CREB signaling pathway

CREB signaling pathway

    Ethanol-induced PGE 2 up-regulates Aβ production through PKA/CREB signaling pathway Amr Ahmed Gabr, Hyun Jik Lee, Xaykham Onphachanh,...

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    Ethanol-induced PGE 2 up-regulates Aβ production through PKA/CREB signaling pathway Amr Ahmed Gabr, Hyun Jik Lee, Xaykham Onphachanh, Young Hyun Jung, Jun Sung Kim, Chang Woo Chae, Ho Jae Han PII: DOI: Reference:

S0925-4439(17)30215-6 doi:10.1016/j.bbadis.2017.06.020 BBADIS 64803

To appear in:

BBA - Molecular Basis of Disease

Please cite this article as: Amr Ahmed Gabr, Hyun Jik Lee, Xaykham Onphachanh, Young Hyun Jung, Jun Sung Kim, Chang Woo Chae, Ho Jae Han, Ethanol-induced PGE2 up-regulates Aβ production through PKA/CREB signaling pathway, BBA - Molecular Basis of Disease (2017), doi:10.1016/j.bbadis.2017.06.020

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ACCEPTED MANUSCRIPT Title: Ethanol-induced PGE2 up-regulates Aβ production through PKA/CREB signaling pathway

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Amr Ahmed Gabr1, 3, #, Hyun Jik Lee1, 2, #, Xaykham Onphachanh1, Young Hyun Jung1, 2, Jun

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Sung Kim1, 2, Chang Woo Chae1, 2 and Ho Jae Han1, 2, *

Affiliation 1

Department of Veterinary Physiology, College of Veterinary Medicine, Research Institute

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for Veterinary Science, Seoul National University, Seoul 08826, Korea. BK21 PLUS Program for Creative Veterinary Science Research Center, Seoul National

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University, Seoul 08826, Korea.

Department of Physiology, Faculty of Veterinary Medicine, Cairo University, Giza 12211,

These authors contributed equally to this work

*Corresponing

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Egypt.

author: Ho Jae Han, D.V.M., Ph.D.

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Department of Veterinary Physiology, College of Veterinary Medicine and Research Institute for Veterinary Science, Seoul National University, Seoul 08826, Korea Tel: +82-02-880-1261 Fax: +82-2-880-2732 E-mail: [email protected]

Author contributions Gabr.AA: Conception and design, Collection and/or assembly of data, Data analysis and interpretation, Manuscript writing

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ACCEPTED MANUSCRIPT Lee.HJ: Conception and design, Collection and/or assembly of data, Data analysis and interpretation, Manuscript writing

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Onphachanh X: Collection and/or assembly of data

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Jung.YH: Data analysis and interpretation

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Kim.JS: Data analysis and interpretation Chae.CW: Data analysis and interpretation

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Han.HJ: Conception and design, Data analysis and interpretation, Manuscript writing

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Key words: Alzheimer’s disease, Ethanol, Amyloid, BACE1, Eukaryotic initiation factor 2α

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(eIF2α), Prostaglandin E2 (PGE2)

Abbreviations

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Running title: Ethanol-induced PGE2 up-regulates Aβ production

Aβ, Amyloid beta; AD, Alzheimer’s disease; APP, amyloid precursor protein; BACE1, beta-

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site APP-cleaving enzyme 1; ROS, reactive oxygen species; ER, endoplasmic reticulum; eIF2α, eukaryotic initiation factor alpha; CHOP, C/EBP-homologous protein; PGE2, prostaglandin E 2; EP, prostaglandin E receptor; COX, cyclooxygenase; NSAID, nonsteroidal anti-inflammatory drugs; mPGES, microsomal PGE2 synthase; CREB, cAMP response element-binding protein; PKA, protein kinase A; MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; ATF4, Activating transcription factor 4; PI3K, phosphatidylinositide 3-kinases; Akt, Protein kinase B

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ACCEPTED MANUSCRIPT Abstract Ethanol abuse aggravates dementia-associated cognitive defects through the progression of Alzheimer’s disease (AD) pathophysiology. Beta-site APP-cleaving enzyme 1 (BACE1) has

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been considered as a key regulator of AD pathogenesis by controlling amyloid beta peptide

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(Aβ) accumulation. In addition, previous studies reported that endoplasmic reticulum (ER)

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stress and neuroinflammation have been proposed in ethanol-induced neurodegeneration. Thus, we investigated the role of ER stress and PGE2, a neuroinflammation mediator, in the ethanol-stimulated BACE1 expression and Aβ production. Using the human-derived

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neuroblastoma cell line SK-N-MC, the results show that ethanol up-regulated BACE1 expression in a dose-dependent manner. Ethanol stimulated reactive oxygen species (ROS) production, which induced CHOP expression and eIF2α phosphorylation. PBA (an ER stress

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inhibitor) attenuated the ethanol-increased cyclooxygenase-2 (COX-2) expression and PGE2 production. By using salubrinal (an eIF2α dephosphorylation inhibitor) or EIF2A siRNA, we found that eIF2α phosphorylation mediated the ethanol-induced COX-2 expression. In

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addition, COX-2-induced BACE1 up-regulation was abolished by NS-398 (a selective COX-

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2 inhibitor). And, PF-04418948 (an EP-2 receptor inhibitor) pretreatment reduced ethanolinduced PKA activation and CREB phosphorylatin as well as ethanol-stimulated Aβ

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production. Furthermore, 14-22 amide (a PKA inhibitor) pretreatment or CREB1 siRNA transfection suppressed the ethanol-induced BACE1 expression. In conclusion, ethanolinduced eIF2α phosphorylation stimulates COX-2 expression and PGE2 production which

pathway.

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induces the BACE1 expression and Aβ production via EP-2 receptor-dependent PKA /CREB

1. Introduction

Alzheimer’s disease (AD), the most common form of dementia, is a progressive neurodegenerative disease [1]. Although the exact cellular mechanism mediating the pathogenesis of AD is not fully understood, it has been found to be associated with the aggregation of amyloid-beta (Aβ) peptides. Beta-site APP-cleaving enzyme 1 (BACE1) is a 3

ACCEPTED MANUSCRIPT rate-limiting enzyme for Aβ production through the proteolytic cleavage of amyloid precursor protein (APP) [2, 3]. Many studies have proposed that an increase in the BACE1 expression pattern could have an essential role in the progression of AD [4-6]. Heavy alcohol

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(ethanol) consumption is one of the possible risk factors contributing to AD [7-10]. A prior

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study suggested an association between alcohol abuse and a molecular mechanism in AD

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pathogenesis [11]. Although chronic alcohol consumption results in elevated BACE1 levels [11], the exact molecular mechanism by which ethanol induces BACE1 remain obscure. Accumulation of misfolded proteins in the lumen of endoplasmic reticulum (ER) triggers a

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cellular response called ER stress [12]. ER stress is one of the assumed mechanisms that mediates the adverse effect of ethanol on neurons [13]. In vivo experiments have revealed the

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ethanol-induced CHOP and eIF2α phosphorylation in the brain [14]. Moreover, ER stress signaling was found to be activated in brain samples from AD patients [15, 16]. A positive correlation between Aβ extracellular oligomerization and ER stress induction also indicates

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the essential role of ER stress in AD [17]. Moreover, it has been postulated that ER stress

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could promote the BACE1 expression mediated by eIF2α phosphorylation [18]. Because the contribution of ethanol-stimulated ER stress in AD pathogenesis has not been discussed in

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detail, further investigation is required. Ethanol abuse has been shown to intensify prostaglandins and overproduce inflammatory cytokines in the rat brain suggesting the involvement of neuroinflammation in alcohol-

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induced neurodegeneration [19]. The role of inflammation in AD progression has been shown by several observations including diminished Aβ deposition in the brain of an AD animal model after long-term NSAID treatment [20]. In AD patients, high COX-2 expression and PGE2 have been found in the brain and cerebrospinal fluid, respectively [21, 22]. Moreover, PGE2 mediates COX-2 induced-Aβ accumulation [23]. The biological activities of PGE2 are mediated by the four subtypes of the PGE2 receptors (EP1-4 receptors) [24]. Nevertheless, the role of EP receptors in ethanol-stimulated Aβ production has not yet been investigated. Moreover, previous reports have revealed the integration of ER stress and inflammation in the pathogenesis of some diseases other than AD [25, 26]. The relation between ethanolinduced ER stress and stimulation of PGE2 signaling has not been thoroughly investigated. Furthermore, on a cellular level, little attention has been given to ethanol-regulated βamyloidogenic effects; therefore, more research is needed to clearly show the contribution of 4

ACCEPTED MANUSCRIPT the ER stress and COX-2-associated pathways in the above effects. Such an investigation could explain how ethanol leads to AD progression. In this study, therefore, we aimed to investigate how ER stress and PGE2 signaling mediate ethanol-induced BACE1 and Aβ

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production.

