Neurochem Res (2014) 39:973–984 DOI 10.1007/s11064-014-1295-1

ORIGINAL PAPER

Cellular Senescence Induced by Prolonged Subculture Adversely Affects Glutamate Uptake in C6 Lineage Mery Ste´fani Leivas Pereira • Kamila Zenki • Marcela Mendonc¸a Cavalheiro Chairini Ca´ssia Thome´ • Eduardo Cremonese Filippi-Chiela • Guido Lenz • Diogo Onofre Gomes de Souza • Diogo Losch de Oliveira

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Received: 28 October 2013 / Revised: 24 February 2014 / Accepted: 22 March 2014 / Published online: 5 April 2014 Ó Springer Science+Business Media New York 2014

Abstract Several researchers have recently used C6 cells to evaluate functional properties of high-affinity glutamate transporters. However, it has been demonstrated that this lineage suffers several morphological and biochemical alterations according to the number of passages in culture. Currently, there are no reports showing whether functional properties of high-affinity glutamate transporters comply with these sub culturing-dependent modifications. The present study aimed to compare the functional properties of high-affinity glutamate transporters expressed in early (EPC6) and late (LPC6) passage C6 cells through a detailed pharmacological and biochemical characterization. Between 60–180 min of L-[3H]glu incubation, LPC6 presented an intracellular [3H] 55 % lower than EPC6. Both cultures showed a time-dependent increase of intracellular [3H] reaching maximal levels at 120 min. Cultures incubated with 3 3 D-[ H]asp showed a time-dependent increase of [ H] until

Electronic supplementary material The online version of this article (doi:10.1007/s11064-014-1295-1) contains supplementary material, which is available to authorized users. M. S. L. Pereira (&)  K. Zenki  M. M. Cavalheiro  C. C. Thome´  D. O. G. de Souza  D. L. de Oliveira (&) Departamento de Bioquı´mica, Instituto de Cieˆncias Ba´sicas da Sau´de (ICBS), Universidade Federal do Rio Grande do Sul (UFRGS), Rua Ramiro Barcelos, 2600 pre´dio anexo, Porto Alegre, RS 90035-003, Brazil e-mail: [email protected] D. L. de Oliveira e-mail: [email protected] E. C. Filippi-Chiela  G. Lenz Departamento de Biofı´sica, Instituto de Biocieˆncias, Universidade Federal do Rio Grande do Sul (UFRGS), Avenida Bento Gonc¸alves, 9500 pre´dio 43422, Porto Alegre, RS 91501-970, Brazil

180 min. Moreover, LPC6 have a D-[3H]asp-derived intracellular [3H] 30–45 % lower than EPC6 until 120 min. Only EAAT3 was immunodetected in cultures and its total content was equal between them. PMA-stimulated EAAT3 trafficking to membrane increased 50 % of L-[3H]glu-derived intracellular [3H] in EPC6 and had no effect in LPC6. LPC6 displayed characteristics that resemble senescence, such as high b-Gal staining, cell enlargement and increase of large and regular nuclei. Our results demonstrated that LPC6 exhibited glutamate uptake impairment, which may have occurred due to its inability to mobilize EAAT3 to cell membrane. This profile might be related to senescent process observed in this culture. Our results suggest that LPC6 cells are an inappropriate glial cellular model to investigate the functional properties of high-affinity glutamate transporters. Keywords C6 glioma  Astroglial model  EAAT3 surface expression Abbreviations b-Gal b-galactosidade CNP Cyclic-nucleotide phosphohydrolase 3 3 D-[ H]asp D-[2,3- H]aspartate DMEM Dulbecco’s modified eagle’s medium EAAT1 Excitatory amino acid transporter 1 EAAT2 Excitatory amino acid transporter 2 EAAT3 Excitatory amino acid transporter 3 EPC6 Early passage C6 cells FBS Fetal bovine serum GS Glutamine synthetase HBSS Hank’s balanced salt solution 3 3 L-[ H]glu L-[3,4- H]glutamate LPC6 Late passage C6 cells NII Nuclear irregularity index NMA Nuclear morphometric analysis

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PDC PI PMA SA-b-gal TBOA

Neurochem Res (2014) 39:973–984 L-trans-Pyrrolidine-2,4-Dicarboxylic

acid Propidium iodide Phorbol 12-myristate 13-acetate Senescence-associated-b-galactosidade LD-threo-b-Benzyloxyaspartic acid

