pathological conditions, such as hypoxia/ischemia, mul-tiple sclerosis, schizophrenia, and epilepsy. This review summarizes these results and provides an overall picture of changes in eAAT3/eAAC1 expression in health and disease.
eAAT3, the human counterpart of the eAAC1 rodent trans-porter, is a member of the eAAT family of high-affinity, sodium-dependent glutamate carriers encoded by genes of the SLC1 family [1–3]. eAAT3/eAAC1 is coded by SLC1A1/Slc1a1 and was the first sodium-dependent mam-malian amino acid transporter to be cloned [4]. when studying the activity of eAAT3/eAAC1 in cultured human fibroblasts before its cloning, early researchers demon-strated that the transporter can accept the D isomer of aspartate but not the D isomer of glutamate [5]. This stere-oselective anomaly has been consistently confirmed for all the transporters for anionic amino acids of the SLC1 family and is one of the functional hallmarks of eAAT carriers.
Like other eAAT carriers, eAAT3/eAAC1 uses the transmembrane gradients of Na+, K+, and H+ electrochem-ical potentials as the driving force to accumulate glutamate from the extracellular space [6, 7]. Glutamate is co-trans-ported with 3 Na+ and 1 H+, while 1 K+ is extruded in the same cycle, ensuring the electrogenicity of the transport cycle and the formation of a high transmembrane gradient of the transported substrates.
The expected molecular weight of the transporter is 64 kDa, but electrophoretic migration yields higher values,
Abstract excitatory amino acid transporters (eAATs) are high-affinity Na+-dependent carriers of major impor-tance in maintaining glutamate homeostasis in the cen-tral nervous system. eAAT3, the human counterpart of the rodent excitatory amino acid carrier 1 (eAAC1), is encoded by the SLC1A1 gene. eAAT3/eAAC1 is ubiqui-tously expressed in the brain, mostly in neurons but also in other cell types, such as oligodendrocyte precursors. while most of the glutamate released in the synapses is taken up by the “glial-type” eAATs, eAAT2 (GLT-1 in rodents) and eAAT1 (GLAST), the functional role of eAAT3/eAAC1 is related to the subtle regulation of glutamatergic trans-mission. Moreover, because it can also transport cysteine, eAAT3/eAAC1 is believed to be important for the syn-thesis of intracellular glutathione and subsequent protec-tion from oxidative stress. In contrast to other eAATs, eAAT3/eAAC1 is mostly intracellular, and several mecha-nisms have been described for the rapid regulation of the membrane trafficking of the transporter. Moreover, the carrier interacts with several proteins, and this interaction modulates transport activity. Much less is known about the slow regulatory mechanisms acting on the expression of the transporter, although several recent reports have identified changes in eAAT3/eAAC1 protein level and activity related to modulation of its expression at the gene level. Moreover, eAAT3/eAAC1 expression is altered in
M. G. Bianchi · D. Bardelli · M. Chiu · O. Bussolati (*) Unit of General Pathology, Department of Biomedical, Biotechnological and Translational Sciences (SBiBiT), University of Parma, via volturno 39, 43125 Parma, Italye-mail: [email protected]
M. G. Bianchi Unit of Occupational Medicine, Department of Clinical and experimental Medicine, University of Parma, Parma, Italy
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ascribed to a high degree of glycosylation. Moreover, west-ern-blot experiments have demonstrated high-molecular-weight bands, referred to as trimers of the transporter. In fact, trimers are thought to constitute the functional unit of eAAT3/eAAC1 in situ [8, 9]. Although experimental data on the carrier structure are incomplete, eAATs have 8–10 transmembrane domains, with glycosylation sites in the extracellular loop between TM3 and TM4 and several puta-tive intracellular consensus phosphorylation sites [10].
eAAT3/eAAC1 is expressed not only in the central nervous system (CNS) but also in Schwann cells [11]. Moreover, the carrier is also expressed in the intestine and kidney, where it accounts for the absorption/reabsorption of dicarboxylic amino acids, and in many other tissues. Defective expression of functional eAAT3/eAAC1 results in dicarboxylic aminoaciduria (OMIM222730) [12]. In the CNS, of the five eAATs characterized so far, eAAT4 [13] and eAAT5 [14] are highly expressed in cerebellar Purkinje cells and in retinal photoreceptors and bipolar cells, respectively. In contrast, eAAT1 and eAAT2 pre-dominately have a glial distribution, while eAAT3/eAAC1 is highly expressed in glutamatergic and GABAergic neu-rons [15], especially in somata and dendrites (with par-ticular abundance in the neck of dendritic spines [16]) but not in synaptic terminals of axons or spine heads [16–18]. Other neuronal populations express eAAT3/eAAC1, such as dopaminergic neurons; these neurons are very sensi-tive to carrier dysfunction [19, 20] or inhibition [21]. eAAT3/eAAC1 is also expressed in cells of glial origin, such as in subsets of oligodendrocytes [22], in immature oligodendrocytes [23], and in oligodendrocyte progenitor cells [24]. An isolated report describes eAAT3/eAAC1 expression also in microglial cells after traumatic brain injury [25]. Mature astrocytes are generally believed to be eAAT3/eAAC1-negative, but it should be noted that astro-glial tumor cells, such as rat C6 glioma cells and several human glioma cell models, express the transporter [26]. At variance with other eAAT transporters [27, 28], the expres-sion of eAAT3/eAAC1 is mainly intracellular [22, 29, 30]; only a minor portion of the carriers are targeted to the plasma membrane under control conditions, while most are associated with cytoplasmic vesicles [15, 16].
