Jan Lewerenz

The Cystine/Glutamate Antiporter System xc− in Health and Disease: From Molecular Mechanisms to Novel Therapeutic Opportunities

The antiporter system x(c)(-) 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 x(c)(-) is a rather evolutionarily new amino acid transport system. In addition, we summarize the current knowledge regarding the molecular mechanisms that regulate system x(c)(-), including the transcriptional regulation of the xCT light chain, posttranscriptional mechanisms, and pharmacological inhibitors of system x(c)(-). Moreover, the roles of system x(c)(-) 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 x(c)(-) 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 x(c)(-) is also present in the eye. Moreover, an elevated expression of xCT has been reported in cancer. We highlight the diverse roles of system x(c)(-) in the regulation of the immune response, in various aspects of cancer and in the eye and the CNS.

A novel non-rapid-eye movement and rapid-eye-movement parasomnia with sleep breathing disorder associated with antibodies to IgLON5: a case series, characterisation of the antigen, and post-mortem study

BACKGROUND Autoimmunity might be associated with or implicated in sleep and neurodegenerative disorders. We aimed to describe the features of a novel neurological syndrome associated with prominent sleep dysfunction and antibodies to a neuronal antigen. METHODS In this observational study, we used clinical and video polysomnography to identify a novel sleep disorder in three patients referred to the Sleep Unit of Hospital Clinic, University of Barcelona, Spain, for abnormal sleep behaviours and obstructive sleep apnoea. These patients had antibodies against a neuronal surface antigen, which were also present in five additional patients referred to our laboratory for antibody studies. These five patients had been assessed with polysomnography, which was done in our sleep unit in one patient and the recording reviewed in a second patient. Two patients underwent post-mortem brain examination. Immunoprecipitation and mass spectrometry were used to characterise the antigen and develop an assay for antibody testing. Serum or CSF from 298 patients with neurodegenerative, sleep, or autoimmune disorders served as control samples. FINDINGS All eight patients (five women; median age at disease onset 59 years [range 52-76]) had abnormal sleep movements and behaviours and obstructive sleep apnoea, as confirmed by polysomnography. Six patients had chronic progression with a median duration from symptom onset to death or last visit of 5 years (range 2-12); in four the sleep disorder was the initial and most prominent feature, and in two it was preceded by gait instability followed by dysarthria, dysphagia, ataxia, or chorea. Two patients had a rapid progression with disequilibrium, dysarthria, dysphagia, and central hypoventilation, and died 2 months and 6 months, respectively, after symptom onset. In five of five patients, video polysomnography showed features of obstructive sleep apnoea, stridor, and abnormal sleep architecture (undifferentiated non-rapid-eye-movement [non-REM] sleep or poorly structured stage N2, simple movements and finalistic behaviours, normalisation of non-REM sleep by the end of the night, and, in the four patients with REM sleep recorded, REM sleep behaviour disorder). Four of four patients had HLA-DRB1*1001 and HLA-DQB1*0501 alleles. All patients had antibodies (mainly IgG4) against IgLON5, a neuronal cell adhesion molecule. Only one of the 298 controls, who had progressive supranuclear palsy, had IgLON5 antibodies. Neuropathology showed neuronal loss and extensive deposits of hyperphosphorylated tau mainly involving the tegmentum of the brainstem and hypothalamus in the two patients studied. INTERPRETATION IgLON5 antibodies identify a unique non-REM and REM parasomnia with sleep breathing dysfunction and pathological features suggesting a tauopathy. FUNDING Fondo de Investigaciones Sanitarias, Centros de Investigación Biomédica en Red de enfermedades neurodegenerativas (CIBERNED) and Respiratorias (CIBERES), Ministerio de Economía y Competitividad, Fundació la Marató TV3, and the National Institutes of Health.

Induction of Nrf2 and xCT are involved in the action of the neuroprotective antibiotic ceftriaxone in vitro.

In amyotrophic lateral sclerosis, down‐regulation of the astrocyte‐specific glutamate excitatory amino acid transporter 2 is hypothesized to increase extracellular glutamate, thereby leading to excitotoxic motor neuron death. The antibiotic ceftriaxone was recently reported to induce excitatory amino acid transporter 2 and to prolong the survival of mutant superoxide dismutase 1 transgenic mice. Here we show that ceftriaxone also protects fibroblasts and the hippocampal cell line HT22, which are not sensitive to excitotoxicity, against oxidative glutamate toxicity, where extracellular glutamate blocks cystine import via the glutamate/cystine‐antiporter system xc−. Lack of intracellular cystine leads to glutathione depletion and cell death because of oxidative stress. Ceftriaxone increased system xc− and glutathione levels independently of its effect on excitatory amino acid transporters by induction of the transcription factor Nrf2 (nuclear factor erythroid 2‐related factor 2), a known inducer of system xc−, and the specific xc− subunit xCT. No significant effect was apparent in fibroblasts deficient in Nrf2 or xCT. Similar ceftriaxone‐stimulated changes in Nrf2, system xc−, and glutathione were observed in rat cortical and spinal astrocytes. In addition, ceftriaxone induced xCT mRNA expression in stem cell‐derived human motor neurons. We conclude that ceftriaxone‐mediated neuroprotection might relate more strongly to activation of the antioxidant defense system including Nrf2 and system xc− than to excitatory amino acid transporter induction.

