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Objective Previously, we reported that inhibition of the astrocytic cystine/glutamate antiporter system xc- (SXC), using sulfasalazine (SAS), decreased evoked excitatory signaling in three distinct hyperexcitability models ex vivo. The current study expands on this work by evaluating the in vivo efficacy of SAS in decreasing astrogliosis-mediated seizure burden seen in the beta-1 integrin knockout (B1KO) model. Methods Video-EEG (electroencephalography) monitoring (24/7) was obtained using Biopac EEG acquisition hardware and software. EEG spectral analysis was performed using MATLAB. SAS was used at an equivalence of doses taken by Crohn's disease patients. Whole-cell patch-clamp recordings were made from cortical layer 2/3 pyramidal neurons. Results We report that 100% of B1KO mice that underwent 24/7 video-EEG monitoring developed spontaneous recurrent seizures and that intraperitoneal administration of SAS significantly reduced seizure frequency in B1KOs compared to B1KOs receiving sham saline. Spectral analysis found an acute reduction in EEG power following SAS treatment in B1KOs; however, this effect was not observed in nonepileptic control mice receiving SAS. Finally, whole-cell recordings from SXC knockout mice had hyperpolarized neurons and SXC-B1 double knockouts fired significantly less action potentials in response to current injection compared to B1KOs with SXC. Significance To devise effective strategies in finding relief for one-in-three patients with epilepsy who experience drug-resistant epilepsy we must continue to explore the mechanisms regulating glutamate homeostasis. This study explored the efficacy of targeting an astrocytic glutamate antiporter, SXC, as a novel antiepileptic drug (AED) target and further characterized a unique mouse model in which chronic astrogliosis is sufficient to induce spontaneous seizures and epilepsy. These findings may serve as a foundation to further assess the potential for SAS or inform the development of more potent and specific compounds that target SXC as a novel treatment for epilepsy. National Institutes of Health [5R01CA227149, 5R01NS036692] Published version National Institutes of Health, Grant/Award Number: 5R01CA227149 and 5R01NS036692

Yuechen Zhao,1 Yanqing Li,2 Ruifeng Zhang,1 Feng Wang,3 Tiejun Wang,1,* Yan Jiao4,* 1Department of Radiation Oncology, The Second Hospital of Jilin University, Changchun, People’s Republic of China; 2Department of Pathophysiology, College of Basic Medical Sciences, Jilin University, Changchun, People’s Republic of China; 3Department of Oral and Maxillofacial Surgery, Hospital of Stomatology, Jilin University, Changchun, People’s Republic of China; 4Department of Hepatobiliary and Pancreatic Surgery, The First Hospital of Jilin University, Changchun, People’s Republic of China*These authors contributed equally to this workCorrespondence: Tiejun WangDepartment of Radiation Oncology, The Second Hospital of Jilin University, No. 218 Ziqiang Street, Changchun 130022, People’s Republic of ChinaTel +8619904449098Email drwangtiejun@yeah.netYan JiaoDepartment of Hepatobiliary and Pancreatic Surgery, The First Hospital of Jilin University, No. 71 Xinmin Street, Changchun 130021, People’s Republic of ChinaTel +8613843101157Email lagelangri1@126.comAbstract: Erastin was initially discovered as a small molecule compound that selectively kills tumor cells expressing ST and RASV12 and was later widely investigated as an inducer of ferroptosis. Ferroptosis is a recently discovered form of cell death caused by peroxidation induced by the accumulation of intracellular lipid reactive oxygen species (L-ROS) in an iron-dependent manner. Erastin can mediate ferroptosis through a variety of molecules including the cystine-glutamate transport receptor (system XC−), the voltage-dependent anion channel (VDAC), and p53. Erastin is able to enhance the sensitivity of chemotherapy and radiotherapy, suggesting a promising future in cancer therapy. We hope that this review will help to better understand the role of erastin in ferroptosis and lay the foundation for further research and the development of erastin-based cancer therapies in the future.Keywords: erastin, ferroptosis, system XC−, p53, VDAC, cancer

Viele neurodegenerative oder auch neuroinflammatorische Krankheiten, unter anderem Alzheimer-Demenz, Chorea Huntington oder Epilepsie, zeigen Anzeichen einer Glutamatdysregulation mit einer System xc--Hochregulierung und einer Herunterregulierung des Glutamattransporters excitatory amino acid transporter (EAAT) 2. Die dabei m��glicherweise entstehende Exzitotoxizit��t und die pathophysiologische Bedeutung von System xc- in diesen Krankheiten konnte bisher jedoch nicht eindeutig nachgewiesen werden. System xc- fungiert als Na+-unabh��ngiger und Cl--abh��ngiger Antiporter, der Glutamat, Cystin und Cystathionin in einem 1:1-Verh��ltnis in beide Richtungen transportiert. Vorstudien haben gezeigt, dass die System xc--Expression durch verschiedene Stimuli stark induzierbar ist. Dabei ist es sehr wahrscheinlich, dass vor allem die Hochregulation der spezifischen Untereinheit xCT eine ��bergeordnete Rolle bei der Induktion der System xc--Aktivit��t in Astrozyten spielt. Die xCT-Regulation in Astrozyten auf transkriptioneller Ebene wird unter anderem ��ber die nuclear factor (erythroid-derived 2)-related factor (Nrf2) und activating transcription factor 4 (Atf4) reguliert. Diese Arbeit zeigt zun��chst, dass die System xc--Aktivit��t gemessen als Homocysteat (HCA)-sensitive [3H]-Glutamataufnahme sowohl durch Lipopolysaccharide (LPS) als auch durch eine synthetische Polyinosin-Polycytidyls��ure (Poly(I:C)) als Ersatz f��r virale, doppelstr��ngige RNA induziert werden kann. Dieser Effekt ist sowohl von der Liganden-Konzentration als auch dessen Wirkdauer abh��ngig. Dabei ist die Aktivit��tssteigerung durch LPS um ein Vielfaches h��her als durch Poly(I:C). In einem weiteren Schritt wird gezeigt, dass LPS bei der h��chsten getesteten Konzentration deutlich st��rker als Poly(I:C) sowohl die Boten-RNA (mRNA) als auch das xCT-Protein erh��ht, w��hrend bei Substanzen in gleichen Konzentrationen angewendet die Promotoraktivit��t ��hnlich stark beeinflusst wird. Diese Diskrepanz zwischen Promotoraktivit��t und mRNA-Spiegel kann eventuell ��ber post-transkriptionelle Mechanismen erkl��rt werden. Im Bereich -226 bp bis -100 bp im proximalen xCT-Promotor liegt eine Region, die in der Hochregulation der xCT-Transkription eine Rolle spielt. Eine Beteiligung der, als funktionell relevant, beschriebenen Nrf2-Bindungsstelle in diesem Bereich kann ausgeschlossen werden. Zudem wird gezeigt, dass weder LPS noch Poly(I:C) einen Effekt auf das intrazellul��re Glutathion (GSH)-Niveau haben. Somit bleibt der Signalweg, ��ber den inflammatorische Stimuli zu einer Erh��hung der System xc--Aktivit��t f��hren, weiter unklar. Als m��glicher Kandidat k��nnte nuclear factor 'kappa-light-chain-enhancer' of activated B-cells (NF-��B) in den Fokus r��cken, der sowohl in den Signalwegen der Toll-��hnlichen Rezeptoren (TLR) eine gewichtige Rolle spielt und auch im xCT-Promotor eine m��gliche Bindungstelle hat.