2. Materials and methods

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2.1. Materials

SK-N-MC human neuroblastoma cells were provided by the Korean cell line bank (Seoul, Republic of Korea). Fetal bovine serum (FBS) and serum replacement (SR) were obtained

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from HyClone (UT, USA) and Gibco (Grand Island, NY, USA), respectively. β-actin, βtubulin, lamin A/C, COX-2, CREB-1, p-CREB (Ser113), CHOP, and cat-PKA antibodies

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were purchased from Santa Cruz biotechnology (Dallas, TX, USA). The eIF2α and p-eIF2α

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antibodies were obtained from Cell signaling technology, Inc. (Danvers, MA, USA). Aβ and BACE1 antibodies were acquired from Abcam (Cambridge, UK). The EP-2 antibody was purchased from Cusabio (Wuhan, China). Ethanol and the antibody for the C99 fragment of

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APP were obtained from EMD Millipore Copr. (Darmstadt, Germany). Secondary horse radish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse antibodies were supplied by Thermo Fisher (Waltham. MA, USA). Small interfering RNAs (siRNAs) for CREB1, EIF2A

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and non-targeting (NT) were purchased from Dharmacon (Lafayette, CO, USA). Acetaldehyde (AA), parafilm, 4-phenyl butyric acid (PBA), N-acetylcysteine (NAC), prostaglandin E2 (PGE2), fomepizole, diallyl disulfide and sodium azide were acquired from Sigma Aldrich (St. Louis, MO, USA). All inhibitors used in this study, such as sodium azide, NAC, PBA, salubrinal, NS-398, 14-22 amide and PF-04418948, did not significantly affect the cell viability (Sup. Fig. 1).

2.2. Cell culture Dishes (60 mm) were seeded with 5×105 SK-N-MC cells in 3 ml of culture medium consisting of

10% FBS

and 1% antibiotic (penicillin and streptomycin)-antimycotic

(amphotericin) mixture solution in Dulbecco's essential medium (DMEM; Gibco). Cells were incubated in a humid atmosphere at 37 °C and 5% CO2 for 24 h, and then, the medium was 5

ACCEPTED MANUSCRIPT exchanged with fresh medium. Subsequently, at 60% confluence, the culture medium was exchanged with DMEM (serum-free medium, 2 ml/60 mm dish) containing 1% SR and 1% antibiotic-antimycotic mixture to eliminate the effect of FBS and synchronize the cell cycle.

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Cells were incubated with the serum free medium for 12 h, and then conditioned medium was

2.3. Ethanol and acetaldehyde (AA) exposure

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changed to another serum-free medium 30 min prior to treatment with reagents.

Ethanol {2×10-1% (34 mM), 4×10-1% (69 mM), 8×10-1% (103 mM)} and AA {(3×10-4% (54

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μM), 6×10-4% (107 μM), 12×10-4% (215 μM)} were added directly to the 2 mL of conditioned media in the dose-dependent experiment. In the other experiments, cells were

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exposed to 4×10-1% ethanol and 6×10-4% AA for 30 min after treatment with the reagent. Dishes were directly sealed with a thin layer of paraffin film to prevent the evaporation of the

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2.4. Western blotting

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ethanol and AA.

Cells were collected after being washed twice with cold PBS and then pelleted by

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centrifugation at 13,200 rpm for 5 min at 4 °C. Afterward, cells were incubated for 30 min on ice with RIPA lysis buffer (Thermo Fisher) supplemented with proteinase and phosphatase inhibitor cocktail before centrifugation at 13,200 rpm for 30 min at 4°C. The protein

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concentration was determined using a bicinchoninic acid (BCA) assay kit (Thermo Fisher). Samples containing 10-20 μg of protein were loaded into 10-15% sodium dodecyl sulfatepolyacrylamide gel (SDS-PAGE) for electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane. Protein-containing membranes were washed with Tris-buffered saline containing 0.1% Tween-20 (TBST) solution and then blocked with 5% skim milk for 30 min. Blocked membranes were washed with TBST (3 times every 10 min), and incubated with primary antibody overnight at 4 °C. Then, the membranes were washed and incubated with HRP-conjugated secondary antibody at room temperature for 2 h. The western blotting bands were visualized by chemiluminescence (Bio-Rad, Hercules, CA, USA). Densitometric analysis was carried out with the Image J software (developed by Wayne Rasband, National Institutes of Health, Bethesda, MD, USA; http://rsb.info.nih. go.kr/ij/).

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2.5. Real time polymerase chain reaction (real time-PCR)

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RNA was extracted from cells using a commercial RNA extraction kit (TaKaRa, Otsu,

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Japan). Using a reverse transcription kit (iNtRON Biotechnology, Seongnam, Republic of Korea), reverse transcription of 1 μg of the extracted RNA was carried out. Reverse

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transcription was performed at 45 °C for 60 min for cDNA synthesis and at 95 °C for 5 min for RTase inactivation. With the forward and reverse primers for EP1-4 and β-actin, cDNA was amplified. Two microliters of the reverse transcription products were then amplified with

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the QuantiSpeed SYBR Kits (Life technologies, Gaithersburg, MD, USA). Real-time quantification of the RNA targets was performed in a Rotor-Gene 6000 real-time thermal

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cycling system (Corbett Research; NSW, Australia). The primer sequences are described in supplementary Table 1. The Real-Time PCR was performed as follows: 15 min at 95 °C for DNA polymerase activation; 15 sec at 95 °C for denaturing; and 40 cycles of 15 sec at 94 °C,

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30 sec at 56 °C, and 30 sec at 72 °C. Data were collected during the extension step (30 sec at

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72 °C), and analysis was performed with the software provided. Following the real-time PCR, a melting curve analysis was conducted to verify the specificity and identity of the PCR

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products.

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2.6. Immunocytochemistry

Cells cultured in confocal dishes (Thermo Fisher) were fixed with 80% acetone in PBS for 10 min at -20 °C followed by washing (3 times) with PBS. Subsequently, cells were blocked with 5% FBS in PBS to reduce the nonspecific binding. Blocked cells were incubated with a 1:100 dilution of primary antibody overnight at 4 °C followed by washing three times with PBS. Cells were incubated for 1 h at room temperature with Alexa Fluor secondary antibody and PI (propidium iodide) and then washed three times with PBS. Images were obtained with a FluoviewTM 300 confocal microscopy (Olympus, Japan).

2.7. Measurements of intracellular ROS levels Determination of the intracellular ROS level was performed using CM-H2DCFDA staining (DCF-DA, Life technology). Detached cells with 0.25% trypsin were counted using a Petroff7

ACCEPTED MANUSCRIPT Hausser Counter. Next, cells (5×105) were incubated with 10 μm DCF-DA in PBS for 1 h at 30 °C in the dark followed by washing twice with PBS. Then, a 100 μl cell suspension was loaded into a 96-well black plate and measured with a luminometer (Victor, Perkin-Elmer,

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Waltham, MA, USA).

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2.8. Transfection of small interfering RNAs

Prior to ethanol exposure, specific siRNAs for EIF2A and CREB1, and a non-targeting siRNA (as a negative control) were transfected into cells for 24 h with the TurboFect™

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transfection Reagent (Thermo Fisher) according to the manufacturer’s instructions. The

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concentration of each transfected siRNA was 25 nM.

2.9. Nuclear fractionation

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Following collection, cells were suspended in nuclear fractionation buffer [1.5 mM KH2PO4,

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2.5 mM EDTA, 1 mM dithiothreitol, 0.1 mM PMSF, and 10 mg/ml leupeptin (pH 7.5)] by pipetting and then incubated on ice for 10 min. Lysates were centrifuged at 3,000 rpm for 5

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min at 4 °C, and the supernatant was collected as a non-nuclear fraction. The remaining pellet was incubated with RIPA lysis buffer on ice for 20 min and then centrifuged at 15,000 rpm

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for 30 min at 4 °C. The supernatant representing the nuclear fraction was collected.

2.10. Determination of prostaglandin E2 and Aβ concentration Aβ and PGE2 production levels in the cell culture media after ethanol exposure were estimated using specific Aβ and PGE2 enzmyme-linked immunosorbent assay (ELISA) Kits obtained from Wako Chemical (Chuo-Ku, Osaka, Japan) and Cayman Chemical (Ann Arbor, MI, USA), respectively. Following the manufacturer’s instructions, the PGE2 concentration was determined.

2.11. Immunoprecipitation

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ACCEPTED MANUSCRIPT To estimate the amount of Aβ in the media using a commercial co-immunoprecipitation kit (Thermo Fisher), Aβ specific antibody was immobilized and conjugated to agarose beads according to the manufacturer’s instructions. After the cell treatment, cell-conditioned media

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were collected and centrifuged at 3,000 rpm for 10 min at 4 °C to remove cell debris.

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Subsequently, a cocktail of proteinase/phosphatase inhibitors was added to clean the media.

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The media were either stored at -80 °C or incubated with the agarose bead-conjugated Aβ specific antibody for 12 h at 4 °C. The agarose beads were spun down by centrifugation at 1,000 rpm for 1 min at 4 °C. The beads were washed six times with washing buffer, and the

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proteins were collected after incubation with elution buffer for 5 min. After that, the protein concentration was measured with the BCA assay, and the same amount of protein for the

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different samples was used to do the western blot analysis as previously mentioned.

2.12. Statistical analysis

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All data shown in the results are showed as a mean ± standard error of mean (S.E.M

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Comparison between the treated and control groups were performed by Student’s t-test for

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3. Results

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two group analysis. A p value < 0.05 was considered statistically significant.

3.1. Effect of ethanol on BACE1 expression and ER stress Previous studies reported that the blood alcohol concentration (BAC) level in alcohol abuser may reaches and exceeds 0.4%. Therefore, we investigated the effect of various concentrations of ethanol (0.2, 0.4, and 0.8%) on BACE1 expression in neuronal cells. As shown in the figure 1a, the expression levels of BACE1 and the C99 fragment of APP (C99) were directly increased with the increment in the ethanol concentration. qPCR was performed to evaluate the BACE1 mRNA transcript level. BACE1 mRNA expression increased in the ethanol-treated cells (Fig. 1b). To confirm this effect we determined the BACE1 expression in ethanol-treated mouse hippocampal neurons; ethanol-induced BACE1 expression in mouse hippocampal neurons (Sup. Fig. 2). Moreover, an increase in the fluorescence intensity of BACE1 in cells treated with the same amount of ethanol as above was observed through 9

ACCEPTED MANUSCRIPT immunofluorescence analysis (Fig. 1c). Based on the immunoprecipitation and ELISA results, ethanol increased Aβ secretion (Figs. 1d and 1e). In addition, we investigated the cytotoxic effect of ethanol on neuronal cell death. Our data showed that the various

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concentrations of ethanol (0 - 8×10-1%) incubation for 24 h did not affect the cell viability

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(Sup. Fig. 3). It has been known that ethanol is mainly metabolized via three metabolic

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enzymes, such as catalase, alcohol dehydrogenase and cytochrome P450IIE1 (CYP) [27]. To evaluate the effect of ethanol metabolism on BACE1 expression, cells were pretreated with inhibitors of ethanol metabolism, such as an alcohol dehydrogenase inhibitor fomepizole, a

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CYP inhibitor diallyl disulfide and a catalase inhibitor sodium azide. As shown in the supplementary figures 4a and 4b, pretreatment of fomepizole or diallyl disulfide did not affect the ethanol-induced BACE1 expression. However, there was a significant decrease in

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the levels of BACE1 and C99 in the sodium azide-pretreated cells (Fig. 1f). These findings indicate that catalase is a major metabolic enzyme involved in BACE1 expression induced by ethanol. To confirm the effect of ethanol metabolite acetaldehyde (AA) on BACE1

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expression, we used different concentrations of AA (0 - 12×10-4%). BACE1 expression was

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induced in response to different acetaldehyde (AA) concentrations (Fig. 1g). To examine whether ER stress contributes to the ethanol-induced BACE1 expression, we determined the

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response of CHOP and the phosphorylation of eIF2α to different ethanol concentrations. As shown in the figure 2a, ethanol-induced eIF2α phosphorylation and CHOP expression occurred in a dose-dependent manner. To investigate the potential mechanism that mediates

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the ethanol-induced ER stress, we investigated the effect of ethanol on the intra-cellular ROS production using the DCF-DA assay. As shown in the figure 2b, The DCF-DA result indicated that ethanol significantly induced intra-cellular ROS production. Hence, we pretreated the cells with NAC, a ROS scavenger, to determine the role of ROS in the ethanolinduced ER stress. Our results showed that NAC pretreatment suppressed ethanol-induced peIF2α and CHOP, as well as BACE1 and C99 (Fig. 2c). To investigate whether ER stress is up-stream of the ethanol-increased BACE1, we pretreated PBA, an ER stress inhibitor, prior to ethanol treatment. And, we observed that PBA pretreatment eliminated the ethanolinduced BACE1 and C99 levels caused by both ethanol and AA (Figs. 2d and 2e). Collectively, our findings suggest that ethanol stimulates ER stress via ROS production, which is involved in BACE1 expression.

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ACCEPTED MANUSCRIPT 3.2. Involvement of eIF2α phosphorylation in ethanol-induced COX-2 expression and PGE2 production

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To explore whether ethanol induces COX-2 in neurons, we performed a concentration-

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response experiment. Our results show that COX-2 expression was elevated in correlation to the ethanol concentration (Fig. 3a). Consistent with this result, the qPCR result showed an

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increase in the COX2 mRNA levels in the ethanol-treated cells compared with that of the non-treated control cells (Fig. 3b). Moreover, ethanol (4×10-1%) significantly promoted COX-2 expression within 12 h (Fig. 3c). To clarify whether ethanol-induced COX-2

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expression is related to ER stress, we investigated the effect of PBA on the ethanol-induced COX-2 expression. Our findings show that PBA ameliorated the ethanol-induced COX-2

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expression and PGE2 production (Figs. 3d and 3e). Moreover, transfection of EIF2A siRNA blocked COX-2 expression in ethanol-treated cells (Fig. 3f). We confirmed that transfection of EIF2A siRNA significantly inhibited mRNA expression of EIF2A (Sup. Fig. 5). As further

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verification, our data also showed that ethanol-induced COX-2 expression and eIF2α

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phosphorylation were augmented in cells treated with salubrinal, an eIF2α dephosphorylation inhibitor (Figs. 3g and 3h). In addition, we confirmed that ethanol-induced PGE2 production

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was potentiated by salubrinal (Fig. 3i). Cells incubated with NS-398, a selective COX-2 inhibitor, prior to ethanol exposure produced a low amount of PGE2 compared to the ethanoltreated cells without NS-398 (Fig. 4a). To determine whether COX-2 has a role in ethanolinduced BACE1, we treated cells with NS-398 followed by AA and ethanol exposure. The

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western blot results showed a significant decrease in BACE1 expression in the NS-398 pretreated cells compared to AA or ethanol-exposed cells without NS-398 (Figs. 4b and 4c). In addition, we confirmed that NS-398 pretreatment abolished BACE1 expression induced by ethanol in mouse hippocampal neuron (Sup. Fig. 6). As an alternative investigation, we treated cells with various concentrations of PGE2 to detect the relation between the PGE2produced from COX-2 overexpression and the ethanol-evoked BACE1. PGE2 increased the BACE1 and C99 expression in a dose-dependent manner (Fig. 4d). To verify this result, we investigated the effect of PGE2 on BACE1 expression at the single cell level using a confocal microscope. As shown in the figure 4e, BACE1 expression was increased in the PGE2-treated cells. Taken together, our findings suggest that ethanol-induced eIF2α phosphorylation stimulates COX-2 expression and PGE2 production, which is associated with ethanol-induced BACE1 expression. 11

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3.3. Involvement of EP-2 receptor signaling in ethanol-induced BACE1 expression and Aβ

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production

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Next, we tested whether PGE2 receptors are involved in the increased BACE1 by ethanol.