This peculiar distribution has relevant consequences for the regulation of transporter activity. In cells of both nerv-ous and non-nervous origin, eAAT3/eAAC1 activity is modulated by post-translational mechanisms that rapidly modify its trafficking and abundance on the plasma mem-brane. eAAT3/eAAC1 abundance on the plasma mem-brane is readily enhanced upon PKC activation [31] or stimulation with PDGF [32]. Several PKC enzymes may be involved in these regulatory effects at different levels [31–34]. In addition, other kinases have been implicated in the regulation of eAAT3/eAAC1 trafficking and activity
in Xenopus oocytes injected with the transporter cDNA, including phosphatidylinositol-3 (PI3)-kinase, the phosph-oinositide-dependent kinase PDK1, the serum- and gluco-corticoid-inducible kinase SGK1, and the phosphatidylino-sitol-3-phosphate-5-kinase PIKfyve [35]. Interestingly, the gene encoding PIKfyve is associated with schizophrenia and the mutant form expressed in patients seems to exert a dominant-negative effect on eAAT3 trafficking in Xeno-pus oocytes [36]. In the same experimental model, mTOR increases eAAT3/eAAC1 activity [37], which, instead, is decreased by the mTOR inhibitor AMPK [38].
These effects may be due to a direct interaction between eAAT3/eAAC1 and the kinase or, more often, to changes in the interaction between the transporter and several intra-cellular proteins, such as the endoplasmic reticulum pro-teins addicsin/glutamate transporter-associated protein 3-18 (GTRAP3-18, [39]) and reticulon (RTN2B, [40]), which are believed to play opposite regulatory roles in the membrane-targeting of the transporter [41, 42]. Inter-estingly, the gene for GTRAP3-18, which is a negative modulator of eAAT3/eAAC1 activity, maps to human chromosome 3p14, a susceptibility locus for cancer and epilepsy [43]. GTRAP3-18 is negatively regulated by ADP-ribosylation factor-like 6-interacting protein 1 (Ar16ip1), which causes a decreased interaction between GTRAP3-18 and eAAT3/eAAC1 in neurons [44]. Moreover, eAAT3/eAAC1 trafficking involves several steps depend-ent on Rab11 [45], SNAP-23 (a SNARe protein) [46], and caveolin-1 [47], although evidence for a direct interac-tion between the transporter and these proteins is lacking. The δ-opioid receptor is a G-protein that also modulates eAAT3/eAAC1 activity [48]. eAAT3/eAAC1 also inter-acts with the cytoskeletal protein α-adducin [49], which is particularly abundant in dendritic spines [50], although, in this case, the functional implications of the interaction have not been defined.
Compared to these multiple sites of regulation at the pro-tein level, the characterization of mechanisms modulating eAAT3/eAAC1 synthesis at the transcriptional level has been much slower. This review aims to recount the infor-mation available on this subject as well as on pathologi-cal situations in which the expression of eAAT3/eAAC1 is altered. Before examining the regulatory mechanisms, a brief summary on the physiological role of eAAT3 is discussed.
EAAT3/EAAC1 functions in physiology and pathology
In general, the activity of eAAT transporters is consid-ered the main mechanism responsible for the termination of glutamatergic transmission. As such, eAAT transport-ers are believed to be important factors for the prevention
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of excitotoxic damage, i.e., neuronal loss due to excessive influx of calcium mediated by the overstimulation of glu-tamate receptors of the NMDA and AMPA/kainate classes.
However, the roles of the three most important eAATs, eAAT1, eAAT2, and eAAT3/eAAC1, are thought to be diverse [51]. This is well exemplified by experiments with knock-out animals. whereas the knock-outs for GLAST (the rodent counterpart of eAAT1) and GLT-1 (the rodent eAAT2) exhibit early signs of severe excitotoxic damage [52], the knock-out for eAAT3/eAAC1 has an apparently normal development [53]. However, these animals present symptoms of precocious neurodegeneration and subtle neu-rologic damage, associated with cell loss in the cerebral cortex, increased aggressiveness and impaired self-groom-ing [54]. In particular, eAAT3/eAAC1-deficient mice exhibit spatial learning/memory dysfunction, loss of dopa-minergic neurons in the substantia nigra, and movement disorders; these disturbances typically appear at senescence [20, 54] but have also been reported in young animals [55]. Moreover, patients affected by dicarboxylic aminoaciduria also display hints of neurologic damage [12, 56].
