The Study On Mechanism Of DA4-JC In Improving Cognitive Behavioral Disorder And Pathological Damage In APP/PS1/tau Transgenic Mice

Objective:To study the effects of GLP-1/GIP double receptor agonist DA4-JC on cognition,emotional behavior and pathological characteristics of 10-month-old APP/PS1/tau Alzheimer’s disease（3xTg-AD）mice,and to further study the possible signal transduction mechanism of DA4-JC in neuroprotection.Methods:The 9-month-old 3xTg-AD mice and C57 mice were randomly divided into four groups:normal control group（WT+PBS）,normal treatment group（WT+DA4-JC）,AD control group（3xTg-AD+PBS）and AD treatment group（3xTg-AD+DA4-JC）.According to the weight of mice,DA4-JC（10 nmol/kg）or PBS with the same volume was given once a day by intraperitoneal injection,and the weight and blood sugar level were weighed once every 7 days before and during the administration until the end of administration.After 30days of continuous administration,the behavioral experiment was started,and the drug was continuously administered during the behavioral period.1.Behavior experiment（1）Open field test:The experimenter gently placed the mouse tail in the central area of the open field,let it move for 5 minutes in a quiet state,and recorded the total distance of the mouse moving in the open field and the percentage of the mouse moving time in the center of the box.（2）New object recognition experiment:On the second day after the open field experiment,put two identical objects in the open field,then put the mouse in and let it explore freely for 10 minutes;After 5 h,one of the objects was replaced with an object with the same material,different color and shape,and it was allowed to explore freely for10 min again,and the time for mice to explore new and old objects was recorded respectively^[1].The detection index is:NOI=time to explore new objects/（time to explore new objects+time to explore old objects）×100%^[2].（3）Y-maze spontaneous alternation experiment:The mice were put in from the intersection of the three arms of the Y-maze,and allowed to explore freely for 8 min.The arm-feeding sequence and total times of mice were recorded,and the correct rate and total times of arm-feeding of mice in each group were calculated^[3].（4）O maze test and elevated cross maze test:In O maze test,mice were put into the central area facing the closed arm and moved freely for 10 min,and the time of mice moving in the open arm within 10 min was recorded^[4],and the percentage of mice moving in the open arm was calculated.The elevated cross maze test is to put mice into the central area facing the closed arm and move freely for 10 min,record the times of mice entering the open arm and the closed arm within 10 min,and calculate the percentage of times of mice entering the open arm and the closed arm in each group^[5].（5）Classic Morris water maze experiment and reversal water maze experiment:Put the mice in the water maze,carry out hidden platform experiment,space exploration experiment and visual platform experiment respectively,record the escape latency of each group of mice within 1 min,and calculate and compare the percentage of time spent swimming in the target quadrant and the times of crossing the platform during the space exploration stage^[6].After the classic water maze experiment,the platform was put into the contraposition quadrant,and the reversal hidden platform experiment and space exploration experiment were carried out again,with the same indexes as the classic water maze experiment.After 6 h,the visual platform experiment stage was carried out,and the swimming speed and the time to reach the visual platform of each group of mice were recorded^[3].（6）Tail suspension test and forced swimming test:In tail suspension test,the tail of the mouse is suspended and fixed in a tail-hanging box,so that the nose tip of the mouse is about 20-25 cm away from the bottom of the tail-hanging box.Keep the camera as close as possible to the mice to get clearer images,record the immobility time of mice within 6 min,and calculate the immobility time percentage of mice in each group after 4 min^[6].In the forced swimming test,the mice were put into a transparent cylinder filled with 15 cm deep tap water,and allowed to move for 6 min.The immobility time of mice after 4 min was recorded,and the percentage of mice after 4 min immobility was calculated^[6].（7）Fear conditioning experiment:It is divided into intensive training and detection stages.In the training stage,the mice were given fear training;The memory of conditioned fear was measured after 24 h interval.What the computer system detects is the change of rigidity rate of mice.2.After the behavioral experiment,electrophysiology,molecular biology and morphology experiments were carried out.（1）Electrophysiological LTP experiment:After behavioral experiment,electrophysiological records were made in vivo.At first,after anesthesia,the mice were fixed on the stereotactic apparatus of the brain,and the electrodes were slowly inserted into the hippocampal CA1 region.After appropriate stimulation,the excitatory postsynaptic potential（f EPSP）of the basal field was continuously recorded for 30 min,then high frequency stimulation（HFS）was given,and then the long-term potentiation（LTP）of f EPSP was recorded for 60 min to evaluate the difference of synaptic plasticity in each group of mice.