The Molecular Mechanism Study Of The Cross-talk Between SNCA/α-synuclein Oligomerization And Autophagy In Manganese Induced Neurocytes Injury

Objective:Manganese（Mn）,as one of necessary trace elements,involves in the constitution of many enzymes and biological reactions,which is important for brain physiology and homeostasis.However,excessive and chronic exposure to Mn cause nerve cell damage,abnormal behavioral changes,resulting in Parkinsonian-like symptoms referred to as manganism.Because of the widespread of Mn in industrial production,Mn becomes an environmental pathogenic factor for the neurodegenerative diseases.Although multiple neurotoxicity of Mn have been studied extensively,including mitochondria damage,endoplasmic reticulum stress,oxidative stress,α-Syn oligomerization,autophagy dysregulation,α-Syn oligomerization is considered be major causative factor in Mn-induced neurotoxicity.Autophagy functions as degrade/recycle regulatory mechanism participating in the sustaining homeostasis of cellular protein.Dysregulation of autophagy is closed correlated with the development of neurodegenerative disorders.Therefore,there is a complicated cross-talk betweenα-Syn oligomerization and autophagy in the process of Mn-induced neurotoxicity.Autophagy is regulated by multiple signalings,such as mTOR-ULK,Beclin1-PIK3C3.Inhibition of mTOR is link with autophagy activation.Besides,the interaction of Beclin1 and HMGB1 in cytosolic fraction involves in autophagic induction.Recently,some studies reported that overexpressedα-Syn interferes with the interaction of HMGB1 and Beclin1.These findings provide a new thought to comprehensive explore the mechanism of Mn-induced autophagic autophagy and neurotoxicity.Cory B has been found neuroprotective role in various nerve cells.However,the exact mechanism of Cory B in ameliorating Mn-induced autophagic dysregulation and neurotoxicity is required illuminated.Based on these findings,the present study conducted the animal test in vivo and cell culture in vitro to overall demonstrate the complicated relationship betweenα-Syn and autophagy in Mn-induced neurotoxicity.Thus,we appliedα-Syn gene knockout mice(α-Syn^-/-)and wild-type mice(α-Syn^+/+)to elucidate the physiological role ofα-Syn on autophagy activation and apoptosis upon Mn exposure.Furthermore,rapamycin（Rap）and 3-methyladenine（3-MA）,autophagy regulators,were introduced into the study to demonstrate the effect of autophagy on Mn-inducedα-Syn oligomerization and neurotoxicity in wild-type mice model of manganism.In addition,we applied SH-SY5Y cell to study the role of cytosolic HMGB1 in Mn-induced autophagic dysregulation and neurotoxicity and also reveal the potential mechanism of Cory B in ameliorating Mn-induced autophagic autophagy and neurotoxicity.The current study provides not only new experimental and theoretical basis foundation,but also a potential therapy target of neurodegenerative disorders.Methods:1.The mice used in this study were generated by homozygousα-Syn knockout male mice purchased from Jackson Laboratory(B6;129X-Snca^tmlRosl,stock#3692,Bar Harbor,MA)crossing with the inbred female mice（C57/BI6J）.Experiments were carried out in 10-week-old homozygousα-Syn^-/-andα-Syn^+/+mice（backcrossed in C57BI6 for at least 6 generation）（25±5 g）（female:male=1:1）from the same generation.