Cystine-glutamate antiporter deletion accelerates motor recovery and improves histological outcomes following spinal cord injury in mice.
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ABSTRACT:
SUBMITTER: Sprimont L
PROVIDER: S-EPMC8190126 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Understanding the molecular basis of the regenerative response following hepatic injury holds promise for improved treatment of liver diseases. Here, we report an innovative method to profile gene expression specifically in the hepatocytes that regenerate the liver following toxic injury. We used the Fah-/- mouse, a model of hereditary tyrosinemia, which conditionally undergoes severe liver injury unless fumarylacetoacetate hydrolase (FAH) expression is reconstituted ectopically. We used translating ribosome affinity purification followed by high-throughput RNA sequencing (TRAP-seq) to isolate mRNAs specific to repopulating hepatocytes. We uncovered upstream regulators and important signaling pathways that are highly enriched in genes changed in regenerating hepatocytes. Specifically, we found that glutathione metabolism, particularly the gene Slc7a11 encoding the cystine/glutamate antiporter (xCT), is massively upregulated during liver regeneration. Furthermore, we show that Slc7a11 overexpression in hepatocytes enhances, and its suppression inhibits, repopulation following toxic injury. TRAP-seq allows cell type-specific expression profiling in repopulating hepatocytes and identified xCT, a factor that supports antioxidant responses during liver regeneration. xCT has potential as a therapeutic target for enhancing liver regeneration in response to liver injury.
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Project description:CD36 is a pleiotropic receptor involved in several pathophysiological conditions, including cerebral ischemia, neurovascular dysfunction and atherosclerosis, and recent reports implicate its involvement in the endoplasmic reticulum stress response (ERSR). We hypothesized that CD36 signaling contributes to the inflammation and microvascular dysfunction following spinal cord injury. Following contusive injury, CD36(-/-) mice demonstrated improved hindlimb functional recovery and greater white matter sparing than CD36(+/+) mice. CD36(-/-) mice exhibited a reduced macrophage, but not neutrophil, infiltration into the injury epicenter. Fewer infiltrating macrophages were either apoptotic or positive for the ERSR marker, phospho-ATF4. CD36(-/-) mice also exhibited significant improvements in injury heterodomain vascularity and function. These microvessels accumulated less of the oxidized lipid product 4-hydroxy-trans-2-nonenal (4HNE) and exhibited a reduced ERSR, as detected by vascular phospho-ATF4, CHOP and CHAC-1 expression. In cultured primary endothelial cells, deletion of CD36 diminished 4HNE-induced phospho-ATF4 and CHOP expression. A reduction in phospho-eIF2? and subsequent increase in KDEL-positive, ER-localized proteins suggest that 4HNE-CD36 signaling facilitates the detection of misfolded proteins upstream of eIF2? phosphorylation, ultimately leading to CHOP-induced apoptosis. We conclude that CD36 deletion modestly, but significantly, improves functional recovery from spinal cord injury by enhancing vascular function and reducing macrophage infiltration. These phenotypes may, in part, stem from reduced ER stress-induced cell death within endothelial and macrophage cells following injury.
| S-EPMC4086463 | biostudies-literature
Project description:The cystine-glutamate antiporter (system xc-) is a Na+-independent amino acid transporter that exchanges extracellular cystine for intracellular glutamate. It is thought to play a critical role in cellular redox processes through regulation of intracellular glutathione synthesis via cystine uptake. In gliomas, system xc- expression is universally up-regulated while that of glutamate transporters down-regulated, leading to a progressive accumulation of extracellular glutamate and excitotoxic cell death of the surrounding non-tumorous tissue. Additionally, up-regulation of system xc- in activated microglia has been implicated in the pathogenesis of several neurodegenerative disorders mediated by excess glutamate. Consequently, system xc- is a new drug target for brain cancer and neuroinflammatory diseases associated with excess extracellular glutamate. Unfortunately no potent and selective small molecule system xc- inhibitors exist and to our knowledge, no high throughput screening (HTS) assay has been developed to identify new scaffolds for inhibitor design. To develop such an assay, various neuronal and non-neuronal human cells were evaluated as sources of system xc-. Human glioma cells were chosen based on their high system xc- activity. Using these cells, [14C]-cystine uptake and cystine-induced glutamate release assays were characterized and optimized with respect to cystine and protein concentrations and time of incubation. A pilot screen of the LOPAC/NINDS libraries using glutamate release demonstrated that the logistics of the assay were in place but unfortunately, did not yield meaningful pharmacophores. A larger, HTS campaign using the 384-well cystine-induced glutamate release as primary assay and the 96-well 14C-cystine uptake as confirmatory assay is currently underway. Unexpectedly, we observed that the rate of cystine uptake was significantly faster than the rate of glutamate release in human glioma cells. This was in contrast to the same rates of cystine uptake and glutamate release previously reported in normal human fibroblast cells.
