Glial Glutamate Transporter-Mediated Plasticity: System x c − /xCT/SLC7A11 and EAAT1/2 in Brain Diseases

Glial cells play an essential role in the complex function of the nervous system. In particular, astrocytes provide nutritive support for neuronal cells and are involved in regulating synaptic transmission. Oligodendrocytes ensheath axons and support information transfer over long distances. Microglial cells constitute part of the innate immune system in the brain. Glial cells are equipped with the glutamate-cystine-exchanger xCT (SLC7A11), the catalytic subunit of system x c − , and the excitatory amino acid transporter 1 (EAAT1, GLAST) and EAAT2 (GLT-1). Thereby, glial cells maintain balanced extracellular glutamate levels that enable synaptic transmission and prevent excitotoxic states. Expression levels of these transporters, however, are not fixed. Instead, expression of glial glutamate transporters are highly regulated in reaction to the external situations. Interestingly, such regulation and homeostasis is lost in diseases such as glioma, (tumor-associated) epilepsy, Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis or multiple sclerosis. Upregulation of system x c − (xCT or SLC7A11) increases glutamate export from the cell, while a downregulation of EAATs decreases intracellular glutamate import. Occurring simultaneously, these reactions entail excitotoxicity and thus harm neuronal function. The release of glutamate via the antiporter system x c − is accompanied by the import of cystine—an amino acid essential in the antioxidant glutathione. This homeostasis between excitotoxicity and intracellular antioxidant response is plastic and off-balance in central nervous system (CNS) diseases. System x c − is highly expressed on glioma cells and sensitizes them to ferroptotic cell death. Hence, system x c − is a potential target for chemotherapeutic add-on therapy. Recent research reveals a pivotal role of system x c − and EAAT1/2 in tumor-associated and other types of epilepsy. Numerous studies show that in Alzheimer’s disease, amyotrophic lateral sclerosis and Parkinson’s disease, these glutamate transporters are dysregulated—and disease mechanisms could be interposed by targeting system x c − and EAAT1/2. Interestingly, in neuroinflammatory diseases such as multiple sclerosis, there is growing evidence for glutamate transporter involvement. Here, we propose that the current knowledge strongly suggest a benefit from rebalancing glial transporters during treatment.


Introduction
Glial cells play a major role in the central nervous system (CNS).Comprising different cell types, glial cells regulate many physiological processes: Astrocytes, participating in the tripartite synapse, remove excess levels of glutamate, thereby enabling proper glutamatergic neurotransmission and preventing extrasynaptic N-methyl-Daspartate (NMDA) receptor-induced excitotoxicity [1,2].Microglia are CNS resident immune cells that regulate the immune response to threatening stimuli [3].Oligodendrocytes ensheath axons to protect and support them and to enable rapid information transfer inside neuronal networks [4].
The excitatory amino acid glutamate plays an important role in the homeostasis of the brain [5], thus its regu-lation by glial transporters is particularly relevant in diseases.In the CNS, neuronal damage can occur through increased extracellular glutamate, which then triggers cell death-inducing cascade by the activation of extrasynaptic NMDA receptors, giving rise to the so-called mechanism of excitotoxicity [6,7].This has strong implications for various diseases of the CNS such as Alzheimer's disease or Huntington disease [8].To regulate glutamate homeostasis, astroglial cell express glutamate exporters and glutamate importers.Through this interplay the level of extracellular glutamate can be regulated.The major glutamate exporter in the CNS is the system x c − .It is mainly expressed in the astroglia [9], and during the development of the rat, the expressions of the system x c − subunits 'xCT' or 'SLC7A11' (light chain) and '4F2hc' (heavy chain) increase until at least 3 months of age [10].With aging, sys-tem x c − is overexpressed in rats [11].These findings suggest that with the growing nervous systems, the need to balance oxidative stress and glutamate homeostastis remains an important task that is fulfilled by system x c − .
Astroglial glutamate importers such as the excitatory amino acid transporters 1 and 2 (EAAT1/2) counteract the extrusion of glutamate.EAATs are sodium-dependent glutamate transporters that are responsible for the removal of extracellular glutamate by importing glutamate into the astrocytes' cytosol [12].The examination of both glutamateexporting and -importing transporters provides important insights into the overall astroglial contribution to glutamate homeostasis and its pathophysiological relevance.The detailed molecular aspects and the physiology of glutamate transporters are beyond the scope of this plasticity-focused review.We refer the reader to excellent reviews that comprehend this knowledge [13][14][15][16].
