title: The mechanistic target of rapamycin (mTOR) pathway and S6 Kinase mediate diazoxide preconditioning in primary rat cortical neurons.
abstract: We examined the role of the mechanistic target of rapamycin (mTOR) pathway in delayed diazoxide (DZ)-induced preconditioning of cultured rat primary cortical neurons. Neurons were treated for 3 days with 500 μM DZ or feeding medium and then exposed to 3 h of continuous normoxia in Dulbecco's modified eagle medium with glucose or with 3 h of oxygen-glucose deprivation (OGD) followed by normoxia and feeding medium. The OGD decreased viability by 50%, depolarized mitochondria, and reduced mitochondrial respiration, whereas DZ treatment improved viability and mitochondrial respiration, and suppressed reactive oxygen species production, but did not restore mitochondrial membrane potential after OGD. Neuroprotection by DZ was associated with increased phosphorylation of protein kinase B (Akt), mTOR, and the major mTOR downstream substrate, S6 Kinase (S6K). The mTOR inhibitors rapamycin and Torin-1, as well as S6K-targeted siRNA abolished the protective effects of DZ. The effects of DZ on mitochondrial membrane potential and reactive oxygen species production were not affected by rapamycin. Preconditioning with DZ also changed mitochondrial and non-mitochondrial oxygen consumption rates. We conclude that in addition to reducing reactive oxygen species (ROS) production and mitochondrial membrane depolarization, DZ protects against OGD by activation of the Akt-mTOR-S6K pathway and by changes in mitochondrial respiration. Ischemic strokes have limited therapeutic options. Diazoxide (DZ) preconditioning can reduce neuronal damage. Using oxygen-glucose deprivation (OGD), we studied Akt/mTOR/S6K signaling and mitochondrial respiration in neuronal preconditioning. We found DZ protects neurons against OGD via the Akt/mTOR/S6K pathway and alters the mitochondrial and non-mitochondrial oxygen consumption rate. This suggests that the Akt/mTOR/S6k pathway and mitochondria are novel stroke targets.
authors.affiliation: Neuroscience Program, Tulane University School of Science and Engineering, New Orleans, Louisiana, USA.|Department of Pharmacology, Tulane University School of Medicine, New Orleans, Louisiana, USA.
authors.forename: Somhrita
authors.initials: S
authors.lastname: Dutta
authors.affiliation: Department of Pharmacology, Tulane University School of Medicine, New Orleans, Louisiana, USA.
authors.forename: Ibolya
authors.initials: I
authors.lastname: Rutkai
authors.affiliation: Neuroscience Program, Tulane University School of Science and Engineering, New Orleans, Louisiana, USA.|Department of Pharmacology, Tulane University School of Medicine, New Orleans, Louisiana, USA.
authors.forename: Prasad V G
authors.initials: PV
authors.lastname: Katakam
authors.affiliation: Neuroscience Program, Tulane University School of Science and Engineering, New Orleans, Louisiana, USA.|Department of Pharmacology, Tulane University School of Medicine, New Orleans, Louisiana, USA.
authors.forename: David W
authors.initials: DW
authors.lastname: Busija
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology / Q000196:embryology; D003470:Culture Media / Q000494:pharmacology; D003981:Diazoxide / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D066298:In Vitro Techniques; D019194:Ischemic Preconditioning; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects / Q000502:physiology; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors / Q000502:physiology*; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D010100:Oxygen / Q000494:pharmacology; D010101:Oxygen Consumption; D010766:Phosphorylation; D061251:Primary Cell Culture; D011499:Protein Processing, Post-Translational; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species; D019893:Ribosomal Protein S6 Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*; D020123:Sirolimus / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*
chemical_list: D003470:Culture Media; D009419:Nerve Tissue Proteins; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; C546845:mTOR protein, rat; C494920:Akt1 protein, rat; D051057:Proto-Oncogene Proteins c-akt; D019893:Ribosomal Protein S6 Kinases; D058570:TOR Serine-Threonine Kinases; D003981:Diazoxide; D010100:Oxygen; D020123:Sirolimus
keywords: S6 Kinase; diazoxide preconditioning; ischemic stroke; mTOR pathway; oxygen glucose deprivation; rapamycin