Epilepsy is a neurological disorder that presents a significant public health burden, with an estimated five million people being newly diagnosed each year. However, current therapeutics designed to modify neuronal processes, provide no relief to 1-in-3 epileptic patients. Additionally, no disease modifying therapies currently exist to treat the underlying pathological processes involved in epileptogenesis. The overarching goal of this project is to further characterize the role astrocytes play in epileptogenesis, in hopes of revealing novel therapeutic targets to benefit patients who otherwise have no effective treatment options. System xc- (SXC), a cystine/glutamate antiporter expressed in astrocytes, is one such target that has been shown to play a critical role in establishing ambient extracellular glutamate levels in both health and disease. SXC has been shown to play a major role in setting ambient glutamatergic tone in the central nervous system (CNS) as pharmacological inhibition of SXC, using (S)-4-carboxyphenylglycine (S-4-CPG) or antisense xCT, resulted in a 60% reduction in extrasynaptic glutamate in the nucleus accumbens. Additionally, investigations in tumor-associated epilepsy revealed that overexpression of SXC seen in glioblastomas lead to higher levels of peritumoral glutamate, neuronal excitotoxicity, and ultimately seizures. These studies also found that SXC inhibition with sulfasalazine (SAS), an FDA approved drug and potent inhibitor of SXC, can ameliorate seizure burden in a glioblastoma mouse model. Therefore, the principal objective of this study is to further investigate the role of astrocytic SXC activity in epileptogenesis and seizure generation. In doing so, we also evaluated the efficacy of SAS in reducing seizure burden in vivo using an astrogliosis-mediated epilepsy mouse model. In this dissertation we show that (1) SXC inhibition, using SAS, is able to decrease induced epileptiform activity in multiple models of chemically induced hyperexcitability (2) this is due to a preferential decrease of NMDAR-mediated currents and (3) SXC inhibition, via SAS, decreases seizure burden in vivo in an astrogliosis-mediated epilepsy model. Doctor of Philosophy Epilepsy, characterized by unpredictable seizures, affects approximately 2.2 million Americans, with 150,000 new cases being diagnosed each year. Seizures typically occur when there is an imbalance between the excitatory and inhibitory processes in the brain. Because neurons are the primary cell in the brain that carry out these processes, clinically used anti-epileptic drugs (AEDs) work by either decreasing neuronal excitation or increasing neuronal inhibition. Despite success with managing seizures, up to 1-in-3 patients with epilepsy do not find any relief with existing AEDs. A statistic that has not changed in over 50 years of drug development. With this in mind, the overarching goal of this dissertation is to explore the efficacy of targeting non-neuronal processes to treat epilepsy and broaden the search for new AED targets by further characterizing a unique mouse model of epilepsy. One such target studied in our lab is system xc- (SXC), a glutamate/cystine antiporter present on astrocytes, a non-neuronal cell that provides maintenance, support and protection for neurons. Investigations in tumor-associated epilepsy from our lab revealed that hyperactivity of SXC in tumor cells was directly related to the development of tumor-associated epilepsy. These studies also revealed that SXC inhibition using sulfasalazine (SAS), an FDA approved drug, can decrease seizure burden in a tumor mouse model. Therefore, the principal objective of this study is to further investigate the role of astrocytic SXC activity in the development of epilepsy and seizure generation. In this dissertation we show that SXC inhibition, using SAS, is able to decrease neuronal hyperactivity and decreases seizure burden in an astrogliosis-mediated epilepsy model.

Given the important functions that glutamate serves in excitatory neurotransmission, understanding the regulation of glutamate in physiological and pathological states is critical to devising novel therapies to treat epilepsy. Exclusive expression of pyruvate carboxylase and glutamine synthetase in astrocytes positions astrocytes as essential regulators of glutamate in the central nervous system (CNS). Additionally, astrocytes can significantly alter the volume of the extracellular space (ECS) in the CNS due to their expression of the bi-directional water channel, aquaporin-4, which are enriched at perivascular endfeet. Rapid ECS shrinkage has been observed following epileptiform activity and can inherently concentrate ions and neurotransmitters including glutamate. This review highlights our emerging knowledge on the various potential contributions of astrocytes to epilepsy, particularly supporting the notion that astrocytes may be involved in seizure initiation via failure of homeostatic responses that lead to increased ambient glutamate. We also review the mechanisms whereby ambient glutamate can influence neuronal excitability, including via generation of the glutamate receptor subunit GluN2B-mediated slow inward currents, as well as indirectly affect neuronal excitability via actions on metabotropic glutamate receptors that can potentiate GluN2B currents and influence neuronal glutamate release probabilities. Additionally, we discuss evidence for upregulation of System xc-, a cystine/glutamate antiporter expressed on astrocytes, in epileptic tissue and changes in expression patterns of glutamate receptors. NIHUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [1R01CA227149-01A1, 1R01NS036692-01A1]; DODUnited States Department of Defense [W81XWH-18-1-0521] This work was supported by NIH grants 1R01CA227149-01A1 and 1R01NS036692-01A1, and DOD grant W81XWH-18-1-0521.

