Effect Of Aerobic Exercise On Recognition Memory And TREM2 Function In Alzheimer’s Disease Rat

As one of the most important type in dementia,Alzheimer’s disease（AD）is an age-related neurodegenerative disease with remarkable progressive cognitive decline.AD not only reduces the patient’s living quality and increases the probability of complications（depression,and so on）,but also brings serious financial burden to the family and society.Therefore,how to deeply understand the mechanism,prevent or ameliorate the occurrence of AD has emerged as an important problem.In the early stage of AD,changes in the interaction between neuron and glial cells is a pathological cascade triggered by Aβ-induced the rare variants of triggering receptor expressed on myeloid cells 2（TREM2）.In addition,substantial animal models and clinical evidence have demonstrated that physical exercise significantly promoted cognition in neurodegenerative disease,such as AD and ischemia.But its mechanism needs to be further explore.Accumulating evidence revealed that physical exercise also modulates multiple systems that are known to regulate inflammation and glial activation,thus to improve cognitive function in AD.However,whether exercise improves cognitive function in AD is associated with TREM2 has not been reported.Therefore,our research aim to explore the role of TREM2 in physical exercise improve recognition memory and it’s mechanism.Experiment 1 The expression of TREM2,microglial activation and inflammation in the development of Aβ-induced AD.[Object]To explore the relationship of TREM2,microglial activation and inflammation in the development of Aβ-induced AD.[Methods]78 Sprague-Dawley（SD）rats were randomly divided into following groups:control group（C group,n=7）,Aβgroup（n=35）and PBS group（n=36）.Aβgroup were received intracerebroventricular（ICV）injection of Aβ_1-42protein;PBS group were injected with the same volume of PBS.Then,all rats received behavior test and molecular experiment.We use novel object recognition test to evaluate recognition memory in behavior test.Use the method of western blot and ELISA to measure the expression of TREM2,M1 microglia marker CD16,M2 microglia marker CD206 and inflammatory factor（IL-1,TNF-α,i NOS,IL-4,IL-10,Arg-1）as well as Aβprotein.[Results]1)Compared with C group,novel recognition index in novel object recognition test significantly impaired in Aβ14 day and Aβ21 day groups（P<0.05）.2)Compared with C group,hippocampal Aβprotein level is significantly increased in Aβ7 day,Aβ14 day and Aβ21 day groups（P<0.05）.3)ELISA revealed pro-inflammation（IL-1β,TNF-α,i NOS）significantly increased（P<0.05,P<0.01）while anti-inflammation（IL-4,IL-10,Arg-1）inverse after Aβintervention（P<0.05）.4)Western blot revealed that hippocampal CD16 significantly increased while CD206 inverse than that in C group after 14 day（P<0.05）.5)Western blot also demonstrated that the level of hippocampal TREM2 protein decreased over time and was significantly decreased than that in C group after 14 day（P<0.05）.[Conclusion]Recognition memory impairment in Aβ-induced AD model is associated with the decrease of hippocampal TREM2 induced microglial activation and inflammation.Experiment 2 Neuroprotective effect of physical exercise on recognition memory in AD model and TREM2-related mechanism[Object]To investigate the underlying neurochemical mechanism of physical exercise on recognition memory in AD rat model.[Methods]52 Sprague-Dawley（SD）rats were randomly divided into four groups:control group（C group,n=13）,Alzheimer’s disease group（AD group,n=13）,4 week of physical exercise and Alzheimer’s disease group（Exe+AD group,n=13）,4 week of physical exercise and blank group（Exercise group,n=13）.Exe+AD group and Exercise group were received 4 weeks of treadmill exercise intervention,then Exe+AD group and AD group were received ICV injection of Aβ_1-42protein.After 3weeks,all rats received behavior test and molecular experiment.1)We use novel object recognition test to evaluate recognition memory in behavior test.2)Golgi staining was used to analyse hippocampal neuronal dendritic complexity of neuron and spine density.Transmission electron microscope was applied to observe synaptic ultrastructure.Hippocampal synaptophysin（SYP）,syntaxin（SYX）,post synaptic density 95 k Da（PSD95）and growth associated protein of 43k Da（GAP43）were detected by western blot.Extracellular dopamine（DA）,glutamic acid（Glu）andγ-aminobutyric acid（GABA）levels were obtained by microdialysis and measured by liquid chromatography.3)Use the method of western blot and ELISA to measure the expression of inflammatory factor（IL-1,TNF-α,i NOS,IL-4,IL-10,Arg-1）and the expression of TREM2,M1 marker CD16,M2 marker CD206 as well as Aβprotein.