Mol Neurobiol DOI 10.1007/s12035-017-0703-3
Ethanol Alters APP Processing and Aggravates Alzheimer-Associated Phenotypes Daochao Huang 1 & Mengjiao Yu 1 & Shou Yang 1 & Dandan Lou 1 & Weitao Zhou 1 & Lingling Zheng 1 & Zhe Wang 2 & Fang Cai 2 & Weihui Zhou 1 & Tingyu Li 1 & Weihong Song 1,2
Received: 3 May 2017 / Accepted: 31 July 2017 # Springer Science+Business Media, LLC 2017
Abstract The majority of Alzheimer’s disease (AD) cases are sporadic with unknown causes. Many dietary factors including excessive alcohol intake have been reported to increase the risk to develop AD. The effect of alcohol on cognitive functions and AD pathogenesis remains elusive. In this study, we investigated the relationship between ethanol exposure and Alzheimer’s disease. Cell cultures were treated with ethanol at different dosages for different durations up to 48 h and an AD model mouse was fed with ethanol for 4 weeks. We found that ethanol treatment altered amyloid β precursor protein (APP) processing in cells and transgenic AD model mice. High ethanol exposure increased the levels of APP and betasite APP cleaving enzyme 1 (BACE1) and significantly promoted amyloid β protein (Aβ) production both in vitro and in vivo. The upregulated APP and BACE1 expressions upon ethanol treatment were at least partially due to the activation of APP and BACE1 transcriptions. Furthermore, ethanol treatment increased the deposition of Aβ and neuritic plaque formation in the brains and exuberated learning and memory impairments in transgenic AD model mice. Taken together, our results demonstrate that excessive ethanol intake facilitates AD pathogenesis. * Weihong Song
[email protected] 1
Chongqing Key Laboratory of Translational Medical Research in Cognitive Development and Learning and Memory Disorders, Ministry of Education Key Laboratory of Child Development and Disorders, China International Science and Technology Cooperation Base of Child Development and Critical Disorders, Children’s Hospital of Chongqing Medical University, Chongqing 400014, China
2
Townsend Family Laboratories, Department of Psychiatry, Brain Research Center, The University of British Columbia, 2255 Wesbrook Mall, Vancouver, BC V6T 1Z3, Canada
Keywords Ethanol exposure . Alzheimer’s disease . APP processing . BACE1 . Aβ . Cognitive deficits
Abbreviations AD Alzheimer’s disease APP Amyloid β precursor protein BACE1 Beta-site APP cleaving enzyme 1 Aβ Amyloid β protein DID Drinking in the dark CHX Cycloheximide
Introduction Alzheimer’s disease (AD) is the most common neurodegenerative disorder leading to progressive cognitive impairment. The characteristic features of AD neuropathology include neuritic plaque, neurofibrillary tangle, and neuronal loss. Neuritic plaques are formed by abnormal deposition of amyloid β protein (Aβ) in the brain [1, 2]. Aβ is derived from sequential cleavages of the amyloid β precursor protein (APP) by βsecretase and γ-secretase complex [3]. The oligomeric forms of Aβ are neurotoxic and have been shown to impair synaptic plasticity, and their accumulation results in the formation of neuritic plaques. APP is a type I integral membrane protein undergoing a complex proteolytic processing. APP is cleaved by the beta-site APP cleaving enzyme 1 (BACE1), the bona fide β-secretase in vivo, at the Asp1 site to produce C-terminal fragment-β (CTFβ or C99 as it consists of the last 99 amino acids of APP), and C99 is subsequently cleaved by the γsecretase complex to release the Aβ protein [4–8]. However, APP is mostly cleaved by α-secretases to generate CTFα (or C83), and C83 is further cleaved by γ-secretase to produce CTFγ and p3 fragment. Alternatively, APP can also be cleaved
Mol Neurobiol
by BACE1 at the Glu11 site to produce CTFβ′ C89 [9], or by BACE2 at the Phe20 site to release CTFθ C80 [10, 11], precluding Aβ production. Increasing Aβ production by enhancing BACE1 and/or γ-secretase activity could facilitate AD pathologies, and preventative and therapeutic inhibition of BACE1 and/or γ-secretase could be an effective approach for AD treatment [12–16]. Less than 1% of AD patients are early-onset familial cases caused by genetic defects including mutations in APP, presenilin-1 (PS1), and presenilin-2 (PS2) gene, as well as trisomy-21. The majority of AD cases are sporadic with unknown causes. The risk factors of AD could be both genetic and environmental [17]. Many dietary factors such as high caloric intake, saturated fatty acids consumption, and excessive alcohol drinking have been reported to increase the risk of developing AD [18, 19]. A number of studies investigated a potential association of ethanol consumption and AD, but the findings remain controversial [20]. Light-to-moderate/regular alcohol drinking could have a protective effect, and wines containing high amount of antioxidant polyphenol molecule display a beneficial effect by preventing AD-type deterioration of Aβ neuropathology and memory deficits [21–23]. Moreover, light-to-moderate/regular alcohol consumption can reduce cholesterol in the cardiovascular system, which may decrease the risk of AD, as well [24]. However, heavy ethanol consumption could impair memory and cognitive functions, and lead to the loss of cortical and subcortical brain structures [25, 26]. Ethanol consumption increases the APP expression [27], causes loss of hippocampal cells [28], and enhances Aβ production [29]. Although these findings suggested a correlation between ethanol and AD or AD-like symptoms, a direct correlation between ethanol consumption and AD has not been established [30]. In this study, we investigated whether ethanol exposure contributed to AD pathogenesis and the underlying mechanism. We showed that ethanol treatment significantly increase APP and expression both in vitro and in vivo. Ethanol feeding to the AD model mice enhanced Aβ production and neuritic plaque formation and aggravated learning and memory deficits. Our work provides evidence that heavy ethanol exposure has a detrimental effect on AD and increases the risk of AD development.
