| BackgroundEpidemiological studies have shown that PD is the second most common neurodegenerative disease after AD.The typical pathologies of PD include abnormal apoptosis of DA neurons in substantia nigra and DA deficiency in striatum.At the same time,abnormal apoptosis of DA neurons in the cytoplasm of neurons was observed Lewy body formed by α-synuclein aggregation.With the progress of the disease,PD patients will have the clinical symptoms of exercise and non-exercise.The pathogenesis of PD is complex and diverse,which is not clear at present.Above all,TRIM31 has been reported to be associated with tumor and inflammatory diseases.In our previous study,we found that TRIM31 deficient mice aggravated MPTP induced dopaminergic degeneration and showed more severe motor dysfunction than wild-type mice.However,the specific mechanism of TRIM31 involved in PD is not clear.Mitochondrial outer membrane channel protein VDAC1 has the key role in mitochondria mediated apoptosis.Under physiological environment,VDAC1 exists in the form of monomer and dimer.Under stimulation conditions,with the occurrence of apoptosis,VDAC1 dimer changes in conformation and assembles into higher oligomer.Therefore,this article will explore the role and mechanism of TRIM31 and VDAC1 in PD.Methods1.To explore the effect of TRIM31 deletion on mice in physiological state.1.1 In 2-12 months old wild-type and TRIM31-/-mice,observing the changes of learning,memory or exercise ability of by using Morris test or Rota-Rod test.1.2 The changes of PD related protein markers in 2,6,8-month-old wild-type mice and TRIM31-/-mice was detected by using immunoblotting.1.3 The changes of TH in SN and STR of 8-month-old wild-type mice and TRIM31-/-mice was detected by using immunohistochemistry.2.To explore the effect of TRIM31 deletion on mitochondrial function in physiological state.2.1 The changes of mtDNA in wild-type mice and TRIM31-/-mice aged 2,6 and 8 months was detected by using RT-PCR.2.2 The level of mitochondrial related proteins in SN and STR of WT mice and TRIM31-/-mice aged 2,6 and 8 months was detected by using immunoblotting.2.3 In 2 or 8 month-old WT and TRIM31-/-mice,using transmission electron microscope to observe the morphology of mitochondria.3.To detect the expression of TRIM31 in PD model.3.1 The PD model was established by transfecting α-synuclein(A53T)plasmid.Using RT-PCR to detect mRNA level of TRIM31.3.2 The changes of TRIM31 in control group and model group was detected by using immunoblotting.4.To detect the expression of PD related protein.4.1 In control or model group,to detect the changes of Tyrosine Hydroxylase and Phosphorylated α-synuclein.4.2 The changes of α-synuclein in SN of wild-type mice and TRIM31-/-mice was detected by using immunohistochemistry.5.To detect the apoptosis related proteins in PD model.5.1 After knocking down TRIM31,the changes of cytochrome C,Bcl-2/BAX,cleaved-cas9 and cleaved-cas3 in the control group and model group was detected by using immunoblotting.5.2 After knockdown of TRIM31,apoptosis was detected by TUNEL fluorescence.5.3 After transfection of GFP-TRIM31 plasmid,using immunoblotting to detect cytochrome C,Bcl-2/BAX,Cleaved-Cas 9 and Cleaved-Cas 3.5.4 In TRIM31-/-mice,the changes of cytochrome C,Bcl-2/BAX,cleaved-Cas 9 and cleaved-Cas 3 was detected by using immunoblotting.5.5 In TRIM31-/-mice,TUNEL histochemistry was used to detect neuronal apoptosis in substantia nigra.6.To explore the regulatory effect of TRIM31 on VDAC1.6.1 After transfection of GFP-TRIM31 plasmid,using immunoblotting to detect VDAC1 protein level.6.2 After transfection of GFP-TRIM31 plasmid or knocking down of TRIM31,the protein level of VDAC 1 was detected by using immunoblotting in PD model.6.3 After