Given the functional importance of Sxc- in cell types both within and outside the CNS, it is not surprising that multiple mechanisms exist through which to regulate its expression. Indeed, amino acid deprivation, xenobiotic exposure and oxidative stress have all been shown to trigger the up-regulation of Sxc- (Bannai, 1984; Bannai et al., 1989; Sato et al., 1995; Ishii et al., 2000). The best-characterized of these mechanisms are those linked to the Nrf2-ARE signalling pathway (Figure 6) in which the transcription of a specific gene is governed by the presence of a cis-acting regulatory DNA element (i.e. the antioxidant response element, ARE) in its promoter. Numerous enzymes associated with the synthesis of GSH, the inactivation of reactive oxygen species (ROS) and phase II detoxification contain AREs (Ishii et al., 2000) (Prochaska et al., 1985; Talalay, 1989; Spencer et al., 1991; Limon-Pacheco et al., 2007), and at least four ARE motifs have been identified in the promoter region sequence of mouse xCT (Sasaki et al., 2002). The expression of these ARE-containing genes is up-regulated by the binding of nuclear factor erythroid-2-related factor (Nrf2), a CNC-bZIP transcription factor (Jaiswal, 2004; van Muiswinkel and Kuiperij, 2005; Vargas and Johnson, 2009; Giudice et al., 2010) following its translocation into the nucleus. Electrophilic or oxidative stress can also lead to direct phosphorylation of Nrf2 by kinases in several cascades [e.g. protein kinase C, phosphatidyl-inositol 3-kinase, PKR-like endoplasmic reticulum kinase, JNK, ERK and p38 MAPK (MK14)] that also facilitates translocation into the nucleus (Kobayashi and Yamamoto, 2006; Kandil et al., 2010). The regulatory control provided by these pathways appears to vary among cell types (Sasaki et al., 2002; Qiang et al., 2004), and several studies point to existence of alternate mechanisms of Sxc- regulation (Sasaki et al., 2002; Sato et al., 2004; Lewerenz et al., 2009).
Figure 6. Nrf-2/ARE activation pathway. Under basal conditions Nrf-2 is dimerized with KEAP1 and continuously targeted for degradation due to ubiquitination by Cul3. Electrophilies and oxidants can disrupt the dimerization of Nrf2 and KEAP directly by modifiying cysteine residues in KEAP1 or through phosphorylation of Nrf2 at Ser40 by protein kinases. Nrf2 is then free to translocate into the nucleus, bind with an adaptor protein (e.g. Mafs and ATF4) and increase ARE-driven transcription.
Download figure to PowerPoint
In CNS tissue, treatment of primary astrocytes with dibutyryl cyclic AMP (dbcAMP) has been shown to differentiate the glial cells into a more in vivo-like, activated phenotype that includes the increased expression of several proteins associated with the glutamate system, including: glutamine synthetase, EAATs and Sxc- (Hertz et al., 1998; Gochenauer and Robinson, 2001; Daginakatte et al., 2008). More recently, a closer examination of these dbcAMP-treated astrocytes reveals that Sxc- expression may also directly be regulated by GSH levels in a phenotype dependent manner (Seib et al., 2011). Sxc- activity is markedly up-regulated in dbcAMP-treated astrocytes in which GSH levels have been depleted with synthesis blocker buthionine sulphoximine (BSO), a response not observed in untreated protoplasmic astrocytes. Interestingly, an Nrf2-dependent increase in Sxc- activity is observed when the electrophile tBHQ is added to the protoplasmic astrocytes, but not in differentiated cultures, suggesting a distinct regulatory mechanism. Changes in Sxc- activity induced by BSO also correlate to increases in both xCT mRNA and protein and are temporally linked to GSH levels. Furthermore, the effect of BSO cannot be mimicked by H2O2 treatment or reversed by the addition of general antioxidants, suggesting that it is a change in GSH level that triggers the induction. Consistent with such a mechanism, co-administration of permeable GSH pro-drug GSH-ethylester and BSO prevents the up-regulation of Sxc- activity.