The prevalent opinion is that the basic mechanism for termination of glutamatergic transmission resides in eAAT1 and eAAT2 function. Consistent with this hypothesis, these transporters are endowed with higher affinity for substrates than eAAT3/eAAC1 and are local-ized in glial cell processes close to synaptic clefts, while eAAT3/eAAC1 is mostly extra-synaptic [16]. Therefore, eAAT3/eAAC1 may be important in situations where glu-tamate spills out from the synaptic zone or reaches abnor-mally high extracellular concentrations, for example under ischemic conditions (see below). However, eAAT3/eAAC1 may play an important role in glutamatergic termination in areas such as the hippocampus, where most synapses are not closely surrounded by astrocytic processes [57]. How-ever, eAAT3/eAAC1 is far less abundant than eAAT2 also in these regions [18].
Although glutamate uptake represents the best-charac-terized activity of the carrier, eAAT3/eAAC1 also trans-ports cysteine [58, 59] and may provide the majority of the influx of the amino acid in neurons [60]. Through cysteine uptake, eAAT3/eAAC1 contributes to the intracellular synthesis of glutathione [59, 61], one of the most impor-tant intracellular antioxidants. eAAT3/eAAC1-mediated uptake may be the major contributor of cysteine to glu-tathione synthesis, because the knock-out mouse for xCT, the catalytic subunit of the sodium-independent transport system for cystine, is apparently normal and shows no changes in brain glutathione content [62]. Indeed, the mod-ulation of eAAT3/eAAC1 activity correlates with neuronal glutathione content [41]. Further, the impaired Rab11-dependent trafficking of eAAT3/eAAC1 is associated with enhanced sensitivity to oxidative stress in a murine model
of Huntington disease [63]. The mouse knocked-out for eAAT3/eAAC1, which presents a decreased glutathione content in neurons and early onset brain aging, exhibits significant biochemical and functional improvements if treated with the antioxidant N-acetyl-cysteine [64].
The high expression of eAAT3/eAAC1 in GABAergic neurons supports its role in the metabolic fueling of GABA synthesis. This role is also supported by the increase in par-oxysmal activity in animals treated with antisense oligonu-cleotides that hinder transporter expression (see below and [65]).
In addition, eAAT3/eAAC1 also has an impor-tant function in the regulation of synaptic transmission. eAAT3/eAAC1 is involved in long-term potentiation and, hence, in memory and higher cerebral functions [66–69]. Acute regulation of eAAT3/eAAC1 membrane expression and activity by volatile anesthetics [70, 71] and carbamaz-epine [72] may represent examples of pharmacological modulation of these mechanisms.
Regulatory mechanisms at the gene and protein levels
The expression of eAAT3/eAAC1 varies during brain development [73, 74], and the transporter is expressed before eAAT1 and eAAT2 both in vitro [75] and in vivo [73]. early expression suggests a role for eAAT3/eAAC1 in neuroprotection of CNS cells during brain development. Moreover, because GABAergic neurons reach maturation before excitatory glutamatergic cells in the hippocam-pus, the early expression of eAAT3/eAAC1 may play an important role in developing the glutamatergic neuronal network [76]. These data are consistent with recent evi-dence showing that the eAATs are differentially regulated during the differentiation of human astroglial progenitors [77]. In particular, eAAT3/eAAC1 is more expressed than eAAT1 and eAAT2 in A2B5-positive cells and decreases upon differentiation to the astrocytic phenotype. Interest-ingly, early precursors of the oligodendroglial cell lineage are A2B5 positive [78].
Notwithstanding these data, studies focused on the modulation of the SLC1A1 gene under differentiating stim-uli are lacking. In Hs683 human oligodendroglioma cells valproic acid (vPA) and trichostatin A, two inhibitors of histone deacetylases (HDACs), strongly increase SLC1A1 mRNA as well as eAAT3/eAAC1 protein expression and transport activity [79]. In the same model, vPA signifi-cantly also enhances the expression of PDGFRA, an early marker of oligodendrocyte precursors cells (OPCs), while it does not affect the expression of either TUBBIII or GFAP, markers of neuronal and glial cells, respectively. vPA is a well-known differentiating agent, and its effects on the dif-ferentiation of CNS cells have been repeatedly reported. In
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particular, vPA promotes neuronal differentiation of multi-potent adult neural progenitor cells and influences the tim-ing of oligodendrocyte differentiation in the developing rat brain [80, 81].
