The Beneficial Effects Of Voluntary Running Exercise On Microglia Metabolism In The Hippocampus Of APP/PS1 Transgenic AD Mice

PART 1: THE EFFECTS OF VOLUNTARY RUNNING EXERCISE ON COGNITIVE FUNCTION OF APP/PS1 TRANSGENIC AD MICEBackground: Alzheimer’s disease(AD)as the most common cause of dementia,has become one of the major public health problems in the world.Running exercise as an important behavioral intervention has been shown to be helpful in improving the cognitive function of AD.In mouse studies,voluntary wheel-running exercise better mimicked human exercise and subjected less stress than treadmill exercise.However,the benefits of long-term voluntary wheel-running exercise on cognitive function in APP/PS1 transgenic mice have not been studied.Objective: To study the effects of long-term(3 months)voluntary wheel-running exercise on cognitive function and AD pathology in APP/PS1 transgenic mice,and to further clarify the therapeutic effect of long-term voluntary wheel-running exercise on cognitive impairment of AD.Methods: Eighty 10-month-old male APP/PS1 mice(AD)and their litter wild-type mice(WT)were randomly divided into sedentary groups and running groups(AD＿Sed,WT＿Sed,AD＿Run and WT＿Run,n =20/group).The mice in the running group were free access to a running wheel for 3 months.The behavioral tests including new object recognition(NOR),Morris water maze(MWM)and Y-maze were performed to assess cognitive function at the age of 13 months.The volume of the hippocampus was measured using the Cavalieri’s principle of stereological methods.The deposition of Aβ plaques in the hippocampus was observed using Thioflavin S staining(Thio S),and the synaptic plasticity of the hippocampus was observed using the immunofluorescence staining of postsynaptic dense protein(PSD95).Results: The body weight of mice among four groups had no significant difference.There was no significant difference in the average daily running distance between WT＿Run mice and AD＿Run mice.In the NOR test,the discrimination index of AD＿Sed mice was significantly lower than WT＿Sed mice(p < 0.01),and the discriminant index of AD＿Run mice was significantly higher than AD＿Sed mice(p < 0.01).In the MWM test,the average distance and escape latency of AD＿Sed mice were significantly longer than WT＿Sed mice(p < 0.01,p < 0.001),and the average distance and escape latency of AD＿Run mice were significantly shorter than AD＿Sed mice(p < 0.05,p < 0.01).The number across the platform in AD＿Sed mice was significantly decreased compared with WT＿Sed mice(p < 0.01),and the number across the platform in AD＿Run mice was significantly increased compared with AD＿Sed mice(p < 0.05).There was no significant difference in the accuracy of Y maze among the four groups.The volume of DG and CA1 regions in AD＿Run mice was significantly increased compared with AD＿Sed mice(p < 0.05,p < 0.01),and the ratio of positive area of Aβ plaque in the hippocampus of AD＿Run mice was significantly decreased compared with AD＿Sed mice(p(≤ 20 μm)< 0.05,p(>20 μm)< 0.001).The fluorescence intensity of PSD95 in the hippocampus of AD＿Sed mice was significantly lower than WT＿Sed mice(p(DG)< 0.01,p(CA1)< 0.05,p(CA3)< 0.01).The fluorescence intensity of PSD95 in the hippocampus of AD＿Run mice was significantly higher than AD＿Sed mice(p(DG)< 0.01,p(CA3)< 0.01).Conclusions: Long-term voluntary running exercise significantly improved spatial and learning memory in APP/PS1 mice,increased the volume of DG and CA1 regions in APP/PS1 mice,increased the density of PSD95 in DG and CA3 regions of APP/PS1 mice and reduced the deposition of Aβ plaques in the hippocampus of APP/PS1 mice.PART 2: THE EFFECTS OF VOLUNTARY RUNNING EXERCISE ON GLUCOSE METABOLISM IN THE HIPPOCAMPUS OF APP/PS1 TRANSGENIC AD MICEBackground: Many studies have demonstrated that running exercise can effectively reduce the pathology of AD and improve the cognitive function of AD.For example,running exercise reduces Aβ deposition and Tau phosphorylation,promotes synaptic plasticity in AD model mice and patients with AD.However,the underlying molecular mechanism are still poorly studied.Objective: To explore the underlying mechanism of running exercise alleviating cognitive decline in AD mice from the transcriptome level and imageology in order to find the intermediate target of running exercise for the treatment of cognitive impairment in AD.Methods: Eighty 10-month-old male APP/PS1 mice(AD)and their litter wild-type mice(WT)were randomly divided into sedentary groups and running groups(AD＿Sed,WT＿Sed,AD＿Run and WT＿Run,n =20/group).The mice in the running group were free access to a running wheel for 3 months.Then,the hippocampus tissues of WT＿Sed,AD＿Sed and AD＿Run mice was isolated for RNA sequencing,and the glucose metabolism of the mouse brain was further observed using [18]F-FDG-PET.Results: Compared with WT＿Sed mice,115 genes were significantly up-regulated and 10 genes were significantly down-regulated in AD＿Sed mice.Some of the upregulated genes were disease-associated microglia(DAM)genes,such as Triggering receptor expressed in myeloid cells 2(Trem2),TYRO protein tyrosine kinase binding protein(Tyrobp),Integrin subunit alpha X(Itgax),Secreted phosphoprotein 1(Spp1),Cd68 and Cd74.q RT-PCR showed that the relative m RNA level of Trem2,Tyrobp,Itgax,Cd68 and Cd74 were significantly up-regulated in the hippocampus of AD＿Sed mice compared with WT＿Sed mice(p < 0.01,p < 0.05,p < 0.01,p< 0.05,p < 0.001).However,there were few significant differentially expressed genes(DEGs)between AD＿Run mice and AD＿Sed mice.q RTPCR confirmed that DAM-related genes did not differ significantly between AD＿Run and AD＿Sed mice,including Trem2,Tyrobp,Itgax,Spp1 and Cd68.Gene set enrichment analysis(GSEA)found that “TCA cycle”,“glycolysis/ gluconeogenesis”,“Pentose phosphate pathway”,“glutamatergic synapse”,and “GABAergic synapse” were significantly reduced in AD＿Sed mice compared with WT＿Sed mice.However,“pyruvate metabolism”,“lycolysis/gluconeogenesis”,“Fructose and mannose metabolism”,and “Pentose phosphate pathway” were significantly upregulated in AD＿Run mice compared with AD＿Sed mice.[18]F-FDGPET results confirmed that long-term running exercise could significantly improve glucose uptake in multiple brain regions of AD mice,including cortex(p < 0.001),hippocampus(p < 0.01),thalamus(p < 0.01),striatum(p< 0.01),hypothalamus(p < 0.001),amygdala(p < 0.001).Conclusions: There was minor DEGs in the hippocampus of AD mice between AD＿Run mice and AD＿Sed mice,suggesting that the regulation of long-term running exercise on hippocampus of AD mice may occur at the post-transcriptional level.The results of GSEA suggested that long-term running exercise could up-regulated glucose metabolism pathways in the hippocampus of AD mice.[18]F-FDG-PET further confirmed that long-term running exercise significantly increased glucose metabolism in multiple brain regions of AD mice.PART 3: THE EFFECTS OF VOLUNTARY RUNNING EXERCISE ON METABOLIC ACTIVITY AND MORPHOLOGICAL PLASTICITY OF MICROGLIA IN THE HIPPOCAMPUS OF APP/PS1 TRANSGENIC AD MICEBackground: Microglia as innate immune cells in the brain play an important role in the pathological process of AD.Improvements of cognitive function in AD by running exercise has been well known.However,microglia-related studies on cognition improvements in AD by running exercise are still rare.TREM2 is a member of the immunoglobulin superfamily mainly expressed on microglia and is known to play an important role in regulating microglia metabolic activity and function,as well as glucose metabolism in the brain.However,the effects of running exercise on microglia and brain glucose metabolism and the relationship between them remain unclear.Objective: To investigate the effects of running exercise on metabolism,function and morphology of microglia in hippocampus of AD mice and assess the relationship between microglia