Introduction The amino acid L-glutamate (L-glu) is considered to be the major mediator of excitatory signals in the mammalian central nervous system (CNS) and is involved in most aspects of normal brain function and pathology [1]. This neurotransmitter is generally restricted to synaptic cleft of glutamatergic synapses, where reaches concentrations in the range of 1 mM [2]. The end of glutamatergic signaling is accomplished primarily by a family of Na?-dependent high-affinity glutamate transporters. Five subtypes of these transporters have been identified in mammals (EAAT1– EAAT5) and they differ in regional, cellular, and developmental distributions [3]. Glial cells play a major role to remove glutamate from synaptic cleft, because the exclusive astroglial high-affinity glutamate transporters EAAT1 and EAAT2 are responsible for most of the glutamate uptake activity in CNS [1]. Malignant gliomas comprise a heterogeneous group of primary CNS tumors that originate from glial cells or their progenitor cells [2]. Gliomas utilize the neurotransmitter glutamate in several ways to gain growth advantage [4]. Unlike their non-malignant counterparts, gliomas release glutamate through a highly express cystine-glutamate antiporter, system xc (SXC), a solute transporter, importing cystine in exchange for glutamate [4]. To contribute to this excitotoxic environment, gliomas also lack functional of Na?-dependent high-affinity transporters [4]. Takano et al. [5], after implanted C6 cells that actively release glutamate in rats, proved that tumors formed had growth advantage, demonstrating that excess of extracellular glutamate create a favorable microenvironment for tumor development. C6 lineage was originally induced in Wistar rats through repetitive administration of N,N0 -nitroso-methyl urea and tumors formed were excised and explanted into cultures [6]. In the last decades, C6 cell cultures have been widely used in neuro-oncology as a model to investigate several aspects of biology of brain tumors [7]. In addition, this lineage has been employed to test the efficacy of a variety of therapeutic modalities including radiation therapy [8], inhibition of proteasomal activity [9] and treatment with angiogenic inhibitors [10]. Similar to other cell lines [11], several factors can influence the morphological and physiological properties

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of C6 lineage in culture, such as the composition of medium [12], the presence of growth factors [13], the type of lineage clone [12] and the treatment of culture with cellular morphogens [14]. Furthermore, another important factor that influences C6 cellular properties in culture is the prolonged subculture practice. C6 cells from later passages experience alterations in cell morphology [15], response to stimuli [16], growth rates [15] and protein expression [15] when compared to early passages. Parker et al. [15] described that early passage C6 cells (EPC6) showed a dark nuclei, scanty cytoplasm and thin cytoplasmic processes. Cultures also presented a reduced activity of glutamine synthetase (GS) (EC 6.3.1.2) [17, 18] and increased expression of cyclic-nucleotide phosphohydrolase (CNP) (EC 3.1.4.37) [13]. In addition, there are evidence showing that EPC6 are less differentiated and more glioblastic in nature than cells from later passages [12, 13, 15, 18]. In vivo, EPC6 injected in rat brain presented cellular features similar to glioblastoma multiforme, which includes nuclear pleomorphism, high mitotic index, foci of tumor necrosis, intra tumoral hemorrhage and parenchymal invasion [19]. In this context, EPC6 has been considered a glioblastoma model and it has been used for a variety of studies that investigate glioma metabolism [20], tumor growth and invasion [5, 21], cellular migration [22], tumor-induced blood–brain barrier disruption [23], and drug-based therapies [24]. In contrast, late passage C6 cells (LPC6) in culture presented larger nuclei, abundant cytoplasm and longer cytoplasmic processes [15]. These cells also showed high levels of GS activity [17, 18] and elevated amounts of S100B secretion [25]. Since these last biochemical features were very similar to those displayed by astrocyte primary cultures, LPC6 has been widely considered as a useful cellular model to study glial cell properties. In fact, these cultures have been used almost interchangeably with primary astrocyte cultures to investigate glutamate–glutamine cycling [13, 15–17, 26–28], glial secretion of extracellular modulators [26–28] and, more recently, to investigate the functional properties of excitatory amino acids transporters [29–32]. In this context, Tramontina et al. [32] have used LPC6 to evaluate the influence of normal and high glucose concentrations (6 and 12 mM, respectively) on glutamate uptake, S100B secretion and GS activity. Moreover, Quincozes-Santos et al. [30] investigated the effects of typical and atypical antipsychotic agents on glutamate uptake and GS activity in LPC6 cultures. Despite being used as a glial model, it is well known that C6 glioma cell line do not express the glial high-affinity glutamate transporters (EAAT1 and EAAT2) and only endogenously expresses the neuronal type (EAAT3) [33–35]. These data per se may compromise the use of this lineage as a glial model.

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Although some researchers still consider LPC6 an astrocytic cellular model, to date there are no studies investigating whether the functional and structural properties of the high-affinity glutamate transporters expressed in LPC6 cultures are really similar to those observed in primary astrocyte cultures [36, 37] and also whether these properties are different of those presented by EPC6. Therefore, in the present study we evaluated the functional properties exhibited by the high-affinity glutamate transporters expressed in both EPC6 and LPC6 cultures through a detailed pharmacological and biochemical characterization. This study may contribute to the idea that C6 lineage may not be considered a cellular model to mimic the physiology of glial cells.

et al. [38] (Supplementary Fig 1). The analysis of the doubling time was required to determine the density of cells which would be seeded for each culture were at 80–90 % confluence when the experimental tests were performed (Supplementary Fig 1). For multinuclear cell quantification, nuclear morphometric analysis (NMA) and senescence-associated-b-galactosidade (SA-b-gal) staining, early and late passages were seeded in 24-multiwell plates at 1.5 9 104 cells/well and 1.35 9 104 cells/well, respectively. These assays were performed after 24 h in culture. For other assays, early (2 9 105 cells/well) and late (1.8 9 105 cells/well) passages cells were seeded in 6-multiwell plates and assays were performed after 48 h in culture (80–90 % of confluence). All cultures were maintained at 37 °C in a 95:5 air/CO2 atmosphere.