Studies seeking drug candidates to induce the up-regulation of EAAT expression as a potential point of intervention in amyotrophic lateral sclerosis (ALS) demonstrated that some β-lactam antibiotics could increase expression of EAAT2 through an action at the promoter level (Rothstein et al., 2005). One of the more effective of these compounds, Ceftriaxone, was recently reported to also produce an increase in Nrf-2 and xCT expression in HT22 hippocampal cells and fibroblasts, as well as protect these cells from l-Glu-induced exicitotoxicty (Lewerenz et al., 2009). Such an induction has also been postulated to provide a novel therapeutic approach in inflammatory neurodegenerative disease (for review, see Chen and Kunsch, 2004; Vargas and Johnson, 2009). In those instances where an increase in Sxc- activity may be of therapeutic benefit (see physiology and pathology section below), pharmacological strategies targeting enhanced expression provide an alternative to screening for potential allosteric activators.
CNS GSH production and oxidative protection via Sxc-
The generation and maintenance of GSH levels in all mammalian cells is critical to the detoxification of xenobiotics and the prevention of oxidative stress and damage. This latter effect is especially relevant to the CNS given its high levels of oxygen consumption, the abundance of enzymes and metabolites that can generate ROS and its limited antioxidant capacity (Halliwell, 2006). As a transporter, Sxc- acts as a key component in GSH production by providing l-Cys2 as a synthetic precursor (Figure 7). The combination of micromolar concentrations of l-Cys2 in the CSF and millimolar concentrations of l-Glu in the astrocyte serve to drive the exchange, after which the intracellular l-Cys2 is rapidly reduced to l-CysH and enzymatically incorporated into GSH (Sagara et al., 1993). While both astrocytes and neurons have been shown to express the xCT subunit of Sxc-, mature neurons exhibit little or no Sxc- activity and appear even less capable of directly mediating the uptake l-Cys2. GSH production in neurons is thus dependent upon the uptake of L-CysH that is provided by astrocytes via the ‘cystine/cysteine cycle’ (Dringen et al., 1999; Wang and Cynader, 2000). Essentially, the l-Cys2 that is transported into the astrocytes by Sxc- is reduced and subsequently released as either l-CysH itself or GSH, which can also serve as an l-CysH source following its extracellular metabolism by γ-glutamyl-transpeptidase (GGT) and aminopeptidase N. As previously mentioned, the observation that QA toxicity in immature cortical neurons could be attributed to blocking Sxc- and the resultant oxidative stress (and not EAA receptor mediated excitotoxicity) highlighted the significance of the transporter in these pathways (Murphy et al., 1989; Cho and Bannai, 1990; Shih et al., 2003; 2006a). Under conditions of primary or secondary oxidative stress, astrocytes also are capable of contributing to neuroprotection by rapidly providing neurons with increased levels of GSH precursors via the Nrf2-dependent induction of the GSH synthesis (Sagara et al., 1993; Dringen et al., 1999; Wang and Cynader, 2000; Guebel and Torres, 2004) (Shih et al., 2006a).
Given that the maintenance of intracellular GSH levels is critical to most cells, those that do not express Sxc-, as is the case with mature neurons, presumably accumulate either l-CysH or l-Cys2 through alternative systems. Other members of SLC7 family are also capable of transporting these precursors. Thus, CysH is a substrate of the 4F2hc-linked SLC7A10 transporter Asc-1. The SLC7A9 transporter b°,+AT (SLC7A9) can mediate the uptake of Cys2, although it is dimerized with rBAT instead of 4F2hc. (Verrey et al., 2003). As its name implies, functionally defined ASC (alanine–serine–cysteine) system members ASCT1 and ASCT2 (SLC1A4 and A5, respectively) transport L-CysH in a sodium-dependent manner (Zerangue and Kavanaugh, 1996a; Kanai and Hediger, 2004). l-CysH is also a substrate of sodium-dependent neutral amino acid transporters SNAT1, SNAT 2 and SNAT4, which are members of the SLC38 family and are typically associated with glutamine and asparagine uptake (Mackenzie and Erickson, 2004). Amongst the alternative routes of entry for l-CysH or l-Cys2 within CNS cells, the most intriguing is associated with the EAATs (Zerangue and Kavanaugh, 1996b; McBean, 2002; Chen and Swanson, 2003). In particular, l-CysH was shown to be transported by the neuronal transporter EAAT3 (EAAC-1) with a maximal flux rate comparable with that of l-Glu (Zerangue and Kavanaugh, 1996b). Consistent with such a role, GSH levels are negatively regulated by the interaction of EAAC-1 with the glutamate transport-associated protein 3–18 (GTRAP3-18) (Watabe et al., 2007) and mice homozygous deficient for EAAT3, exhibit not only increased neurodegeneration with age, but also decreased neuronal levels of GSH concentrations and an increased vulnerability to oxidative stress (Aoyama et al., 2006).
