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Comparative Study
. 2006 Oct 11;26(41):10514-23.
doi: 10.1523/JNEUROSCI.3178-06.2006.

Cystine/glutamate exchange modulates glutathione supply for neuroprotection from oxidative stress and cell proliferation

Andy Y Shih  1 , Heidi Erb, Xiaojian Sun, Shigenobu Toda, Peter W Kalivas, Timothy H Murphy
Affiliations
Comparative Study

Cystine/glutamate exchange modulates glutathione supply for neuroprotection from oxidative stress and cell proliferation (VSports app下载)

Andy Y Shih et al. J Neurosci. .

Abstract (VSports最新版本)

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 beta-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 VSports手机版. .

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Figures

Figure 1.
Figure 1.
Characterization of an anti-xCT polyclonal antibody and xCT regional brain distribution. A, xCT and 4F2hc membrane topology depicting xCT antibody epitope. xCT and 4F2hc heterodimerize through an extracellular disulfide link that can be disrupted with the reducing agents β- mercaptoethanol and dithiothreitol, collectively referred to as β-ME. a.a., Amino acid. B, The anti-xCT antibody detected a major 35 kDa band and a minor 55 kDa band (xCT-mod) that was specific only to xCT and Nrf2-overexpressing astrocyte cultures. Low basal levels of the same bands were detected in the GFP-overexpressing control. The 105 kDa band seen under nonreducing conditions corresponded to the xCT plus 4F2hc heterodimer. Treatment with β-ME disrupted the disulfide link between xCT and 4F2hc, causing the 105 kDa band to disappear, shifting to monomeric 35 kDa xCT. xCT-mod could not be disrupted by reducing conditions, suggesting that it is not a disulfide-linked xCT homodimer. A strong nonspecific band was detected at 95 kDa. Stripping and reprobing with an anti-4F2hc antibody revealed the 105 kDa band (xCT + 4F2hc) under nonreducing conditions and a 80 kDa band (4F2hc only) with reducing conditions. C, Examination of xCT regional distribution in the embryonic rat brain revealed high expression of 35 kDa xCT and xCT-mod in the meninges, whereas all other brain regions examined express primarily xCT-mod. D, In the adult rat brain, 35 kDa xCT and xCT-mod was also highly expressed in the meninges. In contrast to embryonic tissues, other adult brain regions express primarily 35 kDa xCT. Blots are representative results from at least three independent experiments on separately prepared cultures or separate rats.
Figure 2.
Figure 2.
Meningeal cells exhibit higher GSH content and enhanced cystine-dependent GSH synthesis. Characteristics of GSH metabolism in primary meningeal and astrocyte cultures were examined using a previously characterized mCBi labeling assay (Sun et al., 2005). A, Meningeal cells showed an increased initial slope of mCBi fluorescence labeling over time, corresponding to increased glutathione S-transferase activity and rate of GSH–mCBi conjugation. Importantly, a higher plateau of fluorescence in meningeal cells (after 20 min of labeling) indicates higher cellular GSH content, because GSH levels were limiting in this assay. B, After the fluorescence plateau was reached, the mCBi labeling assay could also be used to monitor new GSH synthesis. Application of 100 μm cystine to the culture medium generated a larger increase in GSH–mCBi fluorescence from meningeal cultures compared with astrocytes, indicating that meningeal cells preferentially uptake cystine for GSH synthesis. The HBSS vehicle control had no significant effect on new GSH synthesis in either meningeal cells or astrocytes. The graphs in A and B are representative traces from at least three independent experiments. C, Consistent with preferential meningeal use of cystine to make GSH, Na+-independent l-S35-cystine uptake was threefold higher than astrocytes and fit the pharmacological profile of system xc−. All competitors were introduced at a concentration of 1 mm, except quisqualic acid (300 μm). Data represent the mean ± SEM from three independent experiments on separately prepared cultures. *p < 0.05 compared with astrocytes. Cys, l-Cystine; Glu, l-glutamate; Quis, quisqualic acid; HCA, homocysteic acid; L-Asp, l-aspartate; D-Asp, d-aspartate; Kain, kainic acid.
Figure 3.
Figure 3.
