Cell Mol Neurobiol DOI 10.1007/s10571-015-0160-3
ORIGINAL RESEARCH
Ouabain and BDNF Crosstalk on Ganglion Cell Survival in Mixed Retinal Cell Cultures Gustavo de Rezende Correˆa • Vinicius Henrique Pedrosa Soares Leandro de Arau´jo-Martins • Aline Araujo dos Santos • Elizabeth Giestal-de-Araujo
•
Received: 28 October 2014 / Accepted: 27 January 2015 Ó Springer Science+Business Media New York 2015
Abstract Brain-derived neurotrophic factor (BDNF) is a well-known and well-studied neurotrophin. Most biological effects of BDNF are mediated by the activation of TrkB receptors. This neurotrophin regulates several neuronal functions as cell proliferation, viability, and differentiation. Ouabain is a steroid that binds to the Na?/K? ATPase, inducing the activation of several intracellular signaling pathways. Previous data from our group described that ouabain treatment increases retinal ganglion cells survival (RGC). The aim of the present study was to evaluate, if this cardiac glycoside can have a synergistic effect with BDNF, the classical trophic factor for retinal ganglion cells, as well as investigate the intracellular signaling pathways involved. Our work demonstrated that the activation of Src, PLC, and PKCd participates in the signaling cascade mediated by 50 ng/mL BDNF, since their selective inhibitors completely blocked the trophic effect of BDNF. We also demonstrated a synergistic effect on RGC survival when we concomitantly used ouabain (0.75 nM) and BDNF (10 ng/mL). Moreover, the signaling pathways involved in this synergistic effect include Src, PLC, PKCd, and JNK.
Aline Araujo dos Santos and Elizabeth Giestal-de-Araujo have contributed equally to this work. G. de Rezende Correˆa (&) V. H. P. Soares L. de Arau´jo-Martins A. A. dos Santos E. Giestal-de-Araujo Departamento de Neurobiologia, Programa de Neurocieˆncias, Instituto de Biologia, Universidade Federal Fluminense, Outeiro de Sa˜o Joa˜o Batista s/n, Nitero´i, Rio de Janeiro CEP 24020-140, Brazil e-mail: [email protected] A. A. dos Santos Departamento de Fisiologia e Farmacologia, Instituto Biome´dico, Universidade Federal Fluminense, Nitero´i, Rio de Janeiro CEP 24210-130, Brazil
Our results suggest that the synergism between ouabain and BDNF occurs through the activation of the Src pathway, JNK, PLC, and PKCd. Keywords Ouabain BDNF Retina Neuronal survival Synergistic effect
Introduction Ouabain is a glycoside used for many years to treat cardiac dysfunction. This therapeutic application involves an increase in intracellular calcium concentration observed after ouabain treatment: as ouabain binds to the Na?, K?ATPase a-subunit, an inhibition of ion transport takes place, leading to the accumulation of intracellular Na? and a consequent increase in intracellular calcium through the Na?/Ca2? exchanger (Blanco and Mercer 1998). Interestingly, endogenous ouabain has also been identified (Hamlyn et al. 1991), its plasma concentration ranging from 50 pM to 80 nM (Goto et al. 1992; Gottlieb et al. 1992; Schoner et al. 2003). Many intracellular pathways are activated following ouabain binding to Na?, K?-ATPase. They involve distinct families of enzymes such as PKC, CAM kinase, and Src kinases, as well as the Ras/Raf/MEK/ MAP kinase signaling pathway (Mohammadi et al. 2003). The transactivation of EGF receptors by ouabain treatment can also stimulate the Ras/MAPK cascade (Haas et al. 2002). Since its discovery in 1982 (Barde et al. 1982), BDNF became a well-studied neurotrophin in the nervous system. Data from different groups demonstrate the role of this trophic factor in the control of cell survival, proliferation, and differentiation during the development as well as during the adult life (Uchida et al. 2013; de Almeida et al.
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2013). BDNF actions are carried out by activation of TrkB receptor subtype, two BDNF molecules being required for the receptor activation. After activation, several signaling pathways become effective, among them the phospholipase C (PLC), the PI3K, and the Ras–Raf–MEK–MAPK pathways (Bagayogo and Dreyfus 2009). The effect of several trophic factors on retinal ganglion cell survival has been extensively described. Specifically, the importance of neurotrophins both in vitro and in vivo has been demonstrated in many species, including the rat (Pan and Zhang 2013; Chung et al. 2013; Ebel et al. 2013; Ramos-Languren and Escobar 2013). The effects of BDNF and NT-4 on retinal ganglion cell survival indicate that these neurotrophins play an important role during retinal development. Neurotrophins can also rescue ganglion cells after injury, pointing toward the clinical importance of these studies (Isenmann et al. 2003). Previous results from our group demonstrated that ouabain has a trophic effect on the population of retinal ganglion cells (RGC), increasing its viability after 48 h in culture. The best result was obtained with a concentration of 3 nM (100 % increase when compared to the 48 h control cultures). It was also observed that this effect involves the activation of Src, EGFR, and PKC. Paradoxically, JNK, a protein well known for its effects on the induction of cell death (Sabapathy 2012; Davies and Tournier 2012) is involved in the trophic effect of ouabain (Correˆa et al. 2005, 2010). It is well known that BDNF has a trophic effect in retinal ganglion cells (Chen and Weber 2001). However, in our experimental setup, the BDNF role in triggering or regulating the intracellular pathways involved in the effects has not been investigated. For that reason, in the present work, we initially investigated the intracellular pathways responsible for the trophic effect of BDNF on retinal ganglion cell survival. Our objective was to ascertain that 50 ng/mL of BDNF stimulates the same pathways we had previously identified as activated by ouabain. Moreover, we also evaluated a possible synergistic effect of low doses of BDNF and ouabain, on retinal ganglion cell survival, in order to establish if ouabain could potentiate the trophic effect of BDNF and the intracellular pathways involved.
Experimental Procedures Ethical Aspects Procedures using animals were approved by the local committee for animal care and experimentation (CEPAprojects #00196-10). All efforts were made to minimize the number of experimental animals used and their suffering.