Source for extrasynaptic excitatory signalling
The l-Cys2-coupled export of l-Glu through Sxc- takes on additional significance within the CNS as it represents a non-vesicular route by which this excitatory transmitter can access EAA receptors, particularly those present in extrasynaptic locales. Given the presence of Sxc- on astrocytes, l-Glu released in this manner also represents a novel mechanism for glial-neuronal communication. Work by Kalivas, Baker and co-workers demonstrate that the extracellular l-Glu released through Sxc- has a physiological role in the tonal regulation of extrasynaptic type 2/3 mGluR receptors and the control of neurotransmitter release throughout the CNS (Baker et al., 2002; Xi et al., 2003; Mohan et al., 2011). In both the striatum and nucleus accumbens, extrasynaptic activation of type 2/3 mGluR receptors inhibits the release of L-Glu and/or dopamine. Aberrant changes in Sxc- activity and l-Glu homeostasis at these synapses have been implicated in the pathophysiology of drug addiction (Kalivas et al., 2009; Madayag et al., 2010). In rat models of chronic, self-administered cocaine addiction, a series of neuroadaptations have been identified that underlie the disease including: a decrease in extra-synaptic l-Glu concentrations in the nucleus accumbens, a decrease in l-Cys2 and l-Glu exchange, and an increase in glutamatergic transmission. Restoring Sxc- activity by applying l-CysH or N-acetyl-CysH (NAC) can return extra-synaptic l-Glu to control levels (Baker et al., 2003); restoring normal glutamatergic transmission and in doing so prevents cocaine primed drug seeking and relapse (Baker et al., 2003; Amen et al., 2011). A similar mechanism has been implicated for nicotine addiction and suggests targeting l-Glu homeostasis is a potential method for treating addiction (Knackstedt and Kalivas, 2009; Knackstedt et al., 2009).
The ability of Sxc- to contribute to neuronal signalling through the release of l-Glu is, however, not uniformally accepted. Thus, while a l-Cys2-mediated efflux of l-Glu was shown to be sufficient to activate non-NMDA receptors in cerebellar slices and NMDA receptors in hippocampal slices (Warr et al., 1999; Cavelier and Attwell, 2005), it was concluded that the combination of CSF l-Cys2 levels (typically in the 0.1–0.5 µM range) and the Km for l-Cys2 at Sxc- (typically reported in 50–100 µM range), would preclude Sxc- from contributing to ambient l-Glu levels. Such a conclusion, however, must be tempered somewhat by assay results quantifying the uptake of 35S-l-Cys2 into slices of nucleus accumbens, which yielded Km values in the 2–4 µM range (Baker et al., 2003). More recent studies on primary cultures of differentiated astrocytes also yielded Km values for l-Glu transport that were two- to fivefold lower than observed in many glial cell lines (Seib et al., 2011). l-Cys2 levels in the 0.1–0.3 µM range were able to produce an Sxc--mediated efflux of l-Glu that decreased the synaptic release of l-Glu as a consequence presynaptic mGluR2 activation (Moran et al., 2005). These different findings point to the likelihood that the ability of l-Glu released from Sxc- to activate EAA receptors probably varies among circuits.