Meningeal cells are highly neuroprotective. Various non-neuronal cell types (meningeal cells, astrocytes, and peripheral fibroblasts) (7–10 DIV) were transplanted into immature cortical neuron cultures. To test the neuroprotective potential of each cell type, the cocultures were exposed to oxidative glutamate toxicity (A, B; 3 mm glutamate for 24 h). Meningeal cells and fibroblasts were initially seeded at a three times lower density than astrocytes because of a faster proliferation rate, so that a similar number of transplanted cells were present just before glutamate exposure (as presented on the x-axis). After oxidative glutamate toxicity, neurons were identified by immunocytochemistry using anti-NSE antibodies and counted. Meningeal cells conferred significantly more protection than astrocytes at a similar cell density, whereas fibroblasts were much less neuroprotective. Data represent the mean ± SEM from three independent experiments on separately prepared cultures. *p < 0.05 compared with astrocytes.
Figure 4.
Figure 4.
xCT overexpression in astrocytes enhances system xc− activity, increases GSH synthesis, and provides neuroprotection in a dose-dependent manner. A, Ad-xCT-infected mixed cultures exhibited an approximate fivefold higher Na+-independent l-[3H]glutamate uptake than Ad-GFP-infected control cultures. Enhanced uptake fit the pharmacological profile previously defined for system xc−. CssC, l-Cystine; Glu, l-glutamate; Quis, quisqualic acid; HCA, homocysteic acid; L-Asp, l-aspartate; D-Asp, d-aspartate; Kain, kainic acid. All competitors were introduced at a concentration of 1 mm, except quisqualic acid (300 μm). B, C, Ad-xCT infection increased total intracellular GSH and GSH in the media by ∼1.7-fold and ∼1.6-fold, respectively, compared with Ad-GFP control. D, E, Ad-xCT-infected astrocytes were transplanted into naive immature cortical neuron cultures and exposed to oxidative glutamate (Glu) toxicity. Ad-xCT-infected astrocytes, seeded at 2% of the total cell number, were capable of providing widespread neuronal protection. Ad-GFP-infected astrocytes were relatively not neuroprotective at the same density. F, GSH production was essential for neuroprotection because pretreatment of the by Ad-xCT-infected astrocytes with 200 μm BSO (an irreversible γ-glutamylcysteine synthetase inhibitor) abolished subsequent xCT-mediated neuroprotection. G, Ad-xCT-infected astrocytes (transplanted at 2% of total cell number) also conferred neuroprotection from direct exposure to the reactive O2 species, H2O2 (10 μm for 24 h). Data represent the mean ± SEM from at least three independent experiments on separately prepared cultures. *p < 0.05 or ***p < 0.001 compared with Ad-GFP-infected astrocytes.
Figure 5.
Figure 5.
xCT-overexpressing fibroblasts confer neuroprotection during oxidative glutamate toxicity. Ad-xCT-infected fibroblasts were transplanted into naive immature cortical neuron cultures and exposed to oxidative glutamate toxicity (A, B; as described in Fig. 4D,E). xCT overexpression was sufficient to increase neuroprotection by fibroblasts, a cell type that was not associated with CNS neuroprotection. Ad-GFP-infected fibroblasts were relatively not neuroprotective in comparison. Data represent the mean ± SEM from three independent experiments on separately prepared cultures. *p < 0.05 compared with Ad-GFP-infected fibroblasts.
Figure 6.
Figure 6.
Astrocytes derived from sut/sut mice are unable to proliferate early in culture. Astrocyte cultures were prepared from P0–P2 sut/sut mouse pups (sut/sut) or C3H/HeSnJ controls (+/+). After 7 DIV, total viable cells from each well of a 12-well plate were counted using Trypan blue exclusion. Under normal culture conditions, sut/sut astrocytes were unable to proliferate, whereas +/+ astrocytes grew normally. The application of 55 μm β-ME to the culture medium rescued sut/sut astrocyte growth but had no significant effect on +/+ astrocytes. Data represent the mean ± SEM from three independent experiments on separately prepared cultures. ***p < 0.001 compared with sut/sut astrocytes with β-ME treatment.
Figure 7.
Figure 7.
Adult sut/sut mice exhibit brain atrophy. A, Gross brain morphology was compared between cresyl violet-stained coronal sections from sut/sut mice (sut/sut) (n = 7; 3 males and 4 females) and C3H/HeSnJ controls (+/+) (n = 6; 3 males and 3 females). B, C, sut/sut mice exhibited an overall smaller brain size (B; p = 0.0057) and severely enlarged lateral ventricles (C; p < 0.0001). D, E, Furthermore, we observed cortical thinning (D; p = 0.0059) and reduced striatal area (E; p = 0.023). F, No significant difference, however, was seen in the hippocampal area, although a trend was noted (p = 0.34). Data represent the mean ± SEM. All statistical analyses were performed with two-way ANOVA; p < 0.05 was considered statistically significant.
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