Experimental Procedure

3 L-[ H]glu

Materials

C6 cell cultures were pre-incubated during 15 min at 37 °C in Hank’s balanced salt solution (HBSS) containing (mM): 137 NaCl, 0.60 Na2HPO4, 3.00 NaHCO3, 20 Hepes sodium salt, 5.00 KCl, 0.40 KH2PO4, 1.26 CaCl2, 0.90 MgSO4 and 5.55 glucose; pH 7.4. After, cells were washed once with HBSS and incubated with HBSS (37 °C) containing 100 lM of unlabeled L-glu or D-asp plus 0.35 lCi mL-1 L[3H]glu or 0.49 lCi mL-1 D-[3H]asp. Uptake was stopped at specific times by two washes of ice-cold HBSS (4 °C) immediately followed by addition of 0,5 N NaOH (Supplementary Fig 2). Sodium-independent uptake was determined at 37 °C using HBSS containing N-methyL-Dglutamine instead of NaCl and Na2HPO4. Sodium-dependent uptake was obtained by subtracting sodium-independent from total uptake. For L-[3H]glu and D-[3H]asp uptake inhibition assays, cultures were pre-incubated (15 min) and incubated (120 min) in HBSS containing 100 lM PDC or 100 lM TBOA [37] plus 100 lM of unlabeled L-glu or D-asp with 0.35 lCi mL-1 L-[3H]glu or 0.49 lCi mL-1 D-[3H]asp (Supplementary Fig 2). For PMA-dependent L-[3H]glu uptake stimulation, cultures were pre-incubated (15 min) in HBSS containing 100 nM PMA and after L-[3H]glu uptake assay was performed during 5 min. Control groups were incubated in DMSO (0,01 %) (Supplementary Fig 2). Protein determination was assessed using method described by Peterson et al. [39] and the radioactivity was quantified by scintillation. Data were expressed as nmol of -1 L-glu.mg prot or nmol of D-asp.mg prot-1.

H]glutamate (L-[3H]glu) and D-[2,3-3H]aspartate (D[ H]asp) were purchased from PerkinElmer Inc., USA. Dulbecco’s modified eagle’s medium (DMEM), Fetal bovine serum (REF 12657-029 LOT 210185 K) (FBS), penicillin/streptomycin and fungizone were obtained from GibcoÒ, InvitrogenTM, USA. Propidium iodide (PI), L-glu, D-aspartate (D-asp), monoclonal mouse anti-b-actin IgG, cell culture chemicals, western blot and GS assay reagents and b-galactosidase staining kit were purchased from Sigma-Aldrich Co., USA. Western blot polyclonal antiglutamate transporters (EAAT1, EAAT2 and EAAT3) were purchased from Alpha Diagnostic Intl. Inc., USA. Horseradish peroxidase-conjugated donkey anti-rabbit IgG and horseradish peroxidase-conjugated sheep anti-mouse IgG were obtained from GE Healthcare, USA. L-transPyrrolidine-2,4-Dicarboxylic acid (PDC), DL-threo-bBenzyloxyaspartic acid (TBOA) and Phorbol 12-myristate 13-acetate (PMA) were purchased from Tocris Bioscience, USA. Tissue culture flasks and tissue culture test plates were obtained from Techno Plastic Products AG (TPPÒ), Switzerland. All other chemicals were purchased from local commercial suppliers. L-[3,43

3

Cell Culture C6 glioma cells were obtained from American Type Culture Collection (Rockville, Maryland, USA). Early (6–15 passages) and late (beyond 100 passages) passage C6 cells were grown in culture flasks containing 1 % DMEM, 23.8 mM NaHCO3, 8.39 mM Hepes sodium salt (pH 7.4), 0.25 lg/mL fungizone, 50 U/mL penicillin, 50 lg/mL streptomycin and 5 % FBS. We evaluated the cell proliferation using the sulforhodamine B test according to Vichai

3 L-[ H]glu

and D-[3H]asp Uptake Assay

and D-[3H]asp Release Assay

Release of L-[3H]glu- and D-[3H]asp-derived [3H] was assessed through a similar protocol for amino acids uptake. Cells were incubated during 60 min in HBSS at 37 °C