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First, we determined the effect of ethanol on the expression of PGE2 receptors. Our PCR results show that EP2 and EP3 are mainly expressed in cells (Sup. Fig. 7). With qPCR, we determined the effect of ethanol on the receptor expression of both subtypes. And, our data showed ethanol stimulated EP2 mRNA expression (Fig. 5a). In agreement, immunoblotting

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showed an increased EP-2 expression in response to increasing ethanol concentrations (Fig. 5b). Additionally, immunofluorescence staining showed an increase in the fluorescence

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intensity of EP-2 in the ethanol-treated cells (Fig. 5c). Next, we investigated the role of ER stress in the ethanol-induced EP-2 expression. Our results show that PBA blocked the ethanol-induced up-regulation of the EP-2 receptor at the transcription and translation levels

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(Figs. 5d and 5e). Moreover, pretreatment with PBA also attenuated the AA-induced EP-2

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expression (Fig. 5f). Those findings indicate that ethanol stimulates EP-2 expression via ER stress. Next, our results show that nuclear translocation and phosphorylation of CREB-1 at

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the Ser113 residue were induced in the ethanol-treated cells in a time-dependent manner (Fig. 6a and Sup. Fig. 8). We observed that the phosphorylation of CREB-1 induced by ethanol was abolished by NS-398 pretreatment (Fig. 6b). The cells were pretreated with 14-22 amide,

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a PKA inhibitor, and western blotting showed a more decreased BACE1 expression and CREB-1 phosphorylation (Fig. 6c). In agreement with the above results, BACE1 expression in cells transfected with CREB1 siRNA before ethanol exposure was much lower than that in the ethanol-treated cells, and it is similar to the expression in the NT siRNA transfected control (Fig. 6d). We pretreated cells with PF-04418948, a selective EP-2 antagonist. The western blot results showed the suppression of both ethanol-induced CREB-1 phosphorylation at Ser113 residue and catalytic PKA expressions. Interestingly, the cells pretreated with PF-04418948 showed BACE1 expression levels similar to that in the control group (Fig. 6e). And, PF-04418948 pretreatment decreased the ethanol-increased fluorescence intensity of catalytic PKA in the nuclear region (Fig. 6f). Moreover, PF04418948 pretreatment inhibited ethanol-induced Aβ secretion in the medium (Fig. 6g). The

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ACCEPTED MANUSCRIPT results suggest that the PGE2–induced EP-2 activation leads to BACE1 expression and Aβ

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production through the PKA/CREB-1 pathway.

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4. Discussion

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Previous animal and clinical studies have shown that ethanol-up-regulated BACE1 levels lead to dementia [11, 28]. In contrast, other experimental investigations have shown that ethanol reduces Aβ accumulation suggesting that ethanol at low concentrations might have a neuroprotective effect in AD [29, 30]. Although previous study reported that chronic alcohol

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consumption stimulates Aβ-producing enzyme expressions and tau accumulation [11, 31], there are few evidences showing the influence of alcohol on the Aβ accumulation in the

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human brain. A previous report showed the increased Aβ (1-42) level in the cerebral spinal fluid of patients with Wernicke-Korsakoff syndrome, alcohol-associated dementia [32]. In our approach, we tested a range of ethanol concentrations (0.2, 0.4, and 0.8%) and showed

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that the up-regulation of BACE1 is ethanol concentration dependent; thus, we used an ethanol

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concentration of 0.4%. According to the BAC level, 0.4% is a potentially harmful concentration for a first-time drinker while for those who have been drinking alcohol for

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many years, even if their BAC reaches and exceeds this level, they normally survive [33, 34]. Although, this level is unnoticeable in alcohol abusers, along these lines, heavy alcohol drinking contributes to the onset of AD. Furthermore, our results show that alcohol

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metabolism has a potential role in ethanol-induced BACE1. It is well established that AA is the primary ethanol metabolic product [35] in the brain which mediates the detrimental ethanol effect by inducing ROS production [36]. Moreover, acetaldehyde produced from ethanol metabolism elsewhere other than the brain, for example, in the liver, has limited ability to reach neurons [35]. Therefore, AA detected in the brain is mainly produced from ethanol metabolism in the brain, and 0.1-0.2% ethanol in the blood penetrates the blood brain barrier [37, 38]. In agreement, our result confirmed that AA at these low concentrations increases the BACE1 level in the same manner as the ethanol concentrations. Moreover, inhibition of ethanol metabolism by catalase abolished the BACE1 expression induced by the ethanol treatment. Our findings suggest that ethanol-produced AA stimulates BACE1 expression.

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ACCEPTED MANUSCRIPT Ethanol-induced neurodegeneration is correlated with oxidative stress [13, 14]. Our study revealed that

blocking ethanol-stimulated ROS

alleviated

the

ethanol-potentiated

phosphorylation of eIF2α and expressions of CHOP and BACE1. ROS-induced oxidative

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stress is strongly interrelated with AD pathogenesis through altered BACE1 expression [39].

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Ethanol-produced ROS triggers the accumulation of improperly folded proteins in the ER

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which lead to the induction of ER stress [40]. Cells treated with an oxidative agent showed that oxidative stress-induced BACE1 is mediated by eIF2α phosphorylation [18, 41]. Due to long length and particular AUGs of 5′ UTR in BACE1mRNA, BACE1 translation is constitutively minimized under basal condition, and is activated by eIF2α phosphorylation

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[42, 43]. It has been known that the eIF2α can be phosphorylated by four different kinases: the Heme-regulated eukaryotic initiation factor eIF2α kinase (HRI), the double-stranded

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RNA-activated protein kinase (PKR), the PKR-like endoplasmic reticulum-related kinase (PERK), the general control nonderepressible-2 kinase (GCN2) [44]. In our study, ethanol stimulates oxidative and ER stress which can activate PKR and PERK leading to eIF2α

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phosphorylation, respectively [45, 46]. Therefore, our findings indicate that ethanol-induced

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eIF2α phosphorylation can be a key factor stimulating BACE1 translation. Furthermore, inhibition of eIF2α kinases improves AD-associated memory deficit [47]. Moreover, we

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showed that attenuation of ethanol-induced ER stress ameliorated COX-2 expression as well as stimulated PGE2 secretion. Long-term ethanol treatment induced transcription factor TNFα and up-regulated COX-2 expression in astrocytes [48]. PGE2 is formed by the action of

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microsomal PGE2 synthase (mPGES), COX-1 and COX-2. However, COX-2 is responsible for the production of the main part of PGE2 in neurons and astrocytes [49, 50]. Induction of ER stress using tunicamycin resulted in PGE2 overproduction mediated by COX-2 [25, 51]. We clearly showed the direct effect of ethanol-induced ER stress on the regulation of COX-2 expression. Previous works demonstrated induction of ER stress signaling stimulated COX-2 expression through MAPK, NF-κB, eIF2α/ATF4 pathway [25, 26, 52]. In this study, ethanolenhanced eIF2α phosphorylation up-regulates COX-2 expression. PERK activation, an eIF2α kinase, mediates ER stress-stimulated neuroinflammation [53]. Meanwhile, our data showed salubrinal enhanced ethanol-induced eIF2α phosphorylation and COX-2 expression, whereas PBA pretreatment abolished ethanol-induced COX-2 and BACE1 expressions. Salubrinal inhibits the dephosphorylation of eIF2α [54]. And, PBA interacts with hydrophobic domain of unfolded and misfolded proteins, which increases the chance for correct folding [55, 56]. 14

ACCEPTED MANUSCRIPT Those findings suggest that opposite effects of ER stress inhibitors on the ethanol-induced COX-2 expression in this study may be due to the different action mechanism of inhibitors. In addition, a previous report demonstrated that salubrinal reduced neuronal cell apoptosis

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and ER stress by enhancement of eIFα/ATF4/CHOP signaling down-regulated by chronic ER

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stress in hippocampal neuron [57]. Conversely, another previous report showed that

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salubrinal augmented the free fatty acid-induced ER stress in pancreatic β-cell [58]. Although salubrinal has been known as an ER stress inhibitor, present and previous findings suggest that the effect of salubrinal on the ER stress can be dependent on cell types, experimental condition and cellular microenvironment. Moreover, selective inhibition of COX-2 has been

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found to block the PGE2-induced Aβ aggregation and Aβ-induced memory suppression in AD [23, 59]. Our results show that the ethanol-induced BACE1 was attenuated by blocking

leading to increased BACE1 levels.

