The Neuroprotective Effects And Mechanism Of Adiponectin Alleviates The Anxiety- And Depression-like Behaviors In Triple Transgenic Alzheimer’s Disease Model Mice

Objective:To observe whether adiponectin(APN)could effectively alleviate the anxiety-and depression-like behaviors occurred in 9-month-old APP/PS1/tau triple transgenic Alzheimer’s disease(3xTg-AD)model mice,and investigate the possible mechanisms.Methods:The 9-month-old 3xTg-AD mice and wild type(WT)mice were randomly divided into four groups(n=8-10 for each group): WT+Saline,WT+APN,3xTg-AD+Saline and 3xTg-AD+APN.Each mouse was chronically intracerebroventricular injection of APN(0.1μg/4μl)or saline under free moving condition.Drug administration was performed once daily beginning 10 days before the behavioral tests and continued until the behavioral tests completed.Three different experiments were included in the behavioral tests:(1)Sucrose preference test:In the first day,each mouse was free access to two bottles of sucrose water.In the second day,one bottle of water and one bottle of sucrose water were given to each mouse.In the third day,the diet and water were deprived for 24 h before the test.Then one bottle of water and one bottle of sucrose water with same weight were given to each mouse.The consumption of sucrose water and water in 2 h were recorded,and the ratio of sucrose preference was counted(the consumption of sucrosewater/(the consumption of sucrose water + the consumption of water)*100%).(2)Elevated plus-maze task: The second day after sucrose preference test,each mouse was placed in the center of the maze facing open arm,and allowed to explore freely for 5 min.The total arm entries and time spent in open arms were recorded,with the time percentage of each mouse spent in open arms calculated(time spent in open arms/5min×100%).(3)Tail suspension test: The second day after elevated plus-maze task,mice were suspended by the tail from a white painted box.The immobility time of each mouse in 6 min was recorded,and the percentage of immobility time was calculated((immobility time/6 min)*100%).After the behavioral experiments,some mice were used to do immunohistochemistry staining.Mice were anesthetized with 5% chloral hydrate by intraperitoneal injection and then performed cardiac perfusion with PBS and 4%paraformaldehyde.The brain was taken out,put into 4% paraformaldehyde to fix and 30% sucrose solution to dehydrate.The thickness of brain slice was 25 μm by using frozen sections.The brain slices were blocked with 5% goat serum,followed by incubating with the primary antibody,second antibody and DAB.The staining of astrocyte and microglia in the hippocampus was observed by light microscope.The brain of some mice completed behavioral tests were taken out to do Western blot experiments.The mice were anesthetized with 5% chloral hydrate,and the hippocampal tissues were isolated to extract the total protein.Then,SDS-PAGE electrophoresis was carried,and the protein was transferred to the PVDF membrane.After 5% BSA treatment 2 h,the primary antibody,second antibody and ECL were added in proper order.The relative gray values of target protein(p-AMPK、t-AMPK、SIRT1、p-GSK-3β(ser9)、t-GSK-3β)were analyzed by using Alpha SA software.Results:(1)APN effectively alleviated the anxiety-like behavior observed in 3xTg-AD mice.In the elevated plus maze task,compared to WT+Saline mice,the 3xTg-AD+Salinemice spent less time in open arms(P < 0.01),indicating the anxiety-like behavior occurred in 3xTg-AD mice.More importantly,the time percentage of 3xTg-AD+APN group spent in open arms was significantly longer than that in 3xTg-AD+Saline group(P<0.01),indicating that APN could effectively alleviate the anxiety-like behavior observed in 3xTg-AD mice.In addition,there was no significant difference of total arm entries among four groups,indicating gene mutation and adiponectin treatment did not affect the locomotor performance of mice.(2)APN effectively ameliorated the depression-like behaviors in 3xTg-AD mice.In sucrose preference test,the sucrose preference percentage in 3xTg-AD+Saline group was significantly lower than that in WT+Saline group(P<0.01)and 3xTg-AD+APN group(P<0.05).At the same time,there was no significant difference of total water consumed among four groups.In tail suspension test,the immobility time percentage in 3xTg-AD+Saline group was significantly higher than that in WT+Saline group(P< 0.001)and 3xTg-AD+APN group(P < 0.05).These results indicated that9-month-old 3xTg-AD showed depression-like behaviors,and APN could ameliorate depression-like behavior observed in 3xTg-AD mice.(3)APN inhibited the over activation of microglia and astrocytes in the hippocampus of 3xTg-AD mice.In immunohistochemistry experiments,there were activation of microglia(Iba-1)and astrocytes(GFAP)among four group.Compared with WT+Saline group,the Iba-1 and GFAP immunopositive areas in the hippocampus of 3xTg-AD+Saline group were significantly increased(P < 0.001),indicating the over activation of microglia and astrocytes in the hippocampus of3xTg-AD mice.However,APN treatment effectively decreased the percentage of Iba-1(P<0.001)and GFAP(P<0.001)immunopositive area in the hippocampus of3xTg-AD mice,indicating that APN could inhibit the over activation of microglia and astrocytes in 3xTg-AD mice.(4)APN up-regulated the expression of p-AMPK and p-GSK-3β(ser9)in the hippocampus of 3xTg-AD mice,without affecting the expression of SIRT1.There was no significant difference of the expression of t-AMPK and t-GSK-3β in the hippocampus of mice among four groups.Compared with WT+Saline group,theexpression of p-AMPK and p-GSK-3β(ser9)in the hippocampus of 3xTg-AD+Saline group were significantly decreased(P<0.05).However,APN treatment effectively increased the expression of p-AMPK and p-GSK-3β in the hippocampus of 3xTg-AD mice(P<0.05),indicating that APN could up-regulate the expression of p-AMPK and p-GSK-3β(ser9).In addition,the expression of SIRT1 in the hippocampus of3xTg-AD+Saline mice was significantly lower than that in WT+Saline mice(P <0.05),but APN treatment did not affect the SIRT1 expression in the hippocampus of3xTg-AD mice.Conclusion:(1)9-month-old 3xTg-AD mice showed obvious anxiety-and depression-like behaviors.Adiponectin treatment could effectively alleviate the anxiety-and depression-like behaviors observed in 3xTg-AD mice.(2)The over activation of microglia and astrocytes,and the down-regulation of p-AMPK,p-GSK-3β(ser9)and SIRT1 were observed in the hippocampus of9-month-old 3xTg-AD mice.However,APN treatment effectively inhibited the over activation of microglia and astrocytes,and up-regulated the expression of p-AMPK and p-GSK-3β(ser9)in the hippocampus of 3xTg-AD mice.(3)Adiponectin might alleviate the anxiety-and depression-like behaviors observed in 9-month-old 3xTg-AD mice by up-regulating the expression of p-AMPK and p-GSK-3β(ser9),and inhibiting the neuroinflammation in the hippocampus.