Endoplasmic reticulum stress-mediated neuronal apoptosis by acrylamide exposure

Toxicology and Applied Pharmacology 310 (2016) 68–77

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Endoplasmic reticulum stress-mediated neuronal apoptosis by acrylamide exposure Yuta Komoike ⁎, Masato Matsuoka Department of Hygiene and Public Health I, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan

a r t i c l e

i n f o

Article history: Received 24 June 2016 Revised 29 August 2016 Accepted 10 September 2016 Available online 12 September 2016 Keywords: Acrylamide Endoplasmic reticulum stress eIF2α–ATF4 pathway Reactive oxygen species SH-SY5Y cells Zebrafish

a b s t r a c t Acrylamide (AA) is a well-known neurotoxic compound in humans and experimental animals. However, intracellular stress signaling pathways responsible for the neurotoxicity of AA are still not clear. In this study, we explored the involvement of the endoplasmic reticulum (ER) stress response in AA-induced neuronal damage in vitro and in vivo. Exposure of SH-SY5Y human neuroblastoma cells to AA increased the levels of phosphorylated form of eukaryotic translation initiation factor 2α (eIF2α) and its downstream effector, activating transcription factor 4 (ATF4), indicating the induction of the unfolded protein response (UPR) by AA exposure. Furthermore, AA exposure increased the mRNA level of c/EBP homologous protein (CHOP), the ER stress-dependent apoptotic factor, and caused the accumulation of reactive oxygen species (ROS) in SH-SY5Y cells. Treatments of SH-SY5Y cells with the chemical chaperone, 4-phenylbutyric acid and the ROS scavenger, N-acetyl-cysteine reduced the AA-induced expression of ATF4 protein and CHOP mRNA, and resulted in the suppression of apoptosis. In addition, AA-induced eIF2α phosphorylation was also suppressed by NAC treatment. In consistent with in vitro study, exposure of zebrafish larvae at 6-day post fertilization to AA induced the expression of chop mRNA and apoptotic cell death in the brain, and also caused the disruption of brain structure. These findings suggest that AA exposure induces apoptotic neuronal cell death through the ER stress and subsequent eIF2α–ATF4–CHOP signaling cascade. The accumulation of ROS by AA exposure appears to be responsible for this ER stress-mediated apoptotic pathway. © 2016 Elsevier Inc. All rights reserved.

1. Introduction Acrylamide (AA) is mainly used as an intermediate material for the synthesis of polyacrylamide in the industry. It has been reported that AA is produced in certain carbohydrate-rich foods that are cooked at high temperature (Tareke et al., 2002). The presence of AA in foods has caused worldwide concern because epidemiological and experimental studies have indicated that AA poses a variety of health hazards including neurotoxicity, developmental toxicity, genotoxicity, reproductive toxicity, and carcinogenicity (Xu et al., 2014). Notably, AA is most known to be the neurotoxic compound in humans and animals (Gold and Schaumburg, 2000). Although chemical and biochemical mechanisms of AA neurotoxicity have been extensively studied (Friedman, 2003; LoPachin and Gavin, 2012), intracellular stress signaling pathways leading to the development of AA neurotoxicity remain unclear. The endoplasmic reticulum (ER) is the intracellular organelle responsible for various aspects of the quality control of biologically ⁎ Corresponding author. E-mail addresses: [email protected] (Y. Komoike), [email protected] (M. Matsuoka).

http://dx.doi.org/10.1016/j.taap.2016.09.005 0041-008X/© 2016 Elsevier Inc. All rights reserved.

active proteins, such as synthesis, folding, post-translational modification, and delivery (Ron and Walter, 2007). When cells are exposed to extracellular stress, unfolded proteins accumulate in the lumen of the ER, and this accumulation induces the ER stress and the subsequent adaptive program called the unfolded protein response (UPR) (Schröder and Kaufman, 2005). Three pathways of the UPR, i.e., protein kinase RNA-activated-like ER kinase (PERK)–eukaryotic translation initiation factor 2α (eIF2α) pathway, inositol-requiring enzyme 1 (IRE1)– X-box binding protein 1 (XBP1) pathway, and activating transcription factor 6 (ATF6) pathway, have been identified so far (Wang and Kaufman, 2012). These UPR pathways upregulate the expression of ER chaperones, including glucose-regulated protein 78 (GRP78) and GRP94 (Zhu and Lee, 2015), and activate the ER-associated protein degradation pathway (Vembar and Brodsky, 2008) in addition to the global translation arrest caused by the phosphorylation of eIF2α (Schröder and Kaufman, 2005). In addition, a transcription factor, ATF4 is produced through alternative translation by the eIF2α phosphorylation (Harding et al., 2000). The primary purpose of UPR is to facilitate adaptation of cells to environmental changes and to re-establish normal ER functions by reducing both entry of newly synthesized proteins into the ER and existing unfolded proteins. However, if the UPR fails to resolve the ER overload condition, apoptotic pathways are