In another model of immature CNS cells, the rat glioma cell line C6, the differentiating agent all–trans retinoic acid (ATRA) markedly increases eAAT3/eAAC1 expres-sion at both the mRNA and protein levels. ATRA effect is specific for the Slc1a1 gene because it does not affect either GLT-1 (Slc1a2) or GLAST (Slc1a3) protein levels [82]. RAR-specific but not RXR-specific inhibitors prevent eAAT3/eAAC1 induction after ATRA treatment, while the RAR-specific agonist Am80 (tamibarotene, retinobenzoic acid) mimics the ATRA effect. Conversely, eAAT3/eAAC1 induction is prevented by Rarbeta silencing, demonstrating that the ATRA effect on eAAT3/eAAC1 strictly depends upon the RARβ receptor [83]. A consensus site for RARβ has been identified in the rat Slc1a1 promoter but not in the human SLC1A1 promoter region [83]. Along with Slc1a1 induction, the expression of the oligodendrocytic marker Plp1 is also enhanced by ATRA in C6 cells [82, 84], sug-gesting that retinoids may be involved in the activation of the oligodendrocytic differentiation pathway.
Soon after the cloning of the transporter, increased expression of eAAT3/eAAC1 was reported in bovine NBL-1 renal epithelial cells incubated under hypertonic conditions. Incubation of these cells in medium supple-mented with 200 mM sucrose leads to a marked increase in eAAT3/eAAC1 mRNA and protein, as well as to the stim-ulation of anionic amino acid transport [85]. Although it has not yet been demonstrated, it is likely that, as with other amino acid transporters [86], eAAT3/eAAC1 induction also involves the activation of the osmosensitive transcrip-tion factor NFAT5/ToneBP [87]. Incubation in the pres-ence of tunicamycin, an inhibitor of protein glycosylation that triggers endoplasmic reticulum (eR) stress, increases eAAT3/eAAC1 mRNA and protein [88]. In contrast, incubation of NBL-1 cells in the absence of amino acids, a condition that promotes the expression of other sodium-dependent transporters, such as SNAT2 [89], stimulates aspartate transport but not eAAT3/eAAC1 expression [90]. Thus, it seems that in NBL-1 renal epithelial cells Slc1a1 is induced by several (but not all) forms of stress associated with increased protein unfolding and eR stress. Unfortu-nately, these experiments have not been repeated in CNS-derived cell models.
Ma et al. [91] have found that a binding sequence for the regulatory factor X1 (RFX1) exists in the promoter region of SLC1A1, but not in the promoters of SLC1A2 (encod-ing eAAT2) and SLC1A3 (encoding eAAT1). RFX pro-teins are transcription factors that bind to X-boxes of DNA sequences. In the mouse brain, RFX1 is expressed from the embryonic state (e15) to adulthood, and its knock-out
is lethal in the early embryonic state, suggesting an indis-pensable role in brain development [92]. In rats and mice, RFX1 is highly expressed in several regions of the brain, such as the hippocampus, the cerebellum (Purkinje cells), and the olfactory bulb, with localization in the nuclei of neurons and microglial cells but not in the nuclei of astrocytes [93]. In the study by Feng and Zuo [93], 24 h after transfection of C6 glioma cells with human RFX1, eAAT3/eAAC1 expression increased by 60 % and trans-porter activity increased by 40 %. Conversely, knock-down of RFX1 expression by antisense oligonucleotides decreased eAAT3/eAAC1 expression in rat cortical neu-rons in culture. Moreover, transfection with RFX1 stimu-lated the transcriptional activity of the SLC1A1 promoter in both rat glioma C6 cells and human neuroblastoma SHSY-5Y cells. Thus, although SLC1A1 mRNA was not directly measured in that study, these data suggest that the increase in eAAT3/eAAC1 expression was due to the interaction of RFX1 with the promoter [93]. However, the natural activa-tors of RFX1 are not known, and it is difficult to assess its physiological role.
Although most of the previous studies on eAAT3/eAAC1 function have focused on its role in the regulation of glutamate homeostasis, accumulating evi-dence suggests that the carrier plays an essential function in controlling cell redox potential (see above). Astrocytes sup-port neuronal antioxidant capacity by releasing glutathione, the most important anti-oxidant agent in the CNS, which is cleaved to cysteine in brain extracellular space [94]. Free cysteine supply is the limiting step for glutathione synthesis, and neuronal cysteine influx depends upon eAAT3/eAAC1 activity [95]. The activation of the nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant responsive element (ARe) pathway by oxidative stress pro-motes not only glutathione release from astrocytes but also the transcriptional regulation of neuronal eAAT3/eAAC1 both in vitro and in vivo [95]. Moreover, Nrf2-depend-ent overexpression of eAAT3/eAAC1 protein is associ-ated with an increase in neuronal glutathione content, and these effects are abrogated in mice genetically deficient in either Nrf2 or eAAT3/eAAC1. Selective overexpres-sion of Nrf2 in brain neurons by lentiviral gene transfer is sufficient to up-regulate both neuronal eAAT3/eAAC1 protein and glutathione content. These findings support a mechanism whereby Nrf2 activation can coordinate astro-cyte glutathione release with neuronal glutathione syn-thesis through transcriptional up-regulation of neuronal eAAT3/eAAC1 expression. eAAT3/eAAC1-mediated cysteine uptake has also recently been implicated in the increased neuronal glutathione content of mice treated with caffeine or uric acid [96], although the underlying mech-anism has not yet been elucidated. Interestingly, recent contributions demonstrate that fumarates can activate
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Nrf2-dependent responses and thereby ameliorate the dis-ease course of experimental autoimmune encephalomyeli-tis (eAe), a rodent model of multiple sclerosis (MS) [97, 98]. The protective effect of fumarates is associated with improved preservation of myelin and neurons. Unfortu-nately, no investigation was performed on possible changes in eAAT3/eAAC1 induced by fumarates in these models.
