Regulation of glutamate transporter GLAST and GLT-1 expression in astrocytes by estrogen

Molecular Brain Research 138 (2005) 1 – 7 www.elsevier.com/locate/molbrainres

Research Report

Regulation of glutamate transporter GLAST and GLT-1 expression in astrocytes by estrogen Justyna Pawlaka, Veronica Britob, Eva Kqppersb, Cordian Beyerc,T ¨ sterbergstr. 3, 72074 Tu¨bingen, Germany Anatomisches Institut, Universita¨t Tu¨bingen, O Abteilung Anatomie und zellula¨re Neurobiologie, Universita¨t Ulm, 89069 Ulm, Germany c Institut fu¨r Neuroanatomie, RWTH Aachen, 52074 Aachen, Germany

a b

Accepted 24 October 2004 Available online 17 May 2005

Abstract Estrogen influences neuronal development and a broad spectrum of neural functions. In addition, several lines of evidence suggest a role as neuroprotective factor for estrogen in the CNS. Neuroprotection can result from direct estrogen–neuron interactions or be mediated indirectly involving the regulation of physiological properties of nonneuronal cells, such as astrocytes and microglia. Increased l-glutamate levels are associated with neurotoxic and neurodegenerative processes in the brain. Thus, the removal of l-glutamate from the extracellular space by astrocytes through the astroglial glutamate transporters GLT-1 and GLAST appears essential for maintaining a homeostatic milieu for neighboring neurons. We have therefore studied the influence of 17h-estradiol on l-glutamate metabolism in cultured astrocytes from the neonate mouse midbrain using quantitative RT-PCR and Western blotting for both transporters as well as functional l-glutamate uptake studies. The administration of estrogen significantly increased the expression of GLT-1 and GLAST on the mRNA and protein level. Likewise, specific l-glutamate uptake by astrocytes was elevated after estrogen exposure and mimicked by dbcAMP stimulation. Induction of transporter expression and l-glutamate uptake were sensitive to ICI 182,780 treatment suggesting estrogen action through nuclear estrogen receptors. These findings indicate that estrogen can prevent l-glutamate-related cell death by decreasing extracellular l-glutamate levels through an increased l-glutamate uptake capacity by astrocytes. D 2004 Elsevier B.V. All rights reserved. Theme: Development and regeneration Topic: Glia and other non-neuronal cells Keywords: Estrogen; Midbrain; Astrocyte; Glutamate; GLT-1; GLAST; mRNA

1. Introduction Estrogen influences a wide variety of developmental and functional aspects in the CNS including synaptic plasticity, neural network formation, and neuronal activity [4,11,26]. In addition, several in vitro approaches have proven the protective efficacy of estrogen by rescuing neurons from oxidative stress [3], chemically induced apoptosis [16], and excitotoxic glutamate overflow [31].

* Corresponding author. Fax: +49 7071 29 4041. E-mail address: [email protected] (C. Beyer). 0169-328X/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.molbrainres.2004.10.043

Recent studies have provided additional information that estrogen may protect against distinct neurodegenerative diseases such as Alzheimer’s disease and global ischemiainduced cell death [22,36]. It has been demonstrated that the regulation of proteins of the Bcl-2 family either by stimulating antiapoptotic factors such Bcl-x(L), Bcl-2 [7] or by inducing proapoptotic genes and factors such as nip2, Bax, Bid [27] may represent an important intracellular target of protective estrogen action in neurons. Furthermore, estrogen interacts directly with neuroprotective intracellular signaling pathways, that is, the PI3-kinase/ Akt signaling [20] and MAP-kinase cascades [19], and acts itself as a neuroprotective antioxidant [3]. A third line of

