No abstract

The cystine/glutamate exchanger (system xc −) mediates the transport of cystine into the cell in exchange for glutamate. By releasing glutamate, system xc − can potentially cause excitotoxicity. However, through providing cystine to the cell, it regulates the levels of cellular glutathione (GSH), the main endogenous intracellular antioxidant, and may protect cells against oxidative stress. We tested two different compounds that deplete primary cortical cultures containing both neurons and astrocytes of intracellular GSH, L-buthionine-sulfoximine (L-BSO), and diethyl maleate (DEM). Both compounds caused significant concentration and time dependent decreases in intracellular GSH levels. However; DEM caused an increase in radiolabeled cystine uptake through system xc −, while unexpectedly BSO caused a decrease in uptake. The compounds caused similar low levels of neurotoxicity, while only BSO caused an increase in oxidative stress. The mechanism of GSH depletion by these two compounds is different, DEM directly conjugates to GSH, while BSO inhibits γ-glutamylcysteine synthetase, a key enzyme in GSH synthesis. As would be expected from these mechanisms of action, DEM caused a decrease in intracellular cysteine, while BSO increased cysteine levels. The results suggest that negative feedback by intracellular cysteine is an important regulator of system xc − in this culture system.

The cystine/glutamate antiporter (system xc-) transports cystine into cell in exchange for glutamate. Fibroblast growth factor-2 (FGF-2) upregulates system xc- selectively on astrocytes, which leads to increased cystine uptake, the substrate for glutathione production, and increased glutamate release. While increased intracellular glutathione can limit oxidative stress, the increased glutamate release can potentially lead to excitotoxicity to neurons. To test this hypothesis, mixed neuronal and glial cortical cultures were treated with FGF-2. Treatment with FGF-2 for 48 h caused a significant neuronal death in these cultures. Cell death was not observed in neuronal-enriched cultures, or astrocyte-enriched cultures, suggesting the toxicity was the result of neuron-glia interaction. Blocking system xc- eliminated the neuronal death as did the AMPA/kainate receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX), but not the NMDA receptor antagonist memantine. When cultures were exposed directly to glutamate, both NBQX and memantine blocked the neuronal toxicity. The mechanism of this altered profile of glutamate receptor mediated toxicity by FGF-2 is unclear. The selective calcium permeable AMPA receptor antagonist 1-naphthyl acetyl spermine (NASPM) failed to offer protection. The most likely explanation for the results is that 48 h FGF-2 treatment induces AMPA/kainate receptor toxicity through increased system xc- function resulting in increased release of glutamate. At the same time, FGF-2 alters the sensitivity of the neurons to glutamate toxicity in a manner that promotes selective AMPA/kainate receptor mediated toxicity.

In the central nervous system, cystine import in exchange for glutamate through system xc− is critical for the production of the antioxidant glutathione by astrocytes, as well as the maintenance of extracellular glutamate. Therefore, regulation of system xc− activity affects multiple aspects of cellular physiology and may contribute to disease states. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuronally-derived peptide that has already been demonstrated to modulate multiple aspects of glutamate signaling suggesting PACAP may also target activity of cystine-glutamate exchange via system xc−. In the current study, 24-hour treatment of primary cortical cultures containing neurons and glia with PACAP concentration-dependently increased system xc− function as measured by radiolabeled cystine uptake. Furthermore, the increase in cystine uptake was completely abolished by the system xc− inhibitor, (S)-4-carboxyphenylglycine (CPG), attributing increases in cystine uptake specifically to system xc− activity. Time course and quantitative PCR results indicate that PACAP signaling may increase cystine-glutamate exchange by increasing expression of xCT, the catalytic subunit of system xc−. Furthermore, the potentiation of system xc− activity by PACAP occurs via a PKA-dependent pathway that is not mediated by the PAC1R, but rather the shared vasoactive intestinal polypeptide receptor VPAC1R. Finally, assessment of neuronal, astrocytic, and microglial-enriched cultures demonstrated that only astrocyte-enriched cultures exhibit enhanced cystine uptake following both PACAP and VIP treatment. These data introduce a novel mechanism by which both PACAP and VIP regulate system xc− activity.

The study of the mechanism of β-N-methylamino-L-alanine (BMAA) neurotoxicity originally focused on its effects at the N-methyl-D-aspartate (NMDA) receptor. In recent years, it has become clear that its mechanism of action is more complicated. First, there are certain cell types, such as motor neurons and cholinergic neurons, where the dominate mechanism of toxicity is through action at AMPA receptors. Second, even in cortical neurons where the primary mechanism of toxicity appears to be activation of NMDA receptors, there are other mechanisms involved. We found that along with NMDA receptors, activation of mGLuR5 receptors and effects on the cystine/glutamate antiporter (system x-) were involved in the toxicity. The effects on system x- are of particular interest. System x- mediates the transport of cystine into the cell in exchange for releasing glutamate into the extracellular fluid. By releasing glutamate, system x- can potentially cause excitotoxicity. However, through providing cystine to the cell, it regulates the levels of cellular glutathione (GSH), the main endogenous intracellular antioxidant, and in this way may protect cells against oxidative stress. We have previously published that BMAA inhibits cystine uptake leading to GSH depletion and had indirect evidence that BMAA is transported into the cells by system x-. We now present direct evidence that BMAA is transported into both astrocytes and neurons through system x-. The fact that BMAA is transported by system x- also provides a mechanism for BMAA to enter brain cells potentially leading to misincorporation into proteins and protein misfolding.