Studies On Molecular Mechanisms Of NOX2-ROS Mediation Of AMPK/Akt-mTOR Signaling And Its Regulation In The Process Of Neuronal Apoptosis

The present study,using cellular and molecular biology techniques and methods including western blotting,fluorescent protein labeling,trypan blue exclusion,RNA interference,DNA cleavage,TUNEL and DAPI staining etc.,and employing PC 12 cells,SH-SY5Y cells and primary neurons as experimental subjects,through the establishment of in vitro models of cadmium(Cd)exposure,as well as in vitro model of Parkinson’s disease(PD),we systematically dissected the molecular mechanisms of role and its regulation of NOX2-ROS mediation of AMPK/Akt-mTOR signaling in neuronal apoptosis.The detailed results were summarized as follows:1-Celastrol-regulated AMPK/mTOR signaling is involved in resisting Cd-induced neuronal apoptosisPC 12,SH-SY5Y cells and/or primary neurons,or PC 12 cells infected with shRNA to mTOR and GFP,respectively,were pretreated with/without celastrol(1 μM)for 1 h,followed by exposure to Cd(10 or 20 μM)for 4 h or 24 h.The number of live cells was analyzed by trypan blue exclusion,cellular morphology was observed,cell apoptosis was assessed by DAPI and TUNEL staining,caspase-3/7 was assayed by fluorescence labeling,and expression of ACC,AMPK,mTOR,S6K1,4E-BP1 and cleaved-caspase-3 proteins was detected using western blotting.The results showed that celastrol significantly inhibited Cd-induced cell viability reduction and apoptosis increase,and elevated cleaved-caspase-3 and caspase-3/7 activity were observed in PC12,SH-SY5Ycells and primary neurons induced by Cd.Celastrol obviously reversed Cd-reduced phosphorylation of ACC and AMPK,and blocked Cd-evoked phosphorylation of mTOR,S6K1 and 4E-BP1.Co-treatment with rapamycin or silencing mTOR strengthened celastrol’s prevention against Cd-induced neuronal apoptosis.These data suggest that celastrol-regulated AMPK/mTOR signaling is involved in resisting Cd-induced neuronal apoptosis.2.Celastrol prevents against Cd-induced neuronal apoptosis via AMPK-mediated mTOR signaling pathwayPC 12 cells and/or primary neurons,or PC 12 cells infected with Ad-dn-AMPKa or Ad-AMPKa-ca and Ad-GFP,respectively,were pretreated with/without celastrol(1 p,M)for 1 h,or with/without Compound C(20 μM)or AICAR(2 mM)for 1 h and then exposed to celastrol(1 μM)for 1 h,followed by exposure to Cd(10 or 20 μM)for 4 h or 24 h.The expression of ACC,AMPK,mTOR,S6K1,4E-BP1 and cleaved-caspase-3 proteins was detected using western blotting,and cell apoptosis was assessed by DAPI staining.The results showed that Compound C significantly attenuated celastrol’s prevention aganist Cd-induced neuronal apoptosis,whereas AICAR enhanced the resistance of celastrol to Cd-induced neuronal apoptosis.The expression of dn-AMPKα weakened celastrol’s protection against Cd-induced neuronal apoptosis,but expression of AMPKα-ca potently potentiated celastrol’s reversion of Cd-induced the event.Our findings suggest that celastrol prevents against Cd-induced neuronal apoptosis via AMPK-mediated mTOR signaling pathway.3.Celastrol inhibits Cd-induced mitochondrial ROS and CaMKⅡ signaling against apoptosis in neuronal cellsPC12,SH-SY5Y cells and/or primary neurons,PC12 cells infected with shRNA to CaMKⅡ and GFP,or PC 12 cells infected with Ad-dn-AMPKα,Ad-AMPKα-ca,Ad-GFP and Ad-LacZ,respectively,were pretreated with/without celastrol(1 μM)for 1 h,or with/without rapamycin(0.2 μg/ml)for 48 h,or with/without Compound C(20μM),AICAR(2 mM),NAC(5 mM)or Mito-TEMPO(10 μM)for 1 h and then pretreated with/without celastrol(1 μM)for 1 h,followed by exposure to Cd(10 or 20μM)for 4 h or 24 h.In other cases,PC12,SH-SY5Y cells and/or primary neurons were pretreated with/without celastrol(1 μM)for 1 h,followed by co-treatment of Cd with TTFA(10 μM),rotenone(0.5 μM)or antimycin A(50 μM)for 4 h or 24 h,respectively.Cell apoptosis was assessed by DAPI staining,the CM-H2DCFDA probe was used to analyze the intracellular ROS level,the expression of ACC,AMPK,mTOR,S6K1,4E-BP1 and cleaved-caspase-3 proteins was detected using western blotting.The results showed that celastrol significantly prevented Cd-induced mitochondrial ROS elevation mediation of AMPK/mTOR signaling pathway against neuronal apoptosis.co-treatment of celastrol with NAC、TTFA、Antimycin