Absence of system xc- on immune cells invading the central nervous system alleviates experimental autoimmune encephalitis.

BACKGROUND: Multiple sclerosis (MS) is an autoimmune demyelinating disease that affects the central nervous system (CNS), leading to neurodegeneration and chronic disability. Accumulating evidence points to a key role for neuroinflammation, oxidative stress, and excitotoxicity in this degenerative process. System xc- or the cystine/glutamate antiporter could tie these pathological mechanisms together: its activity is enhanced by reactive oxygen species and inflammatory stimuli, and its enhancement might lead to the release of toxic amounts of glutamate, thereby triggering excitotoxicity and neurodegeneration. METHODS: Semi-quantitative Western blotting served to study protein expression of xCT, the specific subunit of system xc-, as well as of regulators of xCT transcription, in the normal appearing white matter (NAWM) of MS patients and in the CNS and spleen of mice exposed to experimental autoimmune encephalomyelitis (EAE), an accepted mouse model of MS. We next compared the clinical course of the EAE disease, the extent of demyelination, the infiltration of immune cells and microglial activation in xCT-knockout (xCT-/-) mice and irradiated mice reconstituted in xCT-/- bone marrow (BM), to their proper wild type (xCT+/+) controls. RESULTS: xCT protein expression levels were upregulated in the NAWM of MS patients and in the brain, spinal cord, and spleen of EAE mice. The pathways involved in this upregulation in NAWM of MS patients remain unresolved. Compared to xCT+/+ mice, xCT-/- mice were equally susceptible to EAE, whereas mice transplanted with xCT-/- BM, and as such only exhibiting loss of xCT in their immune cells, were less susceptible to EAE. In none of the above-described conditions, demyelination, microglial activation, or infiltration of immune cells were affected. CONCLUSIONS: Our findings demonstrate enhancement of xCT protein expression in MS pathology and suggest that system xc- on immune cells invading the CNS participates to EAE. Since a total loss of system xc- had no net beneficial effects, these results have important implications for targeting system xc- for treatment of MS.

Comparative analysis of antibodies to xCT (Slc7a11): Forewarned is forearmed.

The cystine/glutamate antiporter or system Xc- exchanges cystine for glutamate, thereby supporting intracellular glutathione synthesis and nonvesicular glutamate release. The role of system Xc- in neurological disorders can be dual and remains a matter of debate. One important reason for the contradictory findings that have been reported to date is the use of nonspecific anti-xCT (the specific subunit of system Xc-) antibodies. Often studies rely on the predicted molecular weight of 55.5 kDa to identify xCT on Western blots. However, using brain extracts from xCT knockout (xCT(-/-)) mice as negative controls, we show that xCT migrates as a 35-kDa protein. Misinterpretation of immunoblots leads to incorrect assessment of antibody specificity and thereby to erroneous data interpretation. Here we have verified the specificity of most commonly used commercial and some in-house-developed anti-xCT antibodies by comparing their immunoreactivity in brain tissue of xCT(+/+) and xCT(-/-) mice by Western blotting and immunohistochemistry. The Western blot screening results demonstrate that antibody specificity not only differs between batches produced by immunizing different rabbits with the same antigen but also between bleedings of the same rabbit. Moreover, distinct immunohistochemical protocols have been tested for all the anti-xCT antibodies that were specific on Western blots in order to obtain a specific immunolabeling. Only one of our in-house-developed antibodies could reveal specific xCT labeling and exclusively on acetone-postfixed cryosections. Using this approach, we observed xCT protein expression throughout the mouse forebrain, including cortex, striatum, hippocampus, midbrain, thalamus, and amygdala, with greatest expression in regions facing the cerebrospinal fluid and meninges.

In-depth behavioral characterization of the corticosterone mouse model and the critical involvement of housing conditions.

