Anneleen Schallier

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 xc− 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 xc− (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 xc− 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 xc− inhibitors lowered extracellular glutamate, whereas the system xc− activator N-acetylcysteine elevated extracellular glutamate in the rat hippocampus. This indicates that system xc− 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 xc− is an important source of extracellular glutamate in the hippocampus. System xc− is required for optimal spatial working memory, but its inactivation is clearly beneficial to decrease susceptibility for limbic epileptic seizures.

Dopaminergic neurons of system xc−-deficient mice are highly protected against 6-hydroxydopamine-induced toxicity

Malfunctioning of system xc–, responsible for exchanging intracellular glutamate for extracellular cystine, can cause oxidative stress and excitotoxicity, both important phenomena in the pathogenesis of Parkinsons disease (PD). We used mice lacking xCT (xCT_/_ mice), the specific subunit of system xc˜, to investigate the involvement of this antiporter in PD. Although cystine that is imported via system xc˜ is reduced to cysteine, the rate‐limiting substrate in the synthesis of glutathione, deletion of xCT did not result in decreased glutathione levels in striatum. Accordingly, no signs of increased oxidative stress could be observed in striatum or substantia nigra of xCT_/_ mice. In sharp contrast to expectations, xCT_/_ mice were less susceptible to 6‐hydroxydopamine (6‐OHDA)‐induced neurodegeneration in the substantia nigra pars compacta compared to their age‐matched wild‐type littermates. This reduced sensitivity to a PD‐inducing toxin might be related to the decrease of 70% in striatal extracellular glutamate levels that was observed in mice lacking xCT. The current data point toward system xc˜ as a possible target for the development of new pharmacotherapies for the treatment of PD and emphasize the need to continue the search for specific ligands for system xc˜.—Massie, A., Schallier, A., Kim, S. W., Fernando, R., Kobayashi, S., Beck, H., De Bundel, D., Vermoesen, K., Bannai, S., Smolders, I., Conrad, M., Plesnila, N., Sato, H., Michotte, Y. Dopaminergic neurons of system xc “‐deficient mice are highly protected against 6‐hydroxydopamine‐induced toxicity. FASEB J. 25, 1359–1369 (2011). www.fasebj.org

Region- and Age-Specific Changes in Glutamate Transport in the AβPP23 Mouse Model for Alzheimer's Disease

Using 8- and 18-month-old AβPP23 mice, we investigated the involvement of high-affinity glutamate transporters (GLAST, GLT-1, EAAC1), vesicular glutamate transporters (VGLUT1-3) and xCT, the specific subunit of system x(c)⁻, in Alzheimers disease (AD) pathogenesis. Transporter expression was studied in cortical and hippocampal tissue and linked to extracellular glutamate and glutamate reuptake activity as measured using in vivo microdialysis. In 8-month-old animals, we could not observe plaque formation or gliosis. Yet, in hippocampus as well as cortex GLAST and GLT-1 expression was decreased. Whereas in cortex this was accompanied by upregulated VGLUT1 expression, extracellular glutamate concentrations were decreased. Surprisingly, inhibiting glutamate reuptake with TBOA revealed increased glutamate reuptake activity in cortex of AβPP23 mice, despite decreased GLAST and GLT-1 expression, and resulted in status epilepticus in all AβPP23 mice, contrary to wildtype littermates. In hippocampus of 8-month-old AβPP23 mice, we observed increased EAAC1 expression besides the decrease in GLAST and GLT-1. Yet, glutamate reuptake activity was drastically decreased according to the decreased GLAST and GLT-1 expression. In 18-month-old AβPP23 mice, plaque formation and gliosis in cortex and hippocampus were accompanied by decreased GLT-1 expression. We also showed, for the first time, increased cortical expression of VGLUT3 and xCT together with a strong tendency towards increased cortical extracellular glutamate levels. VGLUT2 expression remained unaltered in all conditions. The present findings support the hypothesis that alterations in transport of glutamate, and more particular via GLT-1, may be involved in AD pathogenesis.