In a report by Molteni et al. [99], eAAT3/eAAC1 was up-regulated in parallel with glutamate NMDA receptor subunits after chronic periods of exercise, providing further support for a functional role of the transporter in the modu-lation of glutamatergic transmission.
Overall, these data suggest that the transcriptional regu-lation of eAAT3/eAAC1 expression has peculiar features because most of the reported mechanisms specifically affect the transporter, which, conversely, appears insensitive to well-characterized inducers of eAAT1 and eAAT2 [100, 101]. Similarly, studies on knock-out models for glutamate receptors demonstrate that eAAT1 and eAAT2 expres-sion is lower in mGluR3−/− mice, while eAAT3/eAAC1 is decreased in mGluR2−/− animals [102]. However, examples of a parallel regulation of eAAT1, eAAT2, and eAAT3/eAAC1 proteins have been also reported [103].
Pathological conditions involving changes in EAAT3/EAAC1 expression
Alterations in eAAT3/eAAC1 have been associated with several pathological conditions. In particular, there is a strong association between single-nucleotide polymor-phisms (SNPs) in SLC1A1 and obsessive–compulsive dis-order [104–107], but these polymorphisms seem more correlated with changes in transporter function than with its expression. Dicarboxylic aminoaciduria is a Mendelian disorder (OMIM 222730) where eAAT3/eAAC1 function is defective due to the expression of a mutant, functionally impaired transporter that is also scarcely sorted to the cell membrane [12]. Clear-cut, although contrasting, changes in eAAT3/eAAC1 expression have been described in Alz-heimer’s disease (AD). Results in mouse models of AD are conflicting. Schallier et al. [108] reported an increase in eAAT3/eAAC1 expression (although overwhelmed by a concomitant decrease in GLT-1 and GLAST levels) in AβPP23 mice, while a decrease in the expression of all three glutamate transporters has been reported by Cas-sano et al. [109] in triple transgenic (3×Tg) AD mice. In humans, aberrant intracellular accumulation of a detergent insoluble eAAT3/eAAC1 was described in hippocampal neurons of AD patients, although overall cortical expres-sion of the transporter was comparable to controls, suggest-ing a defect in transporter processing rather than synthesis [110].
Four conditions in which alterations in eAAT3/eAAC1 expression have been identified will be discussed: hypoxia/ischemia, MS, epilepsy, and schizophrenia.
Ischemia/hypoxia
Due to perturbations of ionic gradients, glutamate uptake through eAAT transporters is impaired during ischemia and the rise of extracellular glutamate is believed to con-tribute significantly to tissue damage. The possibility that changes in the expression of eAAT transporters also occur under ischemic conditions has been investigated for many years.
The potential protective role of eAAT3/eAAC1 was proposed following the cloning of the transporter [4] and discussed in one of the first reviews concerning glu-tamate transporters [111]. The first experimental evi-dence of ischemia-related expression changes was pro-vided by Martin et al. [112]; immunoblotting showed that eAAT3/eAAC1 was reduced by 55 % in the putamen of newborn piglets 24 h after an ischemic stress caused by 30 min of hypoxia (arterial O2 saturation, 30 %) followed by 7 min of airway occlusion (O2 saturation, 5 %). This decrease was possibly secondary to neuronal loss, con-sistent with the Slc1a1 mRNA decrease demonstrated in gerbil CA1 hippocampal neurons 24 h after a transient ischemia [113] and the marked decrease (42–68 %) in eAAT3/eAAC1 protein detected in the same model [114]. The latter study compared the decrease of GLT-1, which precedes neuronal loss, with that of eAAT3/eAAC1, which parallels cell death. Their results suggest that decreased eAAT3/eAAC1 may have a role in GABAergic dysfunc-tion. Decrease of eAAT3/eAAC1 expression as a conse-quence of neuronal loss has been more recently shown by Han et al. [115] following microsphere embolism in rats. However, the possibility that adaptive changes in ischemic cells may directly affect transporter expression should not be excluded a priori [38].