Materials and Methods Transgenic Mice and Ethanol Treatment All animal studies were performed in accordance with the Guide for the Care and Use of Laboratory Animals of the Ethics Committee of Chongqing Medical University. The experimental protocols were approved by the Animal Study Committee of the Children’s Hospital of Chongqing Medical University. APP23 mice overexpressing the Swedish APP751 (KM → NL) mutant
transgene and PS45 mice overexpressing the human G384Amutated presenilin-1(PS1) were crossed to generate APP23/ PS45 double transgenic mice [31]. The genotype of APP23/ PS45 mice was confirmed by PCR using DNA extracted from tail tissues. Ethanol treatment was performed using a modified Bdrinking in the dark^ (DID) procedure [32, 33]. Individually housed APP23/PS45 mice at 6 weeks of age were maintained on a reverse 12-h light/dark cycle. At the first hour into the dark cycle, all the mice had no drink water and then given a 4-h access to drink a sweetened solution of either 20% (v/v) ethanol and 0.066% (w/v) saccharin or 0.066% (w/v) saccharin alone for four consecutive weeks. Ethanol intake, food consumption, and body weight were monitored daily throughout the study. The Morris water maze test was performed 2 days after the last ethanol treatment. Mice were sacrificed after behavioral test, and half brains were immediately homogenized for protein assays, and the other halves of the brains were fixed in 4% paraformaldehyde for immunohistochemical staining. Meanwhile, wild-type (WT) animals were treated with the same amount of ethanol and duration to determine the behavioral performance in Morris water maze test. Measurement of Blood Ethanol Concentration Blood ethanol concentration was determined from blood taken after 4 h of drinking at days 7, 14, 21, and 28. Quantitative determination of ethanol was achieved using an EnzyChrom TM Ethanol Assay Kit (BioAssay Systems). Briefly, 10 μL of blood samples collected from the tail vein was diluted with 190 μL of 0.9% normal saline and centrifuged to obtain serum. Ten microliters of diluted serum, or known ethanol standard ranging from 0 to 785 mg/L, was incubated with 90 μL of the Working Reagent for 30 min at room temperature, and 100 μL Stop Reagent was the added to stop the reaction. The optical density (OD) of the sample was measured at the wavelength of 565 nm. Sample ethanol concentration was calculated by Plot Standard Curve from OD values of the standard wells. Morris Water Maze Test The APP23/PS45 and wild-type mice were subjected to the Morris water maze test 2 days after the last ethanol treatment. The Morris water maze test was performed as previously described [34, 35]. The procedure consisted of three steps: 1 day of visible platform test, 4 days of hidden platform tests, and a probe trial 24 h after the last hidden platform test. In the visible and hidden platform tests, each mouse was allowed 60 s to search for the platform and tested for five continuous trials, with an inter-trial interval of 1 h. In the probe trial, the platform was removed, then, mice were given 60 s to locate the original place of the platform in the pool. Mouse behavior including escape latency, swimming distance, and the times of passes through the platform was automatically recorded by ANY-maze tracking software (ANY-maze, Stoelting).
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Cell Culture and Ethanol Treatment Cells were cultured in DMEM supplemented with 10% fetal bovine serum (complete DMEM) and maintained at 37 °C in an incubator containing 5% CO2. 2EB2 cell line was human embryonic kidney (HEK293) cells stably overexpressing human Swedish APP and BACE1 and was cultured in complete DMEM with 100 μg/mL Zeocin and 50 μg/mL G418. 20E2 cells stably expressing human Swedish APP in HEK293 cell line were cultured in complete DMEM with 50 μg/mL G418. SH105 cells, a cell line of human neuroblastoma cells (SH-SY5Y) stably expressing BACE1, were cultured in complete DMEM with 100 μg/mL Zeocin. The 2EB2, 20E2, SHSY5Y, and SH105 cells were all treated with 0, 0.4, 0.8, 1.6, 3.2, and 6.4 mg/mL ethanol for 48 h, respectively. Luciferase Assay Plasmids, containing the promoter regions of the human 2.94 kb APP or 3.18 kb BACE1 genes, were used for luciferase assay to determine the promoter activity. The human APP or BACE1 promoter plasmids were transfected into SH-SY5Y cells and treated with 0, 0.4, 0.8, 1.6, 3.2, and 6.4 mg/mL ethanol for 24 h. The transfection was performed using Lipofectamine 2000 (Invitrogen), and the luciferase assay was performed according to the protocol for Dual-Luciferase Reporter Assay System (Promega). Quantitative Real-Time PCR SH-SY5Y cells were harvested after treated with 0, 0.4, 0.8, 1.6, 3.2, and 6.4 mg/mL ethanol for 48 h, and total RNA was extracted from cells using TRI Reagent (Sigma). The concentration and purity of total RNA was detected