MitoQ Protects Dopaminergic Neurons In A 6-OHDA Induced PD Model

Background and objective:Parkinson's disease(PD),a progressive disease,is characterized by the degeneration of dopaminergic neurons in the substantia nigra compacta(SNc).The mainly clinical symptoms of PD are static tremor,rigidity,bradykinesia,accompanied and postural instability.It seriously affects the quality of patients' life and their social functions.PD has become the second most common neurodegenerative disease after Alzheimer's Disease(AD)in elder population.Available therapies for Parkinson's disease only treat symptoms of the disease;it doesn't slow the progression.It is very important to explore the underlying molecular mechanisms and treatment methods of Parkinson's disease.Though extensive research has been established,the precise cause of PD remains unclear.There are some pathogenic mechanisms have been proposed,including oxidative stress,mitochondrial dysfunction,impairment of the ubiquitinproteasome system,and neuroinflammation.Mitochondria are dynamic organelles that constantly fuse and divide.These organelles could contribute to sustain a normal function of neurons by regulating calcium homeostasis,apoptosis,energy supply,free radical generation and other metabolic activities.Mitochondrial outer membrane fusion is mediated by mitofusins(Mfn1 and Mfn2)and inner membrane fusion is mediated by optic atrophy 1(OPA1).Mitochondrial fission is mediated by Drp1.Mitochondrial impairment in PD is involved in the pathogenesis of PD,the precise mechanisms remains to be elucidated as well as lacking effective treatment.Therefore,it is of great significance to explore the potential mechanism of mitochondrial dysfuction in Parkinson's disease.MitoQ is the most mitochondria-targeted antioxidant which has a protective effect on mitochondrial oxidative stress related disease model.Although several groups have reported a positive effect of MitoQ in vitro and vivo models of neurodegenerative disease,the precise molecular mechanisms of MitoQ on mitochondrial dynamics require further exploration.In our study,we explored the protective effect of MitoQ on mitochondrial dynamics in 6-OHDA-induced PD models in vitro and in vivo,find the effects of MitoQ on mitochondrial morphology and function against 6-OHDA in DA neurons.Further more,to find the underlining mechanisms of MitoQ on protecting against mitochondrial damage induced by 6-OHDA in a PD.Method:First,we established 6-hydroxydopamine(6-OHDA)-induced PD models in SN4741 cells to detect the change of Mfn1,Mfn2 and Drp1 expression level by western blotting.Secondly,SN4741 cells were pretreated with 50 nM MitoQ for 12 h,and then incubated with 80 ?M 6-OHDA for an additional 12 h to observe the effect of MitoQ on Mfn1 and Mfn2.Meanwhile,by using transfection of siRNA,immunofluorescence,quantitative real-time PCR and other methods to explore the molecular mechanism of MitoQ on PD models in vivo.Mitochondrial staining,JC-1 staining and TMRE staining were used to detect the change of mitochondrial morphology and function in order to study the protective effect of MitoQ on mitochondia.Then,TUNEL staining and western blot were used to observe the cell apoptosis to establish the protective effect of MitoQ on DA neurons.Finally,mice were injected with 6-OHDA into the right MFB to induce PD models in vivo.TH staining and western blot were used to explore the effect of MitoQ in 6-OHDA models and the underlining mechanism.Result: Part one: 6-OHDA enhanced mitochondrial fission in SN4741 cellsSN4741 cells were treated with gradient concentrations of 6-OHDA for 12 h and the expressions of mitochondrial fusion/fission proteins were analyzed by Western blotting.Mitochondrial fusion proteins Mfn1 and Mfn2 were decreased and fission protein Drp1 was increased with varying 6-OHDA concentrations.In a time course of 80 ?M 6-OHDA treatment,Mfn1 and Mfn2 expressions were declined and Drp1 was increased with time.Taken together,these results indicated that 6-OHDA enhanced mitochondrial fission in vitro.Part two: MitoQ promoted mitochondrial fusion by up-regulating Mfn2 protein and mRNA level in SN4741 cells.(1)To determine the effect of the mitochondrial antioxidant MitoQ on mitochondrial fusion and fission,SN4741 cells were treated with varying doses of MitoQ for 12 h.Western blots showed that fusion proteins Mfn1 and Mfn2 were markedly increased,with a peak at 50 nM.The effects of MitoQ on the expression of the mitochondrial proteins(Mfn1,Mfn2 and Drp1)at different time points were also measured.Mitochondrial fusion proteins were notably increased by MitoQ treatment at 6-12 h,especially Mfn2,which was peaked at 12 h.These findings suggest that MitoQ functions by regulating mitochondrial fusion proteins.