（2）Western-blotting:Then quickly take out the fresh hippocampus of mice,extract the protein,detect the content of related protein,make glue,sample,run glue,transfer membrane,seal,then incubate the primary antibody and the secondary antibody,finally expose it,and analyze the gray value of target bands（PINK1,Parkin,P62,PSD95,SYP）with software.（3）Frozen and paraffin-embedded immunohistochemistry experiment:After the behavioral experiment and electrophysiological experiment,the brains of mice in each group were cut from the sagittal line,half of which was used for frozen immunohistochemistry and half for paraffin-embedded immunohistochemistry.The distribution and changes of Aβand p-tau protein in hippocampal CA1 region of mice in each group were observed by microscope.（4）Golgi staining experiment:After the behavioral and electrophysiological experiments,the brain tissues of mice were stained by Golgi staining.In this experiment,FD Rapid Golgi Stain Kit（FD Neuro Technologies）was used.After taking the brain tissue of mice according to the instructions of the kit,the pictures were taken after observation with a microscope,and the number changes of dendritic spines of mice in each group were counted by software.（5）Transmission electron microscope experiment:After the behavioral and electrophysiological experiments,the hippocampal tissue of mice was taken and CA1region was isolated,and sent to the electron microscope room for a series of related experimental treatments,and then observed and photographed,and the number and morphological changes of mitochondria and synapses in hippocampus of mice in each group were counted.Results:（1）Body weight and blood sugar level of mice in each group:Before and after injection of DA4-JC,there was no significant difference in body weight and blood sugar level among the four groups（P>0.05）.（2）Open-field test:Within 5 min,there was no significant difference in the total movement distance of mice in each group and the percentage of time spent moving in the center of the box（P>0.05）.（3）New object recognition test:NOI of mice in 3xTg-AD+PBS group was significantly lower than that of mice in WT+PBS group（P<0.001）,but increased after DA4-JC treatment（P<0.05）.（4）Y-maze test:There was no significant difference in the total arm-feeding times of mice in each group（P>0.05）.The accuracy of spontaneous alternation in 8 min in3xTg-AD+PBS group was significantly lower than that in WT+PBS mice（P<0.001）,but increased after DA4-JC treatment（P<0.01）.（5）O maze test and elevated cross maze test:In O maze test,the percentage of mice entering the open and closed arms within 10 min in 3xTg-AD+PBS group was significantly lower than that in WT+PBS group（P<0.01）,but it did not improve significantly after DA4-JC treatment（P>0.05）.In the elevated maze test,the percentage of time of opening arms in 10 min in 3xTg-AD+PBS group was significantly lower than that in WT+PBS group（P<0.05）,and there was no obvious change and improvement after DA4-JC treatment（P>0.05）.（6）Classic Morris water maze experiment:The time of escaping latency of mice searching for hidden platform decreased for 5 consecutive days.On the 3rd,4th and 5th day of hiding platform,compared with WT+PBS group,the time of finding platform in3xTg-AD+PBS group was significantly longer（P<0.05）.After DA4-JC treatment,the latency on the 4th and 5th day was shortened with statistical significance（P<0.05）.In the exploratory experiment,compared with the WT+PBS group,the percentage of swimming time in the target quadrant and the times of crossing the platform of the transgenic mice were lower（P<0.05）.After DA4-JC treatment,the two indexes were reversed（P<0.05）.Reversal water maze experiment:In the 7-10 days of hidden platform experiment,the latency of searching for underwater hidden platform decreased continuously;On the9th and 10th days of swimming,it can be seen that the time of finding the platform of mice in 3xTg-AD+PBS group is significantly longer than that in WT group（P<0.05）.After DA4-JC intervention,the latency of 3xTg-AD mice on the 9th and 10th day was significantly shortened（P<0.05）.In the para-position exploration experiment,the swimming time ratio and crossing times of 3xTg-AD+PBS mice were significantly lower than those of WT mice（P<0.05）.After DA4-JC treatment,both indexes were improved（P<0.05）.Visual platform test:There was no statistical difference between the time of finding the platform and the swimming speed in water（P>0.05）.（7）Tail suspension test and forced swimming test:In the tail suspension test,the percentage of immobility time of 3xTg-AD mice was significantly higher than that of WT mice（P<0.01）.After administration of DA4-JC,the percentage of immobility time of3xTg-AD mice did not change significantly（P>0.05）.In the forced swimming experiment,the percentage of immobility time of 3xTg-AD mice was significantly higher than that of WT mice（P<0.001）.After administration of DA4-JC,there was no significant improvement in 3xTg-AD mice（P>0.05）.