Mechanism Of Glycogen Synthase Kinase-3β(GSK3β) Activity And Cytoskeleton Remodeling On Methamphetamine-induced Neurotoxicity

Backgroud:Methamphetamine(METH),also known as methamphetamine,belongs to Amphetamine-Typed Stimulant(ATS).Its hydrochloride is a colorless,transparent crystal with an outer shape of ice,commonly known as "ice".A Japaness chemist A.Ogata was first synthesized in 1919.The role of METH involves a wide range of pharmacological toxicological features such as excitatory central nervous system,sympathomimetics and addiction.Long-term ingestion can severely affect their nervous system function.The various forms of violence,death,relapse,and abstinence caused by drug abusers are not only social problems but also are closely related to forensic work-In recent years,the abuse of METH in large and medium-sized cities in China is still very severe.It is the most serious and widespread drug following heroin.At the same time,new types of synthetic methamphetamine-based synthetic drugs have gradually increased.Abuse has also shown rapid growth.trend.METH can lead to loss of dopaminergic neurons and reactive proliferation of glial cells,which may lead to pathological changes associated with a variety of degenerative diseases,and the cytoskeletal abnormalities may be associated with multiple nerves including AD and PD.There is a great relationship between the occurrence and development of degenerative diseases.In recent years,it has been reported in the literature that the GSK3β substrate array indicates that GSK3β is a key kinase that regulates microtubule dynamics,suggesting that GSK3β kinase may participate in the process of neurotoxicity injury of METH through its regulation of cytoskeletal proteins.Glycogen synthase kinase 3β(GSK3β)is a multifunctional serine/threonine protein kinase that is widely expressed in brain tissue and has a wide range of substrates.In our previous research on METH neurotoxicity,we found that METH can increase the expression of a-synuclein and phosphorylate GSK3β by activating GSK3β,It is demonstrated that GSK3β is closely related to the pathogenesis of neurodegenerative diseases such as Parkinson’s disease(PD)as an upstream regulatory molecule of.In addition,it has been reported in the literature that a-Syn can form a polymer with the cytoskeletal protein tau and is phosphorylated by the tau-specific kinase glycogen synthase kinase 3β(GSK3β)and then participates in the neurodegeneration such as Alzheimer’s disease(AD)Degenerative disease pathogenesis.This suggests that GSK3β plays an important role in the neurotoxic mechanism induced by METH and is a key target.The cytoskeleton is a network structure mainly composed of protein fibers,mainly composed of microtubules,actin fibers and intermediate fibers,which plays an important role in maintaining the morphological structure and internal structure of the cells.However,Tube assembly process coordinated with each other to promote the growth and extension of axons.Our previous experimental data showed that the expression of stathmin(op18),a specific phosphorylation product of GSK3β in neurons of striatum in METH-poisoned rats,was significantly increased,further illustrating that METH mediates changes in the activity of GSK3β There may be other related cytoskeletal proteins besides tau.The cytoskeletal related protein changes and damage are the pathological basis of neurodegeneration,suggesting that METH-induced neuronal damage and degenerative changes may be associated with METH-induced changes in GSK3β activity,but the current domestic and international research results are not elucidated The corresponding mechanism needs further research and confirmation.In this study,the toxic cell model was established.The changes of GSK3β activity were verified by the detection of morphological,immunofluorescence,GSK3β and related cytoskeletal proteins.Further,GSK3β inhibitor(LiCL)was used to further verify the effect of changes in GSK3β activity on cytoskeletal proteins in order to further elucidate the mechanisms of neurotoxicity of METH through the study on the possible regulatory mechanisms and effects of GSK3β on cytoskeleton changes,To provide theoretical basis for finding the targets of METH neurotoxicity drug.Purpose:The METH cell model was established to detect the effect of METH on GSK3βactivity and the role of GSK3β in the subsequent related cytoskeleton-related proteins through the detection of immunofluorescence,GSK3β,and related cytoskeletal related proteins.The results obtained by using the GSK3β inhibitor(LiCL)were reversed to further clarify the cytoskeleton remodeling induced by changes in the activity of GSK3β under the influence of METH,thereby investigating the mechanism of neurotoxicity of METH.Neurotoxicity and detoxification drugs provide a theoretical basis for new targets.Therefore,we intend to investigate the possible mechanisms and effects of changes in the activity of GSK3β on cytoskeletal changes induced by METH and further elucidate the mechanism of neurotoxicity of METH.Methods:1.The establishment of METH poisoning cell model in PC12 cell and primary neuronal cells(1)PC 12 cells were first seeded into 6-well plates with 10%fetal bovine serum(FBS)DMEM medium and cultured at 37 ℃ under 5%C02.When the cells grew to 70%confluence,0.5mmol/L,0.1mmol/L,1.5mmol/L and 2.0mmol/L METH for 24 hours.The total protein was extracted and the phosphorylated GSK3β P-GSK3β)expression changes in order to synthesize and select the appropriate concentration of cell poisoning model.