A predominant role for GLT-1 in the protection from ischemic damage was proposed by Rao et al. [116], who showed that knock-down of this transporter, but not of eAAT3/eAAC1, exacerbated the neuronal damage induced by transient focal cerebral ischemia in rat brain. Douen et al. [117] also stressed the importance of GLT-1, using as a model rats that were preconditioned 3 days before ischemia with cortical spreading depression (CSD) and showed relative protection from ischemic damage. These authors noted that extracellular brain glutamate after ischemia was indeed lower in the preconditioned ani-mals and that the change was associated with a decrease of eAAT1 and eAAT2, but not eAAT3/eAAC1, expres-sion in the plasma membrane. The authors suggested that a lower expression of eAAT transporters contributes to
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less glutamate efflux through the reverse operation, origi-nally described as an alternative functional mode of eAAT transporters under hypoxic conditions [118]. A decrease in Slc1a1 expression under comparable conditions was described by Montori et al. [119].
The first experimental contribution pointing to an adap-tive and/or protective role of increased eAAT3/eAAC1 expression in ischemia came in 2000 from the Matute laboratory [120]. These researchers compared the expres-sion of the main eAAT transporters in the rat hippocam-pus and cerebral cortex in both early (8 h and 1 day) and late (28 days) phases of recovery from transient forebrain ischemia. They found a moderate increase in eAAT3/eAAC1 protein early after the insult, followed by a late reduction in both CA1 pyramidal neurons and in layer v pyramidal neurons. Moreover, the authors observed a sustained increase in eAAT3/eAAC1 expression in oli-godendroglial progenitor cells of subcortical white matter [120], although, in a later study, the same group reported that eAAT1 and eAAT2 were also up-regulated in this area [121]. Transient changes in eAAT3/eAAC1 expression during ischemia and the recovery phase were also reported by Hwang [122], who described a fast decrease of trans-porter immunoreactivity in spinal cord neurons just after ischemia/reperfusion, followed by an increase of expres-sion (after 3 h of reperfusion) and a further subsequent decrease. eAAT3/eAAC1 expression was also slowly increased, at both the mRNA and protein levels, under “hypoxia-pure” conditions [123] in rat PC12 cells. In this case, the maximum increase was reached after 18 h of incu-bation at 1 % O2 and corresponded to an eightfold increase at protein level and a four-fold increase in transport activ-ity. However, GLT-1 was also up-regulated under the same conditions.
These results are, at least in part, consistent with the evi-dence obtained by Romera et al. [124] in rat cortical cul-tures exposed to sublethal oxygen-glucose deprivation (an in vitro model for ischemic preconditioning), and by Pra-dillo et al. [125] in preconditioned rat brain in vivo. In both studies, eAAT3/eAAC1 was up-regulated by increased TNF-α signaling. However, in the same studies, TNF-α-independent eAAT2 up-regulation was also reported. A generalized up-regulation of eAAT transporters was described in rat brain upon ischemic pre-conditioning by Bigdeli et al. [126, 127].
Results from eAAC1 knock-out mice support the pro-tective role of the transporter under ischemic conditions [128, 129]. In particular, won et al. demonstrated that eAAC1−/− mice subjected to transient cerebral ischemia exhibit much more widespread hippocampal neuronal death than wild-type mice. These authors suggested that impaired cysteine uptake would severely hinder intracellu-lar glutathione synthesis, cell capability to sustain oxidative
stress (expected under ischemic conditions), and main-tenance of zinc homeostasis. N-acetyl-cysteine restored neuronal glutathione, normalized basal zinc levels, and attenuated ischemia-induced zinc translocation, superoxide production, and neuron death.
Given the putative role of eAAT3/eAAC1 as a protec-tive factor under ischemic conditions, attempts to up-regu-late its expression have been performed as a means to limit ischemic damage. Chen et al. [130] used intrathecal granu-locyte colony-stimulating factor (G–CSF) to improve neu-rological defects in the ischemic spinal cord of wistar rats. Using microdialysis, they found that G–CSF significantly hampers the increase in spinal concentrations of excitatory amino acids induced by ischemia or by exogenous intrath-ecal administration of glutamate. These effects are associ-ated with the increased expression of GLAST, GLT-1 and eAAT3/eAAC1.
Multiple sclerosis
excitotoxicity and oxidative stress have been implicated in MS-related damage that involves not only white mat-ter but also gray matter. However, evidence for a role of the eAAT3/eAAC1 transporter in MS is thus far limited, although, as detailed in the Introduction, the transporter is also expressed in cells of oligodendrocytic lineage and, in particular, in oligodendrocyte precursor cells [23, 24, 131].
Ohgoh et al. [132], prompted by the evidence of the involvement of AMPA/kainate receptor in experimental autoimmune encephalomyelitis (eAe, the animal model of MS) [133], examined the expression of GLT-1, GLAST, and eAAT3/eAAC1 in the spinal cord of the eAe Lewis rat. while eAAT3/eAAC1 was dramatically increased at both the protein and mRNA levels, GLT-1 and GLAST were down-regulated. The AMPA/kainate receptor inhibitor NBQX ameliorated eAe symptoms and suppressed changes in transporter expression. No attempt was made to identify the cell type(s) in which changes in carrier expression occur.
