Gap junctional control of glial glutamate transporter expression

www.elsevier.com/locate/ymcne Mol. Cell. Neurosci. 35 (2007) 130 – 137

Gap junctional control of glial glutamate transporter expression Maciej Figiel, Claudia Allritz, Claudia Lehmann, and Jürgen Engele⁎ Institute of Anatomy, University of Leipzig, Medical Faculty, Liebigstr. 13, 04103 Leipzig, Germany Received 5 October 2006; revised 6 February 2007; accepted 8 February 2007 Available online 15 February 2007 The uptake of glutamate into astroglia is the predominant mechanism to terminate glutamatergic neurotransmission and to prevent neurotoxic extracellular glutamate concentrations. Here, we show that uncoupling cultured cortical astrocytes with the gap junction blocker, propofol, or the Cx43 mimetic peptide, Gap27, inhibits the expression of GLT-1, the major glutamate transporter subtype in the cortex. The dependence of GLT-1 expression on gap junctions was further confirmed by the use of astrocytes in which either the expression of Cx43, the major astrocytic gap junction protein, was inhibited by RNA interference or which were derived from animals carrying an astrocyte-specific deletion of the Cx43 gene. In both cases, reduced astrocytic coupling was associated with a pronounced decline in GLT-1 expression. Finally, a luciferase reporter gene assay demonstrated that blockade of gap junctions/connexins suppressed transcriptional activity of GLT-1 promoter. These observations unravel a previously unrecognized role of gap junctions in the control of glial glutamate transport. © 2007 Elsevier Inc. All rights reserved.

Introduction Glutamate is the main excitatory neurotransmitter in the vertebrate central nervous system. At high extracellular concentrations glutamate represents a potent neurotoxin which leads to neuronal over-stimulation and subsequent excitotoxic cell death. Astroglial cells are long known to play a complex role in the control of extracellular glutamate homeostasis (Nedergaard et al., 2002, for review). On one hand, astrocytes modulate the duration of glutamatergic neurotransmission and synaptic strength by clearing extracellular glutamate through the high affinity sodiumdependent glutamate transporters, excitatory amino acid transporter-2/glutamate transporter-1 (EAAT-2/GLT-1) and EAAT-1/glutamate aspartate transporter (GLAST) (Sims and Robinson, 1999, for review). On the other hand, astrocytes signal to neurons via depolarization- or receptor-induced release of glutamate (Nedergaard et al., 2002; Newman, 2003; Takano et al., 2005). Disturbed

⁎ Corresponding author. Fax: +1 49 341 97 220 09. E-mail address: [email protected] (J. Engele). Available online on ScienceDirect (www.sciencedirect.com). 1044-7431/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2007.02.009

expression of GLT-1 and/or GLAST occurs in various chronic brain diseases such as amyotrophic lateral sclerosis, Alzheimer's disease, and Huntington's disease as well as after several forms of acute brain injuries and seems to contribute in part to the neuronal cell death associated with these pathological conditions (Sims and Robinson, 1999). Despite the clinical importance, the extra- and intracellular mechanisms governing glial glutamate transport have not yet been fully unraveled. Previous attempts to characterize this regulatory network so far resulted in the identification of pituitary adenylate cyclase-activating polypeptide (PACAP), epidermal growth factor (EGF), transforming growth factor α (TGFα) platelet-derived growth factor (PDFG), and fibroblast growth factor-2 (FGF-2) as potent stimulators of glial glutamate transporter expression (Figiel and Engele, 2000; Figiel et al., 2003; Zelenaia et al., 2000) as well as of endothelins (ETs) as negative regulators of GLT-1 and GLAST expression (Rozyczka et al., 2004). Various components of the astrocytic glutamate system, including GLAST, GLT-1, and AMPA-type glutamate receptors seem to be expressed by different subpopulations of astrocytes (Wallraff et al., 2004; Matthias et al., 2003; Perego et al., 2000). This (partially) segregated expression is especially intriguing given the fact that astrocytes are extensively coupled by gap junctions, and hence implies the functional compartmentalization of the astrocytic syncytium. An obvious mechanism to coordinate these different functional compartments would be gap junctions. Gap junctions form closable pores that connect the cytoplasms of neighbouring cells and allow the diffusion of small molecules up to a molecular weight of approx. 1000 Da. The genome of man and rodents contains at least 20 connexin genes which are developmentally regulated and expressed in a tissue-specific manner (Sohl and Willecke, 2004; for review). Connexin43 (Cx43) is the major conjunctional protein in astrocytes (Giaume and McCarthy, 1996). In our present studies, we sought to determine whether astroglial gap junction coupling affects glutamate transporter expression. We have predominantly focused on the GLT-1 transporter subtype which is currently regarded as the major glutamate transporter performing over 90% of total glutamate uptake in the brain (Sims and Robinson, 1999). We show that uncoupling of cultured cortical astrocytes is associated with a substantial loss in GLT-1 expression and reduced glutamate uptake.