Time-dependent changes in striatal xCT protein expression in hemi-Parkinson rats.

Altered glutamate signaling is associated with Parkinsons disease. To study the involvement of the cystine/glutamate antiporter in the pathogenesis of Parkinsons disease, we developed new polyclonal antibodies recognizing xCT, the specific subunit of this antiporter. The striatal xCT protein expression level was investigated in a hemi-Parkinson rat model, using semiquantitative western blotting. We observed time-dependent changes after a unilateral 6-hydroxydopamine lesion of the nigrostriatal pathway with increased expression levels in the deafferented striatum after 3 weeks. Twelve weeks postlesion, expression levels returned to normal. These data suggest, for the first time, an involvement of the cystine/glutamate antiporter in determining the aberrant glutamate neurotransmission in the striatum of a parkinsonian brain.

Time-dependent changes in GLT-1 functioning in striatum of hemi-Parkinson rats

Striatal dopamine loss in Parkinsons disease is accompanied by a dysregulation of corticostriatal glutamatergic neurotransmission. Within this study, we investigated striatal expression and activity of the glial high-affinity Na(+)/K(+)-dependent glutamate transporters, GLT-1 and GLAST, in the 6-hydroxydopamine hemi-Parkinson rat model at different time points after unilateral 6-hydroxydopamine injection into the medial forebrain bundle. Using semi-quantitative Western blotting and an ex vivo D-[(3)H]-aspartate uptake assay, we showed a time-dependent bilateral effect of unilateral 6-hydroxydopamine lesioning on the expression as well as activity of GLT-1. At 3 and 12 weeks post-lesion, striatal GLT-1 function was bilaterally upregulated whereas at 5 weeks there was no change. Even though our data do not allow a straightforward conclusion as for the role of glutamate transporters in the pathogenesis of the disease, they do clearly demonstrate a link between disturbed glutamatergic neurotransmission and glutamate transporter functioning in the striatum of a rat model for Parkinsons disease.

vGLUT2 heterozygous mice show more susceptibility to clonic seizures induced by pentylenetetrazol

Glutamate, the most abundant excitatory neurotransmitter in the central nervous system, is well known to be implicated in epileptic seizures. Therefore, impairments in glutamate transport could have an involvement in the mechanism of epileptogenesis. The uptake of glutamate into synaptic vesicles is mediated by vesicular glutamate transporters (vGLUTs). There are three known vGLUT isoforms, vGLUT1-3. In this study, we are particularly interested in the vGLUT2 isoform. We investigated the possible role of vGLUT2 in pentylenetetrazol (PTZ)-induced seizure generation. Seizure threshold of PTZ was compared in vGLUT2 heterozygous knock out (HET) and wild type (WT) mice. In comparison with their WT littermates a lower dose of PTZ was needed in the vGLUT2 HET mice until the onset of the first myoclonic jerk. The threshold for PTZ-induced clonic seizure activity was also lower in the vGLUT2 HET mice. These results indicate, for the first time, that vGLUT2 is likely involved in the epileptogenesis of generalized seizures.

Assessment of the convulsant liability of antidepressants using zebrafish and mouse seizure models

In the past, antidepressants have been thought to possess proconvulsant properties. This assumption remains controversial, however, because anticonvulsant effects have been attributed to certain antidepressants. To date, it remains unclear which antidepressants can be used for the treatment of patients with epilepsy with depression. The present study was designed to determine the anticonvulsant and/or proconvulsant effects of three antidepressants (citalopram, reboxetine, bupropion) against pilocarpine- and pentylenetetrazole-induced acute seizures in larval zebrafish and mice. In zebrafish, all antidepressants were anticonvulsant in the pentylenetetrazole model. In addition, citalopram was anticonvulsant in the zebrafish pilocarpine model, whereas reboxetine and bupropion were without significant effect. In mice all three antidepressants increased some thresholds for pentylenetetrazole-induced convulsive-like behaviors at varying doses, whereas thresholds for pilocarpine-induced convulsive-like behaviors were generally lowered, particularly at the highest doses tested. In general we conclude that the convulsant liability of antidepressants is model and concentration dependent.

