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System xc- is a plasma membrane amino acid antiporter, of mainly glial origin, that couples the import of cystine with the export of glutamate. System xc- (specific subunit xCT) contributes substantially to ambient extracellular glutamate levels in various regions of the brain, including the striatum and hippocampus. Despite the fact that system xc- is highly expressed in the brain and is a proposed therapeutic target for various neurological disorders, its function under physiological conditions in the central nervous system remains poorly understood. By acting as a source of glial extrasynaptic glutamate, system xc- might modulate synaptic transmission as a mechanism of neuro-glial communication. Previous electrophysiological findings indicate that system xc- delivered glutamate can inhibit excitatory synaptic neurotransmission in the corticoaccumbens pathway and at hippocampal CA3-CA1 synapses. To gain further insight into the proposed function of system xc- as modulator of synaptic transmission we here focus on corticostriatal synapses.

Neuroinflammation is a common feature in the pathogenesis of Parkinson's disease (PD), together with - among others - oxidative stress, proteasomal and mitochondrial dysfunction. Peripheral inflammation-induced microglial activation has been shown to increase the susceptibility of the brain for nigrostriatal degeneration. The aim of this study is therefore to create a dual-hit mouse model for PD, characterized by lipopolysaccharide (LPS)-induced neuroinflammation prior to nigral proteasome inhibition. To do so, we administered repeated low-dose LPS injections (250µg/kg for 4 days; i.p.) or a single high-dose LPS injection (5mg/kg, i.p.), to induce neuroinflammation. Next, we administered a unilateral intranigral injection of lactacystin (LAC; 3µg), a proteasome inhibitor that acutely induces dopaminergic neurodegeneration. Seven days after administration of LAC, mice were evaluated behaviorally before collecting the brain. Nigrostriatal degeneration was analyzed by quantifying striatal dopamine loss as well as loss of nigral dopaminergic neurons. Presence of neuroinflammation was confirmed by labelling Iba1-positive cells in the substantia nigra (SN). Our results show that systemic LPS injection(s) induce(s) an increase in the number of Iba1-positive cells in the SN, without inducing degeneration of the nigrostriatal pathway. However, LPS-induced neuroinflammation increases the susceptibility for LAC-induced degeneration of the nigrostriatal pathway. This dual-hit model might as such represent a relevant mouse model for PD - combining two pathogenic mechanisms - to be used to investigate the potential of therapeutic targets.

Aim: We recently proposed the cystine-glutamate antiporter system xc-as a potential neuroprotective target in Parkinson’s disease (PD), an age-related neurodegenerative disorder. System xc- is an important source of extrasynaptic glutamate and is enhanced in response to inflammation and oxidative stress, thereby decreasing the threshold for excitotoxicity. Furthermore, it modulates neuroinflammation, by favouring the pro-inflammatory, neurotoxic microglial phenotype. Expression of xCT, the specific subunit of system xc-, is increased in the brain of several models for PD and loss of system xc- (xCT-/- mice) is protective in the 6-OHDA mouse model for PD both in young and aged mice. However, it does not affect the susceptibility for MPTP-induced neurotoxicity in young mice. To further explore system xc- as a novel target to treat PD, we investigated the effect of system xc-- deficiency on proteasome-inhibition induced nigrostriatal degeneration

System xc- is a plasma membrane amino acid antiporter, of mainly glial origin, that couples the import of cystine with the export of glutamate. System xc- (specific subunit xCT) contributes substantially to ambient extracellular glutamate levels in various regions of the brain, including the striatum and hippocampus. Despite the fact that system xc- is highly expressed in the brain and is a proposed therapeutic target for various neurological disorders, including Parkinson’s disease, Alzheimer’s disease, multiple sclerosis and epilepsy, its function under physiological conditions in the central nervous system remains poorly understood. By acting as a source of glial extrasynaptic glutamate, system xc- might modulate synaptic transmission as a mechanism of neuro-glial communication. Previous electrophysiological findings indicate that system xc- delivered glutamate can inhibit excitatory synaptic neurotransmission in the cortico-accumbens pathway (Moran et al. J Neurosci. 2005; 25:6389-93) and at hippocampal CA3-CA1 synapses (Williams et al. J Neurosci. 2014; 34:16093-102). In order to gain further insight into the proposed function of system xc- as modulator of synaptic transmission, we carried out single section electron microscopy analyses of excitatory axospinous synapses at the level of the dorsolateral striatum and motor cortex of adult xCT knockout (xCT-/-) and xCT wildtype (xCT+/+) mice. Our findings accommodate the hypothesis that system xc- negatively modulates neurotransmission, as morphological changes in the excitatory synapses in the dorsolateral striatum of xCT-/- mice reflect increased synaptic activity. In particular, we could observe depletion of glutamate immunogold labeling from presynaptic terminals of xCT-/- mice, an increase in the head diameter and area of spines contacted by asymmetric synapses, an increase in the length, thickness and area of the postsynaptic density, an increased occurrence of spinules, and an increase in the average area of synaptic vesicles. The ultrastructural changesobserved in xCT deficient mice suggest the involvement of both presynaptic and postsynaptic forms of synaptic strength regulation via system xc-. In the future we would like to extend our findings on excitatory synapses in the motor cortex, as well as evaluate the expression of AMPA and NMDA receptor expression as a possible contributor to the increased size of the postsynaptic density in xCT deficient mice. Together, these findings shed new light on the re-organization of the glutamatergic system after genetic deletion of system xc-, and confirm the involvement of this antiporter in the control of synaptic strength in vivo.