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Results Since in addition to astrocytes, GLT-1 is also expressed by neurons and microglial cells (see for example, Jacobsson et al., 2006; Suchak et al., 2003), all experiments were performed with cultures of purified astrocytes. In accordance with our previous findings (Figiel and Engele, 2000; Figiel et al., 2003; Rozyczka et al., 2004), GLT-1 expression was readily detectable in untreated control cultures of purified cortical astrocytes by Western blotting (Fig. 1). As previously reported (Swanson et al., 1997), GLT-1 expression level was, however, distinctly lower in vitro as compared to in vivo. If not stated otherwise, a 72-h treatment paradigm was chosen which was previously shown to allow for maximal stimulatory as well as inhibitory effects on glial glutamate transporter expression (Figiel and Engele, 2000; Figiel et al., 2003; Rozyczka et al., 2004). In addition, all compounds were only applied at concentrations initially found to be non-toxic to cultured astrocytes. Maintaining cortical astrocytes for 72 h with the lipophilic gap junction blocker, propofol (300 μM), as well as with the Cx43 mimetic peptide, Gap27 (300 μM), resulted in a loss of basal GLT-1 expression. In average, this loss amounted to 57%, and 43% with propofol, and Gap27, respectively (Fig. 1). The use of propofol and Gap27 at 100 μM and 50 μM, respectively, produced a 20% loss of GLT-1 protein levels which, however, was statistically not significant. To determine how the gap junction blockers interfere with previously identified positive regulators of glial glutamate

Fig. 1. Effects of gap junction blockers on astrocytic glutamate transporter expression. Dissociated cell cultures were established from the cortical hemispheres of postnatal day 1–2 rats and maintained with 10% horse serum to stimulate glial proliferation. Upon reaching confluency, cultured cells were trypsinized and replated at lower density. After the third replate, cultures were further maintained with serum-free N2-medium additionally supplemented with the gap junctions blockers propofol (300 μM) or Gap27 (300 μM) alone or in combination with either TGFα (100 ng/ml) or PACAP (100 nM). After 72 h, cells were lysed, subjected to GLT-1 Western blotting as described under Experimental methods, and integrated optical densities of immunoreactive bands were measured. To control for protein loading, blots were additionally stained with GAPDH antibodies. Numbers represent average changes in GLT-1 levels ± S.D., corrected for protein loading, as determined in 3 independent experiments. GLT-1 levels present in untreated controls were set to 1. Both PACAP and TGFα robustly promoted GLT-1 expression (ap < 0.05, treatment vs. control; Tukey test). Gap junction blockers inhibited basal GLT-1 expression and also abolished the stimulatory influences of positive regulators of glial glutamate transporter expression (bp < 0.05; presence vs. absence of gap junction blockers; Tukey test).

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transporter expression, cortical astrocytes were maintained for 72 h with gap junction blockers in combination with either PACAP (100 nM) or TGFα (100 ng/ml). Corroborating previous observations (Figiel and Engele, 2000; Figiel et al., 2003; Zelenaia et al., 2000), both PACAP and TGFα alone stimulated GLT-1 expression by 2- to 3.7-fold (Fig. 1). These stimulatory influences were completely abolished in the additional presence of either propofol or Gap27 (Fig. 1). Together these findings identify gap junction blockers as potent inhibitors of GLT-1 expression. Lipophilic gap junction blockers seem to mechanically squeeze gap junction channels upon integrating into the cell membrane, an effect eventually also affecting the function of other cell membranebound proteins (Evans and Boitano, 2001). The Cx43 mimetic peptide, Gap27, is homologous to the second extracellular loop of the Cx43 protein and prevents docking of Cx43 hemi-channels, and thus the formation of gap junction pores (Evans and Boitano, 2001). In addition, Gap27 targets to Cx37 (Martin et al., 2005). Moreover, evidence exists that Gap27 directly interferes with the channel closing mechanism (Evans and Boitano, 2001). Due to the apparent lack of selectivity of propofol and Gap27, it seemed necessary to analyze whether the effects seen with these compounds on GLT-1 expression result from their direct interference with gap junctions/ connexins. To address this issue, we made use of the rat hepatoma cell line, H4-II-E-C3 (H4), which shows robust expression of GLT-1 (Pollard and McGivan, 2000; Fig. 2), but lacks detectable levels of both Cx43 and Cx32 (data not shown); Cx32 is the prevailing connexin form in hepatocytes (Iwai et al., 2000). Exposure of H4 cells to propofol or Gap27 for 72 h did not affect GLT-1 protein levels (Fig. 2). In apparent contrast, the hydrophilic gap junction blocker, carbenoxolone, completely abolished GLT-1 expression in H4 cells within 72 h (Fig. 2). These findings imply that propofol and Gap27 affect glutamate transporter expression by a connexindependent mechanism. To further confirm putative regulatory influences of gap junctions on glial glutamate transporter expression, we analyzed GLT-1 expression in cortical astrocytes in which Cx43 expression was inhibited by RNA-interference. Transfection of cultured astrocytes with selective Cx43 siRNA, but not with non-homologous siRNA, resulted in an average 82% decline in Cx43 protein levels after 96 h (Fig. 3A). This decrease in Cx43 was associated with a pronounced loss in astrocytic gap junction coupling as revealed by scrape loading-dye transfer technique (Figs. 3B, C). Subsequent analysis of glial glutamate transporter expression in Cx43-depleted astrocytes demonstrated an almost 40% reduction in basal GLT-1 expression as compared to astrocytes transfected with non-homologous (random) siRNA (Fig. 4A). In an additional set of experiments, we further determined how PACAP (100 nM) and TGFα (100 ng/ml) added for the final 72 h would affect glial glutamate transporter expression in Cx43-depleted astrocytes. Both treatments failed to stimulate GLT-1 expression in Cx43 siRNA-transfected astrocytes (Fig. 4A). Known side effects of RNA interference consist in the activation of cellular immune responses (Sledz et al., 2003). To exclude that these non-specific responses interfere with glial glutamate transporter expression, GLT-1 expression levels were further analyzed in cortical astrocytes derived from transgenic mice in which Cre recombinase under control of the human GFAP promoter deletes the Cx43 coding region, flanked by recombinase recognition sites (loxP sites) (Theis et al., 2003, 2004). In contrast to Cx43 null mice, which die at birth due to cardiac malformation (Reaume et al., 1995), mice carrying a selective ablation of Cx43 in GFAP expressing astrocytes (Cx43fl/fl:hGFAPcre animals) are

