1. 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-D-aspartate (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 regulation 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. It is mainly expressed in the astroglia [9], and during the
development of the rat, the expressions of the system x subunits
‘xCT’ or ‘SLC7A11’ (light chain) and ‘4F2hc’ (heavy chain) increase until at
least 3 months of age [10]. With aging, system x 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.
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 glutamate-exporting 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 imports cystine to fuel the antioxidant response of the cells [17]
in exchange for glutamate, which is exported to the extracellular fluid. System
x 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 [18]. However, while
system x contributes to the antioxidative response [17], its
deficiency in system x 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
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 fuels the formation of antioxidant
glutathione. Inhibiting cystine import through system x 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 thus represents a potential therapeutic target, and tumor cell
death during chemotherapy could potentially be boosted by add-on therapy with
system x 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.
2. Overview of the Function of Glial Glutamate Transporters System
x and EAAT1/2
There is no final consensus yet on the expression levels of the cystine-glutamate
antiporter system x in different CNS cell types. Soria et
al. [26] reported co-expression of system x 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 is
expressed mainly in astrocytes, where it is coexpressed with the sodium-dependent
glutamate transporter EAAT1 [9]. System x expression was not found in
neurons, oligodendrocytes, or microglia. In line with this, Mesci et al.
[28], could not detect system x 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 can be detected [29]. Further studies are
required to confirm in which cell system x may be expressed. While we
wanted readers to bear in mind this uncertainty when evaluating study results,
system x modulation affects every cell type that is in contact with
system x modulated extracellular glutamate level regardless of their
individual expression.
Although system x 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 and to
describe system x expression across the brain with in situ
hybridization [31], the first electrophysiological evidence for system
x′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 inhibitor
(S)-4-carboxyphenylglycine [33]. (S)-4-carboxyphenylglycine is a
rather unspecific blocker for system x 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. There is no indication that the impact of
(S)-4-carboxyphenylglycine on system x 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
inhibitor sulfasalazine could decrease these eEPSCs [39]. In mixed primary
hippocampus cultures with both astrocytes and neurons, treatment with system
x inhibitor erastin led to a reduction of the RRP size without
affecting the recycling pool size [40]. In these cultures, treatment with system
x inhibitors erastin [23] and sorafenib [41] resulted in increased
extracellular glutamate levels. In addition to acting on system x,
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 is not restricted to the presynaptic site. A
study in Drosophila revealed that a reduction in system x
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′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 knock-out of system x leads to more
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors
being expressed at the membrane, and to a strengthened synaptic transmission
[44]. Furthermore, in system x 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
does not influence these basic kinds of behavior [46].
Extracellular glutamate may bind to a variety of receptors, amongst them AMPA
receptors, N-methyl-D-aspartate (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 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 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 knock-outs, 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 glutamate-importing capacities, mortality and seizure susceptibility
increased [50]. Interestingly, during aging an astrocyte-specific 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.
3. System x 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 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 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 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, which evokes epileptic activity in the peritumoral area [65]. High
expression of system x and low expression of EAAT are biomarkers for
glioma-associated seizures [66, 67]. While the primary tumor enhances its
glutamate-releasing capabilities (high system x expression),
astrocytes in the peritumoral area decrease their glutamate-importing 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 further 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 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. 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].
N-acetylcysteine as an activator of system x could not exert
its usually proconvulsive effects in xCT mice – ascribing a
seizure-promoting role to system x 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 inactivation had anticonvulsive and
antiepileptogenic effects, weighing also towards the seizure-promoting role of
system x [82]. In their model, pharmacological system x
inhibition (by sulfasalazine treatment) also prevented seizures. In line with
this, system x null mice (xCT) displayed a reduced
occurrence of seizures in a pentylenetetrazole kindling model, a model in which
inhibition of GABA receptors increases synaptic stimulation and excitation
[83, 84]. Although the same authors, however, later reported lower seizure
thresholds in system x null mice after kainic acid or
pentylenetetrazole and more severe seizures [85]—ascribing a seizure-preventing
role to system x—it generally appears that targeting glial system
x may support therapy of seizures. In a very recent study, the system
x 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 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-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 activity.
4. 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 and EAAT1/2
in Alzheimer’s disease, amyotrophic lateral sclerosis, and Parkinsonian disease.
