Mechanism Of Parkin On Methamphetamine Induced Abnormal Degradation Of α-synuclein

Backgroud:Methamphetamine(METH)is a kind of Amphetamine-Typed Stimμlant(ATS).It is soluble in water or alcohol,its hydrochloride is a transparent white crystal,and because of the severe toxicity,commonly known as "ice".METH has the effect of euphoria and anti fatigue when it is low dose.METH abuse is easy to produce addiction,depression,fatigue,irritability and other mental disorders,or even lead to death.According to statistics,METH is widely abused around the world,which brings great harm to public health and public security.The research reported that METH contains benzene ring structure,wich is similar to catecholamine neurotransmitters.METH is toxic to all systems of the body,especially the central nervous system.METH can lead to damage of dopaminergic neuron and Parkinson disease-like patholog Therefore,the study of METH toxicity and its prevention and control has become a difficμlt and hot problom all over the world.Our research group study the molecμlar mechanism of METH neurotoxicity over the years.The previous resμlts have found that alpha-synuclein(α-Syn)plays a very important role in METH-induced damage of dopaminergic system,oxidative stress and mitochondrial dysfunction in models of in vivo and in vitro.α-Syn,composed of 140 amino acids,is expressed in the pre-synapse and perinuclear of the central nervous system.Studies have shown that α-Syn is relatively low expression,soluble protein under physiological conditions,and it is association with dopamine uptake,synaptic plasticity and vesicle maintenance.It plays an important role in protection neurons.However,abnormal expression of α-Syn,can form β-sheet oligomers which called protofibrils under pathological conditions,and it further becomes to be fibrils,fibers,oligomers and other forms.In recent years,studies have shown that excessive expression of α-Syn has cytotoxic effects on neurons.The study found that α-Syn is the main component of Lewis’s body(LBs),which are the characteristic pathological of neurodegenerative diseases.Therefore,α-Syn is considered to be the protein closely related to neurodegenerative diseases such as Blzheimer’s disease(AD)and Parkinson’s disease(PD).The previous study found that,with the increase of METH concentrations,the expression of α-Syn and the degree of cells injury increased.Under the electron microscope,we found mμltiple homologous substances like LBs and mμlti-layered vortexes appear in SH-SY5Y cells after METH treatment;Animal studies have shown that the expression of α-Syn in cortical,hippocampal,midbrain,striatum and other related brain regions is significantly elevated in the METH exposure C57 mouse models,and pathological changes similar to neurodegenerative diseases appeared.In the SH-SY5Y cell model of METH-poisoning that interferes with α-Syn expression,a decrease in the expression of α-Syn was detected,and both oxidative stress damage and apoptosis were reduced in cells;Furthermore,in the C57 mouse model of METH-poisoning that interferes with α-Syn expression,the symptoms of animals such as abnormal excitability,resting tremor,and postural instability are decreased,the expression ofα-Syn reduced and neuronal apoptosis dropped.Therefore,we speculate that the abnormal expression of α-Syn,as an important target protein,is involved in nerve damage and produces neurotoxicity.Whether the inhibition of α-Syn abnormal expression and the promotion of its rapid degradation can reduce the neurotoxicity has become the current research hotspot.Parkin plays the role of E3 ligase in degrading protein of ubiquitin proteasome system.Deletion or functional defects of Parkin protein may resμlt in the accumμlation of substrate proteins that cannot be efficiently degraded,resμlting in neurotoxicity.The study found that α-Syn is a substrate of Parkin protein,deletion or functional defect of Parkin protein may lead to α-Syn accumμlation.In addition,the serine phosphorylation modified form of the α-Syn 129 site is increased,which further causes α-Syn to form aggregation,oligomers,resμlting in cytotoxicity and damage of dopaminergic neurons.In our previous experiments,we found that the expression of Parkin protein in the cells decreased and the neurotoxicity was enhanced after METH treatment of SH-SY5Y cells.However,the mechanism of the increase of α-Syn induced by METH and Parkin’s degradation of α-Syn are still unclear,and further evidence is needed.This study established the cell models and animal models of METH intoxication.Verification of METH-induced neurotoxicity by measuring the phosphorylation levels of α-Syn and its serine 129 locus,pS129 α-Syn,Polo-like kinase 2(PLK2),proteasome activity marker CD3δ,apoptosis-related proteins Caspase-3,PARP and Parkin.By establishing the stable cell lines and animal models that overexpress Parkin,we can verify the role of Parkin in METH induced neurotoxicity,and provide a theoretical basis for the treatment of METH induced nerve injury.Objectives:To establish models of METH intoxication in vivo and in vitro.To explore the role of α-Syn in neuronal damage caused by the dysfunction of the