using a spectrophotometer NanoDrop 2000 (Thermo). Synthesis of the first-strand complementary DNA (cDNA) was performed with 1 μg of total RNA according to the instruction manual of the ThermoScript™ RT-PCR System Kit (Invitrogen). Quantitative real-time PCR analysis of the cDNA of APP and BACE1 was performed using SYBR® Premix Ex Taq™ II (TaKaRa) with CFX Manager™ software detection system (Bio-Rad). The following primers were used to specifically amplify human APP and BACE1genes: APP forward 5′-atgccgttgacaagtatctcg and reverse 5′-tctgcctcttcccattctctc; BACE1 forward 5′taccaaccagtccttccgc and reverse 5′-ctcccataacagtgcccgt. GAPDH was used as an internal control, GAPDH forward 5′-agtccactggcgtcttcacc and reverse 5′-cagagggggcagagatgatg. All real-time PCR assays were performed in triplicate. The levels of the APP and BACE1 cDNAs were calculated based on standard curve, and data were normalized by the level of GAPDH. Cycloheximide Treatment For APP and BACE1 degradation experiments (half-life measurements), 2EB2 cells were treated with 3.2 mg/mL ethanol for 48 h, harvested at 0, 15, 30, or 60 min after 100 μg/mL cycloheximide (CHX)
treatment, and then RIPA lysis buffer for western blotting analysis. Western Blotting Brain tissues or cells were homogenized and lysed in RIPA lysis buffer supplemented with complete protease inhibitor (Roche Diagnostics). The lysates were diluted in 4× SDS-sample buffer and then boiled and resolved on 10% Tris-glycine or 16% Tris-tricine SDS-PAGE. The immunoblotting procedure was performed as previously described [36]. APP and CTF were detected by rabbit anti-APP C-terminal polyclonal antibody C20, the disintegrin and metalloproteinase domaincontaining protein 10 (ADAM10) as an important αsecretase was detected by anti-ADAM10 antibody (Cell Signaling Technology), β-secretase BACE1 was detected by anti-BACE1 monoclonal antibody D10E5 (Cell Signaling Technology) or by anti-BACE1 C-terminal polyclonal antibody 208, PS1 as a core component of γ-secretase complex was detected by anti-PS1 N-terminal antibody 231, and β-actin was detected by antibody AC15 (Sigma-Aldrich) as the internal control. Immunohistochemical Staining The immunohistochemical staining procedure was performed as previously published [13]. After sacrificing the mice, half of the brains were fixed in 4% paraformaldehyde and sectioned to 30-μm thickness. Every twelfth slice with the same reference position was mounted onto slides for staining. The monoclonal 4G8 antibody was used to detect the senile plaques in the sections using the ABC and DAB methods. Thioflavin-S staining was also used to indicate Aβ aggregates. Plaque depositions in these stains were visualized under a × 40 objective lens and the number of plaques per slice was quantified. Aβ40/42 ELISA Assay The Aβ40/42 assay was performed as the protocol of an Aβ40/Aβ42 Colorimetric ELISA Kit (Invitrogen). The cortical tissues of APP23/PS45 mice were prepared according to the ELISA protocol prior to carrying out the Aβ40/42 detection. The culture media of 20E2 cells were collected after ethanol treatment and centrifuged to precipitate cells in the media. For preventing degradation of Aβ peptides, the tissue lysis buffer and the collected media were added with protease inhibitor and AEBSF (Complete; Roche). The concentration of Aβ40/ 42 was measured according to the manufacturer ’s instructions. Statistical Analysis Software tool of SPSS Statistics 17.0 was employed to assess the statistical significance in different groups. All results were presented as mean ± SEM and analyzed by ANOVA or Student’s t test. P < 0.05 was considered as statistical significance.
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Results Ethanol Altered APP Processing and Increased Aβ Production in Cells To assess the effect of ethanol on APP processing and Aβ production in vitro, 2EB2, a stable cell line expressing both human Swedish mutant APP and BACE1, was treated with increasing concentrations of ethanol for 48 h. The protein levels of APP, APP-CTFs, ADAM10, BACE1, and PS1 in the cell lysates were detected by Western blotting. Ethanol treatment significantly increases the expressions of APP, APP-CTFs, and BACE1 (Fig. 1a). Quantification showed that APP in 2EB2 cells was markedly increased to 141.4 ± 12.4, 147.5 ± 11.1, and 149.3 ± 12.4% (P < 0.01) (Fig. 1a), and BACE1 was significantly increased by 40.3 ± 5.6 (P < 0.01), 51.8 ± 7.2 (P < 0.001), and 52.5 ± 7.8% (P < 0.001) (Fig. 1a). Moreover, the levels of APP-CTFs were also significantly increased to 140.7 ± 11.3, 142.4 ± 10.8, and 143.2 ± 6.8% (P < 0.01) (Fig. 1a). To further confirm these effects, 20E2, a stable cell line expressing human Swedish mutant APP, was also treated with ethanol (Fig. 1c). Quantification showed that APP protein levels were significantly increased to 159.8 ± 23.2 (P < 0.01), 172.6 ± 26.6 (P < 0.001), and 