The Role Of Fbxw7 In Regulating Mitochondrial Dynamics In Parkinson’s Disease And The Exploration Of Its Mechanism

Objective:Loss of dopaminergic neurons due to mitochondrial dysfunction is a widely accepted mechanism in the pathogenesis of Parkinson’s disease(PD).Mitochondrial dynamics refers to the process by which mitochondria maintain mitochondrial homeostasis,provide energy to cells,and regulate cellular autophagy and apoptosis through continuous division and fusion.Abnormal mitochondrial dynamics,which can lead to mitochondrial dysfunction,is closely related to the survival of dopaminergic neurons and may be one of the mechanisms by which mitochondria mediate the pathogenesis of PD.Fbxw7 is an evolutionarily conserved F-box protein that is now widely recognized as a classical tumor suppressor.There is growing evidence that Fbxw7 can be involved in the pathological process of neurodegeneration through the regulation of cell cycle,cell growth and survival,but the specific role of Fbxw7 in PD and its involvement in the pathogenesis of PD remain unclear.It has also been shown that Fbxw7 can bind to STAT3 to affect STAT3 expression and ubiquitination degradation,and regulate the expression of proteins related to mitochondrial dynamics through STAT3.Based on the above background,this study will investigate whether Fbxw7 can regulate mitochondrial division/fusion through mediating the activation of STAT3and thus affect the pathological process of PD,using animal and cellular models of PD,in order to elucidate the possible role of Fbxw7 in the pathogenesis of PD.Methods:1.MPTP was constructed as a mouse PD model.20 male C57BL/6J mice were randomized into two groups(10 pieces per group):normal control group(CON group)and disease group(MPTP group).Baton twirling test and pole climbing test were used to assess the motor ability of mice.After the successful modeling was confirmed by behavioral experiments,the midbrain was taken from each group of mice.Western Blotting technique was used to observe the variation in protein expression levels of Fbxw7,p-STAT3,mitochondrial division-related protein(Drp1),and mitochondrial fusion-related protein(Mfn2,OPA1)in the two groups.2.Different concentrations(100μmol/L,200μmol/L,400μmol/L,800μmol/L,1200μmol/L,1600μmol/L,1800μmol/L)of MPP~+were selected to treat MN9D cells,and CCK8 was used to determine cell viability and select the most suitable concentration to construct PD cell model.3.MN9D cells stably expressing Fbxw7 were established.The cells were treated according to the MPP~+drug concentration mapped in experiment 2,and the test was divided into four groups:control group(CON group),disease group(MPP~+group),disease+transfected empty vector group(MPP~++NC group),and disease+transfected overexpressing Fbxw7 plasmid group(MPP~++OE group),and the transfection efficiency was verified by q PCR technique and Western Blotting technique.4.The DCFH-DA probe was used to detect the level of ROS in four groups of MN9D cells,and the JC-1 fluorescent probe was used to detect the level of mitochondrial membrane potential in four groups of MN9D cells.Western Blotting technique was used to observe the changes of p-STAT3 protein and DRP1,OPA1 and Mfn2 protein expression levels in the four groups.Results:1.Compared with the CON group,the time of pole-climbing experiment was significantly longer(P<0.05)and the time of stick-turning experiment was significantly shorter(P<0.05)in the MPTP group,and the mice in the MPTP group had motor dysfunction,indicating that the acute mouse PD injury model was successfully constructed.2.Compared with the CON group,Fbxw7 protein expression was significantly lower(P<0.05),p-STAT3 protein expression was noteworthily higher(P<0.05),DRP1 protein expression was significantly increased(P<0.05),and OPA1 and Mfn2 protein expression was significantly reduced(P<0.05)in the midbrain of mice in the MPTP group.Changes in Fbxw7 protein,p-STAT3 protein and mitochondrial dynamics-related proteins may be associated with PD.3.The damaging power of MPP~+on MN9D cells increased with its drug concentration.In this experiment,1600μmol/L MPP~+was selected to treat MN9D cells in the subsequent experiment,and the cell survival rate was 72%at this concentration(P<0.05).4.Compared with the CON group,the expression of Fbxw7 m RNA and protein was significantly lower in MN9D cells in the MPP~+group(P<0.05),which is consistent with the animal results;compared with the MPP~++NC group,the expression of Fbxw7 m RNA and protein was significantly higher in the MPP~++OE group(P<0.05),indicating that overexpression of Fbxw7 plasmid transfection was successful.5.Compared with the CON group,DCF fluorescence intensity was significantly enhanced in MN9D cells in the MPP~+group(P<0.05);compared with the MPP~++NC group,DCF fluorescence intensity was significantly weakened and ROS levels were significantly reduced in MN9D cells in the MPP~++OE group(P<0.05),indicating that overexpression of Fbxw7 reduced the increase in ROS caused by MPP~+.6.Compared with the CON group,red fluorescence was diminished,green fluorescence was enhanced,and mitochondrial membrane potential was significantly decreased in MN9D cells of the MPP~+group(P<0.05);compared with the MPP~++NC group,red fluorescence was enhanced,green fluorescence was diminished,and mitochondrial membrane potential was significantly increased in MN9D cells of the MPP~++OE group(P<0.05),indicating that overexpression of Fbxw7 elevated the decrease in mitochondrial membrane potential caused by MPP~+.7.Compared with the CON group,the expression of p-STAT3protein in MN9D cells in the MPP~+group was significantly increased(P<0.05);compared with the MPP~++NC group,the expression of p-STAT3protein in MN9D cells in the MPP~++OE group was significantly decreased(P<0.05),indicating that overexpression of Fbxw7 reduced the elevation of p-STAT3 protein induced by MPP~+.8.Compared with the CON group,DRP1 protein expression was significantly higher(P<0.05)and OPA1 protein and Mfn2 protein expression was significantly lower(P<0.05)in the MN9D cells of the MPP~+group;compared with the MPP~++NC group,DRP1 protein expression was significantly lower(P<0.05)and OPA1 protein and Mfn2protein expression was significantly higher(P<0.05),indicating that overexpression of Fbxw7 restored the abnormal expression of mitochondrial dynamics related proteins caused by MPP~+.Conclusion:1.Fbxw7 plays a protective role in PD;2.Fbxw7 regulates mitochondrial dynamics in PD;3.Fbxw7 may play a protective role in PD by regulating STAT3activation and modulating mitochondrial dynamics.