Study On The Neuroprotection Of Novel GLP-1/GIP Dual Agonist DA5-CH And Its Mechanism In APP/PS1 Transgenic Mice Of Alzheimer’s Disease

Objective:By conducting behavioral experiments at the whole level and subsequent immunofluorescence test of brain tissue section,we can explore whether the novel GLP-1/GIP dual agonist DA5-CH can effectively ameliorate the cognitive impairments and pathology in the APP/PS1 mouse model of Alzheimer’s Disease（AD）.Further,we also carry out in vivo hippocampal late-phase long-term potentiation（L-LTP）recording at the level of organ and synapse and Western-blot to explore the potential cellular and molecular mechanism,so as to open up new ideas and strategies for the prevention and treatment of AD.Methods:A total of fiftysix 9-month-old APPswe/PS1_{d E9}（APP/PS1）transgenic mice and wild type（WT）C57BL/6J（C57）nest control mice were divided into the following four groups:WT+Saline,WT+DA5-CH,APP/PS1+Saline and APP/PS1+DA5-CH.Each group had fourteen mice,half male and half female.Behavioral tests,in vivo hippocampal L-LTP recording,immunofluorescence staining,Western-blotting were carried out in turn after 28 days drug application（DA5-CH or saline）.Behavioral tests included open field test,Y-maze test,Morris water maze test and reversal water maze test.In open field test,each mouse was placed individually in the apparatus from the center for 5 min and the total distance,as well as the percentage of time in center,was recorded.In Y-maze test,the mouse was allowed to explore freely the open arms for 8 min.The total arm entries and arm order were recorded.Morris water maze test includes two phases,acquisition trial and probe trial.The acquisition phase lasted for five training days（days 1–5）for the mice to search for the underwater platform located at the first quadrant.The swimming traces of mice and the escape latencies were automatically recorded by camera and video analysis software.In the probe trial on the day 6,the platform was removed from the pool,and animals were allowed to swim freely for 1 min.The frequency of platform crossing and the swimming time in the target quadrant（the first quadrant）were recorded.Reversal water maze test was performed after that.The platform was moved to the opposite quadrant（the third quadrant）,and mice were placed into the water for similar four days acquisition trials and one day probe trial as mentioned above.To eliminate the effects of exercise and vision on previous experiments,we added visible platform test.The platform was elevated 1cm above the water level and swimming time to target was recorded.In the experiment of in vivo hippocampal L-LTP recording,the mice,under the anesthetization,were placed in a stereotaxic apparatus.The skin was cut open to expose the skull,and a binding stimulating/recording electrode was inserted into the CA1 region of hippocampus through a small hole drilled on the skull.Basic synaptic transmission was observed for 30 min,with stable fEPSPs under test stimulation.Then,short term synaptic plasticity was observed by recording paired-pulse facilitation（PPF）with two paired test stimuli.After PPF recording,L-LTP was induced by three sets of high-frequency stimulation（HFS）and lasted for at least 180min.After the above experiments,some mice were randomly selected for immunofluorescence staining.The brain of the mice were removed and frozen to-80℃.The brain was sectioned at a thickness of 30μm and followed by adding the primary antibody,second antibody,and DAPI.The Aβplaque and phosphorylated tau protein were observed by confocal laser scanning microscopy.The other mice were randomly selected for the Western blotting.The optical density of the target strip（p-PI3K,t-PI3K,p-AKT,t-AKT,p-GSK3βand t-GSK3β）was analyzed.Results:We found that:（1）In open field test,the statistical analysis did not show any difference in total distance covered by the animals and the ratio of time spent in the center.These results suggest that DA5-CH did not affect the locomotor and exploratory performance of APP/PS1 and WT mice.（2）In Y-maze test,the total arm entries of mice were without significant difference between groups.However,the percentage of right alternation in the mice of APP/PS1+Saline group（P<0.001）had a significant decrease compared to that in WT+Saline group,while the percentage in DA5-CH treated APP/PS1 transgenic mice had a significant improvement（P<0.001）.These results indicate that the working memory of APP/PS1 transgenic mice in Y-maze was seriously impaired,which could be improved by DA5-CH treatment.