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Project description:During brain ischemia, an excessive release of glutamate triggers neuronal death through the overactivation of NMDA receptors (NMDARs); however, the underlying pathways that alter glutamate homeostasis and whether synaptic or extrasynaptic sites are responsible for excess glutamate remain controversial. Here, we monitored ischemia-gated currents in pyramidal cortical neurons in brain slices from rodents in response to oxygen and glucose deprivation (OGD) as a real-time glutamate sensor to identify the source of glutamate release and determined the extent of neuronal damage. Blockade of excitatory amino acid transporters or vesicular glutamate release did not inhibit ischemia-gated currents or neuronal damage after OGD. In contrast, pharmacological inhibition of the cystine/glutamate antiporter dramatically attenuated ischemia-gated currents and cell death after OGD. Compared with control animals, mice lacking a functional cystine/glutamate antiporter exhibited reduced anoxic depolarization and neuronal death in response to OGD. Furthermore, glutamate released by the cystine/glutamate antiporter activated extrasynaptic, but not synaptic, NMDARs, and blockade of extrasynaptic NMDARs reduced ischemia-gated currents and cell damage after OGD. Finally, PET imaging showed increased cystine/glutamate antiporter function in ischemic rats. Altogether, these data suggest that cystine/glutamate antiporter function is increased in ischemia, contributing to elevated extracellular glutamate concentration, overactivation of extrasynaptic NMDARs, and ischemic neuronal death.
| S-EPMC4109556 | biostudies-other
Project description:The major cellular antioxidant glutathione is depleted during HIV infection and in obesity. Although the consequence of glutathione depletion on immune function is starting to emerge, it is currently not known whether glutathione dysregulation influences the differentiation and maturation of dendritic cells (DCs). Moreover, the effect of glutathione depletion on DC effector functions, such as Ag presentation, is poorly understood. Glutathione synthesis depends on the cystine/glutamate antiporter, which transports the rate-limiting precursor cystine into the cell in exchange for glutamate. In this paper, we present a detailed study of antiporter function in DCs and demonstrate a role for the antiporter in DC differentiation and cross-presentation. We show that the antiporter is the major mechanism for transport of cystine and glutamate and modulates the intracellular glutathione content and glutathione efflux from DCs. Blocking antiporter-dependent cystine transport decreases intracellular glutathione levels, and these effects correlate with reduced transcription of the functional subunit of the antiporter. We further demonstrate that blocking antiporter activity interferes with DC differentiation from monocyte precursors, but antiporter activity is not required for LPS-induced phenotypic maturation. Finally, we show that inhibiting antiporter uptake of cystine interferes with presentation of exogenous Ag to class II MHC-restricted T cells and blocks cross-presentation on MHC class I. We conclude that aberrant antiporter function disrupts glutathione homeostasis in DCs and may contribute to impaired immunity in the diseased host.
| S-EPMC3100200 | biostudies-literature
Project description:T cell-invigorating cancer immunotherapies have near-curative potential. However, their clinical benefit is currently limited, as only a fraction of patients respond, suggesting that these regimens may benefit from combination with tumor-targeting treatments. As oncogenic progression is accompanied by alterations in metabolic pathways, tumors often become heavily reliant on antioxidant machinery and may be susceptible to increases in oxidative stress. The cystine-glutamate antiporter xCT is frequently overexpressed in cancer and fuels the production of the antioxidant glutathione; thus, tumors prone to redox stress may be selectively vulnerable to xCT disruption. However, systemic inhibition of xCT may compromise antitumor immunity, as xCT is implicated in supporting antigen-induced T cell proliferation. Therefore, we utilized immune-competent murine tumor models to investigate whether cancer cell expression of xCT was required for tumor growth in vivo and if deletion of host xCT impacted antitumor immune responses. Deletion of xCT in tumor cells led to defective cystine uptake, accumulation of reactive oxygen species, and impaired tumor growth, supporting a cancer cell-autonomous role for xCT. In contrast, we observed that, although T cell proliferation in culture was exquisitely dependent on xCT expression, xCT was dispensable for T cell proliferation in vivo and for the generation of primary and memory immune responses to tumors. These findings prompted the combination of tumor cell xCT deletion with the immunotherapeutic agent anti-CTLA-4, which dramatically increased the frequency and durability of antitumor responses. Together, these results identify a metabolic vulnerability specific to tumors and demonstrate that xCT disruption can expand the efficacy of anticancer immunotherapies.
| S-EPMC6511047 | biostudies-literature
Project description:As noted by Warburg, many cancer cells depend on the consumption of glucose. We performed a genetic screen to identify factors responsible for glucose addiction and recovered the two subunits of the xCT antiporter (system xc-), which plays an antioxidant role by exporting glutamate for cystine. Disruption of the xCT antiporter greatly improves cell viability after glucose withdrawal, because conservation of glutamate enables cells to maintain mitochondrial respiration. In some breast cancer cells, xCT antiporter expression is upregulated through the antioxidant transcription factor Nrf2 and contributes to their requirement for glucose as a carbon source. In cells carrying patient-derived mitochondrial DNA mutations, the xCT antiporter is upregulated and its inhibition improves mitochondrial function and cell viability. Therefore, although upregulation of the xCT antiporter promotes antioxidant defence, it antagonizes glutamine metabolism and restricts nutrient flexibility. In cells with mitochondrial dysfunction, the potential utility of xCT antiporter inhibition should be further tested.
