The Effect Of Autophagic Flux Regulation In Alzheimer's Disease And Medication Application

Objectives:To detect the dynamic changes of autophagic flux and its relationship with Amyloid ? peptide(A?)in Alzheimer's disease(AD)patients,AD model mice of different ages and AD model cells.Methods:(1)Human brain tissue:the human brain tissue samples of AD patients after death were obtained from human brain tissue bank.The normal dead human brain tissue samples were used as control,and autophagic flux were observed by immunofluorescence staining.(2)Animal level:the changes of autophagic flux in AD model mice were observed by electron microscopy,immunofluorescence and Western blotting.(3)Cell level:the autophagic flux was observed by immunofluorescence,quantitative PCR and Western blotting,using the SH-SY5Y cell stably expressing mutant APP(APPswe)and SH-SY5Y cell treated with A?25-35 as AD cell model.Results:(1)In AD patients,LC3 was accumulated in large amount,but the expression of Lampl and CTSB decreased,and the co-expression of LC3 and CTSB decreased.(2)In the brain of APP/PS1 double transgenic mice,autophagosomes(APs)and autolysosomes(ALs)accumulation occur with the deposition of senile plaques(SPs),and the levels of lysosomal markers CTSB and Lamp1 protein decreased significantly.(3)In the brain of APP/PS1/LC3 triple transgenic mice,the number of APs increased with the age of mice,but the number of ALs did not increase accordingly.There were more APs but less ALs in the cortex and hippocampus where prone to SP formation,while there were less APs but more ALs in the white matter where SP do not easily form.(4)After overexpression of APPswe,the levels of ATG5,ATG14,LC3,p62 and Lamp1 decreased significantly.Exogenous A?25-35 up-regulated the expression of early autophagy related proteins ATG5,ATG12,ATG16L,BECN1 and down-regulated the levels of middle and late autophagy markers p62,ATG14 and Lamp1.Conclusion:There are a lot of APs accumulation in the brain of AD patients and AD model mice.The fusion of APs and lysosome is blocked,the number and function of lysosomes are decreased in the cortex and hippocampus where prone to SP formation,while autophagic flux work well in the white matter where SP do not easily form.The activation of autophagy is mainly due to the increase of A? rather than the overexpression of mutated APP gene.However,both the treatment with exogenous A?25-35 and the mutation of the endogenous APP gene blocked the fusion of APs with lysosomes and decreased lysosomal functioning.Objectives:To investigate the expression of autophagy associated gene 14(ATG14)in AD patients,AD model mice of different ages and AD model cells,the effect and mechanism of ATG14 on A?,and the effect on the function of AD model mice.Methods:(1)The expression of ATG14 in the brain of postmortem AD patients,APP/PS1 double transgenic AD model mice,SH-SY5Y-APPswe cells and SH-SY5Y cells+A?25-35 AD model cells were detected by immunofluorescence,fluorescence quantitative PCR and western blotting.(2)SH-SY5Y-APP,We cell were divided into blank group,empty group,ATG14 overexpression group and ATG14 inhibition group.The levels of ATG14 mRNA and protein were detected by fluorescence quantitative PCR and Western blot;the number of APs and ALs were detected by morphological examination;the expression level of autophagy markers and the expression of related proteins in the process of A? production were detected by Western blot.(3)The adeno-associated virus carrying ATG14 plasmid was injected into the hippocampus of APP/PS1 double transgenic mice,and normal saline was injected into the hippocampus of mice as the control group.After 30 days,the learning and memory ability of mice was detected by Morris water maze;Western blot was used to detect ATG14 and other autophagic markers,as well as the expression of A? production related proteins;the changes of SPs in cortex and hippocampus were observed by morphological staining.(4)SH-SY5Y-APPswe cells were divided into two groups:empty vector group and ATG14 overexpression group.They were treated with Rapamycin,Bavaramycin or Chloroquine,respectively.Then the number of APs and ALs were measured.Results:(1)The expression of ATG14 did not change significantly in the brain of AD patients,compared with the control group;But there were less ATG14 and a lot of LC3 accumulation in the cells near the area of SPs deposition in AD patients,while there were more ATG14 expression in the area without senile plaque deposition,ATG14 and LC3 coexisted a lot.(2)Compared with wild-type mice in the same nest,there was no significant difference in ATG14 expression in the brain of 3-month-old APP/PS1 AD model mice,while the expression of ATG14 in the brain of 6-month-old and 10-month-old APP/PS1 AD model mice was significantly down-regulated.(3)Compared with APPWT cells,ATG14 was down-regulated in APPsWe cells.After SH-SY5Y cells were treated with A?25-35,ATG14 decreased significantly,and with the increase of A?25-35 concentration,ATG14 decreased gradually.(4)After ATG14 overexpression,the relative expression of LC3-?/? increased and p62 decreased significantly,the levels of APP,BACE1,PS1 and A? in APPswe cells were significantly lower than those in the control group.After ATG14 silencing,the LC3-II/I ratio and the relative expression of p62 increased,the levels of APP,BACE1 and A? were significantly higher than those in the control group,and PS1 did not change significantly.