| Backgroud:Methamphetamine(Methamphetamine,METH)belongs to the amphetamine type stimulants(Amphetamine-Typed Stimulant,ATS),soluble in water and alcohol,its hydrochloride as transparent crystal,like the appearance of ice,known as "ice",was widely abused in the world,is one of the largest drug harm to public health and social.METH is a great harm to health,while it brings people a short period of time.Studies have shown that METH can produce numerous adverse reactions,including neurotoxicity,neuropsychological defects and cardiovascular toxicity.A lot of evidence that METH played an important role in the central nervous system,structure and catecholamine neurotransmitters are similar,mainly for dopamine and other monoamine nerve injury,can appear similar pathological and some neurodegenerative diseases such as Parkinson disease change.Our laboratory has been devoted to the study of the molecular mechanism of neurotoxicity of METH over the years.Previous studies have found that in vitro experiments of ?-synuclein(?-synuclein,?-Syn)caused by METH poisoning in the dopaminergic system plays an important role in damage,oxidative stress and mitochondrial dysfunction in the system imbalance;?-Syn is a kind of soluble,non-folding protein,which is composed of 140 amino acids,which is the main component of Lewy 's body,the characteristic pathological structure of PD.High concentration of a-Syn can form a cytotoxic beta oligomer,This indicates that the abnormal aggregation and fibrosis of this protein are closely related to Parkinson's disease(PD),Alzheimer's disease(AD)and other neurodegenerative diseases.METH can cause the brain to appear similar to the changes of the neurodegenerative diseases,and the ratio of METH to PD is significantly increased.In previous studies,we found for the first time that the expression of a-Syn in the cortex,hippocampus and striatum of METH rats was significantly increased.Establishment a rat model of METH poisoning,when ?-Syn was silenced,the abnormal behavior of animals was reduced,and the related indicators showed that the interference of ?-Syn expression has protective effects on oxidative stress and neuronal apoptosis in rats with RNAi.There is an inevitable link between ?-Syn and neurotoxicity.As an important target protein.?-Syn participates in the process of nerve injury.Recent studies have shown that METH can lead to the inclusion of Lewys like bodies in the substantia nigra of the mouse.Therefore,inhibiting the abnormal aggregation of ?-Syn and accelerating its degradation can reduce the neurotoxicity is the current research hotspot.In the study of clinical pathology in PD patients,Whether familial or sporadic cases,the serine phosphorylation of ?-Syn in the dopaminergic neurons of the substantia nigra pars compacta is very high.Although human ?-Syn has 9,42,87 and 129 serine phosphorylation sites,but the serine phosphorylation of ?-Syn is mainly located at the site of the 129.It is found that the 129 site serine phosphorylation of the?-Syn can promote the formation of cytoplasmic protein aggregates.At present,it is not clear that the normal physiological function of ?-Syn and how to participate in the pathogenesis of PD.Most of the studies suggests that the increase in the level of phosphorylated ?-Syn can cause toxic damage of dopaminergic neurons.However,it is still not clear whether METH induced phosphorylation of a-Syn is the main cause of abnormal aggregation,so it is of great significance to further study the neurotoxic mechanism of METH induced a-Syn phosphorylation.Objectives:In this study,we established in vitro and in vivo model of METH intoxication.Using molecular biology and neurobiology,to investigate the role of serine phosphorylation of a-Syn in METH in the formation of oligomers and neuronal damage.Using siRNA interference technique,western blot and immunofluorescence were used to detect the abnormal aggregation of ?-Syn,pS129 ?-Syn and ?-Syn.At the same time,the expression of Maker gene(Caspase-3 and PARP),mitochondrial membrane potential and DA were detected,In order to study the neurotoxic mechanism of abnormal aggregation induced by METH induced a-Syn phosphorylation,to lay a good foundation for further clarifying the important target of a-Syn in METH neurotoxicity.Method:1.Detection of METH induced abnormal aggregation and phosphorylation of a-SynCell experiment,inoculation of SH-SY5Y cells to the 96 hole plate or 6 well plate,Under the conditions of 37? and 5%CO2,the cells were cultured with 2%FBSmedium containing 0 mmol/L,0.5 mmol/L,1 mmol/L,1.5 mmol/L,2 mmol/L and 2.5 mmol/L METH for about 24 h,respectively.after METH treatment,the LC25 and LC50.Western