Oxidative stress can strongly interfere with physiologic function.System x c − imports cystine to fuel the antioxidant response of the cells [17] in exchange for glutamate, which is exported to the extracellular fluid.System x c − activity in astrocytes is not only regulated by glutamate but also by its second substrate, cystine, since the intracellular glutathione (GSH) levels can regulate the expression of system x c − [18].However, while system x c − contributes to the antioxidative response [17], its deficiency in system x c − does not induce oxidative stress by itself, but may disrupt glutamate homeostasis [19].
Neurons in the CNS are susceptible to oxidative stress due to their high oxygen consumption during metabolism and the high abundance of lipids, which represent targets for oxygen radicals [20].As some neurons are especially vulnerable to reactive oxygen species, it is important for the brain to maintain an antioxidative response [21].The glutamate-cystine antiporter system x c − is involved in the antioxidative response, which has been also strongly investigated in the context of cancer biology [22].On a molecular level, the import of cystine through system x c − fuels the formation of antioxidant glutathione.Inhibiting cystine import through system x c − inhibitors such as erastin promotes ferroptosis, an iron-dependent form of cell death triggered by missing antioxidants [23].In the field of neurooncology, system x c − thus represents a potential therapeutic target, and tumor cell death during chemotherapy could potentially be boosted by add-on therapy with system x c − inhibitors [24,25].
In this review, we will focus on the issue of whether alterations in glutamate transporter activity are a consequence of the disease pathology or are part of the pathogenesis in glioma, tumor-associated epilepsy, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, or multiple sclerosis.We further address the benefits of boosting/inhibiting these proteins and we are going to approach the question of how to selectively target glial cells, because of their various responsabilities in the nervous system.

Overview of the Function of Glial Glutamate Transporters System x c − and EAAT1/2
There is no final consensus yet on the expression levels of the cystine-glutamate antiporter system x c − in different CNS cell types.Soria et al. [26] reported co-expression of system x c − with markers for neurons, as it was shown in other studies [10,27], and for oligodendrocytes.Recent findings questioned these data by claiming that system x c − is expressed mainly in astrocytes, where it is coexpressed with the sodium-dependent glutamate transporter EAAT1 [9].System x c − expression was not found in neurons, oligodendrocytes, or microglia.In line with this, Mesci et al. [28], could not detect system x c − in motor neurons.In a systematic approach to evaluate antibody specificity and technical specifications, it became evident that the antibody production process and the staining protocols can heavily impact if system x c − can be detected [29].Further studies are required to confirm in which cell system x c − may be expressed.While we wanted readers to bear in mind this uncertainty when evaluating study results, system x c − modulation affects every cell type that is in contact with system x c − modulated extracellular glutamate level regardless of their individual expression.
Although system x c − expression in neurons is uncertain, its effect on neuronal activity is undebated.Around the time when Sato et al. [30] used molecular-biological techniques to clone human system x c − and to describe system x c − expression across the brain with in situ hybridization [31], the first electrophysiological evidence for system x c − ′s impact on vesicular release was obtained: in acute brain slices containing the medial prefrontal cortex, bath application of cystine reduced the frequency of miniature excitatory postsynaptic currents (mEPSCs) that are the electrophysiological expression of the readily releasable pool (RRP) of synaptic vesicles [32].The effect was suppressed by system x c − inhibitor (S)-4-carboxyphenylglycine [33].(S)-4-carboxyphenylglycine is a rather unspecific blocker for system x c − and also acts as an antagonist for the group 1 metabotropic glutamate receptors [34].This weak receptor specificity of (S)-4-carboxyphenylglycine could interfere with the inhibition of system x c − .There is no indication that the impact of (S)-4-carboxyphenylglycine on system x c − than its modulation of metabotropic glutamate receptors, since data reveal no direct connection between the RRP size and group I mGluR activity, at least in the functional context of synaptic long-term depression [35].