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Materials Medium 199, fetal bovine serum, PD98050, and LY284002 were purchased from GIBCO (Gaithersburg—USA). Glutamine, streptomycin, penicillin, poly-L-ornithine, ouabain, and peroxidase (HRP) were purchased from Sigma–Aldrich (St. Louis—USA). Dimethyl sulfoxide (DMSO) and glutaraldehyde were purchased from Mallinckrodt Baker (Paris—USA) and Entellan came from Merck (Darmstadt— Germany). EGF and BDNF were purchased from PeproTech (Rocky Hill, NJ, USA). AG1478, SB203580, PDTC, and JNK inhibitor came from Calbiochem–Merck (Darmstaddt—Germany). Chelerythrine chloride, K252a, PP1, and U73122 were purchased from Biomol (Plymouth Meeting, PA, USA). Rottlerin and BAPTA-AM were purchased from Invitrogen (USA). Trypsin was purchased from Worthington (USA). The antibodies used came from Santa Cruz Biotechnology (USA). Labeling of Retinal Ganglion Cells Within the first 24 h after birth, Lister Hooded rats were anaesthetized by hypothermia. One microliter of a solution of 30 % HRP in 2 % DMSO was injected directly into each superior colliculus. This procedure was possible because in newborn rats, the mesencephalic region is not covered by the cerebral cortex (Araujo and Linden 1993). The animals were returned to their mothers and survived for *16 h prior to the procedures for cell culture. Retinal Cultures Primary cultures were prepared using previously described techniques (Santos and Araujo 2000). Briefly, neonatal rats at postnatal day 1 were killed by decapitation and the retinas dissected free from scleral tissue and pigmented epithelium in a calcium- and magnesium-free balanced salt solution (CMF) containing 100 lg/mL streptomycin ? 100 U/mL penicillin. The tissue was then incubated in CMF containing 0.1 % trypsin for approximately 16 min at 37 °C. Trypsin action was stopped by addition of culture medium (199 medium supplemented with 2 mM glutamine, 100 lg/mL streptomycin, and 100 U/mL penicillin) with 5 % FCS. The tissue was then resuspended in complete culture medium and mechanically dissociated using a polished Pasteur pipette. Cells were added to Petri dishes or Petri dishes containing coverslips, both previously treated with poly-L-ornithine (50 lg/mL) at a plating density of 105 cells/cm2. After plating, cultures were incubated in 1 mL of culture medium for 2–4 h to allow cells to attach to the substrate. Then, 1 mL of culture medium or 1 mL of medium containing the drugs to be tested was added to each Petri dish. In our experimental procedure,
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drug treatment was kept over the whole time in culture. Medium-insoluble drugs were previously diluted in DMSO. The effect of diluted DMSO on retinal ganglion cell survival was evaluated as a control, and we did not find any changes using dilutions up to 1:100 (data not shown). Drugs were added once, after 2–4 h in culture. The cultures were then maintained between 45 min and 48 h in vitro at 37 °C in a humidified atmosphere of 5 % CO2 and 95 % air.
phospho-CREB antibody (Ser-133) (1:400). The membranes were washed in TBS and were then exposed to horseradish peroxidase-conjugated secondary anti-rabbit or anti-mouse IgG antibody at 1:2,000 and 1:80,000 dilutions, respectively, at room temperature for 60 min. Detection was performed on X-ray film (hyperfilm—Amersham) using chemiluminescence system (ECL). The density of protein bands was analyzed by densitometry with Scion Image. The mean value for the control was set at 100 %.
Identification of Retinal Ganglion Cells Statistical Analysis The presence of the enzyme HRP in the cytoplasm of retinal ganglion cells was revealed according to the protocol of (Mesulan 1982). Briefly, monolayers were fixed with a mixture of 1 % paraformaldehyde and 2 % glutaraldehyde in 0.1 M sodium phosphate buffer (Karnovsky solution) for 5 min, washed in phosphate buffer and reacted with tetramethylbenzidine and H2O2. After reaction, the coverslips were washed in 0.2 M acetate buffer, dehydrated by air drying, immersed briefly in xylene and mounted in Entellan. Quantification of Retinal Ganglion Cells Retinal ganglion cells were counted using an Olympus BX41 (Tokyo, Japan) microscope at a magnification of 9400, under bright field. As an internal control for the variable percentage of ganglion cells labeled with HRP in distinct experiments, the number of labeled cells at 4 h in culture was taken as 100 % and the results were reported as percentage of this control. Approximately, 800 retinal ganglion cells were labeled in 4 h control coverslips. Independently from the number of labeled cells, the 48 h survival was always in the same range (40–60 %). All data were expressed as mean ± standard error of the mean from four independent experiments. Each individual experiment was performed at least in duplicate. Western Blot Phospho-NFj-B, phospho-CREB, phospho-Src, and phospho-JNK levels were determined by Western blot analysis. Retinal cell cultures were lysed by sonication in lysis buffer (2 % sodium dodecyl sulfate and 0.5 M Tris pH 6.8). After determination of protein concentration by the Bradford method (Bradford 1976), samples (60 lg/ lane) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (9 %) and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were incubated overnight with rabbit anti-phosphoNFj-B antibody (Ser-377) (1:200) or mouse anti-phosphoJNK antibody (Thr-183 and Thr-185) (1:400) or rabbit antiphospho-Src antibody (Tyr-416) (1:400) or rabbit anti-
The overall statistical analysis was first obtained by oneway analysis of variance (ANOVA). The statistical significance of all pairs of multiple groups of data was assessed by Newman–Keuls comparison test. A value of P \ 0.05 was considered significant.
