| BackgroundParkinson's disease(PD)is the most prevalent human neurodegenerative movement disorder after Alzheimer's disease(AD).PD is characterized by the selective and progressive loss of dopaminergic neurons in the substantia nigra pars compacta(SNpc)and the presence of alpha-Synuclein(?-SYN)-rich inclusions,named Lewy bodies(LBs)in the surviving neurons.The pathogenesis of PD is complicated.Many factors such as aging,genetics,infection,immune abnormality,neurotoxin and oxidative stress were invovled in the pathogenesis of PD.In recent years,it has been found that there are active microglial cells and the chronic inflammatory response in the nigrostriatal region of patients with PD.A large number of animal and cellular experiments also revealed that neuroinflammation was involved in the development of PD.These studies demonstrate that the excessive activation of microglia and the neuroinflammation are the important risk factors of PD.Microglial cells are the inherent immune cells in the central nervous system(CNS).In different pathogenetic stages of PD,microglial activation states may change throughout a pathologic process.Microglia has two alternative activation phenotypes,termed the M1(proinflammatory)phenotype and the alternative M2(anti-inflammatory)phenotype.The proinflammatory M1 phenotype favors the production and release of cytokines that exacerbate neural injury.In contrast,the M2 phenotype promotes the release of neurotrophic factors that promote neurorepair.Thus,by regulating the phenotye of microglia and promoting the transformation of microglia from M1 to M2 phenotype may provide a new therapeutic strategy for PD.However,the mechanism of microglia phenotype modulation remains unclear.Triggering receptor expressed on myeloid cells 2(TREM2)is an immunoglobulin-like receptor expressed on myeloid cells,which is highly expressed on microglia in the CNS.TREM2 is known to have anti-inflammatory properties.Several lines of evidence suggest an essential role of TREM2 in modulation of microglia functions including phagocytosis and production of pro-inflammatory cytokines.More recently,studies have demonstrated that rare TREM2 genetic variants significantly increase the risk for AD and PD.However,the expression and biological functions of TREM2 in brain under PD context remain largely unknown.Furthermore,whether TREM2 plays an essential role in modulation of microglial function during PD progression and whether TREM2 represents a potential therapeutic target for PD remain unexplored.We demonstrated that inhibition of TREM2 expression in vivo and in vitro aggravated ?-SYN-induced neurotoxicity and neuroinflammation.These findings suggest that TREM2 plays an important role in the pathogenesis of PD and may represent a potential target for the treatment of PD.ObjectiveTo investigate the role of TREM2 in modulating neuroinflammation in ?-SYN?mediated PD models.Materials and MethodsIn vitro studies1.BV2 microglial cells were exposed to LPS,MPP+,6-OHDA and ?-SYN for 24 h respectively,cells were collected and TREM2 mRNA and protein levels were measured by realtime RT-PCR and Western blot analysis.2.BV2 microglial cells were treated with ?-SYN,and cells were harvested at different time points.Protein and mRNA levels of NOX2,COX2 and iNOS were detected by Western blot and realtime RT-PCR.3.The small interfering RNA of TREM2(TREM2-siRNA)was used to knockdown the expression of TREM2 in BV2 microglial cells.Then cells were exposed to ?-SYN for 4 h.Protein and mRNA levels of iNOS,NOX2 and COX2 were detected by Western blot and realtime RT-PCR.4.TREM2-siRNA was used to knockdown the expression of TREM2 in BV2 microglial cells.Cells were exposed to IL-4 for 24 h.The protein levels of p-STAT6,p-STAT1,STAT1,STAT6 and ARG1 were detected by Western blot and the mRNA level of ARG1 was detected by realtime RT-PCR.5.TREM2-siRNA was used to knockdown the expression of TREM2 in BV2 microglial cells.Cells were exposed to ?