4.1 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 compared to age-matched
controls [92]. In line with this, AD model APP23 mice displayed a
stronger system x expression with aging [93]. These transgenic mice
carry a human amyloid- precursor protein with the so-called Swedish
mutation, a double mutation near the -secretase site. Adding the
Alzheimer’s disease-related peptide amyloid- to
neuron-glia-co-cultures amplified the transcription of system x and
induced neurotoxicity [94]. Similarly, the peptide amyloid-
could evoke system x upregulation in human astroglial cells. This
entailed neuronal cell death, which could be prevented by adding the system
x inhibitor sulfasalazine [95]. Taken together, these studies
indicate that in the course of the disease, system x 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-, 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 detection of glutamate with iGluSnFR
[99], Hefendehl et al. [100] have found that glutamate fluctuates and
EAAT2 is downregulated in the vicinity of Amyloid- 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
APP23 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 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.
4.2 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 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 than healthy
controls [28]. Data from SOD-1 mutated mice also suggest that increased system
x 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 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, a reduced expression of EAAT2, and rather
unaffected levels of EAAT1, indicating that enhanced system x driven
glutamate release and impaired EAAT2-mediated 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.
4.3 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), 6-hydroxydopamine (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 deletion [127], indicating that system x
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—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 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 [135],
implicating that system x is neglectable in some cases of
anti-neurodegenerative therapy. With age, system x 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—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.
4.4 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 and
thereby might contribute to pathological states [28, 142]. We discuss the
contribution of glial glutamate transporters system x 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 compared to controls, potentially leading to
excitotoxic insults [145]. When system x was inhibited by introducing
a mutation in xCT, mice became resistant to the induction of EAE, indicating a
strong role of system x in mediating disease in activity by acting on
immune cells [146]. In another study, xCTmice were as susceptible to EAE
as control mice, but mice after bone marrow transplantation from xCTmice—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 in EAE severity remains rather independent from the chosen
protocol. The increased system x activity in EAE mice has been
confirmed in F-fluorodeoxyglucose PET scans [148]. In addition to solely
investigating xCT, its molecular and cellular interplay is of particular
importance. Interleukin 1 (IL1) is an important factor in EAE
and MS [149, 150], which regulates system x mRNA and thereby may
contribute to excitotoxicity [151]. The glutamate transporters system
x 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
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 expression
[127].
EAAT1/2 is decreased, while system x is positively associated with
MS, and system x 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.
5. Conclusions
In this review we brigded the experimental data with clinical data on glial
glutamate transporters system x 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 (tumor-associated)
epilepsy, Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral
sclerosis, and multiple sclerosis.
Under physiological conditions, the astroglial glutamate transporters system
x 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--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.
Abbreviations
APP23, amyloid-protein precursor 23 gene; ALS, amyotrophic lateral
sclerosis; CA, cornu ammonis; CNPase, 2′,3′-cyclic nucleotide
3′-phosphodiesterase; CNS, central nervous system; EAAT1/2, excitatory amino acid
transporters 1 and 2; EAE, experimental autoimmune encephalomyelitis; EPSC,
excitatory postsynaptic currents; FGF-2, fibroblast growth factor 2; GLAST,
glutamate aspartate transporter 1; GLT-1, glutamate transporter-1; GSH,
glutathione; Iba-1, ionized calcium binding adaptor molecule 1; IL 1,
Interleukin-1 beta; L-DOPA, l-3,4-dihydroxyphenylalanine; mGluR1, metabotropic
glutamate receptor 1; MPTP, 1 methyl 4 phenyl 1,2,3,6-tetrahydropyridine; mRNA,
messenger ribonucleic acid; MS, multiple sclerosis; NMDA receptor,
N-methyl-D-aspartate receptor; 6-OHDA, 6-hydroxydopamine; PET, positron emission
tomography; qPCR, quantitative polymerase chain reaction; RRP, readily releasable
pool; SLC1A2, solute carrier family 1 member 2; SLC1A3, solute carrier family 1
member 3; SLC7A11, solute carrier family 7 member 11; SOD-1, superoxide dismutase
1; TMEV, Theiler’s murine encephalomyelitis virus; TTX, tetrodotoxin; xCT,
cystine/glutamate antiporter; WHO, world health organization.
Author Contributions
EY, NS and MD designed the study. EY, JKD and MD collected and analyzed the
literatures. EY, HHS, JKD, MD and NS wrote, read and approved the final
manuscript.
Ethics Approval and Consent to Participate
Not applicable.
Acknowledgment
We acknowledge financial support by Deutsche Forschungsgemeinschaft and
Friedrich-Alexander-Universität Erlangen-Nürnberg within the funding programme.
Funding
Deutsche Forschungsgemeinschaft and Friedrich-AlexanderUniversität Erlangen-Nürnberg funding
Programme of “Open Access Publication Funding”.
Conflict of Interest
The authors declare no conflict of interest.