Beyond its role in GSH production and oxidative protection within the different cell types of the mammalian eye (Lim et al., 2005; 2007; Li et al., 2007) additional insight into the role of the Sxc- in extrasynaptic signalling comes from a closer examination of retinal circuits (Hu et al., 2008). Electron microscopy studies reveal the ultra-structural localization of Sxc- (based upon xCT expression) to the photoreceptor (rod and cone) ribbon synapse with ON bipolar cells of the outer plexiform layer. Using the cation channel probe agmatine (AGB) as a fluorescent reporter, it was shown that l-Glu released through Sxc- could activate mGluR 6 receptors that, in turn, would close non-selective cation channels on the post synaptic ON bipolar cells. Consistent with the participation of Sxc-, the ability of l-Cys2 to reduce this AGB fluorescence was blocked by co-incubation with 4-S-CPG. The findings suggest that signalling within the photoreceptor synapse incorporates both vesicular release and Sxc--mediated exchange as a source of l-Glu.
Evidence validating Sxc- as a source of ambient l-Glu and its contribution to signalling and plasticity has also come from work with Drosophila melanogaster. Studies on an xCT homologue genderblind in D. melanogaster suggest that the transporter influences courtship behaviour through its regulation of ambient extracellular l-Glu in vivo (Grosjean et al., 2008). The loss of this gene reduces extracellular l-Glu levels by about half. Furthermore, changes in ambient l-Glu levels arising from altered Sxc- activity were shown to influence both the function (i.e. desensitization) and localization (i.e. clustering) of ionotropic glutamate receptors (iGluRs) in the Drosophila CNS (Augustin et al., 2007; Featherstone, 2010).
Source for excitotoxicity
When the efflux of l-Glu through Sxc- becomes excessive, its character within the CNS switches from that of a potential excitatory transmitter to that of a well-characterized and pathologically relevant excitotoxin (Waxman and Lynch, 2005; Lau and Tymianski, 2010). Early recognition that Sxc- could be a source of excitotoxic l-Glu came from an examination of microglia cells that, owing to a marked need for oxidative protection, express high levels of the transporter (Piani and Fontana, 1994). Thus, under conditions of CNS infection or trauma, activation of microglia from a resting state by pro-inflammatory stimuli, as well as migration into localized areas, not only have beneficial neuroprotective and neurothrophic effects, but the ability to release substantial levels of l-Glu (Espey et al., 1998; Qin et al., 2006; Shih et al., 2006b; Barger et al., 2007). Given the ubiquitous involvement of microglia in CNS disease and pathology, it is not surprising that this ability to exacerbate neurodegeneration through the conversion of oxidative stress to excitotoxic stress via Sxc- has been linked to a variety of disorders, including: Alzheimer's disease (Barger and Basile, 2001), Parkinson's disease, AIDS (Zeng et al., 2010), bacterial infection/LPS (Taguchi et al., 2007) and multiple sclerosis (Domercq et al., 2007). Ironically, the observations that microglia express high levels of Sxc- and that transporter-mediated efflux of l-Glu provides a non-synaptic pathway of mGluR signalling also prompted the examination of this mechanism within the non-neural cells of the immune system. These studies revealed that l-Glu released by dendritic cells acts through mGluRs on T cells to modulate T-cell activation and that this release occurs through Sxc- (Pacheco et al., 2006; 2007).
The specific up-regulation of Sxc−- in astrocytes by interleukin-1β (IL-1β) and subsequent activation of mGluR1 receptors has also been demonstrated to enhance hypoxic neuronal injury (Fogal et al., 2005; 2007). Neurons co-cultured with astrocytes were found to be more susceptible to hypoxic neuronal cell death after treatment of cultures with IL-1β, an affect that was mediated through increased expression and efflux of l-Glu through Sxc-. Confirming the role of Sxc-, the IL-1β-induced increase in l-Cys2 uptake (and l-Glu efflux) could be blocked with 4-S-CPG and was absent in astrocytes prepared from mice carrying a mutation in the SLC7A11 gene (Jackman et al., 2010).