Results We started our analyses by evaluating the dependence of the BDNF protective effect on Src activation. We used PP1 (1 lM), a selective inhibitor of this enzyme, in the presence of 50 ng/mL BDNF and evaluated the retinal ganglion cells survival. The results in Table 1 show that the Src pathway is involved in the trophic effect of BDNF on retinal ganglion cells survival since PP1 blocked the effect of this neurotrophin. Since the effect of ouabain on retinal ganglion cells survival involves the phosphorylation of EGF receptors (Correˆa et al. 2010), we treated the cultures with an inhibitor of these receptors (2.5 lM AG1478) in the presence of 50 ng/mL BDNF. Our results indicate that the signaling pathway of BDNF does not involve the activation of EGF receptors as the AG1478 did not block the trophic effect of this neurotrophin (Table 2). The next step was to evaluate the involvement of phospholipase C (PLC) in the BDNF signaling pathway with the use of a selective inhibitor of this enzyme (4 lM U73122). The data present in Table 1 show a complete blockade of the effect of BDNF in retinal ganglion cells survival when this neurotrophin was concomitantly used with U73122. Our previous results demonstrated the involvement of protein kinase C (PKC) signaling pathway in the trophic effect of ouabain in retinal ganglion cells (Correˆa et al. 2005). We demonstrated that PKCd, a member of the novel class of the PKC family, is the isoenzyme involved on the ouabain effect (Correˆa et al. 2010). Thus, we decided to examine whether this protein kinase is involved in the signaling pathway of BDNF. Treatment of retinal cell cultures with 1.25 lM chelerythrine chloride (CC), an
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Cell Mol Neurobiol Table 1 The trophic effect of 50 ng/mL BDNF on RGC survival is mediated by Src, PLC, and PKCd Treatment
(% of control) CT 48 h
BDNF
-
SEM
?
SEM
-
SEM
?
SEM
PP1 (1 lM)
52.43
3.80
51.30
1.48
96.94
2.75
48.05*
4.60
U73122 (4 lM)
52.43
3.80
47.88
1.90
96.94
2.75
46.09*
4.40
CC (1.25 lM) ROT (2 lM)
53.55 52.50
4.50 4.20
58.28 49.12
6.40 6.40
96.92 109.62
6.60 6.70
54.86* 51.67*
3.50 4.60
Cells were kept in culture for 48 h. CT control cultures. Cells were treated with BDNF and inhibitors once, after 2–4 h in culture. The following inhibitors were used: 1 lM PP1 (Src); 4 lM U73122 (phospholipase C); 1.25 lM chelerithrine chloride (CC) (protein kinase C); 2 lM rottlerin (ROT) (PKCd). Data are reported as the mean ± SEM (n = 12). P \ 0.001, compared to the 48 h control * Significantly reverts the BDNF effect
Table 2 The trophic effect of 50 ng/mL BDNF on RGC survival is not mediated by an increase in intracellular calcium levels and EGF receptors activation Treatment
% of control CT 48 h
BDNF
-
SEM
?
SEM
-
SEM
?
SEM
AG1478 (2.5 lm)
59.70
9.70
55.45
9.80
103.74
3.35
100.07
4.50
BAPTA-AM (20 lm)
52.43
4.00
49.62
2.40
109.62
6.70
98.60
4.20
Cells were kept in culture for 48 h in the presence of complete culture medium (CT control cultures), or cells were treated with BDNF and inhibitors once, after 4 h in culture. The following inhibitors were used: 2.5 lM AG1478 (EGF receptor inhibitor) or 20 lM BAPTA-AM (an intracellular calcium chelator). Data are reported as the mean ± SEM (n = 12). P \ 0.001, compared to the 48 h control
inhibitor of PKC, abolished the trophic effect of BDNF (Table 1). To investigate the role of intracellular calcium levels in the BDNF effect, we used 20 lM BAPTA-AM (an intracellular calcium chelator). The results present in Table 2 indicate that an increase in intracellular calcium concentration is not necessary for the BDNF effect. Based on this last result and on the fact that the isoenzymes of the novel class of the PKC family are independent on calcium for their activation, we decided to analyze if PKCd is also involved on the BDNF effect. Our results show that 2 lM rottlerin (a PKCd inhibitor) completely blocked the BDNF effect (Table 1). Since 50 ng/mL BDNF and 3 nM ouabain isolated exert trophic effects on RGC maintained in culture for 48 h, we analyzed if the treatment with low doses of and BDNF (10 ng/mL) and ouabain (0.75 nM) could promote a synergistic action on the survival of this neuronal population. As can be seen in Fig. 1a, when both treatments were used alone, they are not able to increase RGC survival. However, when concomitantly used, they induce a significant increase in RGC survival. This result confirms that there is a synergism between ouabain and BDNF on RGC survival. Based on this result, we decided to investigate the intracellular pathways involved in the synergistic effect. We started by analyzing the role of TrkB receptors on it. The
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cultures were treated with 50 nM K252a, an inhibitor of Trk receptors, and we observed that K252a totally abolished the synergistic effect (Fig. 1b). Based on our previous results, we decided to analyze the pathways that are activated by 3 nM ouabain (Correˆa et al. 2005, 2010) and also by 50 ng/mL BDNF on retinal ganglion cells survival. To ascertain the involvement of Src, we used PP1 (1 lM) and we observed that this inhibitor blocked the synergistic effect (Fig. 2a). Analysis of the Src levels indicated the participation of this protein in the synergistic effect, confirming the results obtained previously. It can be observed in Fig. 2b that the individual treatments with 0.75 nM ouabain or 10 ng/mL BDNF do not affect the phosphorylation levels of this protein. However, the combined treatment, for 45 min, significantly elevates their levels (Fig. 2b, c). To evaluate the involvement of PLC in the synergistic effect, we used 4 lM U73122. As can be observed in Fig. 3a, U73122 completely abolished the synergistic trophic effect. We then analyzed the role of PKCd in the synergistic effect. Data present in Fig. 3b show that 2 lM rottlerin totally blocks the effect of ouabain and BDNF on retinal ganglion cell survival. We also studied a possible involvement of JNK in the synergistic effect using its specific inhibitor at the concentration of 1 lM. The results
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Fig. 1 Synergistic effect of ouabain and BDNF on RGC survival. Cells were kept in culture for 48 h in the presence of complete culture medium. a The simultaneous treatment with 10 ng/mL BDNF and 0.75 nM ouabain significantly increased the RGC survival. However, the treatment with 10 ng/mL BDNF or 0.75 nM ouabain did not
increase the survival of RGC. b The synergistic effect of ouabain and BDNF was totally abolished by the presence of 50 nM K252a (an inhibitor of Trk receptors). CT control, OUA ouabain. Data are reported as the mean ± SEM (n = 12). P \ 0.001, compared to the 48 h control
