The Effect Of Treadmill Exercise On Microglia-mediated Aβ Clearance In The Hippocampus Of APP/PS1 Mice

Objective Microglia is a kind of resident immune cells in central nervous system,and its dysfunction is closely related to the pathogenesis of AD.The present study,using male APPswe/PS1dE9(APP/PS1)mice,aimed to examine the effects of three months of treadmill exercise on the spatial learning and memory ability as well as hippocampal Aβaccumulation.The present study aimed to analyze the cellular mechanisms underlying the effect of treadmill exercise to reduce hippocampal Aβaccumulation from microglia-mediated Aβclearance,phagocytosis and degradation.Methods 3-month-old male APP/PS1 mice were randomly divided into APP/PS1sedentary group(APP/PS1-SED,n=24)and APP/PS1 exercise group(APP/PS1-EX,n=24),while 3-month-old male C57BL/6 mice were randomly divided into wild-type sedentary group(WT-SED,n=21)and wild-type exercise group(WT-EX,n=21).Mice in APP/PS1-EX mice and WT-EX group were subjected to a treadmill exercise training for 3 months,45 min/day,5 days/week.After the treadmill exercise intervention,the spatial learning and memory ability of mice were measured using a Morris water maze test.24 hours after the behavioral experiments,six mice in each group were selected to deep anesthesia and transcardially perfused with 0.1M ice-cold PBS,then the right hemispheres were carefully dissected out and fixed in4%paraformaldehyde and made into paraffin-embedded coronal sections;the left hippocampus were collected and prepared for further Elisa analysis.After deep anesthesia and transcardially perfused with 0.1M ice-cold PBS,the hippocampus of another six mice from each group were rapidly dissected out and prepared for microglia flow cytometry sorting,then the harvested microglia was used for further Elisa and Western blot analysis.Another three mice in each group and additional one APP/PS1-SED mice were intraperitoneal injected methoxy-X04(X04)at a dose of 10mg/kg body weight,three hours later,the mice were deep anaesthetized and the hippocampus were dissected out and prepared for flow cytometry analysis.The remaining mice were deep anaesthetized and the hippocampus were dissected out and prepared for primary microglia separation,which then be used for in vitro fibrillar Aβclearance,phagocytosis and degradation assays,TFEB fluorescent staining as well as lysosomal pH detection.The indicators are presented as follows:1)Indicators for Aβ:Soluble Aβ40 and Aβ42 levels in the hippocampus were measured by Elisa,and fibrillar Aβdeposition was measured by X04 fluorescent staining.2)Indicators for microglial function and Aβlevels in microglia:In in vitro Aβclearance assay,the levels of residual Aβ42 in medium supernatant were measured by Elisa;in in vivo Aβphagocytosis assay,the percentage of Aβ~+microglia and the levels of fibrillar Aβin microglia were determined by the percentage of X04~+Tmem119~+cells account for Tmem119~+cells and mean fluorescent intensities(MFI)of X04 in Tmem119~+cells using flow cytometry analysis;in in vitro Aβphagocytosis assay,the levels of FAM-Aβ42 in microglia were measured by Elisa;in in vitro Aβdegradation assay,the levels of Aβin microglia at t3 and t24 were determined by the MFI of X04 in microglia using fluorescent staining,and degradation index of microglia was calculated by a formula which can be written as degradation index=(X04 MFI at t3-X04 MFI at t24)/X04 MFI at t3;in addition,the levels of soluble Aβ40 and Aβ42 in microglia were measured by Elisa.3)Indicators for lysosome:The activities of microglial lysosomal hydrolase cathepsin B(CTSB)and cathepsin D(CTSD)as well as lysosomal pH were measured by fluorescent Elisa;the protein levels of mature CTSB,mature CTSD,LAMP1,LAMP2 and TFEB in microglia were measured by western blot;microglial TFEB nuclear translocation was measured by fluorescent staining.Results 1)In the navigation test,the escape latency of APP/PS1-SED mice on the fourth and fifth days as well as the travelled distances on the fifth days was significantly higher than that of WT-SED mice,and the percentage of time spent in platform quadrant of APP/PS1-SED mice on the fourth and fifth days was significantly lower than that of WT-SED mice;Compared with APP/PS1-SED mice,the escape latency on the fifth day was significantly decreased in APP/PS1-EX mice.In the probe test,compared with WT-SED mice,the platform crossing times were significantly reduced in APP/PS1-SED mice;compared with the APP/PS1-SED group,the platform crossing times of APP/PS1-EX mice were significantly increased.Moreover,compared with WT-SED mice,the levels of soluble Aβ40 and Aβ42as well as fibrillar Aβdeposition in the hippocampus of APP/PS1-SED mice were significantly increased;compared with APP/PS1-SED mice,the levels of soluble Aβ40 and Aβ42 as well as fibrillar Aβdeposition in the hippocampus were significantly decreased in APP/PS1-EX mice.2)Compared with WT-SED mice,the levels of residual Aβ42 in microglial medium supernatant,the percentage of X04~+Tmem119~+cells account for all Tmem119~+cells,the MFI of X04 in Tmem119~+cells and the levels of microglial soluble Aβ42 were significantly increased in APP/PS1-SED mice,while the levels of FAM-Aβ42 in microglia and microglial degradation index were significantly decreased in APP/PS1-SED mice;compared with APP/PS1-SED mice,the levels of residual Aβ42 in microglial medium supernatant,the percentage of X04~+Tmem119~+cells account for all Tmem119~+cells,the MFI of X04 in Tmem119~+cells and the levels of microglial soluble Aβ42 were significantly decreased in APP/PS1-EX mice,while microglial degradation index was significantly increased in APP/PS1-EX mice,with unchanged levels of FAM-Aβ42 in microglia.3)Compared with WT-SED mice,the levels of microglial nuclear TFEB as well as the activities of microglial lysosomal hydrolase CTSB and CTSD in APP/PS1-SED mice were significantly decreased,while microglial lysosomal pH in APP/PS1-SED mice was significantly increased;compared with APP/PS1-SED mice,the levels of microglial nuclear TFEB,LAMP1,LAMP2 and mature CTSD as well as CTSD activity were significantly increased in APP/PS1-EX mice,while microglial lysosomal pH in APP/PS1-EX mice was significantly decreased.Conclusions 1)APP/PS1 mice exhibited a significant increase in hippocampal Aβaccumulation and a deficit in spatial learning and memory ability,and three months of treadmill exercise significantly decreased Aβaccumulation in the hippocampus of APP/PS1 mice and improved Aβ-related spatial learning and memory deficits.2)Microglia from APP/PS1 mice exhibited reduced capacity to clear,phagocyte and degrade fibrillar Aβ,as well as increased Aβaccumulation.More importantly,three months of treadmill exercise increased the capacity of microglia to clear and degrade fibrillar Aβ,decreased Aβaccumulation in microglia,with no effect on microglial phagocytosis.3)Microglia from APP/PS1 mice exhibited a significant decrease in nuclear translocation of TFEB and deficits in lysosomal function,and three months of treadmill exercise significantly enhanced nuclear translocation of TFEB and its mediated lysosomal function in microglia from APP/PS1 mice.