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activated (Malhotra and Kaufman, 2007). The c/EBP homologous protein (CHOP), also called the growth arrest and DNA damage induced gene-153 (GADD153), is the most important molecule for the ER stress-induced apoptotic cell death (Malhotra and Kaufman, 2007). The UPR branch, the PERK–eIF2α pathway induces the expression of CHOP and plays a crucial role in the neuronal apoptotic cell death in vitro and in vivo (Iurlaro and Munoz-Pinedo, 2015). In the present study, we investigated the effects of AA exposure on the eIF2α–ATF4 signaling pathway and expression of CHOP mRNA and ER chaperone proteins in SH-SY5Y human neuroblastoma cells. The functional role of the ER stress and the accumulation of reactive oxygen species (ROS) in the AA-induced apoptosis was revealed in SH-SY5Y cells treated with the chemical chaperone, 4-phenylbutyric acid (4PBA) and the ROS scavenger, N-acetyl-cysteine (NAC), respectively. Finally, we showed the expression of chop mRNA and apoptotic cell death in the brain of zebrafish larvae following exposure to AA. 2. Materials and methods 2.1. Chemicals AA was purchased from Wako Pure Chemical (Osaka, Japan). 4-PBA, NAC, Solvent Blue 38 (Luxol Fast Blue MBSN), and cresyl violet acetate were purchased from Sigma-Aldrich (St. Louis, MO). Antibodies against phospho-eIF2α (P-eIF2α), eIF2α, poly (ADP-ribose) polymerase (PARP), and horseradish peroxidase (HRP)-conjugated anti rabbit IgG were purchased from Cell Signaling Technology (Beverly, MA). Antibodies against ATF4, GRP78, and HRP-conjugated anti rat IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti β-Actin (ACTB) antibody was purchased from Sigma-Aldrich. Anti GRP94 antibody was purchased from Enzo Life Science (Farmingdale, NY). HRP-conjugated anti mouse IgG was purchased from GE Healthcare (Buckinghamshire, UK). ALXA FLUOR 555-conjugated anti digoxin antibody was purchased from Bioss (Woburn, MA). 2.2. Cell culture and treatments SH-SY5Y cells established from human neuroblastoma were obtained from American Type Culture Collection (Manassas, VA) and European Collection of Authenticated Cell Cultures (Salisbury, UK). Cells were seeded on BD Purecoat carboxyl surface cultureware (Corning, Corning, NY) and grown in Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin (Gibco, Carlsbad, CA) in a humidified atmosphere of 5% CO2 in air at 37 °C. For each experiment, an appropriate number of exponentially growing SH-SY5Y cells were seeded 2 or 3 days before treatment with chemicals. AA and NAC were dissolved in deionized water, sterilized by filtration, and stored at 4 °C or −20 ° C. 4-PBA was dissolved in culture medium and stored at 4 °C. AA solution was added to the culture medium at various concentrations, ranging from 1 to 10 mM, after medium change and cells were incubated for up to 8 h. In the pretreatment experiments, SH-SY5Y cells were incubated in culture medium containing 10 mM 4-PBA or 5 mM NAC for 30 min and then AA was added into the culture medium in the presence of either chemical. 2.3. Cell viability assays Cell viability was determined by measuring the activity of mitochondrial reductases in cells using Cell Count Reagent SF (Nacalai Tesque, Kyoto, Japan), which is an improved alternative to MTT assay, and a microplate reader model 680 (Bio-Rad, Hercules, CA). SH-SY5Y cells were seeded on the poly-L-lysine (PLL)-coated 96-well microplates (Iwaki, Shizuoka, Japan). Cell viability assay was replicated four times with the use of independently cultured cells.

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2.4. Immunoblotting Immunoblotting was performed as described previously (Komoike and Matsuoka, 2013) with some modifications. Briefly, cells were lysed in the cell lysis buffer containing complete Mini EDTA-free protease inhibitor (Roche Applied Science, Mannheim, Germany) and phosphatase inhibitor cocktail 1 & 2 (Sigma-Aldrich). Proteins were separated by Mini-Protean TGX gel (Bio-Rad) and transferred to Immobilon-P membrane (Merk Millipore, Billerica, MA). Membranes were briefly rinsed with 0.1% Tween 20 in Tris-buffered saline (TBST), and non-specific binding of the antibody was blocked by incubating the membranes in 5% skim milk (Nacalai Tesque) in TBST for 1 h at room temperature. Subsequently, the membranes were incubated with working dilutions of primary antibodies overnight at 4 °C. Blots were washed with TBST and incubated with the appropriate HRPconjugated secondary antibodies for 1 h at 37 °C. HRP was detected with SuperSignal West Dura Extended Duration Substrate (Thermo Scientific, Waltham, MA) and an Ez-Capture MG (Atto, Tokyo, Japan). The signal intensity of bands was quantified with ImageJ 1.46r (National Institutes of Health, Bethesda, MD). A series of immunoblotting was replicated four times with the use of cell lysates prepared from independently harvested cells.