Depression and anxiety are disabling and highly prevalent psychiatric disorders. To better understand the neurobiological basis of mood and anxiety disorders, relevant animal models are needed. The corticosterone mouse model is frequently used to study depression. Chronic stress and accompanying glucocorticoid elevation causes pathological changes in the central nervous system, which are related to psychiatric symptoms. Exogenous administration of corticosterone is therefore often used to induce depressive-like behavior in mice and in some cases also features of anxiety-like behavior are shown. However, a thorough characterization of this model has never been conducted and housing conditions of the used subjects often differ between the implemented protocols. We chronically administered a subcutaneous corticosterone bolus injection to single- and group-housed mice, and we subsequently evaluated the face validity of this model by performing a battery of behavioral tests (forced swim test, mouse-tail suspension test, saccharin intake test, novelty-suppressed feeding test, elevated plus maze, light/dark paradigm and open field test). Our results show that corticosterone treatment has a substantial overall effect on depressive-like behavior. Increases in anxiety-like behavior on the other hand are mainly seen in single housed animals, independent of treatment. The current study therefore does not only show a detailed behavioral characterization of the corticosterone mouse model, but furthermore also elucidates the critical influence of housing conditions on the behavioral outcome in this model.

Nigral proteasome inhibition in mice leads to motor and non-motor deficits and increased expression of Ser129 phosphorylated α-synuclein.

Parkinson's disease is a neurodegenerative disorder characterized by motor and non-motor disturbances. Various pathogenic pathways drive disease progression including oxidative stress, mitochondrial dysfunction, α-synuclein aggregation and impairment of protein degradation systems. Dysfunction of the ubiquitin-proteasome system in the substantia nigra of Parkinson's disease patients is believed to be one of the causes of protein aggregation and cell death associated with this disorder. Lactacystin, a potent inhibitor of the proteasome, was previously delivered to the nigrostriatal pathway of rodents to model nigrostriatal degeneration. Although lactacystin-treated animals develop parkinsonian motor impairment, it is currently unknown whether they also develop non-motor symptoms characteristic of this disorder. In order to further describe the proteasome inhibition model of Parkinson's disease, we characterized the unilateral lactacystin model, performed by stereotaxic injection of the toxin in the substantia nigra of mice. We studied the degree of neurodegeneration and the behavioral phenotype 1 and 3 weeks after lactacystin lesion both in terms of motor impairment, as well as non-motor symptoms. We report that unilateral administration of 3 µg lactacystin to the substantia nigra of mice leads to partial (~40%) dopaminergic cell loss and concurrent striatal dopamine depletion, accompanied by increased expression of Ser129-phosphorylated α-synuclein. Behavioral characterization of the model revealed parkinsonian motor impairment, as well as signs of non-motor disturbances resembling early stage Parkinson's disease including sensitive and somatosensory deficits, anxiety-like behavior, and perseverative behavior. The consistent finding of good face validity, together with relevant construct validity, warrant a further evaluation of proteasome inhibition models of Parkinson's disease in pre-clinical research and validation of therapeutic targets.

Lack of effect of Theiler's murine encephalomyelitis virus infection on system xc⁻.

Changes in the expression of xCT, the specific subunit of system xc(-) or the cystine/glutamate antiporter, have been associated with several neurological disorders and system xc(-) was recently proposed as a potential target for the development of new treatment strategies for multiple sclerosis (MS). In this study we used Theiler's murine encephalomyelitis virus (TMEV) infection, both in vitro and in vivo, as a model to further evaluate the involvement of system xc(-) in MS. Protein levels of xCT, as well as activity of system xc(-) were unaffected in RAW264.7 macrophages after infection with the demyelinating DA strain of TMEV. Also, protein expression of xCT remained stable in spinal cord and brain of FVB mice 1-2 and 6 weeks after intracranial injection of the DA strain of TMEV. These results demonstrate that TMEV infection of macrophages or FVB mice has no effect on system xc(-) and as such cannot be used as a model to study the involvement of system xc(-) in MS.

Altered vesicular glutamate transporter expression in human temporal lobe epilepsy with hippocampal sclerosis.

