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2010/2011 Unconjugated Bilirubin (UCB) is the final product of the heme catabolism. The high serum UCB concentrations in the first days of life of the newborns, due to immature mechanisms for hepatic uptake, conjugation and biliary secretion, is called physiological neonatal jaundice. This common condition is generally a benign and transient phenomenon, but in some cases the hyperbilirubinemia can progress to bilirubin encephalopaties ranging from minimally neurological injury to severe and permanent neurodevelopmental dysfunction. In the present thesis the SH-SY5Y neuroblastoma cell line was used to approach the molecular events associated to bilirubin neurotoxicity and highlight the biochemical and molecular events that are induces in the neurons when get contact with the UCB. Depending on the bilirubin concentration and the time of exposure to UCB, we were able to define experimental setups for the study of bilirubin resistance and bilirubin toxicity. Using the model to study bilirubin resistance, it was demonstrated that the resistance is not entirely achieved by limiting the entrance or increasing extrusion of the pigment from the cell, but rather by enhancing the cellular defensive mechanisms, in particular against the oxidative stress. This was achieved by increasing the intracellular glutathione content via the specific induction of the genes and activity of the System Xc-. Furthermore, the cells exposed to bilirubin over-expressed several additional genes that encode for important antioxidant and detoxifying proteins like Heme Oxygenase-1 and NAD(P)H:quinone oxidoreductase 1. As far as the mechanisms of bilirubin neurotoxicity, we showed that UCB exposure lead to the induction of the intracellular ROS accumulation. Moreover, the data presented report evidences that the bilirubin toxicity could be displayed by a mechanism of excitotoxicity carried out by the cellular release of glutamate. Further studies will be necessary to elucidate the molecular mechanisms by which bilirubin produces neurotoxicity and to understand how the cells avoid the damage. The information presented here could contribute to the identification of targets to avoid the bilirubin damage. La bilirubina non coniugata (UCB) è il prodotto finale del catabolismo dell’eme. L’ alta concentrazione di bilirubina nei primi giorni di vita del neonato è conosciuto come ittero neonatale. Questa condizione è generalmente benigna e transitoria, ma in alcuni casi l’iperbilirubinemia può portare a encefalopatie che vanno da minimi danni neurologici a severe e permanenti disfunzioni neuronali. Nella presente tesi la linea cellulare di neuroblastoma umano SH-SY5Y è stata utilizzata per identificare gli eventi molecolari associati alla neurotossicità della bilirubina ed evidenziare gli eventi biochimici e molecolari che sono indotti nelle cellule neuronali quando queste sono esposte alla UCB. A seconda della concentrazione e del tempo di esposizione all’ UCB che viene utilizzato, si sono definiti due modelli sperimentali per studiare sia la resistenza delle cellule alla bilirubina che la sua tossicità. Utilizzando il modello per studiare la resistenza alla bilirubina, è stato dimostrato che la resistenza non è completamente dovuta alla limitazione dell’ entrata o alla rapida eliminazione del pigmento dalla cellula, ma da un aumento dei meccanismi di difesa cellulare, in particolare quelli contro lo stress ossidativo. Si è dimostrato che questo’ultimo è dovuto all’aumento intracellulare di glutatione attraverso l’induzione specifica di alcuni geni e ad un aumento dell’attività del Sistema Xc-. Inoltre, le cellule esposte a bilirubina overesprimono vari geni addizionali che codificano per importanti proteine antiossidanti e detossificanti come l’emossigenasi 1 e la NAD(P)H quinone ossidoreduttasi. Per quanto riguarda ai meccanismi di neurotossicità della bilirubina, è stato dimostrato che l’ esposizione a UCB induce l’accumulo intracellulare di ROS. Inoltre, i dati presentati evidenziano che la tossicità della bilirubina può essere dovuta a meccanismi di esototossicità prodotti dalla liberazione cellulare di glutammato. Ulteriori studi servirebbero per completare l’ identificazione dei meccanismi molecolari cui attraverso i quali la bilirubina produce il danno neuronale e capire come le cellule riescono a evitare il danno. L’informazione presentata in questo lavoro potrebbe contribuire all’identificazione di possibili targets che potrebbero prevenire il danno indotto da bilirubina. XXIV Ciclo 1982