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COX-2 indicating that the ethanol-elevated COX-2 is part of the ethanol-induced ER stress

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In vitro and in vivo experiments have revealed that PGE2-induced Aβ secretion is associated

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with specific G-protein coupled receptors (EP-1 to -4), particularly, the EP-2 and EP-4 receptor subtypes [24]. In an AD animal model, deletion of the EP-2 receptor counteracts the

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aggravation of AD [60]. Another recent study showed the essential role of EP-3 downstream signals which intensify the cognitive deficits in AD [61]. Concerning EP receptors, our results showed that ethanol significantly up-regulates EP-2 receptor compared to the EP-3 receptor. Compatible with our observation, increased G protein activity levels were observed

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in the alcohol-dependent brain [62]. However, further investigation is needed to elucidate how ethanol affects the regulation of the EP receptors. Our findings suggest that ER stress affects the ethanol-elevated EP-2 levels. Furthermore, we showed that ethanol enhances CREB phosphorylation by PKA activation eventually leading to increased BACE1 levels. Previous reports have emphasized that ethanol promotes adenylyl cyclase activity and subsequently cAMP accumulation which substantially mediates ethanol-enhanced PKA activation and nuclear translocation [63, 64]. Ethanol has a regulatory effect on CREB phosphorylation which is mediated by PKA [65]. Moreover, another investigator has shown the involvement of both the PI3K/AKT and PKA/CREB signaling pathways in the regulation of BACE1 expression and Aβ production stimulated by interleukin1β-induced PGE2 [59]. The EP-2 receptor regulates PGE2-induced Aβ secretion through a signaling cascade associated with cAMP [24]. Compatible with the pathway above; our study showed that the 15

ACCEPTED MANUSCRIPT selective blocking of the EP-2 receptor alleviated the ethanol-stimulated CREB/PKA pathway and associated BACE1. Collectively, these findings link ethanol-induced PGE2

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signaling through the EP-2 receptor with the stimulation of BACE1 and Aβ secretion.

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In conclusion, we demonstrated that ethanol-induced ER stress has a regulatory effect on the EP-2-associated PKA/CREB pathway by COX-2-mediated PGE2 production leading to

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BACE1 expression and Aβ production (Fig. 7). Our findings present the detailed mechanism how ethanol-induced ER stress regulates BACE1 expression and Aβ production. Moreover,

Conflict of Interest

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The authors declare no conflict of interest.

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we also suggest the substantial role of the EP-2 receptor in ethanol-induced AD progression.

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Acknowledgements

This research was supported by National R&D Program through the National Research

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Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2013M3A9B4076541), and Next-Generation BioGreen 21 Program (No. PJ011141),

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Rural Development Administration, Republic of Korea.

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ACCEPTED MANUSCRIPT Figure legends:

Figure 1. Effect of EtOH on BACE1 expression and Aβ secretion. (a) Cells were

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incubated with ethanol concentrations (0 - 8×10-1%) for 24 h. BACE1, C99, and β-actin

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expressions were detected by western blotting. n=3. *p<0.05 versus control. (b) Cells were

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incubated with ethanol (4×10-1%). Quantitative analysis of BACE1 and ACTB mRNA expression estimated by real-time PCR. BACE1 mRNA expression was normalized by ACTB mRNA expression. n=3. *p<0.05 versus control. (c) Cells were immunostained with BACE1 (green) and PI (red) after incubation with ethanol (4×10-1%) for 24 h. n=3, Scale bars, 10 um

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(magnification, ×800). *p<0.05 versus control. (d) Cells were exposed to ethanol (4×10-1%). Then, immunoprecipitated Aβ from the medium was analyzed by western blotting. n=3.

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*p<0.05 versus control. (e) Cells were incubated with ethanol (4×10-1%). Aβ concentration in the medium was quantified using specific Aβ ELISA kit. *p<0.05 versus control. (f) Cells were pretreated with sodium azide (5 mM) for 30 min prior to ethanol (4×10-1%) treatment

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for 24 h. BACE1, C99, and β-actin expressions were detected by western blotting. n=3.

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*p<0.05 versus vehicle-treated control. # p<0.05 versus 4×10-1% ethanol treatment. (g) Cells were incubated with AA concentrations (0 - 12×10-4%) for 24 h followed by western blotting

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of BACE1, C99, and β-actin. n=3. *p<0.05 versus vehicle-treated control.

Figure 2. EtOH-induced ROS generation stimulates BACE-1 expression through ER

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stress. (a) CHOP, eIF2α, p-eIF2α and β-actin expressions were analyzed by western blotting. n=3. *p<0.05 versus control. (b) Intracellular ROS was determined after treatment of ethanol (4×10-1%). n=6. *p<0.05 versus control. (c) Cells were pretreated with NAC (5 mM) for 30 min then exposed to ethanol 4×10-1% for 24 h. BACE1, C99, CHOP, eIF2α, p-eIF2α and βactin were detected by western blotting. n=3. *p<0.05 versus vehicle-treated control. # p<0.05 versus 4×10-1% ethanol treatment. (d and e) Cells were pretreated with PBA (5 mM) for 30 min prior to ethanol (4×10-1%) treatment for 24 h. BACE1, C99 and β-actin were detected by western blotting. n=3. *p<0.05 versus vehicle-treated control. # p<0.05 versus 4×10-1% ethanol treatment and 6×10-4% AA treatment, in d and e, respectively.

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ACCEPTED MANUSCRIPT Figure 3. Effect of EtOH-induced eIF2α phosphorylation on COX-2 expression. (a,c) COX-2 and β-actin expressions were detected by immunoblotting using specific antibody. n=3. *p<0.05 versus control. (b) COX2 and ACTB mRNA levels determined by RT-PCR and

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ACTB mRNA used as internal control. n=3. *p<0.05 versus control. (d and e) Cells were

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treated with PBA (5 mM) for 30 min before ethanol (4×10-1%) exposure for 24 h. COX-2 and

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β-actin expressions were measured by western blotting and secretion of PGE2 in cell medium was quantified using specific PGE2 ELISA kit. n=6. *p<0.05 versus vehicle-treated control. # p<0.05 versus 4×10-1% ethanol treatment. (f) Cells were transfected with EIF2A siRNA and non-targeting (NT) siRNA for 24 h prior 4×10-1% ethanol treatment for 12 h. COX-2, eIF2α

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and β-actin were analyzed by western blotting. n=3. *p<0.05 versus NT siRNA-treated control,. #p<0.05 versus 4×10-1% ethanol treatment. (g and h) Cells were pretreated with

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salubrinal (10 μM) for 30 min before ethanol (4×10-1%) treatment for 12 h. (g) p-eIF2α, eIF2α, COX-2 and β-actin expressions were determined by immunoblotting. n=3. *p<0.05 versus vehicle-treated control. #p<0.05 versus 4×10-1% ethanol treatment. (h) Cells were

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immunostained with COX-2 (green) and PI (red). n=3, Scale bars, 10 um (magnification,

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×800). *p<0.05 versus control. #p<0.05 versus 4×10-1% ethanol treatment. (i) Secreted PGE2 in cells medium was estimated by specific PGE2 ELISA kit. n=3. Western blotting data

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represents a three independent experiment. n=6. *p<0.05 versus vehicle-treated control, # p<0.05 versus 4×10-1% ethanol treatment.