2.5. cDNA synthesis Total RNA was extracted from cells and these RNA samples served as the templates for reverse transcription to synthesize cDNA using RNeasy plus Mini Kit and Omniscript RT Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. RNA extraction and consecutive cDNA synthesis were replicated four times with the use of independently harvested cells.

2.6. Semi-quantitative PCR The cDNA samples were subjected to semi-quantitative polymerase chain reaction (PCR) using EmeraldAmp PCR Master Mix (Takara Bio, Otsu, Japan). The primers used in this study are listed in Table 1. The PCR products were separated by electrophoresis on 5% NuSieve 3-1 agarose gel (Lonza, Rockland, ME) in Tris-acetate-EDTA buffer. Gels were stained with 0.5 μg/ml ethidium bromide and recorded with an Image Saver HR (Atto). Semi-quantitative PCR was performed four times with the use of independently synthesized cDNAs.

2.7. Quantitative real time PCR The cDNA samples were also subjected to real time PCR using Universal SYBR Select Master Mix in the StepOne real time PCR system (Applied Biosystems, Carlsbad, CA). The primers used in this study are listed in Table 1. Real time PCR was performed four times with the use of independently synthesized cDNAs.

2.8. ROS detection Intracellular ROS was visualized using CellRox Green Reagent (Molecular Probes, Carlsbad, CA) according to manufacturer's instructions. Cells were seeded on the PLL-coated chamber slides (Iwaki) and exposed to 10 mM AA for 6 h. Nuclei were counterstained with 4′,6diamidino-2-phenylindole (DAPI, Molecular Probes) and resulting specimens were sealed with ProLong Gold antifade reagent (Molecular Probes). Signals were observed under an ECLIPSE E1000 fluorescent/ differential interference contrast (DIC) microscope (Nikon, Tokyo, Japan). ROS detection was replicated three times with the use of independently cultured cells.

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Table 1 Primers used in this study. Target

Accession no.

Application

Primer name

Positiona

Sequence

CHOP

NM_001195053

Semi-qPCR

hchop_F hchop_R hchop-RT_F hchop-RT_R hactb_F hactb_R hactb-RT_F hactb-RT_R

191–211 469–489 530–547 587–607 741–760 981–1000 980–1000 1060–1080

5′-CTCCTGGAAATGAAGAGGAAG-3′ 5′-CCGTTCATTCTCTTCAGCTAG-3′ 5′-CGACTCGCCGAGCTCTGA-3′ 5′-TCCCAATTGTTCATGCTTGGT-3′ 5′-CATCACCATTGGCAATGAGC-3′ 5′-CAGGAGGAGCAATGATCTTG-3′ 5′-TCAAGATCATTGCTCCTCCTG-3′ 5′-CTGCTTGCTGATCCACATCTG-3′

qRT-PCR ACTB

NM_001101

Semi-qPCR qRT-PCR

Semi-quantitative PCR (semi-qPCR), quantitative real time PCR (qRT-PCR). a Nucleotide number in open reading flame. Adenine at start codon is counted as 1.

2.9. TUNEL assay for SH-SY5Y cells Apoptotic cells were visualized by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay using In Situ Cell Death Detection kit, Fluorescein (Roche Applied Science, Mannheim, Germany). Cell preparation, chemical treatment, post-processing of slides, and microscopic observation were performed in the same way as ROS detection. TUNEL assay was replicated three times with the use of independently cultured cells. 2.10. Exposure of zebrafish larvae to AA Adult zebrafish (Danio rerio) were maintained at 28.5 °C under 14-h light/10-h dark cycle condition in tanks of circulating system. Fertilized eggs from natural crosses were collected immediately after spawning and cultured at 28.5 °C in egg culture water (0.006% NaCl, 0.00025% methylene blue in deionized water) in a humidified incubator. At 1-day post fertilization (dpf), embryos were observed under stereomicroscope and healthy embryos were transferred to a small tank of the circulating system and cultured until 6-dpf. At 6-dpf, zebrafish larvae were transferred to 100 mm petri dishes and exposed to 2.5 mM AA in egg culture water (30 ml per dish) at 28.5 °C in a humidified incubator for 24 h. Thirty larvae were used in each exposure and the experiment was replicated four times with the use of independently obtained siblings. All animal experiments were designed according to the institutional ethical code for laboratory animal and were approved by the institutional review committee (Tokyo Women's Medical University, Approve No. 15-95-B).