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containing L-[3H]glu or D-[3H]asp plus unlabeled 100 lM of L-glu or D-asp. In order to begin the [3H] release assay, cultures were washed twice and re-incubated during 60 min in HBSS at 37 °C containing only 100 lM of unlabeled L-glu or D-asp. Assay was stopped by collecting extracellular HBSS medium followed by addition of 0.5 N NaOH to cells. Extracellular and intracellular radioactivity was quantified by scintillation and protein determination was assessed as previously described (Supplementary Fig 2). Data were expressed as nmol of L-glu.mg prot-1 or nmol of D-asp.mg prot-1 (Supplementary Fig 2), and as radioactivity efflux rate. The radioactivity efflux rate (f = D3H/Dt.3Ht) was the fractional rate of loss of radioactivity per unit time (h). D3H represents the total radioactivity lost to extracellular media in the time interval Dt. 3Ht represents the radioactivity released to extracellular media added to the amount of radioactivity extracted from de cells at the end of the experiments (intracellular radioactivity). This rate was evaluated based on Hopkin and Neal [40] and expressed as f (h-1). Membrane Integrity Early and LPC6 were incubated in HBSS medium in absence (controls) or presence of unlabeled 100 lM of Lglu at specific times. After, cells were incubated with 5 lM PI during 30 min. The number of PI positive cells was assessed by Flow Cytometry (FACSCalibur, BD Biosciences) (Supplementary Fig 2). A total of 10.000 events were counted and data were expressed as percentage of cells that incorporate PI. Western Blot Both cultures were treated with HBSS containing 100 lM Lglu at specific times (Supplementary Fig 2). Cells were homogenized in lysis buffer (5 mM Tris base, 1 mM EDTA, 0.1 % SDS and protease inhibitor cocktail; pH 7.0) and protein content was normalized to 2 lg protein/lL. Aliquots were diluted 1:1 in sample buffer (0.01 g % Bromophenol Blue, 60 mM Tris base, 20 % glycerol, SDS 2 % and 2-bmercaptoethanol 5 %; pH 6.8) and were resolved by 8 % SDS-PAGE. Proteins were electro transferred to nitrocellulose membranes (GE Healthcare, USA) using a semi-dry transfer apparatus (Bio-Rad, Trans-Blot SD). After 2 h of incubation in blocking solution containing 5 % powdered milk and 0.1 % Tween-20 in Tris–buffered saline (TBS; 10 mM Tris base, 30 mM NaCl, pH 7.4), membranes were incubated overnight with anti-EAAT3 IgG (0.17 lg/mL), or anti-EAAT2 IgG (0.33 lg/mL), or anti-EAAT1 IgG (1 lg/ mL) or anti-b-actin IgG (1:3,000) at 48 C overnight. Membranes were exposed to horseradish peroxidase-conjugated

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anti-rabbit IgG diluted 1:1,000 overnight or horseradish peroxidase-conjugated anti-mouse IgG (1:3,000) for 2 h at 48 C. The chemiluminescence (ECL, GE Healthcare, USA) was detected using X-ray films (Kodak X-Omat). Films were scanned and the percentage of band intensity was analyzed using Optiquant software. b-actin was used as protein loading control. Glutamine Synthetase Activity Glutamine synthetase (GS) enzymatic activity assay was performed as previously described by Petito et al. [41]. Briefly, early and LPC6 were lysed in 150 mM KCl containing 0.1 % Triton X-100. Samples were adjusted to 1 lg/lL and 100 lL were incubated in 100 lL of reaction medium (10 mM MgCl2, 50 mM L-glu, 10 mM 2-bmercaptoethanol; 50 mM hydroxylamine–HCl; 10 mM ATP and 100 mM imidazole–HCl buffer; pH 7.4) for 15 min at 37 °C. Reaction was stopped by addition of 400 lL of solution containing 370 mM ferric chloride, 0.67 N HCl and 3.3 % trichloroacetic acid. After centrifugation (1,000 g during 10 min), the absorbance of supernatants was measured at 530 nm. Synthetic c-glutamylhydroxamate (c-GH) was used as standard. GS activity was expressed as lmol c -GH.h-1.mg prot-1. Nuclear Morphometric Analysis Nuclear morphometric analysis was performed as described by Filippi-Chiela et al. [42]. Briefly, cells were fixed with 4 % paraformaldehyde diluted in phosphate buffer saline (PBS, pH 7.4) for at least 30 min at room temperature (RT) and subsequently maintained in PBS (pH 7.4). After, fixed cells were marked with a solution containing 300 nM DAPI and 0.1 % Triton X-100 diluted in PBS for 1 h at RT, followed by image acquisition. Nuclear morphometric quantification was performed using the Image Pro Plus 6.0 software (IPP6; Media Cybernetics, Silver Spring, Md., USA). Roundness, aspect, radius ratio and area/box of cell nucleus were quantified and grouped in an index named nuclear irregularity index (NII). Data were presented as a XY plot of area versus NII, through which the percentage of large and regular nuclei was determined. Senescence-Associated-b-galactosidade (SA-b-gal) Staining Cells were washed in PBS and fixed in 4 % paraformaldehyde diluted in PBS (pH 7.4) for at least 30 min at RT. After, cells were washed and incubated with fresh SA-b-gal staining solution containing 1 mg/mL b-gal, 40 mM citric acid/ sodium phosphate (pH 6.0), 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, and 2 mM

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MgCl for 12–16 h at 37 °C [43]. Results were presented as percentage of SA-b-gal-positive cells to total cells. Multinuclear Cells Quantification DAPI stain were performed according to protocol previous described. Images of cells stained by DAPI were merged with images of cells in visible light to provide multinuclear cells quantification. Results were presented as percentage of multinuclear cells to total cells. Statistical Analysis Data from membrane integrity and western blot analysis were presented as mean ± SD from at least three independent experiments. Data from amino acids uptake, radioactive release and GS activity assays were presented as mean ± SEM from at least three independent experiments carried out in triplicate. Two-way Analysis of Variance followed by Bonferroni post hoc test was used to analyze data from uptake assay, release assay, membrane integrity and western blot analysis. GS activity and radioactivity efflux rate were analyzed by Student T test. P \ 0.05 was considered significant.