transfection of GFP-TRIM31 plasmid or knocking down of TRIM31,the changes of VDAC1 protein levels was detected by using MG132,chloroquine and cycloheximide,respectively.7.Explore the interaction between TRIM31 and VDAC 1.7.1 In unprocessed or MPP+induced SH-SY5Y cells,using LSM780 to observe the co-localization of TRIM31 and VDAC1.7.2 In HEK-293Tcells,the exogenous binding was detected by using CO-IP assay after transfection of GFP-TRIM3 1 and Flag-VDAC1 plasmids.7.3 In SH-SY5Y cells,after MPP+stimulation,using CO-IP assay to detect the endogenous binding.7.4 Detecting the binding site of TRIM31 and VDAC1.In HEK-293Tcells,the binding was detected by CO-IP assay after transfection of Myc-VDAC1,Myc-VDAC1(△25),Flag-TRIM31(△R),Flag-TRIM31(△N),Flag-TRIM31(△ C)and Flag-TRIM31(△ CC)plasmids.8.Explore the effect of TRIM31 on the ubiquitination of VDAC1.8.1 In HEK-293T cells,Flag-TRIM31(WT),Flag-TRIM31(C53a,C56a),Myc-VDAC1 and HA-ubiquitin plasmids were transfected.CO-IP assay detected the ubiquitin.8.2 In HEK-293T cells,the ubiquitination was detected by CO-IP assay after transfection of related plasmids.9.To explore the effect of TRIM31 on the oligomerization of VDAC1.After knocking down or overexpressing TRIM31,the oligomerization of VDAC1 was detected in HEK-293T cells using semi denatured agarose gel.Results1.Under physiological conditions,compared with wild-type mice,TRIM31-/-mice showed impairment in learning,memory and motor ability,and the situation became more serious with the increase of mice age.2.Compared with 2-month-old mice,mitochondria in substantia nigra of 8-month-old mice were swollen,vacuolated and cristae blurred.3.Under physiological environment,Tyrosine Hydroxylase decreased and α-syn increased in SN of TRIM31-/-mice compared with WT mice.The IHC of Tyrosine Hydroxylase showed the same result.4.Under physiological environment,no difference was detected of mtDNA deletion ratio in SN,and the level of PGC-1 α,OPA1,MFN1,Bcl-2/BAX,MFN2 in SN and STR decreased,with VDAC1 increased,while DRP1 and TOM20 did not change significantly.5.TRIM31 and phosphorylated α-syn were significantly increased by transfectingα-synuclein(A53T)plasmid compared with the control group.IHC showed thatα-synuclein protein in SN of TRIM31-/-mice increased significantly.6.In SH-SY5Y cells cultured in vitro,TRIM31 silence significantly increased the level of apoptosis index.At the same time,after overexpression of TRIM31,apoptosis related proteins showed the opposite trend.7.In HEK-293T cells,the level of VDAC1 decreased after gradient overexpression of TRIM31.In addition,VDAC1 was increased after silence TRIM31,while decreased after overexpression.In addition,MG132 could inhibit the degradation of VDAC1,but CQ had no obvious effect.Meanwhile,CHX could not inhibit the degradation of VDAC1.8.Co-localization of TRIM31 and VDAC1 in HEK-293T or SH-SY5Y cells was observed by confocal laser scanning.Moreover,TRIM31 and VDAC1 were bound in both exogenous and endogenous CO-IP assays.Furthermore,TRIM31 interacts with the β-barrel domain of VDAC1 through its coil-coiled domain.9.In the experiment of exogenous ubiquitination,TRIM31 can promote the ubiquitination of VDAC1 through its E3 ubiquitin ligase activity.In addition,TRIM31 can catalyze the Lys48(K48)-linked polyubiquitination on VDAC1 to degrade it.10.In the oligomerization experiment,knockdown of TRIM31 significantly increased the VDAC1 oligomerization,while overexpression decreased the oligomerization.ConclusionIn this study,we used TRIM31 knockout mice,MPTP induced PD model and α-syn(A53T)transgenic PD model to explore the effect and molecular mechanism of TRIM31 involved in the pathological process of PD. |
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