Research On The Role Of Mitochondrial Quality Control In Rotenone-induced Dopaminergic Neurodegeneration

Background:Parkinson’s disease(PD)is the second most common age-related neurodegenerative disease,and is a lethal complex disease of progressive development.The effect of a single gene on PD is minimal,both environmental toxins and genetic factors contribute to the progression of PD.Specifically,a great amount of evidence has accumulated focusing on the potential role of mitochondria in PD etiopathogenesis.Rotenone is a naturally occurring compound extracted from the roots of several plant species,and it has been extensively used as an insecticide.It has been found that long-term chronic rotenone exposure can lead to symptoms of PD.As a mitochondrial complex I inhibitor,rotenone has been extensively used to induce parkinsonian models,and it can reproduce most of the motor symptoms and histopathological features of PD.A large number of research suggest that mitochondria play a central role in rotenone-induced dopaminergic neurodegeneration.Mitochondrial dynamics disorder may participate in the pathogenesis of neurodegeneration of PD,and may be involve in rotenone-induced pathogenesis of PD.Therefore,regulation the balance between mitochondrial dynamics and maintaining mitochondrial homeostasis may be the most important protective measures in rotenone-induced neurodegeneration.In the present study,we used rotenone to induce PD model in vivo and vitro to investigate the effect and mechanism of mitochondrial fission/fusion,mitophagy,mitochondrial biogenesis and mitochondrial ATP sensitive potassium channel in the process of rotenone-induced dopaminergic neurodegeneration to provide new ideas and direction for potential therapeutic approach of PD.Content:1.The role of mitochondrial biogenesis and mitochondrial fission/fusion in rotenone-induced dopaminergic neurotoxicity.We firstly established rotenone-induced PC12 cell toxic model.To evaluate the toxic effect of rotenone on PC12 cells,the cell viability was detected by CCK-8 kit.The morphology of mitochondrial in PC12 cells was observed under transmission electron microscope.Mitochondrial mass was analyzed by image acquisition under the confocal laser microscope.At the same time,the changes of mitochondrial membrane potential of PC12 cells were detected by TMRM staining and the mtDNA copy number was quantified by qPCR.Key signal moleculars in mitochondrial biogenesis and fission/fusion were respectively detected with RT-PCR and WB.M1(mitochondrial fusion promoter)and Mdivi-1(mitochondrial fission inhibitor)were used respectively to confirmed the role of mitochondrial fission/fusion in rotenone induced-neurotoxicity.To determine the regulatory role of PGC-1α on mitochondrial fission/fusion,cell models with PGC-1α upregulated and downregulated were established.Further,the phosphorylated Drp1 expression and its translocation to mitochondria was determined by immunofluorescence assay.2.The role of PINK1/Parkin pathway in rotenone-induced neurotoxicity.To evaluate the level of autophagy in PC12 cells exposed to rotenone,the protein levels of LC3 and p62 were detected by WB and the fluorescence intensity of LC3-GFP was assayed.At the same time,mitochondrial total protein and cellular total protein were isolated and extracted,and the protein levels of PINK1,Parkin and p-Parkin were detected respectively.The colocalization efficient of GFP-LC3 and Mito-Tracker Red and Mito-tracker Green and Lyso-tracker Red were analyzed to evaluate mitophagy level in PC12 cells exposed to rotenone.To assess the effect of PINK1/Parkin pathway on mitochondrial biogenesis,PGC-1α and mtTFA directly involved in mitochondrial biogenesis and mtDNA copy number were measured with PINK1 downregulated or upregulated.To evaluate the effect of PINK1 on mitochondrial fission/fusion,the protein levels of MFN2,OPA1,Drp1 and Fis1 were observed with alteration of PINK1 expression.In addition,to better understand the interaction between PINK1/Parkin pathway and PGC-1α,the protein level of PINK1,Parkin and its phosphorylated protein were detected when the expression of PGC-1α was upregulated or downregulated.3.The role of mitochondrial ATP-sensitive potassium channel in rotenone-induced dopaminergic neurodegeneration.Rotenone-induced PD model in vivo and vitro were established,and diazoxide(mitoKATP opener)or 5-HD(mitoKATP inhibitor)was given before rotenone exposure.Rotarod performance test and open field test were performed to evaluate neurobehavior of SD rats.Magnetic Resonance Imaging(MRI)scanning were performed to assay the changes of structure and function of basal ganglia network in rats.WB and immunohistochemistry were performed to detect the protein level of TH.Mitochondrial mass and mitochondrial number were observed with confocal laser microscope and transmission electron microscope respectively.The contents of dopamine and ATP in striatum of SD rats and PC12 cells were detected by HPLC.The levels of ROS and the activity of mitochondrial complex I in striatum and PC12 cells were detected.RT-PCR and WB were used to detect the mRNA and protein levels of PGC-1α,mtTFA,MFN2,OPA1,Drp1 