Ethanol Alters APP Processing And Aggravates Alzheimer-associated Phenotypes

Objective 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 Disease,and we investigated whether ethanol exposure contributed to AD pathogenesis and the underlying mechanism.Methods The 2EB2,20E2 and SH105 cells were all treated with 0,0.4,0.8,1.6,3.2 and 6.4 mg/mL ethanol for 48 hours,respectively.Individually housed APP23/PS45 mice at 6 weeks of age were maintained on a reverse 12-hour light/dark cycle.At the first hour into the dark cycle,all the mice had no drink water,and then given a 4-hour 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 4 consecutive weeks.Ethanol intake,food consumption,and body weight were monitored daily throughout the study.The APP23/PS45 mice were subjected to the Morris water maze test two days after the last ethanol treatment.Luciferase assay were used to determine the promoter activity of APP and BACE1.Quantitative Real-time PCR was performed for analysis of the cDNA of APP and BACE1.The protein levels of APP,APP-CTFs,ADAM10,BACE1 and PS1 in the cell lysates were detected by Western blotting.Immunohistochemical staining and Thioflavin-S staining were used to detect the senile plaques in the brain.The Aβ40/42 assay was performed as the protocol of an Aβ40/Aβ42 Colorimetric ELISA kit.Results Quantification showed that the cell viability of HEK293 cells was significantly decreased with 12,16 and 20 mg/mL ethanol treatment.And Quantification showed that the cell viability of SH-SY5 Y cells was significantly decreased with 8,12,16 and 20 mg/mL ethanol treatment.Quantification showed that APP,BACE1 and CTF in 2EB2 cells were significantly increased with 1.6,3.2 and 6.4 mg/mL ethanol treatment.Quantification showed that ADAM10 and PS1 in 2EB2 cells were no difference between ethanol-treated and control groups.The APP expression was further confirmed in 20E2 cells.Quantification showed that APP level was significantly increased in 1.6,3.2 and 6.4 mg/mL ethanol-treated cells.Aβ40 and Aβ42 levels from the conditioned media of 20E2 cells were markedly increased in 1.6,3.2 and 6.4 mg/mL ethanol-treated groups.The protein levels of APP,ADAM10,BACE1 and PS1 in the cell lysates were detected by Western blotting in SH105 cells.Quantification showed that APP and BACE1 CTF in SH105 cells were significantly increased with 0.8,1.6,3.2 and 6.4 mg/mL ethanol treatment.ADAM10 and PS1 in SH105 cells were no difference between ethanol-treated and control groups.The human APP and BACE1 promoter were respectively transfected into SH-SY5 Y cells and treated with 0,0.4,1.6,and 3.2 mg/mL ethanol for 24 hours.Luciferase assay was performed.Ethanol treatment increased the luciferase activity of APP and BACE1 promoter.SH-SY5 Y cells were treated with different concentration of ethanol for 48 hours,APP and BACE1 mRNA levels were measured by quantitative RT-PCR with specific primers.Ethanol significantly increased the mRNA levels of APP and BACE1.Ethanol treatment affected the APP and BACE1 promoter activities and gene transcriptions.2EB2 cells were treated with 3.2 mg/mL ethanol for 48 hours and harvested at 0,15,30 or 60 minutes after 100 ug/ml CHX treatment.The cell lysates were analyzed by Western blotting.Quantification showed that the percentage of remaining APP and BACE1 in 2EB2 cells were no difference between ethanol-treated and control groups at any time point.Ethanol did not affect APP and BACE1 degradation.The expression levels of APP,APP-CTFs,BACE1,ADAM10,and PS1 in the brain of APP23/PS45 mice were analyzed by Western blotting.APP,BACE1 and CTF were significantly increased in ethanol-treated mice.Quantification showed that there was no difference in the level of PS1 or ADAM10 between ethanol-treated and control mice.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 in ethanol-treated mice relative to controls,respectively.These data confirmed that,consistent with the in vitro results,ethanol treatment increased APP and BACE1 expression and Aβ production in vivo.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.Thioflavin-S staining further confirmed that ethanol treatment dramatically increased the number of neuritic plaques in the brains of APP23/PS45 double transgenic mice.Ethanol treatment increased neuritic plaque formation in AD model mice.The Morris water maze was performed two days after the last ethanol treatment.In the visible platform test,the ethanol-treated and control mice exhibited similar escape latency and swimming distance to the platform.On the third and fourth day of the hidden platform tests,the escape latency was longer for the ethanol-treated groups than that for the control group,and the ethanol-treated mice swam longer distances to reach the platform.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 treatment aggravated learning and memory impairment in AD model mice.Conclusion Ethanol affected the APP and BACE1 promoter activities and gene transcriptions,and increased the synthesis of APP and BACE1 protein,but ethanol does not contribute to APP and BACE1 accumulations through inhibiting degradations.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 beta-site APP cleaving enzyme 1(BACE1)and significantly promoted amyloid β protein(Aβ)production both in vitro and in vivo.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.Ethanol exposure promotes APP processing and aggravates Alzheimer-associated phenotypes.These data suggest that heavy ethanol consumption has an adverse impact on AD and increases risk of AD development.