Neuronal hypoxia-reoxygenation and nitric oxide: A protective role of autophagy

Abstract

In stroke, hypoxia and reoxygenation induces glutamate excitotoxicity, nitric oxide is overproduced and can damage mitochondria and be detrimental to cellular bioenergetics and survival. Macroautophagy of modified proteins and mitophagy of modified mitochondria may provide a mechanism for the neurons to deal with nitric oxide-induced cellular damage. The role of autophagy in hypoxia-reoxygenation induced mitochondrial damage is not clear. We hypothesized that autophagy plays a protective role in neuronal hypoxia-reoxygenation damage and tested this in cultured rat cortical primary neurons. Cellular bioenergetic responses to DetaNONO under hypoxia conditions and during reoxygenation using Seahorse bioscience XF24 extra flux analyzer were examined. We found that 250 μM DetaNONO did not decrease basal oxygen consumption rate (OCR) in the 30 min pre-hypoxia condition. In response to hypoxia untreated cells maintained the same OCR in hypoxia as normoxia, whereas NO exposed cells exhibited a gradual decrease in mitochondrial respiration. Upon reoxygenation, the respiration in DetaNONO-treated cells recovered to similar levels to pre-hypoxia levels. Inhibition of autophagy by 3-MA alone did not affect cellular bioenergetics in hypoxia-reoxgenated conditions. Interestingly, inhibition of autophagy by 3-MA in the DetaNONO-treated cells completely resulted in a severe bioenergetic dysfunction during reoxygenation. These studies provide new insights into understanding how autophagy and NO may play an interactive role in neuroinflammation-induced cellular damage which is pertinent to our understanding of the pathology in stroke.