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Figure 4. Effect of EtOH-induced PGE2 production by COX-2 on BACE1 expression. (a) Cells were pretreated with NS-398 (50 μM) for 30 min before 4×10-1% ethanol exposure for 12 h. Secreted PGE2 in cells medium was estimated by specific PGE2 ELISA kit. n=3. *p<0.05 versus vehicle-treated control, # p<0.05 versus 4×10-1% ethanol treatment. (b and c) Cells were incubated in NS-398 (50 μM) for 30 min before ethanol (4×10-1%) or AA (6×104

%) treatment for 24 h. and BACE1, C99, and β-actin expressions were determined by

western blotting. n=3. *p<0.05 versus vehicle-treated control. #p<0.05 versus 6×10-4% AA. (d) Cells were treated with PGE2 concentrations (0 - 10 μM). BACE1, C99 and β-actin expressions were analyzed by western blotting. n=3. *p<0.05 versus vehicle-treated control. (e) Cells were treated with PGE2 (0 - 10 μM) for 24 h. Cells were immunostained with BACE1 (green) and PI (red) Scale bars, 10 um (magnification, ×800). n=3. *p<0.05 versus vehicle-treated control, # p<0.05 versus 4×10-1% ethanol treatment. 18

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Figure 5. Role of EtOH-induced ER stress signaling in PGE2 receptors expression. (a)

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Cells were treated with ethanol (4×10-1%) for 24 h. EP2, EP3, and ACTB mRNA levels were

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analyzed by quantitative RT-PCR. ACTB mRNA served as internal control. n=3. *p<0.05 versus vehicle-treated control. (b) Cells were incubated in ethanol concentrations (0 – 8×10%). EP-2 expression was detected by western blotting. n=3. *p<0.05 versus vehicle-treated

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1

control. (c) Cells were treated with ethanol (4×10-1%) and immunostained with EP-2 (green) and PI (red). Scale bars, 10 um (magnification, ×800). n=3. *p<0.05 versus vehicle-treated (4×10-1%).

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control. (d and e) Cells were treated with PBA (5 mM) for 30 min before exposing to ethanol EP2, ACTB mRNA levels and EP-2 protein expression were analyzed by

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quantitative RT-PCR and immunoblotting, respectively. n=3. *p<0.05 versus vehicle-treated control. #p<0.05 versus 4×10-1% ethanol treatment. (f) Cells were pretreated with PBA (5 mM) for 30 min prior to AA incubation for 24 h (6×10-4%). EP-2 and β-actin expressions

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were analyzed by western blotting using specific antibody. n=3. *p<0.05 versus vehicle-

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treated control. # p<0.05 versus 6×10-4% AA treatment.

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Figure 6. EtOH-induced PGE2 stimulates Amyloid beta secretion via PKA/ CREB/BACE1 pathway. (a) Cells were treated with ethanol (4×10-1%). Non-nuclear and nuclear expression of CREB-1, p-CREB (Ser113) and β-actin were determined by western

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blotting. n=3. *p<0.05 versus vehicle-treated control. (b) Cells were incubated in NS-398 (50 μM) for 30 min before ethanol exposure (4×10-1%). BACE1, CREB, p-CREB (Ser113) and β-actin expressions were determined by western blotting. n=3. *p<0.05 versus vehicle-treated control. # p<0.05 versus 4×10-1% ethanol treatment. (c) Cells were pretreated of 14-22 amide (1 μM) for 30 min before ethanol (4×10-1%) exposure. CREB-1, p-CREB (Ser113) and BACE1 were analyzed by western blotting. n=3. *p<0.05 versus vehicle-treated control. # p<0.05 versus 4×10-1% ethanol treatment. (d) Cells were transfected with CREB1 siRNA or NT siRNA for 24 h prior to ethanol exposure for 24 h. BACE1 and β-actin expressions were measured by western blotting. n=3. *p<0.05 versus NT siRNA-treated control,. #p<0.05 versus 4×10-1% ethanol treatment. (e) Cells were incubated with PF-04418948 (10 μM) for 30 min prior to 4×10-1% ethanol treatment. Cat-PKA, CREB, p-CREB (Ser113), BACE1 and β-actin expressions were determined by western blotting. n=3. *p<0.05 versus vehicle-treated 19

ACCEPTED MANUSCRIPT control. # p<0.05 versus 4×10-1% ethanol treatment. (f) Cells were immunostained with CatPKA (green) and PI (red). Scale bars, 10 um (magnification, ×800). n=3. *p<0.05 versus vehicle-treated control. # p<0.05 versus 4×10-1% ethanol treatment. (g) Aβ was

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immunoprecipitated from cells medium and detected by western blotting. Western blot

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images represent a three independent experiment. *p<0.05 versus vehicle-treated control. #

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p<0.05 versus 4×10-1% ethanol treatment.

Figure 7. Schematic model illustrates the molecular mechanisms involved in ethanol-

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induced BACE1. Ethanol evokes ROS generation which leads to eIF2α phosphorylation. Then, eIF2α phosphorylation stimulates COX-2 expression. Induced COX-2 stimulates PGE2

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production. PGE2 binds with EP-2 receptor leads to PKA activation and CREB

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phosphorylation. Finally, activated CREB triggers BACE1 expression and Aβ secretion.

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References

[1] R.N. Kalaria, G.E. Maestre, R. Arizaga, R.P. Friedland, D. Galasko, K. Hall, J.A. Luchsinger, A. Ogunniyi, E.K. Perry, F. Potocnik, M. Prince, R. Stewart, A. Wimo, Z.X.

AC

Zhang, P. Antuono, G. World Federation of Neurology Dementia Research, Alzheimer's disease and vascular dementia in developing countries: prevalence, management, and risk factors, Lancet Neurol. 7 (2008) 812-826. [2] Y.W. Zhang, R. Thompson, H. Zhang, H. Xu, APP processing in Alzheimer's disease, Mol Brain. 4 (2011) 3. [3] R.E. Tanzi, L. Bertram, Twenty years of the Alzheimer's disease amyloid hypothesis: a genetic perspective, Cell. 120 (2005) 545-555. [4] H. Fukumoto, B.S. Cheung, B.T. Hyman, M.C. Irizarry, β-secretase protein and activity are increased in the neocortex in Alzheimer disease, Arch Neurol. 59 (2002) 1381-1389. [5] L.B. Yang, K. Lindholm, R. Yan, M. Citron, W. Xia, X.L. Yang, T. Beach, L. Sue, P. Wong, D. Price, R. Li, Y. Shen, Elevated β-secretase expression and enzymatic activity detected in sporadic Alzheimer disease, Nat Med. 9 (2003) 3-4. 20

ACCEPTED MANUSCRIPT [6] J. Zhao, Y. Fu, M. Yasvoina, P. Shao, B. Hitt, T. O'Connor, S. Logan, E. Maus, M. Citron, R. Berry, L. Binder, R. Vassar, Beta-site amyloid precursor protein cleaving enzyme 1 levels become elevated in neurons around amyloid plaques: implications for Alzheimer's

T

disease pathogenesis, J Neurosci. 27 (2007) 3639-3649.

IP

[7] P.A. Saunders, J.R. Copeland, M.E. Dewey, I.A. Davidson, C. McWilliam, V. Sharma, C.

SC R

Sullivan, Heavy drinking as a risk factor for depression and dementia in elderly men. Findings from the Liverpool longitudinal community study, Br J Psychiatry. 159 (1991) 213216.

NU

[8] L. Letenneur, S. Larrieu, P. Barberger-Gateau, Alcohol and tobacco consumption as risk factors of dementia: a review of epidemiological studies, Biomed Pharmacother. 58 (2004) 95-99.

MA

[9] E. Kapaki, I. Liappas, G.P. Paraskevas, I. Theotoka, A. Rabavilas, The diagnostic value of tau protein, β-amyloid (1-42) and their ratio for the discrimination of alcohol-related cognitive disorders from Alzheimer's disease in the early stages, Int J Geriatr Psychiatry. 20

D

(2005) 722-729.

TE

[10] A. Venkataraman, N. Kalk, G. Sewell, C.W. Ritchie, A. Lingford-Hughes, Alcohol and Alzheimer's Disease-Does Alcohol Dependence Contribute to β-Amyloid Deposition,

(2017) 151-158.

CE P

Neuroinflammation and Neurodegeneration in Alzheimer's Disease?, Alcohol Alcohol. 52

[11] S.R. Kim, H.Y. Jeong, S. Yang, S.P. Choi, M.Y. Seo, Y.K. Yun, Y. Choi, S.H. Baik, J.S.

AC

Park, A.R. Gwon, D.K. Yang, C.H. Lee, S.M. Lee, K.W. Park, D.G. Jo, Effects of chronic alcohol consumption on expression levels of APP and Aβ-producing enzymes, BMB Rep. 44 (2011) 135-139.