were counterstained with DAPI and sealed with ProLong Gold antifade reagent, and microscopic observation was performed under the ECLIPSE E1000. TUNEL assay was replicated four times with the use of independently obtained siblings. 2.14. In situ hybridization for zebrafish larvae The digoxigenin (DIG)-labeled RNA probe for zebrafish chop was synthesized as described previously (Komoike and Matsuoka, 2013). The deparaffinized and rehydrated serial cross sections of zebrafish larvae were pre-hybridized with hybridization solution (HBS: 1× salt, 10% dextran sulfate, 50% formamide, 1 mg/ml rRNA, 1× Denhardt's solution; 10× salt: 3 M NaCl, 100 mM sodium phosphate buffer (pH 7.5), 100 mM EDTA, 100 mM Tris-Cl (pH 7.5); 100× Denhardt's solution: 2% bovine serum albumin (BSA), 2% Ficol, 2% polyvinyl pyrrolidone) at 65 °C for 30 min, and then hybridized with DIG-labeled probe overnight at 55 ° C. Slides were washed with wash buffer 1 (50% formamide, 2 × SSC, 0.1% Tween 20) and wash buffer 2 (25% formamide, 1× SSC, 0.5× PBS, 0.1% Tween 20) for 30 min at 65 °C twice each and with 0.1% Tween

2.11. Preparation of serial cross sections of zebrafish larvae AA-exposed and non-exposed zebrafish larvae at 6- or 7-dpf were fixed with 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) overnight at 4 °C with continuous rotation. The fixed embryos were serially dehydrated in Tissue Dehydration Solution (Wako), soaked in xylene, and embedded in Paraplast Plus embedding medium (Sigma-Aldrich). The specimens were cut into serial cross sections of 7 μm, stretched on the MAS-GP typeA micro slide glass (Matsunami Glass, Osaka, Japan) and used in the following experiments. 2.12. Klüver-Barrera's staining Deparaffinized and rehydrated serial cross sections of zebrafish larvae were stained by Klüver-Barrera's method to visualize myelin sheath and neuronal cells according to the original protocol (Klüver and Barrera, 1953). Klüver-Barrera's staining was replicated four times with the use of independently obtained siblings. 2.13. TUNEL assay for zebrafish larvae The deparaffinized and rehydrated serial cross sections of zebrafish larvae were also subjected to the TUNEL assay using the In Situ Cell Death Detection Kit, TMR Red (Roche Applied Science). Specimens

Fig. 1. AA induces cellular damage and cell death. Microscopic images (A) and viability (B) of SH-SY5Y cells exposed to 0, 5, or 10 mM AA for 8 h. Photographs shown are representative of four experiments (A). Cell viability was determined by SF assay (mean ± SD, n = 4). **P b 0.01 compared to 0 mM AA (b).

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20 in PBS (PBST) for 5 min at room temperature. Then, specimens were blocked with 5% BSA in PBST for 30 min at room temperature and incubated with ALXA FLUOR 555-conjugated anti digoxin antibody (Bioss) overnight at 4 °C. Following six times washing with PBST for 10 min each at room temperature, specimens were counterstained with DAPI and sealed with ProLong Gold antifade reagent, and microscopic observation was performed under the ECLIPSE E1000. In situ analysis was replicated four times with the use of independently obtained siblings. 2.15. Statistical analysis Data are expressed as the mean ± standard deviation (SD). Statistical significance was determined by the Student's t-test. A value of P b 0.05 was considered to be statistically significant. 3. Results 3.1. AA induces cellular damage and cell death in SH-SY5Y cells First, to examine whether AA induces damage or death of neuronal cells, SH-SY5Y cells were exposed to 5 or 10 mM AA for 8 h. The untreated SH-SY5Y cells were found to be stuck to the culture dish with short neurite-like protrusions under microscopic observation (Fig. 1A, a). In contrast, small part of and nearly half of the cells exposed to 5 and 10 mM AA, respectively, were round in shape without any protrusions (Fig. 1A, b and c). These observations were in good agreement with the cell viability estimated by the SF assay; e.g. approximately 95% and 60% of the cells exposed to 5 and 10 mM AA, respectively, were viable (Fig. 1B). These results suggest that exposure of SH-SY5Y cells to 10 mM AA causes definite cellular damage and cell death.

