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1.  The Cystine/Glutamate Antiporter System xc− in Health and Disease: From Molecular Mechanisms to Novel Therapeutic Opportunities 
Antioxidants & Redox Signaling  2013;18(5):522-555.
Abstract
The antiporter system xc− imports the amino acid cystine, the oxidized form of cysteine, into cells with a 1:1 counter-transport of glutamate. It is composed of a light chain, xCT, and a heavy chain, 4F2 heavy chain (4F2hc), and, thus, belongs to the family of heterodimeric amino acid transporters. Cysteine is the rate-limiting substrate for the important antioxidant glutathione (GSH) and, along with cystine, it also forms a key redox couple on its own. Glutamate is a major neurotransmitter in the central nervous system (CNS). By phylogenetic analysis, we show that system xc− is a rather evolutionarily new amino acid transport system. In addition, we summarize the current knowledge regarding the molecular mechanisms that regulate system xc−, including the transcriptional regulation of the xCT light chain, posttranscriptional mechanisms, and pharmacological inhibitors of system xc−. Moreover, the roles of system xc− in regulating GSH levels, the redox state of the extracellular cystine/cysteine redox couple, and extracellular glutamate levels are discussed. In vitro, glutamate-mediated system xc− inhibition leads to neuronal cell death, a paradigm called oxidative glutamate toxicity, which has successfully been used to identify neuroprotective compounds. In vivo, xCT has a rather restricted expression pattern with the highest levels in the CNS and parts of the immune system. System xc− is also present in the eye. Moreover, an elevated expression of xCT has been reported in cancer. We highlight the diverse roles of system xc− in the regulation of the immune response, in various aspects of cancer and in the eye and the CNS. Antioxid. Redox Signal. 18, 522–555.
I. Introduction
A. Oxidative stress and antioxidant defense
B. GSH metabolism
C. Glutamate: neurotransmission and neurotoxicity
II. The Cystine/Glutamate Antiporter System xc−
A. Functional and pharmacological characteristics of system xc−
B. The molecular biology of system xc−
C. The phylogeny of xCT, the specific subunit of system xc−
D. Regulation of system xc− by transcriptional regulation of its specific subunit xCT
E. Regulation of system xc− activity by protein trafficking and protein modification
F. Regulation of system xc− activity by substrate availability
III. Expression of System xc− In Vitro and In Vivo and Its Functional Consequences
A. In the absence of disease, system xc− shows a rather restricted expression pattern in vivo
B. System xc− is induced in most cultured cells
C. The role of system xc− in the regulation of GSH synthesis, the extracellular redox milieu, and extracellular glutamate levels
D. Oxidative glutamate toxicity—an in vitro paradigm for neuronal death induced by system xc− inhibition
1. The cell death pathway in oxidative glutamate toxicity
2. Using oxidative glutamate toxicity to identify neuroprotective pathways
3. Using oxidative glutamate toxicity to screen for neuroprotective drugs
4. Oxidative glutamate toxicity in vivo
IV. The Role of System xc− in Health and Disease
A. System xc− in vivo—lessons from xCT-deficient mice
B. The role of system xc− in the immune system and inflammation
C. The role of system xc− in cancer and resistance against anti-cancer drugs
1. System xc− is regulated by potentially oncogenic pathways
2. System xc− mediates the infection of cells by oncogenic Kaposi's sarcoma herpesvirus
3. System xc− plays an important role in the multidrug resistance of cancers
4. Inhibition of system xc− reduces cancer cell replication, tissue invasion, and metastasis
5. System xc− expressed in tumor cells may be used as a target for anticancer drug delivery
6. Up-regulation of system xc− in normal cells provides protection against carcinogenesis—a possible role in cancer prevention
7. Synopsis of the role of system xc− in cancer and resistance against anti-cancer drugs
D. System xc− and diseases of the eye
1. Studies of system xc− in the retina
2. Studies of system xc− in the lens and cornea
3. Synopsis and future directions for system xc− and diseases of the eye
E. The role of system xc− in diseases of the CNS
F. The role of system xc− activity in memory and behavior
V. Conclusion
doi:10.1089/ars.2011.4391
PMCID: PMC3545354  PMID: 22667998
2.  Fumaric Acid Esters Stimulate Astrocytic VEGF Expression through HIF-1α and Nrf2 
PLoS ONE  2013;8(10):e76670.