175.1 ± 20.9% (P < 0.001) in 1.6, 3.2, and 6.4 mg/mL ethanol-treated groups (Fig. 1c). The ELISA was performed to measure the levels of Aβ40 and Aβ42 in the media of 20E2 cells. With 1.6, 3.2, and 6.4 mg/mL ethanol treatment, Aβ40 levels were markedly increased to 139.2 ± 13.1, 144.6 ± 13.7, and 147.1 ± 5.5% (P < 0.01) (Fig. 1d), and the levels of Aβ42 were significantly increased to 132.5 ± 8.9, 136.4 ± 11.9, and 136.6 ± 12.4% (P < 0.01) (Fig. 1e). These data indicated that ethanol treatment altered APP processing and increased Aβ production. Ethanol Increased the Levels of APP and BACE1 in SH105 and SH-SY5Y Cells To further demonstrate the effect of ethanol in neuronal cell lines, we treated SH105 cells, a human neuroblastoma cell line stably expressing human BACE1, with different concentrations of ethanol for 48 h. The Western blotting showed that the low ethanol dosage at 0.8 mg/mL was sufficient to significantly increase APP and BACE1 protein levels. APP in SH105 cells were significantly increased to 138.4 ± 6.6 (P < 0.01), 142.6 ± 9.0 (P < 0.01), 137.2 ± 4.0 (P < 0.01), and 126.0 ± 3.8% (P < 0.05) (Fig. 2a). BACE1 in SH105 cells were also markedly altered to 129.2 ± 3.1 (P < 0.05), 141.8 ± 4.7 (P < 0.01), 141.4 ± 6.9 (P < 0.01), and 131.3 ± 6.6% (P < 0.05) (Fig. 2a). The protein levels of ADAM10 and PS1 were not altered in all ethanol-treated groups (P > 0.05) (Fig. 2b). Additionally, in SH-SY5Y cells without exogenous APP or BACE1 expression, ethanol at the concentrations of 0.8, 1.6, 3.2, and 6.4 mg/mL also increased endogenous APP by 56.0 ± 16.7(P < 0.05), 93.5 ± 32.1(P < 0.01),
66.9 ± 2.4(P < 0.01), and 49.9 ± 11.3% (P < 0.05) and increased endogenous BACE1 by 34.4 ± 6.0(P < 0.05), 48.5 ± 9.4 (P < 0.05), 44.9 ± 2.6(P < 0.05), and 61.2 ± 6.0% (P < 0.01), respectively (Fig. 2c). These data further demonstrated that ethanol treatment elevated endogenous APP and BACE1 expression levels in neuronal cell line in vitro. Ethanol Affected APP and BACE1 Promoter Activities and Gene Transcriptions To investigate whether the increase in APP and BACE1 expressions induced by ethanol were a result of upregulation promoter activities, SH-SY5Y cells were transfected with the human APP promoter or human BACE1 promoter containing plasmid and treated with ethanol. The APP promoter plasmid pAPPLuc 2.94 kb APP and the 3.18 kb BACE1 promoter plasmid pB1-A were previously described [37]. Luciferase assay was performed to examine the activities of the APP and BACE1 promoters after 24 h ethanol treatment at 0, 0.4, 0.8, 1.6, 3.2, and 6.4 mg/mL ethanol. We found that 0.8, 1.6, 3.2, and 6.4 mg/mL ethanol treatments markedly increased the promoter activity of pAPP-Luc in SH-SY5Y cells to 137.2 ± 7.5 (P < 0.01), 147.9 ± 5.4 (P < 0.001), 168.5 ± 4.5 (P < 0.001), and 184.7 ± 8.7%(P < 0.001) (Fig. 3a) and significantly increased the luciferase activity of pB1-A to 142.0 ± 5.4 (P < 0.05), 144.3 ± 9.5 (P < 0.05), 172.2 ± 12.6 (P < 0.001), and 179.7 ± 12.6%(P < 0.001) (Fig. 3b) as compared with the control. These results suggested that ethanol treatment upregulated APP and BACE1 promoter activities. To confirm that the upregulations of APP and BACE1 proteins upon ethanol treatment were due to enhanced gene transcription, we examined the levels of endogenous APP and BACE1 mRNAs in SH-SY5Y cells after 0, 0.4, 0.8, 1.6, 3.2, and 6.4 mg/mL ethanol treatment. Consistent with the promoter assay data, 0.8, 1.6, 3.2, and 6.4 mg/mL ethanol treatments significantly increased the APP mRNA level to 146.9 ± 5.1 (P < 0.05), 155.4 ± 6.1 (P < 0.01), 173.9 ± 13.5 (P < 0.001), and 168.9 ± 14.2% (P < 0.001) (Fig. 3c) and increased BACE1 mRNA level to145 ± 8.6 (P < 0.01), 161.7 ± 9.9 (P < 0.001), 182.8 ± 10.9 (P < 0.001), and 186.1 ± 7.8% (P < 0.001) (Fig. 3d). Taken together, these results demonstrated that ethanol treatment upregulated APP and BACE1 protein levels at least partially via its effect on their promoter activities and transcriptions. We further examined the potential pathways through which ethanol regulates APP and BACE1 gene expressions. In neuronal cells, there are several putative receptors of ethanol including NMDA receptor and GABA receptor [38, 39]. Realtime RT-PCR indicated that pharmaceutical inhibition of GABA but not NMDA receptor significantly suppressed ethanol-induced increase of APP mRNA. However, an agonist of GABA receptor failed to replicate the effect of ethanol on APP expression (data not shown). Therefore, GABA
