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Increased expression of cystine/glutamate antiporter in multiple sclerosis.

Pampliega O, Domercq M, Soria FN, Villoslada P, Rodríguez-Antigüedad A, Matute C - J Neuroinflammation (2011)

Bottom Line: In addition, xCT expression is also increased in EAE and in the disease proper.In the later, high expression of xCT occurs both in the central nervous system (CNS) and in peripheral blood cells.Together, these results reveal that increased expression of the cystine/glutamate antiporter system x(c)⁻ in MS provides a link between inflammation and excitotoxicity in demyelinating diseases.

View Article: PubMed Central - HTML - PubMed

Affiliation: Neurotek-UPV/EHU, Parque Tecnológico de Bizkaia, Zamudio, Bizkaia, Spain.

ABSTRACT

Background: Glutamate excitotoxicity contributes to oligodendrocyte and tissue damage in multiple sclerosis (MS). Intriguingly, glutamate level in plasma and cerebrospinal fluid of MS patients is elevated, a feature which may be related to the pathophysiology of this disease. In addition to glutamate transporters, levels of extracellular glutamate are controlled by cystine/glutamate antiporter x(c)⁻, an exchanger that provides intracellular cystine for production of glutathione, the major cellular antioxidant. The objective of this study was to analyze the role of the system x(c)⁻ in glutamate homeostasis alterations in MS pathology.

Methods: Primary cultures of human monocytes and the cell line U-937 were used to investigate the mechanism of glutamate release. Expression of cystine glutamate exchanger (xCT) was quantified by quantitative PCR, Western blot, flow cytometry and immunohistochemistry in monocytes in vitro, in animals with experimental autoimmune encephalomyelitis (EAE), the animal model of MS, and in samples of MS patients.

Results and discussion: We show here that human activated monocytes release glutamate through cystine/glutamate antiporter x(c)⁻ and that the expression of the catalytic subunit xCT is upregulated as a consequence of monocyte activation. In addition, xCT expression is also increased in EAE and in the disease proper. In the later, high expression of xCT occurs both in the central nervous system (CNS) and in peripheral blood cells. In particular, cells from monocyte-macrophage-microglia lineage have higher xCT expression in MS and in EAE, indicating that immune activation upregulates xCT levels, which may result in higher glutamate release and contribution to excitotoxic damage to oligodendrocytes.

Conclusions: Together, these results reveal that increased expression of the cystine/glutamate antiporter system x(c)⁻ in MS provides a link between inflammation and excitotoxicity in demyelinating diseases.

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xCT expression is increased in the CNS of rats with EAE. A. Histogram showing the neurological score during the course of acute EAE induced in Lewis rats by immunization with myelin basic protein. The peak of neurological disability was at day 14 post-immunization, which was selected for obtaining tissue samples. B. xCT mRNA (left) and protein (right) expression in spinal cord from control and acute EAE rats, as assessed by qPCR and Western blot analysis. Data are referred to mean expression level of controls (n = 5-6). C. Double immunofluorescence for xCT (green) and OX-42 (red), a marker of microglia and infiltrating macrophages. OX42+ cells express high xCT levels in acute EAE. Both meninges (asterisk in top) and infiltrating cells (bottom) in inflammatory foci show high levels of xCT in rat spinal cord with EAE as compared to controls. D. Microglial cells (OX42+ cells) of EAE rats have higher xCT levels in spinal cord than controls. Notice the difference between resting microglia in control rats, with ramified morphology (arrows in control) and microglia in EAE showing round shaped morphology, characteristic of its activated state (arrows in EAE). Scale bar = 20 μm.E. xCT mRNA (left) and protein (right) expression in spinal cord from control and chronic EAE mice, assessed by qPCR and Western blot analysis. Data are referred to mean expression level of controls (n = 5).
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Figure 3: xCT expression is increased in the CNS of rats with EAE. A. Histogram showing the neurological score during the course of acute EAE induced in Lewis rats by immunization with myelin basic protein. The peak of neurological disability was at day 14 post-immunization, which was selected for obtaining tissue samples. B. xCT mRNA (left) and protein (right) expression in spinal cord from control and acute EAE rats, as assessed by qPCR and Western blot analysis. Data are referred to mean expression level of controls (n = 5-6). C. Double immunofluorescence for xCT (green) and OX-42 (red), a marker of microglia and infiltrating macrophages. OX42+ cells express high xCT levels in acute EAE. Both meninges (asterisk in top) and infiltrating cells (bottom) in inflammatory foci show high levels of xCT in rat spinal cord with EAE as compared to controls. D. Microglial cells (OX42+ cells) of EAE rats have higher xCT levels in spinal cord than controls. Notice the difference between resting microglia in control rats, with ramified morphology (arrows in control) and microglia in EAE showing round shaped morphology, characteristic of its activated state (arrows in EAE). Scale bar = 20 μm.E. xCT mRNA (left) and protein (right) expression in spinal cord from control and chronic EAE mice, assessed by qPCR and Western blot analysis. Data are referred to mean expression level of controls (n = 5).