Fig. 2 The synergistic effect of 0.75 nM ouabain and 10 ng/mL BDNF is mediated by Src. Cells were kept in culture in the presence of complete culture medium. a The inhibition of Src (1 lM PP1) abolished the synergistic effect on RGC survival after 48 h in culture. b The graph shows the levels of p-Src in cultures treated with OUA, BDNF and BDNF ? OUA for 45 min. c Representative blot of samples. CT control, OUA ouabain. Data are reported as the mean ± SEM (n = 12). P \ 0.001 compared to the 45 min (blot) or 48 h (survival) control
represented in Fig. 3b show that the inhibition of JNK blocks the synergistic effect and Fig. 3c show that the combined treatment induces a decrease in the levels of phospho-JNK (Fig. 3c, d). To further investigate the signaling pathways, we decided to assess which transcription factor might be involved on the effects of BDNF 50 ng/mL and ouabain 3 nM. We analyzed the role of NFj-B using a selective inhibitor of this protein (PDTC 20 nM). Figure 4a clearly shows that the NFj-B is involved in the effect of ouabain 3 nM on retinal ganglion
cell survival. However, the survival pathways activated by BDNF do not involve the activation of this transcription factor (Fig. 4b). Figure 4c and d shows the levels of phospho-NFj-B after ouabain, BDNF, and the combined treatment. We did not observe changes in its levels in any of these three conditions. Figure 4e shows that NFj-B does not participate in the trophic effect promoted by the treatment with ouabain and BDNF in low doses. We next investigated a possible role of the transcription factor CREB, analyzing the change in the levels of
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Cell Mol Neurobiol Fig. 3 The synergistic effect of 0.75 nM ouabain and 10 ng/mL BDNF is mediated by PLC, PKCd, and JNK. Cells were kept in culture in the presence of complete culture medium. a The inhibition of phospholipase C (4 lM U73122) abolished the synergistic effect after 48 h in culture. b The blockade of PKCd (2 lM ROT) or JNK (1 lM iJNK) also inhibited the synergistic effect after 48 h in culture. c The graph shows the levels of p-JNK in different treatments. Both BDNF and BDNF ? OUA treatments for 45 min induced a decrease in the p-JNK levels. d Representative blot of samples. CT control, OUA ouabain, U73122 phospholipase C selective inhibitor, ROT rottlerin, iJNK JNK inhibitor. Data are reported as the mean ± SEM (n = 12). P \ 0.001 compared to the 45 min (blot) or 48 h (survival) control
phospho-CREB in retinal cells cultures treated for 45 min. Figure 5 shows that BDNF (10 ng/mL) treatment alone decreases phospho-CREB levels, while ouabain treatment (0.75 nM) increases these levels. Accordingly, the combined treatment (OUA ? BDNF) did not significantly affect the phosphor-CREB levels.
Discussion The literature strongly supports the trophic effect of BDNF on retinal ganglion cells (von Bartheld 1998). For this reason we studied, in the present work, if ouabain could potentiate the effect of this classical trophic factor on retinal ganglion cells. Our data show that simultaneous treatment with BDNF 10 ng/mL and ouabain 0.75 nM increases the survival of retinal ganglion cells kept in mixed retinal cell cultures for 48 h. In fact this treatment is able to maintain all retinal ganglion cells initially plated suggesting that this protocol induces a trophic effect on axotomized neurons in vitro. As the isolated treatments with ouabain or BDNF, in such low concentrations, do not increase the survival of ganglion cells, we conclude that the effect occurs by a synergistic action of these two molecules.
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Data from the literature show that ouabain binding to Na?/K?-ATPase induces the activation of intracellular pathways involving protein–protein interactions, promoting responses such as cell proliferation and differentiation (Xie and Askari 2002). This mechanism of signal transduction is triggered by low concentrations of ouabain and does not appear to alter the cell ionic concentration (Scheiner-Bobis and Schoner 2001). Corroborating Correˆa and co-workers, using ouabain in a quite low concentration (3 nM) demonstrated that this treatment induced a trophic effect on retinal ganglion cell population (Correˆa et al. 2005). These results are in agreement with data reported by Dvela, in 2012, showing that the endogenous concentration of ouabain corresponds to nanomolar levels (Dvela et al. 2012). It is interesting to note that the low concentration used by Correˆa et al. is equivalent to the physiological concentration of endogenous ouabain, therefore, in the nanomolar level. From the analysis of the signaling pathways of BDNF in our experimental model, we found some of them in common with those trigged by ouabain and previously described: Src, PLC, and PKCd. According to these data, we decided to check whether these pathways are actually used to elicit the synergism between BDNF and ouabain (Correˆa et al. 2005, 2010). In the present paper, we demonstrate
Cell Mol Neurobiol Fig. 4 The involvement of NFjB in the trophic effect elicited by OUA in RGC survival. a The treatment with 20 nM PDTC (an inhibitor of NFjB) abolished the trophic effect of 3 nM OUA in RGC. b However, the trophic effect of 50 ng/mL BDNF and OUA ? BDNF (e) is not mediated by NFjB. c The graph shows the levels of p-NFjB in different experimental conditions. It was not observed any difference between the conditions analyzed. d Representative blot of samples. e NFj-B does not participate in trophic effect promoted by treatment with ouabain and BDNF in low doses. CT, control; 0.75 nM OUA, ouabain; 20 nM PDTC, NFjB selective inhibitor. Data are reported as the mean ± SEM (n = 12). P \ 0.001 compared to the 45 min (blot) or 48 h (survival) control
that Src is also participating in BDNF effect on retinal ganglion cell survival as well as in the synergistic effect of ouabain and BDNF. Our data concerning the involvement of Src in the BDNF effect are in agreement with previous results of Vries and colleagues. This group demonstrated in 2010 that following BDNF binding to its receptor, Src activation occurs (Vries et al. 2010). Also in 2010, You and colleagues demonstrated in the brain of adult mice the involvement of the Src protein subtype D (SrcD) in signal transduction of TrkB receptors, via SH3 and PTB domains present on these receptors (You et al. 2010). Our previous data show that EGFR was involved in the trophic effect of ouabain on retinal ganglion cells (Correˆa et al. 2010). However, after investigating the possible involvement of EGFR in the BDNF effect, we did not observe any correlation. Based on this result, we can suggest that the EGF receptor does not mediate the effect of BDNF on retinal ganglion cell survival.