In dieser Arbeit wurde experimentell untersucht, ob therapeutische Konzentrationen des stimmungsstabilisierenden Medikaments Lithium zu einer gesteigerten Aktivität und Expression des Glutamat/Cystin-Antiporters System xc- in vitro führen. System xc- ist ein heterodimeres Transportsystem bestehend aus einer schweren und einer leichten Kette, das konzentrationsabhängig Cystin nach intrazellulär und Glutamat in den Extrazellulärraum in einem Verhältnis von 1:1 transportiert. Zwar ist es auf Zellen verschiedener Organsysteme des Körpers exprimiert, jedoch im Wesentlichen auf Zellen des zentralen Nervensystems. Der Transport Cystins nach intrazellulär führt zu einer verstärkten Bildung Glutathions, das als antioxidatives Molekül eine wesentliche Rolle in der Abwehr von oxidativem Stress spielt. Dieser wird durch Zellschädigung als entscheidender Faktor in der Entstehung und Pathogenese insbesondere neurodegenerativer, aber auch psychiatrischer Erkrankungen gesehen. Durch System xc- nach extrazellulär transportiertes Glutamat wird über in der Zellmembran vorhandene exzitatorische Aminosäuretransporter wieder nach intrazellulär überführt. Bei einer gesteigerten System xc--Aktivität scheint der Rücktransport dem gesteigerten Aufkommen Glutamats jedoch nicht nachzukommen, so dass eine erhöhte Glutamatkonzentration im Extrazellulärraum resultiert. Als exzitatorischer Neurotransmitter könnte so ein erhöhtes Glutamat-Vorkommen zu einer Überstimulation und damit zu einer Zellschädigung führen. System xc- gilt durch die positive Beeinflussung von oxidativem Stress aber der gleichzeitigen Steigerung der extrazellulären Glutamatkonzentration als zweischneidiges Schwert. Eine Hochregulation des Transportsystems in neuronalen HT22-Zellen durch 20 mM Lithium über eine Inhibition der Glykogensynthasekinase-3β war bereits bekannt. In dieser Arbeit sollte nun untersucht werden, ob auch therapeutische Lithium-Konzentrationen zu einer System xc--Hochregulierung führen. Lithium ist bekannt und gebräuchlich als stimmungsstabilisierendes Medikament zur Behandlung bipolar affektiver Störungen und zur Augmentationstherapie therapieresistenter Depressionen. Zudem gibt es neue Stimmen, die Lithium eine neuroprotektive Wirkung zuschreiben. Der therapeutische Bereich Lithiums liegt zwischen 0.5 und 1.2 mmol/l. Eine Überdosierung kann unter anderem zu schweren Herzrhythmusstörungen und Krampfanfällen führen. Die Wirkung Lithiums auf System xc- wurde an zwei verschiedenen neuronalen Zellreihen untersucht. Zunächst wurde mittels eines radioaktiven Glutamat-Aufnahmeassays untersucht, ob Konzentrationen von 0,3, 1 und 3 mmol/L Lithium zu einer System xc--Aktivitätssteigerung der immortalisierten neuronalen HT22-Zellreihe führt. Im Anschluss wurde durch eine quantitative Polymeraskettenreaktion untersucht, ob die gleichen Lithium-Konzentrationen auch zu einer verstärkten Expression der mRNA der spezifischen Untereinheit des Transportsystems führen. Im Anschluss wurden die Experimente an primären murinen Astrozyten durchgeführt. Es konnte gezeigt werden, dass therapeutische Lithium-Konzentrationen zu einer verstärkten System xc--Aktivität und xCT-Expression in beiden Zellreihen führen. Gleichzeitig wurde gezeigt, dass therapeutische Konzentrationen Lithiums keine System xc--Aktivitätssteigerung in Fibroblasten bewirken. Die molekulare Interaktion zwischen Lithium und System xc- könnte im Wesentlichen über eine Inhibition der Glykogensynthasekinase-3-β und in der Folge zu einer gesteigerten Aktivität der Transkriptionsfaktoren ATF4 und Nrf2 stattfinden. In der Folge würde eine erhöhte System xc--Aktivität einerseits zu einem gesteigerten Glutathionmetabolismus und damit einer verstärkten Abwehr von oxidativem Stress führen. Die gesteigerte extrazelluläre Glutamatkonzentration könnte andererseits die epileptogene Nebenwirkung Lithiums bei Überdosierung erklären. Jedoch könnte eine System xc--Aktivitätssteigerung durch therapeutische Lithium-Konzentrationen auch Einfluss auf die glutamaterge Neurotransmission nehmen. Über eine Aktivierung metabotroper Glutamatrezeptoren könnte die synaptische Glutamatfreisetzung reguliert werden. Dies könnte eine gesteigerte Langzeitpotenzierung beziehungsweise Langzeitdepression, als wichtiger Hintergrund kognitiver Gedächtnisfaktoren, bewirken. Eine Stimulation beziehungsweise Inhibition verschiedener prä- oder postsynaptischer metabotroper Glutamatrezeptoren hatte in verschiedenen Studien eine antidepressive und anxiolytische Wirkung. Eine gesteigerte System xc--Aktivität durch therapeutische Lithium-Konzentrationen könnte somit möglicherweise einen Teil der stimmungsstabilisierenden Wirkung des Medikaments erklären.

Oxidative damage has been identified as a major causative factor in degenerative diseases of the retina, retinal pigment epithelial (RPE) cells are at high risk. Hence, identifying novel strategies for increasing the antioxidant capacity of RPE cells, the purpose of this study, is important. Specifically, we evaluated the influence of selenium in the form of selenomethionine (Se-Met) in cultured RPE cells on system xc- expression and functional activity and on cellular levels of glutathione, a major cellular antioxidant. ARPE-19 and mouse RPE cells were cultured with and without selenomethionine (Se-Met), the principal form of selenium in the diet. Promoter activity assay, uptake assay, RT-PCR, northern and western blots, and immunofluorescence were used to analyze the expression of xc-, Nrf2, and its target genes. Se-Met activated Nrf2 and induced the expression and function of xc- in RPE. Other target genes of Nrf2 were also induced. System xc- consists of two subunits, and Se-Met induced the subunit responsible for transport activity (SLC7A11). Selenocysteine also induced xc- but with less potency. The effect of Se-met on xc- was associated with an increase in maximal velocity and an increase in substrate affinity. Se-Met increased the cellular levels of glutathione in the control, an oxidatively stressed RPE. The Se-Met effect was selective, under identical conditions, taurine transport was not affected and Na+-coupled glutamate transport was inhibited. This study demonstrates that Se-Met enhances the antioxidant capacity of RPE by inducing the transporter xc- with a consequent increase in glutathione.

System xc- ist ein Cystin-Glutamat-Antiporter, der im Menschen vor allem in Zellen des zentralen Nervensystems vorkommt. Insbesondere in Astrozyten ist er sehr ausgeprägt exprimiert. System xc- tauscht Glutamat (Glu) und Cystin im Verhältnis 1:1 aus. Dieser Austausch ist prinzipiell in beiden Richtungen möglich. In vivo wird aufgrund der Konzentrationsgradienten Glutamat exportiert und Cystin importiert. Cystin ist das limitierende Substrat der Glutathionsynthese. Glutathion (GSH) ist das wichtigste Antioxidanz des Körpers und dient somit dem Schutz der Zelle vor oxidativem Stress - dem Ungleichgewicht zwischen freien Sauerstoffradikalen (ROS) und Antioxidanzien. Oxidativer Stress führt zur Schädigung von Proteinen, Lipiden und DNA und trägt zur Alterung der Zellen bei. Diese führt zur Entstehung neurodegenerativer Erkrankungen wie der Parkinson- und Alzheimer-Erkrankung der Amyotrophen Lateralsklerose und der Multiplen Sklerose. Während eine System xc--Induktion gegen oxidativen Stress durch Steigerung der GSH-Synthese protektiv wirkt, hat die Induktion auch Schattenseiten. Im zentralen Nervensystem ist System xc- hauptverantwortlich für extrasynaptisches Glutamat. Eine zu hohe extrazelluläre Glu-Konzentration kann zu Exzitotoxizität führen. Dabei entsteht durch Übererregung der NMDA-Rezeptoren ein Calcium-Einstrom, der zum Zelltod führt und an der Pathogenese von Epilepsien, Multipler Sklerose, der Amyotophen Lateralsklerose und der Chorea Huntington beteiligt ist. System xc- wird durch oxidativen Stress, Schwermetalle und Elektrophile über den Transkriptionsfaktor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) und bei Aminosäuremangel über den aktivierenden Trankriptionsfaktor 4 (ATF4) hochreguliert. Weiterhin sind einige mit Entzündung assoziierte Induktoren wie Interleukin 1β, Lipoploysaccharide und Tumornekrosefaktor α (TNF-α) bekannt Dabei ist der genaue Mechanismus noch ungeklärt ist. Die Induktion durch TNF-α wurde in dieser Arbeit untersucht. Die Experimente wurden mit primären murinen Astrozyten und mit murinen embryonalen Fibroblasten (MEF) durchgeführt. Letztere wurden als Modell verwendet, da knock-out-Zelllinien in der Regel nur als MEF zur Verfügung standen. Die System xc--Aktivität wurde funktionell mittels eines Glu-Aufnahmeassays mit radioaktiv markiertem L-[3H]-Glu bestimmt. System xc- wird sowohl in primären Astrozyten als auch in MEF über den TNF-α-Rezeptor 1 zeit- und konzentrationsabhängig induziert. Der zeitliche Verlauf deutete auf einen transkriptionellen Mechanismus hin. Der Transkriptionsfaktor Nrf2 ist essentiell für die TNF-α-vermittelte System xc-- Induktion. In Nrf2-/- MEF ließ sich die System xc--Aktivität nicht mehr durch TNF-α induzieren. Die pharmakologische Inhibition von nuclear factor 'kappa-light-chain-enhancer' of activated B-cells (NF-κB) sowie Inhibitor von κB-Kinase beta(IKKbeta)-Defizienz führten zu einer Steigerung der System xc--Aktivität. Dies könnte durch den hemmenden Einfluss von NF-κB auf den Nrf2-Signalweg erklärt werden. Bei p38-Inhibition konnte eine Steigerung der Induktion gesehen werden, die darauf zurückzuführen sein könnte, dass p38 hemmende Effekte auf den Nrf2-Signalweg hat. Gegenteilig dazu erzielte eine Inhibition der durch extrazelluläres Signal regulierten Kinasen (ERK1/2) eine Hemmung der TNF-α vermittelten System xc--Induktion. Dies könnte durch die Nrf2-Induktion durch ERK1/2 zu erklären sein. Auf den ersten Blick scheint die System xc--Induktion ein vorteilhafter Mechanismus, um dem ROS-induzierten oxidativen Stress entgegenzuwirken. Allerdings werden durch die Induktion Probleme in Gestalt der Glu-induzierten Exizitotoxizität geschaffen. In verschiedenen Tiermodellen konnte gezeigt werden, dass die System xc--Induktion die Progression neurodegenerativer Erkrankungen fördert. Als mögliche Gegenmaßnahme zur System xc--induzierten Exzitotoxizität kommt die Induktion exzitatorischer Aminosäuretransporter (EAAT) in Frage. Die System xc--Induktion im Rahmen einer ZNS-Inflammation scheint an der Entstehung und Progression neurodegenerativer Erkrankungen - am ehesten durch Glu-bedingte Exzitotoxizität - beteiligt zu sein. Eine zusätzliche pharmakologische Steigerung scheint deshalb nicht erstrebenswert. System xc--Inhibition kommt bei Neuroinflammation als therapeutisches Ziel in Frage, dazu müsste aber der Signalweg abschließend bekannt sein.

