Inhibition of autophagy and glycolysis by nitric oxide during hypoxia-reoxygenation impairs cellular bioenergetics and promotes cell death in primary neurons.
Excessive nitric oxide (NO) production is known to damage mitochondrial proteins and the autophagy repair pathway and so can potentially contribute to neurotoxicity. Accordingly, we hypothesized that protection against protein damage from reactive oxygen and nitrogen species under conditions of low oxygen by the autophagy pathway in neurons would be impaired by NO and enhance bioenergetic dysfunction. Rat primary cortical neurons had the same basal cellular respiration in hypoxia as in normoxia, whereas NO-exposed cells exhibited a gradual decrease in mitochondrial respiration in hypoxia. Upon reoxygenation, the respiration in NO-treated cells did not recover to prehypoxic levels. Hypoxia-reoxygenation in the presence of NO was associated with inhibition of autophagy, and the inability to recover during reoxygenation was exacerbated by an inhibitor of autophagy, 3-methyladenine. The effects of hypoxia could be recapitulated by inhibiting glycolytic flux under normoxic conditions. Under both normoxic and hypoxic conditions NO exposure induced immediate stimulation of glycolysis, but prolonged NO exposure, associated with irreversible inhibition of mitochondrial respiration in hypoxia, inhibited glycolysis. Importantly, we found that NO inhibited basal respiration under normoxic conditions only when glucose was absent from the medium or glycolysis was inhibited by 2-deoxy-d-glucose, revealing a novel NO-dependent mechanism for the inhibition of mitochondrial respiration that is modulated by glycolysis. Taken together these data suggest an oxygen-dependent interaction between mitochondrial respiration, glycolysis, and autophagy in protecting neuronal cells exposed to NO. Importantly, they indicate that mitochondrial dysfunction is intimately linked to a failure of glycolytic flux induced by exposure to NO. In addition, these studies provide new insights into the understanding of how autophagy and NO may play interactive roles in neuroinflammation-induced cellular damage, which is pertinent to our understanding of the pathology of neurodegenerative diseases in which excessive NO is generated.
Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, AL 35294-0017, USA; Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294-0017, USA.
Gloria A
GA
Benavides
Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, AL 35294-0017, USA; Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294-0017, USA.
Qiuli
Q
Liang
Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, AL 35294-0017, USA; Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294-0017, USA.
Matthew
M
Dodson
Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, AL 35294-0017, USA; Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294-0017, USA.
Victor
V
Darley-Usmar
Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, AL 35294-0017, USA; Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294-0017, USA; Department of Veterans Affairs, Birmingham VA Medical Center, Birmingham, AL 35233, USA. Electronic address: zhanja@uab.edu.
Jianhua
J
Zhang
D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001343:Autophagy / Q000187:drug effects*; D015687:Cell Hypoxia; D002470:Cell Survival; D002478:Cells, Cultured; D003847:Deoxyglucose / Q000494:pharmacology; D042783:Endothelial Cells / Q000378:metabolism; D004734:Energy Metabolism / Q000187:drug effects*; D006019:Glycolysis / Q000187:drug effects*; D008928:Mitochondria / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000037:antagonists & inhibitors; D009474:Neurons / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D026361:Reactive Nitrogen Species / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism
D024101:Mitochondrial Proteins; D026361:Reactive Nitrogen Species; D017382:Reactive Oxygen Species; D009569:Nitric Oxide; C025946:3-methyladenine; D003847:Deoxyglucose; D000225:Adenine
2-DG; 2-deoxyglucose; 3-MA; 3-methyladenine; CQ; DetaNO, (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate; DetaNONOate; ECAR; FCCP; Free radicals; Glucose; LC3; Mitochondria; NOS; OCR; carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; chloroquine; extracellular acidification rate; microtubule-associated protein light-chain 3; nitric oxide synthase; oxygen consumption rate