Probably the strongest links between the Sxc--mediated efflux of l-Glu and CNS pathology have been made during the course of investigation on brain tumours, where Sxc- is a major contributor to the growth, survival and expansion of malignant gliomas (de Groot and Sontheimer, 2010). The high levels of xCT expression in astrocytomas and gliomas, compared with normal astrocytes, likely reflects the increased growth and metabolic rates associated with tumour cells (Ye et al., 1999). More specifically, however, the import of l-Cys2 through Sxc- is needed to meet the increasing demand for cellular GSH as the tumour outgrows its blood supply, becomes hypoxic and is challenged by the accompanying build up of ROS and NO (Ogunrinu and Sontheimer, 2010). From a therapeutic perspective, the increased GSH levels can also represent an enhanced ability of glioma cells to protect themselves from both radiation and chemotherapeutic agents (Dai et al., 2007; Liu et al., 2007). Thus, while not yet induced specifically by the inhibition of Sxc-, depletion of GSH using the synthesis inhibitor l- BSO rendered gliomas more sensitive to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)-based chemotherapy and d-54 human glioma xenografts to radiotherapy (Lippitz et al., 1990; Allalunis-Turner et al., 1991). Even greater attention has focused on the Sxc--mediated efflux of l-Glu from the glial tumours and its impact on brain tumour aetiology. At one level, the released l-Glu can act as an excitotoxin through the over activation of ionotropic EAA receptors, induce neurodegeneration and clear the area for tumour expansion (Ye et al., 1999). Thus, both glioma cultures in vitro and implanted glioma cells in vivo have been shown to release l-Glu (Ye and Sontheimer, 1999; Behrens et al., 2000; Takano et al., 2001). Co-cultures of glioma and neuronal cells demonstrated that the released l-Glu was sufficient to induce excitotoxic damage and that the neuronal loss could be reduced by either by preventing its efflux through Sxc- (4-S-CPG or SSZ) or inhibiting its action at NMDA receptors (MK-801).
Even if the l-Glu does not reach concentrations that are excitotoxic, the excessive activation of these receptors in peritumoural regions provides an explanation for the seizures commonly observed in the early stages of the disease (Sontheimer, 2008). Lastly, the released l-Glu also is capable of mediating an autocrine/paracrine signal that enhances invasive and migratory properties through the activation of Ca2+ permeable AMPA receptors on the glioma cells themselves (Lyons et al., 2007; Lastro et al., 2008). All of these relationships have highlighted the potential of Sxc- as a therapeutic target in tumour cells, particularly gliomas. Blockers could be envisioned as acting by any number of mechanisms, some of which might be synergistic, including: metabolic inhibition, increasing oxidative stress, radio-sensitization, decreasing chemoresistance, reducing invasiveness or preventing peritumoural seizures. Indeed, SSZ (Chart 3), which was originally developed for anti-inflammatory applications before its Sxc- inhibitory actions were discovered (see Pharmacology section), has been shown to reduce GSH and tumour proliferation in both gliomas (Lyons et al., 2007) and small-cell lung cancer (Guan et al., 2009). Inhibition of Sxc- activity also inhibited cell metastasis in esophageal squamous carcinoma cells through caveolin-1/β-catenin pathway (Chen et al., 2009). Treatment of astrocytoma cells with SSZ depleted GSH levels and induced caspase-mediated apoptosis (Chung et al., 2005) Importantly, the antitumour effects of SSZ have been shown to be exclusively via inhibition of Sxc--mediated L-Cys2 uptake and GSH production as opposed to the reported anti-inflammatory effects of SSZ acting via NfkB (Chung and Sontheimer, 2009). Unfortunately SSZ, as well as most of the other Sxc- inhibitors described earlier in this review, suffer from problems related to specificity, potency and pharmacokinetic properties. Progress on mapping out how SSZ is interacting with the domains on the Sxc- binding site should, however, provide insight into optimizing new derivatives.