Aerobic Exercise Ameliorates Autophagy Impairment And Cognitive Dysfunction Via ADRB2/AMPK/mTOR Pathway In APP/PS1 Mice

ObjectiveTo investigate the effect of aerobic exercise（AE）on the improvement of autophagy impairment and cognitive impairment in APP/PS1 mice.To further investigate the role of Adrenoceptor Beta 2（ADRB2）in this and related mechanisms.Materials and MethodsIn this study,we used a mouse treadmill to train 8-month-old APP/PS1 double transgenic mice with AE for two months.The number of Aβplaques in the hippocampus and cortex was observed by using 4G8 and thioflavin staining,and the amount of soluble and insoluble Aβprotein in the hippocampus was also measured.The levels of soluble and insoluble Aβproteins were measured in the hippocampus,and the recruitment of microglia near the Aβplaques after AE training was observed by immunodouble-labeling assay.Further bioinformatics analysis revealed that AE modulates the adrenergic system and activates ADRB2.After pretreatment of mice with theβ-adrenoceptor inhibitor propranolol（Prop）intraperitoneally,the levels of autophagy,pathological damage and cognition were examined.Finally,C57 mice injected with Aβ_1-42 were pretreated with the ADRB2 agonist clenbuterol（Clen）and examined for autophagy levels,pathological damage,and cognition.Results1.AE ameliorates cognitive dysfunction and synaptic damage in APP/PS1 miceWater maze and fear condition test on mice showed that AE significantly improved cognitive impairment in APP/PS1 mice,as evidenced by shorter latency to find hidden platforms,increased number of platform crossings during the testing phase,increased swimming distance in the target quadrant,and increased freeze time and number of freeze times for electrical stimuli.Golgi staining and electron microscopy showed that AE improved synaptic morphology and increased the number of dendritic spines and synapses in APP/PS1 mice,while AE was detected to improve the reduction of postsynaptic membrane-associated proteins GluN1,GluN2A,and GluN2B in APP/PS1 mice.2.AE enhances autophagy levels in APP/PS1 mice,increases Vma21 expression and improves V-ATPase activity to increase Aβclearance4G8 immunofluorescence staining,sulfatoxin staining and immunoprotein blotting experiments showed that AE significantly decreased Aβdeposition,increased Aβclearance and did not affect Aβproduction in APP/PS1 mice.The results of immunodouble-labeling experiments showed that AE enhanced the recruitment of Iba1-labeled microglia around 4G8plaques,while the fluorescence results of CD68 and 4G8 co-labeling showed that AE promoted the endocytosis of Aβ.Immunofluorescence and western blot experiments revealed that AE significantly increased the distribution and expression of autophagy marker protein LC3 in the brains of APP/PS1 mice,and the expression levels of autophagosome assembly proteins ATG5and ATG7 were also significantly increased,while the autophagic substrate p62 was significantly depleted,while transmission electron microscopy results showed that AE increased APP/PS1 mice by increasing the number of autophagic vesicles in the hippocampus.The results of immunofluorescence double-labeling showed that AE increased the number of LC3 autophagy-positive cells near 4G8-labeled Aβplaques.The results of immunofluorescence double-labeling showed that AE increased the number of LC3 autophagy-positive cells near4G8-labeled Aβplaques.The Aβclearance of APP/PS1 mice was significantly inhibited by AE after simultaneous pretreatment with autophagy inhibitors.Finally,bioinformatics analysis suggested and molecular experiments were performed to demonstrate that AE increased Vma21expression improved V-ATPase activity in APP/PS1 mice.3.Aerobic exercise ameliorates autophagy impairment and cognitive dysfunction via ADRB2/AMPK/mTOR pathway in APP/PS1 miceBy analyzing RNA-seq data after aerobic exercise in AD patients in the population,the results suggested that the adrenergic system was activated in AD patients after aerobic exercise,while ADRB2 was correlated with AE regulating 187 differential genes in APP/PS1 mice.The results of real-time fluorescence quantitative PCR and western blot experiments showed that AE ameliorated the decrease of mRNA level and protein content of ADRB2 in APP/PS1 mice.In further studies,pretreatment of mice with Prop revealed that Prop reduced the ameliorative effects of AE on cognitive function and Aβclearance in APP/PS1 mice,while inhibition of AE improved autophagy impairment and activation of AMPK/mTOR signaling pathway in APP/PS1 mice.4.Activation of ADRB2 ameliorates Aβ-induced cognitive dysfunction,pathological impairment,and autophagy impairmentTo investigate the effects of pharmacological activation of ADRB2 on Aβ-induced cognitive impairment,pathological impairment,and autophagic impairment,C57 mice injected with Aβ_1-42 were pretreated with the ADRB2 agonist Clen,and we found that activation of ADRB2ameliorated Aβ-induced pathological and cognitive impairment as well as autophagic impairment.In addition,by using the mCherry-GFP-LC3 dual fluorescent autophagy plasmid,we found that activation of ADRB2 in N2a cells ameliorated Aβ-induced autophagic flow impairment.ConclusionsAE enhanced autophagy by activating the ADRB2/AMPK/mTOR pathway,increased Vma21expression and improved V-ATPase activity as well as increased microglia aggregation near Aβplaques to increase Aβclearance;meanwhile,intraperitoneal injection of ADRB2 agonist improved Aβ-induced cognitive impairment,pathological impairment and impaired autophagy.