A variety of pathological stimuli is able to induce systemic inflammation. When systemic inflammation occurs, soluble mediators released from peripheral immune cells are able to reach the central nervous system (CNS) leading to defective brain homeostasis and activation of central immune cells, further exacerbating brain inflammation. Moreover, peripheral inflammation also leads to acute and chronic effects on cognition and behavior. System xc-, with xCT as specific subunit, exchanges intracellular glutamate for extracellular cystine and is the main source of extracellular glutamate in mouse hippocampus and striatum. In vitro, it has been shown that several pro-inflammatory stimuli increase the expression of xCT. Besides leading to excessive release of glutamate, increased expression and/or activity of system xc- can directly influence microglia phenotypes towards a pro-inflammatory/neurotoxic state. Modulation of system xc- could therefore be beneficial in inflammation-related disorders in two ways: attenuate the glial release of toxic amounts of glutamate and directly influence microglial polarization towards a protective phenotype. In this study we induced peripheral inflammation via a single injection of 5 mg/kg bacterial lipopolysaccharide (LPS) i.p. and we sought to: i) investigate the consequences of peripheral inflammation on glutamate transporters, ii) unveil the central effects of peripheral inflammation in xCT+/+ and xCT-/- mice and iii) evaluate if deletion of xCT dampens acute clinical implications induced by peripheral inflammation. We demonstrated that LPS strongly impairs glutamate homeostasis in vivo leading to significant changes in xCT and GLT1 glutamate transporter protein expression levels in the hippocampus 1 week p.i. Those changes may potentially trigger enhanced hyperexcitability and neuronal death due to toxic extracellular levels of glutamate. We observed that in xCT-/- mice several LPS-induced clinical implications, such as hypothermia, depressive-like behavior in the forced swim and tail suspension tests and increased seizure susceptibility in the pentylenetetrazole model, were significantly attenuated. Our results highlight the importance of system xc- under inflammatory conditions and bring us one step closer in better understanding its potential relevance in neurological disorders characterized by neuroinflammation.

Zonisamide (ZNS), an anti-epileptic drug used in the symptomatic treatment of Parkinson’s disease (PD), has been recently linked with neuroprotective properties in toxin- and genetic-based models of PD. One of the mechanisms proposed to mediate the neuroprotective effects of ZNS involve an increase in expression of the cystine/glutamate antiporter system xc-, leading to enhanced cystine supply for astrocytic glutathione synthesis (Asanuma et al. Ann Neurol 2010 67(2):239-49). At the same time, however, enhancement of system xc- might trigger excitotoxicity due to pathological astrocytic glutamate release, leading to non-cell autonomous neuronal death (Massie et al. FASEB J 2011 25(4):1359-69). In order to gain further insights into the neuroprotective properties of ZNS and elucidate the role of system xc-, we have employed the lactacystin (proteasome inhibition) mouse model of PD. Our findings indicate that chronic treatment with ZNS (30mg/kg; i.p.) protects against lactacystin-induced neurodegeneration, confirming the neuroprotective properties of ZNS, for the first time in a model based on proteasome inhibition. The neuroprotective effects of ZNS were accompanied by an improvement in sensorimotor function, as evaluated using the adhesive removal test. ZNS treatment failed, however, to modulate the expression of system xc- in midbrain and striatum of lactacystin treated mice, indicating that the neuroprotective action of ZNS do not involve changes in system xc- expression. Similarly, we found that chronic treatment with ZNS did not influence system xc- expression or glutathione levels in the basal ganglia of control (untreated) mice. Finally, in vitro studies indicated that ZNS treatment did not change system xc- activity in HT22 cells or primary astrocytes, and did not influence glutathione levels in astroglial C6 cells. In conclusion, our study revealed neuroprotective and symptomatic effects of chronic ZNS treatment in a PD model based on proteasome inhibition. Our top-down approach investigating the effect of ZNS treatment in pathological and physiological conditions and in cell culture, failed to reveal any significant effect on system xc- expression or activity. We thereby propose that the neuroprotective actions of ZNS are not mediated via system xc-. Instead, other pathways are likely to be involved, such as the proposed anti-inflammatory, anti-oxidant and neurotrophic properties of ZNS. Future studies in this regard will be important in understanding the mechanisms of neuroprotection of ZNS. Nevertheless, our findings support the use of ZNS as a symptomatic and possible disease-modifying therapy in PD.

