Studies On The Role And Molecular Mechanisms Of Neuronal Autophagy Inhibition Based On Oxidative Stress In Development Of Experimental Parkinson’s Disease

The present study,using cell biology and molecular biology techniques and methods including cell culture,RNA interference,fluorescent staining and imaging analysis,protein fluorescent labeling,Western blot analysis,and cell viability detection,etc.,and employing PC12 cells,SH-SY5Y cells and primary murine neurons as experimental objects,by establishing cellular models of PD,systematically study molecular mechanisms of neuronal mitochondrial H₂O₂-mediated PTEN/Akt pathway contributing to apoptosis by autophagy inhibition in the PD generation,and explore the importance and its biological significance of parkin in the context of PD.The results are as follows:1 Study on role of autophagy inhibition-induced neuronal apoptosis in the development of PDPC12 cells,SH-SY5Y cells and primary neurons,or PC12 cells infected with lentiviral FLAG-Atg5 or EGFP,as experimental subjects,were treated with different concentrations of 6-OHDA（0-240μM or 120μM）,MPP⁺（0-1 m M or 1 m M）or rotenone（0-1μM or 1μM）for 24 h.Expressions of Atg5,LC3,p62,parkin,and cleaved-caspase-3 proteins were assayed by using Western blotting,fluorescence-labeled autophagosomes were counted by infection with Ad-GFP-LC3,cell viability and apoptosis were detected using MTS experiment and DAPI staining.The results showed that 6-OHDA,MPP⁺or rotenone induced the decreases of Atg5,LC3-I/II,parkin and autophagosomes,but did the increase of p62 expression,in a concentration-dependent manner,in PC12 cells,SH-SY5Y cells and primary neurons.Overexpression of Atg5 significantly reversed the decreases of LC3-I/II,parkin,autophagosomes and cell viability,as well as the increases of p62,cleaved-caspase-3and apoptosis in PC12 cells in response to 6-OHDA,MPP⁺or rotenone.These results suggest that there exists an inhibited autophagy-dependent neuronal apoptosis in the development of PD.2 Study on mechanisms of neuronal PTEN/Akt pathway involved in autophagy inhibition contributing to apoptosis in the development of PDPC12 cells,SH-SY5Y cells and primary neurons,or PC12 cells infected with Ad-PTEN-c/s,Ad-myr-Akt and/or Ad-GFP,as experimental subjects,were treated with different concentrations of 6-OHDA（0-240μM or 120μM）,MPP⁺（0-1 m M or 1m M）or rotenone（0-1μM or 1μM）for 24 h,or pretreated with/without rapamycin（100 ng/ml）for 2 h and then treated with 6-OHDA（120μM）,MPP⁺（1 m M）or rotenone（1μM）for 24 h.Expressions of PTEN,Akt,Atg5,LC3,p62,parkin,and cleaved-caspase-3 proteins were evaluated by using Western blotting,expression of p-PTEN（Thr366）was monitored by immunofluorescence staining,fluorescence-labeled autophagosomes were counted by infection with Ad-GFP-LC3,cell viability and apoptosis were detected using MTS experiment and DAPI staining.The results showed that 6-OHDA,MPP⁺or rotenone induced the decreases of p-PTEN（Thr366）,p-Akt（Ser473）and p-Akt（Thr308）levels in a concentration-dependent manner in PC12 cells,SH-SY5Y cells and primary neurons.Expression of dn-PTEN significantly reversed the decreases of LC3-I/II,parkin,autophagosomes and cell viability,as well as the increases of p62,cleaved-caspase-3and apoptosis in PC12 cells in response to 6-OHDA,MPP⁺or rotenone,and overexpression of Akt potentiated the above events reversed by expression of dn-PTEN.Rapamycin enhanced resistant role of overexpressed Akt.These results suggest that neuronal inactivation of PTEN/Akt pathway involved in inhibition of autophagy-dependent apoptosis in the development of PD.2 Study on neuronal oxidative stress-mediated PTEN/Akt pathway and autophagy inhibition contributing to apoptosis in the development of PDPC12 cells,SH-SY5Y cells and primary neurons,as experimental subjects,were pretreated with/without catalase（CAT,350 U/ml）or Mito-TEMPO（10μM）for 1 h and then treated with 6-OHDA（120μM）,MPP⁺（1 m M）or rotenone（1μM）for 24 h.Expressions of PTEN,Akt,Atg5,LC3,p62,parkin,and cleaved-caspase-3 proteins were evaluated by using Western blotting,cellular H₂O₂ levels were assayed using a fluorescent probe H₂DCFDA,fluorescence-labeled autophagosomes were counted by infection with Ad-GFP-LC3,cell apoptosis were detected using DAPI staining.The results showed that pretreatment with CAT or Mito-TEMPO obviously reversed6-OHDA-,MPP⁺-or rotenone-induced activation of PTEN and inactivation of Akt,the decreases of LC3-I/II and parkin as well as the increases of p62 and cleaved-caspase-3,and attenuated excessive H₂O₂ production induced by 6-OHDA,MPP⁺or rotenone,inhibited the toxins-evoked autophagosomes’decrease and apoptosis in PC12 cells,SH-SY5Y cells and primary neurons.The results indicate that neuronal mitochondrial excessive H₂O₂ production mediates inactivation of PTEN/Akt pathway leading to inhibition of autophagy-dependent apoptosis in the development of PD.4 Study on importance and its biological significance of neuronal parkin in the development PDPC12 cells infected with lentiviral sh RNA Parkin,sh RNA GFP,FLAG-Parkin or EGFP,as experimental subjects,were treated with 6-OHDA（120μM）,MPP⁺（1 m M）or rotenone（1μM）for 24 h.Expressions of PTEN,Akt,Atg5,LC3,p62,parkin,and cleaved-caspase-3 proteins were evaluated by using Western blotting,cellular H₂O₂levels were assayed using a fluorescent probe H₂DCFDA,fluorescence-labeled autophagosomes were counted by infection with Ad-GFP-LC3,cell viability and apoptosis were detected using MTS experiment and DAPI staining.The results showed that downregulation of parkin markedly resisted 6-OHDA-,MPP⁺-or rotenone-induced activation of PTEN and inactivation of Akt,the decreases of LC3-I/II and parkin as well as the increases of p62 and cleaved-caspase-3,and excessive H₂O₂ production,meliorated the status of cell apoptosis in PC12 cells,SH-SY5Y cells and primary neurons.Interestingly,Overexpression of parkin strengthened 6-OHDA-,MPP⁺-or rotenone-induced neurotoxicity.These results suggest that neuronal parkin status has a regulatory effect on PTEN/Akt pathway and consequential autophagy inhibition contributing to apoptosis in the development of PD,undoubtedly,the underlying mechanisms remain to be further studied.