Aβ Deposition-induced Senescence Of Astrocytes In The Pathogenesis Of Alzheimer’s Disease And Its Intervention Research

Objective: Cell senescence plays a vital role in the pathogenesis of Alzheimer’s disease(AD),but the relationship and mechanism with the occurrence and development of AD have not been fully elucidated.Amyloid β(Aβ)is a key factor in the pathological changes of the brain in AD patients.However,whether Aβ can accelerate cellular senescence,especially astrocyte,and cellular senescence will subsequently induce cognitive function impairment are still unclear.The efficacy of senescent cells’ clearance in reversing AD pathological progress as an aging therapy needs to be further investigated.Therefore,the primary cultured astrocytes(Astrocytes,AS),APPswe/PS1 d E9 double transgenic model mice(B6.Cg-Tg)(APP/PS1)and other related materials.were employed as the research objects to observe the cellular senescence and the type of senescent cells in the brain of the APP/PS1 transgenic mice with the increasing months of age.In vivo and in vitro experiments were conducted to explore the role of cellular senescence in the pathogenesis of AD and to ensure whether Aβ exerts its neurotoxicity in inducing or promoting senescence of astrocytes and its involving mechanism.To explore the elimination of ABT263(Also known as navitoclax,a specific inhibitor of anti-apoptotic protein,specifically induces apoptosis of senescent cells)on senescent cells of APP/PS1 transgenic mouse brain and to figure out the reversal of the AD process and possible mechanism after reducing senescent cells provided theoretical bases for AD’s clinical treatment.Targeted elimination of senescent astrocytes utilizing the designed vector system in brain tissues of APP/PS1 mouse,to explore the effect and possible mechanism of targeted elimination on the process of AD and to further examine the role of astrocyte senescence in the pathogenesis of AD,provided an assumption for the pathogenesis of AD and clinical treatment research and the treatment targets basis.Methods: 1.Cytology experiment:(1)Aβ-induced cell senescence: primary cultured AS cells were used for continuous subculture for 90 days,and Aβ oligomers,galactose(Dgal),PBK pathway inhibitor LY294002 was used alone or in combination to treat cultured AS cells.AS cells were isolated,purified,and then identified.β-galactosidase staining(Senescence Associated β-Galactosidase,SA-β-Gal)was used to detect positive cell senescence,Real-time quantitative PCR(Real-Time PCR)measurements were performed to determine m RNA expression levels of the senescence-associated pathways of p16,p21,and p53.Western blotting(WB)method was used to analyze the expression levels of cell senescence pathway-associated proteins p16,p21,p53,phosphorylated protein phosphatidylinositol-3 kinase(PI3K),serine / threonine methyl protein kinase acid(AKT),glycogen synthase kinase-3β(GSK-3β),and apoptosis-related protein Bax and Bcl-2.The levels of interleukin-6(IL-6),interleukin-8(IL-8),matrix metalloproteinase 1(MMP-1)in supernatants were detected using Enzyme-linked immunoassay assay(ELISA).Apoptosis level,cell cycle,and reactive oxygen species(Reactive oxygen species,ROS)were measured by flow cytometry.(2)Construction and verification of the apoptosis-inducing vector system: Based on the connection of FKBP12 and FRB domain induced by rapamycin and the dimerization of FKBP by the dimer AP20187,we respectively constructed FKBP-2A-td Tomato(Red fluorescence)and FRB-Cas8-2A-BSD plasmids,the above plasmids were transfected into 293 T cells,AGS cells,U251 cells,K562 cells with Lip2000,then treated with AP20187(B/B Homodimerizer)and rapamycin,observe the red fluorescent cells with a fluorescence microscope,and detect cell apoptosis by flow cytometry.