Fumaric acid esters (FAE) are oral analogs of fumarate that have recently been shown to decrease relapse rate and disease progression in multiple sclerosis (MS), prompting to investigate their protective potential in other neurological diseases such as amyotrophic lateral sclerosis (ALS). Despite efficacy in MS, mechanisms of action of FAEs are still largely unknown. FAEs are known to activate the transcription factor Nrf2 and downstream anti-oxidant responses through the succination of Nrf2 inhibitor KEAP1. However, fumarate is also a known inhibitor of prolyl-hydroxylases domain enzymes (PhD), and PhD inhibition might lead to stabilization of the HIF-1α transcription factor under normoxic conditions and subsequent activation of a pseudo hypoxic response. Whether Nrf2 activation is associated with HIF-1α stabilization in response to FAEs in cell types relevant to MS or ALS remains unknown. Here, we show that FAEs elicit HIF-1α accumulation, and VEGF release as its expected consequence, in astrocytes but not in other cell types of the central nervous system. Reporter assays demonstrated that increased astrocytic VEGF release in response to FAEs was dependent upon both HIF-1α and Nrf2 activation. Last, astrocytes of transgenic mice expressing SOD1(G93A), an animal model of ALS, displayed reduced VEGF release in response to FAEs. These studies show that FAEs elicit different signaling pathways in cell types from the central nervous system, in particular a pseudo-hypoxic response in astrocytes. Disease relevant mutations might affect this response.
doi:10.1371/journal.pone.0076670
PMCID: PMC3789659  PMID: 24098549
3.  Fluorodeoxyglucose positron emission tomography in anti-N-methyl-D-aspartate receptor encephalitis: distinct pattern of disease 
Background
Patients with encephalitis associated with antibodies against N-methyl-D-aspartate-receptor antibody (NMDAR-ab) encephalitis frequently show psychotic symptoms, amnesia, seizures and movement disorders. While brain MRI in NMDAR-ab encephalitis is often normal, abnormalities of cerebral glucose metabolism have been demonstrated by positron emission tomography (PET) with 18F-fluorodeoxyglucose (FDG) in a few usually isolated case reports. However, a common pattern of FDG-PET abnormalities has not been reported.
Methods
The authors retrospectively identified six patients with NMDAR-ab encephalitis in two large German centres who underwent at least one whole-body FDG-PET for tumour screening between January 2007 and July 2010. They analysed the pattern of cerebral uptake derived from whole-body PET data for characteristic changes of glucose metabolism compared with controls, and the changes of this pattern during the course of the disease.
Results
Groupwise analysis revealed that patients with NMDAR-ab encephalitis showed relative frontal and temporal glucose hypermetabolism associated with occipital hypometabolism. Cross-sectional analysis of the group demonstrated that the extent of these changes is positively associated with clinical disease severity. Longitudinal analysis of two cases showed normalisation of the pattern of cerebral glucose metabolism with recovery.