In electrophysiological recordings, mEPSCs can only be detected when action potential-driven release is blocked, e.g., with the voltage-gated sodium channel blocker tetrodotoxin (TTX).Spontaneous EPSCs (measured in the absence of TTX) are not decreased by cystine [33].Cystine's dampening effect on mEPSCs is mediated through group II metabotropic glutamate receptors that balance glutamate release by suppressing synaptic firing upon above-threshold extracellular glutamate detection [36].The increase in extracellular cystine could elevate extracellular glutamate concentrations, which triggers metabotropic glutamate receptors, which in turn suppress neuronal vesicle release [37,38].When evoked EPSCs (eEPSC) from cortical layer 2/3 neurons were recorded, system x c − inhibitor sulfasalazine could decrease these eEPSCs [39].In mixed primary hippocampus cultures with both astrocytes and neurons, treatment with system x c − inhibitor erastin led to a reduction of the RRP size without affecting the recycling pool size [40].In these cultures, treatment with system x c − inhibitors erastin [23] and sorafenib [41] resulted in increased extracellular glutamate levels.In addition to acting on system x c − , sorafenib inhibits several targets, such as the RAF pathway and the vascular endothelial growth factor receptor family (VEGFR), which is of particular importance since VEGF has direct synaptic effects [42].The broad range of targets on which sorafenib acts makes it difficult to determine which target is particularly relevant in this context.Thus, there is a need for more specific inhibitors to narrow down the targets involved when 'dirty drugs' are used.

The action of system x c
− is not restricted to the presynaptic site.A study in Drosophila revealed that a reduction in system x c − reduced extracellular glutamate levels [43].Specifically, in Drosophila mutants with genetic elimination of the so-called xCT gene "genderblind", extracellular glutamate falls off by 50% compared to wild-type-level [43].In response to this reduced glutamate level, the postsynaptic receptors become more expressed [43].This elevated postsynaptic receptor expression could be reversed by exposing postsynaptic glutamate receptors to normal levels of glutamate [43].System x c − ′s regulation of extracellular glutamate hence has a direct effect on synaptic transmission.This finding was further substantiated by recordings from the Schaffer collaterals, the CA3-to-CA1 synapse in the hippocampus, showing that a knockout of system x c − leads to more α-amino-3-hydroxy-5methyl-4-isoxazolepropionic acid (AMPA) receptors being expressed at the membrane, and to a strengthened synaptic transmission [44].Furthermore, in system x c − deficient sut mice, long-term potentiation and long-term memory were decreased, while the basic synaptic transmission was unaltered [45].In line with that normal synaptic transmission, the assessment of mouse behavior in terms of spontaneous alternation, rotarod, and open field behavior, revealed that a genetic knockout of system x c − does not influence these basic kinds of behavior [46].Extracellular glutamate may bind to a variety of receptors, amongst them AMPA receptors, N-methyl-Daspartate (NMDA) receptors, or metabotropic glutamate receptors (mGluRs), and also to glutamate transporters such as EAAT1/2.While mEPSCs abate upon an excess extracellular glutamate, mGluR1-mediated EPSCs swell [47].This indicates competition for the extracellular glutamate between neuronal receptors and glial transporter.Extracellular glutamate levels cannot only be increased by system x c − activation.Also the inhibition of EAAT1 (GLAST) and of EAAT2 (GLT1) has similar effects [47].The interplay between all different glutamate transporters is complex and essential in determining how the synaptic transmission is affected by the manipulation of one of them.This is illustrated for example by the finding that system x c − deletion led to increased expression of EAAT2 [48], which does not represent a compensatory, but rather a reinforcing mechanism of decreasing extracellular glutamate.The extent to which glial glutamate transporter-mediated glutamate uptake (via EAAT1/2) affects neurotransmission is even brain region-specific: compared with the neonatal rat hippocampus, the expression of EAAT1 and EAAT2 is lower, and the entry of glutamate into neonatal rat cortical astrocytes is slower [49].