Fig. 1 Morphological differences between EPC6 (a) and LPC6 (b) entirely confluent cultures

Results Morphological Differences Between EPC6 and LPC6 and Glutamine Synthetase Activity Since it has been known that C6 lineage exhibits morphological and biochemical changes according to number of passages in culture, we examined cellular morphology and GS activity in our C6 lineage. On visual inspection using phase-contrast light microscopy C6 cells from EPC6 appeared smaller and thinner than cells from LPC6 (Fig. 1) and these cultures showed similar levels of GS activity after 3 days in culture (1.40 ± 0.14 lmol c-GH/h.mg prot, n = 4; 1.11 ± 0.24 lmol c-GH/h.mg prot, n = 4; respectively) (Fig. 2). Both results were in accordance with data showed by Parker et al. [15]. 3 L-[ H]glu-Derived

Intracellular Content of [3H]

To evaluate glutamate uptake profile exhibited by C6 cells from early and late passages, we exposed both cultures to L[3H]glu during 5–180 min. Both EPC6 and LPC6 cells showed a time-dependent increase in intracellular [3H], reaching maximal levels from 120 min. However, L-[3H]gluderived intracellular [3H] in LPC6 was 55 % lower from 60 min (21.94 ± 1.92 nmol/mg protein; n = 13; P \ 0.001, two-way ANOVA) to 180 min of incubation

Fig. 2 Glutamine synthetase activity in EPC6 and LPC6. Data were expressed as mean ± SEM. n = 4 experiments performed in triplicate

(36.55 ± 2.36 nmol/mg protein; n = 5; P \ 0.001, two-way ANOVA) when compared to EPC6 (48.75 ± 5.07 nmol/mg protein, n = 13; 85.93 ± 6.78 nmol/mg protein, n = 5; respectively) (Fig. 3). Membrane Integrity In order to verify if the reduced L-[3H]glu-derived intracellular [3H] exhibited by LPC6 in Fig. 3 could be related to a loss of cell membrane integrity, we quantified the PI

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Fig. 3 Intracellular content of [3H] derived from L-[3H]glu uptake in EPC6 and LPC6. Cells were incubating in HBSS medium containing 100 lM of unlabeled L-glu plus 0.35 lCi.mL-1 L-[3H]glu during 5–180 min. Data were expressed as mean ± SEM. * = P \ 0.001 when compared to respective time in LPC6 cultures (Two-way ANOVA followed by Bonferroni post hoc test). n = 4–13 experiments performed in triplicate

incorporation in both cultures during incubation with 100 lM L-glu. There was no difference of PI incorporation in both cultures when they were or not treated with L-glu (Supplementary Fig 3). Glutamate Transporters Immunocontent Although C6 cells express only the excitatory amino acid transporter 3 (EAAT3) transporter [33, 35, 44], western blot analysis was performed to evaluate if the difference of 3 3 L-[ H]glu-derived intracellular [ H] between EPC6 and LPC6 was related to a distinct profile of Na?-dependent glutamate transporter immunocontent. Only EAAT3 was immunodetected in both cultures and its total immunocontent was the same between them (Fig. 4). 3 D-[ H]asp-Derived

Intracellular Content of [3H]

Once taken up by cells, glutamate can be metabolized in several ways, of which glutamine formation and entry into the tricarboxylic acid cycle are quantitatively most important [45]. In order to evaluate if a low glutamate metabolism was related to the reduced capacity of LPC6 to maintain [3H] derived from L-[3H]glu (Fig. 3), we exposed both cultures to a non-metabolizable glutamate analogue, 3 D-[ H]asp, and evaluated its transport through high affinity ? Na -dependent glutamate transporter. Both cultures showed a time-dependent increase in 3 3 D-[ H]asp-derived intracellular [ H] across time. Intracellular

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Fig. 4 Immunocontent of Na?-dependent glutamate transporters in EPC6 (EP) and LPC6 (LP) after 5–180 min of incubation in HBSS containing 100 lM of unlabeled L-glu. Data were expressed as mean ± SD. n = 4–10 independent experiments. Samples from rat brain were used as positive control

[3H] in LPC6 was approximately 45 and 30 % lower at 60 min (32.71 ± 2.95 nmol/mg protein; n = 4; P \ 0.05, two-way ANOVA) and 120 min of incubation (81.42 ± 10.50 nmol/mg protein; n = 8; P \ 0.05, two-way ANOVA), respectively, when compared to EPC6 (117.05 ± 7.43 nmol/mg protein; n = 8;61.24 ± 1.58 nmol/mg protein, n = 4; respectively) (Fig. 5). This result indicates that [3H] derived from D-[3H]asp uptake was being accumulated in cytosol of both cell cultures, suggesting that L-[3H]glu could have been metabolized during uptake assay (Fig. 3). Release of L-[3H]glu-and D-[3H]asp-Derived [3H] Lower levels of L-[3H]glu-derived intracellular [3H] exhibited by LPC6 in Fig. 3 might be related to an increased release of L-[3H]glu-derived radioactivity to

