Effect Of PTK2B On Aβ、p-Tau In Blood And Brain Tissues Of Cognitively Impaired Mouse

Objective:To uncover the age-based real-time expression pattern of ptk2b and ptk2b-encoded protein,protein tyrosine kinase 2β（PTK2B）,in the brain tissues of transgenic animal models of Alzheimer’s disease（AD）and its relationship with the levels of Aβ_1-42,phosphorylation of Tau（p-Tau）and low-density lipoprotein related protein 1（LRP-1）in blood and brain tissues and the changes in behavioral functions,so as to discover the effect of PTK2B on the pathogenesis and behavioral functions and the underlying mechanism.The study was conducted in two parts.Firstly,we hope to identify the age-based real-time expression pattern of ptk2b and PTK2B in the brain of APP/PS1transgenic animal models and the relationship of PTK2B expression with the levels of Aβ_1-42,Tau phosphorylation and LRP-1 in blood and brain tissues.Then,we explored the correlation of LRP-1 with the expression of ptk2b and PTK2B,aiming to analyze the relationship between changes in the level of Aβand ptk2b expression in blood and brain of cognitively impaired mouse.Methods:Part1:In this study,5-,10-and 15-month-old APPswe/PS1d E9 double-transgenic mice harboring the genotype of AD confirmed by the gene test were divided into the 5-,10-and 15-month-old experiment groups,and simultaneously,age-matched C57BL/6J mice were placed into the corresponding control groups,with 8 mice in each group.All mice were subjected to the Morris Water Maze for test of cognitive and behavioral ability.Expression profiles of PTK2B,Aβ_1-42,p-Tau/Tau and LRP-1 in the hippocampus or serum of mice were quantified by using the Western blot,immunohistochemistry（IHC）staining,or enzyme-linked immunosorbent assay（ELISA）,morphological changes in the hippocampus were depicted by using the hematoxylin&eosin（HE）staining,while the m RNA expression of ptk2b in the hippocampus was quantified by using the real-time quantitative polymerase chain reaction（q RT-PCR）.Part2:A total of 64 3-month-old C57BL/6J mice were divided randomly into the experimental group and control group.All mice underwent the intracerebroventricular（i.c.v.）intubation.Mice in the experimental group received the i.c.v.infusion of oligomeric Aβ_1-42（0.1μg/μL,3.6μL）to construct the cognitively impaired models,and,three days later,those mice were further injected with the PF431396（an inhibitor of PTK2B,15μg/m L,Aβ+PF group）,Phorbol-12-myristate-13-acetate（PMA,an agonist of PTK2B,18.75μg/m L,Aβ+PMA group）,RAP（an inhibitor of LRP-1,0.2μg/m L,Aβ+RAP group）and normal saline（Aβ+NS group）.For mice in the control group,they underwent the i.c.v.infusion of NS,and following 3 days,they would additionally be injected with PF431396（PF group）,PMA（PMA group）,RAP（RAP group）and NS（NS group）in volume of 2μL.One week later,all mice were subjected to the determination of behavioral function in Morris Water Maze and the measurement of expression of Aβ_1-42,LRP-1 and PTK2B in serum and hippocampus using the IHC staining,enzyme-linked immunosorbent assay（ELISA）and Western blot,and the measurement of m RNA expression of ptk2b in hippocampus using the q RT-PCR.Results:Part 1:Results of Morris Water Maze indicated that as compared to the C57 mice,APP/PS1 mice had significant decline in the cognitive functions（P<0.05 or 0.001 in comparison of the average of escape latency to search for the platform;P<0.05 or 0.001 in comparison of the average of times of platform crossing in probe trial;P<0.001 in comparison of the average of time spent in the platform quadrant）.As for the assessment of behavioral functions for APP/PS1 mice,they manifested a progressive decline in the behavioral functions as they aged（P<0.01 or 0.001 in comparison APP/PS1-10 M significantly longer than 5 M of the average of escape latency to search for the platform as15 M than 10 M;P<0.05,0.01 or 0.001 in comparison APP/PS1-10 M significantly more less than 5 M of the average of times of platform crossing in probe trial as well as between15 M and 10 M;P<0.05 or 0.001 in comparison APP/PS1-10 M significantly more less than 5 M of the average of time spent in the platform quadrant as 15 M than 10 M）,while the results of C57 mice presented