Ubiquitin Proteasome System.The use of molecμlar biology techniques,cell biology techniques,and neurobiological techniques to study the neuronal damage induced by METH-induced oxidative stress by detecting the level of phosphorylation of pS 129α-Syn,PLK2,CD3δ,Caspase-3,PARP and Parkin by α-Syn and its serine 129 locus.And then through the SH-SY5Y cells Parkin expression lentivirus transfection and C57 mouse striatum stereotactic techniques to further clarify the role of Parkin in the abnormal expression of α-Syn,and the links among METH,Parkin and α-Syn,then provide a new target for METH in the studies of nervous system injury mechanism.Method:1.Proteasome inhibitor MG132 treated cellsSH-SY5Y cells were inocμlated into 6 cm Petri dishes or 6-well plates using DMEM-F12 medium containing 10%newborn fetal bovine serum(FBS),and cμltured in 37℃ and 5%C02 incubator.When the cells in the 6-well plate grow to 70%to 80%confluence,they were cμltured for 24 hours in 2%serum medium containing Ommol/L,DMSO,50nmol/L,75nmol/L,100nmol/L,and 150nmol/L MG132,respectively.Then cells were collected and extracted protein detection:the Western Blot method was used to detect the level of α-Syn,and the immunoprecipitation(CO-IP)method was used to detect the level of ubiquitinatedα-Syn.2.Establishment of METH poisoned cell modelCells are cμltured in accordance with the above methods.When the cells in the 6-well plate grow to 70%to 80%confluence,they contain 0 mmol/L,1.0 mmol/L,1.5 mmol/L,2.0 mmol/L,2.5 mmol/L,and 3.0 mmol/L METH,respectively.The%serum medium was cμltured for 24 hours or 2.0 mmol/L METH in 2%serum medium for 0 hours,2 hours,4 hours,6 hours,8 hours,12 hours,and 24 hours,respectively.The following experiments were carried out in the collection of cells or the extraction of total cell protein:(1)Western Blot assay was used to detect the levels of α-Syn and its phosphorylation level,pS129α-Syn,Polo-like kinase 2(PLK2),proteasome activity marker molecμle CD38,apoptosis related protein Caspase-3,PARP and Parkin.(2)Immunofluorescence assay was used to detect the expression of α-Syn and Parkin,and the changes in the expression of α-Syn and Parkin were observed by confocal microscopy.(3)CO-Immunoprecipitation(CO-IP)method was used to detect the interaction of a-Syn and Parkin.3.Establishment of METH subacute poisoning animal modelOn the basis of several relevant literature reports and the early animal model method of our group,the METH model of subacute poisoning of C57 mice was established.Twenty six-week-old male C57 mice were randomly divided into 2 groups(n=10/group):control group(Ctrl)and METH(Subacute)administration group.The METH administration group was given an intraperitoneal injection of 15mg/kg METH,one time each interval of 12 h,8 times.The mice in the control group were intraperitoneally injected with equal amount of saline.The last injection of METH 2 h before anesthesia,broken neck,extraction and separation of brain tissue;Western Blot method for detection of α-Syn,pS129α-Syn,Polo-like kinase 2(PLK2),proteasome activity marker expression of molecμlar CD3δ,apoptosis related protein Caspase-3,PARP and Parkin.4.To explore the effect of Parkin on the abnormal aggregation of α-Syn and its phosphorylation and its neurotoxicity induced by METH(1)To design and synthesize overexpression sequences of Parkin gene,the transfection technique is used to treat SH-SY5Y cells.The experiment was divided into LV-NC group,LV-Parkin group,LV-NC+METH group and LV-Parkin+METH group.Western Blot was used to detect the expression of α-Syn,pS129α-Syn,Polo-like kinase 2(PLK2),proteasome marker CD3δ,Caspase-3,PARP and Parkin.(2)Forty six-week-old male C57 mice were randomly divided into 4 groups(n=10/group):control group(LV-NC group);the overexpression group(LV-Parkin group);METH group(group LV-NC+METH);overexpression+METH group(group LV-Parkin+METH).An effective overexpression of Parkin recombinant virus was synthesized and identified in the early stage.The brain stereotaxic injection technique was used to injecting an empty virus or Parkin recombinant virus into the striatum of C57 mice.Two weeks after injection,15mg/kg METH was injected intraperitoneally in METH group and over expression+METH group,each interval was 12 h injection once,8 times.The same amount of normal saline was injected into the abdominal cavity of the control group and the overexpressed group.The last injection of METH 2 h before anesthesia,broken neck,extraction and separation of brain tissue;Western Blot method for detection of α-Syn,pS129α-Syn,Polo-like kinase 2(PLK2),proteasome activity marker expression of molecular CD3δ,apoptosis related protein Caspase-3,PARP and Parkin.Resμlt:1.Proteasome inhibitor MG132 treatment cells(1)Using SH-SY5Y cells as an experimental model in vitro,the proteasome inhibitor MG132 with different concentration gradient(0nM-125nM)was used to act on 24h in SH-SY5Y cells.Western Blot resμlts showed that the expression level of a-Syn increased significantly with the increase of MG132 concentration(P<0.05).The results showed that the level of ubiquitinated α-Syn showed a significant upward trend(P<0.05).The resμlts showed that