When pharmacological action on the glutamate transporters is planned, it is important to assess how this broad inhibition would impact the glutamate transporters on the diverse glial cells.One promising approach to tackle this question is the investigation of cell-type specific knockouts, e.g., of EAAT2.When EAAT-2 was genetically knocked out using neuron-or astrocyte-specific Cre-loxP systems, it turned out that the astrocytic EAAT2-deletion had more severe effects than the neuronal deletion of EAAT2 [50].When astrocytes had lost their glutamateimporting capacities, mortality and seizure susceptibility increased [50].Interestingly, during aging an astrocytespecific EAAT2 knock-out differs from a neuron-specific knock-out on behavioral and transcriptional levels [51], indicating that the different contributions of neuron-and astroglial-mediated glutamate imports are directly linked to behavioral states.These findings stress the fact that it is important to precisely investigate each transporter and its role in physiological and pathophysiological settings.
Altogether, glial glutamate transporters control important aspects of the physiologic neuronal activity that gives rise to behavior.In the following paragraph, we will discuss how these transporters shape astrocytes' pathophysiological role in the context of different CNS diseases.

System x c − and EAAT1/2 in Peritumoral Astrocytes and Their Impact on Epileptic Activity
The knowledge that astrocytes are associated with human disease is growing rapidly [52].One of the most devastating diseases of the human CNS represents malignant glioma.Resections-if even possible at all-are incomplete at best.The tumors are often treatment resistant and can progress to higher CNS WHO grades even after early detection, up to glioma of WHO grade 4 (mostly glioblastoma) [53,54].The expression levels of system x c − in such highly malignant human glioblastoma cells are positively correlated with tumor invasion, and negatively correlated with patient survival [55,56].In contrast to the high expression of system x c − in tumor cells [57,58], the glutamate importers EAAT1 and EAAT2 are abnormally low expressed in glioma cell lines [59], animal models [60] and human glioblastoma [61].It has even been shown that increased EAAT2 expression inhibits tumor growth [62], further underscoring the idea of glutamate as a proponent of malignant growth [63].
In addition to the harms caused by the tumor itself, healthy CNS function is often affected in the peritumoral area as well.In a very recent study, the transcriptome of human astrocytes that stem from tissue surrounding the tumor site was analyzed [64].The authors found that peritumoral astrocytes downregulated genes related to reacting to their microenvironment and synaptic function.Interestingly, amongst those genes were the EAAT2-encoding gene SLC1A2 and the EAAT1-encoding gene SLC1A3 [64].In the peritumoral area of human tumor samples, system x c − expression was not elevated [39], suggesting that solely glutamate importers were downregulated and thereby could contribute to increase extracellular glutamate levels.In a tumor-transplant model performed in mice, it was found that primary CNS tumors release glutamate via system x c − , which evokes epileptic activity in the peritumoral area [65].High expression of system x c − and low expression of EAAT are biomarkers for glioma-associated seizures [66,67].While the primary tumor enhances its glutamatereleasing capabilities (high system x c − expression), astrocytes in the peritumoral area decrease their glutamateimporting capacities (low EAAT1/2 expression).This leads to a higher glutamatergic tone in the peritumoral area.As a result, glioma often coincides with epilepsy [68].
Glutamate transporters are involved in the pathomechanisms of epileptic seizures also without glioma as the underlying cause.In biopsies from patients with pharmacoresistant temporal lobe epilepsy, qPCR analysis revealed an upregulation of xCT mRNA [69].In humans, EAAT1/2 is also involved in epilepsy.Astroglial glutamate transporters EAAT1/2 are investigated in the context of seizures since almost thirty years ago, in mice, the EAAT2 gene was located in a chromosomal region known to modulate neuroexcitability and seizure frequencies [70].In patients suffering from pharmacoresistant temporal lobe epilepsy but without hippocampal sclerosis (i.e., without significant cell loss), EAAT2 was upregulated in hippocampal subregions, as confirmed by immunohistochemical stainings and in situ hybridization from post-mortem tissue [71].In human epileptic foci in the neocortex, EAAT2 protein expression was decreased [72], similar to other cohorts of patients with intractable epilepsy in which EAAT1 and EAAT2 were decreased [73,74].Taken together, it appears that in human patients suffering from epilepsy, the glutamate homeostasis is shifted towards an increase in the extracellular glutamate level.However, human studies on measurable levels of glutamate in epilepsy have yielded mixed results and fur-ther studies using standardized experimental paradigms are needed to elucidate this topic [75].