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3.2. AA activates the eIF2α–ATF4 pathway in SH-SY5Y cells Next, to investigate whether AA exposure induces the activation of the PERK–eIF2α pathway, one of the UPR branches, SH-SY5Y cells were exposed to AA for various incubation periods and at various concentrations. As a positive control, SH-SY5Y cells were exposed to thapsigargin (Tg), the inhibitor of endoplasmic reticular Ca2+-ATPase, and the activation of PERK–eIF2α pathway, i.e. phosphorylation of eIF2α at Ser51 and induction of ATF4 protein, in SH-SY5Y cells was confirmed (Supplemental Fig. S1A–D). When cells were exposed to 10 mM AA, the level of eIF2α protein phosphorylated and that of ATF4 significantly increased at 1 and 4 h, respectively (Fig. 2A and B). When cells were exposed for 8 h, phosphorylation of eIF2α significantly increased at all the concentrations tested and the level of ATF4 increased at 7.5 and 10 mM AA (Fig. 2C and D). In contrast, specific splicing of XBP1 mRNA was not evident at any time points (Supplemental Fig. S2A) and AA concentrations (Supplemental Fig. S2B). Exposure to Tg also failed to induce splicing of XBP1 mRNA (Supplemental Fig. S2C), suggesting that the IRE1–XBP1 pathway of the UPR might not play a major role in the ER stress response in SH-SY5Y cells. These findings indicate that AA exposure primarily induces the phosphorylation of eIF2α followed by the accumulation of its downstream protein, ATF4. 3.3. AA induces the expression of CHOP mRNA in SH-SY5Y cells We examined whether the expression of representative downstream targets of the UPR, including CHOP, GRP78, and GRP94, is upregulated in SH-SY5Y cells exposed to AA. The semi-quantitative PCR analysis (Fig. 3A and C) and the quantitative real time PCR analysis (Fig. 3B and D) showed that the expression of CHOP mRNA significantly

Fig. 2. AA activates the eIF2α–ATF4 pathway. Time- (A, B) and dose- (C, D) response in the activation of the eIF2α–ATF4 pathway in SH-SY5Y cells. Cells were exposed to 10 mM AA for 0–8 h (A, B) or 0–10 mM AA for 8 h (C, D) and phosphorylation of eIF2α and protein expression of ATF4 were detected by immunoblotting. Immunoblots shown are representative of four experiments (A, C). Relative level was calculated from the densitometric data of the immunoblots and is shown graphically (B, D; mean ± SD, n = 4). The values P-eIF2α represent the signal intensity ratio of phosphorylated protein to total protein relative to that of controls (0 mM or 0 h) corrected with ACTB. The values of ATF4 represent the signal intensity relative to the controls corrected with ACTB. *P b 0.05, **P b 0.01 compared to controls.

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Fig. 3. AA induces the expression of CHOP mRNA. Time- (A, B) and dose- (C, D) response in the expression of CHOP mRNA and ER chaperone proteins in SH-SY5Y cells. Cells were exposed to 10 mM AA for 0–8 h (A, B) or 0–10 mM AA for 8 h (C, D), and expression of CHOP mRNA and ER chaperones (GRP94 and GRP78 proteins) were detected by semi-quantitative PCR and immunoblotting, respectively. Images shown are representative of four experiments (A, C). Relative level of CHOP mRNA was determined by quantitative real time PCR and corrected with ACTB mRNA. Relative level of signal intensity of GRP94 and GRP78 was calculated from the densitometric data of the immunoblots and corrected with ACTB. The values representing the relative to controls (0 mM or 0 h) are shown graphically (B, D; mean ± SD, n = 4). For CHOP mRNA level, semilog graphs are used. *P b 0.05, **P b 0.01 compared to controls.

increased at all time points (Fig. 3A and B) and concentrations tested (Fig. 3C and D), and notably up to 100-fold at 7.5 or 10 mM AA after 8 h exposure. On the other hand, protein levels of ER chaperones, GRP78 and GRP94, were not changed (Fig. 3A–D). In contrast, exposure of SH-SY5Y cells to Tg significantly induced the expression of both CHOP mRNA and GRP78 protein (Supplemental Fig. S3A–D). These results suggest that, at least under our experimental conditions, exposure of SH-SY5Y cells to AA induces the expression of the pro-apoptotic CHOP protein but not the cytoprotective ER chaperones.

3.4. AA induces apoptosis in SH-SY5Y cells Dramatic elevation of CHOP mRNA expression raised the possibility that exposure of SH-SY5Y cells to AA induces apoptotic cell death. Therefore, we assessed the cleavage of poly (ADP-ribose) polymerase (PARP), the substrate of caspase-3, as an apoptosis marker by immunoblotting. Following exposure to 10 mM AA, the cleaved PARP was detected at 2 h and thereafter (Fig. 4A and B). In the dose-response experiment, the level of cleaved PARP was found to be increased at 2.5 mM and peaked at 7.5 mM AA (Fig. 4C and D). To confirm the apoptosis, we also performed the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. All of control cells were TUNEL negative (Fig. 4E, c). However, TUNEL positive cells were found in SH-SY5Y cells exposed to 10 mM AA for 6 h (Fig. 4E, g). These results suggest that the neuronal cell death caused by AA exposure involves apoptosis.

3.5. Attenuation of the ER stress protects SH-SY5Y cells from the AA-induced apoptosis To investigate the role of the ER stress in the AA-induced apoptosis, SH-SY5Y cells were treated with 4-PBA, a chemical chaperone that attenuates the ER stress (Kolb et al., 2015), prior to AA exposure. Treatment with 10 mM 4-PBA significantly reduced the levels of ATF4 protein and CHOP mRNA in cells exposed to 10 mM AA for 6 h, but did not clearly change the level of phosphorylated eIF2α (Fig. 5A and B). Furthermore, treatment with 4-PBA reduced the cleavage of PARP (Fig. 5C and D) and the number of TUNEL positive cells (Fig. 5E, c vs. g). These results suggest that attenuation of the ER stress protects cells from AA-induced apoptosis possibly by suppressing CHOP expression.