Conclusions
A characteristic change in cerebral glucose metabolism during NMDAR-ab encephalitis is an increased frontotemporal-to-occipital gradient. This pattern correlates with disease severity. Similar changes have been observed in psychosis induced by NMDAR antagonists. Thus, this pattern might be a consequence of impaired NMDAR function.
doi:10.1136/jnnp-2011-301969
PMCID: PMC3740122  PMID: 22566598
4.  The flavonoid fisetin attenuates postischemic immune cell infiltration, activation and infarct size after transient cerebral middle artery occlusion in mice 
The development of the brain tissue damage in ischemic stroke is composed of an immediate component followed by an inflammatory response with secondary tissue damage after reperfusion. Fisetin, a flavonoid, has multiple biological effects, including neuroprotective and antiinflammatory properties. We analyzed the effects of fisetin on infarct size and the inflammatory response in a mouse model of stroke, temporary middle cerebral artery occlusion, and on the activation of immune cells, murine primary and N9 microglial and Raw264.7 macrophage cells and human macrophages, in an in vitro model of inflammatory immune cell activation by lipopolysaccharide (LPS). Fisetin not only protected brain tissue against ischemic reperfusion injury when given before ischemia but also when applied 3 hours after ischemia. Fisetin also prominently inhibited the infiltration of macrophages and dendritic cells into the ischemic hemisphere and suppressed the intracerebral immune cell activation as measured by intracellular tumor necrosis factor α (TNFα) production. Fisetin also inhibited LPS-induced TNFα production and neurotoxicity of macrophages and microglia in vitro by suppressing nuclear factor κB activation and JNK/Jun phosphorylation. Our findings strongly suggest that the fisetin-mediated inhibition of the inflammatory response after stroke is part of the mechanism through which fisetin is neuroprotective in cerebral ischemia.
doi:10.1038/jcbfm.2011.189
PMCID: PMC3345911  PMID: 22234339
fisetin; flavonoids; macrophages; microglia; stroke
5.  Effects of dimethyl fumarate on neuroprotection and immunomodulation 
Background
Neuronal degeneration in multiple sclerosis has been linked to oxidative stress. Dimethyl fumarate is a promising novel oral therapeutic option shown to reduce disease activity and progression in patients with relapsing-remitting multiple sclerosis. These effects are presumed to originate from a combination of immunomodulatory and neuroprotective mechanisms. We aimed to clarify whether neuroprotective concentrations of dimethyl fumarate have immunomodulatory effects.
Findings
We determined time- and concentration-dependent effects of dimethyl fumarate and its metabolite monomethyl fumarate on viability in a model of endogenous neuronal oxidative stress and clarified the mechanism of action by quantitating cellular glutathione content and recycling, nuclear translocation of transcription factors, and the expression of antioxidant genes. We compared this with changes in the cytokine profiles released by stimulated splenocytes measured by ELISPOT technology and analyzed the interactions between neuronal and immune cells and neuronal function and viability in cell death assays and multi-electrode arrays. Our observations show that dimethyl fumarate causes short-lived oxidative stress, which leads to increased levels and nuclear localization of the transcription factor nuclear factor erythroid 2-related factor 2 and a subsequent increase in glutathione synthesis and recycling in neuronal cells. Concentrations that were cytoprotective in neuronal cells had no negative effects on viability of splenocytes but suppressed the production of proinflammatory cytokines in cultures from C57BL/6 and SJL mice and had no effects on neuronal activity in multi-electrode arrays.
Conclusions
These results suggest that immunomodulatory concentrations of dimethyl fumarate can reduce oxidative stress without altering neuronal network activity.
doi:10.1186/1742-2094-9-163
PMCID: PMC3419128  PMID: 22769044
Dimethyl fumarate; Oxidative stress; Neuroprotection; Neuromodulation
6.  p62, Ref(2)P and ubiquitinated proteins are conserved markers of neuronal aging, aggregate formation and progressive autophagic defects 
Autophagy  2011;7(6):572-583.
