| Backgroud:Methamphetamine(METH),known as an amphetamine-type stimulant,has strong neurotoxic effects on the central nervous system(CNS).METH abusers are more likely to damage the dopamine neurons,with high risk of developing neurodegenerative symptoms like Parkinson's disease(PD)and Alzheimer's disease(A)D).Abnormal accumulation of normally soluble proteins into insoluble deposit bodies is a typical pathological characteristics shared by many neurodegenerative diseases.The abnormal aggregation and accumulation of alpha-synuclein(a-syn)in Lewy bodies(LBs)is a hallmark of PD.Normally,a-syn is a 140-amino acid protein encoded by the SCNA gene,and may keep balance between unfolded monomers and folded polymers.It exits in the presynaptic and perinuclear regions of neurons to maintain synaptic vesicles required for neurotransmission.Many factors contribute to decrease the solubility and increasing the toxicity of a-syn,such as mutation,post-translational modification,oxidative stress and distorted protein clearance mechanisms.Pathological forms of a-syn occur as oligomers,fibrils or fibers resulted in aggregation and formation of inclusions,such as LBs that eventually lead to the death of nerve cells.Cerebral accumulation of hyperphosphorylation of microtubule-associated protein Tau into neurofibrillary tangles(NFTs)is a common feature of Alzheimer's disease(AD).Tau is a protein of440 amino acids at full length encoded by the MAPT gene for regulating microtubule assembly and stability.Several factors contribute to the aggregation process,but it is widely recognized that hyperphosphorylation plays a key role in mediating Tau aggregation.Abnormal phosphorylation of Tau can further oligomerize,eventually forming fibrils known as paired helical filaments(PHFs)and even NFTs.Recent studies showed that internalized Tau oligomers significantly promote the development of neurofibrillary pathology and resulted in reduced nerve cell viability.Though PD and AD have different characteristics,a large number of AD cases show LBs,and AD pathologies,particularly NFTs,are commonly found in the brains of PD patients.More importantly,multiple stusies showed that a-syn and Tau can synergistically promote the aggregation and accumulation of each other,a-syn directly interacts with Tau and can induce the pathological Tau to aggregate and fibrillate.Moreover,a-syn may indirectly modulate Tau aggregation by promoting GSK3?-mediated phosphorylation of Tau at AD-associated phosphoepitopes Serine 396(pS396).Conversely,Tau overexpression alters a-syn aggregation pattern,reducing the number but increasing the size and toxicity of a-syn.In our previous studies,we have demonstrated that METH promotes the overexpression of a-syn,Tau and the hyperphosphorylation of Tau in Ser396 in vitro and in vivo.Now that METH induces the pathologies like PD and AD,as well as the co-occurrence of a-syn and Tau aggregates in the same models,we hypothesized that a-syn and Tau interact,accelerate the overexpression of each other and increased the toxicity of each other after the exposure of METH both in vitro and in vivo.Therefore,we aimed to investigate whether a-syn can influence the expression and phosphorylation of Tau and whether Tau can promote the overexpression and accumulation of a-syn induced by METH exposure.Objectives:Alpha-synuclein(a-syn)and Tau are highlight and co-occurrence in neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease,and similar neurotoxicity could be found after methamphetamine(METH)abuse.In this research,we designed precise experiments mainly to investigate whether a-syn and Tau work synergistically in METH exposure.This research could provide potential therapeutic approaches targeting the vicious circle between a-syn and Tau in METH abusers and neurodegenerative disorders.Method:1.Explored the expression of a-syn and pSer396 Tau protein in METH abusersThe striatums of METH abusers were from the cases of Judicial Expertise Center of Southern Medical University.The striatums of dead died in a traffic accident were used as a contral.Immunohistochemistry(IHC)were used to detect the level of a-syn and pSer396 Tau.2.Establishment of METH subacute poisoning animal modelHealthy adult male C57 BL/6J mice were divided randomly into the saline control group(Ctrl)and the subacute exposure group(15 mg/kg × 8 injections,at 12 h intervals)(4-Days)(n = 10/group).The last injection of METH 2 h before anesthesia,broken neck,extraction and separation of brain tissue:the Western Blot method and Immunohistochemistry(IHC)were used to detect the level of a-syn,total Tau,pSer396 Tau.We targeted the prefrontal cortex,midbrain,hippocampus and striatum because ?