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extracellular medium. Thus, both cultures were submitted to a radioactivity release assay. After 60 min of L-[3H]glu uptake, approximately 50 % of L-[3H]glu-derived intracellular [3H] was released to extracellular medium by EPC6 and LPC6 cultures (Fig. 6a). Radioactivity efflux rate derived from L-[3H]glu was higher in LPC6 [0.88 ± 0.03 f (h-1), n = 8] than in EPC6 [0.76 ± 0.024 f (h-1), n = 8] (Fig. 6c). It was not observed a significant release of D-[3H]aspderived intracellular [3H] in both cultures (Fig. 6b) and efflux rate of radioactivity derived from D-[3H]asp was similar between LPC6 [0.10 ± 0.01 f (h-1), n = 3] and EPC6 [0.08 ± 0.006 f (h-1), n = 3] (Fig. 6d). These results indicates that the extracellular radioactivity observed in Fig. 6a was probably derived from an 3 L-[ H]glu metabolite. Uptake Inhibition

Fig. 5 D-[3H]asp-derived intracellular content of [3H] in EPC6 and LPC6. D-[3H]asp uptake assay was performed incubating cells in HBSS containing 100 lM of unlabeled D-asp plus 0.49 lCi mL-1 3 D-[ H]asp during 60–180 min. Data were expressed as mean ± SEM. * = P \ 0.05 (Two-way ANOVA followed by Bonferroni post hoc test). n = 4–13 experiments performed in triplicate

Fig. 6 Release of intracellular content of [3H] derived from L-[3H]glu or D-[3H]asp. Cultures taken up L-[3H]glu or D-[3H]asp plus unlabeled 100 lM of L-glu or D-asp, respectively, during 60 min. After, cells were washed and re-incubated during 60 min only with HBSS containing 100 lM of unlabeled L-glu or D-asp. Intracellular and extracellular radioactivities were quantified by scintillation. (a) 3 3 3 L-[ H]glu-derived intracellular and extracellular [ H]. (b) D-[ H]asp-

Although glutamate is taken up primarily by high affinity Na?-dependent glutamate transporters [1], other low affinity transporter systems could be involved in extracellular glutamate uptake [1, 46]. Therefore, in order to verify whether transport is mediated by the low affinity transporters, we treated cultures with specific inhibitors (PDC and TBOA) of the high affinity Na?-dependent glutamate transporters.

derived intracellular and extracellular [3H]. (c) Radioactivity efflux rate derived from L-[3H]glu. (d) Radioactivity efflux rate derived from D-[3H]asp. Data were expressed as mean ± SEM. * = P \ 0.05 and ** = P \ 0.001 (Two-way ANOVA followed by Bonferroni post hoc test for a and b; Student T test for c and d). n = 3–8 experiments performed in triplicate

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Fig. 7 Inhibition of EAAT3dependet uptake in EPC6 and LPC6 cultures by 100 lM PDC or 100 lM TBOA. Uptake was assessed after 120 min of incubation in HBSS containing inhibitors and (a) L-[3H]glu or (b) D-[3H]asp. Data were expressed as mean ± SEM. * = P \ 0.01 and ** = P \ 0.001; a and b = P \ 0.01 when compared to respective control groups (without blockers) (Two-way ANOVA followed by Bonferroni post hoc test). n = 3–8 experiments performed in triplicate

Treatment with PDC and TBOA reduced approximately 70 % (26.60 ± 0.95 nmol/mg protein; n = 6; P \ 0.001, two-way ANOVA) and 80 % (14.19 ± 1.60 nmol/mg protein; n = 6; P \ 0.001, two-way ANOVA) of intracellular 3 3 L-[ H]glu-derived [ H] in EPC6 cultures, respectively (Fig. 7a). For LPC6 cultures, uptake blockade by PDC and TBOA decreased approximately 40 % (20.00 ± 1.56 nmol/ mg protein; n = 6; P \ 0.001, two-way ANOVA) and 60 % (12.57 ± 1.61 nmol/mg protein; n = 6; P \ 0.001, twoway ANOVA) the L-[3H]glu-derived intracellular [3H], respectively (Fig. 7a). During D-[3H]asp uptake assay, EPC6 exhibited a reduction of 50 % (57.13 ± 2.07 nmol/mg protein; n = 4; P \ 0.001, two-way ANOVA) and 80 % (20.06 ± 0.98 nmol/mg protein; n = 3; P \ 0.001, two-way ANOVA) of its intracellular content of [3H] when treated with PDC and TBOA, respectively (Fig. 7b). However, in LPC6 cultures, only TBOA treatment was able to reduce significantly the intracellular content of D-[3H]asp-derived [3H] (28.33 ± 0.8 nmol/mg protein; n = 3; P \ 0.001, two-way ANOVA) (Fig. 7b). Taken together, these results suggest that L-[3H]glu (Fig. 3) and D-[3H]asp (Fig. 5) uptake occurred mainly through the high affinity Na?-dependent glutamate transporter EAAT3. 3 L-[ H]glu

Uptake Stimulation

For primary astrocyte cultures, it was shown that extracellular glutamate can stimulate glutamate uptake in a dose

