Cystine/Glutamate Exchange Modulates Glutathione Supply for Neuroprotection from Oxidative Stress and Cell Proliferation

Abstract

The cystine/glutamate exchanger (xCT) provides intracellular cyst(e)ine for production of glutathione, a major cellular antioxidant. Using xCT overexpression and underexpression, we present evidence that xCT-dependent glutathione production modulates both neuroprotection from oxidative stress and cell proliferation. In embryonic and adult rat brain, xCT protein was enriched at the CSF-brain barrier (i.e., meninges) and also expressed in the cortex, hippocampus, striatum, and cerebellum. To examine the neuroprotective role of xCT, various non-neuronal cell types (astrocytes, meningeal cells, and peripheral fibroblasts) were cocultured with immature cortical neurons and exposed to oxidative glutamate toxicity, a model involving glutathione depletion. Cultured meningeal cells, which naturally maintain high xCT expression, were more neuroprotective than astrocytes. Selective xCT overexpression in astrocytes was sufficient to enhance glutathione synthesis/release and confer potent glutathione-dependent neuroprotection from oxidative stress. Moreover, normally nonprotective fibroblasts could be re-engineered to be neuroprotective with ectopic xCT overexpression indicating that xCT is a key step in the pathway to glutathione synthesis. Conversely, astrocytes and meningeal cells derived from sut/sut mice (xCT loss-of-function mutants) showed greatly reduced proliferation in culture attributable to increased oxidative stress and thiol deficiency, because growth could be rescued by the thiol-donor ␤-mercaptoethanol. Strikingly, sut/sut mice developed brain atrophy by early adulthood, exhibiting ventricular enlargement, thinning of the cortex, and shrinkage of the striatum. Our results indicate that xCT can provide neuroprotection by enhancing glutathione export from non-neuronal cells such as astrocytes and meningeal cells. Furthermore, xCT is critical for cell proliferation during development in vitro and possibly in vivo.

Cocaine-Induced Adaptations in Cellular Redox Balance Contributes to Enduring Behavioral Plasticity

“…The finding that constitutive genetic deletion of GSTP1P2 did not alter accumbens levels of xCT, the catalytic subunit of system Xc- (Shih et al, 2006), is not supportive of a linkage between the previously reported cocaine-induced downregulation of system Xc- (Baker et al, 2003) and the changes produced in GSTP1P2 and cellular redox shown herein. Downregulation of xCT reduces extracellular glutamatergic tone on synaptic mGluRs and this is thought to contribute to the vulnerability to reinstate cocaineseeking (Kalivas, 2009).…”

“…Samples were denatured, reduced in sample buffer, and loaded on a 10% Criterion XT Bis-Tris gel with MOPS running buffer (Bio-Rad). The proteins were transferred onto a PVDF membrane with an iBlot dry blotting system (Invitrogen, Carlsbad, CA), blocked in 5% milk in TBS-Tween, and incubated overnight at 4 1C in rabbit anti-GSH reductase (Abcam, 1 : 1000), rabbit antiglutaredoxin 1 (Abcam, 1 : 250), rabbit anti-GSTpi (MBL, 1 : 1000), rabbit anti-xCT (Shih et al, 2006;Szumlinski et al, 2004; 1 : 500), or rabbit anti-thioredoxin (Cell Signaling, 1 : 1000). After extensive washing in TBS-Tween (3 Â 10 min), the membranes were probed with goat antirabbit HRP-conjugated antibodies (Millipore, 1 : 10 000).…”

Dissection of mitogenic and neurodegenerative actions of cystine and glutamate in malignant gliomas

“…Although glioma cells appear to be highly dependent on system X c À -mediated cystine incorporation required for glutathione production and thus the scavenging of reactive oxygen species (Chung et al, 2005;Chung and Sontheimer, 2009;Pham et al, 2010), normal cells might be less vulnerable. This notion is supported by genetic deletion studies, that is, xCT-deficient mice are healthy in appearance and fertile (Sato et al, 2005;Shih et al, 2006;Liu et al, 2007). Moreover, resistance of tumor cells to a variety of anticancer drugs is often associated with increased glutathione levels (Huang et al, 2005).…”

Regulation of Neuronal Glutathione Synthesis

“…Cystine, an oxidized form of two cysteines with a disulfide linkage, is utilized as a substrate for GSH synthesis in some types of brain cells (61). Cystine is taken up into the cell in exchange for glutamate via system xc-, which can be inhibited by a high extracellular glutamate concentration (62).…”