α-Syn^+/+andα-Syn^-/-mice were randomly divided into four groups respectively（n=6 per group）:controls ofα-Syn^+/+andα-Syn^-/-mice,and different dosages of MnCl₂-treatedα-Syn^+/+andα-Syn^-/-mice.I.p.injection with NaCl,50,100 and 200μmol/kg MnCl₂ for 5consective days every week.After Mn exposure for six weeks,relevant indicators were able to collect for detection.Open field test,forced swimming test,static weight bearing assessment and grasping strength were used to observe abnormal behavioural changes.Next,we measured Mn contents in brain andα-Syn oligomerization assay.FCM was conducted to analyze the apoptosis and autophagy occurrence.Transmission electron microscopye was use to observe the ultrastructure of autophagic vesicles.The protein expression levels of autophagic associated protein expression(（Beclin1,LC3II/I,p62）,Lamp 2A,HSC70 were detected by Western blot.Lamp 2A and HSC70 mRNA levels were measured by Real time PCR.The colocalization and interaction of Lamp 2A and HSC70,Lamp 2A andα-Syn were evaluated by Immunofluorescence and Co-immunoprecipitation.2.Total 36 C57/B16 mice（female:male=1:1）were obtained from the Laboratory Animal Center,China Medical University.These mice were randomly divided into six groups:control groups（NaCl control,Rap control and 3-MA control）,200μmol/kg MnCl₂-treated group and Rap or 3-MA pretreated groups（Rap+Mn and 3-MA+Mn）.The manner of pretreatment（5 mg/kg Rap or 3-MA）was conducted by subcutaneously injection（s.c.）for 3 intermittent days,2h before i.p.injection with NaCl solution or 200μmol/kg MnCl₂.After expousure to Mn for six weeks,these mice were subject to determine the relevant indicators.Open field test,forced swimming test,static weight bearing assessment and grasping strength were used to observe abnormal behavioural changes.Next,we also measured Mn contents in brain andα-Syn oligomerization assay in pretreatments.FCM was conducted to analyze the apoptosis and autophagy occurrence.Autophagic associated protein expression levels（Beclin1,LC3II/I,p62）were detected by Western blot andα-Syn mRNA transcription levels was determined by Real time PCR.3.SH-SY5Y cells were individually treated with 50,100 and 200μM MnCl₂ and culture solution（control）for 24 h,and then to carry out the relative indicators detection in this study.In addition,we applied Cory B autophagy inducer to explore the molecular mechanism in Mn-induced neurotoxicity.In detail,SH-SH5Y cells were set up six groups:culture solution（control）,200μM Mn treatment,100μM Cory B alone treatment and three different dosages of Cory B（25,50 and 100μM）pretreatments which incubated for2h before 200μM Mn treatment.After incubation for 24 h at 37℃and 5%（v/v）CO₂,cells were collected to detect relative indicators.We measured the LDH release level and CCK8 level and extracted the nuclear and cytoplasmic proteins by Nuclear and Cytoplasmic Extraction Reagents Kit.The subcellular localization of HMGB1 and colocalization of HMGB1 and Beclin1,HMGB1 andα-Syn,Beclin1 and Bcl2 were observed by Laser confocal microscope.Inverted fluorescence microscope was used to observe MDC-labeled autophagic vesicles.FCM was conducted to detect apoptosis and autophagy occurrence.The interactions of HMGB1 and Beclin1,HMGB1 andα-Syn,Beclin1 and Bcl2 were measured by Co-immunoprecipitation.Real time PCR was used to determine the HMGB1 mRNA level.Western blot was conducted to detect the protein expression levels of HMGB1 in the nuclear and cytosolic fraction,Beclin1,LC3II/I,p62,α-Syn,Bcl2,mTOR,p-mTOR,4EBP1,p-4EBP1,S6K1,p-S6K1.Ad-mCherry-CFP-LC3B transfection was used to analyze the autophagy flux.Results:1.Mn exposure cause abnormal behavioural changes both inα-Syn^-/-andα-Syn^+/+mice.The significant decrease of total distance,rearing number and the increase of immobility time,grasping strength and the contralateral hind paw weight difference in Mn-treatedα-Syn^+/+andα-Syn^-/-mice compared to controls.Moreover,α-Syn^-/-mice performed more sensitivity to Mn-induced