A or Mito-TEMPO,respectively,strengthened the resistance to Cd-induced excessive production of mitochondrial ROS,thereby preventing against neuronal apoptosis.Silencing CaMKⅡ potentiated the inhibitory effects of celastrol on Cd-induced Akt,S6K1 and 4E-BP1 activation and neuronal apoptosis.These findings imply that celastrol inhibits Cd-induced mitochondrial ROS and CaMKⅡ signaling against apoptosis in neuronal cells.4.Upregulation of NOX2 and its regulatory proteins is associated with excessive H2O2-induced apoptosis in neuronal cellular model of PDPC 12 cells and/or primary neurons were treated with different concentration of 6-OHDA(0-120 μM),MPP+(0-1.5 mM)or rotenone(0-2 μM)for 24 h,or treated with 6-OHDA(120 μM),MPP+(1 mM)or rotenone(1 μM)for different time(0-24 h).Cell apoptosis was assessed by DAPI staining,the H2DCFDA probe was used to analyze the intracellular H2O2 level,the expression of NOX2,p22phox,p40phox,p47phox,p67phox and Rac1 proteins was detected using western blotting.The results showed that 6-OHDA,MPP+ and rotenone upregulated expression of NOX2,p22phox,p40phox,p47phox,p67phox and Rac1,and elicited excessive H2O2 production and apoptosis in neuronal cells in a concentration-and time-dependent manner.The findings suggest that upregulation of NOX2 and its regulatory proteins is associated with excessive H2O2-induced apoptosis in neuronal cellular model of PD.5.Cellular and its mitochondrial H2O2 mediated dysfunction of AMPK/Akt-mTOR pathway contributing to apoptosis in neuronal cellular model of PDPC 12 cells and/or primary neurons were treated with different concentration of 6-OHDA(120 μM),MPP+(1 mM)or rotenone(1 μM)for 24 h,or pretreated with/without CAT(350 U/ml),Mito-TEMPO(10 μM)for 1 h and then exposed to 6-OHDA(120 μM),MPP+(1 mM)or rotenone(1 μM)for 24 h.The H2DCFDA probe was used to analyze the intracellular H2O2 level,the expression of NOX2,p22phox,p40phox,p47phox,p67phox,Rac1,AMPK,Akt,mTOR,S6K1,4E-BP1 and cleaved-caspase-3 proteins was detected using western blotting,cell apoptosis was assessed by DAPI staining.The results showed that pretreatment with CAT and Mito-TEMPO significantly reduced excessive H2O2 production and reversed activation of AMPK and inactivation of Akt-mTOR in neuronal cells induced by 6-OHDA,MPP+ and rotenone.In addition,CAT and Mito-TEMPO markedly inhibited 6-OHDA-,MPP+-and rotenone-induced upregulation of NOX2 and its regulatory proteins including p22phox,p40phox,p47phox,p67phox and Racl expression,thereby diminishing neuronal apoptosis.Our data suggesting that induction of cellular and its mitochondrial H2O2 by 6-OHDA,MPP+ and rotenone is involved in upregulation of NOX2 and its regulatory proteins,thereby mediating dysfunction of AMPK/Akt-mTOR pathway contributing to neuronal apoptosis.6.Interaction of NOX2/H2O2 with AMPK/Akt signaling contributes to apoptosis in neuronal cellular model of PDPC 12 cells and/or primary neurons were treated with different concentration of 6-OHDA(120 μM),MPP+(1 mM)or rotenone(1 μM)for 24 h,or pretreated with/without NOX2 inhibitor apocynin(50 μM)or Compound C(10 μM)for 1 h and then exposed to 6-OHDA(120 μM),MPP+(1 mM)or rotenone(1 μM)for 24 h.In addition,PC12 cells infected with shRNA to NOX2 and GFP,respectively,or PC12 cells infected with Ad-myr-Akt,Ad-dn-AMPKa,Ad-GFP and Ad-LacZ,respectively,were treated with/without 6-OHDA(120 μM),MPP+(1 mM)or rotenone(1 μM)for 24 h.Cell apoptosis was assessed by DAPI staining,the H2DCFDA probe was used to analyze the intracellular H2O2 level,the expression of AMPK,Akt,mTOR,S6K1,4E-BP1,cleaved-caspase-3,NOX2,p22phix,p40phox,p47phox,p67phox and Racl proteins was detected using western blotting.The results showed that pharmacological inhibition of NOX2 or silencing NOX2 by RNA interference significantly reduced 6-OHDA-,MPP+-and rotenone-evoked excessive H2O2-mediated dysfunction of AMPK/Akt-mTOR contributing to neuronal apoptosis.Pharmacology inhibition of Akt or expression of myr-Akt or dn-AMPKa by RNA interference significantly blocked upregulation of NOX2,p22phox,p40phox,p47phox,p67phox and Racl,excessive H2O2 production and apoptosis in neuronal cells induced by 6-OHDA,MPP+ and rotenone.The data suggest that interaction of NOX2/H2O2 with AMPK/Akt signaling contributes to apoptosis in neuronal cellular model of PD.