no obvious changes.Besides,an age-dependent decline was found in the level of Aβ_1-42in the serum of APP/PS1 mouse（APP/PS1-10 M is lower than 5 M as 15 M than 10 M,all P<0.05）,while the expression or level of PTK2B（APP/PS1-10 M is higher than 5 M as 15 M than 10 M,P<0.05,0.01 or 0.001）,ptk2b m RNA（APP/PS1-10 M is higher than 5M as 15 M than 10 M,all P<0.01）,Aβ_1-42（APP/PS1-10 M is higher than 5 M as 15 M than 10 M,P<0.05 or 0.01）and p-Tau/Tau（APP/PS1-10 M is higher than 5 M as 15 M than 10 M,all P<0.001）was upregulated gradually in the hippocampus,and LRP-1 in the hippocampus was also downregulated（APP/PS1-10 M is lower than 5 M as 15 M than 10 M,P<0.05,0.01 or 0.001）.Also,results of HE staining also implicated that as APP/PS1 mice,they had more evident morphological changes and decline in the cell quantity of hippocampus.Furthermore,significant differences were observed in comparison of these indicators between APP/PS1 mice and age-matched C57 mice（P<0.05,0.01 or 0.001）.Comparison among the control groups only showed an age-dependent decrease in the expression of LRP-1 in hippocampus（APP/PS1-10 M is lower than 5 M as 15 M than 10 M,P<0.05 or 0.01）,while no evident differences were shown in comparison of other indicators.Part 2:Results of Morris Water Maze showed that in comparison with Control group,C57 mice in the experiment groups presented with significant declines in the behavioral functions,including the obvious prolongation of escape latency of mice to search for the platform in spatial navigation test（P<0.001）and significant decreases in the times or time spent in platform crossing（all P<0.001）.Treatment of PF431396,an inhibitor of PTK2B,for the lateral ventricles could improve the cognitive behaviors in mouse with Aβ_1-42-induced cognitive dysfunction（Escape latency of mice in spatial navigation was shortened,P<0.001;the times of mice crossing the platform increased,P<0.01;time of mice spent in the quadrant of platform increased,P<0.05）,while the administration of PMA,an agonist of PTK2B,or LRP-1,an inhibitor of RAP,resulted in the opposite results（Escape latency of mice in spatial navigation was prolonged,all P<0.001;the times of mice crossing the platform decreased,all P<0.01;time of mice spent in the quadrant of platform was shortened,all P<0.05）.Results of ELISA indicated no significant difference in comparison of the levels of Aβ_1-42in the serum of C57 mice in control groups,while for experiment groups,the level of Aβ_1-42in serum was much lower（P<0.001）;furthermore,administration of PF431396 could further increase the level of Aβ_1-42in the serum of mice with Aβ-induced cognitive dysfunction（P<0.01）,but treatment of PMA（P<0.05）or RAP（P<0.05）could reverse such changes.Results of IHC staining,Western blot and q RT-PCR implicated that the expression of PTK2B,Aβ_1-42,LRP-1 or ptk2b m RNA had no significant variation in the hippocampus of mice in the control groups;however,mice in the experiment groups had significant upregulation of Aβ_1-42（P<0.001）,PTK2B（all P<0.001）and ptk2b m RNA（P<0.001）but downregulation of LRP-1（all P<0.001）as compared to their counterparts in the control groups.For comparison among the experiment groups,administration of PF431396 could downregulate the expression of PTK2B and ptk2b m RNA in the hippocampus of mice（all P<0.05）,with a sharp decline in the level of Aβ_1-42in the hippocampus（P<0.01）,while LRP-1 was upregulated（all P<0.05）;on the contrary,administration of PMA resulted in the quite different changes（P<0.001 or 0.05）;after treatment of RAP,LRP-1 was downregulated in the hippocampus（all P<0.001）,but Aβ_1-42level had an increase（P<0.001）,while the expression of PTK2B and ptk2b m RNA in the hippocampus presented with no obvious changes（all P>0.05）.Conclusion:1)In the hippocampus of APP/PS1 double-transgenic mice,PTK2B,Aβ_1-42and p-Tau/Tau are upregulated,LRP-1 is downregulated,while cognitive and behavioral ability declines,and such changes are presented in a time-dependent manner.2)In cognitively impaired mice,PTK2B,possibly via downregulating the LRP-1 transporter,increases the level of Aβ_1-42in brain,but decreases the level in blood,thereby deteriorating the cognitive and behavioral functions of mice.