toxicology thanα-Syn^+/+mice.There was a dose-dependent increase in the Mn concentration in striatum of Mn-treatedα-Syn^+/+andα-Syn^-/-mice,compared to controls.The increase of autophagosomes and autolysosomes were observed in Mn-treatedα-Syn^-/-andα-Syn^+/+mice by electron microscopy.Mn exposure significantly increased apoptosis and autophagy in bothα-Syn^-/-andα-Syn^+/+mice.However,there were significant differences in apoptosis and autophagy between200μmol/kg Mn-treatedα-Syn^+/+andα-Syn^-/-mice.The levels of Lamp 2A and HSC70mRNA and protein expression both significantly increased in Mn-treatedα-Syn^-/-andα-Syn^+/+mice compared to controls.Additionally,the colocalization and interaction of Lamp 2A and HSC70,Lamp 2A andα-Syn were obviously enhanced Mn-treatedα-Syn^+/+andα-Syn^-/-mice,compared to their controls.The western blot analysis revealed a significant increase of Beclin1,increase of LC3-II/I and decrease of p62 in Mn-treatedα-Syn^+/+andα-Syn^-/-mice,compared to the controls.Furthermore,α-Syn^-/-mice performed more excessive activation of autophagy thanα-Syn^+/+mice.2.Exposure to Mn for six weeks,mice in 200μmol/kg Mn treatment,Rap pretreatment and 3-MA pretreatment performed abnormal behavioral changes.Compared to control,there were significant decreases of total distance,rearing number,and significant increases of immobility time,grasping strength and the contralateral hind paw weight difference in 200μmol/kg Mn treatment,Rap pretreatment and 3-MA pretreatment.The Mn concentration in striatum was increased in 200μmol/kg Mn treatment,Rap pretreatment and 3-MA pretreatment.The pretreatments with Rap or3-MA had no effects on the accumulation of Mn.Compared to 200μmol/kg Mn treatment,Rap pretreatment could activate autophagy,promoteα-Syn oligomers degradation,resulting in ameliorating apoptosis;in contrast,3-MA aggravated apoptosis and inhibited autophagy accompanied byα-Syn oligomers accumulation.3.Mn-induced overexpressedα-Syn impaired the cytosolic HMGB1-dependent autophagy in SH-SY5Y cells.Mn exposure stimulated the increase of HMGB1 mRNA level and cytosolic translocation,increase of protein expressions（cytosolic HMGB1,α-Syn and Bcl2）,the increase of HMGB1-α-Syn binding,Beclin1-Bcl2 binding,the decrease of HMGB1-Beclin1 binding in the cytosolic fraction,resulting autophagic dysregulation.Cory B ameliorated Mn-induced autophagic dysregulation and neurotoxicity via dissociating HMGB1 from overexpressedα-SYN and inhibiting mTOR signaling.100μM Cory B pretreatment effectively decreased the HMGB1-α-SYN binding,subsequently increased HMGB1-Beclin1 binding and reduced Beclin1-Bcl2binding,resulting in restoring autophagy activation and ameliorate apoptosis,compared to 200μM Mn treatment.Besides,Cory B decreased of protein expressions of downstream effectors of mTOR（the ratio of p-mTOR/t-mTOR,p-4EBP1/t-4EBP1,p-S6K1/t-S6K1）,inhibiting mTOR signalings.Conclusion:1.Mn activates CMA independent ofα-Syn.And activation of CMA involves in Mn-inducedα-Syn degradation.2.Mn activates macroautophagy.And mice lack ofα-Syn performed more susceptible and hypersensitivity to Mn than wild type mice.3.Activation autophagy by Rap promoting Mn-inducedα-Syn oligomers degradation,ameliorates neurotoxicity of Mn in vivo.4.Mn induces autophagic dysregulation and neurotoxicity via overexpressedα-Syn binding HMGB1 to restrict cytosolic HMGB1-dependent autophagic induction,subsequently to increase of Beclin1and Bcl2 binding exacerbating apoptosis in SH-SY5Y cells.5.Cory B ameliorates autophagic dysregualtion and neurotoxicity partly attributing to dissociating HMGB1fromα-SYN and inhibiting mTOR signalings in SH-SY5Y cells.