Aim: The cystine/glutamate antiporter or system xc- is a membrane-bound Na+-independent amino acid transporter that is structurally composed of a heavy chain subunit common to all amino acid transporters, 4F2, and a light chain specific subunit, xCT [1]. We here provide the first evidence that system xc- is the major source of extracellular hippocampal glutamate in mice. Methods: Semi-quantitative western blotting was performed on hippocampal tissue of young and old xCT+/+ and xCT-/- mice [2] using guinea pig antibody to VGLUT1 (1:2000; Millipore); mouse antibody to VGLUT2 (1:5000; Millipore); rabbit antibody to VGLUT3 (1/1000; Synaptic Systems); rabbit antibody to GLT-1 (1:30000) [3]; rabbit antibody to GLAST (1:4000)[4]; rabbit antibody to EAAC1 (1:1000; Alpha Diagnostic); rabbit antibody to xCT (1:10000) [5]; mouse antibody to synaptophysine (1:5000; Stressgen); rabbit antibody to GAPDH (1:5000; Santa Cruz); mouse antibody to GAPDH (1:15000; Millipore). In vivo microdialysis was performed in mouse hippocampus by implanting a microdialysis guide (CMA/7) 2 mm above final probe membrane location with the following coordinates relative to bregma: L +3.0, AP -2.7 and DV -1.5. Samples were collected at flow rate of 2 µl/min every 20 min. Glutamate and aspartate content were determined using HPLC [6]. Results: We observed significantly lower glutamate concentrations in baseline dialysis samples obtained from young as well as old xCT-/- mice (young: 0.079 ± 0.022 µM; old: 0.084 ± 0.015 µM) compared to their age-matched xCT+/+ littermates (young: 0.208 ± 0.036 µM; old: 0.191 ± 0.034 µM). Our findings demonstrate that protein expression levels of the major glial glutamate transporters GLT-1 and GLAST, the neuronal EAAC1 transporter and the vesicular glutamate transporters (VGLUT1-3) are unaffected by genotype and that no compensatory up- or down-regulations are observed due to the loss of xCT protein. Conclusion: These novel findings sustain that system xc- is an important source of extracellular glutamate in mouse hippocampus. [1] Sato H et al. (1999) J Biol Chem 247: 11455-11468. [2] Sato H et al. (2005) J Biol Chem. 280(45):37423-37429 [3] Yamada K et al. (1998) J Neurosci 18: 5706-5713. [4] Shibata T et al. (1997) J Neurosci 17: 9212-9219. [5] Massie A et al. (2008) Neuroreport 19: 1589-1592. [6] Van Hemelrijck A et al. (2005); 144(1):63-71.

Breast cancers are the most common source of metastases to bone of which cancer-induced bone pain is a frequent pathological feature. Cancer-induced bone pain is a unique pain state with a multiplicity of determinants that remains to be well understood and managed. Current standard treatments are limited by dose-dependent side effects that can depress the quality of life of patients. Glutamate is a neurotransmitter and bone cell-signalling molecule that has been found to be released via the system xC-cystine/glutamate antiporter on cancer cells of types that frequently metastasize to bone, including breast cancers. This project examines the hypothesis that limiting glutamate release from cancer cells metastasized to bone will reduce bone tissue disruption and cancer-induced bone pain. A mouse model of cancer-induced bone pain was established with intrafemoral human breast cancer cells (MDA-MB-231), and behavioural measurements were taken for weight bearing and induced paw withdrawal thresholds. The system xC- inhibitors sulfasalazine and (S)-4-carboxyphenylglycine both attenuated glutamate release from cancer cells in a dose-dependent manner in vitro. Treatment with sulfasalazine induced a moderate delay in the onset of behavioural indicators of pain in mouse models, and treatment with (S)-4-carboxyphenylglycine had no apparent results. This data suggests that the limitation of extracellular glutamate released from cancers in bone with sulfasalazine may provide some alleviation of the often severe and intractable pain associated with bone metastases. Master of Science (MSc)