Metastatic bone cancers cause severe symptoms including pain that compromises patient functional status, quality of life, and survival. Current treatment strategies have limited efficacy and dose-limiting side effects. Cancer-induced bone pain (CIBP) is a unique pain state that shares features with but is distinct from the pathology of neuropathic and inflammatory pain. This dissertation investigates how CIBP is generated and maintained by the direct effects of cancer cells on their metastatic microenvironment and the peripheral nervous system, including unique signaling properties and gene expression changes. In particular, we found that genetic knockdown of the functional subunit xCT of the system xC- cystine/glutamate antiporter can reduce CIBP, further elucidating this as a therapeutic of interest. We found that the neuroprotective voltage-gated calcium channel inhibitors progesterone and pregabalin markedly reduce mechanical hypersensitivity and excitability in sensory neurons of the dorsal root ganglion (DRG) in male rat models of neuropathic pain, but that these effects and less pronounced in females. In cancer pain, these sex differences are reversed, with females but not males demonstrating a delay in time-to-onset of mechanical hypersensitivity. We also analyzed gene expression at the DRG by RNA-Sequencing of rat models of CIBP. Our findings uncovered differential gene expression between CIBP and sham controls and between ipsilateral and contralateral DRGs in CIBP model rats. These studies have identified several promising avenues for therapeutic research for CIBP. Dissertation Doctor of Philosophy (PhD) The tools we have right now to manage severe and chronic pain are insufficient. Patients with advanced cancers including bone cancer can suffer from very severe pain. This pain is generated in a number of ways including by the tumour itself releasing chemicals that activate pain-sensing nerves, by the destruction of the bone in and around the tumour, and by the sensitization of the nervous system, which can make pain worse and longer lasting. We have taken three approaches to researching cancer pain and to investigating new treatments. We have found that by reducing the amount of glutamate that cancer cells can release into their environment, we can reduce cancer pain in mice. We also found that treating rats with pregabalin and progesterone can change nerve signaling and reduce neuropathic pain, but that this effect is most pronounced in male rats with neuopathic pain and smaller in female rats with neuropathic pain, and even smaller in rats with cancer pain. We also analyzed expression of all the protein-coding genes in dorsal root ganglia from rats with cancer pain and found that there are many differences from rats without pain. Some of these differences may be promising new research targets. Going forward this research has provided important evidence necessary for next steps to develop new therapies and research strategies for cancer pain.

Rationale: Although molecular investigations of regulator of G-protein signaling 4 (RGS4) alterations in schizophrenia patients yielded partially inconsistent findings, the previous studies suggested that RGS4 is both a positional and functional candidate gene for schizophrenia and is significantly decreased in the prefrontal cortex. However, the exact role of RGS4 in the pathophysiology of schizophrenia is unclear. Moreover, a whole genome transcription profile study showed the possibility of RGS4-regulated expression of SLC7A11(xCT), a component of cysteine/glutamate transporter or system xc-. We hypothesized that system xc- is a therapeutic target of RGS4 deficit-mediated schizophrenia. Methods: Pharmacological and genetic manipulation of RGS4 in organotypic brain slice cultures were used as an ex vivo model to investigate its role in system xc- and glutamatergic function. Lentiviral-based mouse models with RGS4 deficit in the prefrontal cortex and treatment with system xc- activator, N-acetyl cysteine (NAC), were utilized to observe their impacts on glutamatergic function and schizophrenic behaviors. Results: Genetic and pharmacological inhibition of RGS4 resulted in a significant decrease in SLC7A11 (xCT) expression and hypofunction of system xc- and reduced glutamatergic function in organotypic brain slice cultures. However, NAC restored the dysregulation of RGS4-mediated functional deficits of glutamate. Moreover, knockdown of RGS4 specifically in the prefrontal cortex caused mice to exhibit behaviors related to schizophrenia such as increased stereotypy, impaired prepulse inhibition, deficits in social interactions, working memory, and nesting behavior, while enhancing sensitivity to the locomotor stimulatory effect of MK-801. These mice displayed glutamatergic dysfunction in the prefrontal cortex, which may have contributed to the behavioral deficits. RGS4 knockdown mice that received NAC treatment had improved glutamatergic dysfunction and schizophrenia behaviors. Conclusion: Our results suggest that RGS4 deficit induces dysregulation and dysfunction of system xc-, which further results in functional deficits of the glutamatergic system and subsequently to schizophrenia-related behavioral phenotypes. Activation of system xc- offers a promising strategy to treat RGS4 deficit-mediated schizophrenia.