[Results]Compared with C group,the change of AD group are shown in:1)Novel recognition index in novel object recogniton test significantly decreased（P<0.001）,as well as hippocampal Aβprotein significantly increased（P<0.001）.2)Neuronal dendritic complexity of neuron and spine density significantly decreased（P<0.05）.Significant impairment synaptic ultrastructure were observed in AD rat;Hippocampal synaptic-related protein（SYX,SYP,GAP43,PSD95）and neurotransmitter（（DA,Glu,GABA）was also significantly decreased（P<0.01）.3）Hippocampal pro-inflammation factor（IL-1β,TNF-α,i NOS）was significantly increased（P<0.05）and anti-inflammation factor（IL-4,IL-10,Arg-1）was significantly decreased（P<0.05）;hippocampal M1 marker CD16 significantly increased（P<0.001）while M2 marker CD206 inverse（P<0.05）;western blot also demonstrate that hippocampal HSP60,TREM2 protein is significantly decreased（P<0.001）,while NF-κB was significantly increased（P<0.01）,but we did not observe any significantly change of DAP12.Neuroprotective effects of aerobic exercise on AD are mainly as follows:1)Compared with AD group,a significantly increased novel preference index of Exe+AD group（P<0.001）in novel object test,but significantly decreased in hippocampal Aβprotein（P<0.001）.2)Compared with AD group,the neuron from Exe+AD group exhibited a more complex branching pattern（P<0.05）.And healthier synaptic ultrastructure was observed in Exe+AD group;hippocampal synaptic-related protein（SYX,SYP,GAP43,PSD95）and neurotransmitter（DA,Glu,GABA）was also significantly increased（P<0.01,P<0.001）in Exe+AD group.3)Compared with AD group,hippocampal pro-inflammation factor（IL-1β,TNF-α,i NOS）was significantly decreased（P<0.05）and anti-inflammation factor（IL-4,IL-10,Arg-1）was significantly increased（P<0.05）in Exe+AD group.Hippocampal CD16 significantly decreased（P<0.01）while CD206 inverse（P<0.05）in Exe+AD group;The protein level of HSP60,TREM2 was significantly increased（P<0.05）in Exe+AD group,while NF-κB was significantly decreased（P<0.05）,but no significantly change of DAP12.[Conclusion]Up-regulation of hippocampal TREM2 by physical exercise can ameliorate the damage of neuronal dendritic complexity,synaptic ultrastructure,decrease hippocampal synapses-related protein,typical neurotransmitter with recognitive memory impairment induced by Aβin AD model.The neuroprotection of physical exercise are associated with the inhibition of hippocampal microglial activation and inflammation by HSP60/TREM2-DAP12/NF-κB pathway.Experiment 3 Brain protective effect of exercise reduced after down-expression of TREM2 in AD model.[Object]To further verify the neuroprotective effect of physical exercise after AD when hippocampal TREM2 was down-regulated.[Methods]33 Sprague-Dawley（SD）rats were randomly divided into three groups:4 week of physical exercise and Alzheimer’s disease group（Exe+AD group,n=11）,AAV-Control and physical exercise and Alzheimer’s disease group（AAV-Control+Exe+AD group,n=11）,AAV-TREM2 and physical exercise and Alzheimer’s disease group（AAV-TREM2+Exe+AD group,n=11）.Stereotactic intracerebral injection in the bilateral hippocampus was performed to achieve microglial TREM2 downexpression by using adeno-associated virus（AAV）with CD68 promoter.After 4 weeks treadmill exercise and 3 weeks Aβinjection,all rats received behavior test and molecular experiment,which the same with experiment 2.[Results]1)Novel object test showed that the novel recognition index significantly decreased（P<0.01）,as well as hippocampal Aβprotein significantly increased（P<0.05）in AAV-TREM2+Exe+AD group compared with AAV-Control+Exe+AD group.2)Compared with AAV-Control+Exe+AD group,hippocampal neuronal dendritic complexity and spine density in AAV-TREM2+Exe+AD group significantly decreased（P<0.05）,and impaired synaptic ultrastructure were observed in AAV-TREM2+Exe+AD group;Hippocampal synaptic-related protein（SYX,SYP,GAP43,PSD95）and neurotransmitter（DA,Glu,GABA）was also significantly decreased（P<0.05）in AAV-TREM2+Exe+AD group.3)Compared with AAV-Control+Exe+AD group,AAV-TREM2+Exe+AD group’s hippocampal pro-inflammation factor（IL-1β,TNF-α,i NOS）was significantly increased（P<0.05）and anti-inflammation factor was significantly decreased（P<0.05）after downregulate TREM2.Western blot demonstrate that AAV-TREM2+Exe+AD group’s hippocampal M1 microglia marker CD16 significantly increased（P<0.05）while M2 microglia marker CD206 inverse（P<0.05）after downregulate TREM2.Western blot also demonstrated that AAV-TREM2+Exe+AD group’s hippocampal TREM2 is significantly decreased（P<0.01）after downregulate TREM2,while NF-κB was significantly increased（P<0.01）,but HSP60,DAP12 revealed no significant difference.[Conclusion]blockade of hippocampal TREM2 aggravated microglial activation and inflammation,increased the damage of neuronal dendritic complexity,synaptic ultrastructure,and the decrease of hippocampal dendsynapses-related protein,typical neurotransmitter.Blockade of hippocampal TREM2 reduced brain protective effect of exercise in AD rat model.Take together,physical exercise improve recognition memory by protect neuronal synaptic and typical neurotransmitter.This phenomenon is associated with physical exercise inhibit hippocampal microglial activation and inflammation by HSP60/TREM2-DAP12/NF-κB pathway.