（3）The escape latencies in the APP/PS1+Saline group were significantly higher compared to the WT+Saline group（day 2,P<0.05;day 3-5,P<0.01）.Importantly,DA5-CH treatment significantly decreased the values in APP/PS1 mice,significantly lower than that of the APP/PS1+Saline group（day 3,P<0.01;day 4-5,P<0.05）.This indicates that chronic treatment with DA5-CH can significantly ameliorate spatial learning in APP/PS1 mice.The frequency of platform crossings and the percentage of swimming time in target quadrant in APP/PS1+Saline group were significantly lower than that in WT+Saline group（respectively,P<0.01,P<0.01）,but not in the DA5 treated APP/PS1 mice（respectively,P<0.05,P<0.01）,indicating that the spatial memory disorder in the AD mice could be effectively ameliorated by the chronic treatment with DA5-CH.There was no significant difference in the escape latency between all groups in the visible test,thus excluding the impact of motor ability and vision impairments on the above tests.（4）In the reversal MWM test,the mean escape latency of mice in the APP/PS1 model group was significantly higher than that in the normal WT group（day 9,P<0.01;day 10,P<0.05）.However,DA5-CH decreased the escape latency in APP/PS1 mice compared with the APP/PS1+Saline group（day 8-10,P<0.05）.In the probe trials,the significant decrease in the percentage of swimming time in target quadrant and frequency of platform crossing in the APP/PS1 mice（respectively,P<0.001,P<0.05）compared with the WT mice had an obvious recovery in the DA5 treated APP/PS1 mice（respectively,P<0.05,P<0.05）,compared with that in the APP/PS1+Saline group.This indicates that chronic treatment with DA5-CH can alleviate the impairment in the relearning ability and cognitive flexibility of APP/PS1 transgenic mice.（5）In the experiment of in vivo hippocampal L-LTP recording,there was a significant decline in the maintenance of L-LTP at 60 min（P<0.05）,120 min（P<0.05）and 180 min（P<0.05）after HFS in the APP/PS1 model group,compared with that in the control group.In contrast,in DA5-CH injected APP/PS1 transgenic mice,L-LTP was maintained at higher levels（P<0.05 for each one）.The results indicate that DA5-CH could effectively reverse the impairment of hippocampal L-LTP in the APP/PS1 AD mice.Besides,no significant difference in the PPF ratio（fEPSP2/fEPSP1）was found between all groups.This suggests that effects of DA5-CH and APP/PS1 genes on hippocampal L-LTP are mainly associated with postsynaptic changes.（6）In immunofluorescence staining studies,a large number of Aβpositive plaques and phosphorylated tau were observed in the hippocampal slices of APP/PS1+Saline group（respectively,P<0.001,P<0.001）,significantly more than in the WT+Saline.Importantly,the numbers of Aβplaques and phosphorylated tau had a significant reduction in the hippocampus of APP/PS1+DA5-CH group mice（respectively,P<0.001,P<0.001）compared to that in the APP/PS1 model group.These results demonstrate that nine-month-old APP/PS1 transgenic mice have shown distinct Aβplaques and neurofibrillary tangles in the hippocampus and the treatment with DA5-CH can effectively reduce these pathological biomarkers.（7）In Western-blotting,the relative protein expression of total PI3K/β-actin,total AKT/β-actin and total GSK3β/β-actin did not change in all groups.However,the levels of p-PI3K,p-AKT in APP/PS1+Saline group mice showed down-regulation（respectively,P<0.05,P<0.05）,while the level of p-GSK3β（P<0.05）showed up-regulation,compared to the values in the WT+Saline group.Importantly,compared with the APP/PS1+Saline group,the levels of the p-PI3K and p-AKT were increased（respectively,P<0.05,P<0.05）,and the levels of p-GSK3βhad been decreased（P<0.05）in the group of APP/PS1 mice injected with DA5-CH,suggesting that DA5-CH may plays a neuroprotective role via ameliorating the PI3K/AKT/GSK3βsignaling pathway in the hippocampus of APP/PS1 mice.Conclusion:The present study confirmed for the first time the neuroprotective roles of the novel GLP-1/GIP dual agonist DA5-CH on the cognitive impairments and neuropathological processes in APP/PS1 transgenic AD mice.The neuroprotection of DA5-CH was associated with the improvement of hippocampal synaptic plasticity and PI3K/AKT/GSK3βsignaling pathway.Therefore,this study provides a novel and promising strategy to treat neurodegenerative disease such as AD.