Background Chronic pain is a major symptom that develops in cancer patients, most commonly emerging during advanced stages of the disease. The nature of cancer-induced pain is complex, and the efficacy of current therapeutic interventions is restricted by the dose-limiting side-effects that accompany common centrally targeted analgesics. Methods This review focuses on how up-regulated glutamate production and export by the tumour converge at peripheral afferent nerve terminals to transmit nociceptive signals through the transient receptor cation channel, TRPV1, thereby initiating central sensitization in response to peripheral disease-mediated stimuli. Results Cancer cells undergo numerous metabolic changes that include increased glutamine catabolism and over-expression of enzymes involved in glutaminolysis, including glutaminase. This mitochondrial enzyme mediates glutaminolysis, producing large pools of intracellular glutamate. Up-regulation of the plasma membrane cystine/glutamate antiporter, system xc-, promotes aberrant glutamate release from cancer cells. Increased levels of extracellular glutamate have been associated with the progression of cancer-induced pain and we discuss how this can be mediated by activation of TRPV1. Conclusion With a growing population of patients receiving inadequate treatment for intractable pain, new targets need to be considered to better address this largely unmet clinical need for improving their quality of life. A better understanding of the mechanisms that underlie the unique qualities of cancer pain will help to identify novel targets that are able to limit the initiation of pain from a peripheral source–the tumour.

Jennifer Fazzari,Matthew D Balenko,Natalie Zacal,Gurmit Singh Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON, Canada Abstract: The cystine/glutamate antiporter has been implicated in a variety of cancers as a major mediator of redox homeostasis. The excess glutamate secreted by this transporter in aggressive cancer cells has been associated with cancer-induced bone pain (CIBP) from distal breast cancer metastases. High-throughput screening of small molecule inhibitors of glutamate release from breast cancer cells identified several potential compounds. One such compound, capsazepine (CPZ), was confirmed to inhibit the functional unit of system xc− (xCT) through its ability to block uptake of its radiolabeled substrate, cystine. Blockade of this antiporter induced production of reactive oxygen species (ROS) within 4 hours and induced cell death within 48 hours at concentrations exceeding 25 μM. Furthermore, cell death and ROS production were significantly reduced by co-treatment with N-acetylcysteine, suggesting that CPZ toxicity is associated with ROS-induced cell death. These data suggest that CPZ can modulate system xc− activity in vitro and this translates into antinociception in an in vivo model of CIBP where systemic administration of CPZ successfully delayed the onset and reversed CIBP-induced nociceptive behaviors resulting from intrafemoral MDA-MB-231 tumors. Keywords: glutamate, breast cancer, cancer-induced bone pain, system xc&minus

The inflammatory response in the central nervous system involves activated microglia. Under normal conditions they remove damaged neurons by phagocytosis. On the other hand, neurodegenerative diseases are thought to involve chronic microglia activation resulting in release of excess glutamate, proinflammatory cytokines and reactive oxygen species, leading to neuronal death. System xC- cystine/glutamate antiporter (SXC), a sodium independent heterodimeric transporter found in microglia and astrocytes in the CNS, imports cystine into the cell and exports glutamate. SXC has been shown to be upregulated in neurodegenerative diseases including multiple sclerosis, ALS, neuroAIDS Parkinson's disease and Alzheimer's disease. Consequently, SXC inhibitors could be of use in the treatment of diseases characterized by neuroinflammation and glutamate excitotoxicity. We report on the optimization of a primary microglia-based assay to screen for SXC inhibitors. Rat primary microglia were activated using lipopolysaccharides (LPS) and glutamate release and cystine uptake were monitored by fluorescence and radioactivity respectively. LPS-induced glutamate release increased with increasing cell density, time of incubation and LPS concentration. Conditions to screen for SXC inhibitors were optimized in 96-well format and subsequently used to evaluate SXC inhibitors. Known SXC inhibitors sulfasalazine, S-4CPG and erastin blocked glutamate release and cystine uptake while R-4CPG, the inactive enantiomer of S-4CPG, failed to inhibit glutamate release or cystine transport. In addition, several erastin analogs were evaluated using primary microglia and found to have EC50 values in agreement with previous studies using established cell lines., Highlights • Conditions to screen for SXC inhibitors were optimized in 96-well format. • Assay enables higher throughput screens for SXC inhibitors with primary microglia. • Screening assay was used to evaluate prototype SXC inhibitors and erastin analogs.

Cancer-induced pain (CIP) is a debilitating condition that accompanies late-stage cancer for the majority of patients. The work presented in this dissertation addresses the multifaceted role of glutamate in cancer cell-induced pain signalling and provides several potential therapeutic directions. Several cell types, including breast cancer cells and microglia, release glutamate via the system xC- antiporter. To limit the excitotoxic tendency of breast cancer cells to release glutamate in excess, we first indirectly inhibited xCT, the active subunit of system xC-, with the TrkA inhibitor AG879. We demonstrated that the system xC- antiporter is functionally influenced by the actions of nerve growth factor on its cognate receptor, TrkA, and that inhibiting this complex reduced CIP via downstream actions on xCT. Co-culture studies then demonstrated the direct effect of glutamate released by wildtype MDA-MB-231 carcinoma cells on microglial activation, as well as functional system xC- activity, while knockdown of xCT in MDA-MB-231 cells mitigated microglial activation and cystine uptake. Blockade of system xC- with sulfasalazine attenuated nociception in an immunocompetent murine model of CIP and inhibited tumour-induced microglial activation in the dorsal horn of the spinal cord. Finally, tumour-induced nociceptive behaviours appeared to progress in parallel with microglial activation in the hippocampus, and ablating microglia delayed the onset and severity of tumour-induced nociceptive behaviours, confirming that microglia are implicated in CIP and regional microglia are influenced by this pain. This is the first experimental evidence to demonstrate the effects of peripheral tumour on hippocampal microglial activation in relation to cancer-related nociception. These data collectively demonstrate that the system xC- antiporter is functionally implicated in CIP and may be particularly relevant to pain progression through spinal microglia. Upregulated xCT in chronically activated microglia may be one pathway to central glutamate cytotoxicity. Therefore, microglial xCT may therefore be a valuable target for mitigating CIP. Thesis Doctor of Philosophy (Medical Science) Cancer-induced pain (CIP) is a debilitating condition that accompanies late-stage metastatic cancer. Clinically, achieving analgesia often comes at the expense of patients’ quality of life, as current therapeutics fail to adequately manage this pain and induce dose-dependent side effects. Cancer cells secrete excess amounts of glutamate, a signalling molecule involved in CIP, which can activate immune cells called microglia within the spinal cord. Mice that demonstrate tumour-induced pain exhibit an amplified immune response that manifests through the activation pattern and quantity of microglia within the spinal cord, as well as brain regions implicated in pain and distress. Pharmacologically blocking glutamate release from cancer cells limits this pain response, in addition to several physiological indicators of pain, including microglial activation in the central nervous system. Changes in microglia-related glutamate signalling may reflect the emotional problems reported by patients with CIP. Better understanding the mechanisms of CIP will help generate more comprehensive treatment approaches.

