The Effects And Mechanisms Of Microglial TRIF Signaling Pathway In Parkinson’s Disease

Parkinson’s disease(PD) is a chronic degenerative movement disorder characterized by the selective degeneration of the substantia nigra in midbrain, and its etiology remains unknown. Autopsy, genetics investigation, retrospective studies and molecular imaging all point to a fact that inflammation plays a key role in the onset and development of PD. Microglia are recognized as the most important resident brain immune cells in the central nervous system(CNS) and are key players in immune surveillance, immune regulation and phagocytic recognition. The classical M1 phenotype of microglia may exacerbate the damages to dopaminergic neurons by releasing large amounts of inflammatory cytokines and reactive oxygen species. Whereas, the alternative M2 phenotype of microglia are intended to antagonize the pro-inflammatory functions of M1 microglia, thus inhibiting the inflammatory responses and alleviate the dopaminergic neuron damage. Toll-like receptors(TLRs) are important receptors that mainly expressed in the microglia of CNS, and activation of their associated downstream pathways may be intimately correlated with potential regulatory mechanism underlying polarization of microglia M1/M2 phenotypes. ObjectiveTo investigate the relationship between TLR3-TRIF signaling pathway and microglial activation as well as polarization of distinct microglia M1/M2 phenotypes. To explore the effect of microglia conditioned medium on dopaminergic neuronal injury and expression profiles of specific proteins. Method a. Interfering TRIF expression in microglia was achieved using transfection of theindicated si RNA into BV2 cell line. The downstream proteins in the TLR3-TRIFsignaling pathway and changes in cytokines expression were detected usingWestern blot and real-time q PCR. b. The co-culture system was utilized to observe whether TLR3-TRIF signalingpathway could impact the migration potential of microglia. c. The effect of si TRIF in microglia on polarization of microglia M1/M2 phenotypeswere explored using real-time q PCR. d. The impact of microglia conditioned medium on apoptosis of MN9 D cells wereassessed applying immunofluorescence and flow cytometry. e. Western blot was used to detect the influence of co-culture system of MN9 D andBV2 on expression levels of tyrosine hydroxylase and dopamine transporter inMN9D cells. Results a. Western blot showed that the expression of both TRIF and P-IRF3 in TLR3-TRIFsignaling pathway was unregulated 24 hours after si RNA-transfection in microgliaBV2 cells(P<0.05). Meanwhile, q PCR demonstrated that IFN-β secretion in BV2cells presented an upward trend in the control group. However, an stable secretionof IFN-β in BV2 cells was observed in the si TRIF group, which had a significantdifferences as compared with that of the control group(P <0.05). b. The transwell system proved that,as compared to the control group,the migrationpotential of BV2 cells in the si TRIF group was profoundly decreased as evidencedby the numbers of migrated BV2 cells after being activated by damaged MN9Dcells(P <0.01). c. q PCR experimental results displayed that, after being triggered by damaged MN9Dcells or LPS(inducer of M1microglia)/ IL-4(inducer of M2 microglia), the markersof M1 phenotype such as TNF-α, IL-6, CD86 and i NOs as well as markers of M2phenotype including IL-10, CD206, Arg1 and YM1 in the control BV2 cell groupincreased significantly over time, respectively, which showed significant differencecompared with that of the si TRIF cell group(P <0.01)d. Immunofluorescence staining and flow cytometry unveiled that the microgliaconditioned medium in the si TRIF group exerted more robust pro-apoptotic anddamage effect on MN9 D cells as compared with the control group(P <0.05).Western blot showed that, after being treated with microglia conditioned mediumfor 24 h, the expression levels of both tyrosine hydroxylase and dopaminetransporter in MN9 D cells in the control group were significantly different fromthat in the si TRIF group(P <0.01). Main conclusions a. TRIF interfered in BV2 cells may reduce the migration activity under stimulationof dopaminergic neurons damage. b. TRIF interfered in BV2 cells may affect the polarization of microglia M1/M2phenotypes following stimulation of both damaged dopaminergic neurons andLPS/ IL-4 inducers, thereby inhibiting the microglial activation. c. TRIF interfered in BV2 cells might exacerbate the damage to MN9 D cells in theco-culture system. Meanwhile, it may affect the expression of tyrosine hydroxylaseand dopamine transporter in MN9 D cells..