Protective Mechanism Of Parkin-mediated Mitochondrial Autophagy In Manganese-induced Neurotoxicity

Parkinson’s disease(PD)is the second most common neurodegenerative disease.Currently,there is no specific drug to treat Parkinson’s disease.The main manifestatio ns are tremor,rigidity and dyskinesia.As an essential trace element,manganese exists in soil,water and air.Studies have shown that excessive manganese intake can lead t o Parkinson-like disease,but the specific mechanism is rarely reported.Previous studi es in vivo and in vitro confirmed that with the increase of manganese concentration,P arkin m RNA levels in the whole blood,striatum and cortex of rats decreased,and Par kin protein expression in human bone marrow neuroblastoma cells decreased.Therefo re,this study further explored the mechanism of manganese induced nervous system i njury on the basis of previous studies.This study is divided into two parts.In the first part,apoptosis,DA content,mitochondrial autophagy function related indexes and Par kin signaling pathway related protein expression levels of SK-N-SH cells were detect ed by staining SK-N-SH cells with different doses of manganese for 24 h,to explore th e effects of manganese on SK-N-SH cell activity,mitochondrial autophagy function a nd Parkin protein expression level.In the second part,SK-N-SH cells were transfecte d with lentivirus to construct Parkin and OPTN gene overexpression/knockdown cell model.After 24 h of manganese staining,related indexes of mitochondrial autophagy f unction and Parkin and OPTN protein expression levels were detected to investigate t he role of Parkin in mitochondrial autophagy induced by manganese in SK-N-SH cell s.Part I: Effects of manganese on mitochondrial autophagy and Parkin in SK-N-SH cells Objective: To investigate the effects of different manganese concentration on mitochondrial autophagy and Parkin protein in SK-N-SH cells.Methods: After the proliferation toxicity of manganese to SK-N-SH cells was detected by CCK8 method,0,300,900 and 1500μmol/L manganese chloride were selected as the toxic dose of SK-N-SH cells.After 24 hours of infection,apoptosis was measured by Annexin V-PE / 7-AAD,intracellular autophagosomes and mitochondrial morphology were observed by transmission electron microscopy,intracellular ROS levels were detected by fluorescent probe,mitochondrial membrane potential change was detected by JC-1 probe,relevant target proteins(α-syn,Parkin,Pink1,OPTN,LC3,and P62)were detected by WB,and DA content in cell culture medium was detected by Elisa.Results: Compared with the control group,intracellular autophagosomes increased f irst and then decreased,and with the increase of manganese concentration,mitochond rial morphology became round,crest blurred,and its outer membrane was not smooth.The apoptosis rate and ROS content increased with the increase of manganese concen tration(P < 0.05).Mitochondrial membrane potential and DA content decreased with t he increase of manganese concentration(P < 0.05).α-SYN protein increased in medi um and high dose groups(P < 0.05);The expression of OPTN protein in the high dose group increased(P=0.02);Parkin protein expression decreased in low,medium and hi gh dose groups(P < 0.05);The expression of Pink1 protein decreased in the high dose group(P=0.043);LC3Ⅱ/Ⅰ increased in low dose group(P < 0.001),and decreased in medium and low dose groups(P < 0.05).Conclusion: The injury of SK-N-SH cells induced by manganese may be related to the block of mitophagy and the decrease of Parkin protein expression.Part II: Role of Parkin in mitochondrial autophagy in manganese induced SK-N-SH cellsObjective: To investigate the role of Parkin in mitochondrial autophagy induced by manganese in SK-N-SH cells by overexpression/knockdown of Parkin and OPTN genes.Methods: Lentivirus-transfected cells were used to construct Parkin and OPTN over expression/knockdown cell models,which were divided into(1)no-load plasmid grou p,Parkin overexpression group,Parkin knockdown group and(2)no-load plasmid gro up,OPTN overexpression group,OPTN knockdown group.After the cells were infect ed with 900μmol/L Mn2+ for 24 h,the number of autophagosomes in the cells was obs erved by electron microscopy,the ROS level was detected by fluorescence probe,the related order proteins(Parkin,OPTN,LC3 and P62)were detected by WB,and the co ntent of DA in the cell culture medium was detected by Elisa.Results:(1)Compared with no-load plasmid groups,the intracellular DA content and LC3Ⅱ/Ⅰ value in Parkin overexpression group increased(P < 0.05),while ROS conten t,P62 and OPTN protein expression levels decreased(P < 0.05).In knockdown group s,the intracellular DA content and LC3Ⅱ/Ⅰ value decreased(P < 0.05),while ROS con tent,P62 and OPTN protein expression levels increased(P < 0.05).(2)Compared with the no-load plasmid groups,the intracellular DA content and LC3Ⅱ/Ⅰ value in OPTN overexpression groups increased(P < 0.05),P62 protein expression decreased(P < 0.05),and Parkin protein expression unchanged(P > 0.05).In knockdown groups,the int racellular DA content and LC3Ⅱ/Ⅰ value decreased(P < 0.05),P62 and OPTN protein expression increased(P < 0.05),and Parkin protein expression unchanged(P > 0.05).Conclusion: Parkin can mediate the increase of mitophagy to relieve manganese to xicity in SK-N-SH cells,and OPTN protein can participate in mitophagy as Parkin’s a utophagy receptor protein.