Aims: Current treatment options for ovarian clear cell carcinoma (OCCC) are limited to combination of platinum-based and other cytotoxic agents to which patients respond poorly due to intrinsic chemoresistance. There is therefore an urgent need to develop alternative therapeutic strategies for OCCC. Results: Cysteine deprivation suppresses OCCC growth in vitro and in vivo with no apparent toxicity. Modes of cell death induced by cysteine deprivation in OCCC are determined by their innate metabolic profiles. Cysteine-deprived glycolytic OCCC is abolished primarily by oxidative stress-dependent necrosis and ferroptosis, which can otherwise be prevented by pretreatment with antioxidative agents. Meanwhile, OCCC that relies on mitochondria respiration for its bioenergetics is suppressed through apoptosis, which can otherwise be averted by pretreatment with cysteine precursor alone, but not with antioxidative agents. Cysteine deprivation induces apoptosis in respiring OCCC by limiting iron-sulfur (Fe-S) cluster synthesis in the mitochondria, without which electron transport chain may be disrupted. Respiring OCCC responds to Fe-S cluster deficit by increasing iron influx into the mitochondria, which leads to iron overload, mitochondria damage, and eventual cell death. Innovation/Conclusion: This study demonstrates the importance of cysteine availability in OCCC that is for its antioxidative property and its less appreciated role in mitochondria respiration. Regardless of OCCC metabolic profiles, cysteine deprivation abolishes both glycolytic and respiring OCCC growth in vitro and in vivo. Conclusion: This study highlights the therapeutic potential of cysteine deprivation for OCCC.

Ferroptosis is a newly described form of regulated cell death, distinct from apoptosis, necroptosis and other forms of cell death. Ferroptosis is induced by disruption of glutathione synthesis or inhibition of glutathione peroxidase 4, exacerbated by iron, and prevented by radical scavengers such as ferrostatin-1, liproxstatin-1, and endogenous vitamin E. Ferroptosis terminates with mitochondrial dysfunction and toxic lipid peroxidation. Although conclusive identification of ferroptosis in vivo is challenging, several salient and very well established features of neurodegenerative diseases are consistent with ferroptosis, including lipid peroxidation, mitochondrial disruption and iron dysregulation. Accordingly, interest in the role of ferroptosis in neurodegeneration is escalating and specific evidence is rapidly emerging. One aspect that has thus far received little attention is the antioxidant transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2). This transcription factor regulates hundreds of genes, of which many are either directly or indirectly involved in modulating ferroptosis, including metabolism of glutathione, iron and lipids, and mitochondrial function. This potentially positions Nrf2 as a key deterministic component modulating the onset and outcomes of ferroptotic stress. The minimal direct evidence currently available is consistent with this and indicates that Nrf2 may be critical for protection against ferroptosis. In contrast, abundant evidence demonstrates that enhancing Nrf2 signaling is potently neuroprotective in models of neurodegeneration, although the exact mechanism by which this is achieved is unclear. Further studies are required to determine to extent to which the neuroprotective effects of Nrf2 activation involve the prevention of ferroptosis.

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.

xCT protein is the specific subunit of System xc-, a multi-proteic complex importing cystine into cell while releasing extracellular glutamate. Although xCT protein expression is detected in brain glial cells, its expression in the spinal cord and during spinal cord disorders remain elusive. The aim of this study is to characterize the contribution of xCT to functional and histological outcomes following SCI, using wild-type mice (xCT+/+) and genetically-invalidated mice (xCT-/-). In situ hybridization allowed to detect xCT mRNA in astrocyte subpopulations and in meningeal fibroblasts in the normal spinal cord. During the early phase of spinal cord injury, xCT mRNA could also be detected in microglial cells and overall mRNA levels were upregulated, peaking at 4 days post-injury. While both injured xCT+/+ and xCT-/- mice recovered partly their motor functions, xCT-/- mice recovered muscular grip strength as well as pre-SCI weight substantially faster than xCT+/+ mice. Histology of injured spinal cords revealedincreased number of motor neurons in xCT-/- mice at multiple distances around lesion epicenter. As xCT has been demonstrated as a regulator of microglial function (Mesci et al., Brain, 2015), we assessed markers of microglial activation. At 2 weeks post-SCI, the number of type A Iba1+ cells was unexpectedly much higher in contused xCT-/- than in xCT+/+ spinal cords. Analysis of M1/M2 polarization showed that contused xCT-/- spinal cords contained higher mRNA levels of Ym1 and IGF-1 (M2) while lower levels of NOX2 and TNF-a (M1). Additionally, the number of astrocytes and oligodendrocytes were unchanged between the two injured groups. This study suggests that, following SCI trauma, an early xCT upregulation (if confirmed at the protein level) exacerbates microglia-driven inflammation and influences motor neuron survival.

Despite the lack of robust clinical response, treatment strategies for cancer-induced depression (CID) are currently limited to those developed for non-cancer-related depression. The work presented in this dissertation conceptualizes CID as a pathophysiologically distinct form of depression. To investigate CID at the most basic level, we first developed a preclinical model that was validated by comparison to an established model of stress-induced depressive-like behaviours. The positive control model was developed by chronically treating female BALB/c mice with oral corticosterone (CORT). The CID model was developed using subcutaneous inoculation with 4T1 mammary carcinoma cells. Anhedonia, behavioural despair, and dendritic atrophy in the medial prefrontal cortex (mPFC) were observed in both models. Similar to many human cancer cell lines, 4T1 cells were shown to secrete significant amounts of glutamate, which was markedly attenuated using the system xc- inhibitor sulfasalazine (SSZ). In CID mice, oral treatment with SSZ was at least as effective as fluoxetine, a popular clinical antidepressant, at preventing depressive-like behaviours. This effect was primarily attributable to intact SSZ, rather than its anti-inflammatory metabolite. RNA-sequencing was performed on hippocampal samples from CID and CORT animals. Analysis of differential expressed genes (DEGs) revealed significant overlap between the two models. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and biological process gene ontologies (GO:BP) terms related to ion homeostasis and neuronal communication were enriched for both models. CID was associated with additional DEGs that were not identified in the CORT model. These DEGs were enriched in KEGG pathways and GO:BP terms related to neuronal development, intracellular signalling cascade, learning, and memory. These studies suggest that CID may involve a distinct aetiology, and that glutamate secretion by cancer cells presents a viable target for antidepressant treatment. The development of mechanism-based therapeutics for CID will dramatically improve the quality of life for cancer patients. Thesis Doctor of Philosophy (PhD) Cancer patients are at a high risk of developing depression. In addition to the psychological stress caused by a cancer diagnosis, there is evidence that cancer causes depression through biological pathways. To investigate these pathways, a mouse model of cancer-induced depression (CID) was developed. This model showed comparable behavioural and structural brain deficits to those observed in a stress model of depression. Cancer cells secrete elevated levels of glutamate, a signalling molecule that is involved in depression. In CID mice, inhibiting glutamate release had an antidepressant effect similar to that of fluoxetine, a standard clinical antidepressant. A genetic analysis on brain samples from the CID model revealed significant overlap with the stress model of depression. CID mice had additional changes relevant to learning, memory, and brain cell development that were not detected in the stress model. A better understanding of CID will lead to better treatment strategies developed specifically for cancer patients.

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.

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.