Zhang and colleagues recently demonstrated the involvement of PLC in the effect promoted by BDNF on glutamate release in cortical neurons (Zhang et al. 2013). Bagayogo and Dreyfus, in 2009, indicated the participation of the PLC pathway in the effect of BDNF on oligodendrocytes (Bagayogo and Dreyfus 2009). Our previous data showed the involvement of PLC in the signaling pathway activated by ouabain treatment in neonatal rats RGC (Correˆa et al. 2010), and in the present work, we observe that this enzyme is also activated in BDNF effect and in the synergism between BDNF and ouabain. Protein kinase C family plays an important role controlling proliferation, differentiation, survival, neuritogenesis, and apoptosis in the nervous system (Zhao et al. 2012; Pinzon-Guzman et al. 2011; Santos and Araujo 2000). In 2008, Tomimatsu and Arakawa demonstrated that BDNF induces a decrease in the damage induced by glutamatergic excitotoxicity in motoneurons via PKC activation (Tomimatsu and
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Fig. 5 The dual effect of 0.75 nM OUA and 10 ng/mL BDNF in p-CREB levels. a Levels of p-CREB levels are increased in cultures treated for 45 min with OUA and decreased in cultures treated with BDNF. However, the combined treatment with OUA ? BDNF did not significantly influence the levels of p-CREB. b Representative blot of samples. CT control, OUA ouabain. Data are reported as the mean ± SEM (n = 4). P \ 0.001 compared to the 45 min control
Arakawa 2008). In accordance, our results show that both BDNF effect and the synergistic effect of ouabain and BDNF depend on PKC activation, specifically of PKCd. It was previously demonstrated by our group that PKCd was involved in the survival of retinal ganglion cells stimulated either by ouabain or by phorbol 12-myristate 13-acetate (PMA), an activator of the conventional and novel isoforms of PKC (Correˆa et al. 2010). Literature shows the involvement of JNK in progressive phenomena of nervous system development such as neuritogenesis and cell survival. NGF induces neuritogenesis in PC12 cells via activation of JNK (Waetzig et al. 2008), and in HepG2 cells, the JNK activation mediates their survival (Granado-Serrano et al. 2009). JNK can be activated by PKC and calcium-calmodulin, and is involved in the activation of the transcription factor AP-1. This factor regulates cellular survival, proliferation, differentiation, and gene expression (Schoner and Scheiner-Bobis 2007). Sanna and co-workers in 2002 demonstrated that JNK regulates the survival of human embryonic kidney cells via the activation of the proteins that inhibits the apoptosis (IAP) (Sanna et al. 2002). Our data are in accordance with these previous results since
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JNK is involved in the synergistic effect of BDNF and ouabain. We have demonstrated the role of the transcription factor NFj-B on the ouabain effect. However, this transcription factor does not mediate the trophic effect on retinal ganglion cells of BDNF alone neither that of ouabain and BDNF in low doses. Literature shows that CREB is an important transcription factor involved in the BDNF synthesis in neurons (Nibuya et al. 1996). Data from our group already demonstrated that a chronic treatment of retinal cell cultures with BDNF, for 48 h, decreases the levels of BDNF, phospho-TrkB, and phospho-CREB (Perı´golo-Vicente et al. 2013). Our results presented here are in agreement with this previous one and also show that the treatment with ouabain induces an increase in the phospho-CREB levels. Future work will be performed in order to elucidate the intracellular pathways involved in the increase of phospho-CREB levels following ouabain treatment. In this work, we showed for the first time that ouabain can potentiate the trophic effect of BDNF in axotomized neuronal cells. We also described that either BDNF or ouabain treatments induce the activation of some intracellular proteins. The mechanisms involved in the crosstalk between ouabain and BDNF are presently not completely known. As this is the first evidence of the crosstalk, data from the literature cannot conveniently support further discussions on the mechanism involved in this effect. Further experiments will be addressed to further investigate which molecules are mediating this crosstalk. The results presented in this study demonstrate a classic synergistic effect of ouabain and BDNF treatments in increasing the survival of retinal ganglion cells maintained in cultures for 48 h, after axotomy, through the activation of the Src, PLC, PKC, and JNK. As ouabain was already used clinically in the treatment of congestive heart failure and in our work its presence potentiates the trophic effect of a classical trophic factor for retinal ganglion cells, ouabain could be used in order to increase neuronal survival following neuronal injuries. Indeed, it was demonstrated that ouabain has a neuroprotective effect after traumatic brain injury (Dvela-Levitt et al. 2014). Treatment with low concentrations of ouabain, in combination with low concentrations of trophic factor, could increase neuronal survival and decrease the side effects observed when drugs were used in higher concentrations. In the future, our results can contribute to the establishment of new therapeutic protocols able to decrease retinal ganglion cells damage following several insults. Acknowledgments We would like to thank Alexandre Jose´ Fernandes, Bernardino Matheus dos Santos and Arnaldo de Sa´ for technical assistance. We also thank Arnaldo Paes de Andrade for
Cell Mol Neurobiol critical reading of our manuscript. Gustavo de Rezende Correˆa, Vinı´cius Henrique Pedrosa Soares and Leandro de Araujo Martins received fellowships from CAPES. This work was supported by Grants from CAPES, PRONEX-MCT, PROPPi and FAPERJ. Conflict of interest interests.