(3)Construction of a vector system for targeted elimination of senescent astrocytes: on the basis of the above-mentioned apoptosis-inducing vector system,cell senescence-specific protein P16 and astrocyte marker protein Aldh1l1 were used to initiate downstream protein expression to construct two lentiviral vectors(Lenti p16-FKBP-2A-td Tomato,Lenti Aldh1l1-FRB-Cas8-2A-BSD).They were used to infect D-gal-induced senescent mouse astrocytes(Mouse Astrocytes,m AS),then treated with Pamycin and AP20187,the red fluorescent cells were observed under a fluorescence microscope,and cell apoptosis was detected by flow cytometry.2.Zoological experiments:(1)APP/PS1 double transgenic mouse brain cell senescence and related index detection: immunohistochemical methods were used to detect the expression and distribution of Aβ in the brains of APP/PS1 double transgenic mice and the control group,Morris water maze was performed to measure the spatial learning and memory capability of mice in each group,pathological changes of mice brain tissue in each group were evaluated by hematoxylin and eosin(Hematoxylin-Eosin,HE)staining,senescence cells were stained positive by SA-β-Gal staining,Real-Time PCR was used to detect the m RNA expression level of the aging related pathways proteins of p16,p21,p53 in the hippocampus and cortex of APP /PS1 double transgenic mice and wild type mice.WB was performed to determine the expression levels of senescence pathway-associated proteins(p16,p21,p53,PI3K-AKT-GSK-3β),the ratio of total and phosphorylated proteins(PI3K,AKT,GSK-3β),and apoptosis-related proteins(Bax and Bcl-2)in tissues as described above.ELISA measured the changes in levels of IL-6,IL-8,MMP-1 in the hippocampus and cortex of the mice in each group.(2)ABT263 clearing aging cells in the brain tissue of APP/PS1 double transgenic mice: 6 and 12 month-old APP/PS1 double transgenic mice were used to give 50mg/kg/day ABT263 to the gavage for 5 days first,rest for two weeks,and then gavage for 5 days.Immunohistochemical methods were performed to prove the existence of Aβ in the brain tissue of APP / PS1 double transgenic mice and the control group.HE staining evaluated the pathologic changes of each group of mice brain tissue.To measure the spatial learning and memory capability of mice,the morris water maze was performed in both groups.SA-β-Gal staining was expressed in senescence-positive cells.Real-time quantitative PCR was carried out quantitating the m RNA levels of senescence pathway-associated proteins(p16,p21,p53)in the hippocampus of APP/PS1 double transgenic mice.WB method was used to analyze the expression of the senescence pathway-associated proteins(p16,p21,p53),phosphorylated proteins(PI3K,AKT,GSK-3β),apoptosis-related proteins(Bax,Bcl-2,Bcl-x L)in the hippocampus tissue of APP/PS1 double transgenic mice.To detect the expression levels of IL-6,IL-8,and MMP-1 in the hippocampus of mice in each group was performed with the ELISA method.(3)Targeting clearance of senescence astrocytes cell in the brain of APP/PS1 double transgenic mice.Two adeno-associated viruses(AAV p16-FKBP-2A-td Tomato,AAV Aldh1l1-FRB-Cas8-2A-BSD)were constructed.Infected by the viruses,the twelve-month-old APP/PS1 double transgenic mice were then treated with AP20187 and rapamycin successively.And detecting the expression of Aβ in the brain of APP/PS1 double transgenic mice and the control group used immunohistochemical methods.Immunofluorescence was performed to analyze the co-localized expression of cells with Red fluorescent and Aldh1l1,Neu N,Iba1,Olig2 in the brain tissue of mice in both groups.And then to detect senescence-positive cells with SA-β-Gal staining.WB method was used to detect the expression levels of proteins as follows,the senescence-related pathway-proteins,p16,p21,p53,phosphorylated proteins,PI3 K,AKT,GSK-3β,and apoptosis-associated proteins,Bax,Bcl-2 in the cortex of mice in each group.To determine the levels of IL-6,IL-8,and MMP-1 in the cortex of mice in each group by using the ELISA method.Results: 1.Cell experiment results:(1)Aβ-induced cell senescence test: The results showed that the number of senescence-positive cells increased significantly after Aβ treatment of primary culture AS,the expression level of P16 and Bax increased,the expression level of p PI3 K,p AKT,p GSK-3β,and Bcl-2 protein decreased.In addition,the levels of IL-6,IL-8,and MMP-1 increased to varying degrees,the rate of cell apoptosis and the level of ROS also increased,and the cell cycle was mainly blocked in the G2/M phase.At the same time,Aβ can further enhance the cell senescence induced by D-gal and the senescence of natural senescent AS cells.When combined with 10 u M PI3K/AKT/GSK3β signaling pathway inhibitor LY294002 and 1μmol/L Aβ to treat primary cultured astrocytes,compared with the control group,the expression levels of p16 and p21 proteins in the cells of the LY294002 treatment group decreased,there was no significant change in p53 protein expression level.Compared with the 12 Days+Aβ group,the p16 and p21 protein expression levels in the 12 Days+LY294002+Aβ group decreased,but the p53 protein expression level did not change significantly.