Suppression of macroautophagy, due to mutations or through processes linked to aging, results in the accumulation of cytoplasmic substrates that are normally eliminated by the pathway. This is a significant problem in long-lived cells like neurons, where pathway defects can result in the accumulation of aggregates containing ubiquitinated proteins. The p62/Ref(2)P family of proteins is involved in the autophagic clearance of cytoplasmic protein bodies or sequestosomes. These unique structures are closely associated with protein inclusions containing ubiquitin as well as key components of the autophagy pathway. In this study we show that detergent fractionation followed by western blot analysis of insoluble ubiquitinated proteins (IUP), mammalian p62 and its Drosophila homologue, Ref(2)P can be used to quantitatively assess the activity level of aggregate clearance (aggrephagy) in complex tissues. Using this technique we show that genetic or age-dependent changes that modify the long-term enhancement or suppression of aggrephagy can be identified. Moreover, using the Drosophila model system this method can be used to establish autophagy-dependent protein clearance profiles that are occurring under a wide range of physiological conditions including developmental, fasting and altered metabolic pathways. This technique can also be used to examine proteopathies that are associated with human disorders such as frontotemporal dementia, Huntington and Alzheimer disease. Our findings indicate that measuring IUP profiles together with an assessment of p62/Ref(2)P proteins can be used as a screening or diagnostic tool to characterize genetic and age-dependent factors that alter the long-term function of autophagy and the clearance of protein aggregates occurring within complex tissues and cells.
doi:10.4161/auto.7.6.14943
PMCID: PMC3127048  PMID: 21325881
p62; Ref(2)P; insoluble ubiquitinated proteins; aggregates; neural degeneration; Alzheimer disease; aging; macroautophagy
7.  Dementia and leukoencephalopathy due to lymphomatosis cerebri 
BMJ Case Reports  2009;2009:bcr08.2008.0752.
Lymphomatosis cerebri (LC) is a rare variant of primary central nervous system lymphoma (PCNSL). Clinically, the disease typically presents with a rapidly progressive dementia and unsteadiness of gait. Its presentation on cerebral MRI, which is characterised by diffuse leukoencephalopathy without contrast enhancement, often causes diagnostic confusion1 with suspected diagnoses ranging from Binswanger’s disease to leukoencephalopathy or encephalomyelitis. Here we report a patient with subacute dementia and diffuse bilateral white matter changes in the cerebral hemispheres and additional involvement of the brainstem, basal ganglia and thalamus on MRI. Initially, she was considered to suffer from an autoimmune encephalitis, transiently responded to immunosuppression but then developed multiple solid appearing cerebral lymphomas.
doi:10.1136/bcr.08.2008.0752
PMCID: PMC3028137  PMID: 21686648
9.  A Novel Approach to Enhancing Cellular Glutathione Levels 
Journal of neurochemistry  2008;107(3):690-700.
Glutathione (GSH) and GSH-associated metabolism provide the major line of defense for the protection of cells from oxidative and other forms of toxic stress. Of the three amino acids that comprise GSH, cysteine is limiting for GSH synthesis. Since extracellularly cysteine is readily oxidized to form cystine, cystine transport mechanisms are essential to provide cells with cysteine. Cystine uptake is mediated by system xc−, a Na+-independent cystine/glutamate antiporter. Inhibition of system xc− by millimolar concentrations of glutamate, a pathway termed oxidative glutamate toxicity, results in GSH depletion and nerve cell death. Recently, we described a series of compounds derived from the conjugation of epicatechin with cysteine and cysteine derivatives that protected nerve cells in culture from oxidative glutamate toxicity by maintaining GSH levels. In this paper, we characterize an additional epicatechin conjugate, cysteamine-epicatechin, that is 5-10 fold more potent than the earlier conjugates. In addition, we show that these epicatechin conjugates maintain GSH levels by enhancing the uptake of cystine into cells through induction of a disulfide exchange reaction, thereby uncoupling the uptake from system xc−. Thus, these novel epicatechin conjugates have the potential to enhance GSH synthesis under a wide variety of forms of toxic stress.
doi:10.1111/j.1471-4159.2008.05620.x
PMCID: PMC2644427  PMID: 18702664
oxidative stress; cysteine; cystine; cysteamine; epicatechin

Results 1-9 (9)