metabolism and brain glucose metabolism modulated by TREM2.Methods: Eighty 10-month-old male APP/PS1 mice(AD)and their litter wild-type mice(WT)were randomly divided into sedentary groups and running groups(AD＿Sed,WT＿Sed,AD＿Run and WT＿Run,n =20/group).The mice in the running group were free access to a running wheel for 3 months.The mice were killed at 13 months of age.Using immunofluorescence,western blotting and ELISA for detecting the expressions of microglia-associated glucose transporter(GLUT5),TREM2,soluble TREM2(s TREM2),TYROBP,SPP1 and p-SYK etc.The number,morphology,proliferation and activity of microglia were observed with immunohistochemistrical technique,stereological method and immunofluorescence.Results: The protein expression of GLUT5 protein in the hippocampus of AD＿Run mice was significantly higher than AD＿Sed mice(p < 0.05),and the proportion of GLUT5+/IBA1+ microglia in the DG was significantly increased in AD＿Run mice compared with AD＿Sed mice(p <0.05).Meanwhile,the level of TREM2 protein in the hippocampus of AD＿Run mice was significantly higher than AD＿Sed mice(p < 0.01),and the level of s TREM2 in plasma was significantly lower than AD＿Sed mice(p < 0.05).The decrease of s TREM2 level was negatively correlated with glucose uptake in the hippocampus of mice(p < 0.01).SPP1,a TREM2-dependent protein,was significantly upregulated in the hippocampus of AD＿Run mice compared with AD＿Sed mice(p < 0.001),but there was no significant difference in TREM2 adaptor protein TYROBP.More Neu N fragments were found in TREM2+/IBA1+ microglia in the DG of AD＿Run mice,and p-SYK(Tyr525/526)was significantly increased in AD＿Run mice compared with AD＿Sed mice(p < 0.01).In addition,the total number of microglia in the DG and CA1 regions was significantly increased in AD＿Sed mice compared with WT＿Sed mice(p(DG)< 0.01,p(CA1)< 0.05),and the total number of microglia in the DG of AD＿Run mice was significantly increased compared with AD＿Sed mice(p < 0.001).The total number of 5-bromodeoxyuridine(Brd U)positive cells in the DG was significantly increased in AD＿Run mice compared with AD＿Sed mice(p < 0.05),but there was no significant difference in the number of Brd U+/IBA1+ microglia between AD＿Run mice and AD＿Sed mice.The endpoints and length of microglia branch in AD＿Sed mice were significantly decreased compared with WT＿Sed mice(p < 0.001,p < 0.001),but the endpoints and length of microglia branch in AD＿Run mice were significantly increased compared with AD＿Sed mice(p < 0.01,p < 0.001).In addition,compared with AD＿Sed mice,the fluorescence intensity of IBA1+ microglia and the ratio of CD68/IBA1 positive area around hippocampal Aβ plaques in AD＿Run mice was significantly decreased(p < 0.05,p < 0.05).Conclusions: Long-term running exercise promoted microglia glucose metabolism in the hippocampus of AD mice,accompanied by the increase of TREM2 in the hippocampus and the decrease of s TREM2 levels in plasma.Long-term running exercise upregulated TREM2/SYK pathway in the hippocampus to promote phagocytosis in AD mice.These results suggested that running exercise promotes hippocampal microglia glucose metabolism and TREM2-dependent microglia function in AD mice,negatively associated with the shed of TREM2.In addition,long-term running exercise significantly increased the number of microglia,the endpoints and length of microglia branch in the DG of AD mice,and decreased the immunoactivity of IBA1 and CD68 around the Aβ plaques.These results suggested that running exercise restored the morphological plasticity of microglia and inhibited microglial overactivation.Although running exercise increased the number of Brd U+ cells in the DG of AD mice,it did not increase the number of Brd U+/IBA1+ microglia,suggesting that running exercise promotes neurogenesis other than microglia,and the increase of microglia number is not caused by promoting microglial proliferation but due to the promotion of microglial survival.