| S-EPMC5413954 | biostudies-literature
Project description:PurposeThe purpose of this study was to determine the importance of the xCT is a subunit. The cystine/glutamate antiporter is actually system xc-xCT subunit of the cystine/glutamate antiporter in maintaining redox balance by investigating the effects of the loss of xCT on lens transparency and cystine/cysteine balance in the aqueous humour.MethodsC57Bl/6 wild-type and xCT knockout mice at five age groups (6 weeks to 12 months) were used. Lens transparency was examined using a slit-lamp and morphological changes visualized by immunolabelling and confocal microscopy. Quantification of glutathione in lenses and cysteine and cystine levels in the aqueous was conducted by liquid chromatography tandem mass spectrometry (LC-MS/MS).ResultsSlit-lamp examinations revealed that 3-month-old wild-type mice and xCT knockout mice lenses exhibited an anterior localized cataract. The frequency of this cataract significantly increased in the knockout mice compared to the wild-type mice. Morphological studies revealed a localized swelling of the lens fiber cells at the anterior pole. Glutathione levels in whole lenses were similar between wild-type and knockout mice. However, glutathione levels were significantly decreased at 3 months in the knockout mice in the lens epithelium compared to the wild-type mice. Aqueous cysteine levels remained similar between wild-type and knockout mice at all age groups, whereas cystine levels were significantly increased in 3-, 9-, and 12-month-old knockout mice compared to wild-type mice.ConclusionsLoss of xCT resulted in the depletion of glutathione in the epithelium and an oxidative shift in the cysteine/cystine ratio of the aqueous. Together, these oxidative changes may contribute to the accelerated development of an anterior cataract in knockout mice, which appears to be a normal feature of aging in wild-type mice.
| S-EPMC8237109 | biostudies-literature
Project description:BackgroundTreatments immediately after spinal cord injury (SCI) are anticipated to decrease neuronal death, disruption of neuronal connections, demyelination, and inflammation, and to improve repair and functional recovery. Currently, little can be done to modify the acute phase, which extends to the first 48 hours post-injury. Efforts to intervene have focused on the subsequent phases - secondary (days to weeks) and chronic (months to years) - to both promote healing, prevent further damage, and support patients suffering from SCI.MethodsWe used a contusion model of SCI in female mice, and delivered a small molecule reagent during the early phase of injury. Histological and behavioral outcomes were assessed and compared.ResultsWe find that the reagent Pifithrin-μ (PFT-μ) acts early and directly on microglia in vitro, attenuating their activation. When administered during the acute phase of SCI, PFT-μ resulted in reduced lesion size during the initial inflammatory phase, and reduced the numbers of pro-inflammatory microglia and macrophages. Treatment with PFT-μ during the early stage of injury maintained a stable anti-inflammatory environment.ConclusionsOur results indicate that a small molecule reagent PFT-μ has sustained immunomodulatory effects following a single dose after injury.
| S-EPMC6488873 | biostudies-literature
Project description:The astrocytic cystine/glutamate antiporter system xc- represents an important source of extracellular glutamate in the central nervous system, with potential impact on excitatory neurotransmission. Yet, its function and importance in brain physiology remain incompletely understood. Employing slice electrophysiology and mice with a genetic deletion of the specific subunit of system xc-, xCT (xCT-/- mice), we uncovered decreased neurotransmission at corticostriatal synapses. This effect was partly mitigated by replenishing extracellular glutamate levels, indicating a defect linked with decreased extracellular glutamate availability. We observed no changes in the morphology of striatal medium spiny neurons, the density of dendritic spines, or the density or ultrastructure of corticostriatal synapses, indicating that the observed functional defects are not due to morphological or structural abnormalities. By combining electron microscopy with glutamate immunogold labeling, we identified decreased intracellular glutamate density in presynaptic terminals, presynaptic mitochondria, and in dendritic spines of xCT-/- mice. A proteomic and kinomic screen of the striatum of xCT-/- mice revealed decreased expression of presynaptic proteins and abnormal kinase network signaling, that may contribute to the observed changes in postsynaptic responses. Finally, these corticostriatal deregulations resulted in a behavioral phenotype suggestive of autism spectrum disorder in the xCT-/- mice; in tests sensitive to corticostriatal functioning we recorded increased repetitive digging behavior and decreased sociability. To conclude, our findings show that system xc- plays a previously unrecognized role in regulating corticostriatal neurotransmission and influences social preference and repetitive behavior.
| S-EPMC7609546 | biostudies-literature