Mol Neurobiol Fig. 1 Ethanol regulates APP processing and increases Aβ production in 2EB2 and 20E2 cells. 2EB2 cells stably expressing exogenous human APP and BACE1 were treated with 0, 0.4, 0.8, 1.6, 3.2, and 6.4 mg/mL ethanol for 48 h, and the cell lysates were analyzed by Western blotting. a Full-length APP and CTF were detected by antibody C20, BACE1 was detected by anti-BACE1 C-terminal polyclonal antibody 208, and βactin was detected by antibody AC-15 as the internal control. Quantification showed that exogenous APP, BACE1, and CTF in 2EB2 cells were significantly increased with 1.6, 3.2, and 6.4 mg/ mL ethanol treatment. N = 4 ~ 5, *P < 0.01, **P < 0.001, by ANOVA. b ADAM10 was detected by anti-ADAM10 antibody, and PS1 was detected by anti-PS1 N-terminal antibody 231 in Western blotting analysis. Quantification showed that endogenous ADAM10 and PS1 in 2EB2 cells were no difference between ethanol-treated and control groups. N = 4, by ANOVA. c The APP expression was further confirmed in 20E2 cells. Quantification showed that exogenous APP level was significantly increased in 1.6, 3.2, and 6.4 mg/ mL ethanol-treated cells. N = 4, *P < 0.01. d Aβ40 and e Aβ42 levels from the conditioned media of 20E2 cells. The Aβ production was markedly increased in 1.6, 3.2, and 6.4 mg/mL ethanoltreated groups. Values are expressed as mean ± SEM. N = 4, *P < 0.01, by ANOVA
receptor is required for the enhanced APP transcription by ethanol, but the activation of GABA by ethanol, if any, is not insufficient to upregulate APP mRNA. Ethanol Did Not Affect APP and BACE1 Degradations The level of a protein in cells depends on the counterbalance between synthesis and degradation. To further investigate whether the increase in APP and BACE1 expressions induced by ethanol was due to impaired degradations, we used cycloheximide (CHX) to stop protein synthesis and measured the amount of residual APP and BACE1 proteins at various time
points after 3.2 mg/mL ethanol treatment in 2EB2 cells (Fig. 4a, b). Quantification showed that ethanol made little difference in APP and BACE1 catabolisms (P > 0.05) (Fig. 4c, d). Hence, ethanol does not contribute to APP and BACE1 accumulations through inhibiting degradations. Ethanol Did Not Affect the Food Consumption and Body Weight in AD Model Mice To study the effect of ethanol on Alzheimer-associated cognitive impairment and neuropathology, APP23/PS45 and wildtype mice at 6 weeks of age were treated with 20% (v/v)
Mol Neurobiol Fig. 2 Ethanol treatment increased the levels of APP and BACE1 in SH105 and SH-SY5Y cells. A SH-SY5Y cell line stably expressing human BACE1 was treated with 0, 0.4, 0.8, 1.6, 3.2, and 6.4 mg/mL ethanol for 48 h, and the cell lysates were analyzed by Western blotting. a Full-length APP and BACE1 were respectively detected by antibody C20 and 9E10, and β-actin was detected by antibody AC-15 as the internal control. Quantification showed that endogenous APP and exogenous BACE1 were significantly increased in 0.8 1.6, 3.2, and 6.4 mg/mL ethanol-treated cells. N = 4, *P < 0.05, **P < 0.01, by ANOVA. b ADAM10 was detected by antiADAM10 antibody, and PS1 was detected by anti-PS1 N-terminal antibody 231. Quantification of endogenous ADAM10 and PS1 levels showed that ethanol did not impact on these two enzymes in SH105 cells. Values are expressed as mean ± SEM. N = 4 ~ 5, by ANOVA. c Endogenous APP and BACE1 in SH-SY5Y cells were Western blotted after ethanol treatment at 0.8, 1.6, 3.2, and 6.4 mg/mL. N = 3, *P < 0.05, **P < 0.01, by ANOVA
ethanol and 0.066% (w/v) saccharin or 0.066% (w/v) saccharin alone for 4 h/day in the dark. Ethanol intake, blood ethanol concentration, food consumption, and body weight were monitored throughout the 4-week treatment period. APP23/PS45 and wild-type mice consumed 4.62–7.57 g/kg and 4.37– 7.15 g/kg per day (Fig. 5a), resulting in average blood ethanol concentrations ranging from 136 to 192 mg/dL and 155– 184 mg/dL, respectively (Fig. 5b). Food consumption and body weight displayed no significant differences between the saccharin and the ethanol-treated groups at any time point (Fig. 5c, d). Ethanol Altered APP Processing and Increased Aβ Production In Vivo To examine whether ethanol treatment altered APP and APPcleaving secretases in vivo, we examined the expression levels
of APP, APP-CTFs, BACE1, ADAM10, and PS1 in the brain of APP23/PS45 mice by Western blotting analysis (Fig. 6a, c). Ethanol treatment significantly increased the APP levels in the brain from control 100% to 152.0 ± 9.5% (P < 0.001) and BACE1 from control 100% to 140.9 ± 8.7% (P < 0.001) (Fig. 6b). Meanwhile, the levels of CTFβ fragments C99 and C89 were increased to 146.4 ± 9.3% in ethanol-treated mice comparing with the controls (P < 0.001) (Fig. 6b). We further examined the expression levels of ADAM10 and PS1 and observed no difference by ethanol (P > 0.05) (Fig. 6d). Next, an ELISA assay was performed to measure Aβ40 and Aβ42 levels in the transgenic brain tissues with or without ethanol treatment. The levels of Aβ40 and Aβ42 were increased by 60.0 ± 20.5 and 90.2 ± 20.7% in ethanol-treated mice relative to controls, respectively (P < 0.01) (Fig. 6e). These data confirmed that, consistent with the in vitro results, ethanol treatment increased APP and BACE1 expressions and Aβ production in vivo.