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estrogen-mediated neuroprotection may be founded on the capability of neurons to secrete several trophic growth factors (BDNF, GDNF, IGF-I) that are known to play an important role for the development and protection of neurons throughout the brain [1,4,7,12,21]. Besides such direct interactions with apoptotic signaling processes in neurons, there is growing evidence that estrogen can interfere with a number of physiological properties of nonneuronal cells such as astrocytes and microglial cells. This might also contribute to estrogendependent neuronal survival and/or rescue from toxic processes [7,23]. In particular, astrocytes may help mediating the neuroprotective effect of estrogen. Astroglial cells produce growth factors, secrete cytokines, and most importantly, are the key cellular compartment for the uptake and metabolism of the excitatory neurotransmitter l-glutamate [1]. The removal of l-glutamate from the extracellular compartment mainly occurs through the activity of two high-affinity, sodium-dependent astroglial glutamate transporters termed GLAST (EAAT1) and GLT-1 (EAAT2) [13]. There is increasing evidence that the perturbation of glutamatergic neurotransmission by a decrease/loss of glutamate transporter activity/expression can be regarded as a major cause for a number of neurological disorders and to be crucial for distinct neurodegenerative diseases [14]. In spite of the significant importance of l-glutamate metabolism for normal neuronal function and for neuroprotection, we evaluated the effect of estrogen on the expression of the two astroglial glutamate transporters GLAST and GLT-1 in astrocyte cell cultures derived from the neonate mouse midbrain using quantitative RT-PCR analysis and Western blotting to monitor the glutamate uptake capacity of astrocytes, we measured the changes in the concentration of exogenously applied l-glutamate by a modified colorimetric method [28].

2. Materials and methods

passage, cells were maintained with serum-free NBM medium. Cells were then treated for 48 h with 17hestradiol (E, 10 7 M, Sigma,) alone or together with the estrogen receptor antagonist ICI 182,780 (ICI, 10 6 M, Tocris). ICI was added simultaneously with estrogen. For positive control experiments, GLT-1 and GLAST expression was stimulated with dibutryl cyclic AMP (dbcAMP, 250 Ag/ml, 48 h, Sigma). 2.2. Immunoblotting Protein isolation was performed in lysis buffer (65.2 mM Tris–HCl, 2% SDS, 10% saccharose, 0.5 mM PMSF, 2 Ag/ Al aprotinin, and 0.5 Ag/Al leupeptin). Samples were sonicated and denaturated at 95 8C for 5 min. Protein content was determined using the BCA protein estimation kit (Perbi Science). 20 Ag protein of each sample were loaded onto SDS-polyacrylamide gels (10%). After electrophoretic separation, proteins were transferred to nitrocellulose membranes, blocked for 1 h with 5% non-fat dried milk in TBS containing 0.5% Tween 20 at room temperature, and then incubated with polyclonal anti-GLT-1 (1:1,000) or polyclonal anti-GLAST (1:1,000, both Chemicon) together with polyclonal anti-actin (1:1,500, Santa Cruz Biotech.) overnight at 4 8C. Filters were washed in TBS-T (3  10 min) at room temperature and further incubated with the following secondary antibodies: peroxidase-conjugated anti-guinea pig IgG (1:2000, Jackson ImmunoResearch) for GLT-1 and GLAST, and donkey anti-goat IgG-HRP (1:3000, Santa Cruz Biotech.) for actin, (1 h, RT). Blots were washed three times in TBS-Tween, and immunoreactive proteins were visualized with an enzyme-linked chemiluminescence kit (ECL, Amersham). Quantitative analysis of Western blots was accomplished densitometrically with a fluorescence scanner (ImageMaster, Pharmacia) using the manufacturer’s software (ImageMaster, USD version 2,0). Absolute optical densities (OD) of GLAST/GLT-1 bands were normalized to the OD of the corresponding h-actin band and expressed as relative abundance in arbitrary units.

2.1. Cell culturing and hormone treatment 2.3. Light cycler PCR Highly enriched astroglial cultures were established from the midbrain of 1- to 3-day-old BALB/c mice killed by decapitation according to a recently established protocol [18,20,21]. These cultures consisted of approximately 95% astrocytes and few oligodendrocytes (precursors) and were virtually free of neurons and microglial cells. In brief, tissues were dissociated using 0.1% trypsin and 0.02% EDTA in Dulbecco’s phosphate-buffered saline (20 min), then transferred to Hank’s-balanced salt solution containing 10% fetal calf serum, filtered through a 50-Am nylon mesh, centrifuged at 400  g for 5 min, resuspended in MEM, and finally seeded at a density of 1–2  10 6 cells/cm2. Upon reaching confluency, cultured cells were trypsinized and replated. After the second