Mentions: Rats immunized with myelin basic protein showed maximal motor deficits at around 14 days postimmunization (Figure 3A). At that stage, expression of mRNA encoding xCT was more abundant in spinal cord samples from rats with EAE than in control animals (n = 6; p = 0.013; Figure 3B left). Accordingly, xCT protein levels were significantly increased in EAE rats (n = 5; p < 0.05; Figure 3B right). We also analysed the expression of xCT in the spinal cord by double immunofluorescence using antibodies to OX-42, a marker of microglia lineage. No immunolabeling was detected after preincubation xCT antibodies with the corresponding peptide (Additional file 3, Figure S2), demonstrating the specificity of the immunofluorescence signal. xCT expression was dramatically increased in the spinal cord of rats with EAE. The expression was particularly increased in meninges in EAE animals, showing clear signs of inflammation (asterisk in Figure 3C top right). There was also a clear increase of xCT in OX42+ infiltrating cells, which were organized in clusters around vessels (Figure 3C bottom). In addition, activated microglial cells in EAE rats showed a massive upregulation of xCT (Figure 3D). Activated microglia was identified by their characteristic ameboid shape in contrast with the ramified appearance of resting microglia in control animals (arrows in Figure 3D). Increases in xCT expression at the level of RNA and protein were also detected in the chronic EAE mice (n = 5 for RNA and protein; p < 0.05; Figure 3E), a model that reproduces the chronic and progressive phase of MS.


Increased expression of cystine/glutamate antiporter in multiple sclerosis.

Pampliega O, Domercq M, Soria FN, Villoslada P, Rodríguez-Antigüedad A, Matute C - J Neuroinflammation (2011)