Blot was used to detect cell Expression of pS129 a-Syn protein and a-Syn aggregates,also to observe the changes of nerve cell apoptosis.METH Subacute poisoning model:It was established by reference to the literature and the early modeling method.6 week old male C57 mice were randomly divided into 2 groups(n=10/group),control group and METH group.METH group was given intraperitoneal injection of METH(15mg/kg),12 h interval injection,8 times.Mice in control group were injected with normal saline.After the last administration,the brain tissues were isolated,separated and extracted,and the brain tissues were isolated.Western Blot was used to detect the expression of a-Syn,pS129 a-Syn,cleaved and PARP in Caspase-3 cells,2.To investigate the effects of METH induced a-Syn phosphorylation on abnormal aggregation and neurotoxicity(1)silencing PPP2R1a reduces the expression of pS129 ?-Syn:Three siRNA interference sequences were designed and synthesized according to PPP2Rla gene.RNA interference(RNAi)technology to process cells.Western blot method for detecting interference efficiency of pS129 ?-Syn interference sequence.The most effective interference sequence was used to detect the expression of ?-Syn,pS129?-Syn,cleaved,PARP,and cleaved in caspase-3 cells after silencing SH-SY5Y cells by using Western and Blot methods;The expression of ?-Syn and pS129 ?-Syn in the cells was observed by fluorescence confocal microscopy.(2)BI2536 inhibits the phosphorylation of a-Syn:SH-SY5Y cells were inoculated to 6 well plates and the cells were divided into 5 groups:control group(Ctrl),+DMSO control group(Ctrl+DMSO),+BI2536 control group(Ctrl+BI2536)and drug group(METH),BI2536+ group(BI2536+METH),After 4h,the cell protein was collected.Western blot method was used to detect the changes of a-Syn,pS129?-Syn,cleaved caspase-3,cleaved PARP and so on.The abnormal aggregation of?-Syn was detected by immunofluorescence assay;METH subacute poisoning model:6 week old male C57 mice were randomly divided into 4 groups(n=10/group):control group,BI2536 treatment group,METH treatment group and BI2536 with METH treatment group(BI2536+METH).Before METH was administered 24 h for the first time,the BI2536 group and the BI2536+METH group were given a single time in BI2536(caudal vein injection,50mg/).The drug administration group and B12536+ group were given intraperitoneal injection of METH(15mg/kg,a 12 h intervals,once,a total of 8 times).The control group and the control group were injected with normal saline under the same conditions +BI2536.The last time after administration of 2 h anesthesia,neck,brain tissue:Western Blot method was used to detect the changes of ?-Syn,pS129 ?-Syn,cleaved Caspase-3 and PARP,and the activity of DA was detected by enzyme chemistry method;3.To investigate the interaction between METH induced phosphorylation of a-Syn and phosphorylation of Tau and its neurotoxicityThe expression of p-Tau,pS129 ?-Syn and p-Tau were observed by immunofluorescence staining and confocal microscopy,and the expression of p-Tau was detected by the expression of pS 129 a-Syn after using inhibitor BI2536.Result:1.The expression of phosphorylation of a-Syn and abnormal aggregation by METH induced(1)0-2.5 mnol/L METH treated SH-SY5Y cells,the survival rate decreased gradually with the increase of METH concentration,in addition to the 0.5 mmol/L treatment group compared with the control group had no significant difference(p>0.05),the concentration of treatment group compared with the control group there were significant differences(p<0.05).(2)compared to the control group,SH-SY5Y cell pS129 a-Syn treatment METH expression gradually increased with the increase of METH concentration,METH concentration greater than 1 mmol/L,pS129 ?-Syn increased compared with the control group there were significant differences(p<0.05).When the concentration of METH was 2 mmol/L,the increase was the most significant(p<0.0/1)so in the latter experiment,the concentration of mmol/L of 2 METH was chosen to establish the model of toxic cells.(3)2 mmol/L METH after administration of SH-SY5Y cells,using specific mouse anti human 5G4 antibody labeling a-Syn oligomers,Western Blot detection method and immunofluorescence results showed that after administration of METH a-Syn oligomers abnormal expression increased(p<0.01).(4)compared with the blank control group,the Maker cells(Caspase-3 and PARP)were detected in METH treated SH-SY5Y cells,and the expression of METH was significantly increased after administration.