Potassium And Calcium Homeostasis Involved In Methamphetamine Induced Neurotoxicity

BackgroundMethamphetamine（METH）,known as“ice”for its crystalline and ice-like appearance,belongs to amphetamine-type stimulants.It has become one of the most popular and drugs whichbrought about great hazard to the society.METH is characterized by its pharmacological and toxicological featuresof central excitability.Chronic METHabuse leads to pathological,praxiological and neurochemistry changes of the central nerve system.For those who abuse METH,it is not only the mental state but also the structures and the functions of the CNS that have been affected.Numerous studies have provided evidence that chronic METH use is associated with substantial neurotoxicity and cognitive impairment.To date,it is known that oxidative stress,disturbance of energy metabolism,inflammation,disorder of proteasomes and apoptosis have beenrecognized as the potential mechanisms of METH induced neurotoxicity.However,the available evidence is still not enough to elusidate the precise mechanism of METH neurotoxicity.ObjectiveTo explore the relationship between ionic homeostasis disorders and METH-induced neurotoxicity.MethodsC57BL/6J male mice were randomly divided in Control,Saline,METH groups,10 for each group.Mice in METH group were given intraperitoneal injection of10mg/kg of METH,twice a day（8:00,20:00）,for 7 days.The weight and temperature of the mice were measured every day,and Dodd and Hoffman method was used to score for the behavior of the mice.The function of learning and memory of the mice were measured by Y-maze test 12 hours after the last injection.After the mice were sacrificed,Golgi stain was performed in brain slices to characterize the mice dendritic changes in brain neurons.NOS activity detection kit was used to detected NOS activity.CCK-8 cell counting kits were used to detect the effects of different concentrations of METH on neuronal viability on various time points.The DNA and cell nucleus of neurons were observed by comet assay and Hoechst staining.Mitochondrial membrane potential assay kit（JC-1）was used to detect changes in mitochondrial membrane potential.Apoptosis protein Cyt-c,Bax,Bcl-2,Caspase3,neurodegenerative protein APP,Tau,MAPK signaling pathway,calcium related protein CaM,CaMKII,potassium-related protein Kv4.2,KChIPs were detected by western blot.The intracellular calcium ion concentration was detected with.Fluo4-AM probe,whilepotassium currents were recorded bythe whole-cell patch clamp.Moreover,with the specific inhibitor PD98059,SP600125,SB203580,nifedipine,KN62,KN93,W7which respectively inhibited the expression or activity of ERK,JNK,p38,L-type calcium channels,CaMKII,CaM,the cell injury was evaluated.Furthermore,the Lentiviral packet Kv4.2 plasmid was used in the present work with the aim to investigate the prominent role of Kv4.2 in neural damage.Results1.Effects of METH on mouse behaviorIn vivo study,the body temperature was increased after METH injection.The mice showed curled after 3h.The weight and organ coefficient were not obviously changed.Score of stereotyped behavior was increased,while the activity was reduced.2.Effects of METH on mouse neuronsThe results showed a significant decrease in the cortical neuron dendritic length and so wasdendritic density.The activity of TNOS and iNOS was significantly increased,In parallel,with which expression of Bax,cleaved-caspase 3,APP and Tau protein in brain tissue was enhanced.3.Effects of METH on cortical neuronsThe cell viability was decreased by METH in a concentration-and time-dependent manner.Uptaked nuclei,fragmentation of DNA,depolarization of mitochondrial membrane,released Cyt-c,high expression of Bax,Cleaved-caspase3,APP,Tau,and low expression of Bcl-2 were found in METH exposure cells.4.MAPK signaling pathways were involved in METH-induced neural apoptosisThe western-blot results showed that the levels of phosphorylated ERK1/2,JNK and p38 MAPK were obviously up-regulated by METH.JNK and p38 MAPK rather than ERK1/2 pathway were demonstrated to be participated in METH-induced apoptosis.5.Calcium homeostasis was involved METH-induced apoptosisMETH mediated extracellular Ca²⁺influx via L-type calcium channel,and Ca²⁺downstream proteinsincluding CaM,CaMKII were activated.With the treatment of the L-type calcium channel blocker nifedipine,it indicated that METH induced DNA fragmentation,nuclei concentration and Caspase3 hydrolysis,while the up-regulated expression of APP,Tau were markedly attenuated.In addition,CaM,CaMKII were also demonstrated to be involved in METH-mediated apoptosis since KN62,KN93,the CaMKII inhibitor and W7,the CaM inhibitor,significantlyreduced METH-induced Caspase 3 hydrolysis.6.METH-mediated intracellular potassium efflux contributed to neuronal apoptosisMETH significantly prompted potassium efflux in neural cells,which was manifested as the increment of the transient outward potassium currents.Furthermore,the K⁺channel subtype Kv4.2 was proved to migrate from cytoplasm to the plasma membrane,which might be attributed to the up-regulation of KChIPs.Further results revealed that The up-regulation Kv4.2 and KChIPs mediated by METH was modulated by CaM and CaMKII.Conclusions1.METH has an impact on mice behavior,neuron synaptic plasticity,and the pathological proteins are markedly increased.2.The effects of METH on neuronal apoptosis mainly depend on mitochondrial pathway.3.MAPK signaling pathways are involved in METH-induced neuronal apoptosis.4.L-type calcium channel-mediated intracellular calcium homeostasis disorders and the activation of calcium-related proteinsare participated in METH-induced neuronal apoptosis.5.Kv4.2 mediated potassium efflux partiallycontributes to METH-induced neuronal apoptosis.