Parkinson's disease (PD) is an age-related neurodegenerative disorder in which nigrostriatal dopamine (DA)-ergic neurons are progressively lost. The cause of DA-ergic cell death is incompletely understood but seems to involve activation of pathogenic pathways related to oxidative stress, mitochondrial dysfunction, glutamate excitotoxicity, and neuroinflammation. Furthermore, patients with PD demonstrate impaired proteostasis, as they show decreased proteasomal activity in the substantia nigra, as well as hallmark signs of protein aggregation. Despite important advances in understanding the pathogenesis and clinical profile of PD, therapeutic approaches targeting disease progression remain limited. System xc- is a plasma membrane antiporter that brings cystine inside the cell, and exports glutamate in the extracellular environment. As the imported cystine can be used for GSH synthesis, system xc- is believed to bridge oxidative stress and glutamate excitotoxicity, pathways that co-occur in PD. We have previously demonstrated that nigral DA-ergic neurons of system xc- deficient mice are protected against 6-hydroxydopamine, pointing to this glutamate transporter as a potential new therapeutic target in PD (Massie et al. FASEB J 2011 25(4):1359-1369). Still, promising disease-modifying targets failed to translate so far to the clinic, potentially due to repeated use of animal models with limited mechanisms of toxicity. In order to better understand the validity of system xc- as point of pharmacological intervention, we investigated the reaction of system xc- deficient mice to proteasomal inhibition-induced cell death. Our recent data demonstrate that aged system xc- deficient mice are resistant against lactacystin-induced neurodegeneration, both at the level of the nigral DA-ergic neurons, and at the level of striatal DA content, when compared to age-matched wild-type littermates. Interestingly, however, adult system xc- deficient mice demonstrate similar degrees of nigrostriatal DA-ergic neurodegeneration compared to wild-type littermates, highlighting a complex interplay between ageing, proteasomal dysfunction, and glutamate homeostasis. Our observations therefore support system xc- as a novel therapeutic target for neuroprotection against age-related nigrostriatal DA-ergic neurodegeneration in PD.

The cystine/glutamate transporter, designated as system xc(-), is important for maintaining intracellular glutathione levels and extracellular redox balance. The substrate-specific component of system xc(-), xCT, is strongly induced by various stimuli, including oxidative stress, whereas it is constitutively expressed only in specific brain regions and immune tissues, such as the thymus and spleen. Although cystine and glutamate are the well established substrates of system xc(-) and the knockout of xCT leads to alterations of extracellular redox balance, nothing is known about other potential substrates. We thus performed a comparative metabolite analysis of tissues from xCT-deficient and wild-type mice using capillary electrophoresis time-of-flight mass spectrometry. Although most of the analyzed metabolites did not show significant alterations between xCT-deficient and wild-type mice, cystathionine emerged as being absent specifically in the thymus and spleen of xCT-deficient mice. No expression of either cystathionine β-synthase or cystathionine γ-lyase was observed in the thymus and spleen of mice. In embryonic fibroblasts derived from wild-type embryos, cystine uptake was significantly inhibited by cystathionine in a concentration-dependent manner. Wild-type cells showed an intracellular accumulation of cystathionine when incubated in cystathionine-containing buffer, which concomitantly stimulated an increased release of glutamate into the extracellular space. By contrast, none of these effects could be observed in xCT-deficient cells. Remarkably, unlike knock-out cells, wild-type cells could be rescued from cystine deprivation-induced cell death by cystathionine supplementation. We thus conclude that cystathionine is a novel physiological substrate of system xc(-) and that the accumulation of cystathionine in immune tissues is exclusively mediated by system xc(-).

System xc- or the cystine/glutamate antiporter imports one cystine in exchange for one glutamate molecule. Cystine is intracellularly reduced to cysteine, the rate-limiting building block of glutathione. Malfunctioning of system xc- can thus cause oxidative stress as well as excitotoxicity, important phenomenons in the pathogenesis of Parkinson's disease, epilepsy, multiple sclerosis and many other neurological disorders. However, deletion of xCT (xCT-/- mice) did not affect striatal or hippocampal glutathione levels and no signs of increased oxidative stress were seen in xCT-/- mice. Interestingly, extracellular hippocampal and striatal glutamate levels were decreased by >60% in xCT-/- mice compared to controls. In addition, intrahippocampal perfusion with system xc- inhibitors lowered extracellular glutamate whereas the system xc- activator N-acetylcysteine elevated extracellular glutamate in rat hippocampus, suggesting that system xc- might be an interesting target for pathologies associated with excessiveextracellular glutamate release. Indeed, we observed increased expression levels of xCT, the specific subunit of system xc-, in brain samples of rodent models for several neurological disorders as well as patients. To investigate the relevance of this altered expression of xCT, we used xCT-/- mice to study in vivo the effect of system xc- deficiency on the behavioral and/or neurochemical outcome in models for Parkinson's disease, limbic seizures, epilepsy and multiple sclerosis. xCT deletion resulted in protection of the mice in models for Parkinson's disease, limbic seizures and epilepsy. Loss of xCT did not affect the susceptibility of mice in the experimental autoimmune encephalomyelitis (EAE) model for multiple sclerosis. The current data sustain that system xc- is an important source of hippocampal and striatal extracellular glutamate that might become activated in pathological conditions and as such cause accumulation of extracellular glutamate to toxic levels. Inhibition of system xc- might thus represent an innovative strategy for the future development of drugs for the treatment of neurological disorders that are characterized by dysregulated glutamatergic neurotransmission.

Unravelling the Role of System xc- in Epileptogenesis G.Albertini Faculty of Medicine & Pharmacy, Center for Neurosciences, Department FASC Vrije Universiteit Brussel Abstract: Epilepsy is a common neurodegenerative disorder which affects about 65 million people worldwide. Approximately, 30% of patients are drug refractory and this data sheds light on the urgent need for innovative antiepileptic therapies to prevent the progression of the disease. In order to find new antiepileptic strategies, two main objectives should be achieved: the identification of new pharmacological targets and the validation of these targets on reliable animal models that mimic the process of epileptogenesis. It is well understood that oxidative stress and excessive glutamatergic activity play a key role in the induction of the neuronal pathology that leads to a variety of neurological diseases, such as epilepsy. The system xc-, a plasma membrane antiporter that imports cystine inside the cell, later to be used for glutathione synthesis, and exports, in turn, glutamate in the extracellular environment, seems to be the conjunction between the attempt of cells to produce antioxidant molecules and increased levels of glutamate, leading potentially to excitotoxicity. Since system xc- is one of the main source of extracellular glutamate in rodent hippocampus and it has been demonstrated that xCT-/- mice are less susceptible to chemoconvulsant drugs, this antiporter became the subject of our interest. The aim of this work is to provide a further investigation on the involvement of system xc- in the generation of seizures and in altering the epileptogenetic process. Therefore we will investigate the hippocampal xCT expression during the various phases of epileptogenesis and we will study the anticonvulsant effects of a knockdown of xCT using Vivo-Morpholino injections. Moreover, we will investigate the seizure susceptibility of both mice overexpressing xCT and xCT-/- mice in the amigdala kindling model and in a post-status epilepticus model. Lastly, since epilepsy is a multifactorial disease, we will investigate the involvement of system xc- in mechanisms of depression.

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 Today, stress is a major causative factor for a variety of psychiatric disorders. Depression 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, 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 important depression-related brain areas. In this study we investigated the effect of loss of 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 system xc- in the stress response, since the involvement of the antiporter in regulating the response to acute versus chronic stress seems to differ.

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.