3.6. AA induces the accumulation of ROS in SH-SY5Y cells We investigated whether AA exposure increases intracellular ROS in SH-SY5Y cells. The control cells were faintly stained with CellRox ROS detection reagent (Fig. 6, c). In contrast, most of the cells exposed to 10 mM AA for 6 h were positively stained with CellRox (Fig. 6, g). When cells were treated with 5 mM NAC, a ROS scavenger, green signals were almost completely disappeared following exposure to AA (Fig. 6, k). Treatment with NAC alone did not affect ROS level (Fig. 6, o). These results indicate that AA exposure induces the accumulation of ROS in SH-SY5Y cells.

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Fig. 4. AA induces apoptosis. Time- (A, B) and dose- (C, D) response in the induction of apoptosis and TUNEL assay (E) in SH-SY5Y cells. Cells were exposed to 10 mM AA for 0–8 h (A, B) or 0–10 mM AA for 8 h (C, D), and cleavage of PARP was detected by immunoblotting. Gray arrowhead indicates the cleaved PARP. Immunoblots shown are representative of four experiments (A, C). Relative level was calculated from the densitometric data of the immunoblots and is shown graphically (B, D; mean ± SD, n = 4). The values represent the signal intensity relative to controls (0 mM or 0 h) corrected with ACTB. *P b 0.05, **P b 0.01 compared to controls. Cells were exposed to 10 mM AA for 6 h and apoptosis was detected by TUNEL assay (E). White arrows indicate TUNEL positive cells. Nuclei were counterstained with DAPI. DIC, TUNEL, and DAPI images were merged (Merge). Photographs shown are representative of three experiments.

3.7. Elimination of ROS suppresses the AA-induced ER stress and apoptosis in SH-SY5Y cell We examined whether ROS accumulation caused by AA exposure is responsible for the ER stress and the subsequent apoptosis. Treatment with 5 mM NAC lowered the AA-induced increase in the levels of phosphorylated eIF2α protein, ATF4 protein, and CHOP mRNA in SH-SY5Y cells exposed to 10 mM AA for 6 h (Fig. 7A and B). NAC treatment also suppressed the cleavage of PARP (Fig. 7C and D) and the number of TUNEL positive cells (Fig. 7E, c vs. g). These results suggest that elimination of ROS suppresses the activation of eIF2α–ATF4 pathway and protects cells from the AA-induced apoptosis. 4. AA induces chop mRNA expression and apoptosis in zebrafish larval brain Finally, we examined the expression of chop mRNA and apoptosis in the brain of zebrafish larvae following exposure to AA. Prior to detailed

analysis of brain, we investigated the abnormalities in whole body of AA-exposed larvae and found that AA exposure caused the slightly short body length, canopy-like morphology of head, and eye edema (Supplemental Fig. S4). Head cross sections of zebrafish larvae exposed to 2.5 mM AA for 24 h from 6- to 7-dpf were prepared and analyzed by Klüver-Barrera's staining, TUNEL assay, and in situ hybridization for chop expression. The Klüver-Barrera's staining revealed that AA exposure severely disrupted the brain structures in the forebrain, midbrain, and hindbrain (Fig. 8A). This abnormality appears to be caused mainly by disruption but not growth retardation of the brain; because the formation of basic structures of zebrafish larval brain is completed until 6-dpf, and therefore, normal brain of 7-dpf larvae is closely similar to that of 6-dpf except its size (Supplemental Fig. S5). In accord with this abnormality, a large number of TUNEL positive cells were observed throughout the brain (Fig. 8B, d–f, Fig. 8C). TUNEL positive cells did not distribute equally throughout the larval body, but accumulated in whole brain, olfactory bulb, branchial arches, and pectoral and tail fins (Supplemental Fig. S6). In addition, the chop-expressing cells were

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Fig. 5. Attenuation of the ER stress protects cells from the AA-induced apoptosis. Expression of the ER stress markers (A, B) and cleaved PARP (C, D) and TUNEL assay (E) of SH-SY5Y cells treated with or without 10 mM 4-PBA for 30 min prior to the exposure to 10 mM AA for 6 h. Gray arrowhead in (C) indicates the cleaved PARP. Immunoblots shown are representative of four experiments (A, C). The densitometric data of the immunoblots (P-eIF2α, ATF4, PARP, and ACTB) were used for the calculation of relative level of the signals and are shown graphically (B, D; mean ± SD, n = 4). The values of P-eIF2α represent the signal intensity ratio of phosphorylated protein to total protein corrected with ACTB (A). The values of ATF4 (A) and cleaved PARP (C) represent the relative signal intensity corrected with ACTB. In addition to semi-quantitative PCR (A), relative level of CHOP mRNA was determined by quantitative real time PCR and corrected with ACTB mRNA (B; mean ± SD, n = 4). *P b 0.05, **P b 0.01 compared to controls (0 mM 4-PBA, 10 mM AA). Apoptotic cell death was detected by TUNEL assay (E). White arrows indicate TUNEL positive cells. Nuclei were counterstained with DAPI. DIC, TUNEL, and DAPI images were merged (Merge). Photographs shown are representative of three experiments.