Total RNA was isolated from cultures using peqGOLD RNApure (Peqlab) according to the manufacturer’s instructions. Reverse transcription was carried out with 2 Ag total RNA (1 h). Real time PCR was performed using the Light Cycler system (Roche Molecular Biochemicals, Germany) and applying the ready-to-use bhot startb reaction mix from QuantiTect SYBR Green PCR kit (QIAGEN). The mix contains Taq DNA polymerase and SYBR Green I dye. PCR reactions were carried out in a reaction mixture consisting of 2 Al cDNA, 6 Al RNase-free water, 10 Al bhot startb reaction mix, and 1 Al of each primer (10 pmol). Reactions were conducted in glass capillaries (Roche Molecular Biochem.) in the Light Cycler system,

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subjected to a 15 min initial bhot startQ activation of the Taq DNA polymerase at 95 8C, followed by 45 cycles of denaturation at 94 8C for 15 s, annealing at 58 8C for 20 s, and elongation at 72 8C for 20 s. External standard curves were generated by amplification of 10-fold dilutions of purified PCR products of target genes. Samples were analyzed in the log-linear phase where amplification efficiency is constant. The cycle numbers of the log-linear phase were plotted against the logarithm of the concentration of template DNA. The concentrations of target genes were calculated by comparing the cycle numbers of the log-linear phase of the samples with the cycle numbers of an external standard. Data were expressed as the ratio between the amounts of each transcript of interest versus the amount of HPRT transcript (housekeeping gene). Melting curves were analyzed to determine the specificity of PCR reactions. Forward/reverse primers were: GLT-1 5VGGA AGA TGG GTG AAC AGG C-3V/5V-TTC CCA CAA ATC AAG CAG G-3V; GLAST V-ACG GTC ACT GCT GTC ATT G-3V/5V-TGT GAC GAG ACT GGA GAT GA-3V; HPRT 5V-GCT GGT GAA AAG GAC CTC T-3V/5V CAC AGG ACT AGA ACA CCT GC-3V.

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2.6. Statistics In total, seven independent experiments (culturings for PCR, Western blotting and l-glutamate uptake) were carried out consisting each of 3–5 culture dishes per treatment and type of experimental analysis. All values in the text and graphs represent the means F SEM. Quantitative data are expressed in arbitrary units or as percent of controls. Differences between experimental groups were analyzed by one-way analysis of variance (ANOVA) followed by the post hoc Newman–Keuls multiple range test with P b 0.05 considered as criterion for statistical significance.

3. Results In order to analyze the effect of estrogen on the expression and activity of the two glutamate transporters, GLT-1 and GLAST, we used highly pure GFAP-positive astrocyte cultures (Fig. 1A). Astrocytes generally exhibited a flattened morphological shape. Cultured midbrain astrocytes express both types of nuclear estrogen receptors (ER-a and ER-h) as shown by RT-PCR (Fig. 1B). Levels of

2.4. Glutamate uptake Extracellular glutamate levels were measured by a fluorimetric method using the Amplex Red Glutamic Acid assay kit (Molecular Probes) with minor modifications as described [28]. 50 Al of supernatants were transferred into 96-well microplates, mixed with 50 Al substrate mixture containing 100 mM Amplex Red, 0.25 U/ml horseradish peroxidase, 0.08 U/ml l-glutamate oxidase, 0.5 U/ml lglutamate pyruvate transaminase, 200 Al l-alanine, 1 reaction buffer, and incubated at 37 8C for 30 min. Detection of fluorescence reaction was determined using an automated microplate reader at a wavelength of 570 nm (vs. reference wavelength of 655 nm). In a previous study, starting concentrations of 200 AM l-glutamate yielded optimum uptake kinetics [28]. This was also established for our culture system. Glutamate concentrations were then estimated from the standard curve using known l-glutamate amounts. Specific l-glutamate uptake was yielded by performing experiments in a sodium-supplemented Tris buffer (150 mM NaCl). 2.5. Immunocytochemistry For immunocytochemistry, cells were fixed in 4% paraformaldehyde (30 min) and permeabilized with 0,05% saponine (30 min). Then, cells were blocked with 1% bovine serum albumin (1 h) and incubated with monoclonal mouse anti-GFAP (BD Biosciences, Germany, 1:250) overnight at 4 8C followed the next day by exposing to biotinylated anti-mouse IgGs (Jackson ImmunoResearch, Germany, 1:500). Immunoreactivity was visualized using the ABC standard method and DAB.