This issue was instead addressed by vallejo-Illarramendi et al. [134] who found an increase of SLC1A3 and SLC1A2 mRNA in optic nerves from MS patients, while SLC1A1 mRNA did not change. The expression increase was con-firmed at the protein level for both eAAT1 (in oligodendro-cytes) and eAAT2 (in astrocytes), while eAAT3/eAAC1 expression was not investigated. Increased expression of the rodent counterparts of eAAT1 and eAAT2 (at the mRNA, protein and function levels) were also observed in rat optic nerves incubated with excitotoxic glutamate concentrations.
More recently, Newcombe et al. [135] studied gluta-mate receptors, transporters, and enzymes in human MS lesions. They found that the AMPA GluR-1 receptor subu-nit was overexpressed in oligodendrocytes at the borders of MS active lesions, while reactive astrocytes, macrophages,
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and neuronal structures expressed a different receptor rep-ertoire. Unexpectedly, eAAT3/eAAC1 was expressed in macrophages and reactive astrocytes, leading the authors to propose that this ectopic expression may have a protective, although not completely effective, function.
The positive effects of retinoids on eAAT3/eAAC1 expression (see above) may be important in light of the evidence for a positive effect of retinoids in eAe mice [136, 137]. Klemann et al. attributed the protective effect of the synthetic retinoid Am80 to immunomodulation. On the other hand, Huang et al. [137] attributed the protec-tive effect of the natural compound 9-cis-retinoic acid to a RXR-γ-dependent stimulation of oligodendrocytic differ-entiation. Neither study, however, investigated the expres-sion of eAAT3/eAAC1 or other glutamate transporters.
In conclusion, while some evidence suggests a role for eAAT3/eAAC1 in the pathophysiology of MS, a definite demonstration is still lacking.
epilepsy
The role of eAAT3/eAAC1 dysregulation in epilepsy was proposed in the pivotal paper in which antisense eAAT-knock-out models were investigated for the first time [52]. while the eAAT1 and eAAT2 knock-outs produced severe neurotoxicity and progressive paralysis, the phe-notype of the eAAT3/eAAC1 knock-out was milder but associated with epilepsy. Using the same experimental approach, it was then demonstrated that the repression of eAAT3/eAAC1 expression was associated with decreased tonic inhibition and to 50 % loss of hippocampal GABA levels, attributable to the impairment of GABA synthesis from extracellular glutamate [65].
These early findings spurred many investigations with experimental models of epilepsy. Compared to controls, Slc1a1 mRNA was markedly increased in dentate granule cells from rats with pilocarpine-induced epilepsy and in human dentate granule cells from patients with temporal lobe epilepsy (TLe) [138]. In cortical dysplasia, the trans-porter mRNA was elevated in human dysplastic neurons compared with non-dysplastic neurons. These changes were confirmed at the protein level and were interpreted as an attempt to compensate for the higher-than-normal extra-cellular glutamate levels caused by recurrent seizures [138]. Pilocarpine-induced status epilepticus has been associated with an increase in eAAT3/eAAC1 protein in rat hip-pocampus and with a ~15-fold increase in Slc1a1 mRNA in rat synaptoneurosomes [139], although the authors attrib-uted the change more to the targeting of the messenger to dendrites than to an effect on transcription [140].
Doi et al. [141] examined the induction of epilepsy (kindling) by pentylenetetrazol (PTZ) in rats. They found that the expression of hippocampal glutamate transporters
(not only eAAT3/eAAC1 but also GLAST and GLT-1) increased within 24 h by kindling but returned to basal levels 30 days after the last seizure. These findings led the authors to suggest that glutamate transporters may contribute to the occurrence of seizures. Interestingly, easily-kindled rats had levels of eAAT3/eAAC1 and GAT-1 (the GABA transporter) that were 30 % lower com-pared to epilepsy-resistant animals, again suggesting that eAAT3/eAAC1 level is negatively associated with the con-vulsive threshold [141].
voutsinos-Porche et al. reported the temporal depend-ence of epilepsy-associated changes in transporter expres-sion, demonstrating that eAAT3/eAAC1 increased in sev-eral portions of the hippocampus during the acute period of status epilepticus, but returned to control levels in the CA1, 2, and 3 layers during the latent period. The authors con-cluded that “it is not clear to what extent the overexpression of eAAT3/eAAC1 contributes to epileptogenesis and in which area it may represent a preventive or compensatory or response to injury” [142].
Using the model of kainate-induced convulsions, Sim-antov et al. [143] reached apparently divergent conclusions. Kainate acutely lowered eAAT3/eAAC1 in the stratum lacunosum moleculare, and the decrease in protein level was associated with a decrease in mRNA. Opposite effects were reported for GLT-1, leading the authors to conclude that dif-ferential regulation of neuronal and glial eAAT may contrib-ute to kainate-induced seizures. In another model of experi-mental epilepsy due to prenatal exposure to the teratogen methylazoxymethanol, heterotopic hippocampal neurons exhibited striking reductions in eAAT3/eAAC1, as well as in GluR1 NMDA receptor subunit expression [144]. In contrast, a significant increase in the carrier expression was detected in hippocampal and motor cortex area neurons of rats with chest compression-induced audiogenic epilepsy [145].