Chronic Glutamate Toxicity in Neurodegenerative Diseases—What is the Evidence?

Together with aspartate, glutamate is the major excitatory neurotransmitter in the brain. Glutamate binds and activates both ligand-gated ion channels (ionotropic glutamate receptors) and a class of G-protein coupled receptors (metabotropic glutamate receptors). Although the intracellular glutamate concentration in the brain is in the millimolar range, the extracellular glutamate concentration is kept in the low micromolar range by the action of excitatory amino acid transporters that import glutamate and aspartate into astrocytes and neurons. Excess extracellular glutamate may lead to excitotoxicity in vitro and in vivo in acute insults like ischemic stroke via the overactivation of ionotropic glutamate receptors. In addition, chronic excitotoxicity has been hypothesized to play a role in numerous neurodegenerative diseases including amyotrophic lateral sclerosis, Alzheimers disease and Huntingtons disease. Based on this hypothesis, a good deal of effort has been devoted to develop and test drugs that either inhibit glutamate receptors or decrease extracellular glutamate. In this review, we provide an overview of the different pathways that are thought to lead to an over-activation of the glutamatergic system and glutamate toxicity in neurodegeneration. In addition, we summarize the available experimental evidence for glutamate toxicity in animal models of neurodegenerative diseases.

Effects of dimethyl fumarate on neuroprotection and immunomodulation

BackgroundNeuronal 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.FindingsWe 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.ConclusionsThese results suggest that immunomodulatory concentrations of dimethyl fumarate can reduce oxidative stress without altering neuronal network activity.

Basal Levels of eIF2α Phosphorylation Determine Cellular Antioxidant Status by Regulating ATF4 and xCT Expression

eIF2α is part of a multimeric complex that regulates cap-dependent translation. Phosphorylation of eIF2α (phospho-eIF2α) is induced by various forms of cell stress, resulting in changes to the proteome of the cell with two diametrically opposed consequences, adaptation to stress or initiation of programmed cell death. In contrast to the robust eIF2α phosphorylation seen in response to acute insults, less is known about the functional role of basal levels of eIF2α phosphorylation. Here we show that mouse embryonic fibroblasts expressing a nonphosphorylatable eIF2α have enhanced sensitivity to diverse toxic insults, including amyloid β-(1–42) peptide (Aβ), a key factor in the pathogenesis of Alzheimer disease. This correlates with impaired glutathione metabolism because of down-regulation of the light chain, xCT, of the cystine/glutamate antiporter system X-c. The mechanistic link between the absence of phospho-eIF2α and xCT expression is nuclear factor ATF4. Consistent with these findings, long term activation of the phospho-eIF2α/ATF4/xCT signaling module by the specific eIF2α phosphatase inhibitor, salubrinal, induces resistance against oxidative glutamate toxicity in the hippocampal cell line HT22 and primary cortical neurons. Furthermore, in PC12 cells selected for resistance against Aβ, increased activity of the phospho-eIF2α/ATF4/xCT module contributes to the resistant phenotype. In wild-type PC12 cells, activation of this module by salubrinal ameliorates the response to Aβ. Furthermore, in human brains, ATF4 and phospho-eIF2α levels are tightly correlated and up-regulated in Alzheimer disease, most probably representing an adaptive response against disease-related cellular stress rather than a correlate of neurodegeneration.

Mechanisms of Oxidative Glutamate Toxicity: The Glutamate/Cystine Antiporter System xc¯ as a Neuroprotective Drug Target

The glutamate/cystine antiporter system x(c)- transports cystine into cells in exchange for the important neurotransmitter glutamate at a ratio of 1:1. It is composed of a specific light chain, xCT, and a heavy chain, 4F2, linked by a disulfide bridge. Both subunits are localized prominently in the mouse and human brain especially in border areas between the brain and periphery including vascular endothelial cells, ependymal cells, choroid plexus, and leptomeninges. Glutamate exported by system x(c)- is largely responsible for the extracellular glutamate concentration in the brain, whereas the imported cystine is required for the synthesis of the major endogenous antioxidant, glutathione. System x(c)- thus connects the antioxidant defense with neurotransmission and behavior. Disturbances in the function of system x(c)- have been implicated in nerve cell death due to increased extracellular glutamate and reduced intracellular glutathione. In vitro, inhibition of cystine import through system x(c)- leads to cell death by a mechanism called oxidative glutamate toxicity or oxytosis, which includes depletion of intracellular glutathione, activation of 12-lipoxygenase, accumulation of intracellular peroxides, and the activation of a cyclic guanosine monophosphate (cGMP)-dependent calcium channel towards the end of the death cascade. Cell death caused by oxytosis is distinct from classical apoptosis. In this contribution, we discuss the function of system x(c)- in vitro and in vivo, the role of xCT as an important but due to its dual role probably ambivalent drug target, and the relevance of oxytosis as an in vitro assay for the identification of novel neuroprotective proteins and signaling pathways.

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.

Regulation of xCT expression and system \( x_{\text{c}}^{ - } \) function in neuronal cells

The glutamate/cystine antiporter system