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Fig. 2. Effects of gap junction blockers on GLT-1 expression in H4-II-E-C3 cells. The connexin-deficient hepatoma cell line, H4-II-E-C3 (H4), was maintained for 72 h in serum-free N2-medium supplemented with either propofol (Prop; 300 μM), carbenoxolone (Cbx; 40 μM), or Gap27 (300 μM). GLT-1 expression was determined by Western blotting as described in Fig. 1. Numbers represent average changes in GLT-1 levels ± S.D., corrected for protein loading, as determined in 3 independent experiments. Both propofol and Gap27 remained without effects on GLT-1 expression levels, indicating that the inhibitory effects seen with both compounds on cortical astrocytes involved gap junctions. In contrast, carbenoxolone abolished GLT-1 in H4 cells pointing to non-specific, gap junction-independent inhibitory influences of this compound on GLT-1 expression.

viable and show increased exploratory behavior and impaired motor capacities (Frisch et al., 2003). Western blotting revealed that in comparison to astrocytes derived from Cx43fl/fl animals, GLT-1 levels in astrocytes cultured from Cx43fl/fl:hGFAPcre animals were reduced by 65% (Fig. 4B). In all cultures established from Cx43fl/fl:hGFAPcre animals, the successful (> 95%; see Theis et al., 2004) inactivation of astrocytic Cx43 expression was confirmed by Western blotting (data not shown). Collectively, these data establish that GLT-1 expression in cortical astrocytes correlates with Cx43 protein levels/gap junction coupling. To assess the existence of selective regulatory influences of gap junctions/connexins on GLT-1 expression, we transfected the firefly luciferase gene under control of the human GLT-1 promoter into primary cortical astrocytes and examined how cellular uncoupling affects reporter gene expression. Corroborating a previous report (Su et al., 2003), luciferase activity increased 3-fold upon treating transfected astrocytes with EGF (100 ng/ml) for 72 h (Fig. 5). Uncoupling cultured astrocytes with propofol (300 μM; 72 h) resulted in a statistically significant 35% decline in basal reporter

Fig. 4. Depletion of Cx43 expression in cortical astrocytes is associated with a loss in GLT-1 expression. (A) Western blot analysis of GLT-1 expression in cortical astrocytes in which Cx43 expression was inhibited by RNA interference after 96 h. Numbers represent average changes in GLT-1 levels ± S.D., corrected for protein loading, as determined in 3–5 independent experiments. Note the reduced GLT-1 levels in cultures transfected with specific Cx43 siRNA as compared to transfection with non-homologous siRNA (nh siRNA). Further note that additionally maintaining cells for the final 72 h with PACAP (100 nM or TGFα (100 ng/ml) did not affect GLT-1 expression. ⁎p < 0.05; ⁎⁎p < 0.01; treatment vs. control; Tukey test. (B) Determination of GLT-1 levels by Western blotting in astrocytes derived from mice carrying an astrocytespecific deletion of Cx43 expression (Cx43fl/fl:hGFAP-cre). Cx43fl/fl: hGFAP-cre animals exhibit a clear reduction of astrocytic GLT-1 expression when compared to Cx43fl/fl animals. ⁎⁎p < 0.01; Tukey test, n = 3.

