Resistance against Oxidative Glutamate Toxicity: Functional Characterization of Amino Acid Antiporter Subunit xCT, Purinergic ATP Receptor P2X3 and Mitochondrial Fission Factor GDAP1

Oxidative stress is involved in neurodegenerative diseases. High glutamate concentrations lead to a cell death program distinct from excitotoxicity called oxidative glutamate toxicity. Here, excessive extracellular glutamate blocks the activity of the cystine/glutamate antiporter system Xc- resulting in depletion of the important antioxidant glutathione (GSH), increased levels of reactive oxygen species (ROS), the activation of 12/15-lipoxygenase (12/15-LOX), and the opening of cyclic guanosine monophosphate (cGMP)-gated Ca2+ channels. The hippocampal cell line HT22 is an excellent model to study oxidative glutamate toxicity as it lacks ionotropic glutamate receptors. Furthermore, a glutamate-resistant cell line (HT22R) has been generated providing a useful tool to identify protective proteins and pathways. HT22R cells withstand high glutamate concentrations and exhibit increased GSH and reduced ROS levels. Moreover, they differ from glutamate-sensitive HT22 cells at the transcriptional level. Affymetrix array analysis identified several transcripts, including those for xCT, the functional subunit of Xc-, the ionotropic P2X receptor P2X3, and the mitochondrial protein Ganglioside-induced differentiation protein 1 (GDAP1), which were further analyzed in this study. xCT remarkably protected HT22 cells against glutamate toxicity by elevating cellular GSH concentration, which was confirmed by cell death assays with the GSH precursor N-acetyl-L-cysteine. Both xCT silencing and pharmacological inhibition increased the susceptibility of HT22R cells to glutamate and reduced elevated GSH levels indicating that the upregulation of xCT accounts to a large part for the protective phenotype of HT22R cells. Preliminary experiments further indicated a role of xCT in cell differentiation, inflammation, and ischemic stroke. Despite the high upregulation of P2X receptors P2X3 and P2X7 in HT22R cells, their overexpression did not result in protection against oxidative glutamate toxicity. These factors were therefore not further analyzed. GDAP1 is a mitochondrial protein and fission factor. Mutations in GDAP1 cause the hereditary peripheral neuropathy Charcot-Marie-Tooth disease CMT4A. GDAP1 overexpression protected HT22 cells against glutamate toxicity and NSC-34 motor neuron cells against buthionine sulfoximine (BSO)-induced glutathione depletion. Moreover, GDAP1 rescued HT22 cells from cell death induced by overexpression of 12/15-LOX and truncated Bid. Increased GDAP1 expression caused an increase in the mitochondrial membrane potential (Ψm) suggesting high respiratory capacities. At the same time, a GDAP1-induced shift in energy metabolism towards glycolysis was observed together with an increase of the expression of hexokinase-2 and its binding to mitochondria - phenotypes that resemble the Warburg effect, namely elevated glycolysis in the presence of oxygen, a main feature of cancer cells. On the other hand, GDAP1 increased the expression and activity of the mitochondrial complex II or succinate dehydrogenase, but the effect on oxidative phosphorylation was not further studied. The disease-associated point mutation R310Q attenuated some of the functions of GDAP1, including mitochondrial fission, protection against glutamate- and BSO-induced cell death, and the increase of Ψm and complex II activity. Furthermore, fibroblasts derived from two CMT4A patients exhibited reduced Ψm and complex II activities suggesting that, in addition to the impairment of mitochondrial fission, deficiencies in energy production account for the pathology of CMT4A. Taken together, these results prove the involvement of oxidative stress in the pathogenesis of CMT4A, a hereditary neurodegenerative disease.