Mol Neurobiol
Fig. 3 Ethanol treatment affected the APP and BACE1 promoter activities and gene transcriptions. The human APP and BACE1 promoters were respectively transfected into SH-SY5Y cells and treated with 0, 0.4, 0.8, 1.6, 3.2, and 6.4 mg/mL ethanol for 24 h. Luciferase assay was performed. Ethanol treatment increased the luciferase activity of APP (a) and BACE1 (b) promoters. All the promoter data shown are results of three independent experiments. Values are expressed as
mean ± SEM. N = 3, *P < 0.05, **P < 0.01, ***P < 0.001, by ANOVA. SH-SY5Y cells were treated with different concentration of ethanol for 48 h, APP and BACE1 mRNA levels were measured by quantitative RT-PCR with specific primers. Ethanol significantly increased the mRNA levels of APP (c) and BACE1 (d). Values are expressed as mean ± SEM. N = 3, *P < 0.05, **P < 0.01, ***P < 0.001, by ANOVA
Ethanol Treatment Increased Neuritic Plaque Formation in AD Model Mice
performed 2 days after the last ethanol treatment. In the visible platform test, the ethanol-treated and control mice exhibited similar escape latency (49.84 ± 2.80 and 48.30 ± 2.06 s) (P > 0.05) (Fig. 8a) and swimming distance to the platform (7.65 ± 0.67 and 8.20 ± 0.60 m) (P > 0.05) (Fig. 8b). The results indicated that ethanol treatment had no effect on mouse mobility or vision in the behavioral test. However, on the third and fourth day of the hidden platform tests, the escape latency was longer for the ethanol-treated groups (38.18 ± 2.47 and 35.51 ± 3.03 s on the third and fourth day, respectively) than that for the control group (30.38 ± 2.42 and 26.11 ± 3.17 s on the third and fourth day, respectively) (P < 0.05) (Fig. 8c), and the ethanol-treated mice swam longer distances to reach the platform (5.86 ± 0.47 and 5.68 ± 0.39 m on the third and fourth day, respectively) as compared to the control mice (4.38 ± 0.50 and 4.29 ± 0.49 m on the third and fourth day, respectively) (P < 0.05) (Fig. 8d). In the probe trial on the last day of the Morris water maze test after the hidden platform tests, the ethanol-treated mice spent less time in the quadrant where the platform was originally placed (ethanol-treated vs. control, 2.4 ± 0.3 and 4.4 ± 0.5 times) (P < 0.01) (Fig. 8e). These data clearly suggest that ethanol treatment exuberated learning and memory deficits in the AD model mice. By contrast, ethanol treatment did not affect the water maser performance of wild type mice (Fig. 8c–e). Therefore, ethanol may impose faster damage to neuronal functions through amyloidosis.
To assess the effect of ethanol treatment on Aβ deposition and neuritic plaque formation, APP23/PS45 double transgenic mice at 6 weeks of age were daily treated with ethanol for 4 weeks, whereas age-matched control mice received control vehicle solution. Ethanol-treated and control mice were sacrificed after treatment and behavioral tests. The 4G8 antibody and thioflavin-S staining were used to detect Aβ-containing neuritic plaques in the transgenic mouse brains. 4G8 immunostaining showed that ethanol treatment significantly enhanced neuritic plaque formation in the transgenic mice (Fig. 7a, b), and plaque numbers were increased by approximately 48.1% (38.4 ± 2.5 vs. 25.9 ± 2.9 per slice) (P < 0.01) (Fig. 7c). Thioflavin-S staining further confirmed that ethanol treatment dramatically increased the number of neuritic plaques in the brains of APP23/PS45 double transgenic mice (Fig. 7d, e). These results clearly demonstrated that ethanol exposure facilitated amyloid plaque accumulation in the transgenic Alzheimer’s model mice in vivo. Ethanol Treatment Aggravated Learning and Memory Impairment in AD Model Mice To investigate the impact of ethanol treatment on spatial learning and memory in AD model mice, the Morris water maze was
Mol Neurobiol
Fig. 4 Ethanol did not affect APP and BACE1 degradations. To determine APP and BACE1 degradations in the presence of ethanol, 2EB2 cells were treated with 3.2 mg/mL ethanol for 48 h and harvested at 0, 15, 30, or 60 min after 100 μg/mL CHX treatment. The cell lysates were analyzed by Western blotting. a, b Full-length APP were detected by antibody C20, BACE1 was detected by anti-BACE1 C-terminal polyclonal antibody 208, and β-actin was detected by antibody AC-15 as
the internal control in Western blotting analysis. c, d APP and BACE1 protein levels were plotted as a percentage of the amount at 0 min. Quantification showed that the percentage of remaining APP and BACE1 in 2EB2 cells were no different between ethanol-treated and control groups at any time point. Values are expressed as mean ± SEM. N = 4, by ANOVA
Discussion
that ethanol treatment regulated BACE1 gene expression and APP processing in vitro. It is interesting that in our stable cell lines, the expression of exogenous APP and BACE1 are driven by the CMV promoter, and ethanol still elevated the protein levels of APP and BACE1.The response of CMV promoter to ethanol is presumably due to the putative ATF4 and NFκB targeting sequences in CMV (analysis by Genomatix Software v3.9) [41]. ATF4 and NFκB are both transcription factors that can be activated by ethanol [42–44] and also have targets in the promoters of APP and BACE1 genes [45–47]. There could be more ethanol-activated transcription factors upregulating endogenous APP and BACE1 gene expressions. Since the discovery that ethanol consumption may be harmful to dementia and AD development, different animal models have been utilized to investigate the mechanisms. However, the majority of these animal models involved procedures that may produce additional stress. In these procedures, ethanol was administered by gavage, injection, or ethanol vapor exposure [48–50]. Animal models were forced to drink ethanol by long-term water deprivation or by providing a liquid ethanol diet for the sole source of nutrients [51]. The passive and forced methods of ethanol administration may produce potential pain to the animals and introduce confounding variables for results. In the present study, we subjected