The hypothesis that glutamate excess is contributing to epileptic seizures [76] was tested in several animal models with blocked system x c − function: at first, it was described that xCT −/− mice have an elevated threshold for seizures induced by pilocarpine or kainic acid [19].Thus, fewer seizures occur in the absence of system x c − .In these experiments, pilocarpine applications induce seizures in the temporal lobe via its action on the M1 receptor and represent a widely used experimental model to study epileptic seizures [77].Seizures are induced by kainic acid because of its analogism with glutamate and the agonistic action on ionotropic glutamate receptors [78].Nacetylcysteine as an activator of system x c − could not exert its usually proconvulsive effects in xCT −/− mice -ascribing a seizure-promoting role to system x c − through its glutamate-extruding activity [19].In hippocampal rat brain slices, convulsive agents such as kainic acid, pilocarpine, and veratridine reduced the EAATs activity and could therefore be rescued by the application of a mGluR III agonist [79].In this study, the injection of an EAAT inhibitor into rats exacerbated kainic acid-induced seizures.Thus, more seizures occured in the absence of EAATs because blockage of glutamate transporters increased their extracellular concentrations.Involving mGluRs, EAAT inhibition in hippocampal pyramidal cells activated mGluR group I and II receptors and led to epileptic-like activity [80].Even in zebrafish, EAAT2 deficiency led to increased glutamate levels as well as light-induced seizures in neurons and glial cells [81].
In a model of self-sustained status epilepticus-where amygdala-implanted electrodes were used for stimulation that results in ongoing seizure events-genetic system x c − inactivation had anticonvulsive and antiepileptogenic effects, weighing also towards the seizure-promoting role of system x c − [82].In their model, pharmacological system x c − inhibition (by sulfasalazine treatment) also prevented seizures.In line with this, system x c − null mice (xCT sut/sut ) displayed a reduced occurrence of seizures in a pentylenetetrazole kindling model, a model in which inhibition of GABA A receptors increases synaptic stimulation and excitation [83,84].Although the same authors, however, later reported lower seizure thresholds in system x c − null mice after kainic acid or pentylenetetrazole and more severe seizures [85]-ascribing a seizure-preventing role to system x c − -it generally appears that targeting glial system x c − may support therapy of seizures.In a very recent study, the system x c − inhibitor sulfasalazine could ameliorate seizures that were elicited by astrogliosis [86], ruling out pharmacological manipulations that potentially may occur during seizure induction.
In summary, increased expression of system x c − and reduced expression of EAATs promote epileptic activity and seizures in peritumoral astrocytes and in temporal lobe epilepsy.From zebrafish to humans, a decrease in astroglial EAAT activity promotes seizures.This raises the hypothesis that a system x c − -driven increase in glutamate promotes seizures, while the EAAT-driven decrease in extracellular glutamate prevents seizures.In conclusion, epilepsy treatment might benefit from selectively targeting astrocytic glutamate transporters in a manner that promotes the activity of EAATs but decreases system x c − activity.

The Roles of Glutamate Transporters in Neurodegenerative Diseases and Multiple Sclerosis
Neurodegenerative diseases like Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis, is heavily impacting human society.Despite years of research efforts, our understanding of the pathophysiology of neurodegeneration is still ill-defined [87].Moreover, up to now there is no cure for diseased patients.Amongst many cellular and molecular suspects, astrocytes have been implicated in neurodegeneration and in neuroprotection [88,89].In the following, we specifically discuss the roles of system x c − and EAAT1/2 in Alzheimer's disease, amyotrophic lateral sclerosis, and Parkinsonian disease.

Alzheimer's Disease
It is well established that excitotoxicity can lead to severe neuronal cell death in the central nervous system.In particular, excitotoxicity has been suspected to be also involved in Alzheimer's disease (AD) pathology [90,91], and the reduction of extracellular glutamate by targeting glutamate transporters could then prove beneficial for neuronal survival.