None of the authors declares competing
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Granado-Serrano AB, Martı´n MA, Goya L, Bravo L, Ramos S (2009) Time-course regulation of survival pathways by epicatechin on HepG2 cells. J Nutr Biochem 2:115–124 Haas M, Wang H, Tian J, Xie Z (2002) Src-mediated inter-receptor cross-talk between the Na ?/K ? -ATPase and the epidermal growth factor receptor relays the signal from ouabain to mitogen-activated protein kinases. J Biol Chem 277(24): 18694–18702 Hamlyn JM, Blaustein MP, Bova S, DuCharme DW, Harris DW, Mandel F, Mathews WR, Ludens JH (1991) Identification, and characterization of a ouabain-like compound from human plasma. Proc Natl Acad Sci 88:6259–6263 Isenmann S, Kretz A, Cellerino A (2003) Molecular determinants of retinal ganglion cell development survival, and regeneration. Prog Retin Eye Res 22:483–543 Mesulan MM (1982) Tracing neural connections with horseradish peroxidase, 2nd edn. Willey, Hoboken, p 251 Mohammadi K, Liu L, Tian J, Kometiani P, Xie Z, Askari A (2003) Positive inotropic effect of ouabain on isolated heart is accompanied by activation of signal pathways that link Na ?/ K ? -ATPase to ERK1/2. J Cardiovasc Pharmacol 41(4): 609–614 Nibuya M, Nestler EJ, Duman RS (1996) Chronic antidepressant administration increases the expression of cAMP response element binding protein (CREB) in rat hippocampus. J Neurosci 16(7):2365–2372 Pan M, Zhang C (2013) Stimulatory effect of gonadal hormones on fetal rat hippocampal neural proliferation requires neurotrophin receptor activation in vitro. Neurosci Lett 24(546):1–5 Perı´golo-Vicente R, Ritt K, Pereira MR, Torres PM, Paes-deCarvalho R, Giestal-de-Araujo E (2013) IL-6 treatment increases the survival of retinal ganglion cells in vitro: the role of adenosine A1 receptor. Biochem Biophys Res Commun 430(2): 512–518 Pinzon-Guzman C, Zhang SS, Barnstable CJ (2011) Specific protein kinase C isoforms are required for rod photoreceptor differentiation. J Neurosci 31(50):18606–18617 Ramos-Languren LE, Escobar ML (2013) Plasticity and metaplasticity of adult rat hippocampal mossy fibers induced by neurotrophin-3. Eur J Neurosci 37(8):1248–1259 Sabapathy K (2012) Role of the JNK pathway in human diseases. Prog Mol Biol Trans Sci 106:145–169 Sanna MG, Correia JS, Ducrey O, Lee J, Nomoto K, Schrantz N, Deveraux QL, Ulevitch RJ (2002) IAP suppression of apoptosis involves distinct mechanisms: the TAK1/JNK1 signaling cascade and caspase inhibition. Mol Cell Biol 22:1754–1766 Santos AA, Araujo EG (2000) The effect of PKC activation on survival of rat retinal ganglion cells in culture. Brain Res 853:338–343 Scheiner-Bobis G, Schoner W (2001) A fresh facet for ouabain action. Nat Med 7:1288–1289 Schoner W, Scheiner-Bobis G (2007) Endogenous and exogenous cardiac glycosides: their roles in hypertension, salt metabolism, and cell growth. Am J Physiol Cell Physiol 293:509–536 Schoner W, Bauer N, Mu¨lle-Ehmsen J et al (2003) Ouabain as a mammalian hormone. Ann N Y Acad Sci 986:678–684 Tomimatsu N, Arakawa Y (2008) Protein kinase C-mediated protection of motoneurons from excitotoxicity. Neurosci Lett 439:143–146 Uchida H, Matsushita Y, Ueda H (2013) Epigenetic regulation of BDNF expression in the primary sensory neurons after peripheral nerve injury: implications in the development of neuropathic pain. Neuroscience 14(240):147–154 von Bartheld CS (1998) Neurotrophins in the development and regenerating visual system. Histol Histopathol 13:437–459
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ORIGINAL RESEARCH
Ouabain and BDNF Crosstalk on Ganglion Cell Survival in Mixed Retinal Cell Cultures Gustavo de Rezende Correˆa • Vinicius Henrique Pedrosa Soares Leandro de Arau´jo-Martins • Aline Araujo dos Santos • Elizabeth Giestal-de-Araujo
•
Received: 28 October 2014 / Accepted: 27 January 2015 Ó Springer Science+Business Media New York 2015
Abstract Brain-derived neurotrophic factor (BDNF) is a well-known and well-studied neurotrophin. Most biological effects of BDNF are mediated by the activation of TrkB receptors. This neurotrophin regulates several neuronal functions as cell proliferation, viability, and differentiation. Ouabain is a steroid that binds to the Na?/K? ATPase, inducing the activation of several intracellular signaling pathways. Previous data from our group described that ouabain treatment increases retinal ganglion cells survival (RGC). The aim of the present study was to evaluate, if this cardiac glycoside can have a synergistic effect with BDNF, the classical trophic factor for retinal ganglion cells, as well as investigate the intracellular signaling pathways involved. Our work demonstrated that the activation of Src, PLC, and PKCd participates in the signaling cascade mediated by 50 ng/mL BDNF, since their selective inhibitors completely blocked the trophic effect of BDNF. We also demonstrated a synergistic effect on RGC survival when we concomitantly used ouabain (0.75 nM) and BDNF (10 ng/mL). Moreover, the signaling pathways involved in this synergistic effect include Src, PLC, PKCd, and JNK.