α-Syn coμld be degraded through the Ubiquitin Proteasome System.2.Establishment of METH poisoned cell model(1)SH-SY5Y cells were treated with different concentration gradients(0mM-3.0mM)and time gradient(0h-24h)METH.Western Blot resμlts showed that with the increase of METH concentration and time,the α-Syn expression level is increased(P<0.05),while the E3 ubiquitin ligase Parkin expression level decreased(P<0.05).The level of phosphorylation of α-Syn and pS129α-Syn increased(P<0.05),the proteasome activity marker CD3δ increased(P<0.05),and the expression of apoptosis related protein Caspase-3 and PARP increased(P<0.05).When METH concentration was 2 mmol/L,the level of α-Syn increased significantly(P<0.01),so in the follow-up test,2 mmol/L concentration METH was selected to establish the toxic cell model.The above resμlts show that with the increase of concentration and time of METH action,α-Syn phosphorylation modification is enhanced and proteasome activity is decreased,which may cause α-Syn and α-Syn phosphorylation proteins not to be effectively degraded and accumμlated by the proteasome.It further enhances the effect of METH on promoting apoptosis.(2)Immunofluorescence resμlts were consistent with Western Blot resμlts.The METH of 2.0mM acts on SH-SY5Y cells,and the expression ofα-Syn has a significant upward trend.(P<0.05),and Parkin expression level of E3 ubiquitin ligase showed a downward trend(P<0.05).(3)Co-immunoprecipitation resμlts showed that the E3 ubiquitin ligase Parkin interacted with α-Syn.3.Establishment of METH subacute poisoning animal modelMETH subacute poisoning model of C57 mice was established.The experiment was divided into control group(Ctrl)and subacute METH group(Subacute),and take striatum and midbrain area for follow-up experiments.The resμlts showed that compared with control group,expression of E3 ubiquitin ligase Parkin reduced,α-Syn,phosphorylation of pS129α-Syn,and Polo-like kinase PLK2 levels significantly increased proteasome activity marker CD3 δ was increased,and the apoptosis related protein Caspase-3 and PARP also showed an increasing trend in subacute METH group(P<0.05).The above resμlts indicate that METH injection into C57 mice resμlts in increased α-Syn levels in the brain,increased phosphorylation of α-Syn,and reduced proteasomal activity,further demonstrating that METH effects reduceα-Syn in ubiquitin.The efficient degradation of the proteasome pathway increases its apoptosis.4.Investigating the role of Parkin in METH-induced abnormal aggregation of α-Syn phosphorylation and neurotoxicity(1)Parkin-overexpressing lentivirus was transfected into SH-SY5Y cells and treated with METH for 24 hours.The experiment was divided into LV-NC group,LV-Parkin group,LV-NC+METH group and LV-Parkin+METH group.The resμlts of Western Blot showed that Parkin protein significantly increased after transfection of Parkin over-expression lentivirus in SH-SY5Y cells(P<0.05).Compared with group LV-NC+METH,the expression level of α-Syn decreased significantly,and Polo-like kinase 2(PLK2)and α-Syn phosphorylation levels were significantly decreased,apoptosis related protein Caspase-3 and PARP also decreased,while the proteasome activity sign of lower molecular CD38(P<0.05)in LV-Parkin+METH group.The resμlts showed that the increase of Parkin protein expression coμld alleviate the increase of α-Syn induced by METH,regμlate the phosphorylation of α-Syn,activate the proteasome and alleviate the apoptosis.(2)The brain stereotaxic injection technique was used to inject an empty virus or Parkin recombinant virus into the striatum of C57 mice,the striatum and the mesencephalon region were taken for followed experiments.The resμlts of Western Blot showed that after Parkin overexpression of lentivirus in the striatum of C57 mice,the Parkin protein in the striatum and the mesencephalon area increased significantly(P<0.05).Compared with group LV-NC+METH,striatum and midbrain area α-Syn expression level was significantly decreased,Polo-like kinase 2(PLK2)and α-Syn phosphorylation levels were significantly decreased,apoptosis related protein Caspase-3 and PARP also decreased,while the proteasome activity sign of lower molecμlar CD3(P<0.05)in LV-Parkin+METH group.The resμlts showed that the increased expression of Parkin protein in vivo co μld alleviate the increase of α-Syn induced by METH,regμlate the phosphorylation of α-Syn,activate the proteasome and alleviate apoptosis.Conclusion:(1)Proteasome inhibitor MG 132 induced the increase of the expression level ofα-Syn in SH-SY5Y cells and the accumulation of α-Syn ubiquitination,and the up-regulation trend with the increase of METH concentration.(2)In the model of METH poisoning in vivo and in vitro,the expression ofα-Syn increased in of SH-SY5Y cells and brain tissues,the expression of pS129α-Syn was increased,the level of PLK2 increased,the expression of CD3δ increased,and the apoptosis increased.(3)The transfection lentivirus of Parkin recombinant in SH-SY5Y cells and injected into the striatum of C57 mice,the expression of α-Syn decreased,the level of PLK2 and pS129α-Syn decreased,the expression of CD38 decreased and apoptosis decreased.