Mitochondrial function and energy metabolism in neuronal HT22 cells resistant to oxidative stress

Abstract

BACKGROUND AND PURPOSEThe hippocampal cell line HT22 is an excellent model for studying the consequences of endogenous oxidative stress. Extracellular glutamate depletes cellular glutathione by blocking the glutamate/cystine antiporter system xc−. Glutathione depletion induces a well-defined programme of cell death characterized by an increase in reactive oxygen species and mitochondrial dysfunction. EXPERIMENTAL APPROACHWe compared the mitochondrial shape, the abundance of mitochondrial complexes and the mitochondrial respiration of HT22 cells, selected based on their resistance to glutamate, with those of the glutamate-sensitive parental cell line. KEY RESULTSGlutamate-resistant mitochondria were less fragmented and displayed seemingly contradictory features: mitochondrial calcium and superoxide were increased while high-resolution respirometry suggested a reduction in mitochondrial respiration. This was interpreted as a reverse activity of the ATP synthase under oxidative stress, leading to hydrolysis of ATP to maintain or even elevate the mitochondrial membrane potential, suggesting these cells endure ineffective energy metabolism to protect their membrane potential. Glutamate-resistant cells were also resistant to oligomycin, an inhibitor of the ATP synthase, but sensitive to deoxyglucose, an inhibitor of hexokinases. Exchanging glucose with galactose rendered resistant cells 1000-fold more sensitive to oligomycin. These results, together with a strong increase in cytosolic hexokinase 1 and 2, a reduced lactate production and an increased activity of glucose-6-phosphate dehydrogenase, suggest that glutamate-resistant HT22 cells shuttle most available glucose towards the hexose monophosphate shunt to increase glutathione recovery. CONCLUSIONS AND IMPLICATIONSThese results indicate that mitochondrial and metabolic adaptations play an important role in the resistance of cells to oxidative stress.

Sequential Upregulation of Superoxide Dismutase 2 and Heme Oxygenase 1 by tert-Butylhydroquinone Protects Mitochondria during Oxidative Stress

“…Even though DCF is a general indicator of the level of intracellular oxidative stress, as it is routinely used for this indication Tobaben et al, 2011;Kang et al, 2014), DCF does not identify the species of reactive oxygen that is elevated. Similarly, MitoSOX is routinely used to assay superoxide (Mukhopadhyay et al, 2007;Pfeiffer et al, 2014), and as it is taken up by mitochondria, it assays superoxide in this organelle (Robinson, et al, 2008). However, the MitoSOX indicator does not determine if the superoxide originates in the mitochondria.…”

“…MitoSOX Red (Molecular Probes) is a fluorogenic dye targeted to mitochondria and generates red fluorescence after oxidation by superoxide (Mukhopadhyay et al, 2007;Pfeiffer et al, 2014). The fluorescence signal was measured by fluorescenceactivated cell sorting (FACS) analysis.…”

Discovery of lipoic acid-4-phenyl-1H-pyrazole hybrids as novel bifunctional ROCK inhibitors with antioxidant activity

“…24 And neuroprotective drugs are able to protect cells against Glu-induced damage. 25 Therefore, firstly, we examined neuroprotective effects of compounds against Glu-induced cell death by MTT assay.…”

Mitochondrial pharmacology: energy, injury and beyond

“…Indeed, Pfeiffer et al . (Pfeiffer et al ., ) suggest that mitochondrial and metabolic adaptations play an important role in neuronal resistance against chronic oxidative stress. Grubman et al .…”

“…Mitochondrial dysfunction and its consequence, oxidative stress, have long been considered contributory factors in many acute and chronic disorders of the CNS. Indeed, Pfeiffer et al (Pfeiffer et al, 2014) suggest that mitochondrial and metabolic adaptations play an important role in neuronal resistance against chronic oxidative stress. Grubman et al (Grubman et al, 2014) point out that transition metals (Cu 2+ , Zn 2+ , Mn 2+ & Fe 2+ ) are localized to various mitochondrial compartments, with Fe 2+ and Cu 2+ particularly important for the electron transport chain and ATP production.…”