A study in post-mortem tissue from AD patients revealed an increased expression of the light-chain subunit of system x c − compared to age-matched controls [92].In line with this, AD model AβPP23 mice displayed a stronger system x c − expression with aging [93].These transgenic mice carry a human amyloid-β precursor protein with the so-called Swedish mutation, a double mutation near the βsecretase site.Adding the Alzheimer's disease-related peptide amyloid-β 1−40 to neuron-glia-co-cultures amplified the transcription of system x c − and induced neurotoxicity [94].Similarly, the peptide amyloid-β 25−35 could evoke system x c − upregulation in human astroglial cells.This entailed neuronal cell death, which could be prevented by adding the system x c − inhibitor sulfasalazine [95].Taken together, these studies indicate that in the course of the disease, system x c − becomes upregulated.AD patients displayed a reduction of EAAT1 and EAAT2 in the hippocampus or the medial frontal lobe, brain regions being affected in early disease stages [96].Application of Amyloid-β 1−42 , which is central to AD pathology [97] as a neurotoxic agent, induced a decrease in the expression of EAAT1 and EAAT2 in cultured astrocytes [98].Using an elegant approach of in vivo two-photon microscopical de-tection of glutamate with iGluSnFR [99], Hefendehl et al. [100] have found that glutamate fluctuates and EAAT2 is downregulated in the vicinity of Amyloid-β plaques.Increasing the glutamate transporter EAAT2 expression by gain-of-function gene targeting [101,102] ameliorated this phenotype [100].These data display that glutamate levels are normalized due to its increased import.In the transgenic AβPP23 mice, hippocampal expression of EAAT1 and EAAT2 were also decreased [93].In conclusion, it can be stated that AD is accompanied by a decrease in astrocytic glutamate import capabilities.
Therefore, the question appears how the extracellular glutamate level develops in human AD patients.While magnetic resonance spectroscopy in the bilateral posterior cingulate gyrus showed less glutamate content in AD patients [103], analysis of the cerebrospinal fluid from patients with probable AD indicated that glutamate is more prevalent than in control individuals [104].Given the discrepancy between AD research performed in mice and what could be translated to humans in the past [105,106], it would be important to further examine glutamate levels in human patients to receive consistent results that are based on standardized experimental conditions, i.e., at which disease stage glutamate is measured, in which brain region or fluid it is measured, and by which method.However, it appears that glutamate transporter disturbances occur over the course of the disease-with increased system x c − activity and impeded EAAT activity-and could be potentially suited to ameliorate symptoms in patients by selectively targeting them in astrocytes to not interfere with intrinsic neuronal function.

Amyotrophic Lateral Sclerosis
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of motor neurons with the final stage of paralysis, whose pathogenesis is still unclear and treatment options are strongly limited to symptomatic approaches [107].
Recently, it was found for sporadic ALS patients, that system x c − in their astrocytes is significantly stronger expressed than in those of healthy subjects [108].In the SOD-1 mutation model of ALS, and also in human samples, spinal cord-residing microglia expressed more system x c − than healthy controls [28].Data from SOD-1 mutated mice also suggest that increased system x c − activity is a major contributor to excitotoxicity-mediated disease activity at early time points in ALS [109].Interestingly, a cystine-rich dietary supplement in the transgenic hSOD1(G93A) mice has been shown to delay the onset of ALS symptoms, and survival was even extended when riluzole, a medication for ALS patients targeting glutamate release [110], was added to the dietary supplement [111].This finding demonstrates that targeting both the oxidative stress and the glutamate effects proves beneficial for therapy.In summary, system x c − activity is increased in ALS.
In post-mortem samples of the spinal cord and motor cortex of ALS patients, glutamate importer EAAT2 was dramatically decreased [112].The loss of function of EAAT2 during ALS has also been demonstrated experimentally on different levels: in an ALS rat model with SOD-1 mutation, glutamate transport was impaired [113] and EAAT2 expression was diminished [114].In several studies performed in SOD-1 mutated mice, a reduced expression of EAAT2 in the spinal ccord was observed [115][116][117][118][119]. EAAT1, however, was not changed over the course of the disease in the motor cortex from ALS patients or in transgenic rats expressing the human SOD-1 mutant G93A [112,119].Providing a potential therapeutical approach to the downregulation of glutamate importers, activation of metabotropic glutamate receptors has proven beneficial [120].Based on all this knowledge and technological advances, in a recent study, EAAT2-based gene therapy in an ALS mouse model with SOD-1 mutation improved motor function and survival [121].A cellular model of ALS with SOD-1 mutation showed that EAAT2 undergoes enhanced internalization and degradation, which limits its function to import glutamate [122].An early study suggested that defects during the processing of EAAT2 mRNA is the cause for the lack of protein expression found in the aforementioned studies [123].In contrast to EAAT2, alterations in EAAT1 do not play a major role in ALS [112,124].Taken together, ALS is characterized by an increased expression of system x c − , a reduced expression of EAAT2, and rather unaffected levels of EAAT1, indicating that enhanced system x c − driven glutamate release and impaired EAAT2mediated glutamate uptake contribute to ALS pathology.Both effects appear to occur as a consequence of the disease and are most probably not the causal agent.Targeting glutamate homeostasis may represent a beneficial option to ameliorate ALS symptoms, because neurodegeneration occuring due to excitotoxicity could be slowed down.Future studies are required to judge this hypothesis and to shed light on the definite impact of glutamate-driven excitotoxicity on the ALS disease course.

