Damage Of Hippocampal Neurons And Regulation Of P-tau Induced By Methamphetamine Exposure And The Corresponding Mechanism

Backgroud: Methamphetamine(METH),one of the most devastating and widespread drug,is a central psychostimulant of amphetamines.METH-induced neurotoxicity,addiction and dependence are among the major public health issues and research hotspots all over the world.With a variety of pharmacological and toxicological properties,METH long-term abuse can case obvious changes in neurobiology,pathology and behavior.In recent years,studies have shown that METH has strong neurotoxicity.It can cause damage to nerve structure and function,and induce pathological changes in the brain similar to neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease.Current research suggests that the neurotoxic mechanisms of METH include: oxidative stress,imbalance of body temperature,excitotoxicity and inflammatory response.However,the current research results are still inconsistent,and it is difficult to clarify the neurotoxic mechanism of METH.This study intends to investigate the viability of primary cultured hippocampal neurons exposure to METH,and examine the damage of hippocampal neurons induced by METH with different concentration gradients,and the potential toxicological mechanism of insulin pathway involved in mediating METH neurotoxicity in vitro was also explored.Therefore,the present study will provide a novel theoretical direction for the METH neurotoxic mechanism,which provides a theoretical reference for further drug intervention in METH-induced neurotoxicity.Objectives: To establish a METH exposure model of primary cultured hippocampal neuron and examine the effects of METH on the morphology and viability of neuronal cells,In molecular level,we investigate the changes of pathological protein expression and phosphorylation site activation of insulin signaling pathway after METH exposure,Moreover,the insulin pathway in the mechanism of AD-like pathological changes was evaluated after METH exposure and indicate the neurotoxic effects of METH exposure.Methods: In vitro experiments,fetal rat hippocampal neurons were isolated and cultured to identify the purity of primary hippocampal neurons.CCK-8 cell counting kit was used to detect neuronal viability at different concentrations of METH(0?10?20?30?100?300?900?2000?3000?M).The effect of different concentrations of METH on the apoptosis of hippocampal neurons was observed by TUNEL assay.The mouse neuroblastoma Neuro2A(N2a)was selected for culture,exposure of N2 a cells to different concentrations of METH(0,100,300,900?M)for 12 hours,and the changes of insulin pathway associated protein were detected by Western blot.The neural cell line N2 a was exposure to a concentration of 900?M METH for different hours(0,6,12,24),and the expression of GSK3? and tau phosphorylation sites p S199-tau,p S214-tau,and p S396-tau were detected by Western blot.After incubation with different concentrations of TWS119(0,50,200,1000 n M)and N2 a cells for12 hours using TSK119;its effect on the expression of GSK3? and tau phosphorylation was observed.To validate the specific effects of METH on the insulin signaling pathway,the insulin sensitizer rosiglitazone was applied to examine the effect of METH on tau protein expression.Exposure of N2 a cells to 50 ?M insulin sensitizer rosiglitazone for 12 hours,the expression of IRS1,AKT,GSK3? and tau phosphorylation sites p S199-tau,p S214-tau,and p S396-tau were detected by Western blot.Results: 1.METH exposure contributes to primary hippocampal neural damage The purity of primary hippocampal neurons cultured on the 10 th day of culture was determined by the anti-MAP-2 antibody via immunofluorescence,and the purity of the obtained hippocampal neurons was over 95%.The morphological contour is clear and the stereoscopic effect is strong.The results showed that METH exposure resulted in neural damage in a dose dependent manner,which manifested that 100?M METH treatment for 12 hours began to damage neurons,and the cell viability decreased more significantly in the 3000?M dose group.By the TUNEL assay,we found that with the increase of METH treatment concentration,apoptotic cells was significantly increased,and the effect of METH on neuronal apoptosis was dose-dependent.2.Effect of METH exposure on insulin pathway protein 100 ?M METH increased IRS1 Ser307 expression,300,900?M dose significantly increased IRS1 Ser307 expression;METH(300,900?M)decreased the expression of AKT.Then we moved to investigate the time course of METH action.Exposure to METH significantly increased the expression of IRS-1 Ser307 at 6,12 hours,after which the protein expression continued at a maintained level.METH exposure significantly reduced the expression of AKT,with a peak effect at the time point of 12 hours.3.Effect of METH on GSK3?,tau protein expression and phosphorylation METH significantly increased phosphorylation of p Y-GSK3?,p S199-tau,p S214-tau,and p S396-tau,with a peak response occurring at the time point of 12 hour.4.TWS119 inhibits the effect of GSK3? phosphorylation on tau protein induced by METH exposure TWS119 showed a dose-dependent decrease in phosphorylation of GSK3?.mediated by METH.Meanwhile TWS119 treatment also significantly reduced the expression of phosphorylation of p Y216-GSK3?,p S199-tau,p S214-tau,p S396-tau proteins caused by METH.5.Effect of insulin sensitizer rosiglitazone on tau expression and phosphorylation induced by METH exposure METH exposure significantly increased the phosphorylation expression of p S307-IRS1 and p Y216-GSK3?,and decreased the expression of p S473-AKT.After treatment of the insulin sensitizer rosiglitazone,the phosphorylation expression of p Y216-GSK3?,p S199-tau,p S214-tau,and p S396-tau substantially was decreased by METH exposure.Conclusions: 1.METH is neurotoxic to hippocampal neurons,which can cause morphological changes in neuronal cells and affect neuronal viability.2.METH exposure can induce high expression of tau protein in neurons,facilitating the hyperphosphorylated tau is,which triggers the neurodegenerative like changes,i.e.AD-like changes.3.The key nodes of the insulin signaling pathway,IRS-1,AKT and GSK3?,are involved in AD-like changes induced by METH exposure.