(2)Construction of the apoptosis-inducing vector system and verification in cells:a.Induction of apoptosis vector system to induce cell apoptosis: the constructed FKBP-2A-td Tomato and FRB-Cas8-2A-BSD plasmids were co-transfected into 293 T,AGS,U251,K562 cells with Lip2000,and under a fluorescence microscope to confirm that the cells emitted red fluorescence(td Tomato).after the transfected cells were treated with AP20187 and rapamycin,observed with a fluorescence microscope that compared with the transfected group(not treated with AP20187 and rapamycin),the number of red fluorescent cells is significantly reduced,and the results of flow cytometry showed that the rate of cell apoptosis increased significantly.b.Targeted elimination of senescent astrocytes: After infection of D-gal-induced m AS cells with Lenti p16-FKBP-2A-td Tomato,the cells emit apprent red fluorescence,indicating that the plasmid can mark senescent cells;D-gal-induced m AS cells,293 T and AGS cells were infected with Lenti p16-FKBP-2A-td Tomato and Lenti Aldh1l1-FRB-Cas8-2A-BSD plasmids,red fluorescence was observed in all groups.After co-treatment with AP20187 and rapamycin,red fluorescence in m AS cells was obvious decrease,the apoptotic rate increased,but the number of 293 T and AGS cells that emit red fluorescence did not change significantly,and the apoptosis rate did not change significantly.It showed that the carrier system could induce apoptosis of senescent astrocytes to achieve the purpose of targeted elimination.2.Animal experiment results:(1)Aging Results of Brain Cells in APP/PS1 Double Transgenic Mice:(1)Immunohistochemistry results showed 4 months old age WT group and APP/PS1 mice no significant difference,from 6 months old,the APP/PS1 double transgenic mouse hippocampus and cortex in the emergence of Aβ deposition,and with the increase of age,gradually sizes,ranging from the number of plaques.Wild mice of corresponding months old did not have a small amount of Aβ deposition until 18-month-old,HE staining showed no obvious histopathological changes.Immunofluorescence results showed that astrocytes and oligodendrocytes were senescent,but neurons and microglia were not senescence.(2)The results of the Morris water maze showed that there was no significant difference between the 4-month-old WT group and the APP/PS1 group in terms of escape latency,time stay in the target platform,and platform crossing times.Starting from 6 months of age,APP/PS1 double transgenic mice showed a decline in learning and memory.The results of SA-β-Gal staining showed that senescent positive cells appeared in the brain tissue of APP/PS1 mice at the age of 4 months.From the age of 6 months,a small amount of plaques appeared in the brain tissue of APP/PS1 double transgenic mice and earlier than the appearance of Aβ deposition.with the increase of months,senescence-positive cells further increased.Wild mice began to appear senescence-positive cells from 6 months of age,and with the increase of months,the number of senescence-positive cells increased,but There are fewer plaque-like senescence-positive cells,and they are significantly reduced compared with APP/PS1 mice of the same-month-old.(4)The ELISA results showed that there was no significant difference in the expression levels of IL-6,IL-8 and MMP-1 in the hippocampus of the 4-month-old WT group and the APP/PS1 group.The expressions of IL-6,IL-8 and MMP-1 were increased at 6 months of age,and increase significantly with the increase of age,in addition,wild-type mice start from 12 months of age,IL-6,IL-8,MMP-1 expression level also increased.(5)QPCR results showed that in 4-month-old mice,compare to the APP/PS1 double transgenic mice with WT mice,p16 m RNA levels showed a tendency to increase,but no significant difference.At 6 months old,the expression level of p16 m RNA was increased significantly and further increase with age.The expression of p21 m RNA only increased in APP/PS1 double transgenic mice at the age of 18 months.The level of p53 m RNA increased at the age of 12 months,and the level of p53 m RNA increased significantly with the increase of age.The expression levels of p16,p21,and p53 m RNA in hippocampus and cortex are consistent.