(2)When the cells reached 70%confluence,the cells were treated with 2%serum medium containing 0 mmol/L,0.2 mmol/L,0.4 mmol/L,0.6 mmol/L,0.8 mmol/L and 1.0 mmol/After cultured for 24 hours,the total cellular protein was extracted.The expression of phosphorylated GSK3β(P-GSK3β)was detected by Western Blot.The cell toxicity model was established by selecting appropriate concentrations.2.Study the mechanism of GSK3P on METH-induced cytoskeletal remodelingThe neonatal SD rat cortical neurons were seeded in a 6-well plate with fetal calf serum(FBS)specific neuronal culture medium,the cell density was 70%,cultured at 37℃ and 5%C02,to be cultured When neuronal morphology reached to maturity on day 5-7,cells were treated with serum-free neurons containing Ommol/L,0.2mmol/L,0.4mmol/L,0.6mmol/L,0.8mmol/L and 1.0mmol/Culture medium was cultured for 24 hours,and then the total cell protein was extracted.The phosphorylation of cytoskeleton related proteins including Tau,CRMP2,OP 18 and APC levels were detected by Western Blot.PC 12 cells and primary neuronal cells were inoculated into 6-well plates and divided into 4 groups:blank control group(CON),control + inhibitor group(LiCL),administration group(METH)+ Inhibitor group(METH + LiCL),which was treated with inhibitor(LiCL)for 30 minutes in the control + inhibitor group(LiCL)and the administration + inhibitor group(METH + LiCL)METH for drug treatment,were cultured for 24 hours,the cells or cytological collected for the following experiments:(1)Morphological changes of PC 12 cells were observed under an inverted microscope.(2)The expression of phosphorylated CRMP2(P-CRMP2)and phosphorylated STATHMIN(P-OP18)of cytoskeleton related protein CLASP2,phosphorylated TAU(P-TAU)were detected by Western Blot.(3)Confocal microscopy was used to observe the changes of intracellular microfilaments and microtubules.Results:1.The establishment of METH poisoning cell model in PC12 cell and primary neuronal cellsCompared with the blank control group,the expression of GSK3β in METH-treated PC 12 cells and neuronal primary cells increased gradually with the increase of METH concentration.Among them,the phosphorylation of GSK3β(P-GSK3β)in PC 12 cells was significantly higher than that in the control group(p<0.05)when the METH concentration was greater than 1.0 mmol/L.When GSK3βinhibitor LiCL was added,P-GSK3β expression was significantly inhibited by METH.In the primary cells of neurons,when the concentration of METH was greater than 0.4 mmol/L,the P-GSK3β level was increased compared with the control group There was significant difference(p<0.05).When adding GSK3β inhibitor LiCL,METH inhibited the expression of GSK3βincreased significantly.2.Study the mechanism of GSK3βon METH-induced cytoskeletal remodeling(1)Western Blot results showed that the phosphorylation levels of Tau,CRMP2,and OP 18,and the expression of APC in MTH-treated PC 12 cells and primary neuronal cells,as well as the expression of APC,were all higher than those of the control group.The concentration increases and gradually increases.(2)The morphological changes of PC 12 cells were observed under inverted microscope after 24 hours in the 4 groups of cell models.It was seen that the morphology of the cells in the METH group was significantly different from that in the CON group,showing that the cell bodies became round and the processes became shorter.Even disappeared,some of the cells were floating off the wall;the CON+LiCL group had no significant changes in cell morphology compared with the CON group,while the METH+LiCL group had significantly improved cell body or neurite lesions compared to the METH group.(3)The expression of cytoskeleton-associated proteins in PC 12 cells and primary neuronal cells was detected by Western Blot.The expression of APC total protein and phosphorylation proteins of TAU,CRMP2 and OP 18 in CON+LiCL group was compared with CON There was no significant change in the group,and the APC total protein and the phosphorylation levels of TAU,CRMP2,and OP 18 in the METH group were significantly higher than those in the CON group(p<0.05),whereas the APC total protein in the METH+LiCL group was The phosphorylation protein levels of TAU,CRMP2,and OP 18 were significantly lower than those of the METH group(p<0.05).(4)It was observed under confocal microscope that in PC 12 cells,the intracellular microfilaments and microtubules in the METH group were more disordered than in the CON group.In the primary neuronal cells,in addition to the abnormal changes in the arrangement of intracellular microfilaments and microtubules in the METH group,axon shortening and edge-like burr-like changes were also observed.Conclusions1.P-GSK3β was significantly up-regulated in METH-treated PC 12 cells and primary neurons,and the up-regulation tended to increase with increasing METH concentration.LiCL,a GSK3β inhibitor,can effectively inhibit the expression of P-GSK3p.2.METH can induce the phosphorylation of GSK3(3 in cells and lead to the increase of GSK3(3 activity.Activation of GSK3β can make the downstream cytoskeletal proteins APC,TAU,CRMP2,OP 18 increase or phosphorylate,leading to the disorder of cytoskeleton.Reconstruction,causing neuronal degeneration.The GSK3β inhibitor LiCL can effectively inhibit this process therefore inhibit the neurotoxicity of METH.