and Fis1 in striatum and PC12 cells.Mitochondrial total protein and cellular total protein were isolated respectively from striatum and PC12 cells,and the protein levels of Kir6.1,Kir6.2,SUR1,SUR2 A and SUR2 B was detected with WB.In addition,mtDNA copy number,mitochondrial fragmentation and key signal molecular in mitochondrial fission and fusion were detected in cell model with Kir6.1 silencing.4.The role of diazoxide in rotenone-induced chronic toxicity and acute toxicity.Combined with the results of rotenone-induced neurodegeneration of PD(which we named it as chronic injury),rotenone-induced acute toxic model in vivo was established,all treatment factors were similar to rotenone-induced neurodegeneration of PD except for rotenone exposure time.And then,the survival rate of rats,the concentration of ATP and DA,the level of ROS and activity of mitochondrial complex I were detected respectively.Meanwhile,uric acid,blood glucose and hsCRP were also be assayed.Results:1.The interaction of mitochondrial biogenesis and fission/fusion mediated by PGC-1α regulates rotenone-induced dopaminergic neurotoxicity.Cell viability,MMP and TH protein expression were reduced obviously in rotenoneinduced dopamine neurons compared with the control group.Additionally,ROS formation were increased in rotenone-treated neurons.The mitochondrial length,area and cristae were also markedly reduced compared with the control group.A large number of fragmented mitochondria were observed which evidenced by round,small or dot-like dyeing patterns.These results indicated that abnormal mitochondrial number and mass contribute to rotenoneinduced dopamine neurotoxicity.Further study suggested that OPA1,MFN2,PGC-1α and mtTFA were all decreased,while the mitochondrial fission related molecular p-Drp1 increased and another two moleculars such as Drp-1 and Fis1 were declined.Meanwhile,promotion of mitochondrial fusion and inhibition of mitochondrial fission respectively were found to correct the mitochondrial fragmentation,improve mitochondrial mass,and reduce mitochondrial impairment.In addition,PGC-1α over-expression significantly improved the death of PC12 cells,increased both mt DNA copy number and mitochondrial mass.Conversely,PC12 cells with PGC-1α knockdown were more sensitive to the neurotoxin rotenone,with increased mitochondrial fragmentation and decreased cell viability and mt DNA copy number.Furthermore,it could be concluded that PGC-1α might participate in mitochondrial fusion and mitochondrial fission through regulating MFN2 and Drp1.In addition,immunofluorescence experiments showed that p-Drp1 expression were promoted and translocated from cytoplasm to mitochondria.Silencing of PGC-1α promoted the increase and translocation of p-Drp1 from cytoplasm to mitochondria,which could be reversed by PGC-1α overexpression.2.PINK1/Parkin interacted with PGC-1α to regulate mitochondrial biogenesis,fission/fusion and mitophagy in rotenone-induced dopamine neurotoxicity.LC3-2/LC3-1 ratio was increased and the expression of p62 was decreased during the process of rotenone-induced neurotoxicity in PC12 cells.Moreover,rotenone exposure could not only increase the expression of PINK1 and the phosphorylation of Parkin,but also led to the translocation of PINK1 and Parkin from cytoplasm to mitochondria.Further study showed that silencing of PINK1 led to a significant increase in the protein levels of PGC-1α and its target gene mtTFA,with a significant increase of mt DNA copy number.By contrast,PGC-1α,mtTFA and mt DNA copy number could be decreased when PINK1 was overexpressed.These results indicated that PINK1/Parkin pathway had inhibitory effect on mitochondrial biogenesis.What’s more,PGC-1α downregulation led to a significant increase in the protein levels of PINK1,Parkin and p-Parkin,which could be decreased when PGC-1α was overexpressed.The level of mitophagy changed significantly when the expression of PGC-1α was altered.In turn,PGC-1α might inhibit PINK1/Parkin mediated-mitophagy.The results above suggested that there was an interplay between PINK1/Parkin pathway mediated-mitophagy and PGC-1α mediated-mitochondrial biogenesis.Further experiments in our research found that not only the expression of MFN2 was negatively correlated with the expression level of PINK1,but also the expression level of phosphorylated Drp1 was positively correlated with PINK1.These results suggested that MFN2 and Drp1 might be downstream nuclear transcription factors of PINK1 and PGC-1α,which indicated that mitochondrial fission/fusion was affected by the interaction between PINK1/Parkin pathway and PGC-1α.3.Mitochondrial ATP-sensitive potassium channel regulates mitochondrial dynamics to participate in neurodegeneration of Parkinson’s disease.Rotenone induced-PD model in vitro and vivo were established.The role of mitoKATP and its relationship with mitochondrial quality control were investigated.Neurobehavioral tests,pathological experiment,the expression of TH and the concentration of DA verified the establishment of the rotenone-induced PD model.The results of neurobehavior test and histochemistry showed that diazoxide pretreatment aggravated rotenone-induced