Topic 1: Construction Of APP/PS1/LC3 Triple Transgenic Mice And Research On The Dynamic Changes Of Autophagy Flow. Topic: The Protective Effect Of Two Rapamycin On Alzheimer's Disease And Its Mechanism

Objective: To establish a new APP/PS1/LC3 triple-transgenic mice model and to study its dynamic autophagy flux changes.Methods: APP/PS1 double-transgenic Alzheimer’s disease(AD)model mouse was breed with CAG-mRFP-eGFP-LC3 transgenic autophagy flux model mouse,then their filial generations were identified by genotyping,triple-transgenic mice expressing both CAG-mRFP-eGFP-LC3 gene and APP/PS1 gene were selected.Eighteen APP/PS1/LC3 triple-transgenic mice and eighteen CAG-mRFP-eGFP-LC3 transgenic mice were involved in this study,in their third,sixth,tenth month.The Morris water maze was performed to detect learning and memory ability of mice.The activities of autophagy flux were observed under fluorescence microscope.Immunohistochemical staining was used to detect the senile plaques formation;autophagy related proteins were tested by Western blot.Results: The genotyping confirmed that the APP/PS1/LC3 triple-transgenic mouse model was successfully established.Morris water maze test showed that,compared with those of CAG-mRFP-eGFP-LC3 transgenic mice,the escape latency and path length of APP/PS1/LC3 triple-transgenic mice were significantly increased,and the passing times were decreased.The staining showed that sixth month triple-transgenic mice had apparently senile plaques deposition in brains,while the CAG-mRFP-eGFP-LC3 transgenic mice had none.Western blot showed that,compared with that of brood CAG-mRFP-eGFP-LC3 transgenic mice,APP protein in the brain of APP/PS1/LC3 triple-transgenic mice increased significantly.Compared to third month CAG-mRFP-eGFPLC3 transgenic mice,triple-transgenic mice had more autophagosomes in hippocampus area,but there were no difference between autophagosomes in cortex region,or autolysosomes in hippocampus area and cortex region.Compared with those of sixth month CAG-mRFP-eGFP-LC3 transgenic mice,both autophagosomes and autolysosomes were increased in the same age triple-transgenic mice in hippocampus area.Autophagosomes were significantly elevated in the cortex region of sixth month APP/PS1/LC3 triple-transgenic mice,but showed no difference in autolysosome number.Autophagosomes and autolysosomes in hippocampus area of tenth month APP/PS1/LC3 triple-transgenic mice were increased contrasted to tenth month CAG-mRFP-eGFP-LC3 transgenic mice,and both of them elevated in cortex region.The proteins related with autophagy including LC3,Beclin1,P62 also increased in brains of third month,sixth month,tenth month APP/PS1/LC3 triple-transgenic mice,but lysosome membrane protein LMAP1 decreased in sixth month and tenth month APP/PS1/LC3 triple-transgenic mice,compared with brood CAG-mRFP-eGFP-LC3 transgenic mice.Conclusion: Our results show that,compared with CAG-mRFP-eGFP-LC3 transgenic mice,the APP/PS1/LC3 mice impair in cognitive function,and deposit senile plaque.Autophagy is induced in APP/PS1/LC3 triple-transgenic mice,and the degradation of autolysosomes is blocked.Sixth month and tenth month APP/PS1/LC3 triple-transgenic mice shows impaired lysosomal function.Taken together,our studied indicate that APP/PS1/LC3 triple transgenic mice is an ideal mice model for researching autophagy flux function in AD.Objectives: To research the effect of rapamycin(Rapa)on Alzheimer’s Disease(AD)and to discuss its underlying mechanism preliminarily.Methods: in vivo experiment,we divide 10 male APP/PS1 transgenic mice(4-month-old)randomly into rapamycin group and control group.Rapamycin treatment group was intragastricly given rapamycin 2mg/kg per day,and control group was given the same volume of dissolvent with the rapamycin treatment group.After 4 weeks,elevated high-plus maze test,open field test,novel object recognition,Barnes maze were performed to observe behavioral changes on APP/PS1 transgenic mice of rapamycin medication.Thioflavin S staining and 4G8 staining were used to detect senile plaque(SP)aggregation and deposition.The effect of rapamycin on Aβ generation and degradation,Tau hyperphosphorylation and autophagy activation were tested by western blot.In vitro experiment,differentiated SH-SY5 Y cells stable transfected APPsw gene was employed and rapamycin was given with dosage of 50 n M and 100 n M.Cell proliferation was detected by Ed U assay and western blot.Autophagy activity of cells were illustrated by immunofluorescence after transfected with m RFP-GFP-LC3 adenoviral vector.Results: In vivo,rapamycin treatment had no positive effect on cognitive and memorized deficits of APP/PS1 transgenic mice.After rapamycin treatment,the number and area of SP decreased,Aβ expression decreased,β-secrete(BACE1)and presenilin-1(PS1)level declined,and Aβ degradetic enzyme insulin degrading enzyme(IDE)expression increased,but rapamycin has no influence on Amyloid-β protein precursor(APP)level.Rapamycin can decreased expression level of tau and hyperphosphorylated tau.Autophagy related protein LC3 II/I and beclin1 were elevated by rapamycin medication,p62 level decreased,all of them demonstrated activation of autophagy.Wnt and β-Catenin got activate by rapamycin,GSK-3β expression decreased and its activation inhibited.In addition,rapamycin can increase expression level of Neu N.In vitro,50 n M and 100 n M rapamycin increased the number of Ed U incorporated cells.Immunofluorescence result showed that the number of m RFP-GFP-LC3 puncta dramatically increased in the SH-SY5 Y cells stable transfected APPsw gene upon 50 n M and 100 n M rapamycin exposure.