Parkinson's Disease
Parkinson's disease (PD) is a disease of the basal ganglia with strong motor signs that are the result of neurodegeneration [125].In mice, PD can be pharmacologically modeled using substances such as substance 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 6hydroxydopamine (6-OHDA), or the direct application of alpha-synuclein, which enables more detailed investigations of the underlying mechanism of neurodegenerative changes in PD [126].
After MPTP application to induce parkinsonism in mice, it was found that xCT expression was increased in the striatum, but reduced in the substantia nigra, however, MPTP could induce similar phenotypes in wild-type mice and mice with a system x c − deletion [127], indicating that system x c − responds to neurodegeneration but does not affect its induction, at least in this case.Following acute MPTP application in mice, EAAT2 immunolabeling in the dorsolateral striatum decreased [128].
In 6-OHDA-lesioned parkinsonian mice, treatment with levetiracetam-upregulating system x c − -had neuroprotective effects in nigrostriatal dopaminergic neurons [129].In another study, 6-OHDA treatment induced less neurodegeneration: a comparison of the substantia nigra pars compacta of xCT −/− mice with their wild-type littermates suggests that deletion of the glutamate extrusion system x c − provides protection against this form of neurodegeneration [130].When 6-OHDA was applied to the bilateral substantia nigra pars compacta of rats, after two weeks EAAT1 expression decreased [131].To counteract this decrease in the expression of glutamate importers, ceftriaxone was able to increase EAAT2 expression in the 6-OHDA model of Parkinsonian disease [132], similar to what was found in the MPTP model [133].Furthermore, EAAT2 expression was increased inside the basal ganglia following the clinically relevant L-DOPA application [134].
When nigrostriatal lesions in mice were induced through the application of the proteasome inhibitor lactacystin, treatment with the anticonvulsant zonisamide ameliorated this phenotype without any modulation of system x c − [135], implicating that system x c − is neglectable in some cases of anti-neurodegenerative therapy.With age, system x c − deficient mice were less prone to this lactacystin-induced nigrostriatal degeneration [136].
After the application of alpha-synuclein onto astrocytes in cultures, or in a synucleinopathy mouse model, the expression levels of EAAT1 and EAAT2 were increased [137].These findings indicate that the glutamate transporter system is responsive to the induction of the disease as well as the subsequent therapy, making glutamate homeostasis a promising target for the modulation of PD symptoms.
In an interesting and novel approach, PD was modeled in mice by directly targeting the EAATs.When EAAT2 expression was genetically abrogated in the substantia nigra pars compacta, mice developed PD-related phenotypes including cell death.These observations were associated with aberrant calcium signaling [138].
In summary, astroglial glutamate transporters-such as EAAT1/2 and system x c − -are dysregulated in diverse neurodegenerative diseases.They are implicated in the pathophysiology underlying those diseases, and they are responsive to treatment approaches.This underlines the importance of the sensitive regulation of extracellular glutamate for proper CNS function.

Multiple Slcerosis
In the previous paragraphs, we have highlighted the contribution of the glial glutamate transporters to CNS neurodegenerative diseases.Neurodegeneration is often accompanied by neuroinflammatory components [139], which can be cell-mediated by microglia, the CNS resident immune cells [140], or also T cells [141].As previously mentioned, microglia might express system x c − and thereby might contribute to pathological states [28,142].We discuss the contribution of glial glutamate transporters system x c − and EAAT1/2 to neuroinflammation in more detail using multiple sclerosis (MS) as an example.