In Parkinson's disease (PD), the progressive degeneration of the nigrostriatal dopamine (DA) pathway leads to loss of striatal DA content, dysregulation of the basal ganglia motor circuit, and inhibition of movement. In addition, PD patients present with non-motor symptoms that can be debilitating in terms of impact on quality of life. From a molecular perspective, various factors have been implicated in the loss of DA neurons in PD, including failure of protein degradation pathways leading to aberrant protein accumulation. The aim of the current study was to evaluate molecular and behavioral abnormalities in a mouse model of PD, based on the intranigral infusion of proteasome inhibitor lactacystin (LAC). C57BL/6J mice, 12 weeks of age, were stereotaxically injected with 3 µg LAC or saline in the left substantia nigra pars compacta. One or three weeks following surgery, mice were tested in various behavioral paradigms, and brain tissue further processed for evaluating molecular and neurodegenerative changes. Our findings demonstrate that LAC-treated mice show loss of nigral DA-ergic neurons and concurrent striatal DA depletion, and develop behavioral (motor and non-motor) deficits, including impaired motor coordination and balance, motor asymmetry, hyperactivity, anxiety-like behavior, somatosensory dysfunction, and response perseveration. Furthermore, LAC infusion led to accumulation of Ser129-phosphorylated α-synuclein and increased VGLUT2 immunoreactivity in the ipsilateral substantia nigra. Our findings indicate that mice treated with proteasome inhibitor LAC develop parkinsonian features, including motor and non-motor impairment, pathological α-synuclein accumulation, and glutamatergic dysfunction that could be linked with increased activity of the subthalamic nucleus.

In Parkinson's disease (PD), the progressive degeneration of the nigrostriatal dopamine (DA) pathway leads to loss of striatal DA content, dysregulation of the basal ganglia motor circuit, and inhibition of movement. Various pathogenic pathways drive DA neuron death in PD, including oxidative stress, mitochondrial dysfunction, neuroinflammation, glutamate excitotoxicity, and failure of protein degradation pathways. The aim of the current study was to evaluate glutamatergic and behavioural abnormalities in a mouse model of PD, based on the intranigral infusion of proteasome inhibitor lactacystin (LAC). C57BL6/J mice, 12 weeks of age, were stereotaxically injected with 1.5uL LAC 2ug/uL in saline (n=8, LAC group) or 1.5uL saline (n=8, SHAM group) in the left substantia nigra pars compacta. One week following surgery, mice were tested in various behavioural paradigms (rotarod, open field, tail suspension, Y maze), and brain tissue further processed for cresyl violet staining, VGLUT2 immunohistochemistry, and glutamate immunogold electron microscopy. Our findings demonstrate that LAC-treated mice show loss of nigral neurons and behavioural deficits, including impaired motor coordination and balance, motor asymmetry, hyperactivity, and response perseveration. Furthermore, LAC infusion led to an increase in VGLUT2 immunoreactivity in the ipsilateral substantia nigra pars compacta. Our findings at a behavioural and molecular level indicate a possible compensatory mechanism by which the surviving DA neurons at the site of the lesion become hyperactive, as recently observed in mice lesioned with proteasome inhibitor epoxomicin (Subramaniam et al. 2014 Eur J Neurosci). In conclusion, we report that mice treated with proteasome inhibitor LAC develop parkinsonian behavioural features, and glutamatergic dysfunction that could be linked with increased activity of the subthalamic nucleus. In the future we aim at performing ultrastructural analysis of excitatory synapses, as well as evaluating expression of glutamate transporters, levels of extracellular glutamate, and the activity of the STN after LAC lesion.