Aline Araujo dos Santos and Elizabeth Giestal-de-Araujo have contributed equally to this work. G. de Rezende Correˆa (&) V. H. P. Soares L. de Arau´jo-Martins A. A. dos Santos E. Giestal-de-Araujo Departamento de Neurobiologia, Programa de Neurocieˆncias, Instituto de Biologia, Universidade Federal Fluminense, Outeiro de Sa˜o Joa˜o Batista s/n, Nitero´i, Rio de Janeiro CEP 24020-140, Brazil e-mail: [email protected] A. A. dos Santos Departamento de Fisiologia e Farmacologia, Instituto Biome´dico, Universidade Federal Fluminense, Nitero´i, Rio de Janeiro CEP 24210-130, Brazil
Our results suggest that the synergism between ouabain and BDNF occurs through the activation of the Src pathway, JNK, PLC, and PKCd. Keywords Ouabain BDNF Retina Neuronal survival Synergistic effect
Introduction Ouabain is a glycoside used for many years to treat cardiac dysfunction. This therapeutic application involves an increase in intracellular calcium concentration observed after ouabain treatment: as ouabain binds to the Na?, K?ATPase a-subunit, an inhibition of ion transport takes place, leading to the accumulation of intracellular Na? and a consequent increase in intracellular calcium through the Na?/Ca2? exchanger (Blanco and Mercer 1998). Interestingly, endogenous ouabain has also been identified (Hamlyn et al. 1991), its plasma concentration ranging from 50 pM to 80 nM (Goto et al. 1992; Gottlieb et al. 1992; Schoner et al. 2003). Many intracellular pathways are activated following ouabain binding to Na?, K?-ATPase. They involve distinct families of enzymes such as PKC, CAM kinase, and Src kinases, as well as the Ras/Raf/MEK/ MAP kinase signaling pathway (Mohammadi et al. 2003). The transactivation of EGF receptors by ouabain treatment can also stimulate the Ras/MAPK cascade (Haas et al. 2002). Since its discovery in 1982 (Barde et al. 1982), BDNF became a well-studied neurotrophin in the nervous system. Data from different groups demonstrate the role of this trophic factor in the control of cell survival, proliferation, and differentiation during the development as well as during the adult life (Uchida et al. 2013; de Almeida et al.
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2013). BDNF actions are carried out by activation of TrkB receptor subtype, two BDNF molecules being required for the receptor activation. After activation, several signaling pathways become effective, among them the phospholipase C (PLC), the PI3K, and the Ras–Raf–MEK–MAPK pathways (Bagayogo and Dreyfus 2009). The effect of several trophic factors on retinal ganglion cell survival has been extensively described. Specifically, the importance of neurotrophins both in vitro and in vivo has been demonstrated in many species, including the rat (Pan and Zhang 2013; Chung et al. 2013; Ebel et al. 2013; Ramos-Languren and Escobar 2013). The effects of BDNF and NT-4 on retinal ganglion cell survival indicate that these neurotrophins play an important role during retinal development. Neurotrophins can also rescue ganglion cells after injury, pointing toward the clinical importance of these studies (Isenmann et al. 2003). Previous results from our group demonstrated that ouabain has a trophic effect on the population of retinal ganglion cells (RGC), increasing its viability after 48 h in culture. The best result was obtained with a concentration of 3 nM (100 % increase when compared to the 48 h control cultures). It was also observed that this effect involves the activation of Src, EGFR, and PKC. Paradoxically, JNK, a protein well known for its effects on the induction of cell death (Sabapathy 2012; Davies and Tournier 2012) is involved in the trophic effect of ouabain (Correˆa et al. 2005, 2010). It is well known that BDNF has a trophic effect in retinal ganglion cells (Chen and Weber 2001). However, in our experimental setup, the BDNF role in triggering or regulating the intracellular pathways involved in the effects has not been investigated. For that reason, in the present work, we initially investigated the intracellular pathways responsible for the trophic effect of BDNF on retinal ganglion cell survival. Our objective was to ascertain that 50 ng/mL of BDNF stimulates the same pathways we had previously identified as activated by ouabain. Moreover, we also evaluated a possible synergistic effect of low doses of BDNF and ouabain, on retinal ganglion cell survival, in order to establish if ouabain could potentiate the trophic effect of BDNF and the intracellular pathways involved.
Experimental Procedures Ethical Aspects Procedures using animals were approved by the local committee for animal care and experimentation (CEPAprojects #00196-10). All efforts were made to minimize the number of experimental animals used and their suffering.
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Materials Medium 199, fetal bovine serum, PD98050, and LY284002 were purchased from GIBCO (Gaithersburg—USA). Glutamine, streptomycin, penicillin, poly-L-ornithine, ouabain, and peroxidase (HRP) were purchased from Sigma–Aldrich (St. Louis—USA). Dimethyl sulfoxide (DMSO) and glutaraldehyde were purchased from Mallinckrodt Baker (Paris—USA) and Entellan came from Merck (Darmstadt— Germany). EGF and BDNF were purchased from PeproTech (Rocky Hill, NJ, USA). AG1478, SB203580, PDTC, and JNK inhibitor came from Calbiochem–Merck (Darmstaddt—Germany). Chelerythrine chloride, K252a, PP1, and U73122 were purchased from Biomol (Plymouth Meeting, PA, USA). Rottlerin and BAPTA-AM were purchased from Invitrogen (USA). Trypsin was purchased from Worthington (USA). The antibodies used came from Santa Cruz Biotechnology (USA). Labeling of Retinal Ganglion Cells Within the first 24 h after birth, Lister Hooded rats were anaesthetized by hypothermia. One microliter of a solution of 30 % HRP in 2 % DMSO was injected directly into each superior colliculus. This procedure was possible because in newborn rats, the mesencephalic region is not covered by the cerebral cortex (Araujo and Linden 1993). The animals were returned to their mothers and survived for *16 h prior to the procedures for cell culture. Retinal Cultures Primary cultures were prepared using previously described techniques (Santos and Araujo 2000). Briefly, neonatal rats at postnatal day 1 were killed by decapitation and the retinas dissected free from scleral tissue and pigmented epithelium in a calcium- and magnesium-free balanced salt solution (CMF) containing 100 lg/mL streptomycin ? 100 U/mL penicillin. The tissue was then incubated in CMF containing 0.1 % trypsin for approximately 16 min at 37 °C. Trypsin action was stopped by addition of culture medium (199 medium supplemented with 2 mM glutamine, 100 lg/mL streptomycin, and 100 U/mL penicillin) with 5 % FCS. The tissue was then resuspended in complete culture medium and mechanically dissociated using a polished Pasteur pipette. Cells were added to Petri dishes or Petri dishes containing coverslips, both previously treated with poly-L-ornithine (50 lg/mL) at a plating density of 105 cells/cm2. After plating, cultures were incubated in 1 mL of culture medium for 2–4 h to allow cells to attach to the substrate. Then, 1 mL of culture medium or 1 mL of medium containing the drugs to be tested was added to each Petri dish. In our experimental procedure,
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drug treatment was kept over the whole time in culture. Medium-insoluble drugs were previously diluted in DMSO. The effect of diluted DMSO on retinal ganglion cell survival was evaluated as a control, and we did not find any changes using dilutions up to 1:100 (data not shown). Drugs were added once, after 2–4 h in culture. The cultures were then maintained between 45 min and 48 h in vitro at 37 °C in a humidified atmosphere of 5 % CO2 and 95 % air.