Fig. 1. Morphology of cultured GFAP-positive astrocytes derived from the neonatal midbrain (A) and expression of estrogen receptor ER-a/ER-h mRNAs (B) in astrocytes analyzed by RT-PCR. Real-time quantification of GLT-1 (black bars) and GLAST (hatched bars) mRNAs in cultured midbrain astrocyte cultures. Obtained numbers of mRNA copies for glutamate transporters were normalized against the housekeeping gene HPRT in each sample. Control (C) cultures were then set to 1, and data from estrogen (E) and E plus ICI 182,780 (ICI)-treated cultures were expressed as relative amounts compared to controls (data are given in arbitrary units). Positive stimulation was performed with dbcAMP. Treatment period was 72 h. aP b 0.005 vs. control (GLT-1), bP b 0.001 vs. control (GLAST), cP b 0.01 vs. Etreated (applies for GLT-1 and GLAST).

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glutamate transporter mRNAs were examined by real-time PCR quantification. The amounts of mRNAs (cDNAs) were normalized against the housekeeping gene HPRT. The ratios obtained in controls were then set to 1. Exposure to estrogen (10 8 M) resulted in a 74 F 10 (GLT-1, aP b 0.005 vs. control) and 84 F 11 (GLAST, bP b 0.001 vs. control) percent increase compared to untreated controls (Fig. 1C). The simultaneous application of the highly specific nuclear estrogen receptor antagonist ICI 182,780 completely prevented this induction. ICI 182,780 alone had no influence on mRNA levels (not shown). No differences in the expression levels were seen between controls and estrogen plus ICI 182,780-treated cells. In comparison to estrogen application, a significant (cP b 0.01 vs. estrogen) reduction in mRNA levels was measured in cells exposed to estrogen plus ICI 182,780. Positive control experiments using cAMP stimulation yielded highest mRNA levels for both glutamate transporters. Effects of estrogen on GLT-1 and GLAST mRNA levels were measured using different steroid concentrations ranging from 10 6 M to 10 10 M. No clear-cut dose-dependency was observed (not shown). Significant changes in expression were only seen at estrogen doses ranging from 10 7–10 8 M. Lower estrogen concentrations were ineffective. To study the effect of estrogen on the expression of GLT1 and GLAST, we examined transporter protein levels similar to the PCR findings, treatment of astroglial cultures

Fig. 2. Effect of estrogen (E), E plus ICI 182,780 (E+ICI), and dbcAMP treatment (48 h) on GLT-1 (black bars) and GLAST (gray bars) protein levels in midbrain neonatal astroglial cultures. (A) Shows representative immunoblots. (B) Quantitative evaluation of Western blots. Data are given in arbitrary units and normalized to the corresponding h-actin levels. Estrogen significantly (aP b 0.05 vs. control (C) applies for both transporters) increased the amounts of both glutamate transporters similar to dbcAMP stimulation (bP b 0.01 vs. C, applies for both transporters), whereas the estrogen receptor antagonist ICI blocked the estrogen effect ( P b 0.09 vs. C). Protein loading was controlled by additionally screening for h-actin.

Fig. 3. l-glutamate uptake by cultured midbrain astrocytes analyzed indirectly by quantifying the clearance of extracellular added l-glutamate from the culture medium. (A) Shows the time-course of specific (sodiumdependent) l-glutamate uptake at different time intervals (from 4 to 120 min) after the application of 200 AM l-glutamate. Note the linearity of lglutamate uptake between 4 and 60 min yielding a regression coefficient of r 2 = 0.97746. (B) Estrogen (E) and dbcAMP administration (72 h) resulted in a reduction of extracellular l-glutamate by 56% (aP b 0.05 vs. starting levels (C)) and 67% (cP b 0.01 vs. C), respectively, whereas ICI 182,780 and E-treatment was not significantly different from C (bP b 0.12).

