title: Rotenone induction of hydrogen peroxide inhibits mTOR-mediated S6K1 and 4E-BP1/eIF4E pathways, leading to neuronal apoptosis.
abstract: Rotenone, a common pesticide and inhibitor of mitochondrial complex I, induces loss of dopaminergic neurons and consequential aspects of Parkinson's disease (PD). However, the exact mechanism of rotenone neurotoxicity is not fully elucidated. Here, we show that rotenone induced reactive oxygen species (ROS), leading to apoptotic cell death in PC12 cells and primary neurons. Pretreatment with catalase (CAT), a hydrogen peroxide-scavenging enzyme, attenuated rotenone-induced ROS and neuronal apoptosis, implying hydrogen peroxide (H₂O₂) involved, which was further verified by imaging intracellular H₂O₂ using a peroxide-selective probe H2DCFDA. Using thenoyltrifluoroacetone (TTFA), antimycin A, or Mito-TEMPO, we further demonstrated rotenone-induced mitochondrial H₂O₂-dependent neuronal apoptosis. Rotenone dramatically inhibited mTOR-mediated phosphorylation of S6K1 and 4E-BP1, which was also attenuated by CAT in the neuronal cells. Of interest, ectopic expression of wild-type mTOR or constitutively active S6K1, or downregulation of 4E-BP1 partially prevented rotenone-induced H₂O₂ and cell apoptosis. Furthermore, we noticed that rotenone-induced H₂O₂ was linked to the activation of caspase-3 pathway. This was evidenced by the finding that pretreatment with CAT partially blocked rotenone-induced cleavages of caspase-3 and poly (ADP-ribose) polymerase. Of note, zVAD-fmk, a pan caspase inhibitor, only partially prevented rotenone-induced apoptosis in PC12 cells and primary neurons. Expression of mTOR-wt, S6K1-ca, or silencing 4E-BP1 potentiated zVAD-fmk protection against rotenone-induced apoptosis in the cells. The results indicate that rotenone induction of H₂O₂ inhibits mTOR-mediated S6K1 and 4E-BP1/eIF4E pathways, resulting in caspase-dependent and -independent apoptosis in neuronal cells. Our findings suggest that rotenone-induced neuronal loss in PD may be prevented by activating mTOR signaling and/or administering antioxidants.
authors.affiliation: *Jiangsu Key Laboratory for Molecular and Medical Biotechnology, Jiangsu Key Laboratory for Microbes and Functional Genomics, College of Life Sciences, Nanjing Normal University, Nanjing 210023, P. R. China, Department of Biochemistry and Molecular Biology and Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932.
authors.forename: Qian
authors.initials: Q
authors.lastname: Zhou
authors.affiliation: *Jiangsu Key Laboratory for Molecular and Medical Biotechnology, Jiangsu Key Laboratory for Microbes and Functional Genomics, College of Life Sciences, Nanjing Normal University, Nanjing 210023, P. R. China, Department of Biochemistry and Molecular Biology and Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932.
authors.forename: Chunxiao
authors.initials: C
authors.lastname: Liu
authors.affiliation: *Jiangsu Key Laboratory for Molecular and Medical Biotechnology, Jiangsu Key Laboratory for Microbes and Functional Genomics, College of Life Sciences, Nanjing Normal University, Nanjing 210023, P. R. China, Department of Biochemistry and Molecular Biology and Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932.
authors.forename: Wen
authors.initials: W
authors.lastname: Liu
authors.affiliation: *Jiangsu Key Laboratory for Molecular and Medical Biotechnology, Jiangsu Key Laboratory for Microbes and Functional Genomics, College of Life Sciences, Nanjing Normal University, Nanjing 210023, P. R. China, Department of Biochemistry and Molecular Biology and Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932.
authors.forename: Hai
authors.initials: H
authors.lastname: Zhang
authors.affiliation: *Jiangsu Key Laboratory for Molecular and Medical Biotechnology, Jiangsu Key Laboratory for Microbes and Functional Genomics, College of Life Sciences, Nanjing Normal University, Nanjing 210023, P. R. China, Department of Biochemistry and Molecular Biology and Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932.
