a density of 4000 cells/well in growth medium and cultured to about 60–70% confluence prior to the experimental treatment. Photomicrographs of each culture were taken by phase contrast microscopy (IX71, Olympus, Tokyo, Japan) before the MTT assay. Cells were washed three times with phosphate-buffered saline (PBS) and 10 µL of MTT solution (5 mg/mL stock) was added to the cells, and then incubated for 1 h at 37°C. The medium was removed carefully and 150 µL of dimethyl sulfoxide (DMSO) was then added to resolve the blue formazan in living cells. Finally, the absorbance at 570 nm was read with an enzyme-linked immunosorbent assay (ELISA) reader (Bio-Tek). Results were expressed as the percentage of MTT reduction and the absorbance of control cells was set as 100%.

Flow Cytometric Analysis of Apoptosis

After treatment with acrolein, cells were harvested by trypsinization and washed once with PBS (pH 7.4). Cells were stained with annexin-V and propidium iodide (PI) using the annexin V-FITC apoptosis detection kit (BD Biosciences, San Jose, CA, U.S.A.) as a measurement of early apoptotic (lower right quadrant, annexin V+/PI− cells), late apoptotic (upper right, annexin V+/PI+ cells), and necrotic (upper left, annexin V−/PI+ cells) cell populations; the lower left quadrant (annexin V−/PI− cells) depicts live cells. For annexin-V and PI double staining, the procedure was rigidly performed according to the instructions of the manufacturers.

Western Blotting

Cells were washed twice with ice-cold PBS, and then suspended in 100 µL of lysis buffer (20 mM Tris–HCl, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1% Triton X-100, protease inhibitor cocktail, 2 mM Na₃VO₄, and 10 mM NaF, pH 7.5). The protein concentration was determined using BCA assay kit (Beyotime, Jiangsu, China). An equal amount of protein (20 µg) was loaded in each lane. Proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel electrophoresis and electrically transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore). After blocking the membrane with 5% skim milk, target proteins were immunodetected using specific antibodies. The horseradish peroxidase (HRP)-conjugated anti-rabbit immunoglobulin G (IgG) or anti-mouse IgG were applied as the secondary antibody, and visualized using an ECL plus kit (Pierce) and exposed to autoradiographic films according to the manufacturer’s protocol. Membranes were stripped and reprobed for total ERK, p38, JNK, Akt, and GSK-3β. The intensities of bands were performed using Quantity One Software (provided by Bio-Rad, Hercules, CA, U.S.A.).

Reproducibility of Experiments and Statistical Analysis

All quantitative data and experiments described in this study were repeated at least three times. Data were presented as mean ± standard deviation (S.D.) of multiple independent experiments. Statistics were analyzed with one-way ANOVA followed by multiple comparisons with Dunnett’s test (SPSS 17.0 software). Statistical difference was considered at p < 0.05.

RESULTS

Acrolein Induces Apoptosis in HT22 Cells

Previous studies showed acrolein induced cell death via apoptosis in other cell lines.¹⁶⁾ To identify the neurotoxicity of acrolein on neuronal cells, we investigated cells viability and apoptosis in HT22 hippocampal cells. Exposure of HT22 cells to acrolein for 24 h reduced cell viability (MTT assay) in a concentration-dependent manner (Fig. 1A). At 25 µM for 24 h exposure, acrolein inhibited cell viability by approximately 40%, and at 100 µM, it reduced cell viability to below 20%. As we described previously,¹⁷⁾ 25 µM of acrolein was applied in following study because the concentration corresponds to that in AD patients’ brains, which is calculated based on average protein content of each culture dish in our experiments. Phase contrast microphotographs showed obviously cell morphological damages and amount loss by acrolein (25 µM) exposure for 24 h (Fig. 1B). The results of flow cytometric analysis showed that treatment with 25 µM of acrolein for 3 or 6 h induced HT22 cells death by apoptosis (Fig. 1C). Acrolein led to apoptosis, which began at 3 h with a percentage of (16.43 ± 5.81)% (the percentage of cells at lower right and upper right quadrants) and boosted at 6 h with (76 ± 10.62)% in HT22 cells.

Fig. 1. Acrolein Induces Apoptosis in HT22 Cells

Acrolein treatment resulted in significant cell death in a concentration-dependent manner, and cells death was mediated by apoptosis. (A) Effects of indicated concentrations of acrolein treatment on HT22 cell viability. Cell viability was measured using the MTT assay. Data are expressed as mean ± S.D., * p < 0.05, ** p < 0.01 versus respective controls. (B) Phase contrast microphotographs (200×) of HT22 cells treated with 25 µM of acrolein for 24 h. (C) Acrolein induces HT22 cells death through apoptosis which was measured by flow cytometric analysis. Apoptosis began at 3 h with a percentage (16.43 ± 5.81)% (the percentage of cells at lower right and upper right quadrants) and boosted at 6 h with (76 ± 10.62)% in HT22 cells.