-syn level was significantly increased in those four brain regions.3.Establishment of METH poisoned cell modelSH-SY5Y cells,a human neuroblastoma cell line,were initially treated with METH at a range of concentrations(0.5-2.5 mM)for 24 h and 2.0 mM METH for 0,2,4,8,16 or 24 hours.Primary cultured neurons were isolated from E18 C57 mouse embryos were treated with 0,0.2,0.4,0.6,0.8 or 1.0 Mm METH for 24 h and 1.0 Mm METH for 0,2,4,8,12,24,36,or 48 h.Then cells were collected and extracted protein detection:the Western Blot method was used to detect the level of a-syn,Tau,pSer396 Tau,and the immunofluorescence(IF)and immunoprecipitation(CO-IP)were used to detect the relationship between a-syn and Tau.4.To determine wheather a-syn influences the expression of pS396 and totalTau in vitro and in vivoHealthy adult male C57 BL/6J mice and the a-syn knockout mice were used to detect the interaction of a-syn and Tau.There were four groups:the saline control group(Ctrl),the subacute exposure group(METH),the a-syn knockout mice group(SNCA-/-),the a-syn knockout mice with subacute exposure group(SNCA-/-METH).The last injection of METH 2 h before anesthesia,broken neck,extraction and separation of brain tissue:the Western Blot method and Immunohistochemistry(IHC)were used to detect the level of a-syn,total Tau,pSer396 Tau.We targeted the prefrontal cortex,midbrain,hippocampus and striatum because a-syn level was significantly increased in those four brain regions.The siRNA sequences targeting human and mouse a-syn were added to the SH-SY5Y cells and primary cultured neurons for another 4-6 h of incubation.Subsequently,the complex medium was replaced with regular culture medium.After 24 h of incubation,the cells were treated with METH.A negetive siRNA sequenc was used as control.Then cells were collected and extracted protein detection:the Western Blot and the immunofluorescence(IF)were used to detect the level of ?-syn,Tau,pSer396 Tau.5.To determine wheather Tau regulates the expression of ?-syn in MAPT knockout miceHealthy adult male C57 BL/6J mice and the a-syn knockout mice were used to verify the result of cells.There were four groups:the saline control group(Ctrl),the subacute exposure group(METH),the Tau knockout mice group(MAPT-/-),the Tau knockout mice with subacute exposure group(MAPT-/-METH).The last injection of METH 2 h before anesthesia,broken neck,extraction and separation of brain tissue:the Western Blot method and Immunohistochemistry(IHC)were used to detect the level of a-syn,total Tau,pSer396 Tau.We targeted the prefrontal cortex,midbrain,hippocampus and striatum because a-syn level was significantly increased in those four brain regions.6.Exploring the effect of P-GSK-3-? in METH treatment in SH-SY5Y cells and primary cultured neuronsImmunoprecipitation(CO-IP)were used to detect the relationship between a-syn and P-GSK-3?.And the siRNA sequences targeting human and mouse a-syn were used to determine the effect of P-GSK-3?between a-syn and Tau.Result:1.Explored the expression of a-syn and pSer396 Tau protein in METH abusersMETH significantly increased the expression of ?-syn and pSer396 Tau in METH abusers striatums,?-syn and pSer396 Tau can be showed the co-location in the same brain areas of abusers.2.Establishment of METH subacute poisoning animal modelC57 B6/J mice model treated with METH was used to ascertain whether METH induce ?-syn,pS396 and Total Tau overexpression in vivo.Western blot analysis showed that the a-syn,pS396 and Total Tau expression were significantly higher after treatment with METH than in the control group(p<0.05).As we all known that the most dopaminergic neurons exist in the nigrostrnatal region,the immunohistochemistry staining of midbrain and corpus striatum was used to examine the expression of ?-syn,pS396 and Total Tau in dopaminergic neurons and similar results were observed.3.Establishment of METH poisoned cell modelIn SH-SY5Y cells,according to the western blot analysis,METH increased the expression of a-syn,pS396 and Total Tau in a time-dependent.Compared with the control cells,the expression of ?