Data from human tissues are contradictory. Proper et al. [146] reported an increase in eAAT3/eAAC1 in individual neurons of the hippocampus of TLe patients, although the functional consequences of the reported changes are unclear. Increased eAAT3/eAAC1 expres-sion was also reported by Mathern et al. [147], but only in epileptic patients with hippocampal sclerosis. On the other hand, Rakhade and Loweb [148] measured the differential expression of eAAT1–4 at the mRNA and protein levels in electrically mapped human neocortical tissues and found significant reductions of eAAT3/eAAC1 and eAAT4) at both the mRNA and protein level, not associated with changes in the relative neuronal density. The authors con-cluded that “regional reductions in eAAT expression at human neocortical epileptic foci could produce increased local glutamate levels that in turn may contribute to both hyperexcitability and the spontaneous generation of epilep-tic discharges that characterize human epileptic foci” [148].
M. G. Bianchi et al.
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As far as genetic forms of epilepsy are concerned, Dutuit et al. [149] described a 32 % decrease in eAAT3/eAAC1 expression in the thalamus of young genetic absence epi-lepsy rats from Strasbourg (GAeRS) compared to con-trols. However, the expression of GLT-1 and GLAST also decreased, and the changes were not observed in adult animals.
Interestingly, in human gangliogliomas, which are neuronal-glial tumors strongly associated with epilepsy, SLC1A1 is among the genes that are down-regulated com-pared to normal tissue [150]. This alteration has been attributed to a true transcriptional change rather than to a compensatory response to high extracellular glutamate due to repeated seizures. In contrast, the high expression of SLC1A1 mRNA detected in the lesions of patients affected by tuberous sclerosis, another condition exhibiting high sei-zure incidence, has been considered compensatory [151].
Schizophrenia
The possibility that glutamatergic transmission is altered in schizophrenic patients has been debated for several years. Changes in the expression of eAAT1 and eAAT2, but not eAAT3/eAAC1, have been detected in the thalamus [152] or the superior temporal gyrus and the hippocam-pus [153]. while these studies do not support a role for eAAT3/eAAC1 in schizophrenia, the findings suggest that, in addition to glutamate receptors, alterations in glutamate
transport may also contribute to the glutamatergic dysfunc-tion implicated in schizophrenia.
Other reports present evidence for a role of eAAT3/eAAC1 in schizophrenia. Increased carrier tran-scripts and proteins were reported in schizophrenic subjects (dorsolateral prefrontal and anterior cingulated cortex) by Bauer et al. [154]; these findings were, more recently, con-firmed by Rao et al. [155] in the frontal cortex at both the protein and mRNA levels. Conversely, Lauriat et al. [156] did not observe differences in eAAT3/eAAC1 expression in either the dorsolateral prefrontal or the primary visual cortex. However, the authors also highlighted the pos-sibility that the results were influenced by antipsychotic treatment that is often prolonged for many years in these patients [156].
On the contrary, decreased SLC1A1 transcript expres-sion in the striatum was observed in schizophrenia (and in bipolar disorder) by other authors [157] and confirmed in schizophrenic patients in a later study [158].
Conclusions and perspectives
The regulation of eAAT3/eAAC1 expression at the gene level is far less characterized than mechanisms acting on transporter trafficking. Nevertheless, accumulating data yield an increasingly complex picture (Fig. 1), indicating that at least three levels of control modulate the expression
Fig. 1 Sites of regulation of eAAT3/eAAC1 expression on the plasma membrane. The sketch depicts the three documented sites of regulation of eAAT3/eAAC1 expression: changes in SLC1A1 tran-scription, modulation of carrier interaction with binding proteins and
modification of transporter trafficking to and from the plasma mem-brane. For changes in SLC1A1 transcription, transcription factors, chromatin-modifying enzymes, and extracellular stimuli known to be involved in expression changes are detailed
Changes in the expression of the glutamate transporter
1 3
of the transporter on the plasma membrane: transcriptional changes in SLC1A1 expression (Table 1), changes in protein interaction, and trafficking modulation. Overall, this infor-mation suggests that eAAT3/eAAC1 can be finely regu-lated at the gene level; however, detailed information is not yet available on regulations acting at post-transcriptional or translational sites. with respect to the well-known exam-ples of fast trafficking modulation, transcriptional regula-tions are clearly slower but may be more relevant to explain the somewhat contradictory changes in eAAT3/eAAC1 expression described in situations of pathophysiological interest and associated with chronic alterations in nerv-ous functions. Improved knowledge of these mechanisms
is necessary to clarify the role of eAAT3/eAAC1 in these conditions and, more importantly, to envisage novel thera-peutic approaches.
Acknowledgments Research in the authors’ laboratory has been funded by FISM (Fondazione Italiana Sclerosi Multipla onlus), grant no. 2010/R/9 to OB.
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