Aerobic Exercise Training Regulates GSK3β Activity To Improve The Interaction Between Neurons And Glial Cells In AD Mouse Model

Objective: At different stages of Alzheimer’s disease（AD）,the pathological characters are different.In the early stage of AD,changes in the interaction between neurons and glial cells is a pathological cascade triggered by Aβ.Using early stage of AD mouse model,the present study observed the effects of regular aerobic exercise training on the interaction between neurons and glial cells.We aimed to explore the biological mechanisms of exercise training in delaying the development of AD.Methods: 6-month-old wild mice（C57BL/6×129 mouse,totally 30）and AD mice（APP/PS1/tau mouse totally 60）were randomly divided into six groups.1)C57BL/6×129 sedentary group（CS,n=15）;2)APP/PS1/tau sedentary group（AS,n=15）;3)C57BL/6×129 exercise group（CE,n=15）;4)APP/PS1/tau exercise group（AE,n=15）;5)APP/PS1/tau mice of drug（GSK3β inhibitor: ARA-014418）injection group（AA,n=15）;6)APP/PS1/tau mice of drug injection control group（AC,n=15）.animals were provided with standard chow and tap water ad libitum.CE group and AE group were subjected to 12 weeks of treadmill exercise training.The treadmill slope was 0°,and the speed was 10m/min for the first 10 minutes,and then increased to 12m/min for 50 minutes.The training was 60 min per day,and 5 days per week.The AA group was injected with 1ul ARA-014418（1.2μM）through the lateral ventricle,twice per day with 6h apart for three consecutive days.The AC group was injected with the same volume of solvent.All the mice were killed at the time when the 12 weeks exercise training was finished.Brain tissues of the frontal cortex and hippocampus were isolated and collected for protein analyzing using Western Blot and Dot Blot.Proteins of Aβ42,Aβ*8,Aβ*12,Aβ*56,Aβos,p-GS3β-Ser9/GSK3β,p-GS3β-Tyr216/GSK3β,CD68,GFAP,p-CRMP2-Thr514/CRMP2,PSD95,Syn were measured.The brain tissues were analyzed using immunofluorescence,immunohistochemistry,and Elisa to visualize Aβ plaque,activated state of MG and AG,level of inflammatory factors（IL-1β,IL-6,TNFα）.The brain tissues were also evaluated using Gogli staining,electron microscopy,and Whole-cell patch clamp to analyze axon length,number of dendritic,dendritic spine,and synaptic,synaptic structural parameters as well as mEPSCs and mIPSCs.Results: In AS mice,no Aβ plaques or Aβ*8 protein was detected;however,protein content of Aβ42,Aβ*12,Aβ*56,and Aβos,and fluorescence intensity of Aβ as well as phosphorylation level of GSK3β at Tyr216 were significantly higher（P<0.05）,while phosphorylation level of GSK3β at Ser9 was significantly lower compared to CS mice（P<0.05）.In AE mice,the training significantly reduced protein content of Aβ42,Aβ*12,Aβ*56,and Aβos,fluorescence intensity of Aβ,phosphorylation level of GSK3β at Tyr216 site（P<0.05）,but increased phosphorylation level of GSK3β at the Ser9 site（P<0.05）compared to CE mice.In AA mice,protein content of Aβ42,Aβ*12,Aβ*56,and Aβos,fluorescence intensity of Aβ,phosphorylation level of GSK3β at Tyr216 were significantly decreased,while phosphorylation level of GSK3β at Ser9 site was significantly increased compared with that of AC group（P<0.05）.In AS mice,immunoreactivity of CD68 and GFAP,protein levels of CD68 and GFAP,inflammatory factor levels of IL-1β,IL-6,and TNFα were significantly higher than in that of CS mice（P<0.05）.In AE mice,immunoreactivity of CD68 and GFAP,protein levels of CD68 and GFAP,inflammatory factor levels of IL-1β,IL-6,and TNFα were significantly reduced compared with that in CE mice（P<0.05）.In AA mice,levels of inflammatory factors IL-1β,IL-6,and TNFα were significantly decreased compared with that in AC mice（P<0.05）.Compared with CS mice,AS mice had significantly higher