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and time-dependent manner (ED50 of 40 lM of glutamate) and that this uptake stimulation occurs mainly by an increase of translocation of actin-dependent glutamate transporters to cell surface [47]. Therefore, in order to verify if the low capacity of LPC6 cells to maintain intracellular radioactivity taken up as L-[3H]glu (Fig. 3) was related to an impairment of EAAT3 translocation, we evaluated glutamate uptake after a treatment with PMA, a compound that stimulates EAAT3 translocation to C6 plasma membrane [48]. For EPC6, treatment with PMA increased in approximately 50 % (14.66 ± 1.80 nmol glu/ mg prot, n = 3, P \ 0.05, two-way ANOVA) the intracellular [3H] derived from L-[3H]glu when compared to DMSO treated cultures (Fig. 8). However, PMA treatment did not alter L-[3H]glu-derived intracellular [3H] in LPC6. These results suggest that LPC6 exhibit an impaired PMAdependent translocation of EAAT3 to cell surface and that it could be contributing to the diminished capacity of these cells to take up L-[3H]glu (Fig. 3). Cellular Senescence and Multinuclear Cells As shown on Fig. 1, LPC6 cells presented some morphological features that resemble the well-known characteristics of senescent cells, mainly in comparison to EPC6 cells, such as flat and large cell shape [49]. Moreover, since some reports have shown that replicative senescent cells exhibit an impaired actin-dependent translocation of intracellular proteins [50, 51], we investigated the cellular senescence in

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both EPC6 and LPC6 cultures using SA- b-gal assay and NMA [42]. While only 2 % of EPC6 were labeled for b-gal and only 4 % of cells in this culture showed enlarged nuclei (Fig. 9a), about 20 % of LPC6 were b-gal positive and 44 % showed large nucleus area (Fig. 9b). These results suggest that LPC6 cells presented higher levels of basal senescence in comparison to EPC6 cells. Furthermore, we observed more multinuclear cells in LPC6 cultures (2.57 %) than in EPC6 (0,16 %) (Fig. 9a, b). Taken together, these results may suggest that LPC6 cultures showed a large percentage of cells undergoing a basal senescence process.

Discussion

Fig. 8 PMA-mediated stimulation of L-[3H]glu uptake. PMA treatment (100 nM) was performed during 15 min before L-[3H]glu uptake assay. Data were expressed as mean ± SEM. * = P \ 0.05 (Twoway ANOVA followed by Bonferroni post hoc test). n = 3 experiments performed in triplicate

C6 lineage has been widely used as a model to investigate several aspects of cell biology. However, depending on the number of passages in culture, this lineage presents variations in their morphological (Fig. 1) and biochemical characteristics [12, 13, 15, 18]. EPC6 have been widely used as a glioblastoma model for both in vivo and in vitro studies [5, 7, 52], whereas LPC6 have some biochemical and physiological characteristics that resemble mature astroglia [17, 18, 25]. Despite the fact that C6 cells do not express glial high affinity Na?-dependent glutamate transporters [33–35], the

Fig. 9 Evaluation of multinuclear cells and cell senescence by b-galactosidase stain (b-gal) and nuclear morphometric analysis (NMA) in EPC6 (a) and LPC6 (b). Data were expressed as percentage of the total number of cells. NII = Nuclear irregular index

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latter cellular features, associated to an easy cultivation procedure, led some authors to consider LPC6 as an useful cellular model to study several glial cell properties and functions [29, 32, 53], including the effect of chemical modulators on Na?-dependent high-affinity glutamate uptake [30, 32]. However, to date there are no reports in literature evaluating structural and functional properties of glutamate transporters in LPC6 as well as comparing these properties to those exhibited by EPC6 and primary astrocyte cultures. Here we showed that when C6 cells were incubated with 3 L-[ H]glu, both EPC6 and LPC6 displayed a time-dependent increase of L-[3H]glu-derived intracellular [3H], which reached maximal levels from 120 min of incubation (Fig. 3). However, EPC6 presented approximately a 2-fold higher intracellular [3H] derived from L-[3H]glu from 60 min of incubation when compared to LPC6 (Fig. 3). Since LPC6 cultures have been considered a cellular model of mature astroglia, it would be expected that these cells had shown higher rates of glutamate uptake when compared to EPC6, the glioblastoma model. These rates of C6 glutamate uptake were remarkably lower than those of primary astrocyte cultures [36]. One factor that could be involved in this LPC6 reduced glutamate uptake capacity could be the loss of plasma membrane integrity, however both cultures displayed a similar profile of PI incorporation in the absence or presence of glutamate (Supplementary Fig. 3). Although glutamate is taken up primarily by high affinity Na?-dependent glutamate transporters [1], there are others low affinity transporter systems that could be capturing extracellular glutamate [1, 46]. Treatment with PDC and TBOA, two specific inhibitors of high affinity Na?-dependent glutamate transporters, reduced L-[3H]glu uptake in EPC6 and LPC6 (Fig. 7a), which was similar to data observed in astrocyte cultures and hippocampal slices [37]. This result indicates that other low affinity glutamate uptake systems are not involved in the elevated capacity of EPC6 to accumulate [3H] (Fig. 3). In addition, it points to EAAT3 as the main high affinity Na?-dependent glutamate transporter responsible for the uptake of L-[3H]glu in EPC6 and LPC6 (Fig. 4) [33, 35, 44]. Another factor that could be related to the ability of LPC6 to maintain lower intracellular levels of L-[3H]gluderived [3H] (Fig. 3) could be an increased capacity to release it to extracellular medium. After 60 min of L[3H]glu uptake, EPC6 and LPC6 cultures released 50 % of their intracellular [3H] (Fig. 6a). However, LPC6 have a higher efflux rate of radioactivity derived from L-[3H]glu than EPC6 (Fig. 6c). This result indicates that LPC6 could release this intracellular radioactivity a little faster than EPC6, which contributes to the 2-fold lower intracellular levels of L-[3H]glu-derived [3H] in these cells, but not being responsible for all of this difference (Fig. 3).