with estrogen significantly increased the protein levels of both GLT-1 (aP b 0.05) and GLAST (aP b 0.05) as determined by Western blot analysis (Fig. 2). Immunoblots yielded two specifically stained bands with apparent molecular weights of 66 kDa for GLAST and 71 kDa for GLT-1. Again, the application of ICI 182,780 completely antagonized the effect of estrogen ( P b 0.09 vs. estrogentreatment), and dbcAMP administration resulted in an equal increase in both glutamate transporter proteins (bP b 0.01) as observed for estrogen. l-glutamate uptake activity of cultures astrocytes was evaluated by the clearance of exogenously applied lglutamate. First, we measured the specific l-glutamate uptake at different time intervals as an index of the sodium presence at each stage indicating a specific uptake mechanism and a linear regression (r 2 = 0.97746) of glutamate clearance from the culture medium between 4 and 60 min of incubation (Fig. 3A). Astroglial cultures were then treated with estrogen or dbcAMP for 48 h. After that, the assay was started by the application of 200 AM l-glutamate and run for 30 min. In estrogen-treated cultures, extracellular l-glutamate levels were reduced by 56 F 4% (aP b 0.05 vs. control levels) after 30 min of incubation. In dbcAMP-exposed cultures, extracellular lglutamate concentrations were lowered by 67 F 3% (cP b 0.01 vs. control levels).

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4. Discussion We investigated the effects of estrogen on the expression of the astroglial glutamate transporters GLAST and GLT-1 as well as l-glutamate uptake activity in cultured midbrain astrocytes prepared from newborn mice. Our results demonstrate that estrogen increased mRNA and protein levels of both glutamate transporters. This effect was mimicked by dbcAMP stimulation and inhibited by the estrogen receptor antagonist ICI 182,780 suggesting nuclear estrogen receptors to be involved. l-glutamate uptake activity in astrocytes was comparably promoted in estrogenand dbcAMP-treated cultures. Clinical and basic science studies have highlighted the potency of estrogen to decrease the risk and delay the onset as well as the progression of distinct neurodegenerative disorders, and may in addition enhance the recovery from traumatic neurological injuries, that is, stroke (reviewed in Ref. [12]). A number of in vitro studies have deciphered several routes how estrogen-mediated neuroprotection may occur at the cellular level. This includes the supply of trophic factors such as growth factors [21], the stimulation of anti-apoptotic signaling cascades [20], the induction/ inhibition of pro/antiapoptotic proteins, respectively, the regulation of neuronal plasticity and synaptic activity, the control of mitochondrial function, and the antioxidative properties of estrogen itself (reviewed in Ref. [23]). It is worth mentioning that estrogen can exert neuroprotective effects through different intracellular signal transduction mechanisms including classical nuclear receptor activation as well as nonclassically via membrane/cytoplasmatic estrogen receptors coupled to survival-promoting signal transduction cascades (PI-3 kinase, MAP-kinase, NFkappaB) [3,6,12,19,20,21,35]. Another important molecule implicated in neurodegeneration and neuronal cell death is the excitatory neurotransmitter l-glutamate that is predominantly taken up, metabolized, and recycled by astrocytes through the glutamate transporters GLAST/GLT-1 and glutamine synthetase catalytic activity. Reduced glutamate transporter expression is described for neurodegenerative diseases such as Alzheimer’s disease and ALS, after hypoxic or ischemic conditions, as well as traumatic brain injuries and is thought to contribute to neuronal cell death observed under various pathological conditions (reviewed in Ref. [30]). Several of these neuropathological processes and neurotoxic processes are known to be estrogen-sensitive in such a way that estradiol may counteract and/or reduce the degree of neuronal damage and loss [12,15,25,33,34]. Despite the broad knowledge of the excitotoxic potency of l-glutamate and the neuroprotective nature of estrogen, only preliminary information is at present available about interactions between estrogen and glutamate activity/metabolism in the CNS. Cyr et al. [8] have recently described that estrogen can interfere with NMDA receptor function by regulating NMDA receptor subunit expression (NR1 and NR2B) in