[12] C. Hetz, B. Mollereau, Disturbance of endoplasmic reticulum proteostasis in neurodegenerative diseases, Nat Rev Neurosci. 15 (2014) 233-249. [13] F. Yang, J. Luo, Endoplasmic Reticulum Stress and Ethanol Neurotoxicity, Biomolecules. 5 (2015) 2538-2553. [14] Z. Ke, X. Wang, Y. Liu, Z. Fan, G. Chen, M. Xu, K.A. Bower, J.A. Frank, M. Li, S. Fang, X. Shi, J. Luo, Ethanol induces endoplasmic reticulum stress in the developing brain, Alcohol Clin Exp Res. 35 (2011) 1574-1583. [15] L.D. Stutzbach, S.X. Xie, A.C. Naj, R. Albin, S. Gilman, P.S.P.G.S. Group, V.M. Lee, J.Q. Trojanowski, B. Devlin, G.D. Schellenberg, The unfolded protein response is activated 21

ACCEPTED MANUSCRIPT in disease-affected brain regions in progressive supranuclear palsy and Alzheimer's disease, Acta Neuropathol Commun. 1 (2013) 31. [16] J.J. Hoozemans, E.S. van Haastert, D.A. Nijholt, A.J. Rozemuller, P. Eikelenboom, W.

IP

disease hippocampus, Am J Pathol. 174 (2009) 1241-1251.

T

Scheper, The unfolded protein response is activated in pretangle neurons in Alzheimer's

SC R

[17] A.I. Placido, C.M. Pereira, A.I. Duarte, E. Candeias, S.C. Correia, C. Carvalho, S. Cardoso, C.R. Oliveira, P.I. Moreira, Modulation of endoplasmic reticulum stress: an opportunity to prevent neurodegeneration?, CNS Neurol Disord Drug Targets. 14 (2015) 518-

NU

533.

[18] F. Mouton-Liger, C. Paquet, J. Dumurgier, C. Bouras, L. Pradier, F. Gray, J. Hugon, Oxidative stress increases BACE1 protein levels through activation of the PKR-eIF2α

MA

pathway, Biochim Biophys Acta. 1822 (2012) 885-896.

[19] S. Alfonso-Loeches, M. Pascual-Lucas, A.M. Blanco, I. Sanchez-Vera, C. Guerri,

Neurosci. 30 (2010) 8285-8295.

D

Pivotal role of TLR4 receptors in alcohol-induced neuroinflammation and brain damage, J

TE

[20] G.P. Lim, F. Yang, T. Chu, P. Chen, W. Beech, B. Teter, T. Tran, O. Ubeda, K.H. Ashe, S.A. Frautschy, G.M. Cole, Ibuprofen suppresses plaque pathology and inflammation in a

CE P

mouse model for Alzheimer's disease, J Neurosci. 20 (2000) 5709-5714. [21] L. Ho, C. Pieroni, D. Winger, D.P. Purohit, P.S. Aisen, G.M. Pasinetti, Regional distribution of cyclooxygenase-2 in the hippocampal formation in Alzheimer's disease, J

AC

Neurosci Res. 57 (1999) 295-303. [22] T.J. Montine, K.R. Sidell, B.C. Crews, W.R. Markesbery, L.J. Marnett, L.J. Roberts, 2nd, J.D. Morrow, Elevated CSF prostaglandin E2 levels in patients with probable AD, Neurology. 53 (1999) 1495-1498. [23] L.A. Kotilinek, M.A. Westerman, Q. Wang, K. Panizzon, G.P. Lim, A. Simonyi, S. Lesne, A. Falinska, L.H. Younkin, S.G. Younkin, M. Rowan, J. Cleary, R.A. Wallis, G.Y. Sun, G. Cole, S. Frautschy, R. Anwyl, K.H. Ashe, Cyclooxygenase-2 inhibition improves amyloid-β-mediated suppression of memory and synaptic plasticity, Brain. 131 (2008) 651664. [24] T. Hoshino, T. Nakaya, T. Homan, K. Tanaka, Y. Sugimoto, W. Araki, M. Narita, S. Narumiya, T. Suzuki, T. Mizushima, Involvement of prostaglandin E2 in production of amyloid-β peptides both in vitro and in vivo, J Biol Chem. 282 (2007) 32676-32688. 22

ACCEPTED MANUSCRIPT [25] J.H. Hung, I.J. Su, H.Y. Lei, H.C. Wang, W.C. Lin, W.T. Chang, W. Huang, W.C. Chang, Y.S. Chang, C.C. Chen, M.D. Lai, Endoplasmic reticulum stress stimulates the expression of cyclooxygenase-2 through activation of NF-κB and pp38 mitogen-activated

T

protein kinase, J Biol Chem. 279 (2004) 46384-46392.

IP

[26] B. Luo, Y. Lin, S. Jiang, L. Huang, H. Yao, Q. Zhuang, R. Zhao, H. Liu, C. He, Z. Lin,

SC R

Endoplasmic reticulum stress eIF2α-ATF4 pathway-mediated cyclooxygenase-2 induction regulates cadmium-induced autophagy in kidney, Cell Death Dis. 7 (2016) e2251. [27] D. Villalobos-Garcia, R. Hernandez-Munoz, Catalase increases ethanol oxidation

NU

through the purine catabolism in rat liver, Biochem Pharmacol. (2017). [28] K.J. Anstey, H.A. Mack, N. Cherbuin, Alcohol consumption as a risk factor for dementia and cognitive decline: meta-analysis of prospective studies, Am J Geriatr Psychiatry. 17

MA

(2009) 542-555.

[29] D. Ormeno, F. Romero, J. Lopez-Fenner, A. Avila, A. Martinez-Torres, J. Parodi, Ethanol reduces amyloid aggregation in vitro and prevents toxicity in cell lines, Arch Med

D

Res. 44 (2013) 1-7.

TE

[30] J. Parodi, D. Ormeno, L.D. Ochoa-de la Paz, Amyloid pore-channel hypothesis: effect of

48 (2015) 13-18.

CE P

ethanol on aggregation state using frog oocytes for an Alzheimer's disease study, BMB Rep.

[31] T.F. Gendron, S. McCartney, E. Causevic, L.W. Ko, S.H. Yen, Ethanol enhances tau

71.

AC

accumulation in neuroblastoma cells that inducibly express tau, Neurosci Lett. 443 (2008) 67-

[32] S. Matsushita, T. Miyakawa, H. Maesato, T. Matsui, A. Yokoyama, H. Arai, S. Higuchi, H. Kashima, Elevated cerebrospinal fluid tau protein levels in Wernicke's encephalopathy, Alcohol Clin Exp Res. 32 (2008) 1091-1095. [33] R.A. Deitrich, V.G. Erwin, Pharmacological effects of ethanol on the nervous system, CRC Press, Place Published, 1996. [34] D.E. Kouzoukas, G. Li, M. Takapoo, T. Moninger, R.C. Bhalla, N.J. Pantazis, Intracellular calcium plays a critical role in the alcohol-mediated death of cerebellar granule neurons, J Neurochem. 124 (2013) 323-335. [35] R. Deitrich, S. Zimatkin, S. Pronko, Oxidation of ethanol in the brain and its consequences, Alcohol Res Health. 29 (2006) 266-273.

23

ACCEPTED MANUSCRIPT [36] C. D'Addario, Y. Ming, S.O. Ogren, L. Terenius, The role of acetaldehyde in mediating effects of alcohol on expression of endogenous opioid system genes in a neuroblastoma cell line, FASEB J. 22 (2008) 662-670.

T

[37] O.V. Chumakova, A.V. Liopo, S.A. Chizhik, V.V. Tayurskaya, L.L. Gerashchenko,

IP

O.Y. Komkov, Effects of ethanol and acetaldehyde on isolated nerve ending membranes:

SC R

study by atomic-forced microscopy, Bull Exp Biol Med. 130 (2000) 921-924. [38] J.A. Hernandez, R.C. Lopez-Sanchez, A. Rendon-Ramirez, Lipids and Oxidative Stress Associated with Ethanol-Induced Neurological Damage, Oxid Med Cell Longev. 2016 (2016)

NU

1543809.

[39] H.J. Lee, J.M. Ryu, Y.H. Jung, S.J. Lee, J.Y. Kim, S.H. Lee, I.K. Hwang, J.K. Seong, H.J. Han, High glucose upregulates BACE1-mediated Aβ production through ROS-

MA

dependent HIF-1α and LXRα/ABCA1-regulated lipid raft reorganization in SK-N-MC cells, Sci Rep. 6 (2016) 36746.