The cystine/glutamate antiporter or system xc- is a membrane-bound Na+-independent amino acid transporter that is structurally composed of a heavy chain subunit common to all amino acid transporters, 4F2, and a light chain specific subunit, xCT [1]. System xc- exchanges intracellular glutamate for extracellular cystine, thereby contributing to intracellular glutathione (GSH) synthesis and non-vesicular glutamate release [2]. The role of system xc- in non-vesicular hippocampal glutamate release was never investigated. We here provide the first evidence that system xc- is the major source of extracellular hippocampal glutamate in mice. Semi-quantitative western blotting was performed on hippocampal tissue of young and old xCT+/+ and xCT-/- mice [3] using guinea pig antibody to VGLUT1 (1:2000; Millipore); mouse antibody to VGLUT2 (1:5000; Millipore); rabbit antibody to VGLUT3 (1/1000; Synaptic Systems); rabbit antibody to GLT-1 (1:30000) [4]; rabbit antibody to GLAST (1:4000)[5]; rabbit antibody to EAAC1 (1:1000; Alpha Diagnostic); rabbit antibody to xCT (1:10000) [6]; mouse antibody to synaptophysine (1:5000; Stressgen); rabbit antibody to GAPDH (1:5000; Santa Cruz); mouse antibody to GAPDH (1:15000; Millipore). In vivo microdialysis was performed in mouse hippocampus by implanting a microdialysis guide (CMA/7) 2 mm above final probe membrane location with the following coordinates relative to bregma: L +3.0, AP -2.7 and DV -1.5. Samples were collected at flow rate of 2 µl/min every 20 min. Glutamate and aspartate content were determined using HPLC. We observed a significant effect of genotype on glutamate dialysate concentrations with significantly lower glutamate concentrations in baseline dialysis samples obtained from young as well as old xCT-/- mice (young: 0.079 ± 0.022 µM; old: 0.084 ± 0.015 µM) compared to their age-matched xCT+/+ littermates (young: 0.208 ± 0.036 µM; old: 0.191 ± 0.034 µM). We measured in the same dialysates the levels of aspartate, which is not a substrate for system xc- [7], asa negative control. No difference in extracellular aspartate levels could be observed between xCT+/+ and xCT-/- mice, independent of age. We studied possible compensatory changes in hippocampal protein expression of the major glial glutamate transporters GLT-1 and GLAST, the neuronal EAAC1 transporter that can also function as a cysteine transporter, and the vesicular glutamate transporters (VGLUT1-3) in response to xCT gene deletion. Our findings demonstrate that, although as a result of ageing all transporters related to glutamate reuptake as well as two vesicular glutamate transporters are downregulated in hippocampus, protein expression levels of all glutamate transporters are unaffected by genotype and that no compensatory up- or down-regulations are observed due to the loss of xCT protein. In conclusion these novel findings sustain that system xc- is an important source of extracellular glutamate in the hippocampus. [1] Sato H et al. (1999) J Biol Chem 247: 11455-11468. [2] Bannai S. (1986) J Biol Chem 261: 2256-2263. [3] Sato H et al. (2005) J Biol Chem. 280(45):37423-37429 [4] Yamada K et al. (1998) J Neurosci 18: 5706-5713. [5] Shibata T et al. (1997) J Neurosci 17: 9212-9219. [6] Massie A et al. (2008) Neuroreport 19: 1589-1592. [7] Patel SA et al. (2004) Neuropharmacology 46: 273-284.