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the rat brain, thus allowing estrogen to modulate glutamate activity at the receptor level. Two other recent research reports have analyzed directly the effect of estrogen on glutamate transporter activity and achieved conflictive findings. Cultured cortical rat astrocytes responded to the application of 17h-estradiol and estrogen-related compounds (xenoestrogens) with an inhibition of l-glutamate uptake [28]. This estrogen effect was transmitted nonclassically through the binding to and activation of membraneassociated estrogen receptors. The authors assumed that the stimulation of PI3-kinase coupled to NO production could be involved in this inhibitory regulation of glutamate transporter activity by estrogen. The rapid activation of Akt/PI-3 kinase signaling in the CNS by estrogen in a calcium-dependent way has already reported by our group [20]. On the other hand, another study presented convincing data that estrogen increased rather than decreased glutamate transporter activity using human cultured astrocytes derived from cortex of Alzheimer’s patients [24]. These findings are in line with the data presented in this study, which describes an estrogen-mediated induction of glutamate transporters mediated through nuclear estrogen receptors. Others and we have demonstrated the presence of nuclear estrogen receptors in astrocytes in vitro and in vivo [1,10,12]. Interestingly, reactive astrocytes and microglia appear to express estrogen receptors at high levels several days after brain injury, thus enabling them to respond maximally to estrogen [10]. There are several potential explanations for the ostensible discrepancy in the regulation of glutamate transporters by estrogen, that is, increase in our study and in Ref. [24] and decrease in Ref. [28]. One may simply be the difference in applied steroid concentration. Only high estrogen amounts (N10 AM) but not lower levels in the nM range revealed a significant estrogen inhibitory effect on l-glutamate uptake and may have direct modulatory consequences for transporter function by activating posttranslational regulatory mechanisms. In contrast, estrogen levels in the physiological range of 10 8 M and lower which were administered in our experiments and in the second study by Liang et al. [24] may stimulate exclusively nuclear estrogen receptors and thereby affect GLT-1 and GLAST function at the transcriptional level. Another possibility may arise from the cellular composition of astroglial cultures. The culturing method applied by Sato et al. [28] generates astrocyte cultures consisting mainly of type 1 cells, whereas our experimental protocol predominantly yields mixed type 1 and 2 astrocyte cultures. Thus, different types of astrocytes may respond to estrogen diversely or interactions between varying glial cells may determine the estrogen response. In the midbrain, it is apparent that estrogen plays an important role for dopamine neurons by regulating developmental processes, such as neurite growth, synaptic plasticity, and dopamine synthesis by controlling TH expression, and later in adulthood the activity of the nigrostriatal dopamine system at the striatal level [2,4,5,18]. Additionally, estradiol may serve as a neuro-

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protective factor for mesencephalic dopamine cells in vitro as well as in vivo. In embryonic midbrain cultures, estrogen protects against pharmacologically stimulated apoptosis most likely via estrogen receptor-h activation [29]. In estrogen receptor-h knockout mice, morphological abnormalities were reported in the mesencephalon [34]. Taken into account that estrogen has been shown to attenuate the degree of striatal dopamine depletion to known dopaminergic toxins, that is, MPTP, 6-OHDA, and methamphetamine, and the reported higher incidence rate for Parkinson’s disease in males [9], it seems conceivable that estrogen provides neuroprotection for nigral dopamine neurons to a certain extent. Our findings now may provide a first clue to understand at least one potential physiological mechanism that may be implicated in the estrogen-mediated rescue of midbrain dopaminergic neurons from cell death. Despite the importance of glutamate transporter function and regulation for mediating neuroprotection in the CNS, another intriguing property of GLT1 and GLAST activity is coming to the fore. The metabolic crosstalk between developing cortical neurons and astrocytes reflected in the glucose utilization and lactate release seems to be in part influenced by glutamate transporter activity as shown in distinct knockout models for both glial transporters [32]. In addition, the functional status of GLT1 and GLAST determines the activity of hippocampal interneurons by reinforcing mGluR1 responsiveness and, in turn, leading to an increased firing rate [17]. Since the activity of interneurons is also implicated in the regulation of synaptic plasticity, estrogen-dependent control of glutamate transporter function thus might contribute to brain plasticity. In summary, we have demonstrated that estrogen promotes the expression and function of the two glutamate transporters GLT-1 and GLAST in neonatal midbrain astrocytes through nuclear receptor activation. The regulation of extracellular glutamate levels represents another yet underestimated mechanism accounting for better understanding the complexity of estrogen-mediated neuroprotection in the CNS.

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