phosphorylation level of CRMP2 at the Thr514,and synaptic gap width of both excitatory-and inhibitory synapses（P<0.05）,but significantly lower expression level of Syn and dendritic,axon length,number of dendritic spine（the frontal cortex: Stubby spines,thin spines,mushroom spines;hippocampus: thin spines）,number of synaptic,number of excitatory synapse,and number of inhibitory synapse,synaptic interface curvature,length of active area,PSD thickness of excitatory synapses,synaptic interface curvature of inhibitory synapses（hippocampus）,and length of active area of inhibitory synapses（P<0.05）.Compared with CE mice,AE mice presented significantly lower phosphorylation level of CRMP2 at Thr514,synaptic gap width of excitatory-and inhibitory synapses（P<0.05）,but higher expression levels of PSD95 and Syn,axon length,level of dendritic,numbers of dendritic spine（the frontal cortex: Stubby spines,thin spines,mushroom spines;hippocampus: thin spines）and synaptic（excitatory synapse,inhibitory synapse）,synaptic interface curvature,length of active area,PSD thickness of excitatory synapses,synaptic interface curvature of inhibitory synapses（hippocampus）,and length of the active area of inhibitory synapses（P<0.05）.Compared with CS mice,CE mice presented significantly higher values in axon length,level of dendritic,numbers of dendritic spine（the frontal cortex: Stubby spines,thin spines,mushroom spines;hippocampus: thin spines）and synaptic（excitatory synapse,inhibitory synapse）,and length of active region of excitatory synapse（hippocampus）（P<0.05）.Compared with AC group,AA group had significantly lower phosphorylation level of CRMP2 at Thr514,synaptic gap width of both excitatory-and inhibitory synapses（P<0.05）,but higher expression levels of PSD95 and Syn,axon length,level of dendritic,numbers of dendritic spine（the frontal cortex: Stubby spines,thin spines,mushroom spines;hippocampus: thin spines）and synaptic（excitatory synapse,inhibitory synapse）,synaptic interface curvature,length of active area,PSD thickness of excitatory synapses,synaptic interface curvature of inhibitory synapses（hippocampus）,and length of active area of inhibitory synapses（P<0.05）.Compared with CS mice,AS mice had significantly lower frequency and amplitude of mEPSCs and mIPSCs in the frontal cortex pyramidal neurons,frequency of mEPSCs and amplitude of mIPSCs in the hippocampus（P<0.05）,but significantly higher amplitude of mEPSCs and frequency of mIPSCs in pyramidal neurons in hippocampus（P<0.05）.Compared with xxx,AE mice presented significantly higher frequencies and amplitudes of mEPSCs and mIPSCs in the frontal cortex pyramidal neurons,and in the hippocampal brain（P<0.05）,but lower amplitude of mEPSCs,and frequency of mIPSCs in pyramidal neurons in the hippocampus（P<0.05）.Compared with xxx,CE mice had significantly higher frequency and amplitude of mEPSCs and mIPSCs in the frontal cortex and hippocampal neurons（P<0.05）.Compared with the AC group,AA group had higher frequencies and amplitudes of mEPSCs and mIPSCs in the frontal cortex,and in the hippocampus（P<0.05）.Conclusion: 12-week regular aerobic exercise training induced significant down-regulation in activation of GSK3β,inhibition of Aβ42 production and of Aβ*12,Aβ*56,and Aβos accumulation,reduction in releasing of neuroinflammatory factors IL-1β,IL-6,and TNFα,down-regulation of microglia and astrocytes activation,improvement in neuronal synapse structure of（axon length,dendritic,dendritic spine,synapse）and status（PSD95,Syn,CRMP2,synaptic structural parameters）,and enhancement in synaptic transmission efficiency（mIPSCs,mEPSCs）.Taken together,the 12 weeks exercise training in the early stage of AD mice improved interaction between neurons and glial cells,through which to delay the development of AD.