In the cerebrospinal fluid of MS patients, the glutamate level is increased [143], which raises the question of the underlying mechanism.To investigate MS in animal models, experimental autoimmune encephalomyelitis (EAE) can be induced to bring key features of MS into the model animal [144].Investigations of human monocytes and EAE revealed an upregulation of system x c − compared to controls, potentially leading to excitotoxic insults [145].When system x c − was inhibited by introducing a mutation in xCT, mice became resistant to the induction of EAE, indicating a strong role of system x c − in mediating disease in activity by acting on immune cells [146].In another study, xCT −/− mice were as susceptible to EAE as control mice, but mice after bone marrow transplantation from xCT −/− mice-with therefore xCT-deficient immune cells-displayed attenuation of EAE [147].Differences between both studies could have occurred through the usage of different antigens for immunization, however, the main finding of system x c − in EAE severity remains rather independent from the chosen protocol.The increased system x c − activity in EAE mice has been confirmed in 18 Ffluorodeoxyglucose PET scans [148].In addition to solely investigating xCT, its molecular and cellular interplay is of particular importance.Interleukin 1β (IL1β) is an important factor in EAE and MS [149,150], which regulates system x c − mRNA and thereby may contribute to excitotoxicity [151].The glutamate transporters system x c − and EAATs are involved in the microglial toxicity to oligodendrocytes [142], providing evidence for a complex interplay between different cell types that are involved in EAE/ MS.
In addition to the many studies that revealed increased system x c − activity in EAE, the protein expression of EAAT1/2 in MS patients was found to be decreased in the vicinity of cortical lesions [152].EAE rats display an initial upregulation of EAAT1 and EAAT2 mRNAs, but respective proteins do not follow this pattern [153].This has been confirmed in an independent EAE rat study [154].In mouse spinal cord from EAE mice, EAAT2 protein is less expressed [155].Glutamate transporters are not affected in all neuroinflammatory models, though: in contrast to results obtained from EAE, infection with Theiler's murine encephalomyelitis virus (TMEV) did not modulate system x c − expression [127].
EAAT1/2 is decreased, while system x c − is positively associated with MS, and system x c − inhibition has been proven to be beneficial for disease activity.These findings highlight the important role of astrocytes in the regulation of glutamate homeostasis through plasticity of transporter expressions during neuroinflammation.

Conclusions
In this review we brigded the experimental data with clinical data on glial glutamate transporters system x c − and EAAT1/2.We present evidence for the described plasticity as a consequence of the disease pathology as well as part of the pathogenic process.We took a closer look at (tumorassociated) epilepsy, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and multiple sclerosis.
Under physiological conditions, the astroglial glutamate transporters system x c − and EAAT1/2 control neuronal function through pre-and postsynaptic modulation of synaptic transmission.
For epileptic seizures induced by adjacent tumor tissue, an aberrant glutamate transporter activity appears as a causative.In contrast, the resulting glutamate levels in epileptic patients with non-tumor related epileptic disorders are yet unclear.It would be interesting to take a further look at the many different pharmacological models that are used to induce epileptic seizures with many underlying signaling cascades.
The evidence presented in Alzheimer's disease showed that glial glutamate transporter plasticity can be directly induced via Amyloid-β-induced changes.
In ALS, studies showed how restoring glutamate transporter works, strongly indicates their pathogenetic role since those interventions proved beneficial to ameliorate symptoms and prolong survival.Similarly, the role of glutamate transporters in Parkinson's disease may be causative for the disease since treatment approaches directly to these transporters were beneficial for the disease outcome.Since multiple sclerosis is the interplay of many cell types and molecular events that are not fully understood yet, it is more difficult to categorize changes in glutamate transporters as causative or consequential.
The role of glial glutamate transporter-mediated plasticity is still ambiguous.The relevance of these proteins in some brain diseases is increasing and worth for further studies.Approaches addressing the selectivity of inducers or inhibitors to glutamate transporters could further deepen our knowledge of the pathophysiological processes of these brain disorders in order to improve treatment management.