gene expression and in addition attenuated the EGF-induced increase in luciferase activity by approximately 60% (Fig. 5). Complementary real-time RT-PCR showed an average 16 ± 3.5% (p < 0.05, Student's t-test, n = 3) loss of GLT-1 mRNA in propofol-treated cultures (300 μM; 72 h) as compared to untreated controls (data not shown). In cultures treated with a combination of propofol (300 μM; 72 h) and EGF (100 ng/ml; 72 h) this loss in GLT-1 mRNA levels amounted to 68 ± 5% (p < 0.001; n = 3) as compared to EGF alone. Collectively, these findings establish that blockade of gap junctions/ connexins suppresses transcriptional activity of the GLT-1 promoter. The observed inhibitory action of gap junctions/connexins on GLT-1 expression prompted us to determine whether a similar mechanism applies for GLAST, an additional glutamate transporter subtype expressed by astrocytes (Sims and Robinson, 1999). Western blot analysis unraveled that in contrast to GLT-1, uncoup-

Fig. 3. Uncoupling of cultured cortical astrocytes by Cx43 RNA interference. (A) Western blot analysis of Cx43 in cortical astrocytes 96 h after transfection with Cx43 siRNA and non-homologous (nh) siRNA. Numbers represent average Cx43 levels ± S.D. corrected for protein loading as determined by measuring integrated optical densities of immunoreactive protein bands in blots from 3 independent experiments. Note the massive loss of Cx43 in cultures transfected with Cx43 siRNA as compared to transfection with nh siRNA. ⁎p < 0.01; Tukey test. (B and C) Assessment of intercellular gap junction coupling by the scrape loading-dye transfer technique. Cultures were transfected with (B) nh siRNA or (C) Cx43 siRNA. After 96 h, the astrocytic cell layer was cut with a razor blade (delineated by asterisks) in the presence of gap junction-permeable Lucifer yellow. Spreading of the dye was taken as an indicator for intercellular gap junction coupling. Note the marked decrease of intercellular coupling in Cx43 siRNA-transfected cultures. Bar, 20 μm.

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carboxylate transporter 1 (MCT-1), other major cell membraneassociated transporters expressed by astrocytes (Vannucci et al., 1997; Hertz and Dienel, 2005). Western blot analysis showed that uncoupling of cultured cortical astrocytes with propofol (300 μM; 72 h) results in a small, 1.7- to 1.8-fold increase in Glut-1 and MCT-1 protein levels (Figs. 6B, C). A similar increase in Glut-1 expression upon astrocytic uncoupling has been recently reported by Sanchez-Alavarez et al. (2004). Together these observations suggest that blockade of astrocytic gap junctions/connexins selectively inhibits GLT-1 expression. Discussion Fig. 5. Uncoupling inhibits the activity of the human GLT-1 promoter. Cultured cortical astrocytes were transfected with the luciferase reporter gene under control of the putative full-length human GLT-1 promoter and maintained with either propofol (300 μM), EGF (100 ng/ml), or a combination of both factors as indicated. After 72 h, cells were lysed and luciferase activity was determined. Mean luciferase activity (±S.D.) from 3 independent experiments is shown. Uncoupling cells with propofol inhibited basal reporter gene activity and in addition partially prevented the EGFinduced increase in luciferase activity. ⁎p < 0.02; ⁎⁎p < 0.001; Tukey test.

ling of cultured astrocytes with propofol increases GLAST protein levels (Fig. 6A). Real-time RT-PCR further demonstrated that propofol also leads to a 4.5 ± 1.9-fold (n = 3; p < 0.05, t-test) increase in GLAST mRNA levels as compared to untreated controls. To subsequently assess the overall consequences of astrocytic uncoupling on glial glutamate uptake, we performed uptake experiments using tritiated glutamate. In astrocytes maintained for 72 h with propofol (300 μM), specific glutamate uptake was reduced by 58% as compared to untreated controls (control, specific uptake, 73814 + 9740 dpm/min/mg protein; propofol treatment, specific uptake, 31073 + 2905 dpm/min/mg protein; n = 3, p < 0.05; t-test). Additional uptake experiments performed in the presence of the selective GLT-1 inhibitor, dihydrokainate (DHK) showed that in control cultures 32 + 3.4% of total uptake is mediated by GLT-1, whereas in propofol-treated cultures GLT-mediated uptake slightly decreased to 22 + 3.7%. These findings establish that uncoupling impairs glutamate uptake into astroglia. In an additional set of experiments, we determined whether uncoupling of astrocytes would solely affect the expression of proteins involved in glial glutamate transport. To this end, we focused on the glucose transporter 1 (Glut-1) and the mono-