(5)METH subacute poisoning model group,the results of Western Blot showed that a-Syn,pS129 a-Syn,cleaved caspase-3 and PARP subacute administration group was significantly higher than the control group.2.METH induced a-Syn phosphorylation promotes abnormal aggregation and neurotoxicity2.1 silencing of PPP2R1 a in METH induced phosphorylation of ?-Syn and its role in abnormal aggregation and neurotoxicity(1)when the expression of PPP2Rla was silenced,the results showed that all the three siRNA sequences could effectively inhibit the increase of pS129 a-Syn METH expression induced by a-Syn.The expression of PPP2Rla gene using the most effective interference sequence of siRNA#1 silencing in SH-SY5Y cells after 24 h,then the cells treated with METH 4h,Western Blot assay showed that the PPP2Rla detection after silencing SH-SY5Y cells,abnormal increase of pS129 a-Syn induced by METH was inhibited in the confocal microscope observation results are consistent with the former.To detect the expression of a-Syn.(2)Western Blot was expressed in SH-SY5Y cells,compared with Ctrl group,METH group,pS129 ?-Syn increased significantly(p<0.05),and METH+siRNA#1 group of intracellular pS129 ?-Syn compared to the METH group decreased significantly(p<0.05).Under confocal microscope,we can see that the a-Syn in the cells of METH group is significantly clustered,and silencing PPP2Rla can effectively inhibit METH induced pS 129 a-Syn and inhibit the aggregation of a-Syn.(3)TUNEL method was used to detect cell apoptosis:cell apoptosis in Ctrl+siRNA#1 group and Ctrl group had no significant difference(p>0.05),apoptosis cells in METH group increased significantly(p<0.05),decreased METH cell apoptosis in +siRNA#1 group compared with METH group,the expression(p<0.05);detection of mitochondrial membrane potential conversion method JC-1 situation:cells in Ctrl+siRNA#1 group than in Ctrl group had no significant change(p>0.05),mitochondrial membrane group METH potential decreased,red and green fluorescence ratio lower than Ctrl group significantly decreased(p<0.05),while METH +siRNA#1 was significantly higher than that in METH group was increased(p<0.05).2.2 Role of BI2536 in METH induced phosphorylation of a-Syn in abnormal aggregation and neurotoxicity(4)BI2536 can significantly reduce the pS129 a-Syn METH induced expression increased,when the concentration of BI2536 is 0.4?M,the inhibitory effect was the most significant(p<0.01),the concentration of 0.4?M BI2536 inhibited the phosphorylation of a-Syn,METH subacute poisoning model of intravenous injection of BI2536(50mg/kg)to establish animal poisoning model.(5)cells and various brain regions were detected by Western Blot,results showed that:the expression of SH-SY5Y in cells of METH group pS129 ?-Syn compared to the Ctrl group increased significantly(p<0.05),compared to the BI2536+METH group of pS129 a-Syn in METH group decreased significantly(p<0.05).In the observation of confocal microscopy results are consistent with the Western Blot results:METH group SH-SY5Y intracellular a-Syn oligomers expression was significantly increased(p<0.05),pretreatment with BI2536 significantly reduced METH induced expression of a-Syn oligomers(p<0.05).3.METH induced phosphorylation of a-Syn promotes phosphorylation ofTau(1)compared with the blank control group,the expression of p-Tau was significantly higher in METH treated SH-SY5Y cells(P<0.05).Co localization of pS129-Syn and p-Tau under confocal fluorescence microscopy.(2)METH treated SH-SY5Y cells,when BI2536 was added,the expression of pS129 was significantly reduced and the expression of a-Syn was significantly reduced under confocal microscope,and the expression of p-Tau was also decreased.Conclusion:(1)in METH infected cells and animal models,METH induced an abnormal aggregation of a-Syn,and the expression of phosphorylated a-Syn was significantly up-regulated and neurotoxicity.(2)silencing PPP2Rla gene or using PLK inhibitor BI2536 can effectively reduce the expression of phosphorylated a-Syn,thereby inhibiting the METH induced neurons to produce abnormal aggregation of a-Syn and its neurotoxic effects.Thus,it is demonstrated that phosphorylation of a-Syn plays a major role in promoting the neurotoxicity of a-Syn induced abnormal aggregation of METH induced neurons.(3)In the poisoning of METH SH-SY5Y cells,the expression of the phosphorylation of Tau was significantly increased,and the phosphorylation of a-Syn and phosphorylated Tau were colocalized,when BI2536 inhibits the expression of phosphorylated a-Syn,phosphorylated Tau expression also decreased significantly.It is suggested that phosphorylation of a-Syn is associated with phosphorylation of Tau.Phosphorylation of a-Syn may be an important factor to promote Tau phosphorylation. |
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