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

System xc- exchanges intracellular glutamate for extracellular cystine, giving it a potential role in intracellular glutathione synthesis and regulating extracellular glutamate levels. We used mice lacking the xCT subunit of system xc- (xCT-/-) to investigate the possible involvement of this transporter in the susceptibility for limbic seizures. In a first set of experiments we could not detect alterations in hippocampal glutathione content, oxidative stress or brain atrophy in the xCT-/- mice. On the other hand, extracellular hippocampal glutamate levels were significantly decreased. Consequently, xCT deletion elevated the threshold for limbic seizures. xCT-/- mice were less susceptible to the well known chemoconvulsants pilocarpine, kainic acid and NMDA. Furthermore, N-acetylcysteine, an activator of system xc-, was proconvulsant in the pilocarpine and 6 Hz model. This proconvulsant effect was absent in the xCT-/- mice. These findings sustain that system xc- is the major hippocampal source of extracellular glutamate and that impairing system xc- is clearly beneficial to decrease susceptibility for limbic seizures.

Malfunctioning of system xc-, responsible for exchanging intracellular glutamate for extracellular cystine, can cause oxidative stress as well as excitotoxicity, both important phenomenons in the pathogenesis of Parkinson's disease (PD). We used mice lacking xCT (xCT-/- mice), the specific subunit of system xc-, to investigate the involvement of this antiporter in PD. Although cystine that is imported via system xc-, is reduced to cysteine, the rate-limiting substrate in the synthesis of glutathione, deletion of xCT did not result in decreased glutathione levels in striatum. Accordingly, no signs of increased oxidative stress could be observed in striatum or substantia nigra of xCT-/- mice. In sharp contrast to the expectations, xCT-/- mice were less susceptible to 6-hydroxydopamine-induced neurodegeneration in the substantia nigra pars compacta compared to age-matched wildtype littermates. This reduced sensitivity to a PD inducing toxin, might be related to the decrease of 70% in striatal extracellular glutamate that was observed in mice lacking xCT. Our data point towards system xc- as a possible target for the development of new pharmacotherapies for treatment of PD and emphasizes the need to continue searching for specific ligands for system xc-. FASEB J (2011) 25, 1359-69

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 and epilepsy. We used mice lacking xCT (xCT-/- mice), the specific subunit of system xc-, 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 a Parkinson's disease inducing toxin (6-hydroxydopamine, 6-OHDA) as well as for chemoconvulsants evoking limbic seizures. Although cystine, imported via system xc-, is intracellularly reduced to cysteine, the rate-limiting substrate 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 the rat hippocampus. This indicates that system xc- may 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, in sharp contrast to the expectations, 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 and an innovative target for the future development of antiepileptic and/or neuroprotective drugs.

Introduction: Alzheimer's disease (AD) is an irreversible neurodegenerative disorder causing dementia and is characterized by neuronal loss as well as the formation of amyloid plaques and neurofibrillary tangles. To date, few drugs are available and they can only alleviate the symptoms of the disease. In the search for new therapies that could halt or slow down the progression of the disease, it is important to study the mechanisms underlying the pathology of AD. Glutamate, the most important excitatory neurotransmitter in the mammalian central nervous system, plays major roles in normal brain functioning [1]. We here used the APP23 mouse model to investigate the possible involvement of high-affinity Na+/K+-dependent glutamate transporters (GLAST, GLT-1 and EAAC1) as well as vesicular glutamate transporters (vGLUT1-3) in the pathogenesis of AD. Methods: Distribution patterns and expression levels of the above described glutamate transporters were examined in 8 (mature) and 18 (aged) month old APP23 mice and their WT littermates by means of immunohistochemistry and Western blotting. Moreover, using in vivo microdialysis and a specific inhibitor of the excitatory amino acid transporters, we investigated the functional relevance of these changes in protein expression by measuring extracellular glutamate levels as well as glutamate reuptake activity in hippocampus and frontal cortex. Results and discussion: Whereas GLAST expression was only decreased in cortical and hippocampal samples of mature APP23 mice, GLT-1 downregulation was age-independent (8 and 18-month-old mice). In all conditions this downregulated expression in glial reuptake transporters could be linked to a decreased in vivo reuptake activity, except for frontal cortex of mature APP23 mice. In this brain region, status epilepticus was induced when glutamate reuptake was inhibited, because of the increased cortical expression of vGLUT1. In frontal cortex of aged APP23 mice, the decreased reuptake activity for glutamate together with the increased expression level of vGLUT3 resulted in increased basal glutamate levels. Even though in hippocampus of mature APP23 mice glutamate reuptake activity was strongly diminished, we could not detect an effect on extracellular glutamate concentrations, suggesting a deficiency in glutamate release. This finding points towards an important role of vGLUT3, which showed a decreased expression in hippocampal tissue of APP23 mice, in determining extracellular glutamate levels. The present findings support the hypothesis that alterations in transport of glutamate may be involved in the pathogenesis of AD. However, more profound research is needed to point towards the most promising target for the development of new therapeutic strategies. References [1] Walton H.S., Dodd P.R. (2007). Glutamate-glutamine cycling in Alzheimer's disease. Neurochem. Int. 50, 1052-1066