structural change and function disorder of the basal ganglia,which was partially reversed by 5-HD pretreatment.Through further analysis,we determined that the opening state of the mitoKATP channel could aggravate the rotenone-induced neurotoxicity,including the survival rate of rats,the reduction of ATP,Complex I activity and dopamine concentration,abnormal morphology and ultrastructure of mitochondrial in the rat striatum,mitochondrial ROS release,the reduction of mtDNA copy number and the imbalance of mitochondrial fission/fusion,whereas the blocking state of mitoKATP channel exhibited a significant neuroprotective effect on rotenone-induced dopaminergic neurodegeneration.Further results showed that the subunits of the mitoKATP channel in our PD model were K6.1/SUR2 B.Meanwhile,rotenone could reduce the expression of Kir6.1 which indicated that Kir6.1 may be regulated by rotenone and mitoKATP channel modulators,while SUR2 B was not.In addition,silencing of Kir6.1 exhibited a significant protective effect on rotenone-induced neurotoxicity,significant improvements in cell viability,mitochondrial DNA copy number,mitochondrial fragmentation and a reduction of mitochondrial mass.It may be concluded that the component Kir6.1 of mitoKATP channel was the key regulatory subunit in the protection of dopaminergic neurodegeneration.At the same time,Kir6.1 silencing could increase the protein levels of PGC-1α and MFN2,and decrease the protein level of p-Drp1.Therefore,the subunit protein Kir6.1 of the mitoKATP channel is involved in rotenone induced dopaminergic neurodegeneration via regulating mitochondrial biogenesis and mitochondrial fission/fusion.4.Different effect of diazoxide in rotenone-induced acute injury and chronic injury.Combined with the results of chronic injury induced by rotenone,we further investigated the role of diazoxide in rotenone induced acute injury.In the acute damage model induced by rotenone,diazoxide has significant protective effects on the dopaminergic neuron.Diazoxide pretreatment aggravated the decrease of survival rate,mitochondrial dysfunction,uric acid,blood glucose and hsCRP and the effects can be blocked by 5-HD.Conclusions:1.Long-term chronic exposed to rotenone can induce parkinsonism disorder of neuroethology and pathomorphology in rats.2.Rotenone impaired mitochondrial biogenesis,fission/fusion and mitophagy to ultimately induced abnormal morphology,structure and function of mitochondria in dopamine neurons.The intervention of the balance of mitochondrial biogenesis,fission/fusion and mitophagy has a protective effect on rotenone induced dopaminergic neurodegeneration.Therefore,maintaining mitochondrial quality is important in rotenone-induced neurodegeneration.3.MFN2 and Drp1 may be the downstream target protein of PGC-1α.PGC-1α might participate in mitochondrial fusion and mitochondrial fission through regulating Drp1 and MFN2.PGC-1α played an important role in rotenone-induced dopaminergic neurodegeneration through regulating the crosstalk between mitochondrial biogenesis and fission/fusion.4.PINK1/Parkin pathway was involved in the mitophagy induced by rotenone in dopaminergic neurons.Downregulated or upregulated of PINK1 gene could affect the protein levels of mitochondrial biogenesis(PGC-1α),fission(Drp1)and fusion(MFN2).Meanwhile,PGC-1α downregulation or upregulation could affect the protein levels of PINK1/Parkin pathway.Hence,the PINK1/Parkin pathway and PGC-1α antagonized each other to form a balance to further regulate mitochondrial biogenesis,fission/fusion and mitophagy in maintaining mitochondrial homeostasis.5.The composition of mitoKATP channel were subunits of Kir6.1/SUR2 B in dopaminergic neurons.MitoKATP was involved in rotenone induced dopaminergic neurodegeneration by regulating key signal moleculars involved in mitochondrial biogenesis and fission/fusion,and Kir6.1 subunit played a major role in this process.Opening of mitoKATP channels could aggravate the neurotoxicity induced by rotenone,while blocking of mitoKATP channels had a significant protective effect on rotenone induced dopaminergic neurodegeneration.6.The mechanisms of the effect of diazoxide on acute injury and chronic injury may be absolutely different.In the rotenone-induced acute injury,diazoxide pretreatment had significant protective effects on the mitochondrial dysfunction and blood glucose metabolism disorder induced by rotenone,while 5-HD pretreatment had no significant effect on rotenone-induced toxicity.In summary,rotenone may induce dopaminergic neurodegeneration through impairing the balance of mitochondrial dynamics,including mitochondrial biogenesis,mitochondrial fission/fusion and mitophagy.In this process,PGC-1α and PINK1/Parkin pathway antagonized each other to form a balance to further regulate mitochondrial biogenesis,fission/fusion and mitophagy in maintaining mitochondrial homeostasis;In addition,the composition of mitoKATP channel in dopaminergic neurons were subunits of Kir6.1/SUR2 B,and Kir6.1 subunit was involved in rotenone induced dopaminergic neurodegeneration through regulating key signal moleculars involved in mitochondrial biogenesis and fission/fusion.