Effects of Acrolein on MAPKs and PI3K/Akt Pathways

Previous studies reported exposure of acrolein activated MAPKs and Akt signaling pathways in A549 cells, Chinese hamster ovary cells and SK-N-SH cells,^10–13) but we still do not know the time–courses of acrolein on MAPKs and Akt/GSK-3β pathway and whether the two pathways would induce AD-like pathologies in HT22 cells. To clarify the above two questions, we firstly examined the phosphorylated protein (the activated forms) levels of JNK, p38, ERK and Akt (ser473)/GSK-3β by Western blot. Interestingly, all these proteins were rapidly phosphorylated in HT22 cells to their first pikes at either 0.5 or 3 h after exposure to acrolein (Fig. 2), and then gradually dephosphorylated. Some of these proteins, including p38, Akt, and JNK1 were re-phosphorylated to their secondary pikes at 24 h after exposure to acrolein except GSK-3β at 12 h but ERK1/2 was without secondary phosphorylation. Giving that acrolein-induced neurotoxicity in human beings is a chronic process, we chose the time-point of 24 h of acrolein treatment to our following experiments.

Fig. 2. Acrolein Exposure Activates MAPKs and Akt Signaling Pathways in HT22 Cells

HT22 cells were treated with 25 µM acrolein for indicated times. Protein levels were detected by Western blot analysis. Densitometric measurements of band intensity in the Western blots were performed using Quantity One Software. Data are expressed as mean ± S.D., * p < 0.05, ** p < 0.01 versus control. (A, B) p38, JNK1 and ERK1/2 were rapidly phosphorylated to their first pikes at either 0.5 or 3 h after exposure to acrolein and then gradually dephosphorylated. p38 and JNK1 were re-phosphorylated to their secondary pikes at 24 h after exposure to acrolein. ERK1/2 was without secondary phosphorylation. (C, D) Akt (Ser473) and GSK-3β were also phosphorylated rapidly at 0.5 h of acrolein exposure. Akt was re-phosphorylated at 24 h, and GSK-3β from 12 to 24 h of exposure to acrolein.

Effect of MAPKs and PI3K/GSK-3β Inhibitors on Acrolein-Induced Cell Toxicity

Next, we used selective inhibitors of MAPKs and PI3K/GSK-3β signaling pathways to further explore their roles on acrolein-induced cell death. As shown in Fig. 3A, MAPKs inhibitors, CEP11004 (inhibitors of JNK-1, -2 and -3. 10 µM), SB202190 (an inhibitor of p38 MAPK, 10 µM), and PD98059 (an inhibitor of ERK1/2, 50 µM) significantly improved acrolein-induced cell death, respectively (p < 0.05). In addition, the protective effect of LiCl (a GSK-3β inhibitor, 1 mM) was especially more obvious than that of others, while wortmannin (a specific PI3K inhibitor, 1 µM) promoted neurotoxicity of acrolein.

Fig. 3. Effects of Inhibitors of MAPKs and PI3K/GSK-3β Signaling Pathways on Acrolein Induced-Neurotoxicity and AD-Associated Proteins in HT22 Cells

(A) HT22 cells were pre-treated with each of the inhibitors at indicated concentration (SP600125-10 µM and CEP11004-10 µM as a JNK inhibitor; SB202190-10 µM as a p38 inhibitor; PD98059-50 µM as an ERK inhibitor; wortmannin-1 µM as PI3K inhibitors and LiCl-1 mM as GSK3β inhibitors) for 1 h and then incubated with acrolein (25 µM) for additional 24 h. Cell viability was analyzed using the MTT assay. Data are expressed as mean ± S.D., ** p < 0.01 versus untreated control cells; ^# p < 0.05 and ^## p < 0.01 versus acrolein-treated cells in the absence of inhibitors. (B) The protein levels were detected by Western blot analysis. Acrolein alone decreased protein levels ADAM-10, and significantly increased levels of BACE-1 as well as RAGE. When MAPKs signal was inhibited for 24 h, the protein level of ADAM-10 increased significantly. Meanwhile, acrolein-induced increased levels of BACE-1 were markedly restored to control-group level by the 4 inhibitors of MAPKs, but inhibitors of PI3K/GSK-3β did not show the similar effect. Protein level of RAGE was markedly restored by SB202190 (p38 inhibitor) and LiCl (GSK-3β inhibitor). (C) Densitometric measurements of band intensity were performed using Quantity One Software. * p < 0.05 and ** p < 0.01 versus untreated control cells; ^# p < 0.05 and ^## p < 0.01 versus acrolein-treated group in the absence of inhibitors.

Effect of MAPKs and PI3K/GSK-3β Inhibitors on AD-Associated Proteins

Amyloid β peptide (Aβ) and its plaque in the brain tissue is a hallmark of AD. Proteins ADAM-10, BACE-1 and RAGE were reported to be the key proteins to cause dysfunction of Aβ metabolism.^2,18,19) Our very recently studies have reported that 24 h exposure to acrolein increased the proteins levels of BACE-1 and RAGE, while decreased the level of ADAM-10 in HT22 cells.⁹⁾ However, we are uncertain that whether the proteins alterations are relevant to MAPKs signal activation. So, we assessed the proteins levels of ADAM-10, BACE-1 and RAGE when MAPKs pathways were inhibited. As shown in Fig. 3B, acrolein alone decreased protein levels of ADAM-10, and significantly increased levels of BACE-1 as well as RAGE. When MAPKs and PI3K/GSK-3β signal were inhibited for 24 h, the protein level of ADAM-10 increased significantly. Meanwhile, increased level of BACE-1 that was induced by acrolein was markedly restored to control-group level by the 4 inhibitors of MAPKs, but inhibitors of PI3K/GSK-3β did not show the similar effect. Protein level of RAGE was obviously recovered by SB202190 (p38 inhibitor) and lithium chloride (LiCl, GSK-3β inhibitor).

