Effect Of PLTP Over Expression On Learning And Memory In 3×Tg-AD Mice And Its Mechanism

Objective:Alzheimer’s disease（AD）is a degenerative disease of the central nervous system and is the most common type of dementia.At present,the research on AD has attracted extensive attention from scholars at home and abroad,and has proposed a number of pathogenic mechanisms,and developed a targeted drug with certain therapeutic effects,but its pathogenesis has not yet been fully elucidated,and there is still no effective therapeutic drug.Phospholipid transfer protein（PLTP）is a glycoprotein widely expressed in the central nervous system.In recent years,in vitro and in vivo studies have found that PLTP is closely related to AD.Our previous study found that PLTP deficiency can promote the decline of cognitive function in aged mice,so whether high expression of PLTP can improve AD,domestic and foreign studies have not been reported.In this study,3×Tg-AD（APPSwe/PS1d E91/Tau P301L triple transgenic）mice were used as the AD model,and the brain tissue-specific PLTP overexpression model was constructed by adenovirus-mediated PLTP gene lateral ventricular transfection.Shuttle passive avoidance experiments,pathological staining,western blot and other techniques and methods,through the detection of memory,senile plaques,Aβ-related protein,total Tau protein,p Tau protein,GSK-3βpathway-related protein detection,explore the PLTP overexpression of AD Prevention and treatment and possible mechanisms,in order to provide an important basis for the search for new targets and new ideas of AD treatment through PLTP.Methods:1.Brain-specific PLTP overexpression model was constructed using 3×Tg-AD mice as an AD model.Twenty-four male rats of 3×Tg-AD at 10 months were randomly divided into model group（12 rats）and experimental group（12 rats）.Male C57BL/6J（Wild Type,WT）with the same genetic background at the same age was used as a control.Group（12）.The lateral ventricle（1.2 mm from the anterior iliac crest posterior 0.6 mm sagittal suture）was injected with adeno-associated virus（AAV-PLTP-EGFP）carrying the PLTP gene-enhanced green fluorescent protein reporter gene to induce high expression of PLTP.Both the control group and the model group were injected with adeno-associated virus（AAV-EGFP）carrying the PLTP gene and carrying only the enhanced green fluorescent protein reporter gene.2.To observe the effect of PLTP overexpression on learning and memory ability of3×Tg-AD miceTwo weeks after injection of AAV-PLTP-EGFP virus into the lateral ventricle of mice,water maze test and shuttle passive avoidance test were used to detect the learning and memory ability of mice.The water maze experiment is divided into two parts:the positioning navigation experiment and the space exploration experiment.The cognitive function of the mouse is evaluated by the latency of the platform and the number of times the platform enters the platform.The shuttle passive avoidance experiment was carried out in the light and dark shuttle box,and the learning and memory ability was evaluated by the incubation period of the mouse entering the dark box and the staying time in the black box.3.Pathological observation of the protective effect of PLTP overexpression on3×Tg-AD miceThe pathological lesions of each group were detected by HE staining and Nissl staining.The changes of senile plaques（SP）were observed by immunehistochemical staining of Aβ1-42.The neurofibrillary tangles（neurofibrillary tangles）were observed by Bielschowsky silver staining.NFT)changes.4.To observe the regulation of PLTP overexpression on Aβproduction and its related markersAfter behavioral experiments,the mice were sacrificed,and the cerebral cortex and hippocampus of the mice were isolated to prepare tissue homogenate.The expression levels of APP,PS1 and BACE1 related to Aβproduction were detected by Western blot.ELISA was used to detect the expression of Aβ_1-40 and Aβ_1-42levels.5.To observe the regulation of total treat protein and phosphorylated Tau protein by PLTP overexpression.Western blot was used to detect the expression of total Tau protein and p Tau protein in p Ser199,p Ser214,p Thr231 and p Ser404.6.To observe the effect of PLTP overexpression on GSK-3βand GSK-3β（p Ser9）The protein expression levels of GSK-3βand GSK-3β（p Ser9）were detected by western blot.7.The experimental data were statistically analyzed with SPSS16.0 software.One-way analysis of variance（ANOVO）was used for comparison among groups of samples.Statistical significance was found at P<0.05.Result:1.PLTP overexpression can improve the learning and memory ability of 3×Tg-AD miceThe results of the water maze showed that in the navigation experiment and the space exploration experiment,the escape latency was significantly prolonged in the 3×Tg-AD mice compared with the WT mice,and the number of access to the platform was significantly reduced in the exploration experiment.The incubation period increased significantly.The escape latency of 3×Tg-AD mice with high expression of PLTP was significantly shortened.The number of access to the platform was significantly increased during the experiment,and the latency of entering the platform was significantly shortened.The difference was statistically significant.The results of shuttle passive avoidance experiments showed that compared with WT mice,the latency of 3×Tg-AD mice entering the dark box was significantly shortened,and the dark box residence time was significantly prolonged;while the 3×Tg-AD mice with high expression of PLTP entered the dark box.The incubation period was significantly prolonged,and the residence time was significantly shortened.The difference was statistically significant.This indicates that PLTP overexpression can significantly improve the learning and memory ability of 3×Tg-AD mice.2.PLTP overexpression can improve the pathological damage,senile plaque and neurofibrillary tangles in 3×Tg-AD mice.By HE staining and Nissl staining,the cortical neurons of 3×Tg-AD mice were reduced,the nucleus was concentrated,and apoptosis was observed.Aβ_1-42 immunohistochemical staining showed obvious SP,while silver staining found obvious NFT;PLTP over After expression,cortical neuron cells increased,SP decreased,and NFT was also significantly reduced.3.Regulation of PLTP overexpression on Aβlevels3.PLTP overexpression can reduce AβformationThe results of ELISA showed that the expression of Aβ_1-40and Aβ_1-42 protein increased in hippocampus and cortex of 3×Tg-AD mice.After overexpression of PLTP,the expression of Aβ_1-40and Aβ_1-42protein decreased.4.PLTP overexpression can reduce the expression of proteins related to Aβproduction.The results of Western blot showed that the expressions of APP,PS1 and BACE1 were increased in hippocampus and cortex of 3×Tg-AD mice.The expressions of APP,PS1 and BACE1 were decreased after overexpression of PLTP.5.PLTP overexpression can inhibit the expression of Tau protein and its p Tau protein.The results of Western blot showed that the expression of total Tau and its p Tau protein in p Ser199,p Ser214,p Thr231 and p Ser404 increased in hippocampus and cortex of3×Tg-AD mice;total Tau and its p Tau after PLTP overexpression The expression of the protein at the four sites of p Ser199,p Ser214,p Thr231,and p Ser404 was decreased.6.PLTP overexpression can promote the phosphorylation of GSK-3βat the 9th position to inactivate GSK-3β.The results of Western blot showed that the expression of GSK-3βwas increased in the hippocampus and cortex of 3×Tg-AD mice,and the expression of GSK3β（p Ser9）was decreased in phosphorylation of threonine residues at 9;after GTP overexpression,GSK The expression of-3βis decreased,and the expression of GSK3β（p Ser9）produced by phosphorylation of the threonine residue at the 9th position is increased.Conclusion:1.PLTP overexpression can improve the learning and memory ability of 3×Tg-AD2.The improvement of learning and memory ability of 3×Tg-AD by PLTP over expression may be related to the reduction of Aβproduction and Tau protein phos-phorylation by promoting the inactivation of GSK-3β.