[40] G. Chen, C. Ma, K.A. Bower, X. Shi, Z. Ke, J. Luo, Ethanol promotes endoplasmic

D

reticulum stress-induced neuronal death: involvement of oxidative stress, J Neurosci Res. 86

TE

(2008) 937-946.

[41] L. Devi, M. Ohno, PERK mediates eIF2α phosphorylation responsible for BACE1

CE P

elevation, CREB dysfunction and neurodegeneration in a mouse model of Alzheimer's disease, Neurobiol Aging. 35 (2014) 2272-2281. [42] S. Lammich, S. Schobel, A.K. Zimmer, S.F. Lichtenthaler, C. Haass, Expression of the

620-625.

AC

Alzheimer protease BACE1 is suppressed via its 5'-untranslated region, EMBO Rep. 5 (2004)

[43] T. O'Connor, K.R. Sadleir, E. Maus, R.A. Velliquette, J. Zhao, S.L. Cole, W.A. Eimer, B. Hitt, L.A. Bembinster, S. Lammich, S.F. Lichtenthaler, S.S. Hebert, B. De Strooper, C. Haass, D.A. Bennett, R. Vassar, Phosphorylation of the translation initiation factor eIF2α increases BACE1 levels and promotes amyloidogenesis, Neuron. 60 (2008) 988-1009. [44] T.E. Dever, Gene-specific regulation by general translation factors, Cell. 108 (2002) 545-556. [45] G. Li, C. Scull, L. Ozcan, I. Tabas, NADPH oxidase links endoplasmic reticulum stress, oxidative stress, and PKR activation to induce apoptosis, J Cell Biol. 191 (2010) 1113-1125. [46] Z.W. Liu, H.T. Zhu, K.L. Chen, X. Dong, J. Wei, C. Qiu, J.H. Xue, Protein kinase RNAlike endoplasmic reticulum kinase (PERK) signaling pathway plays a major role in reactive 24

ACCEPTED MANUSCRIPT oxygen species (ROS)-mediated endoplasmic reticulum stress-induced apoptosis in diabetic cardiomyopathy, Cardiovasc Diabetol. 12 (2013) 158. [47] T. Ma, M.A. Trinh, A.J. Wexler, C. Bourbon, E. Gatti, P. Pierre, D.R. Cavener, E.

IP

memory deficits, Nat Neurosci. 16 (2013) 1299-1305.

T

Klann, Suppression of eIF2α kinases alleviates Alzheimer's disease-related plasticity and

SC R

[48] S.L. Valles, A.M. Blanco, M. Pascual, C. Guerri, Chronic ethanol treatment enhances inflammatory mediators and cell death in the brain and in astrocytes, Brain Pathol. 14 (2004) 365-371.

NU

[49] K.G. Peri, P. Hardy, D.Y. Li, D.R. Varma, S. Chemtob, Prostaglandin G/H synthase-2 is a major contributor of brain prostaglandins in the newborn, J Biol Chem. 270 (1995) 2461524620.

Pharmacol Ther. 49 (1991) 153-179.

MA

[50] W.L. Smith, L.J. Marnett, D.L. DeWitt, Prostaglandin and thromboxane biosynthesis,

[51] T. Hosoi, M. Honda, T. Oba, K. Ozawa, ER stress upregulated PGE(2)/IFNgamma-

D

induced IL-6 expression and down-regulated iNOS expression in glial cells, Sci Rep. 3

TE

(2013) 3388.

[52] Z. Rasheed, T.M. Haqqi, Endoplasmic reticulum stress induces the expression of COX-2

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through activation of eIF2α, p38-MAPK and NF-κB in advanced glycation end products stimulated human chondrocytes, Biochim Biophys Acta. 1823 (2012) 2179-2189. [53] G.P. Meares, Y. Liu, R. Rajbhandari, H. Qin, S.E. Nozell, J.A. Mobley, J.A. Corbett,

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E.N. Benveniste, PERK-dependent activation of JAK1 and STAT3 contributes to endoplasmic reticulum stress-induced inflammation, Mol Cell Biol. 34 (2014) 3911-3925. [54] M. Boyce, K.F. Bryant, C. Jousse, K. Long, H.P. Harding, D. Scheuner, R.J. Kaufman, D. Ma, D.M. Coen, D. Ron, J. Yuan, A selective inhibitor of eIF2α dephosphorylation protects cells from ER stress, Science. 307 (2005) 935-939. [55] U. Ozcan, E. Yilmaz, L. Ozcan, M. Furuhashi, E. Vaillancourt, R.O. Smith, C.Z. Gorgun, G.S. Hotamisligil, Chemical chaperones reduce ER stress and restore glucose homeostasis in a mouse model of type 2 diabetes, Science. 313 (2006) 1137-1140. [56] L. Ozcan, A.S. Ergin, A. Lu, J. Chung, S. Sarkar, D. Nie, M.G. Myers, Jr., U. Ozcan, Endoplasmic reticulum stress plays a central role in development of leptin resistance, Cell Metab. 9 (2009) 35-51.

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ACCEPTED MANUSCRIPT [57] J.S. Kim, R.W. Heo, H. Kim, C.O. Yi, H.J. Shin, J.W. Han, G.S. Roh, Salubrinal, ER stress inhibitor, attenuates kainic acid-induced hippocampal cell death, J Neural Transm (Vienna). 121 (2014) 1233-1243.

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[58] M. Cnop, L. Ladriere, P. Hekerman, F. Ortis, A.K. Cardozo, Z. Dogusan, D. Flamez, M.

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Boyce, J. Yuan, D.L. Eizirik, Selective inhibition of eukaryotic translation initiation factor 2

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α dephosphorylation potentiates fatty acid-induced endoplasmic reticulum stress and causes pancreatic β-cell dysfunction and apoptosis, J Biol Chem. 282 (2007) 3989-3997. [59] P. Wang, P.P. Guan, T. Wang, X. Yu, J.J. Guo, Z.Y. Wang, Aggravation of Alzheimer's

neuron cells, Aging Cell. 13 (2014) 605-615.

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disease due to the COX-2-mediated reciprocal regulation of IL-1β and Aβ between glial and

[60] X. Liang, Q. Wang, T. Hand, L. Wu, R.M. Breyer, T.J. Montine, K. Andreasson,

MA

Deletion of the prostaglandin E2 EP2 receptor reduces oxidative damage and amyloid burden in a model of Alzheimer's disease, J Neurosci. 25 (2005) 10180-10187. [61] V. Maingret, G. Barthet, S. Deforges, N. Jiang, C. Mulle, T. Amedee, PGE2-EP3

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signaling pathway impairs hippocampal presynaptic long-term plasticity in a mouse model of

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Alzheimer's disease, Neurobiol Aging. 50 (2017) 13-24. [62] R.S. Jope, L. Song, C.A. Grimes, M.A. Pacheco, G.E. Dilley, X. Li, H.Y. Meltzer, J.C.

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Overholser, C.A. Stockmeier, Selective increases in phosphoinositide signaling activity and G protein levels in postmortem brain from subjects with schizophrenia or alcohol dependence, J Neurochem. 70 (1998) 763-771.

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[63] S. Kumar, Q. Ren, J.H. Beckley, T.K. O'Buckley, E.D. Gigante, J.L. Santerre, D.F. Werner, A.L. Morrow, Ethanol Activation of Protein Kinase A Regulates GABA(A) Receptor Subunit Expression in the Cerebral Cortex and Contributes to Ethanol-Induced Hypnosis, Front Neurosci. 6 (2012) 44. [64] D.P. Dohrman, H.M. Chen, A.S. Gordon, I. Diamond, Ethanol-induced translocation of protein kinase A occurs in two phases: control by different molecular mechanisms, Alcohol Clin Exp Res. 26 (2002) 407-415. [65] O. Asher, T.D. Cunningham, L. Yao, A.S. Gordon, I. Diamond, Ethanol stimulates cAMP-responsive element (CRE)-mediated transcription via CRE-binding protein and cAMP-dependent protein kinase, J Pharmacol Exp Ther. 301 (2002) 66-70.

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ACCEPTED MANUSCRIPT Highlights Ethanol induces BACE1 expression and Aβ production.

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Ethanol-produced ROS stimulates eIF2α phosphorylation and CHOP expression. ER stress enhanced by ethanol stimulates COX-2-mediated PGE2 production, which is

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critical for BACE1 expression.

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