Vesicular glutamate transporters (VGLUTs) are responsible for loading glutamate into synaptic vesicles. Altered VGLUT protein expression has been suggested to affect quantal size and glutamate release under both physiological and pathological conditions. In this study, we investigated mRNA and protein expression levels of the three VGLUT subtypes in hippocampal tissue of patients suffering from temporal lobe epilepsy (TLE) with hippocampal sclerosis (HS), International League Against Epilepsy type 1 (ILAE type 1) compared to autopsy controls, using quantitative polymerase chain reaction and semi-quantitative western blotting. mRNA expression levels of the VGLUTs are unaffected in hippocampal epileptic tissue compared to autopsy controls. At the protein level, VGLUT1 expression remains unaltered, while VGLUT2 is significantly decreased and VGLUT3 protein is significantly increased in hippocampal biopsies from TLE patients compared to controls. Our findings at the protein level can be explained by previously described histopathological changes observed in HS. Although VGLUTs have been repeatedly investigated in distinct rodent epilepsy models, their expression levels were hitherto not fully unraveled in the most difficult-to-treat form of epilepsy: TLE with HS ILAE type 1. We here, demonstrate for the first time that VGLUT2 protein expression is significantly decreased and VGLUT3 protein is significantly increased in the hippocampus of patients suffering from TLE with HS ILAE type 1 compared to autopsy controls.

Absence of system xc- in mice decreases anxiety and depressive-like behavior without affecting sensorimotor function or spatial vision.

There is considerable preclinical and clinical evidence indicating that abnormal changes in glutamatergic signaling underlie the development of mood disorders. Astrocytic glutamate dysfunction, in particular, has been recently linked with the pathogenesis and treatment of mood disorders, including anxiety and depression. System xc- is a glial cystine/glutamate antiporter that is responsible for nonvesicular glutamate release in various regions of the brain. Although system xc- is involved in glutamate signal transduction, its possible role in mediating anxiety or depressive-like behaviors is currently unknown. In the present study, we phenotyped adult and aged system xc- deficient mice in a battery of tests for anxiety and depressive-like behavior (open field, light/dark test, elevated plus maze, novelty suppressed feeding, forced swim test, tail suspension test). Concomitantly, we evaluated the sensorimotor function of system xc- deficient mice, using motor and sensorimotor based tests (rotarod, adhesive removal test, nest building test). Finally, due to the presence and potential functional relevance of system xc- in the eye, we investigated the visual acuity of system xc- deficient mice (optomotor test). Our results indicate that loss of system xc- does not affect motor or sensorimotor function, in either adult or aged mice, in any of the paradigms investigated. Similarly, loss of system xc- does not affect basic visual acuity, in either adult or aged mice. On the other hand, in the open field and light/dark tests, and forced swim and tail suspension tests respectively, we could observe significant anxiolytic and antidepressive-like effects in system xc- deficient mice that in certain cases (light/dark, forced swim) were age-dependent. These findings indicate that, under physiological conditions, nonvesicular glutamate release via system xc- mediates aspects of higher brain function related to anxiety and depression, but does not influence sensorimotor function or spatial vision. As such, modulation of system xc- might constitute the basis of innovative interventions in mood disorders.

Modulation of viral replication in macrophages persistently infected with the DA strain of Theiler's murine encephalomyelitis virus.

BACKGROUND: Demyelinating strains of Theiler's murine encephalomyelitis virus (TMEV) such as the DA strain are the causative agents of a persistent infection that induce a multiple sclerosis-like disease in the central nervous system of susceptible mice. Viral persistence, mainly associated with macrophages, is considered to be an important disease determinant that leads to chronic inflammation, demyelination and autoimmunity. In a previous study, we described the establishment of a persistent DA infection in RAW macrophages, which were therefore named DRAW. RESULTS: In the present study we explored the potential of diverse compounds to modulate viral persistence in these DRAW cells. Hemin was found to increase viral yields and to induce cell lysis. Enviroxime and neutralizing anti-TMEV monoclonal antibody were shown to decrease viral yields, whereas interferon-alpha and interferon-gamma completely cleared the persistent infection. We also compared the cytokine pattern secreted by uninfected RAW, DRAW and interferon-cured DRAW macrophages using a cytokine protein array. The chemokine RANTES was markedly upregulated in DRAW cells and restored to a normal expression level after abrogation of the persistent infection with interferon-alpha or interferon-gamma. On the other hand, the chemokine MCP-1 was upregulated in the interferon-cured DRAW cells. CONCLUSION: We have identified several compounds that modulate viral replication in an in vitro model system for TMEV persistence. These compounds now await further testing in an in vivo setting to address fundamental questions regarding persistent viral infection and immunopathogenesis.