authors.forename: Ruijie
authors.initials: R
authors.lastname: Zhang
authors.affiliation: *Jiangsu Key Laboratory for Molecular and Medical Biotechnology, Jiangsu Key Laboratory for Microbes and Functional Genomics, College of Life Sciences, Nanjing Normal University, Nanjing 210023, P. R. China, Department of Biochemistry and Molecular Biology and Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932.
authors.forename: Jia
authors.initials: J
authors.lastname: Liu
authors.affiliation: *Jiangsu Key Laboratory for Molecular and Medical Biotechnology, Jiangsu Key Laboratory for Microbes and Functional Genomics, College of Life Sciences, Nanjing Normal University, Nanjing 210023, P. R. China, Department of Biochemistry and Molecular Biology and Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932.
authors.forename: Jinfei
authors.initials: J
authors.lastname: Zhang
authors.affiliation: *Jiangsu Key Laboratory for Molecular and Medical Biotechnology, Jiangsu Key Laboratory for Microbes and Functional Genomics, College of Life Sciences, Nanjing Normal University, Nanjing 210023, P. R. China, Department of Biochemistry and Molecular Biology and Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932.
authors.forename: Chong
authors.initials: C
authors.lastname: Xu
authors.affiliation: *Jiangsu Key Laboratory for Molecular and Medical Biotechnology, Jiangsu Key Laboratory for Microbes and Functional Genomics, College of Life Sciences, Nanjing Normal University, Nanjing 210023, P. R. China, Department of Biochemistry and Molecular Biology and Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932.
authors.forename: Lei
authors.initials: L
authors.lastname: Liu
authors.affiliation: *Jiangsu Key Laboratory for Molecular and Medical Biotechnology, Jiangsu Key Laboratory for Microbes and Functional Genomics, College of Life Sciences, Nanjing Normal University, Nanjing 210023, P. R. China, Department of Biochemistry and Molecular Biology and Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932 *Jiangsu Key Laboratory for Molecular and Medical Biotechnology, Jiangsu Key Laboratory for Microbes and Functional Genomics, College of Life Sciences, Nanjing Normal University, Nanjing 210023, P. R. China, Department of Biochemistry and Molecular Biology and Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932.
authors.forename: Shile
authors.initials: S
authors.lastname: Huang
authors.affiliation: *Jiangsu Key Laboratory for Molecular and Medical Biotechnology, Jiangsu Key Laboratory for Microbes and Functional Genomics, College of Life Sciences, Nanjing Normal University, Nanjing 210023, P. R. China, Department of Biochemistry and Molecular Biology and Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932 lchen@njnu.edu.cn.
authors.forename: Long
authors.initials: L
authors.lastname: Chen
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D061945:Caspase Inhibitors / Q000494:pharmacology; D018797:Cell Cycle Proteins; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D039561:Eukaryotic Initiation Factor-4E / Q000378:metabolism*; D039642:Eukaryotic Initiation Factors; D006861:Hydrogen Peroxide / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D009474:Neurons / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D018384:Oxidative Stress / Q000187:drug effects; D016716:PC12 Cells; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D051381:Rats; D019893:Ribosomal Protein S6 Kinases / Q000235:genetics / Q000378:metabolism*; D038744:Ribosomal Protein S6 Kinases, 90-kDa / Q000235:genetics / Q000378:metabolism*; D012402:Rotenone / Q000633:toxicity*; D015398:Signal Transduction / Q000187:drug effects; D058570:TOR Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D015854:Up-Regulation
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000975:Antioxidants; D002352:Carrier Proteins; D061945:Caspase Inhibitors; D018797:Cell Cycle Proteins; C499527:Eif4ebp1 protein, mouse; C499526:Eif4ebp1 protein, rat; D039561:Eukaryotic Initiation Factor-4E; D039642:Eukaryotic Initiation Factors; D047908:Intracellular Signaling Peptides and Proteins; D010750:Phosphoproteins; C000601282:eIF4E protein, mouse; D012402:Rotenone; D006861:Hydrogen Peroxide; C546843:mTOR protein, mouse; C546845:mTOR protein, rat; D019893:Ribosomal Protein S6 Kinases; D038744:Ribosomal Protein S6 Kinases, 90-kDa; C512964:Rps6ka1 protein, mouse; C526901:Rps6kb1 protein, rat; D058570:TOR Serine-Threonine Kinases
keywords: Parkinson’s disease; apoptosis; hydrogen peroxide; mammalian target of rapamycin; neuronal cells; rotenone