Inactivation of the Constitutively Active Ghrelin Receptor Attenuates Limbic Seizure Activity in Rodents

Ghrelin is a pleiotropic neuropeptide that has been recently implicated in epilepsy. Animal studies performed to date indicate that ghrelin has anticonvulsant properties; however, its mechanism of anticonvulsant action is unknown. Here we show that the anticonvulsant effects of ghrelin are mediated via the growth hormone secretagogue receptor (GHSR). To our surprise, however, we found that the GHSR knockout mice had a higher seizure threshold than their wild-type littermates when treated with pilocarpine. Using both in vivo and in vitro models, we further discovered that inverse agonism and desensitization/internalization of the GHSR attenuate limbic seizures in rats and epileptiform activity in hippocampal slices. This constitutes a novel mechanism of anticonvulsant action, whereby an endogenous agonist reduces the activity of a constitutively active receptor.

Biphasic and bilateral changes in striatal VGLUT1 and 2 protein expression in hemi-Parkinson rats

Parkinsons disease is characterized by disturbed glutamatergic neurotransmission in the striatum. Important mediators of extracellular glutamate levels are the vesicular glutamate transporters VGLUT1 and VGLUT2 in respectively corticostriatal and thalamostriatal afferents, next to the high-affinity Na(+)/K(+)-dependent glutamate transporters and the cystine/glutamate antiporter. In the present study, we compared bilateral striatal VGLUT1 and VGLUT2 protein expression as well as VGLUT1 and VGLUT2 transcript levels in the neocortex and parafascicular nucleus of hemi-Parkinson rats at different time intervals post unilateral 6-OHDA injection into the medial forebrain bundle versus controls. Three weeks post-injection we detected increased striatal VGLUT1 expression together with decreased VGLUT2 expression. On the other hand, after twelve weeks, the expression of VGLUT1 was decreased in hemi-Parkinson rats whereas the striatal expression of VGLUT2 was comparable to control rats. No effect could be seen on VGLUT transcript levels in the respective projection areas at any time. In conclusion, we observed a biphasic and bilateral change in the protein expression levels of both VGLUTs in the striatum of hemi-Parkinson rats indicative for a different and time-dependent change in glutamatergic neurotransmission from the two types of striatal afferents.

Deletion of insulin-regulated aminopeptidase in mice decreases susceptibility to pentylenetetrazol-induced generalized seizures

The peptide angiotensin IV (Ang IV) influences seizure susceptibility in rat and mouse models. Indeed, Ang IV has been shown to protect rats from limbic seizures in the focal pilocarpine model. Moreover, both anticonvulsive and antiepileptogenic effects of Ang IV have been reported in the acute pentylenetetrazol (PTZ) and kindling model of generalized seizures in mice. It has been hypothesized that the latter effects on seizures could be established via a modulatory effect on dopamine receptors in the basal ganglia or via an indirect interaction between Ang IV and adenosine A1 receptors. However, a possible role for insulin-regulated aminopeptidase (IRAP), the high affinity binding site for Ang IV, has not been studied yet. To unequivocally unravel the involvement of IRAP in generalized seizure generation, we investigated the susceptibility of male IRAP wild-type (IRAP(+/+)) and knock-out (IRAP(-/-)) mice to PTZ-induced seizures. Challenging these mice intravenously with PTZ resulted in significantly increased thresholds for myoclonic twitch and generalized clonic seizures with loss of righting reflexes in IRAP(-/-) mice compared to their IRAP(+/+) littermates. These behavioural data were confirmed by video-electrocorticography monitoring. Our study shows that IRAP(-/-) mice are less sensitive to the development of PTZ-induced seizures and suggests that IRAP is involved in generalized seizure generation.