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.

Caloric Restriction Protects against Lactacystin-Induced Degeneration of Dopamine Neurons Independent of the Ghrelin Receptor.

Parkinson's disease (PD) is a neurodegenerative disorder, characterized by a loss of dopamine (DA) neurons in the substantia nigra pars compacta (SNc). Caloric restriction (CR) has been shown to exert ghrelin-dependent neuroprotective effects in the 1-methyl-4-phenyl-1,2,3,6-tetrathydropyridine (MPTP)-based animal model for PD. We here investigated whether CR is neuroprotective in the lactacystin (LAC) mouse model for PD, in which proteasome disruption leads to the destruction of the DA neurons of the SNc, and whether this effect is mediated via the ghrelin receptor. Adult male ghrelin receptor wildtype (WT) and knockout (KO) mice were maintained on an ad libitum (AL) diet or on a 30% CR regimen. After 3 weeks, LAC was injected unilaterally into the SNc, and the degree of DA neuron degeneration was evaluated 1 week later. In AL mice, LAC injection significanty reduced the number of DA neurons and striatal DA concentrations. CR protected against DA neuron degeneration following LAC injection. However, no differences were observed between ghrelin receptor WT and KO mice. These results indicate that CR can protect the nigral DA neurons from toxicity related to proteasome disruption; however, the ghrelin receptor is not involved in this effect.

L-Theanine intake increases threshold for limbic seizures but decreases threshold for generalized seizures.

L-Theanine, an ethylamide derivate of glutamate found in abundance in green tea, has been shown to exert beneficial actions in animal models for several neurological disorders. We here investigated for the first time the effect of L-theanine intake on seizure susceptibility using acute pilocarpine and pentylenetetrazol (PTZ) mouse models for studying, respectively, limbic seizures or primarily generalized seizures. Moreover, we studied the effect of l-theanine intake on extracellular hippocampal and cortical glutamate and gamma-aminobutyric acid (GABA) levels, using in vivo microdialysis. Feeding mice with a 4% L-theanine solution significantly decreased their susceptibility to pilocarpine-induced seizures whereas susceptibility to PTZ-induced seizures was increased. The latter effect was linked to decreased extracellular GABA concentrations in frontal cortex.

Loss of system x(c)- does not induce oxidative stress but decreases extracellular glutamate in hippocampus and influences spatial working memory and limbic seizure susceptibility.

System x(c)- exchanges intracellular glutamate for extracellular cystine, giving it a potential role in intracellular glutathione synthesis and nonvesicular glutamate release. We report that mice lacking the specific xCT subunit of system x(c)- (xCT(-/-)) do not have a lower hippocampal glutathione content, increased oxidative stress or brain atrophy, nor exacerbated spatial reference memory deficits with aging. Together these results indicate that loss of system x(c)- does not induce oxidative stress in vivo. Young xCT(-/-) mice did however display a spatial working memory deficit. Interestingly, we observed significantly lower extracellular hippocampal glutamate concentrations in xCT(-/-) mice compared to wild-type littermates. Moreover, intrahippocampal perfusion with system x(c)- inhibitors lowered extracellular glutamate, whereas the system x(c)- activator N-acetylcysteine elevated extracellular glutamate in the rat hippocampus. This indicates that system x(c)- may be an interesting target for pathologies associated with excessive extracellular glutamate release in the hippocampus. Correspondingly, xCT deletion in mice elevated the threshold for limbic seizures and abolished the proconvulsive effects of N-acetylcysteine. These novel findings sustain that system x(c)-) is an important source of extracellular glutamate in the hippocampus. System x(c)(-) is required for optimal spatial working memory, but its inactivation is clearly beneficial to decrease susceptibility for limbic epileptic seizures.

Zonisamide attenuates lactacystin-induced parkinsonism in mice without affecting system xc.