distributed to similar to those of TUNEL positive cells in the structurally disrupted brain (Fig. 8D, d–f, Fig. 8E). These results suggest that exposure of zebrafish larvae to AA causes the ER stress response, and results in the apoptosis and structural disruption in the brain. 5. Discussion

Fig. 6. AA induces the accumulation of ROS. SH-SY5Y cells were treated with or without 5 mM NAC for 30 min and then exposed to 10 mM AA for 6 h, and intracellular ROS was detected by CellRox Green reagent. Nuclei were counterstained with DAPI. DIC, CellRox, and DAPI images were merged (Merge). Photographs shown are representative of three experiments.

While the signaling pathways leading to AA neurotoxicity are not known, it has been reported that AA exposure induces apoptosis in SH-SY5Y cells (Sumizawa and Igisu, 2007), rat cortical neurons (Zhang et al., 2014), cerebral cortex of rats (Lakshmi et al., 2012), and zebrafish (Parng et al., 2007). Consistently, we found the cleavage of PARP in SH-SY5Y cells and the TUNEL positive cells in SH-SY5Y cells and brain of zebrafish larvae following exposure to AA. Furthermore, AA exposure induced the phosphorylation of eIF2α and its downstream effector ATF4 protein expression in SH-SY5Y cells. To the best of our knowledge, we show for the first time that AA activates the eIF2α–ATF4 signaling pathway in the neuronal cells, indicating the ER stress response induced by AA exposure. In response to ER stress, PERK, an ER-resident transmembrane protein, is activated by the autophosphorylation (Shi et al., 1998), and phosphorylates eIF2α at serine 51 (Schröder and Kaufman, 2005). Exposure of SH-SY5Y cells to AA induced the phosphorylation of PERK at threonine 980 (data not shown). Therefore, one of the UPR branches, the PERK–eIF2α pathway may be activated by AA exposure. Treatment with 4-PBA, a chemical chaperone that attenuates the ER stress mainly by preventing misfolded protein aggregation (Kolb et al., 2015), reduced the AA-induced apoptosis without substantially suppressing the phosphorylation of eIF2α in

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Fig. 7. Elimination of ROS suppresses the AA-induced ER stress and apoptosis. Expression of the ER stress markers (A, B) and cleaved PARP (C, D), and TUNEL assay (E) of SH-SY5Y cells treated with or without 5 mM NAC for 30 min prior to the exposure to 10 mM AA for 6 h. Gray arrowhead in (C) indicates the cleaved PARP. Immunoblots shown are representative of four experiments (A, C). The densitometric data of the immunoblots (P-eIF2α, ATF4, PARP, and ACTB) were used for the calculation of relative level of the signals and are shown graphically (B, D; mean ± SD, n = 4). The values of P-eIF2α represent the signal intensity ratio of phosphorylated protein to total protein corrected with ACTB. The values of ATF4 (A) and cleaved PARP (C) represent the relative signal intensity corrected with ACTB. In addition to semi-quantitative PCR (A), relative level of CHOP mRNA was determined by quantitative real time PCR and corrected with ACTB mRNA (B; mean ± SD, n = 4). *P b 0.05, **P b 0.01 compared to controls (0 mM 4-PBA, 10 mM AA). Apoptotic cell death was detected by TUNEL assay (E). White arrows indicate TUNEL positive cells. Nuclei were counterstained with DAPI. DIC, TUNEL, and DAPI images were merged (Merge). Photographs shown are representative of three experiments.