xCT expression is increased in the CNS of rats with EAE. A. Histogram showing the neurological score during the course of acute EAE induced in Lewis rats by immunization with myelin basic protein. The peak of neurological disability was at day 14 post-immunization, which was selected for obtaining tissue samples. B. xCT mRNA (left) and protein (right) expression in spinal cord from control and acute EAE rats, as assessed by qPCR and Western blot analysis. Data are referred to mean expression level of controls (n = 5-6). C. Double immunofluorescence for xCT (green) and OX-42 (red), a marker of microglia and infiltrating macrophages. OX42+ cells express high xCT levels in acute EAE. Both meninges (asterisk in top) and infiltrating cells (bottom) in inflammatory foci show high levels of xCT in rat spinal cord with EAE as compared to controls. D. Microglial cells (OX42+ cells) of EAE rats have higher xCT levels in spinal cord than controls. Notice the difference between resting microglia in control rats, with ramified morphology (arrows in control) and microglia in EAE showing round shaped morphology, characteristic of its activated state (arrows in EAE). Scale bar = 20 μm.E. xCT mRNA (left) and protein (right) expression in spinal cord from control and chronic EAE mice, assessed by qPCR and Western blot analysis. Data are referred to mean expression level of controls (n = 5).
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Figure 3: xCT expression is increased in the CNS of rats with EAE. A. Histogram showing the neurological score during the course of acute EAE induced in Lewis rats by immunization with myelin basic protein. The peak of neurological disability was at day 14 post-immunization, which was selected for obtaining tissue samples. B. xCT mRNA (left) and protein (right) expression in spinal cord from control and acute EAE rats, as assessed by qPCR and Western blot analysis. Data are referred to mean expression level of controls (n = 5-6). C. Double immunofluorescence for xCT (green) and OX-42 (red), a marker of microglia and infiltrating macrophages. OX42+ cells express high xCT levels in acute EAE. Both meninges (asterisk in top) and infiltrating cells (bottom) in inflammatory foci show high levels of xCT in rat spinal cord with EAE as compared to controls. D. Microglial cells (OX42+ cells) of EAE rats have higher xCT levels in spinal cord than controls. Notice the difference between resting microglia in control rats, with ramified morphology (arrows in control) and microglia in EAE showing round shaped morphology, characteristic of its activated state (arrows in EAE). Scale bar = 20 μm.E. xCT mRNA (left) and protein (right) expression in spinal cord from control and chronic EAE mice, assessed by qPCR and Western blot analysis. Data are referred to mean expression level of controls (n = 5).
Mentions: Rats immunized with myelin basic protein showed maximal motor deficits at around 14 days postimmunization (Figure 3A). At that stage, expression of mRNA encoding xCT was more abundant in spinal cord samples from rats with EAE than in control animals (n = 6; p = 0.013; Figure 3B left). Accordingly, xCT protein levels were significantly increased in EAE rats (n = 5; p < 0.05; Figure 3B right). We also analysed the expression of xCT in the spinal cord by double immunofluorescence using antibodies to OX-42, a marker of microglia lineage. No immunolabeling was detected after preincubation xCT antibodies with the corresponding peptide (Additional file 3, Figure S2), demonstrating the specificity of the immunofluorescence signal. xCT expression was dramatically increased in the spinal cord of rats with EAE. The expression was particularly increased in meninges in EAE animals, showing clear signs of inflammation (asterisk in Figure 3C top right). There was also a clear increase of xCT in OX42+ infiltrating cells, which were organized in clusters around vessels (Figure 3C bottom). In addition, activated microglial cells in EAE rats showed a massive upregulation of xCT (Figure 3D). Activated microglia was identified by their characteristic ameboid shape in contrast with the ramified appearance of resting microglia in control animals (arrows in Figure 3D). Increases in xCT expression at the level of RNA and protein were also detected in the chronic EAE mice (n = 5 for RNA and protein; p < 0.05; Figure 3E), a model that reproduces the chronic and progressive phase of MS.

Bottom Line: In addition, xCT expression is also increased in EAE and in the disease proper.In the later, high expression of xCT occurs both in the central nervous system (CNS) and in peripheral blood cells.Together, these results reveal that increased expression of the cystine/glutamate antiporter system x(c)⁻ in MS provides a link between inflammation and excitotoxicity in demyelinating diseases.

View Article: PubMed Central - HTML - PubMed

Affiliation: Neurotek-UPV/EHU, Parque Tecnológico de Bizkaia, Zamudio, Bizkaia, Spain.

ABSTRACT

Background: Glutamate excitotoxicity contributes to oligodendrocyte and tissue damage in multiple sclerosis (MS). Intriguingly, glutamate level in plasma and cerebrospinal fluid of MS patients is elevated, a feature which may be related to the pathophysiology of this disease. In addition to glutamate transporters, levels of extracellular glutamate are controlled by cystine/glutamate antiporter x(c)⁻, an exchanger that provides intracellular cystine for production of glutathione, the major cellular antioxidant. The objective of this study was to analyze the role of the system x(c)⁻ in glutamate homeostasis alterations in MS pathology.

Methods: Primary cultures of human monocytes and the cell line U-937 were used to investigate the mechanism of glutamate release. Expression of cystine glutamate exchanger (xCT) was quantified by quantitative PCR, Western blot, flow cytometry and immunohistochemistry in monocytes in vitro, in animals with experimental autoimmune encephalomyelitis (EAE), the animal model of MS, and in samples of MS patients.

Results and discussion: We show here that human activated monocytes release glutamate through cystine/glutamate antiporter x(c)⁻ and that the expression of the catalytic subunit xCT is upregulated as a consequence of monocyte activation. In addition, xCT expression is also increased in EAE and in the disease proper. In the later, high expression of xCT occurs both in the central nervous system (CNS) and in peripheral blood cells. In particular, cells from monocyte-macrophage-microglia lineage have higher xCT expression in MS and in EAE, indicating that immune activation upregulates xCT levels, which may result in higher glutamate release and contribution to excitotoxic damage to oligodendrocytes.

Conclusions: Together, these results reveal that increased expression of the cystine/glutamate antiporter system x(c)⁻ in MS provides a link between inflammation and excitotoxicity in demyelinating diseases.

Show MeSH
Related in: MedlinePlus