Effects Of HSP90 Modulate PTK2B On The Brain Histopathology And Learning And Memory Behavioral Ability In The Cognitive Dysfunction Mice

Objective:To investigate the expression changes of heat shock protein 90（HSP90）in brain tissue of mice in Alzheimer disease（AD）with age;And the effects of ptk2b on AD pathologic markers such as amyloid-beta（Aβ）and Tau phosphorylation level（p-Tau）as well as the learning and memory behavioral ability in cognitive dysfunction mice.The study was divided into two parts:（1）To investigate the changes of expression levels with HSP90,PTK2B,Aβ_1-42and p-Tau in the hippocampus as well as the changes of learning and memory behavioral ability at different ages of APP/PS1 transgenic mice.（2）To investigate the effect of HSP90 regulation on the expression of PTK2B on the hippocampal histopathology together with the learning and memory behavior in the Aβ-induced cognitive impairment mice.Methods:Part I:APP/PS1 transgenic mice（6-month-old mice,APP/PS1-6 M group;9-month-old mice,APP/PS1-9 M group;12-month-old mice,APP/PS1-12 M group）and C57BL/6J mice（6-month-old mice,C57BL/6J-6 M group;9-month-old mice,C57BL/6J-9 M group;12-month-old mice,C57BL/6J-12 M group）at different months of age were the experimental subjects.There were 6 groups with 8 animals in each group.After gene identification of APP/PS1 transgenic mice,mice matching PS1 genotype were used in the experiment.Morris water maze was used to test the learning and memory ability of mice in each group.Western Blot and immunohistochemistry were used to detect the phosphorylation levels of HSP90,PTK2B,Aβ_1-42and Tau protein in hippocampus.The morphological and quantitative changes of hippocampal cells in each group were observed by Nissl’s staining.Real-time quantitative PCR（q RT-PCR）was used to detect hsp90m RNA and ptk2b m RNA levels in hippocampus.Part II:Eighty 9-week-old C57BL/6J mice were divided into 6 experimental groups and 4control groups.intracerebroventricular（i.c.v.）was injected with Aβ_1-42（2μg/m L,2.5μL）in the experimental group and i.c.v.same amount of normal saline（NS）in the control group.After 3 days,the experimental mice were injected with HSP90 inhibitor Luminespib（5μg/m L,Aβ+Lum group）or agonist Tamoxifen（10μg/m L,Aβ+TX group）,PTK2B inhibitor PF431396（15μg/m L,Aβ+PF group）,pre-application of PTK2B inhibitor followed by HSP90 agonist（Aβ+PF+TX group）,pre-application of PTK2B inhibitor followed by injection of HSP90 inhibitor（Aβ+PF+Lum group）and NS（Aβ+NS group）;The control group was injected with HSP90 inhibitor（Lum group）or agonist（TX group）,PTK2B inhibitor（PF group）and NS（NS group）.The dose in each group was 2μL.5 days later,Morris water maze was used to detect the learning and memory behavior ability of each group of mice.Western Blot and immunohistochemistry were used to detect the phosphorylation levels of HSP90,PTK2B,Aβ_1-42and Tau protein in the hippocampus of mice,and q RT-PCR was used to detect the expression of ptk2b m RNA in hippocampus.Results:Part I:The water maze results showed that compared with C57BL/6J mice of the same age,the average incubation period of APP/PS1 mice seeking escape platform was prolonged on the fourth day（6 M,P<0.05;9 M,P<0.01;12 M,P<0.01）,on the fifth day,the average incubation period of searching for escape platform was prolonged（all P<0.01）;The number of crossing the original position of the escape platform and the stopping time in the original quadrant were significantly decreased（all:6 M,P<0.05;9 M,P<0.01;12 M,P<0.01）.It was found that the average incubation period of APP/PS1 mice seeking escape platform was gradually prolonged with the increase of the age of APP/PS1 mice（9 M vs.6M,P<0.05;12 M vs.9 M,P<0.01）,the number of times crossing the original position of the escape platform and the stop time in the original quadrant were decreased（9 M vs.6 M and 12 M vs.9 M were:all P<0.05）.There was no significant difference in the above indexes among C57BL/6J mice in the control group（P>0.05）.Western Blot,immunohistochemistry and q RT-PCR results showed that HSP90 and hsp90 m RNA expression levels in hippocampus of APP/PS1 mice in all experimental groups were decreased compared with C57BL/6J mice of the same age（all:6 M,P<0.05;9M,P<0.01;12 M,P<0.01）,while the expression levels of PTK2B,ptk2b m RNA,Aβ_1-42and p-Tau were increased(PTK2B:P<0.01 for all groups.ptk2b m RNA and Aβ_1-42:6 M,P<0.05;9 M,P<0.01;12 M,P<0.01.p-Tau:P<0.05 for all groups).It was found that the expression levels of HSP90 and hsp90 m RNA in hippocampus of APP/PS1 mice decreased gradually with the increase of the age of APP/PS1 mice（HSP90:9 M vs.6 M,P<0.05;12M vs.9 M,P<0.01.hsp90 m RNA:9 M vs.6 M and 12 M vs.9 M,all P<0.05）;However,the