Previous report has shown that alcoholism was associated with an increased risk of AD [20, 40]. However, the role of ethanol in regulating of AD neuropathology remains unclear. Therefore, we first examined the effect of high ethanol exposure on AD-type neuropathology in cell culture and animal models of AD. We found that high ethanol treatment altered APP processing, significantly increased Aβ production and promoted neuritic plaque formation, and aggravated learning and memory impairment in AD model. A previous study has shown an association between ethanol consumption and pathological processing of APP in AD [27], but the underlying mechanism remains elusive. We first examined the effect of ethanol exposure on APP processing in stable cell lines 2EB2 and 20E2 and found that the levels of APP, Aβ production, and BACE1 were significantly increased by ethanol treatment, while the expression of αsecretase ADAM10 and γ-secretase PS1 were not altered. Similar effects were also observed in neuronal-like cell line SH105 cells. Moreover, we demonstrated that ethanol treatment affected the protein synthesis but not degradation to upregulate APP and BACE1 expressions via its effect on promoter activity and transcription. Our data further confirmed
Mol Neurobiol Fig. 5 Ethanol did not affect the food consumption and body weight in AD model mice. APP23/PS45 mice at 6 weeks of age were administered daily for 4 weeks with a solution of either 20% (v/v) ethanol and 0.066% (w/ v) saccharin, or 0.066% (w/v) saccharin alone for 4 h/day in the dark. Ethanol intake (a) and blood ethanol concentration (b) were monitored. Blood ethanol concentration was determined from blood taken after 4 h of drinking at the 7, 14, 21, and 28 days. No significant difference was found between control and ethanoltreated mice at any time point for either food consumption (c) or body weight (d). Values represent means ± SEM. N = 12 for each group, P > 0.05 by Student’s t test
APP23/PS45 double transgenic mice to a limited access procedure calledBdrinking in the dark^ (DID). The DID procedure was relatively simple, eliminated the need for lengthy access periods, and alleviated the potential stress for animal models. Moreover, APP23/PS45 mice have ingested 4.62– 7.57 g/kg ethanol per day and produced a relative high level of blood ethanol concentrations ranging from 136 to 192 mg/ dL. This level of ethanol consumption did not impact on the appetite of the models, and no significant difference was found between control and ethanol-treated mice at any time point for either food consumption or body weight. Thus, the DID procedure may closely mimic the pattern and type of ethanol consumption characteristic of at least some people. The DID on APP23/PS45 mice may provide a useful model for studying the effects of ethanol exposure on AD development. Epidemiological studies estimated that 7% of adults and 19% of adolescents are alcohol abuse or problem drinkers in the USA [52]. Heavy alcohol consumption has emerged as an important risk factor for cerebrovascular diseases, and an increasing body of evidence suggested that ethanol can cause lasting cognitive impairment [53]. In human studies on the impacts of alcohol, many brain areas including hypothalamus, cerebellum, hippocampus, amygdala, and locus coeruleus presented neuronal loss, and the damage is dose-related especially with heavy alcohol consumption [54, 55]. Mental Health Services Administration defines heavy alcohol consumption as binge drinking for five or more days in the past month. A
Bbinge drinking^ was defined as a pattern of drinking that brings blood alcohol concentration levels to 80 mg/dL by the National Institute on Alcohol Abuse and Alcoholism. In mice, however, a criterion of 100 mg/dL or greater has been suggested because of the high rate of ethanol metabolism in mice [56]. In present study, APP23/PS45 mice drank daily with the average blood ethanol concentrations ranging from 136 to 192 mg/dL that is beyond the100 mg/dL criterion, and therefore, the drinking pattern may mimic heavy alcohol consumption in human. Excessive ethanol consumption is detrimental to the brain. It could cause the ability of memory and cognitive impairment. However, the mechanism of ethanol-induced impairment of cognitive function remains elusive. It was reported that ethanol led to neurodegeneration as indicated by the loss of cortical and subcortical brain structures [57]. Several studies indicated that ethanol has interactions with various signal transduction cascades, including cholinergic system [26], allosteric modulators [39, 58], and second messengers [59]. These signal pathways are closely associated with learning and memory [60, 61]. To evaluate the effect of ethanol treatment on learning and memory deficits in AD mice, the Morris water maze was performed. Our work revealed that ethanol treatment markedly impaired spatial learning skills and memory deficits in APP23/PS45, which is consistent with others [62, 63]. Previous work by Kim et al. [27] demonstrated that chronic alcohol consumption increased the expression of APP and
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Fig. 6 Ethanol treatment altered APP processing and increased Aβ production in vivo. Hemi-brains from control and ethanol-treated APP23/PS45 mice were homogenized in RIPA lysis buffer and separated with 10% Tris-glycine or 16% Tris-tricine SDS-PAGE. a APP and CTF were detected by antibody C20, BACE1 was detected by anti-BACE1 antibody D10E5, and β-actin was detected by antibody AC-15 as the internal control. b Quantification showed that APP, BACE1, and CTF were significantly increased in ethanol-treated mice. N = 12 for each group,*P < 0.001 by Student’s t test. c PS1 was detected by anti-PS1
