The Role Of STIM1 In Methamphetamine-induced Neurotoxicity

Background:Methamphetamine(METH),also known as deoxyephedrine,is a new type of synthetic drug.METH abuse has multiple organ damages,clinical studies have shown that METH can concentrate in human brain tissues and leading to neuronal degeneration and necrosis.Stromal interaction molecule 1(STIM1)is located in the endoplasmic reticulum(ER),its function is to detect Ca2+concentration in the ER and regulate intracellular Ca2+ concentration to maintain the intracellular ion homeostasis.STIM1 plays an important role in regulating cell apoptosis,migration and proliferation.STIM1 can induce the release of dopamine in neurons with METH exposure,but the specific mechanism of action is unclear.Objectives:This study mainly investigated the role of STIM1 in METH-induced apoptosis and autophagy,and providing the molecular mechanism of METH-induced neurotoxicity.Methods:Chapter 1:METH exposure models in vitro(SH—SY5Y cells and Primary cultured neuronal cells)were constructed,Western Blot and Immunofluorescence were used to observe the expression of STIM1.Autophagy marker protein were detected after STIM1 was knocked by siSTIM1.GFP-mRFP-LC3 adenovirus was used to track the flow of autophagy in SH-SY5Y cells treated with METH.Chapter 2:Western Blot was used to detect METH-induced ER stress and mitochondrial apoptosis when STIM1 protein was silenced.Ca2+fluorescence probe was used to detect intracellular Ca2+concentration after STIM1 and Orail were knocked out.Autophagy marker protein were detected after Orai1 was silencing to explore the relationship between autophagy and apoptosis.Chapter 3:METH exposure models in vivo were constructed,Western Blot was used to observe the expression of STIM1.After injecting lentivirus into the hippocampus tissues of mice interfering with STIM1 STIM1 was verified to be involved in the pathway of autophagy and apoptosis induced by METH in vivo.Results:Chapter 1:1.The expression of STIM1 was increased with the increase of METH exposure concentration and time in cells model.2.The expression of p-AKT/AKT and p-mTOR/mTOR were increased,Beclinl and LC3 Ⅱ were decreased after STIM1 expression was inhibited in vitro.This indicates that STIM1 is involved in meth-induced autophagy.Chapter 2:1.Inhibited STIM1 protein expression can reverse mitochondrial dysfunction and apoptosis marker protein were significantly reduced.2.After METH exposure,STIM1 depolymerizes induced ER stress and CHOP protein increased.Inhibition of STIM1 protein can reverse METH-induced ER stress,and the expression of CHOP protein was decreased.3.After METH exposure,STIM1 depolymerizes induced ER migration to the cell membrane and opened Orai1 channel on the cell membrane,resulting in intracellular Ca2+ overload,ER stress and mitochondrial apoptosis.ER stress and mitochondrial apoptosis pathway were significantly inhibited after Orai1 knockout.However,auotpagy marker protein were not changed after Orai1 protein was silenced.Chapter 3:1.The expression of STIM1 in hippocampus and striatum was increased in different METH-treated models.2.In vivo model verified that STIM1 through Orai1 participated in ER stress,mitochondrial apoptosis and autophagy in METH-treated mice.Conclusion:STIM1 may play an important role in METH-induced neurotoxicity.METH exposure leads to STIM1 depolymerize and ER migrates to the cell membrane.STIM1 binds to Orail,which open the Ca2+ channels in the cell membrane and mediate extracellular Ca2+inflow.Thus cause intracellular Ca2+disorders and ER stress,triggering classical mitochondrial apoptosis.Meanwhile,STIM1 is also involved in METH-induced autophagy.