Depression and anxiety disorders are some of the most common and important health problems worldwide. The current therapeutic approaches for these psychiatric conditions are often limited in efficacy, and may be associated with serious side effects, such as sedation, memory impairment, or physical dependence. Therefore, novel pharmacological targets with an improved therapeutic profile are currently of urgent need. System xc- is a plasma membrane transporter that imports cystine and exports glutamate in the extracellular space. Although system xc- can act as a source of extrasynaptic glutamate release, the physiologic role played by this transporter in neurotransmission and behavior has been difficult to unravel due to the lack of selective inhibitors. In order to elucidate the role played by system xc- in vivo, we have recently characterized the behavioral changes observed after transgenic loss of xCT (the specific subunit of system xc-) in mice. Interestingly, preliminary data indicate that xCT knock-out mice show an anxiolytic and antidepressant-like phenotype, as evidenced by significant changes in parameters obtained from anxiety-based tests (open-field and light/dark tests), and depression-based test (forced swim test). This shift in behavior occurred in the absence of significant changes in motor behavior, as evidenced by a battery of motor tests, such as the open-field, rotarod, nest building, and adhesive removal tests, and was stable across ageing, indicating the presence of an endophenotype in the xCT knock-out mice. These novel findings extend the previous observations that targeting the glutamatergic system might form the basis of novel therapeutic interventions for mood disorders. Furthermore, they confirm an active participation of nonsynaptic glutamate release, to behavioral traits in physiological conditions. In conclusion, our data strongly suggest that targeting system xc- might represent a novel therapeutic intervention for mood disorders.

Epilepsy is one of the most common neuro­logical dis­orders char­acter­ized by uncontrolled glu­tam­atergic neuro­trans­mission and oxi­dative stress (Costello & Delanty 2004). System xc- or the cystine/glu­tam­ate anti­porter ex­changes an in­tra­cellular glu­tam­ate mole­cule for an extra­cellular cystine mole­cule, thereby con­tri­buting to in­tra­cellular glutathione synthesis as well as non-vesicular glu­tam­ate release (Bannai 1986). System xc- could be of particular in­ter­est as a new target for treat­ment of epilepsy because of this dual role. Using respectively in vivo micro­dialysis and a quanti­tative colorimetric method, we in­ves­tigated the effect of xCT deletion on baseline cortical glu­tam­ate dialysate con­cen­trations and total glutathione content. Secondly, we studied the possible involvement of system xc- in generalized epilepsy by com­paring the susceptibility for PTZ-induced seizures be­tween xCT-/- and xCT+/+ mice. Basal extra­cellular glu­tam­ate levels in the frontal cortex of xCT-/-mice did not differ from those measured in xCT+/+ littermates, possibly in part due to the com­pen­satory in­crease in ex­pression level of VGLUT1 in the frontal cortex of the xCT-/- mice, as ob­served by semi-quanti­tative Western blotting. Total glutathione content is decreased in the frontal cortex of xCT-/- mice com­pared to their wildtype littermates. More­over, xCT-/- mice are more seizure-pro­ne in the PTZ model com­pared with their xCT+/+ littermates. To strengthen the find­ing that system xc- is implicated in generalized seizures, we used N-Ac, a pro­drug of cystine and activator of system xc- (De Bundel and Schallier et al. 2011). We ob­served that higher doses of PTZ are necessary to induce seizures in N-Ac-treated animals, com­pared with the control mice. It is known that non-vesicular glu­tam­ate release from system xc- stimulates extra­synaptic group II metabotropic glu­tam­ate (mGlu2/3) receptors (Baker et al. 2002) which func­tion as auto­receptors (Mateo & Porter 2007). How­ever, LY341495, apo­tent mGlu2/3 receptor antagonist, pre­treat­ment could not abolish the effect of N-Ac in the PTZ model, sug­gesting that activation of mGlu2/3 receptors is not the most important mecha­nism of altered seizure susceptibility in the N-Ac treated mice. Taken together, our results sug­gest a possible involvement of system xc- in the gene­ration of generalized seizures by regu­lating the synthesis of endo­genous glutathione in the pre­frontal cortex.

Glutamate uptake is affected in the central nervous system of system xc--deficient mice T. Demuyser1, S. Goursaud2, E. Bentea1, J. Van Liefferinge1, E. Merckx1, I. Smolders1, A. Massie1 and E. Hermans2 1Vrije Universiteit Brussel, Brussels, 1090, Belgium and 2Université Catholique de Louvain, Woluwe Saint-Lambert, 1200, Belgium We recently characterized system xc- (cystine/glutamate antiporter, xCT as specific subunit) as the major source of extracellular glutamate in the hippocampus/striatum by showing significantly decreased dialysate glutamate levels in system xc--deficient mice (xCT-/-) compared to wildtype littermates (xCT+/+). In order to exclude that this difference in glutamate levels is linked to compensatory changes in expression of the glutamate reuptake transporters (EAATs), we measured EAAT protein expression in hippocampal/striatal protein extracts of xCT-/- mice and wildtype littermates. No difference was observed. However, glutamate reuptake activity might be affected without changesin expression levels. Therefore, in this study, we conducted a D-[3H]-aspartate uptake assay on synaptosomes of different brain structures of the xCT-/- and xCT+/+ mice. Freshly dissected brain tissue was subjected to the assay that measures the activity of the EAATs by scintillation counting of the radioactive signal (expressed as pmol/min/mg protein). The specific values for the different EAATs were obtained by measuring the D-[3H]-aspartate uptake in the presence and absence of specific GLAST or GLT-1 inhibitors (WAY213613 and UCPH101). Our data show that there is no genotype-dependent effect on D-[3H]-aspartate uptake in prefrontal cortex, striatum and midbrain. On the other hand, we show that uptake of aspartate is significantly increased in the hippocampus and decreased in the spinal cord of xCT-/- versus xCT+/+ mice. Both changes are due to a change in activity of the GLT-1 transporter. These results contribute to the characterization of our transgenic model and will be subject for further research.

Malfunctioning of system xc-, responsible for exchanging intracellular glutamate for extracellular cystine, can cause oxidative stress as well as excitotoxicity, important phenomenons in the pathogenesis of Parkinson's disease, epilepsy and many other neurological disorders. We observed increased expression levels of xCT, the specific subunit of system xc-, in brain samples of rodent models for several neurological disorders. Therefore, we used mice lacking xCT (xCT-/- mice) to study in vivo the effect of system xc- deficiency on striatal and hippocampal glutathione content and extracellular glutamate concentrations. Next, we investigated the sensitivity of xCT-/- mice for Parkinson's disease inducing toxins as well as for chemoconvulsants evoking limbic seizures. Although cystine, imported via system xc-, is intracellularly reduced to cysteine, the rate-limiting building block in glutathione synthesis, deletion of xCT did not affect striatal or hippocampal glutathione levels. Accordingly, no signs of increased oxidative stress were seen in xCT-/- mice. However, extracellular hippocampal and striatal glutamate levels were decreased with >60% in xCT-/- mice compared to controls. In addition, intrahippocampal perfusion with system xc- inhibitors lowered extracellular glutamate whereas the system xc- activator N-acetylcysteine elevated extracellular glutamate in rat hippocampus, suggesting that system xc- might be an interesting target for pathologies associated with excessive extracellular glutamate release. Correspondingly, xCT deletion in mice elevated the threshold for limbic seizures and abolished the proconvulsive effects of N-acetylcysteine. Moreover, xCT-/- mice were less susceptible to 6-OHDA-induced neurodegeneration in substantia nigra pars compacta compared to wildtype littermates. The current data sustain that system xc- is an important source of hippocampal and striatal extracellular glutamate that might become activated in pathological conditions and as such cause accumulation of extracellular glutamate. Inhibition of system xc- might thus represent an innovative strategy for the future development of drugs for the treatment of neurological disorders that are characterized by dysregulated glutamatergic neurotransmission.