System xc- or the cystine/glutamate antiporter exchanges extracellular cystine for intracellular glutamate. Intracellularly cystine is reduced to cysteine, the rate-limiting substrate in the synthesis of glutathione. Increased activity of system xc- can thus protect cells against oxidative stress. Yet, at the same time, an increased release of glutamate via this antiporter can induce excitotoxicity. We used mice lacking xCT, the specific subunit of system xc-, to investigate the possible involvement of system xc- in the pathogenesis of Parkinson's disease. Interestingly, xCT deficient mice showed a decreased sensitivity towards 6-OHDA induced neurodegeneration. At 3 and 10 days after striatal 6-OHDA injection, young (3 month) and old (12-18 month) wildtype mice showed significant neurodegeneration (young 3d: ~10%; young 10d: ~25.0%; old 3d and 10d: ~25%) whereas young and old xCT knockout littermates are devoid of neurodegeneration at 3 days and showed a significant smaller loss of neurons at 10 days (~5%). xCT knockout mice do not show a loss of striatal glutathione or any signs of increased oxidative stress in the striatum or substantia nigra, compared to their wildtype littermates. Yet, in vivo microdialysis experiments revealed that the xCT knockout mice show a decrease of ~75% in striatal extracellular glutamate concentration. This loss of striatal extracellular glutamate is not linked to compensatory changes in expression level of other glutamate transporters as investigated using semi-quantitative Western blotting. We here provide for the first time a clear link between system xc- and neurodegeneration in a model for Parkinson's disease. Moreover, we have in vivo evidence that the major source of extracellular glutamate in the mouse striatum is glutamate release via system xc- as described before by Baker et al. (2002) in the nucleus accumbens of the rat. Our data suggest that preventing release of glutamate via system xc- under pathological conditions might protect the brain against neurodegeneration.

The antiporter system xc− imports the amino acid cystine, the oxidized form of cysteine, into cells with a 1:1 counter-transport of glutamate. It is composed of a light chain, xCT, and a heavy chain, 4F2 heavy chain (4F2hc), and, thus, belongs to the family of heterodimeric amino acid transporters. Cysteine is the rate-limiting substrate for the important antioxidant glutathione (GSH) and, along with cystine, it also forms a key redox couple on its own. Glutamate is a major neurotransmitter in the central nervous system (CNS). By phylogenetic analysis, we show that system xc− is a rather evolutionarily new amino acid transport system. In addition, we summarize the current knowledge regarding the molecular mechanisms that regulate system xc−, including the transcriptional regulation of the xCT light chain, posttranscriptional mechanisms, and pharmacological inhibitors of system xc−. Moreover, the roles of system xc− in regulating GSH levels, the redox state of the extracellular cystine/cysteine redox couple, and extracellular glutamate levels are discussed. In vitro, glutamate-mediated system xc− inhibition leads to neuronal cell death, a paradigm called oxidative glutamate toxicity, which has successfully been used to identify neuroprotective compounds. In vivo, xCT has a rather restricted expression pattern with the highest levels in the CNS and parts of the immune system. System xc− is also present in the eye. Moreover, an elevated expression of xCT has been reported in cancer. We highlight the diverse roles of system xc− in the regulation of the immune response, in various aspects of cancer and in the eye and the CNS. Antioxid. Redox Signal. 18, 522–555.