The Neuroprotective Effects Of Fluoxetine On Middle-aged APP/PS1 Double Transgenic Mice Of Alzheimer's Disease

Part oneThe effects of fluoxetine on the cognitive abilities and characteristicpathological changes in middle-aged APP/PS1 transgenic AD miceObjective: In this study,we aim at demonstrating that whether fluoxetine(FLX)affects the cognitive abilities and characteristic pathological changes in the APP/PS1 mice in the middle-aged stage of AD.The outcomes of the study might provide the feasibility bases for the further studies on the neurobiological bases underlying the neuroprotective effects of FLX on AD.Methods: Forty 16-17 month-old male APP/PS1 mice were respectively randomly assigned to control group(APP/PS1)or treated group(APP/PS1+FLX).The wild-type mice of the same age were considered as normal control group(WT).All mice in three groups were randomly assigned to received systemic administration of either fluoxetine(10 mg/kgi.p.dissolved in 0.9 % Na Cl)or vehicle(equivalent 0.9 % Na Cl i.p.),and all mice were treated once per day in the morning between 9 AM and 10 AM for 5 consecutive weeks.After 5-week treatment,the Morris water maze was performed to assess the spatial learning and memory abilities of mice in three groups.Immunohistochemical staining was used to examine the effect of FLX on the deposition of beta amyloid in middle-aged APP/PS1 mice.Western blotting technique was conducted to investigate the effect of FLX on the protein expression of beta amyloid,Tau and Phospho-Tau(Ser396)in middle-aged APP/PS1 mice.Results: 1.The Morris water maze results showed that,in the hidden platform trials,the middle-aged APP/PS1 mice revealed significantly higher increasing escape latencies(p < 0.01)compared with the WT mice,while the escape latencies of the APP/PS1 mice in the FLX-treated(APP/PS1+FLX)group were markedly lower than the latencies of the mice in the APP/PS1 group(p < 0.05).In the subsequent probe task,the frequency at which the mice crossed the platform location was not statistically different between the APP/PS1+FLX group and their control(APP/PS1)group,and there was also no significant difference between the WT and APP/PS1 groups.Despite these results,the FLX-treated APP/PS1 mice were found to follow tracks that were more similar to those of the WT mice than the APP/PS1 controls.Additionally,the health of the mice was closely supervised,and the body weights of the mice were measured dailyduring treatment.No significant differences were observed among the three groups of mice.2.Immuohistochemical staining showed that there was hardly any aggregation of beta amyloid in the hippocampus in the WT mice,but in the APP/PS1 and APP/PS1+FLX mice,beta amyloid was found to have aggregated in the hippocampus.Moreover,the density of beta amyloid aggregates in the hippocampus was higher and the labeling for beta amyloid was more intense in the APP/PS1 group than in the APP/PS1+FLX group.Western blotting showed that the levels of beta amyloid and Phospho-Tau(Ser396)were significantly lower in the FLX-treated APP/PS1 mice than in the APP/PS1 mice(p < 0.05),while there was no significant diffirence in the level of Tau among the three groups.Conclusions: 1.FLX can restore the cognitive abilities of middle-aged APP/PS1 mice.FLX can reduce the deposition of beta amyloid in late stage of AD.FLX can inhibit the hyperphosphorylation level of Tau in the late stage of AD.2.These results suggested that FLX may provide new hope as a therapy for AD.Part twoThe effects of fluoxetine on detritic spine synapses andsynapse-assiociated protein in the hippocampus of the middle-agedAPP/PS1 transgenic AD miceObjective: The major aim of the present work is to investigate whether fluoxetine(FLX)affects the number of dentritic spine synapses and the expression of synapse-assiociated protein in the hippocampus of the middle-aged APP/PS1 transgenic AD mice.The results of the study might provide morphological and molecular basis for the effects of FLX on the cognitive abilities of transgenic AD mice and provide the bases for clinical application of FLX on AD.Methods: After a 5-week treatment,six mice were randomly chosen from each group for the following stereological investigation.Firstly,all of the chosen mice were perfused with cold 4 % paraformaldehyde(PFA,p H= 7.4)in phosphate-buffered saline(PBS),and their brains were then removed and fixed in 4% PFA in PBS.Before being cut into serial frozen coronal sections at a thickness of 50 ?m,the brains were cryoprotected by immersion in series of sucrose solutions(10 %,20 % and 30 %)for 24 h per solution.All sections were collected in sequence.Immunohistochemical staining and the stereology analyses were performed to investigate