DISCUSSION

Recent studies suggested an important neurotoxic role of acrolein in the development of AD.^20–22) In the present study, our data demonstrated that acrolein induced neurotoxicity in HT22 hippocampal cells and the mechanisms might be related to its activating MAPKs signaling pathways to mediated cells apoptosis, then resulted in abnormal ADAM-10, BACE-1 and RAGE metabolism and might further induce Aβ aggregation/plaque, a hallmark of pathologic character in AD brain. Inhibition of MAPKs signaling pathways restored the above three proteins levels in varying degrees.

MAPKs signaling pathways are involved in cell apoptosis, as well as cell growth and differentiation. ERK is mainly activated by growth factors or mitogens leading to cell differentiation, growth, and survival.²³⁾ JNK and p38 are activated resulting from a series of cytotoxic stresses, causing inflammation and apoptosis.^24,25) In our study, JNK, p38, and ERK were all found to be activated rapidly at 0.5 h after exposure to acrolein. Furthermore, specific inhibitors indicated that activation of JNK, p38, and ERK is involved in acrolein-induced cell death (Figs. 2, 3A). Similarly, other studies also documented acrolein activated MAPKs signaling pathways to mediated cells apoptosis in primary cultured rat astrocytes, SH-SY5Y, SK-N-SH, A549 or Chinese hamster ovary cell lines.^10–13) The inhibitors for each of the MAPKs attenuated acrolein-induced cytotoxicity in varying degrees (Fig. 3A), and level of RAGE was obviously recovered by p38 inhibitor, suggesting that the three kinases of MAPKs might play different roles in acrolein-induced cell death in HT22 cells. Further studies are warranted to delineate the exact mechanisms of three kinases of MAPKs on acrolein-induced neurons death.

The accumulation of Aβ in the brain is a central process leading to the development of AD.²⁶⁾ Amyloid-beta precursor protein (APP), the source of Aβ, can be proteolytically processed by α-secretase (mainly ADAM-10) and β-secretase (BACE-1) in neurons.^19,27) RAGE is the main transporter for Aβ across the blood–brain barrier from blood to brain.^28,29) It is well established that MAPKs signaling pathways are involved in AD process. Aβ-induced MAPKs activation in neurons may trigger tau hypephosphorylation and self-assembly, which underlie the pathogenesis of AD.^14,30) We found that when MAPKs pathways were activated by acrolein, the proteins levels of ADAM-10, BACE-1 and RAGE changed. Interestingly, the above three proteins levels restored when specific inhibitors inhibited MAPKs pathways. These data strongly supported the role of MAPKs pathway on AD pathologies. Early study also demonstrated HNE, another aldehyde may play a pathogenetic role in AD by selectively activating MAPKs pathways and BACE-1 that regulate the proteolytic processing of APP in NT₂ neurons.³¹⁾

Akt is activated by the PI3K survival pathway to mediate growth factor-induced neuronal survival and inhibit apoptosis by phosphorylating a number of downstream targets.³²⁾ We found that treatment of HT22 cells with medium concentration of acrolein resulted in a sustained Akt activation, and GSK-3β inactivation at 0.5, 12 and 24 h. The Akt activation and GSK-3β inactivation might play neuroprotective roles against acrolein-induced cytotoxicity, which was confirmed by using LiCl, a GSK-3β inhibitor and also wortmannin, a PI3K inhibitor (Fig. 3A). Our present results are similar to the earlier studies that sub-lethal concentration of acrolein was also found to activate Akt kinase to prevent cell death,³³⁾ and lithium protects acrolein-induced neurotoxicity which indicating GSK-3β inhibitor might be rescuers of cognitive impairments in AD patients.^17,34) The results in present study need to be further verified by the in vivo study, and data on the drugs safety and adverse effects also should be reported in future animal study.

CONCLUSION

In conclusion, we have showed that inhibition of MAPKs signaling pathways prevents acrolein-induced neurotoxicity and restored the proteins levels of ADAM-10, BACE-1 and RAGE in HT22 cells. Inhibition of MAPKs pathways might be a promising strategy for acrolein-related neuronal disorders. Analogs of MAPKs inhibitors combined with other neuroprotective drugs, or agents with multi-target mechanisms that covered the effect of inhibition of MAPKs pathway might be considered as potential treatment for acrolein-related neuronal disorders, such as AD.

Acknowledgments

This study was supported by National Natural Science Foundation of China (No. 81403444) and Guangdong Provincial Project of Science & Technology (No. 2014A020221007) to YJ. Huang.

Conflict of Interest

The authors declare no conflict of interest.

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