(2)To determine the potential effect of MitoQ promoting mitochondrial fusion against 6-OHDA though Mfn2,SN4741 cells were transfected with siRNA targeting the MFN2 gene or with control siRNA.After siRNA transfection for 24 h,cells were pretreated with 50 nM MitoQ for 12 h,and then incubated with 80 ?M 6-OHDA for an additional 12 h.Western blotting showed that MitoQ pretreatment in control siRNA cells markedly rescued the decrease of mitochondrial fusion Mfn2 expression from 6-OHDA treatment,while there was no significant difference in siMFN2 cells.Conversely,there was no significant difference of Mfn1 expression in MitoQ+6-OHDA group compared with 6-OHDA groupneither in siCON nor siMFN2 cells.Fluorescence staining revealed that fragmented mitochondria clustered in siMFN2 cells compared with the control cells.These results indicate that the effect of MitoQ protecting mitochondrial fusion against 6-OHDA might be mainly depended on Mfn2.(3)PGC-1? is a transcriptional coactivator which plays an important role in mediating mitochondrial function.MitoQ treatment increased PGC-1? expression in dose-and time-dependent manners.Cells were treated with 50 nM MitoQ for 12 h and PGC-1? expression in DA neurons was significantly increased.Consistently,50 nM MitoQ also increased PGC-1? mRNA levelsand MFN2 mRNA levels.To further explore the potential role of PGC-1? in MitoQ regulating the expression of Mfn2,SN4741 cells were transfected with siRNA targeting PGC-1? gene or the control siRNA.After transfection for 24 h,cells were pretreated with 50 nM dose of MitoQ 12 h before 80 ?M 6-OHDA treatment for 12 h.The results showed that 6-OHDA induced a significant decrease of PGC1-? and Mfn2 expression compared with control group in siCON cells.MitoQ pretreatment in control siRNA cells markedly rescued the PGC-1? protein expression compared with 6-OHDA treatment alone as well as the Mfn2 protein expression level.There was no significant difference of both PGC-1? and Mfn2 protein level in siPGC-1? cells.The above results demonstrate that PGC-1? might contribute to the Mfn2 transcriptional up-regulation induced by MitoQ.Part three: MitoQ exerts protective effect on mitochondrial morphology and function against 6-OHDA.(1)Mito-red staining was used to evaluatethe effect of MitoQ on the mitochondrial morphology.The confocal microscopy analysis showed that the 6-OHDA treatment evidently induced mitochondrial fragmentation compared with the control group.Additional MitoQ pretreatment for 12 h showed a rescued change of mitochondrial integrity against 6-OHDA,as indicated by the appearance of longer mitochondrial tubules.JC-1,TMRE and CellROX staining were performed to evaluate mitochondrial function in vitro.MitoQ pretreated cells showed an amelioration of the repression of mitochondrial membrane potential after 6-OHDA exposure.MitoQ pretreatment also decreased the ROS generation induced by 6-OHDA.These data indicated that MitoQ stabilized mitochondrial morphology and function against 6-OHDA in vitro.(2)TUNEL staining confirmed that 6-OHDA induced a significant increase in percentage of cells that are undergoing apoptosis in SN4741 cells,which was attenuated by MitoQ pretreatment.Consistent with these findings,cells treated with 6-OHDA exhibited increased expression of cleaved caspase-3 compared to control cells and pretreatment with MitoQ attenuated this increase.Furthermore,the up-regulation of cleaved caspase-3 was enhanced by 6-OHDA treatment in siMFN2 cells,while no significant difference was noticed in MitoQ+6-OHDA group compared with 6-OHDA group.These results indicated that MitoQ prevented neuronal apoptosis induced by 6-OHDA in vitro might through Mfn2.(3)The protective effect of MitoQ in vivo was determined in a 6-OHDA induced PD model of mouse.Mice were pretreated with MitoQ for 3 days,and then stereooritically injected with 6-OHDA into the right MFB first.Following with additional MitoQ treatment for 14 days,Western boltting confirmed that Mfn2 protein level was significantly decreased in 6-OHDA treated mice.MitoQ treatment alone markedly up-regulated the Mfn2 protein expression and rescued the decrease of Mfn2 in6-OHDA injected mice.Fluorescence staining revealed TH positive DA neurons were notably reduced in 6-OHDA injected group compared with control group,and the reduction was abolished by MitoQ pretreatment against 6-OHDA.Taken together,MitoQ exerted neuroprotective effect in PD models in vivo Conclusion:In summary,our study confirmed that MitoQ exerted protective effect against 6-OHDA induced PD models by alleviating mitochondrial damage.Additionally,MitoQ might mediate mitochondrial fusion in PD models by increasing Mfn2 expression via activating PGC1-?.These data suggested that MitoQ might be a beneficial therapeutic strategy in mitochondrial dysfunction related neurodegenerative diseases including PD.