Although astrocytes are most extensively coupled by gap junctions, and hence form a syncytial-like structure, the exact role of gap junction coupling in astroglial function remains widely elusive. We have now addressed this issue by determining how astrocytic gap junction coupling modulates glutamate transport. Clearance of extracellular glutamate by astrocytic glutamate transporters is the essential step for terminating glutamatergic neurotransmission and for keeping extracellular glutamate at subtoxic concentrations (Sims and Robinson, 1999; for review). We demonstrate that uncoupling of cultured cortical astrocytes decreases expression of GLT-1, the major astroglial glutamate transporter, by inhibiting the transcriptional activity of the GLT-1 promoter. We further demonstrate that although this loss in GLT-1 is partially compensated by an increase in GLAST expression, astrocytic uncoupling is associated with an almost 60% reduction in glutamate uptake. These findings unravel a previously unrecognized role of gap junctions in the modulation of glial glutamate transport. Classical tools of gap junction research include various gap junction blockers which seem to induce closure of gap junctions by either phosphorylation-dependent processes (hydrophilic gap junction blockers) or by mechanically squeezing gap junction pores (lipophilic gap junction blockers; Evans and Boitano, 2001). Other, more recently developed tools are connexin mimetic peptides, which are homologous to the extracellular portion of distinct connexin proteins, and seem to uncouple cells by preventing the docking of hemi-channels (Evans and Boitano, 2001). Unfortunately, all these gap junction blockers either lack selectivity, in terms that they potentially affect other cellular processes or their selectivity has not yet been firmly established.

Fig. 6. Effects of uncoupling on the expression of other major glial transporters. Cell lysates (GLAST) or membrane fractions (Glut-1, MCT-1) were prepared from astrocytic cultures maintained for 72 h with propofol (300 μM) and analyzed for GLAST (A) Glut-1 (B) and MCT-1 (C) expression levels by Western blotting. Integrated optical densities of immunoreactive bands were measured and corrected for β-actin levels. Numbers represent average changes ± S.D. in GLAST, Glut-1, and MCT-1 protein levels as determined in 3 independent experiments. Note that in contrast to GLT-1, astrocytic uncoupling slightly increases (⁎p < 0.05; Student's t-test) GLAST, Glut-1, and MCT-1 expression.

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Confirming that the observed inhibitory action of gap junction blockers on astrocytic GLT-1 expression involves a gap junction/ connexin-dependent mechanism, we found that propofol and GAP27, but not carbenoxolone, do not attenuate GLT-1 expression in the connexin-deficient hepatoma cell line, H4. Additional evidence for the dependence of GLT-1 expression on gap junctions/ connexins emerged from the use of astrocytes in which either expression of Cx43, the major astrocytic connexin (Giaume and McCarthy, 1996), was inhibited by RNA interference or which were derived from animals carrying an astrocyte-specific deletion of the Cx43 gene. In both cases, experimental depletion of Cx43 levels was associated with a robust decline in GLT-1 expression. Finally, two lines of evidence presently favor the assumption that this dependence of GLT-1 expression on astrocytic gap junction coupling is not simply an epiphenomenon, but rather reflects the existence of a selective regulatory mechanism. Firstly, uncoupling did not decrease, but increased expression levels of the glutamate transporter subtype, GLAST, as well as of other cell membrane associated transporters present on astrocytes, such as Glut-1 and MCT-1 (Vannucci et al., 1997; Hertz and Dienel, 2005). Secondly, gap junctions/connexins interfered with the activity of the (human) GLT-1 promoter as established by luciferase reporter gene assay. A putative upstream mechanism recruited by gap junctions/connexins to control GLT-1 expression might be gap junction intercellular communication (GJIC), e.g. the exchange of small molecules up to approximately 1000 Da between coupled cells (Wei et al., 2004). It is, however, equally possible that connexins control transcription of the GLT-1 gene in a direct, GJIC-independent manner, as previously reported for other cellular processes, such as cell proliferation and survival (see Jiang and Gu, 2005; for review). The observed differential effects of astrocytic uncoupling on GLT-1 and GLAST protein levels are consistent with several other findings implying that the expression of both glutamate transporter subtypes is controlled by distinctly different mechanisms. In fact, glucocorticoids only promote the expression of GLT-1, but not of GLAST (Zschocke et al., 2005). Likewise, PACAP stimulates the expression of GLAST at distinctly higher concentrations as compared to GLT-1 (Figiel and Engele, 2000). Provided that our in vitro findings apply to the in vivo situation, the increases in GLAST expression seen upon astrocytic uncoupling could at least in part explain why animals with reduced (Cx43 ko animals; Theis et al., 2003) or lacking astrocytic gap junction coupling (Cx43/ Cx30 ko animals; Wallraff et al., 2006) show, if unchallenged, no obvious phenotypic abnormalities or major functional deficits (but see also below). Whether the increases in GLAST expression, but also in the expression of Glut-1 and MCT-1 occurring in uncoupled astrocytic result from direct influences of gap junction/connexins on the transcription of these transporter proteins remains to be established. The direct gap junction/connexin-dependent control of GLT-1 expression as unraveled in our present studies might represent an essential regulatory component of glutamatergic neurotransmission under both physiological and pathophysiological conditions. Due to the relatively large size of the astrocytic syncytium, the observed gap junctional control of GLT-1 expression could principally allow to modulate glutamatergic neurotransmission within large synaptic networks and might, thus, interfere with processes involved in memory formation (Fries et al., 2003; Pascual et al., 2005). The dependency of GLT-1 expression on gap junctions/connexins could further represent a common mechanistic base for the (transient) decline or loss of GLT-1 seen in various forms of acute and chronic