（8）Fear conditioning experiment:In the training stage,the stiffness ratio of WT mice and 3xTg-AD mice increased gradually with the training duration,but there was no significant difference between the four groups of mice（P>0.05）.However,in the detection stage of fear memory,compared with WT+PBS group,the stiffness ratio of3xTg-AD+PBS group was significantly lower than that of WT group（P<0.001）.After DA4-JC treatment,the stiffness ratio of 3xTg-AD mice under conditioned stimulation increased significantly（P<0.01）.（9）Electrophysiological LTP experiment:There was no significant difference in I/O curve among the mice in LTP groups in vivo,and there was no significant difference in f EPSPs amplitude among the groups under the same suitable stimulation intensity（P>0.05）.There is no significant difference in PPF（f EPSP2/f EPSP1）ratio among the four groups（P>0.05）.LTP was successfully induced after HFS.Compared with WT+PBS group,30min and 60 min after HFS stimulation,the percentage change of f EPSP slope in hippocampal CA1 area of mice in 3xTg-AD+PBS group decreased significantly（P<0.05）,and it was reversed after DA4-JC treatment（P<0.05）.（10）Western-blotting:The average gray values of PINK1,Parkin,PSD95 and SYP in hippocampus of mice in 3xTg-AD+PBS group were significantly lower than those in WT+PBS group（P<0.001）,and the average gray value of P62 was significantly higher than that in WT+PBS group（P<0.001）.Compared with 3xTg-AD+PBS group,the average gray values of PINK1,Parkin,PSD95 and SYP in hippocampus of mice in3xTg-AD+DA4-JC group were significantly increased（P<0.05）,while the average gray value of P62 was significantly decreased（P<0.001）.（11）Frozen/paraffin immunohistochemical experiment:Compared with WT+PBS group,the area percentage of Aβand p-tau in 3xTg-AD+PBS group increased significantly（P<0.001）,while the area percentage of Aβand p-tau in 3xTg-AD mice decreased significantly after DA4-JC treatment（P<0.001）.（12）Golgi staining experiment:The number and morphology of dendritic spines perμm were statistically analyzed by golgi staining.it was found that the number of dendritic spines in 3xTg-AD+PBS group was lower than that in WT+PBS group（p<0.001）.Compared with 3xTg-AD+PBS group,the number of dendritic spines in3xTg-AD+DA4-JC group increased significantly（P<0.001）.（13）Transmission electron microscope:The number and morphology of mitochondria were observed by transmission electron microscope.it was found that the number of small and round mitochondria in 3xTg-AD+PBS group was significantly higher than that in WT+PBS group（P<0.001）,and mitochondria were mostly characterized by cristae rupture and mitochondria swelling.After treatment with DA4-JC,it can be seen that the number of small and round mitochondria in 3xTg-AD+DA4-JC group decreased significantly（P<0.001）,and the morphology of mitochondria was relatively intact.At the same time,observing the number and morphology of synapses under statistical electron microscope,it was found that the number of synapses in 3xTg-AD+PBS group was significantly less than that in WT+PBS group,and the morphology of synapses was abnormal.Compared with3xTg-AD+PBS group,the number and structure of synapses in 3xTg-AD+DA4-JC group were improved（P<0.001）.Conclusion:（1）DA4-JC partially reversed the exploratory ability,spatial working memory ability,long-term learning and memory cognitive ability,cognitive flexibility and fear memory ability of 3xTg-AD mice,but it could not improve their anxiety and depression.（2）DA4-JC can protect the plasticity of hippocampal synapses in 3xTg-AD mice.DA4-JC can increase the number of dendritic spines in hippocampal neurons.DA4-JC can increase the number of synapses in hippocampal CA1 region of 3xTg-AD mice under electron microscope,and make the size and structure of synapse tend to normalize.DA4-JC can increase the levels of PSD95 and SYP protein in hippocampus of 3xTg-AD mice.（3）DA4-JC reduced the deposition of Aβand p-tau protein in the brain of 3xTg-AD mice.（4）DA4-JC can increase the levels of PINK1 and Parkin in hippocampus of 3xTg-AD mice,and at the same time decrease the level of P62,thus improving the damaged mitochondrial autophagy pathway.（5）DA4-JC can increase the number of normal mitochondria and improve the damage of mitochondria morphology and structure.Based on the above results,it can be concluded that GLP-1/GIP/double receptor agonist DA4-JC can improve the cognitive behavior disorder of learning and memory,the pathological features of Aβand p-tau,the structural and functional damage of synapses and dendritic spines in 3xTg-AD mice,which may be related to the up-regulation of PINK1and Parkin levels in hippocampus and the reduction of P62 levels in order to improve autophagy and mitochondrial autophagy.Therefore,GLP-1/GIP double receptor agonist DA4-JC may have positive significance in preventing and treating Alzheimer’s disease.