-SYN for 8 h,then DCF-CA probe was added for 1 h,the ROS level were detected by flow cytometry analysis.6.TREM2-siRNA was used to knockdown the expression of TREM2 in BV2 microglial cells.Cells were exposed to ?-SYN for 4 h.The conditioned medium(CM)were collected and added to SH-SY5Y cells.The proliferation and apoptosis of SH-SY5Y cells were measured by CCK-8 kit and flow cytometry analysis.In vivo studies1.The animal model of PD was established by stereological injection of AAV-SYN into the mice SN.Protein levels of TH and TREM2 in the SN at different time points were detected by Western blot analysis.The mRNA level of TREM2 was detected by realtime RT-PCR.2.The animal model of PD was established by stereotaxic injection of 6-OHDA into the striatum of wild type C57BL/6(WT)mice.Brain tissues were collected at 1 d,1 w,2 w,3 w and 4 w after 6-OHDA injection.The protein levels of TH and TREM2 were detected by Western blot.The mRNA level of TREM2 was detected by realtime RT-PCR.3.The animal model of PD was established by stereological injection of AAV-SYN into the mice SN.Frozen sections were taken from brain tissue at 3 w,8 w and 10 w after injection.TH,GFAP and IBA1 staining were performed.The number of TH-positive cells and the number and morphology of microglia and astrocytes in TREM2 knockout(TREM2-/-)mice and WT mice were observed.4.The animal model of PD was established by stereological injection of AAV-SYN into the mice SN.Brain sections were collected at 3 w after injection for frozen sections and Western blot analysis respectively.Immunofluorescence staining was used to detect the expression of iNOS and ARG1.The Protein levels of iNOS,NOX2,COX2,p-STAT1,p-STAT6,STAT1 and STAT6 were detected by Western blot analysis.Results1.The protein and mRNA levels of TREM2 were decreased after LPS,MPP+,6-OHDA and a-SYN stimulation in BV2 microglial cells,whereas the protein and mRNA levels were increased initially and decreased subsequently in both 6-OHDA and AAV-SYN PD mice models.2.The protein and mRNA levels of iNOS,COX2 and NOX2 were increased at 4 h after ?-SYN stimulation in BV2 microglial cells in vitro.3.TREM2 knockdown aggravated a-SYN-induced inflammatory response compared with scramble siRNA group.The mRNA and protein levels of M1 markers such as iNOS,NOX2 and COX2 were increased after treatment with TREM2-siRNA.4.IL-4 can induce the M2 phenotype of microglia and increase the expression of ARG1 in microglia.The mRNA and protein levels of ARG1 were significantly decreased after TREM2 knockdown by TREM2-siRNA in BV2 microglial cells.5.Compared with WT mice,TREM2-/-mice showed more activated microglia and astrocytes at 3 w,8 w and 10 w after AAV-SYN injection.6.The protein levels of M1 markers such as iNOS,NOX2 and COX2 was significantly increased and the expression of M2 marker ARG1 was decreased in TREM2-/-mice compared with WT mice 3 w after AAV-SYN injection.7.TREM2 knockdown increased the level of ROS caused by ?-SYN in BV2 microglial cells.8.The CM collected from the TREM2-siRNA-treated BV2 microglial cells can increase the apoptosis and decrease the proliferation of SH-SY5Y cells compared with the CM collected from scramble siRNA-treated BV2 cells.9.Compared with WT mice,TREM2-/-mice had less TH-positive neurons in SN after AAV-SYN injection.10.IL-4 can increase the expression of p-STAT6 and decrease the expression of p-STAT1 in microglia.However,after TREM2 knockdown by TREM2-siRNA3 the protein level of p-STAT6 were decreased and the protein level of p-STAT1 was increased compared with scramble siRNA group.11.Compared with WT mice,the protein level of p-STAT1 was increased while p-STAT6 protein level was decreased in TREM2-/-mice after AAV-SYN injection.ConclusionTREM2 may regulate a-SYN-induced neruoinflammation and neurodegenration by modulating microglia phenotype in the immune pathogenesis of PD. |
PDF Full Text Request