brain diseases (Sims and Robinson, 1999; for review). In fact, a combined loss of Cx43 and GLT-1 occurs in the hypoxic/ischemic brain (Rouach et al., 2002; Fukamachi et al., 2001; Rao et al., 2000, 2001; Chen et al., 2005). GLT-1 expression further decreases under several pathological condition, such as traumatic brain injuries and Alzheimer's disease (see Sims and Robinson, 1999; Gegelashvili et al., 2001; for review), which are associated with neuroinflammatory processes. Although in vivo data on the opening/closing status of astrocytic gap junctions in the inflamed brain are not available, it is intriguing to note that co-culturing astrocytes with activated microglia leads to a decline in Cx43 expression and a subsequent loss of astrocytic gap junction coupling (Rouach et al., 2002, Faustmann et al., 2003). Finally, it is of interest that mice carrying astrocyte-directed Cx43 inactivation show accelerated hippocampal spreading depression (Theis et al., 2003) as well as an increased ischemic stroke volume (Nakase et al., 2003). At least in part, these responses could be a consequence of the observed decrease in GLT-1 expression and the associated reduction in glial glutamate uptake. One mechanism involved in gating gap junction pores is pH (Bukauskas and Verselis, 2004; for review). Since prolonged glutamate uptake into astroglia leads to intracellular acidification (Bouvier et al., 1992), intracellular pH could well represent a mechanism inducing or at least orchestrating the injury-related loss of GLT-1 expression. Other potential candidate factors gating gap junctions in the injured brain are endothelins. This family of peptides as well as their receptors are up-regulated after brain trauma (Siren et al., 2000; Tsang et al., 2001; Hama et al., 1997; Loo et al., 2002; Nakagomi et al., 2000; Rogers et al., 1997; SakuraiYamashita et al., 1997), and are known to trigger the closure of gap junctions (Blomstrand et al., 1999) and to inhibit glial glutamate transporter expression and subsequently glial glutamate uptake ((Rozyczka et al., 2004; Matsuura et al., 2002; Leonova et al., 2001). It is, however, of note, that despite these seemingly conclusive facts, we have been unable so far to demonstrate that closure of gap junction is an essential intermediate step in the inhibitory action of endothelins on glial glutamate transporter expression (Engele, unpublished observations). In summary, the observed dependency of glial glutamate transport on astrocytic gap junction coupling opens new insights into the coordination of glutamatergic neurotransmission within large synaptic networks. In addition these findings might provide an important base for the future therapeutic prevention of glutamate-induced brain damage. Experimental methods Animals Sprague–Dawley rats were maintained at a 12 h:12 h dark–light cycle with food and water provided ad libitum. Animals were mated for 12 h, the day of birth was defined as postnatal day (P)0. Mice lacking astrocytic Cx43 were generated by interbreeding mice carrying two floxed alleles of Cx43 (Cx43fl/fl) with hGFAP-cre transgenes (Theis et al., 2003, 2004). For the current study, offspring were obtained from Cx43fl/fl × Cx43fl/fl: hGFAP-cre crosses. Cx43fl/fl animals served as controls which have a similar mixed 129P2/OlaHsd, FVB/N, and C57BL/6 background (Theis et al., 2001, 2004). Animals were phenotyped by PCR as described in detail by Theis et al. (2003, 2004). Glial cultures Purified astroglial cultures were established from the cerebral hemispheres of P1–2 Sprague–Dawley rats, as well as of P1–2 Cx43fl/fl:hGFAP-