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Moreover, these results may explain the maximal levels of radioactivity from 120 min observed in Fig. 3 through the establishment of a steady-state between L-[3H]glu uptake and release of intracellular [3H]. In addition to the role of glutamate as a neurotransmitter, for several cells types, including C6 cells, glutamate plays an important role as a metabolic substrate [54]. Exposure of EPC6 and LPC6 to D-[3H]asp, a non metabolizable analogue of L-[3H]glu [37], resulted in a timedependent increase in D-[3H]asp-derived intracellular [3H] in both cultures (Fig. 5) and resulted in a lower efflux rate of radioactivity derived from D-[3H]asp in both cultures (Fig. 6d). Since D-[3H]asp accumulate within cells (Fig. 6b), this result indicates that which was being released in Fig. 6a is a metabolite that contains [3H] derived from L-[3H]glu. Although our results suggest that 3 L-[ H]glu was metabolized in Fig. 3, this data did not explain the lower capacity of LPC6 to take up glutamate when compared to EPC6. Slightly increase in D-[3H]asp-derived intracellular [3H] in EPC6 at 60-120 min when compared to LPC6 (Fig. 5) may suggest that EPC6 can capture more D-[3H]asp since the beginning of uptake assay, which could be related to distinct profile of EAAT3 surface expression in these cultures. This hypothesis was confirmed after analysis of PMA-stimulation of EAAT3 vesicle trafficking to cell surface, which increased in approximately 50 % the intracellular [3H] in EPC6 and had no effect in LPC6 (Fig. 8). Since total EAAT3 immunocontent was the same for both cultures (Fig. 4), this result indicates that LPC6 exhibit an impaired PMA-dependent EAAT3 vesicle trafficking and that this profile may be related to the lower intracellular levels of [3H] showed in LPC6 cultures (Figs. 3, 5). Some studies have demonstrated that a compromised intracellular protein trafficking is one characteristic of senescent cells [50, 51]. Lim et al. [51] demonstrated that senescent human diploid fibroblasts as well as premature senescent cells, induced by H-Ras double mutants, fail to export actin fiber from nucleus to cytoplasm and fail to translocate pErk 1/2 proteins to cytoplasm in response to MEK activity. In our study, we observed that LPC6 presented an enlarged phenotype when compared to EPC6 (Fig. 1). Furthermore, the majority of C6 cells at later passages showed enlarged nuclei (Fig. 9b), a characteristic of cells entering in senescence, as well as increased b-gal staining, a widely used marker for cellular senescence (Fig. 9b) [42]. Our results suggest that LPC6 culture presented a large amount of cells entering in a basal senescence process, which probably is related to its prolonged cultivation. Due to the fact that some studies have demonstrated that ageing can interfere in organization of actin cytoskeleton [50, 51], which could compromise vesicular

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transport, one of the possible causes of deficiency of EAAT3 vesicle translocation to cell membrane in LPC6 (Fig. 8) is the senescence entry that is triggered along the subculture of C6 cells. Taken together, our findings demonstrated that EPC6 and LPC6 cells highly differed in their capacity to accumulate intracellular [3H] derived from L-[3H]glu and D[3H]asp and that this difference is probably due to a distinct profile of EAAT3 translocation to membrane surface. Besides, lower capacity of LPC6 to mobilize EAAT3 to membrane could be related to higher amount of cells entering in a basal senescence process in these cultures, which could have an impaired vesicular transport due to the low capacity of senescent cells to reorganize their actin cytoskeleton [50, 51]. Since our experiments confirmed that LPC6 did not express glial high affinity Na?-dependent glutamate transporters and that this culture exhibited impairment in glutamate transport, we suggest that C6 lineage is not an adequate cellular model to investigate glial-like functions related to glutamatergic system. Acknowledgments This work was supported by Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES), Instituto Nacional de Cieˆncia e Tecnologia em Excitotoxicidade e Neuroprotec¸a˜o (INCT-EN), FAPERGS and by the FINEP research Grant ‘‘Rede Instituto Brasileiro de Neurocieˆncia (IBN-Net)’’ # 01.06.084200. We are grateful to Elizandra Braganhol for her initial assistance in cultivating C6 glioma cells, Sı´lvia Terra and Rafael Zanin for assistance in flow cytometer, Eduardo Rico for assistance in GS enzymatic activity, Fa´bio Klamt for assistance in sulforhodamine B test and Prof. Renato Dutra Dias for his critical review of the manuscript.

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