SH-SY5Y cells. Additional experiments such as gene knockdown of ATF4 and expression analyses of the genes in other UPR branches, IRE1–XBP1 and ATF6 pathways (Wang and Kaufman, 2012), are required to clarify the direct contribution of eIF2α-ATF4 pathway and their concomitant involvement in the AA-induced neuronal apoptotic cell death. On the other hand, CHOP, the downstream target of the transcription factors ATF4 and ATF6, plays a pivotal role in the ER stress-mediated apoptosis (Malhotra and Kaufman, 2007; Iurlaro and Munoz-Pinedo, 2015). In the present study, AA exposure induced the expression of CHOP mRNA in both SH-SY5Y cells and brain of zebrafish larvae. In contrast, the level of cytoprotective ER chaperones, GRP78 and GRP94 proteins (Zhu and Lee, 2015), was not elevated. Thus, the CHOP-dependent ER stress pathway is dominant over the survival pathway in SH-SY5Y cells exposed to AA. It has also been reported that treatment with aluminum (Mustafa Rizvi et al., 2014), cadmium (Kim et al., 2013), manganese (Seo et al., 2013), and tributyltin (Inageda and Matsuoka, 2009) induced the expression of CHOP and apoptosis in SH-SY5Y cells. These findings suggest that CHOP expression might be not only an ER stress marker but also a pro-apoptotic signaling molecule in the neuronal cells exposed to the neurotoxic chemicals including AA. Further molecular studies are therefore required to reveal the involvement of CHOP expression in the AA-induced apoptotic cell death in SH-SY5Y cells and zebrafish larvae brain. Intracellular production of ROS has been linked to ER stress and the UPR (Cao and Kaufman, 2014). The ER stress results in the accumulation of ROS that promotes a state of oxidative stress (Cullinan and Diehl, 2006). Additionally, ROS generated accelerate further the dysfunction of ER (Chaudhari et al., 2014). In SH-SY5Y cells, the level of ROS was

found to be elevated following exposure to AA. Consistently, it has been reported that AA exposure increased the level of ROS in PC12 rat pheochromocytoma cells (Pan et al., 2015) and rat cortical neurons (Zhang et al., 2014). Rats administered with AA also showed increased levels of hydroxyl radical and oxidative stress markers in the cerebral cortex (Lakshmi et al., 2012). Furthermore, we found that treatment with the ROS scavenger, NAC suppressed the activation of eIF2α–ATF4 signaling pathway, CHOP expression, and apoptosis in SH-SY5Y cells. These findings indicate that intracellular ROS produced by AA exposure are responsible for the ER stress response and subsequent apoptosis in the neuronal cells. It remains to be determined the mechanism of intracellular ROS accumulation by AA and its reactive epoxide metabolite, glycidamide (Calleman et al., 1990). We used zebrafish larvae at 6-dpf as an animal model for the investigation of AA neurotoxicity and found the expression of chop mRNA, TUNEL positive cells, and structural disruption in the brain following exposure to 2.5 mM AA for 24 h. Other investigators also have reported that exposure of 4-dpf zebrafish to 6.25 mM AA for 8 h caused loss of myelin in the forebrain and midbrain, and neuronal apoptosis (Parng et al., 2007). On the other hand, treatment with Tg, a widely-used ER stressor, activates the PERK and IRE1 pathways, and induces apoptosis in the brain and spinal cord of zebrafish embryos (Pyati et al., 2011). Because these findings suggest that zebrafish are a useful model for the analysis of the ER stress-mediated neuronal apoptosis, long-term exposure to much lower concentrations of AA will be of value in the further study. In summary, this study has shown that exposure of SH-SY5Y neuronal cells to AA induced the ER stress response accompanied with the

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Fig. 8. AA induces the UPR and apoptotic cell death in zebrafish larval brain. Klüver-Barrera's staining (A), TUNEL assay (B, C), and in situ hybridization of chop (D, E) in the coronal sections of forebrain, midbrain, and hindbrain of 7-dpf zebrafish larvae. Larvae were treated without (top) or with (bottom) 2.5 mM AA for 24 h from 6- to 7-dpf. Nuclei in (B) and (D) were counterstained with DAPI. White arrowheads in (D) indicate chop expressing cells. Photographs shown are representative of four experiments. Number of TUNEL positive cells (C) and chop-expressing cells (E) in each section of fore-, mid-, and hindbrain was shown graphically (mean ± SD, n = 12). Three sections were randomly selected from the three brain regions of four individuals and number of TUNEL positive cells and chop-expressing cells in the brain were counted. *P b 0.05, **P b 0.01 compared to controls (0 mM AA).

activation of eIF2α–ATF4 signaling pathway, one of the UPR branches. AA exposure increased the mRNA level of pro-apoptotic CHOP and induced apoptosis, and those were suppressed by the chemical chaperone 4-PBA. In addition, NAC suppressed the AA-induced activation of eIF2α–ATF4 pathway, CHOP expression, and apoptosis, indicating the involvement of ROS production in this ER stress response. Furthermore, exposure of zebrafish larvae to AA induced the chop mRNA expression and apoptosis in the brain. These in vitro and in vivo findings suggest that AA exposure induces apoptotic neuronal cell death through the ER stress response. A growing body of evidence has revealed that the ER stress and the UPR contribute to the pathogenesis

of neurodegenerative diseases (Scheper and Hoozemans, 2015). Investigations on the ER stress response may help understand the toxicological mechanisms of AA neurotoxicity.

Funding sources This study was supported in part by Grant-in-Aid for Young Scientists (B) from the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant number 26740025 (Y. K.) and Grant-in-Aid for Scientific Research (C) from JSPS KAKENHI Grant number 26460175 (M. M.).

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