M. Figiel et al. / Mol. Cell. Neurosci. 35 (2007) 130–137 cre, and Cx43fl/fl mice according to a recently established protocol (Figiel et al., 2003; Franke et al., 1998). The resulting cultures predominantly contained GFAP-positive astrocytes (> 90%) and small numbers of oligodendrocytes and glial progenitors. The cultures were virtually free of neurons and microglial cells (Franke et al., 1998). In brief, dissected tissue pieces were trypsinized (0.1%) for 20 min in Ca2+- and Mg2+-free Dulbecco's phosphate buffered saline (Invitrogen, Carlsbad, CA) supplemented with 0.02% EDTA. Tissue pieces were subsequently dissociated in 90% Hank's balanced salt solution (Invitrogen) and 10% fetal calf serum (Invitrogen) by trituration through 10 ml plastic pipettes. Cells were pelleted at 400 g for 5 min, resuspended in 90% minimum essential medium (MEM; Invitrogen) and 10% horse serum (Invitrogen), and seeded into 100 mm culture dishes (hemispheres from 3 animals per dish; TPP, Trasadingen, Switzerland) previously coated with poly-D-ornithine (0.1 mg/ ml; molecular weight, 30–70 kDa; Sigma; Deisenhofen, Germany). Upon reaching confluency, cells were trypsinized and replated. After the third passage, cells were either seeded into 60 or 35 mm culture dishes (TPP) and were further maintained with serum free N2-medium additionally supplemented with TGFα (100 ng/ml; Pepro Tech; Rocky Hill, NJ), PACAP-38 (100 nM; Calbiochem; Schwalbach, Germany), as well as propofol (300 μM; Fresenius Kabi, Bad Homburg, Germany), carbenoxolone (40 μM; Sigma), and Gap-27 (300 μM; Peptide Speciality Laboratories, Heidelberg, Germany) or a combination of these factors as indicated. H4-II-E-C3 cells The rat hepatoma cell line, H4-II-E-C3 (H4; ECACC) was propagated with MEM supplemented with 10% fetal calf serum and 1% non-essential amino acids (NEAA; Sigma). For experiment, cultures were switched to serum-free N2-medium containing 1% NEAA.

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FCS. Transfection was performed with the siPORT™ Amine Transfection Agent (Ambion, Huntingdon, UK) according to the manufacturer's recommendations. For transfection, cultures additionally received 200 μl of serum-free MEM-medium containing 100 nM of Cx43 or nh siRNA and 6 μl of the transfection agent, and were incubated overnight. During the following 24 h, transfected cultures were maintained with MEM supplemented with 10% FCS, and subsequently switched to serum-free N2 medium. Glutamate uptake Glutamate uptake was performed in sodium- or lithium-supplemented Tris buffer, containing 5 mM Tris base, 10 mM HEPES, 140 mM NaCl or LiCl, 2.5 mM KCl, 1.2 mM CaCl2, 1.2 mM MgCl2, 1.2 mM K2HPO4, 10 mM dextrose and the glutamine synthetase inhibitor, methionine sulfoximine (1 mM; Sigma) as described previously (Figiel and Engele, 2000). For uptake experiments, buffer was additionally supplemented with 1 μCi/ml of tritium-labeled glutamate (specific activity, 63–65 Ci/mmol; Amersham; Braunschweig, Germany) and total glutamate concentration was adjusted to 960 μM with unlabeled glutamate. In selected experiments, uptake buffer was adjusted to 160 μM of glutamate and additionally supplemented with the selective GLT-1 inhibitor, dihydrokainate (DHK; 500 μM). Uptake was terminated after 15 min, which is within the linear range of glutamate uptake in cultured cortical astroglia (Figiel and Engele, 2000), by removing the radioactive solution and rinsing cultures three times with ice-cold lithium-containing Tris buffer. Cells were subsequently lysed in 0.1 M NaOH and the amount of incorporated 3[H]glutamate was determined by liquid scintillation counting of the cell lysate. Specific (sodium-dependent) glutamate uptake was defined to be the difference of the amount of radioactivity incorporated by glia in the presence of sodiumand lithium-containing buffer and was referred to the amount of protein determined in sister cultures by using the BCA protein estimation kit (Perbio Science, Bonn, Germany) and bovine serum albumin as a standard.

Western blot analysis Cell–cell communication assay Western blot analysis was either performed with cell lysates (GLT-1, GLAST, Cx43) or membrane fractions (Glut-1, MCT-1). Cultured cells were lysed by ultrasonication in 60 mM Tris–HCl, containing 2% SDS and 10% sucrose. Cell lysates were diluted 1:1 in sample buffer (250 mM Tris–HCl, pH 6.8 containing 4% SDS, 10% glycerol, and 2% β-mercaptoethanol) and denatured at 95 °C for 5 min. For preparing membrane fractions, cell lysate was centrifuged for 10 min at 1000×g. The supernatant was respun for 1 h at 100,000 × g, and the obtained pellet was resuspended in sample buffer. Proteins (5 μg/lane) were separated by SDS-(10%) polyacrylamide gel electrophoresis and transferred to nitrocellulose by electroblotting. Following blockade of nonspecific binding sites with 5% non-fat milk for 30 min, blots were incubated overnight at 4 °C with anti-GLT-1 (1:4000; Chemicon, Temecula, CA), anti-GLAST (1:1000; Chemicon), anti-Cx43 (1:8000; Sigma), anti Glut-1 (1:3000; Chemicon), or anti MCT-1 antibodies (1:1000; Chemicon). Blots were then exposed for 2 h at room temperature to appropriate horseradish peroxidase-labeled secondary antibodies (Dianova, Hamburg, Germany) and immunolabeled protein bands were visualized using the enhanced chemiluminescence kit (Amersham Pharmacia, Freiburg, Germany). Protein loading was controlled by additionally staining blots with GAPDH (1:4000; Research Diagnostics) or β-actin antibodies (1:1000; Santa Cruz Biotechnologies, Santa Cruz, CA). Integrated optical densities of immunoreactive protein bands were measured using the Image Master VDS software (Pharmacia) and normalized to GAPDH or β-actin values. RNA interference Rat Cx43 (accession number, NM 012567) siRNA (5′-AAAGTTGCTGCTGGACATGAA-3′), and non-homologous (nh) siRNA (5′AATTCTCCGAACGTGTCACGT-3′) were designed using the Protein Lounge database (www.proteinlounge.com/sirna_home.asp), and doublestranded RNAs were custom synthesized by Qiagen (Hilden, Germany) For transfection, astrocytes were grown on 100 mm dishes. Two hours prior to transfection, culture medium was replaced by 800 μl MEM containing 2%