(5)After adeno-associated virus overexpression ATG14 was injected into the hippocampus of APP/PS1 double transgenic mice,the learning and memory of the 5-month-old APP/PS1 double transgenic mice were partially improved,the senile plaques in the brain were reduced,the expression of APP,BACE1,PS1 and A? protein were significantly lower than that of the control group.However,overexpression of ATG14 did not reduce A? in the brain of 9-month-old APP/PS1 double transgenic mice.(6)Compared with autophagy activator alone,the combination of RAPA and overexpression ATG14 reduced the number of APs and increased the number of fully acidified ALs;compared with autophagy inhibitors alone,the combination overexpression ATG14 and Baf-A1 or CQ increased the number of APs and ALs in cells.(7)ATG14 overexpression up-regulated the expression of autophagy related proteins ATG5,ATG12,ATG16L,BECN1 and the level of lysosomal marker Lampl,while the expression of Rab7 and its effector molecule RILP decreased.Conclusion:There is less ATG14 expression in the area of SPs in AD patients,but high in the area without senile plaques.The increase of exogenous A? and the overexpression of endogenous APPswe resulted in the down-regulation of ATG14.ATG14 overexpression significantly can reduce the production of A? in the brain of APPswe cells and APP/PS1 double transgenic mice,improve the learning and memory of AD model mice in the early stage of SP formation,and reduced the deposition of SP in the brain.However,there was no significant effect on the decrease of A? in the brain of elder AD model mice.The combination of ATG 14 and autophagy activator can promote autophagic flux better than that of autophagy activator alone.However,when the fusion of AP and lysosome is blocked or the degradation of AL is seriously blocked,the over expression of ATG14 may also lead to more AP aggregation.ATG14 can play a neuroprotective role in early AD by activating autophagy,promoting the fusion of autophagy and lysosome,increasing lysosomal degradation,reducing the production of A? and enhancing the degradation of A?.Objectives:To study the effect and mechanism of Valproic acid(VPA)and Rapamycin(RAPA)on AD model mice.Methods:(1)APP/PS1 double transgenic mice were divided into VPA group,RAPA group,VPA+RAPA group and control group.VPA group was given 30mg/kg/d VPA intraperitoneal injection,RAPA group was given 1mg/kg/d RAPA intragastric injection,VPA+RAPA group was given 30mg/kg/d VPA intraperitoneal injection and lmg/kg/d RAPA intragastric injection,the control group was injected with the same amount of normal saline.After 4 weeks of treatment,Barnes maze was used to detect the learning and memory ability of mice;blood routine and blood biochemical tests were used to evaluate the side effects of the drug;immunohistochemistry and Thioflavin S staining were used to observe the changes of senile plaques in cortex and hippocampus.(2)SH-SY5Y-APPswe cells were used as AD model cells.The effects of VPA and RAPA on cell proliferation,tubulin and autophagic flux were detected in vitro.(3)The number of APs and ALs in the brain were observed by immunofluorescence,and the expression of autophagy marker protein and its mechanism pathway were detected by Western blot.Results:(1)Blood routine and blood biochemical tests showed that there was no significant difference in the effects of treatment group and control group on blood,liver and kidney system.(2)The results of Barnes maze showed that except the combined group,VPA and RAPA group showed better learning and memory ability than the control group.(3)Thioflavin S staining showed that the number and volume of senile plaques decreased in treatment group,and the effect of VPA group was the most significant,RAPA group was the second,VPA combined with RAPA group was the worst.(4)Western blot showed that the expression of APP protein and A? protein in treatment group all decreased,and the downregulated effect in VPA group was the most significant;the expression of Tau protein and PHF-1 protein in VPA and RAPA group reduced significantly,while Tau protein and PHF-1 protein in VPA+RAPA group were only slightly down regulated.(5)Compared with the control group,there were no significant changes in Akt,mTOR and p70s6K in the brain of VPA and RAPA groups,but the phosphorylation levels of Akt(Ser473),mTOR(Ser 2448)and p70s6K(thr389)were significantly reduced,and the inhibitory effect of RAPA on Akt/mTOR/p70s6K pathway was powerful than that of VPA.(6)Immunofluorescence staining showed that VPA significantly increased the expression of lysosomal enzyme CTSB,while LC3 slightly increased.RAPA significantly increased LC3,but CTSB had no significant change.Western blot showed that compared with the control group,LC3 in VPA group increased,p62 decreased,and Lamp1 increased significantly,while LC3 and p62 in RAPA group increased,but the expression of Lampl did not change significantly.Conclusion:VPA and RAPA alone can improve the learning and memory of AD model mice,reduce the deposition of senile plaques,as well as reduce the production of A? and the expression of phosphorylated Tau protein.VPA and RAPA exert neuroprotective effect on AD by activating autophagy.Akt/mTOR/p70s6K mediates VPA and RAPA induced autophagy.However,VPA and RAPA cannot be combined to treat AD.VPA can promote the fusion of autophagy and lysosome,increase the degradation function of lysosome and reduce the accumulation of autophagy.RAPA can activate autophagy but can not enhance lysosomal degradation.