Mutations in GDAP1 lead to recessively or dominantly inherited peripheral neuropathies (Charcot-Marie-Tooth disease, CMT), indicating that GDAP1 is essential for the viability of cells in the peripheral nervous system. GDAP1 contains domains characteristic of glutathione-S-transferases (GSTs), is located in the outer mitochondrial membrane and induces fragmentation of mitochondria. We found GDAP1 upregulated in neuronal HT22 cells selected for resistance against oxidative stress. GDAP1 over-expression protected against oxidative stress caused by depletion of the intracellular antioxidant glutathione (GHS) and against effectors of GHS depletion that affect the mitochondrial membrane integrity like truncated BH3-interacting domain death agonist and 12/15-lipoxygenase. Gdap1 knockdown, in contrast, increased the susceptibility of motor neuron-like NSC34 cells against GHS depletion. Over-expression of wild-type GDAP1, but not of GDAP1 with recessively inherited mutations that cause disease and reduce fission activity, increased the total cellular GHS content and the mitochondrial membrane potential up to a level where it apparently limits mitochondrial respiration, leading to reduced mitochondrial Ca(2+) uptake and superoxide production. Fibroblasts from autosomal-recessive CMT4A patients had reduced GDAP1 levels, reduced GHS concentration and a reduced mitochondrial membrane potential. Thus, our results suggest that the potential GST GDAP1 is implicated in the control of the cellular GHS content and mitochondrial activity, suggesting an involvement of oxidative stress in the pathogenesis of CMT4A.

The glutamate/cystine antiporter system x(c)- transports cystine into cells in exchange for the important neurotransmitter glutamate at a ratio of 1:1. It is composed of a specific light chain, xCT, and a heavy chain, 4F2, linked by a disulfide bridge. Both subunits are localized prominently in the mouse and human brain especially in border areas between the brain and periphery including vascular endothelial cells, ependymal cells, choroid plexus, and leptomeninges. Glutamate exported by system x(c)- is largely responsible for the extracellular glutamate concentration in the brain, whereas the imported cystine is required for the synthesis of the major endogenous antioxidant, glutathione. System x(c)- thus connects the antioxidant defense with neurotransmission and behavior. Disturbances in the function of system x(c)- have been implicated in nerve cell death due to increased extracellular glutamate and reduced intracellular glutathione. In vitro, inhibition of cystine import through system x(c)- leads to cell death by a mechanism called oxidative glutamate toxicity or oxytosis, which includes depletion of intracellular glutathione, activation of 12-lipoxygenase, accumulation of intracellular peroxides, and the activation of a cyclic guanosine monophosphate (cGMP)-dependent calcium channel towards the end of the death cascade. Cell death caused by oxytosis is distinct from classical apoptosis. In this contribution, we discuss the function of system x(c)- in vitro and in vivo, the role of xCT as an important but due to its dual role probably ambivalent drug target, and the relevance of oxytosis as an in vitro assay for the identification of novel neuroprotective proteins and signaling pathways.

Selecting neuronal cell lines for resistance against oxidative stress might recapitulate some adaptive processes in neurodegenerative diseases where oxidative stress is involved like Parkinson's disease. We recently reported that in hippocampal HT22 cells selected for resistance against oxidative glutamate toxicity, the cystine/glutamate antiporter system x c À , which imports cystine for synthesis of the antioxidant glutathione, and its specific subunit, xCT, are upregulated. (Lewerenz et al., J Neurochem 98(3):916-25). Here, we show that in these glutamate-resistant HT22 cells upregulation of xCT mediates glutamate resistance, and xCT expression is induced by upregulation of the transcription factor ATF4. The mechanism of ATF4 upregulation consists of a 13 bp deletion in the upstream open reading frame (uORF2) overlapping the ATF4 open reading frame. The resulting uORF2-ATF4 fusion protein is efficiently translated even at a low phosphorylation levels of the translation initiation factor eIF2a, a condition under which ATF4 translation is normally suppressed. A similar ATF4 mutation associated with prominent upregulation of xCT expression was identified in PC12 cells selected for resistance against amyloid b-peptide. Our data indicate that ATF4 has a central role in regulating xCT expression and resistance against oxidative stress. ATF4 mutations might have broader significance as upregulation of xCT is found in tumor cells and associated with anticancer drug resistance.

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.