-syn,pS396 and Total Tau in METH-treated groups at the concentration of 2.0 mM for 8h was significantly increased by 8.39-fold,2.34-fold and 3.32-fold,respectively(p<0.05).In addition,the immunoflurescence staining results showed that the expression of a-syn and pS396 was significant upregulated at 2.0 mM for 8 h in METH-treated cells.The immunoflurescence staining and immunoprecipitation results showed the co-location of a-syn and pSer396 Tau.Similar results were also observed in the primary cultured neurons.Western blot analysis showed that the expression of a-syn,pS396 and Total Tau in METH-treated groups at the concentration of 1.0 mM for 24 h was significantly increased by 2.06-fold,1.62-fold and 1.61-fold,respectively(p<0.05).These results were consistent with the results observed in the immunoflurescence staining.The immunoflurescence staining and immunoprecipitation results showed the co-location of a-syn and pSer396 Tau.4.To determine wheather a-syn influences the expression of pS396 and total Tau in vitro and in vivoFirstly,we examine the expression of a-syn,pS396 and Total Tau in the ?-syn knockout mice in the prefrontal cortex,midbrain,hippocampus and striatum.Likewise,western blot analysis showed that ?-syn knockout significantly reduced the levels of pS396 and Total Tau in SNCA-/-METH group compared with METH group in all four brain regions(p<0.05).The immunohistochemistry staining of midbrain and corpus striatum was used to examine the expression of a-syn,pS396 and Total Tau in dopaminergic neurons and similar results were observed.To confirm the a-syn silencing effect in vivo,we respectively used siRNA targeting a-syn to reduce its expression in SH-SY5Y cells and primary cultured neurons.Western blot analysis showed that METH exposure increased a-syn expression significantly,and then siRNA attenuated?41%and?25%of a-syn expression after cotreatment with METH in SH-SY5Y cells and primary cultured neurons(p<0.05).We also examined the expression of pS396 and Total Tau,and it is noted that METH exposure increased pS396 and Total Tau expression significantly.The siRNA only group showed little influence compared with control group.More importantly,the expression of pS396 and Total Tau were significantly decreased in the siRNA+ METH group compared with the METH group by?50%and?47%in both SH-SY5Y cells and primary cultured neurons(p<0.05).Similar results were observed in the immunoflurescence staining.5.To determine wheather Tau regulates the expression of a-syn in MAPT knockout miceTo determine whether knockout of Tau protects against the overexpression of a-syn after treatment of METH,we used Tau knockout mice to examine the level of a-syn in the prefrontal cortex,midbrain,hippocampus and striatum.Similarly,western blot analysis showed that METH exposure significantly increased a-syn,pS396 and Total Tau levels as before.However,Tau knockout significantly decreased the expression of a-syn in MAPT-/-METH group compared with METH group in all four brain regions(p<0.05).Besides,the immunohistochemistry staining of midbrain and corpus striatum was used to further examine the expression of a-syn,pS396 and Total Tau in dopaminergic neurons and similar results were observed.These results suggest that knockour of Tau can effectively inhibit METH-induced overexpression of a-syn in mice brains.6.Exploring the effect of P-GSK-3-? in METH treatment in SH-SY5Y cells and primary cultured neuronsImmunoprecipitation showed that the interaction between a-syn and P-GSK-3?.Silence the expression of a-syn can reduce the overexpression of P-GSK-3?by METH.As to the phosphorylation of pSer396 Tau by P-GSK-3?,this result suggested that P-GSK-3? plays an important role in the interaction between a-syn and Tau.Conclusion:The main conclusions of this research are as follows:(1)METH significantly increased the expression of a-syn and pSer396 Tau in MOETH abusers striatums;(2)the expressions of a-syn,pS396 and tau were significantly increased with METH treatment in vitro and in vivo:(3)down-regulating a-syn expression can reduce the expression and accumulation of Tau and pS396 after METH exposure in vitro and in vivo;(4)silencing Tau can decrease METH-induced overexpression of a-syn in Tau knockout mice;(5)Silence the expression of a-syn can reduce the overexpression of P-GSK-3?by METH.These results suggested that NDs-related proteins a-syn and Tau accelerate the overexpression and accumulation of each other in the METH exposure.A preliminary discussion suggested that P-GSK-3? plays an important role in the interaction between a-syn and Tau. |
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