Zonisamide (ZNS), an anticonvulsant drug exhibiting symptomatic effects in Parkinson's disease (PD), was recently reported to exert neuroprotection in rodent models. One of the proposed neuroprotective mechanisms involves increased protein expression of xCT, the specific subunit of the cystine/glutamate antiporter system xc-, inducing glutathione (GSH) synthesis. Here, we investigated the outcome of ZNS treatment in a mouse model of PD based on intranigral proteasome inhibition, and whether the observed effects would be mediated by system xc-. The proteasome inhibitor lactacystin (LAC) was administered intranigrally to male C57BL/6J mice receiving repeated intraperitoneal injections of either ZNS 30mgkg-1 or vehicle. Drug administration was initiated three days prior to stereotaxic LAC injection and was maintained until six days post-surgery. One week after lesion, mice were behaviorally assessed and investigated in terms of nigrostriatal neurodegeneration and molecular changes at the level of the basal ganglia, including expression levels of xCT. ZNS reduced the loss of nigral dopaminergic neurons following LAC injection and the degree of sensorimotor impairment. ZNS failed, however, to modulate xCT expression in basal ganglia of lesioned mice. In a separate set of experiments, the impact of ZNS treatment on system xc- was investigated in control conditions in vivo as well as in vitro. Similarly, ZNS did not influence xCT or glutathione levels in naive male C57BL/6J mice, nor did it alter system xc- activity or glutathione content in vitro. Taken together, these results demonstrate that ZNS treatment provides neuroprotection and behavioral improvement in a PD mouse model based on proteasome inhibition via system xc- independent mechanisms.

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.

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.

Validation of the 6 Hz refractory seizure mouse model for intracerebroventricularly administered compounds.

The six hertz (6 Hz) refractory seizure model is considered an indispensable chain of the Anticonvulsant Screening Project. We here describe an adapted protocol using the intracerebroventricular (i.c.v.) delivery route, which will allow researchers to perform targetvalidation or proof-of-principle studies using promising compounds with unknown or limited blood-brain barrier permeability (e.g. neuropeptides and peptidomimetics) in this model. Seizures were induced by single application of a current intensity of 49 mA to i.c.v.-implanted NMRI mice using an ECT Unit 57800 Ugo Basile stimulator. By applying these key parameters, c-Fos immunohistochemistry revealed the recruitment of the dentate gyrus, ratifying this model as a valuable tool for testing i.c.v. administered compounds against therapy-resistant seizures. This finding was further strengthened, since i.c.v. administration of levetiracetam suppressed 6 Hz-evoked seizure severity but sodium phenytoin did not. We also propose to use "seizure duration" as an alternative, accurate parameter to express the results within this model.

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.

MPTP-induced parkinsonism in mice alters striatal and nigral xCT expression but is unaffected by the genetic loss of xCT.

Nigral cell loss in Parkinson's disease (PD) is associated with disturbed glutathione (GSH) and glutamate levels, leading to oxidative stress and excitotoxicity, respectively. System xc- is a plasma membrane antiporter that couples cystine import (amino acid that can be further used for the synthesis of GSH) with glutamate export to the extracellular environment, and can thus affect both oxidative stress and glutamate excitotoxicity. In the current study, we evaluated the involvement of system xc- in a progressive 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. Our results indicate that the expression of xCT (the specific subunit of system xc-) undergoes region-specific changes in MPTP-treated mice, with increased expression in the striatum, and decreased expression in the substantia nigra. Furthermore, mice lacking xCT were equally sensitive to the neurotoxic effects of MPTP compared to wild-type (WT) mice, as they demonstrate similar decreases in striatal dopamine content, striatal tyrosine hydroxylase (TH) expression, nigral TH immunopositive neurons and forelimb grip strength, five weeks after commencing MPTP treatment. Altogether, our data indicate that progressive lesioning with MPTP induces striatal and nigral dysregulation of system xc-. However, loss of system xc- does not affect MPTP-induced nigral dopaminergic neurodegeneration and motor impairment in mice.

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.

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.

An ultrasensitive nano UHPLC-ESI-MS/MS method for the quantification of three neuromedin-like peptides in microdialysates.

AIM: An ultrasensitive nano UHPLC-ESI-MS/MS method is developed to simultaneously monitor three low-concentration neuromedin-like peptides in microdialysates. RESULTS: Peptide preconcentration and sample desalting is performed online on a trap column. A shallow gradient slope at 300 nl/min on the analytical column maintained at 35°C, followed by two saw-tooth column wash cycles, results in the highest sensitivity and the lowest carryover. The validated method allows the accurate and precise quantification of 0.5 pM neurotensin and neuromedin N (2.5 amol on column), and of 3.0 pM neuromedin B (15.0 amol on column) in in vivo microdialysates without the use of internal standards. CONCLUSION: The assay is an important tool for elucidating the role of these neuromedin-like peptides in the pathophysiology of neurological disorders.

Improved sensitivity of the nano ultra-high performance liquid chromatography-tandem mass spectrometric analysis of low-concentrated neuropeptides by reducing aspecific adsorption and optimizing the injection solvent.

