The Role Of Microglial TREM2 In Diabetic Cognitive Impairment Mice And Related Mechanism

ObjectCognitive impairment is one of the common complications of central nervous system(CNS)damage caused by diabetes.However,the exact mechanism of its occurrence and development has not been fully elucidated.Epidemiological evidence has demonstrated that type 2 diabetes mellitus(T2DM)patients are prone to Alzheimer’s disease(AD);on the other hand,AD patients also have a higher risk of developing T2 DM.Furthermore,T2 DM and AD share several common age-related pathophysiological features,such as amyloid deposition,insulin resistance,oxidative stress damage,mitochondrial dysfunction,abnormal cholesterol metabolism,inflammatory cytokines infiltration.In recent years,the rare variants of triggering receptor expressed on myeloid cells 2(TREM2)have been identified as an important mechanism for the development of AD based on the data of Genome-Wide Association Studies(GWAS)from population-based research,and the rare variants of TREM2 have been confirmed to cause microglial dysfunction.However,it remains unclearwhether microglial TREM2 signalling plays an important role in diabetic cognitive impairment progress.In the present study,we aim to explore the role of TREM2 on inflammatory response and polarized phenotype of microglia in vivo and in vitro,and then investigate the role and mechanism of microglial TREM2 in diabetic cognitive impairment mice.MethodsIn vitro: The cell model of TREM2 overexpression and interference were established by transfection of BV-2 cells with plasmids,and then lipopolysaccharide(LPS)was used to create inflammatory stimulation microenvironment.Experimental BV-2 cells were divided into four groups:empty control(Con),LPS+vector,LPS+TREM2-OE,LPS+TREM2-shRNA.Quantitative real-time polymerase chain reaction(qPCR)was applied to measure the changes of mRNA expression of inflammatory cytokines and the markers of M1 and M2 microglial phenotype.The expression of related proteins of NF-κB pathway were detected by using western blot.The double-labelling immunofluorescence was used to determine the changes of protein expression of M1 and M2 microglial phenotype.In vivo: Male C57BL/6J mice were fed on high-fat diet(HFD)for50 weeks to establish the diabetic cognitive impairment rodent model and the control group mice were fed on normal chow diet(NCD).Stereotactic intracerebral injection in the bilateral hippocampus was performed 38 weeks after diet intervention to achieve microglial TREM2 overexpression by using adeno-associated virus(AAV)with CD68 promoter.Experimental rodents were divided into three groups: NCD-con,HFD-con and HFD-TREM2.Morris water maze(MWM),Y maze,and novel object recognition test(NOR)were performed 10 weeks after Stereotactic intracerebral injection to evaluate the function of learning,spatial memory and recognitive memory.Glucose metabolism of periphery was detected by using intraperitoneal glucose tolerance test(ipGTT).Golgi staining was used to analyse the dendritic complexity of neuron and spine density of apical dendrite in CA1 region of hippocampus.Transmission Electron Microscope(TEM)was applied to observe the Ultrastructural of synapse.The protein expression levels of synaptophysin,PSD95 and spinophilin of hippocampus were detected by western blot.Simultaneously,qPCR was performed to determine the mRNA expression of inflammatory cytokines and the markers of M1 and M2 microglia in the hippocampus.The activation of NF-κB pathway were measured by western blot.The polarization of microglia in the hippocampus was examined by using double-labelling immunofluorescence,and the morphology of microglial cells were analyzed by using Image J to estimate the activation of microglia.ResultsIn vitro cell experiments showed that the expression of TREM2 decreased and the mRNA expression of TNF-α,IL-1β,IL-6 and iNOS increased in BV-2 cells stimulated by LPS.In LPS+TREM2-OE group,the mRNA expression levels of TNF-α 、 IL-1β 、 IL-6 and iNOS were significantly down-regulated,Arg-1 and YM1/2,the markers of M2 microglia phenotype were up-regulated.However,the opposite results were observed in LPS+TREM2-shRNA group.Overexpression of TREM2 decreased the expression of M1 marker iNOS and increased the expression of M2 marker CD206 in Immunofluorescence experiment.Furthermore,under LPS stimulation,TREM2 overexpression significantly inhibited the expression of p-p65 and p-ikb-α in BV-2 cells,however increased the expression of p-p65 and p-ikb-α when TREM2 knock down.In the rodents experiments,HFD-TREM2 mice showed significant amelioration of learning,spatial memory and recognitive memory compared with that of HFD-con mice,a shorter latency in the acquisition phase,and a greater number of platform crosses and longer path length in the target quadrant in the MWM,an increased alternation rate in the Y maze,an increased preference index in the NOR test.Persistent HFD causes the damage of hippocampal neuron,however the pyramidal cells from HFD-TREM2 mice exhibited a more complex branching pattern than those from HFD-con mice.Similarly,there were more spines on the apical dendrites of pyramidal cells from HFD-TREM2 mice than on those from HFD-con mice.Healthier synaptic ultrastructure was observed inHFD-TREM2 mice.Many microglia were activated and induced detrimental inflammatory response in hippocampus when HFD intervention.However,the mRNA expression of IL-1β and TLR-4 were decreased in the hippocampus of HFD-TREM2,meanwhile,the M2 markers Arg-1 and YM1/2 were increased.Moreover,the activation of NF-κB pathway was inhibited when TREM2 overexpressed,as the expression of p-p65 and p-ikb-α were decreased.Overexpression of TREM2 reduced the number of amoebic morphology microglia and increased the number of ramified morphology microglia,decreased the number of iba-1/iNOS positive microglial cells and increased iba-1/Arg-1 microglial cells.ConclusionOverexpression of TREM2 in the hippocampus can rescue the damage of dendrites and synapses in hippocampal pyramidal neurons of mice with cognitive impairment induced by HFD,increase the expression of synaptic proteins,and ultimately improve the cognitive impairment.The neuroprotective effects of TREM2 are related to the promotion of microglial M2 polarization,the inhibition of microglial activation and the inhibition of NF-κB pathway and inflammatory response.