The Role And Mechanism Of TRIM31 In Parkinson's Disease

BackgroundParkinson's disease(PD)is the second most common neurodegenerative disease and its incidence is increasing rapidly.The two main pathological features of PD are the progressive loss of dopaminergic neurons in the substantia nigra(SN)pars compacta and the appearance of aggregation of Lewy body with a-synuclein as the main component in injured neurons.The pathogenesis of PD is still unclear,and many studies have shown that PD is a comprehensive disease caused by multiple factors.Ubiquitin proteasome system disorders and mitochondrial dysfunction play important roles in the progression of PD.E3 ubiquitin ligase TRIM31(Tripartite motif-containing protein 31)is mainly localized to mitochondria in cells.It belongs to the TRIM family with ubiquitin ligase activity which can ubiquitinate specific target molecules and participate in cell proliferation,cell differentiation,development,antiviral,cancerous apoptotic and many other cell biological processes.Studies have shown that TRIM31 is associated with cancer and inflammatory diseases,but whether TRIM31 is involved in the pathogenesis of PD has not been reported yet.Recent studies have found that in antiviral innate immunity,TRIM31 can activate the K63 ubiquitination modification of MAVS by its E3 ubiquitin ligase activity promoting the production of IFN-?.Moreover,IFN-? has been shown to play an important role in the normal function and survival of dopaminergic neurons.IFN-? deficient mice spontaneously develop motor disorders and learning cognitive impairment.Therefore,this paper will explore the role of TRIM31 on PD and its preliminary mechanism.Methods1.Explore the effects of TRIM31 deficiency on mice.1.1 Changes in motor coordination and learning memory ability of TRIM31-/-mice of different age groups were detected by Morris water maze and Rota-rod behavioral test.1.2 Morphological changes of mitochondria in SN of TRIM31 knockout(TRIM31-/-)mice at 2,4,and 6 months old were visualized by electron microscopy.2.Determine the expression of TRIM31 after PD models and its localization.2.1 Mouse PD model was established by MPTP and 6-OHDA.The SN and striatum(STR)tissues were taken at different times after injecting.The expressions of Tyrosine hydroxylase(TH)were detected by Western blot and Immunohistochemical,and the TRIM31 protein levels were detected by Western blot.2.2 Immunofluorescence assay for the co localization of TRIM31 with microglia,astrocytes and neurons in the SN.2.3 Human neuroblastoma cell line SH-SY5Y cells were cultured in vitro and PD model were established in vitro using MPP+ and 6-OHDA.Cells were harvested 24 h after treatment to detect changes in TRIM31 protein levels.2.4 Localization changes in cells after MPP+ and 6-OHDA were detected by Immunofluorescence.3.Determine the role of TRIM31 in mouse PD models.3.1 PD models were established in wild type and TRIM31-/-mice using MPTP and 6-OHDA.The ethology of two genotype mice and the survival rate of the model were measured.3.2 The changes of TH protein levels and TH positive cells in the two genotype mice were measured by Western blot and Immunohistochemical.4.The role of TRIM31 in the regulation of 6-OHDA and MPTP induced PD models and its mechanisms were investigated in vivo and in vitro.4.1 Real-time PCR was used to detect the mRNA levels of IFN-? and IL-1?.4.2 Real-time PCR was used to detect mtDNA deletion rate.4.3 Western blot analysis of mitochondrial homeostasis-related proteins peroxisome proliferator-activated receptor y coactivator-1 alpha(PGC1?),mitofusin 2(MFN2)and optic atrophy type 1(OPA1).Results1.Ethology results showed that the coordination ability of 4 months old TRIM31-/-mice was significantly damaged compared with wild type mice.Significant difference of memory ability began to appear at 6 months old TRIM31-/-mice.Mitochondrial morphology damages were observed by electron microscopy in SN of 4 and 6 months old TRIM31-/-mice,such as mitochondrial swelling,vacuolization and mitochondrial cristae rupture and disappearance.2.TRIM31 is highly expressed in the brain of WT mice,especially in substantia nigra(SN)and striatum(STR).TRIM31 protein expressions were significantly elevated in 6-OHDA and MPTP induced mouse PD models.3.Immunofluorescence results showed that the up-regulation of TRIM31 proteins mainly occurred in the SN neurons.It also occurred in microglia after 6-OHDA treatment.But the expression of TRIM31 proteins did not change in astrocytes.4.In vitro,up-regulation of TRIM31 was also observed after MPP+ and 6-OHDA stimulation in SH-SY5Y.Laser con-focal showed that TRIM31 was mainly localized to mitochondria.MPP+ and 6-OHDA stimulation could promote TRIM31 to translocate to mitochondrial.5.TRIM31 deficiency significantly aggravated the motor dysfunction and damage of DA neurons in MPTP and 6-OHDA induced PD models.The TH protein levels and the number of TH positive neurons decreased more significantly in TRIM31-/-mice.TRIM31-/-mice had a 27%reduction in survival rate after 6-OHDA treatment and a 25%reduction after MPTP treatment compared to wild-type mice.6.In the mouse PD models induced by MPTP and 6-OHDA,the mRNA levels of IFN-? were significantly increased in SN of wild type mice,and the increase of IFN-?in TRIM31-/-mice was significantly inhibited.In SH-SY5Y cultured in vitro,although MPP+ stimulation did not cause an increase in IFN-?,knockdown of TRIM31 significantly reduced the mRNA level of IFN-p.TRIM31 deficiency did not change the up-regulation of the mRNA level of IL-1? after 6-OHDA treatment in vivo and in vitro.7.In the MPTP and 6-OHDA induced mouse PD models,TRIM31 deficiency aggravated the down-regulation in PGC1? and OPA1 protein levels induced by MPTP and 6-OHDA,but the MFN2 protein levels did not change in the two genotype mice.In TRIM31-/-mice,the mitochondrial DNA deletion rate increased under normal conditions but the deletion rate did not change after MPTP treatment.ConclusionIn the 6-OHDA and MPTP induced PD models,TRIM31 protects DA neurons by regulating mitochondrial homeostasis and IFN-? levels.