(6)Results of WB: a.In 4-month-old mice,APP/PS1 double transgenic mice had higher expression of aging-related pathway protein p16 than WT group,and increased significantly with the increase of age,while p21 protein was only increased in18-months-old APP/PS1 double transgenic mice.There was no significant difference in p53 protein.The expression levels of p16,p21,and p53 proteins in hippocampus and cortex tissues were consistent.b.From 4 months of age,the expression of p PI3 K,p AKT and p GSK-3β protein in the hippocampus of APP/PS1 double transgenic mice decreased,and decreased significantly with the increase of months.c.from 6 months old,the expression level of apoptosis-related protein Bcl-2was decreased in APP/PS1 double transgenic mice than WT mice and decreased significantly with the age increases.On the contrary,the expression level of Bax protein increases significantly with the increase of the month age.the expression levels of Bcl-2 and Bax in the hippocampus and cortex are roughly the same.(2)Results of ABT263 Clearing aging cells in APP / PS1 cells double transgenic mice brain: In 6 month,12 month-old mice,comparing with the APP/PS1 group of the same month,the ABT263 gavage treatment group had stronger learning and memory ability,the number of senile plaques was relatively reduced,and the degree of Aβaggregation was reduced either;The number of positive cells in the brain decreases,and the degree of aggregation of positive plaques decreases;the expression levels of IL-6,IL-8,and MMP-1 in the hippocampus decrease;the expression levels of p16 and p21 m RNA Decrease;phosphorylated PI3 K,AKT and GSK-3β protein expression levels increased;Bcl-2 protein expression levels did not change significantly,Bcl-x L protein expression level is further reduced,Bcl-x L protein expression levels further decreased,and Bax protein levels further increased.These results indicate that ABT263 can delay the course of APP/PS1 and the changes of related indicators,and improve the learning and memory ability of mice.(3)targeted elimination of senescent astrocytes cells in APP/PS1 mice: After intracerebroventricular injection of adeno-associated virus and treatment with AP20187 and Rapamycin,compared with the APP/PS1+AAV group(without AP20187 and Rapamycin treatment),Aβ deposition in the brain was significantly reduced,senile plaques were reduced,and SA-β-Gal staining results showed that the number of senescence-positive cells was significantly reduced;IL-6 and IL-8expression levels were reduced,and there was no significant difference in MMP-1expression;WB results found that p16,The expression level of p21 protein decreased,the expression level of p PI3 K,p AKT,and p GSK-3β increased,the expression level of Bcl-2 protein increased,and the expression level of Bax protein decreased.These results indicate that targeted elimination of senescent astrocytes in APP/PS1 mice can also delay the course of APP/PS1.Conclusions:(1)Aβ can induce the senescence of primary cultured astrocytes,which may be related to the up-regulation of pl6 gene expression,an increase of ROS,cell apoptosis and regulation of PI3K-AKT-GSK3β signaling pathway.(2)With the increase of months,APP/PSⅠ mice brain senescent astrocytes and oligodendrocytes increased,SASP senescence phenotype secretion increased,p16 expression increased,astrocyte senescence was deposited in Aβ It has appeared before,indicating that astrocyte senescence is the main type of AD brain cell senescence,and may play an important role in the pathogenesis of AD.(3)The designed apoptosis-inducing vector system can activate caspase8 to form dimers through self-cleavage through the combination of FKBP and FRB and FKBP dimerization,thereby inducing cell apoptosis.The vector system can be introduced into cells by a plasmid,lentivirus and other carriers,and comprising a target cell-specific promoter(Aldh1l1 promoter: astrocyte-specific promoter promoter)in the case of the particular cell(p16 promoter: Senescent cell-specific promoter)expresses and presents a red fluorescent label.At the same time,through the combination of FKBP and FRB and FKBP dimerization,Caspase8 is activated,and apoptosis signals are activated to mediate cell apoptosis,activates the apoptotic signal to mediate cell apoptosis,acts as a fluorescent label and is specifically targeted elimination of specific cells.(4)ABT263 eliminates senescent cells in APP/PS1 mouse brain and targeted elimination of senescent astrocytes in APP/PS1 mouse brain can reduce Aβaggregation and inflammatory response in the brain,and activate P3K-AKT-GSK-3B Pathway,delay the course of AD.