Introduction: In modern society, stress is a major causative factor for a variety of psychiatric disorders. Depression, one of the main causes of disability worldwide, is a multimodal disease with chronic stress considered as a ‘trigger’ for depressive episodes. Depression and comorbid anxiety are usually related to a malfunctioning monoaminergic system, nowadays however compelling evidence points at an important role of glutamate in the etiology of the ‘depressed/anxious brain’. Being the major excitatory neurotransmitter in the central nervous system, glutamate can potentially have important excitotoxic effects. System xc- is the cystine/glutamate antiporter and the major source of extrasynaptic glutamate in some important depression-related brain areas, where it can be an interesting new target for improved psychopharmacological treatment. Methods: In this study we investigated the effect of loss of functional system xc- (e.g. deletion of the specific light chain subunit xCT; xCT-/-), on chronic stressinduced depression and anxiety in a validated animal model. Therefore we subjected xCT-/- and xCT+/+ mice, treated with chronic corticosterone injections (excessive chronic stress), to a battery of acute stress-based tests for depressive- and anxiety- like behavior and compared their behavior to vehicle treated and naïve animals. Results and discussion: Interestingly we found decreased depressive- and anxiety- like behavior in the naïve xCT-/- mice in all of the tests conducted. Unexpectedly however the decrease in depressive- and anxiety- like behavior faded and disappeared after vehicle and corticosterone treatment. These findings support further research for the role of system xc- in the stress response, since the involvement of the antiporter in regulating the response to acute versus chronic stress seems to differ. Research support: This research was supported by the Fund for Scientific Research Flanders (FWO, grant G.038412N), the Queen Elisabeth Medical Foundation (G.S.K.E.), and the Vrije Universiteit Brussel (Strategic Research Program, grant SRP40).

System xc- or the cystine/glutamate antiporter is responsible for exchanging intracellular glutamate for extracellular cystine. Cystine is intracellularly reduced to cysteine, the rate-limiting building block in the synthesis of glutathione. As such, malfunctioning of system xc- can cause oxidative stress as well as excitotoxicity (due to elevated glutamate levels). Both phenomena are common mechanisms in neurological disorders, including multiple sclerosis (MS). To explore the contribution of system xc- to the pathogenesis of MS, we investigated on one hand xCT (specific subunit of system xc-) expression in mouse models for MS as well as brain samples of MS patients, and on the other hand we studied the effect of genetic deletion of xCT on the outcome of mice in the EAE (experimental autoimmune encephalomyelitis) mouse model for MS. No changes in xCT expression were observed in the brain and spinal cord of the TMEV (Theiler’s murine encephalomyelitis virus) mouse model for MS. On the other hand, xCT protein expression was significantly increased in brain, spinal cord and spleen samples of EAE mice as well as in normal appearing white matter of MS patients compared to healthy control subjects. Whereas EAE induction in xCT-/- mice (genetic deletion of xCT and as such deficiency for system xc-) resulted in a slightly more severe clinical outcome compared to xCT+/+ littermates, the loss of system xc- in immune cells (after xCT-/- bone marrow transplantation to irradiated mice), resulted in a moderate, but significant, protection in the EAE model compared to their proper controls (xCT+/+ bone marrow transplantation to irradiated mice). Our findings confirm the involvement of system xc- in the pathogenesis of MS and suggest a pathological contribution of enhanced system xc- on immune cells infiltrating the central nervous system.

LOSS OF SYSTEM XC- INDUCES ANTIDEPRESSANT- AND ANXIOLYTIC- LIKE EFFECTS IN MICE Thomas Demuyser, Eduard Bentea, Lauren Deneyer, Giulia Albertini, Joeri Van Liefferinge, Ellen Merckx, Ann Massie, Ilse Smolders Center for Neurosciences, Vrije Universiteit Brussel, Brussels, Belgium In modern society, stress is a major causative factor for a variety of psychiatric disorders. Depression, one of the main causes of disability worldwide, is a multimodal disease with chronic stress considered as a 'trigger' for depressive episodes. Depression and comorbid anxiety are usually related to a malfunctioning monoaminergic system, nowadays however compelling evidence points at an important role of glutamate in the etiology of the 'depressed/anxious brain'. Being the major excitatory neurotransmitter in the central nervous system, glutamate can potentially have important excitotoxic effects. System xc- is the cystine/glutamate antiporter and the major source of extrasynaptic glutamate in some important depression-related brain areas, where it can be an interesting new target for improved psychopharmacological treatment. In this study we investigated the effect of loss of functional system xc- (e.g. deletion of the specific light chain subunit xCT; xCT-/-), on chronic stress induced depression and anxiety. Therefore we subjected xCT-/- and xCT+/+ mice, treated with chronic corticosterone injections (excessive chronic stress), to a battery of acute stress-based tests for depressive- and anxiety- like behavior and compared their behavior to vehicle treated and naïve animals. Interestingly we found decreased depressive- and anxiety- like behavior in the naïve and vehicle treated xCT-/- mice in most of the tests conducted. Unexpectedly however the decrease in depressive- and anxiety- like behavior was lost in the corticosterone treated xCT-/- mice, in comparison to their xCT+/+ littermates. These findings support further research for the role of system xc- in the stress response, since the involvement of the antiporter in regulating the response to acute versus chronic stress seems to differ.