N-terminal antibody 231, and ADAM10 was detected by anti-ADAM10 antibody. d Quantification showed that there was no difference in the level of PS1 or ADAM10 between ethanol-treated and control mice. N = 14 for each group, P > 0.05 by Student’s t test. e ELISA was performed to measure Aβ40 and Aβ42 levels from the brain tissues of APP23/PS45 mice treated with or without ethanol. Values are expressed as mean ± SEM. N = 8 for control and N = 11 for ethanol-treated mice, *P < 0.01 by Student’s t test
APP processing enzymes, including β-secretase and γsecretase in rat. However, another report showed that moderate ethanol treatment attenuates Aβ neuropathology in a Tg2576 mouse model [21]. The difference in animal models and different procedures of ethanol administration may result in the different effects of ethanol on APP processing and Aβ
production in the two studies. To further examine heavy ethanol consumption effect on APP processing and Alzheimerassociated phenotypes, we treated APP23/PS45 double transgenic mice with ethanol. Our data showed that heavy ethanol consumption aggravated learning and memory impairment, affected APP processing and neuritic plaque formation,
Fig. 7 Ethanol treatment increased neuritic plaque formation in AD model mice. Neuritic plaques of control (a) and ethanol-treated mice (b) were detected using Aβ specific monoclonal antibody 4G8. The plaques were visualized by microscopy with × 40 magnification and the arrows pointed to plaques. c Quantification of neuritic plaques in APP23/ PS45 mice with treatment starting at the age of 6 weeks and sacrificed
immediately after behavioral analysis, N = 12 for control and N = 21 for ethanol-treated mice, *P < 0.01 by Student’s t test. Values are expressed as mean ± SEM. Neuritic plaques of control (d) and ethanol-treated mice (e) were further confirmed using thioflavin-S fluorescent staining. There were significantly more neuritic plaques in ethanol-treated mice
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and various procedures has found that ethanol treatment affected amyloidogenic pathway of AD, suggesting that heavy ethanol consumption increase risk of developing AD. In conclusion, we have found that high ethanol exposure promotes APP processing and aggravates Alzheimerassociated phenotypes. These data suggest that heavy ethanol consumption has an adverse impact on AD and increases risk of AD development. Future study will be carried out to clarify the underlying mechanism, especially to determine through which pathway ethanol affects APP and APP processing enzymes, which will facilitate the identification of therapeutic targets for the treatment of AD induced by ethanol exposure. Acknowledgements We sincerely thank Philip T.T. Ly, Zhifang Dong, and Mingjing Liu for their helpful comments. This work was supported by grants from the National Natural Science Foundation of China (NSFC) Grant 30972461, 81161120498 (T.L.) and the Canadian Institutes of Health Research (CIHR) Grant TAD-117948 (W.S). W.S. is the holder of the Tier 1 Canada Research Chair in Alzheimer’s Disease. Authors’ Contributions DH and WS conceived and designed the experiments; DH, MY, SY, DL, WZ, LZ, and FC performed the experiments; DH, MY, SY, DL, WZ, LZ, ZW, WZ, TL, and WS analyzed and contributed reagents/materials/analysis tools; and DH, ZW, and WS wrote the paper. All authors reviewed the manuscript. Compliance with Ethical Standards All animal studies were performed in accordance with the Guide for the Care and Use of Laboratory Animals of the Ethics Committee of Chongqing Medical University. The experimental protocols were approved by the Animal Study Committee of the Children’s Hospital of Chongqing Medical University. Fig. 8 Ethanol treatment exuberated learning and memory impairment in AD model mice. APP23/PS45 (12 AD-control and 19 AD-ethanol) and wild-type (12 WT-control and 12 WT-ethanol) mice at 6 weeks of age were treated daily for 4 weeks with ethanol and subjected to the Morris water maze test. ANY-maze tracking software was used for recording mice movement. During the first day of visible platform tests, the ethanol-treated and control mice exhibited the similar escape latency (a) and swimming distances (b) to escape onto the visible platform. During the 4 days of hidden platform tests, c ethanol-treated AD mice showed a longer latency to escape onto the hidden platform on the third and fourth day. aP < 0.05 (comparison with WT-control group), bP < 0.05 (comparison with WT-ethanol group), cP < 0.001 (comparison with WT-control group), dP < 0.05 (compared with AD-control group), eP < 0.001 (comparison with WT-ethanol group). d Meanwhile, its swimming distance was also longer than other groups. aP < 0.05 (comparison with WTethanol group), bP < 0.01 (comparison with WT-control group), c P < 0.05 (comparison with AD-control group), dP < 0.01(comparison with WT-ethanol group), eP < 0.001 (comparison with WT-control group), by ANOVA. e In the probe trial on the last day of testing, the ethanol-treated AD mice traveled into the quadrant of hidden platform previously placed, significantly less times than controls. aP < 0.001 (comparison with WT-ethanol group), bP < 0.01 (comparison with WT-control group), cP < 0.01 (comparison with AD-control group), by ANOVA. Values represent means ± SEM
Conflict of Interest The authors declare that they have no conflict of interest.
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