levels of PTK2B,ptk2b m RNA,Aβ_1-42and p-Tau increased gradually(PTK2B:9 M vs.6 M,P<0.01;12 M vs.9 M,P<0.05.ptk2b m RNA:9 M vs.6 M,P<0.05;12 M vs.9 M,P<0.01.Aβ_1-42:P<0.01 for all groups.p-Tau:9 M vs.6 M,P<0.01;12 M vs.9 M,P<0.05).There was no significant difference in the above indexes among the control groups（P>0.05）.Nissl’s staining results showed that compared with C57BL/6J mice of the same age,APP/PS1 mice of all experimental groups had disordered and shriveled nerve cells in hippocampal tissue,and the number of nerve cells increased and the number of nerve cells decreased（6 M,P<0.05;9 M,P<0.05;12 M,P<0.01）.At the same time,with the increase of APP/PS1 mice months,the arrangement of nerve cells became more chaotic,the intercellular space gradually increased and the number of nerve cells decreased（9 M vs.6M,P<0.05;12 M vs.9 M,P<0.05）.There was no significant change in the number of nerve cells among control groups（P>0.05）.Part II:Water maze results showed that compared with the control group（NS group）,the average incubation period of Aβ-induced cognitive dysfunction mice（Aβ+NS group）seeking escape platform was prolonged from the third day,and increased from the fourth day to the fifth day（all P<0.01）.The number of mice crossing the original position of the escape platform and the stopping time in the original quadrant were significantly decreased（all P<0.01）.The average latency of i.c.v.mice injected with HSP90 agonist Tamoxifen（Aβ+TX group）or PTK2B inhibitor PF431396（Aβ+PF group）was shortened（all P<0.01）.The number of crossing the original position of the escape platform and the stopping time in the original quadrant were all increased（all P<0.01）;But i.c.v.injection of HSP90 inhibitor Luminespib（Aβ+Lum group）caused the opposite effect（P<0.05）.After inhibition PTK2B and applying HSP90 inhibitor（Aβ+PF+Lum group）,the average latency of seeking escape platform in mice treated with HSP90 inhibitor alone（Aβ+Lum group）was significantly shortened（P<0.01）.At the same time,the number of mice crossing the original position of the escape platform and the stopping time in the original quadrant were increased（all P<0.01）.Similarly,inhibition PTK2B and then applying HSP90 agonist（Aβ+PF+TX group）significantly shortened the enhanced effect of HSP90 agonist alone（Aβ+TX group）on the mean incubation period of mice seeking escape platform（P<0.01）.The number of mice crossing the original position of the escape platform and the stopping time in the original quadrant were also significantly increased（P<0.05 or P<0.01）.There was no significant difference in the above indexes among the control groups（P>0.05）.Western Blot,immunohistochemistry and q RT-PCR results showed that the expression level of HSP90 in the hippocampus of mice with cognitive impairment induced by Aβwas decreased compared with the control group（P<0.01）.The expression levels of PTK2B,ptk2b m RNA,Aβ_1-42and p-Tau were significantly increased（all P<0.01）.After comparing the experimental groups,the expression levels of PTK2B,ptk2b m RNA,Aβ_1-42and p-Tau in hippocampal tissues of mice treated were increased with HSP90 inhibitor（P<0.05 or P<0.01）.HSP90 agonists caused the opposite effect（P<0.05 or P<0.01）.After treatment with PTK2B inhibitor,the expression levels of Aβ_1-42and the phosphorylation levels of Tau were decreased（P<0.01 or P<0.05）,but the expression of HSP90 was not significantly changed（P>0.05）.The expressions of Aβ_1-42and p-Tau in hippocampal tissue of mice treated with HSP90 inhibitor after pre-inhibition of PTK2B were significantly lower than those of mice treated with HSP90 inhibitor alone（Aβ+PF+Lum group vs.Aβ+PF group,all P<0.01）;However,the expression of HSP90 was not affected（P>0.05）.Similarly,the levels of Aβ_1-42and p-Tau protein in hippocampus of mice treated with HSP90 agonist after pre-inhibition of PTK2B were significantly lower than those of mice treated with HSP90 agonist alone（Aβ+PF+TX group vs.Aβ+TX group,all P<0.05）;However,the expression of HSP90 was not affected（P>0.05）.There were no significant differences in the above indexes in hippocampal tissues among control groups（all P>0.05）.Conclusions:（1）The expression levels of HSP90 protein and hsp90 m RNA and the number of nerve cells in the hippocampus of APP/PS1 transgenic mice were decreased,and the expression levels of PTK2B,ptk2b m RNA,Aβ_1-42and phosphorylation Tau were increased,which showed progressive development with the decrease of learning and memory behavioral ability of mice.（2）HSP90 may affect the phosphorylation levels of Aβ_1-42and Tau protein and the changes of learning,memory and behavioral ability in hippocampus of mice with cognitive dysfunction through the expression of PTK2B.