the effect of FLX on the number of dentritic spine synapses in thehippocampus of middle-aged APP/PS1 mice.Three mice were randomly chosen from each group for the following Western blotting test.The mice were deeply anesthetized and then killed.The brains of the mice in each group were promptly removed.Afterwards,the brains were split into two hemispheres along the midsagittal plane,and the entire hippocampus and cortex were dissected from each hemisphere while on ice.Finally,each dissected hippocampus was frozen through liquid nitrogen transiently.Western blotting technique was conducted to investigate the effect of FLX on the expression of synaptic marker proteins synapsin-1 and PSD-95.Results: 1.Immunohistochemical staining and the stereology analyses showed that the numbers of spinophilin-immunoreactivity dentritic spines in DG and in CA1 and CA2/3 of hippocampus in middle-aged APP/PS1 mice were significantly reduced compared with WT mice,while in FLX treated middle-aged APP/PS1 mice,the numbers of spinophilin-immunoreactivity dentritic spines in DG and in CA1 and CA2/3 of hippocampus were significantly increased compared with untreated APP/PS1 mice.2.Western blotting results revealed that the expresstion of synaptic marker proteins,synapsin-1 and PSD-95,were dramatically increased in the hippocampus of FLX treated middle-aged APP/PS1 mice when compared to the untreated APP/PS1 mice.Conclusions: FLX can ameliorate the hippocampal synaptic changesthrough remarkably remodeling the hippocampal synapses at both the morphological and molecular levels in APP/PS1 transgenic mice in the late stage of AD.Part threeThe effects of fluoxetine on the neurons in the hippocampus of the middle-aged APP/PS1 transgenic AD mice and its possible underlyingmechanismObjective: This work focuses on revealing that whether fluoxetine(FLX)affects the number of neurons in the the hippocampus in APP/PS1 transgenic mice in the hippocampus of the middle-aged APP/PS1 transgenic AD mice,as well as exploring its possible underlying mechanism.The results of the study might provide neurobiological basis for the effects of FLX on the cognitive abilities of transgenic AD mice and provide the basis for clinical application of FLX on AD.Methods: After a 5-week treatment,six mice were randomly chosen from each group for the following stereological investigation.Firstly,all of the chosen mice were perfused with cold 4% paraformaldehyde(PFA,p H =7.4)in phosphate-buffered saline(PBS),and their brains were then removed and fixed in 4% PFA in PBS.Before being cut into serial frozen coronal sections at a thickness of 50 ?m,the brains were cryoprotected by immersion in series of sucrose solutions(10%,20% and 30%)for 24 h per solution.All sections were collected in sequence.Toluidine blue statining and the stereology analyses were performed to investigate the effect of FLX on the number of neurons in the hippocampus of middle-agedAPP/PS1 mice.Three mice were randomly chosen from each group for the following Western blotting test.The mice were deeply anesthetized and then killed.The brains of the mice in each group were promptly removed.Afterwards,the brains were split into two hemispheres along the midsagittal plane,and the entire hippocampus and cortex were dissected from each hemisphere while on ice.Finally,each dissected hippocampus was frozen through liquid nitrogen transiently.Western blotting technique was then conducted to investigate the effect of FLX on the expression of marker proteins(GSK-3?,Phospho-GSK-3?(Ser9)and ?-catenin)in classic Wnt signaling pathway.Results: 1.Immunohistochemical staining and the stereology analysis results showed that the number of neurons in DG of hippocampus in middle-aged APP/PS1 mice were significantly reduced compared with WT mice,while in FLX treated middle-aged APP/PS1 mice,the number of neurons in DG of hippocampus was significantly increased compared with untreated APP/PS1 mice.However,there was no significant diffirence in the numbers of neurons in CA1 and CA2/3 of hippocampus among three groups of mice.2.Western blotting results revealed that the expressions of marker proteins(Phospho-GSK-3?(Ser9)and ?-catenin)in classic Wnt signaling pathway were dramatically increased in the hippocampus of FLX treated middle-aged APP/PS1 mice,while there was no significantdiffirence in the expression of marker protein(GSK-3?)of classic Wnt signaling pathway in the hippocampus among three groups of mice.Conclusions: FLX can prevent the neuron loss in the DG of hippocampus of the APP/PS1 transgenic mice in the hippocampus of the middle-aged APP/PS1 transgenic AD mice and its possible underlying mechanism might be involved in the activation of the classic Wnt signaling pathway.