The mechanistic target of rapamycin (mTOR) pathway and S6 Kinase mediate diazoxide preconditioning in primary rat cortical neurons

• Published in: Journal of neurochemistry Vol. 134; no. 5; pp. 845 - 856
• Main Authors: Dutta, Somhrita, Rutkai, Ibolya, Katakam, Prasad V. G., Busija, David W.
• Format: Journal Article
• Language: English
• Published: England Blackwell Publishing Ltd 01-09-2015
• Subjects: Animals; Cells, Cultured; Cerebral Cortex - cytology; Cerebral Cortex - embryology; Culture Media - pharmacology; Diazoxide - pharmacology; diazoxide preconditioning; Enzyme Activation - drug effects; In Vitro Techniques; Ischemic Preconditioning; ischemic stroke; Kinases; Membrane Potential, Mitochondrial - drug effects; Membrane Potential, Mitochondrial - physiology; Mitochondria - drug effects; Mitochondria - physiology; mTOR pathway; Nerve Tissue Proteins - antagonists & inhibitors; Nerve Tissue Proteins - physiology; Neurochemistry; Neurons - drug effects; Neurons - metabolism; Oxygen - pharmacology; Oxygen Consumption; oxygen glucose deprivation; Phosphorylation; Primary Cell Culture; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-akt - metabolism; rapamycin; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Ribosomal Protein S6 Kinases - antagonists & inhibitors; Ribosomal Protein S6 Kinases - genetics; Ribosomal Protein S6 Kinases - physiology; RNA Interference; RNA, Small Interfering - pharmacology; Rodents; S6 Kinase; Signal Transduction - physiology; Sirolimus - pharmacology; Stroke; TOR Serine-Threonine Kinases - antagonists & inhibitors; TOR Serine-Threonine Kinases - physiology; oxygen glucose deprivation; mTOR pathway; S6 Kinase; ischemic stroke; diazoxide preconditioning; rapamycin
• ISSN: 0022-3042; 1471-4159
• DOI: 10.1111/jnc.13181

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.
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.