Obtaining maximal sensitivity of nano UHPLC-MS/MS methods is primordial to quantify picomolar concentrations of neuropeptides in microdialysis samples. Since aspecific adsorption of peptides to Eppendorf tubes, pipette tips and UHPLC vials is detrimental for method sensitivity, a strategy is presented to reduce adsorption of these peptides during standard preparation. Within this respect, all procedural steps from dissolution of the lyophilized powder until the injection of the sample onto the system are investigated. Two peptides of the neuromedin family, i.e. neuromedin B and neuromedin N, and a neuromedin N-related neuropeptide, neurotensin, are evaluated. The first part of this study outlines a number of parameters which are known to affect peptide solubility. The main focus of the second part involves the optimization of the sample composition in the UHPLC vial by using design of experiments. Contradictory findings are observed concerning the influence of acetonitrile, salts and matrix components. They are found important for injection of the peptides into the system, but crucially need to be excluded from the dilution solvent. Furthermore, the type of surface material, temperature and the pipetting protocol considerably affect the adsorption phenomenon. Statistical analysis on the results of the central composite design reveals that the highest peptide responses are obtained with the injection solvent consisting of 13.1% V/V ACN and 4.4% V/V FA. This aspect of the optimization strategy can be identified as the main contributor to the gain in method sensitivity. Since the reduction of peptide adsorption and the optimization of the injection solvent resulted in a clear and quantifiable signal of the three peptides, optimization of both issues should be considered in the early stage of method development, in particular when the analysis of low-concentration peptide solutions is envisaged.

Phosphoinositide 3-kinases upregulate system xc(-) via eukaryotic initiation factor 2α and activating transcription factor 4 - A pathway active in glioblastomas and epilepsy.

AIMS: Phosphoinositide 3-kinases (PI3Ks) relay growth factor signaling and mediate cytoprotection and cell growth. The cystine/glutamate antiporter system xc(-) imports cystine while exporting glutamate, thereby promoting glutathione synthesis while increasing extracellular cerebral glutamate. The aim of this study was to analyze the pathway through which growth factor and PI3K signaling induce the cystine/glutamate antiporter system xc(-) and to demonstrate its biological significance for neuroprotection, cell growth, and epilepsy. RESULTS: PI3Ks induce system xc(-) through glycogen synthase kinase 3ß (GSK-3ß) inhibition, general control non-derepressible-2-mediated eukaryotic initiation factor 2α phosphorylation, and the subsequent translational up-regulation of activating transcription factor 4. This pathway is essential for PI3Ks to modulate oxidative stress resistance of nerve cells and insulin-induced growth in fibroblasts. Moreover, the pathway is active in human glioblastoma cells. In addition, it is induced in primary cortical neurons in response to robust neuronal activity and in hippocampi from patients with temporal lobe epilepsy. INNOVATION: Our findings further extend the concepts of how growth factors and PI3Ks induce neuroprotection and cell growth by adding a new branch to the signaling network downstream of GSK-3ß, which, ultimately, leads to the induction of the cystine/glutamate antiporter system xc(-). Importantly, the induction of this pathway by neuronal activity and in epileptic hippocampi points to a potential role in epilepsy. CONCLUSION: PI3K-regulated system xc(-) activity is not only involved in the stress resistance of neuronal cells and in cell growth by increasing the cysteine supply and glutathione synthesis, but also plays a role in the pathophysiology of tumor- and non-tumor-associated epilepsy by up-regulating extracellular cerebral glutamate.

Are vesicular neurotransmitter transporters potential treatment targets for temporal lobe epilepsy?

The vesicular neurotransmitter transporters (VNTs) are small proteins responsible for packing synaptic vesicles with neurotransmitters thereby determining the amount of neurotransmitter released per vesicle through fusion in both neurons and glial cells. Each transporter subtype was classically seen as a specific neuronal marker of the respective nerve cells containing that particular neurotransmitter or structurally related neurotransmitters. More recently, however, it has become apparent that common neurotransmitters can also act as co-transmitters, adding complexity to neurotransmitter release and suggesting intriguing roles for VNTs therein. We will first describe the current knowledge on vesicular glutamate transporters (VGLUT1/2/3), the vesicular excitatory amino acid transporter (VEAT), the vesicular nucleotide transporter (VNUT), vesicular monoamine transporters (VMAT1/2), the vesicular acetylcholine transporter (VAChT) and the vesicular γ-aminobutyric acid (GABA) transporter (VGAT) in the brain. We will focus on evidence regarding transgenic mice with disruptions in VNTs in different models of seizures and epilepsy. We will also describe the known alterations and reorganizations in the expression levels of these VNTs in rodent models for temporal lobe epilepsy (TLE) and in human tissue resected for epilepsy surgery. Finally, we will discuss perspectives on opportunities and challenges for VNTs as targets for possible future epilepsy therapies.