Gap junction-dependent cell–cell communication was assessed by the scrape loading-dye transfer technique. Confluent astrocytic cultures were preincubated for 9 min with Ca2+ (1 mM)-containing HEPES-buffered Hank's balanced salt solution (HHBSS; 130 mM NaCl, 2.8 mM KCl, 2 mM MgCl2, 10 mM HEPES, pH 7.2) and subsequently rinsed for 1 min in Ca2+free HHBSS. The cell monolayer was cut with a razor blade in the presence of Ca2+-free HHBSS containing 0.5 mg/ml Lucifer yellow (Molecular Probes). After 2 min, cultures were rinsed three times with Ca2+-containing HHBSS and incubated for another 8 min with Ca2+-containing HHBSS. Cultures were examined using a fluorescence microscope equipped with a UV-light source and a digital camera. Luciferase reporter gene assay The putative human full-length 2.4 kb-promoter of the GLT-1 gene (Su et al., 2003) was cloned into the pGL3-basic luciferase reporter vector (Promega; Mannheim, Germany). Transfection of primary cortical rat astrocytes was performed in MEM supplemented with 2% fetal calf serum for 8 h using the jetPEI transfection reagent (Qbiogene, Carlsbad, CA). Upon switching to serum free N2-medium, transfected cultures were treated with either propofol (300 μM) or EGF (100 ng/ml; Pepro Tech) alone or in combination. After an additional 72 h, cells were lysed and luciferase activity was analyzed using the firefly luciferase detection kit (Promega). Luciferase activity was subsequently measured with a luminometer and corrected for protein content. RNA isolation and real-time RT-PCR analysis Total RNA was isolated from primary glial cultures using the PeqGold isolation kit (PeqLab, Schwalbach, Germany) according to the manufacturer's instructions. RNA concentration was measured by spectrophotometric absorbance at 260 nm. A total of 5 μg of RNA was reverse

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transcribed using 200 U per μl of M-MLV (Promega, Madison WI) and 1 μg random hexamer primers (Thermo Electron, Ulm, Germany). RNA levels were quantified by real-time polymerase chain reaction (PCR) using the following primers: GLT-1 (accession number, X67857); sense, 5′-AGG AGC CAA AGC ACC GAA AC-3′; antisense, 5′-CCC GGG AAG GCT ATC AAC AT-3′; product size, 169 bp. GLAST (accession number, NM_019225); sense, 5-ACC GTC AGC GCT GTC ATTG-3′; antisense, 5′TGT GAC AAG ACT GGA GAT GA-3′; product size, 159 bp. β-Actin (internal standard); sense, 5′-CTA CAA TGA GCT GCG TGT GGC-3′; antisense, 5′-CAG GTC CAG ACG CAG GAT GGC-3′; product size, 271 bp (all from Thermo Electron). Thermocycling for each reaction was done in a final volume of 10 μl containing 1 μl of cDNA sample (or standard), 1 μl LightCycler-DNA Master SYBR-Green I (Roche Diagnostics, Mannheim, Germany), MgCl2 (2.5 mM), and sense and antisense primers (5 pmol each). The thermal cycling conditions were 95 °C for 90 s followed by 45 cycles of 55 °C for 5 s and 72 °C for 20 s. Data were collected using the LightCycler Instrument (Roche Diagnostics). To confirm the specificity of the amplified products, melting curves were performed at the end of the amplification by cooling samples at 20 °C/s to 65 °C for 15 s and then increasing temperature to 95 °C at 0.1 °C/s with continuous fluorescence measurement. For generation of standard curves, PCR products were purified using QIAquick (Qiagen, Hilden, Germany) spin columns and serially diluted in double-distilled water. The second derivative maximum method as provided by the manufacturer's software was applied to determine the maximum acceleration of the amplification process and the respective cycle number was used as a crossing-point value. The concentrations of unknown samples were determined by setting their crossing-points to the standard curve and were normalized to β-actin.

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