phospho-CREB antibody (Ser-133) (1:400). The membranes were washed in TBS and were then exposed to horseradish peroxidase-conjugated secondary anti-rabbit or anti-mouse IgG antibody at 1:2,000 and 1:80,000 dilutions, respectively, at room temperature for 60 min. Detection was performed on X-ray film (hyperfilm—Amersham) using chemiluminescence system (ECL). The density of protein bands was analyzed by densitometry with Scion Image. The mean value for the control was set at 100 %.
Identification of Retinal Ganglion Cells Statistical Analysis The presence of the enzyme HRP in the cytoplasm of retinal ganglion cells was revealed according to the protocol of (Mesulan 1982). Briefly, monolayers were fixed with a mixture of 1 % paraformaldehyde and 2 % glutaraldehyde in 0.1 M sodium phosphate buffer (Karnovsky solution) for 5 min, washed in phosphate buffer and reacted with tetramethylbenzidine and H2O2. After reaction, the coverslips were washed in 0.2 M acetate buffer, dehydrated by air drying, immersed briefly in xylene and mounted in Entellan. Quantification of Retinal Ganglion Cells Retinal ganglion cells were counted using an Olympus BX41 (Tokyo, Japan) microscope at a magnification of 9400, under bright field. As an internal control for the variable percentage of ganglion cells labeled with HRP in distinct experiments, the number of labeled cells at 4 h in culture was taken as 100 % and the results were reported as percentage of this control. Approximately, 800 retinal ganglion cells were labeled in 4 h control coverslips. Independently from the number of labeled cells, the 48 h survival was always in the same range (40–60 %). All data were expressed as mean ± standard error of the mean from four independent experiments. Each individual experiment was performed at least in duplicate. Western Blot Phospho-NFj-B, phospho-CREB, phospho-Src, and phospho-JNK levels were determined by Western blot analysis. Retinal cell cultures were lysed by sonication in lysis buffer (2 % sodium dodecyl sulfate and 0.5 M Tris pH 6.8). After determination of protein concentration by the Bradford method (Bradford 1976), samples (60 lg/ lane) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (9 %) and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were incubated overnight with rabbit anti-phosphoNFj-B antibody (Ser-377) (1:200) or mouse anti-phosphoJNK antibody (Thr-183 and Thr-185) (1:400) or rabbit antiphospho-Src antibody (Tyr-416) (1:400) or rabbit anti-
The overall statistical analysis was first obtained by oneway analysis of variance (ANOVA). The statistical significance of all pairs of multiple groups of data was assessed by Newman–Keuls comparison test. A value of P \ 0.05 was considered significant.
Results We started our analyses by evaluating the dependence of the BDNF protective effect on Src activation. We used PP1 (1 lM), a selective inhibitor of this enzyme, in the presence of 50 ng/mL BDNF and evaluated the retinal ganglion cells survival. The results in Table 1 show that the Src pathway is involved in the trophic effect of BDNF on retinal ganglion cells survival since PP1 blocked the effect of this neurotrophin. Since the effect of ouabain on retinal ganglion cells survival involves the phosphorylation of EGF receptors (Correˆa et al. 2010), we treated the cultures with an inhibitor of these receptors (2.5 lM AG1478) in the presence of 50 ng/mL BDNF. Our results indicate that the signaling pathway of BDNF does not involve the activation of EGF receptors as the AG1478 did not block the trophic effect of this neurotrophin (Table 2). The next step was to evaluate the involvement of phospholipase C (PLC) in the BDNF signaling pathway with the use of a selective inhibitor of this enzyme (4 lM U73122). The data present in Table 1 show a complete blockade of the effect of BDNF in retinal ganglion cells survival when this neurotrophin was concomitantly used with U73122. Our previous results demonstrated the involvement of protein kinase C (PKC) signaling pathway in the trophic effect of ouabain in retinal ganglion cells (Correˆa et al. 2005). We demonstrated that PKCd, a member of the novel class of the PKC family, is the isoenzyme involved on the ouabain effect (Correˆa et al. 2010). Thus, we decided to examine whether this protein kinase is involved in the signaling pathway of BDNF. Treatment of retinal cell cultures with 1.25 lM chelerythrine chloride (CC), an
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Cell Mol Neurobiol Table 1 The trophic effect of 50 ng/mL BDNF on RGC survival is mediated by Src, PLC, and PKCd Treatment
(% of control) CT 48 h
BDNF
-
SEM
?
SEM
-
SEM
?
SEM
PP1 (1 lM)
52.43
3.80
51.30
1.48
96.94
2.75
48.05*
4.60
U73122 (4 lM)
52.43
3.80
47.88
1.90
96.94
2.75
46.09*
4.40
CC (1.25 lM) ROT (2 lM)
53.55 52.50
4.50 4.20
58.28 49.12
6.40 6.40
96.92 109.62
6.60 6.70
54.86* 51.67*
3.50 4.60
Cells were kept in culture for 48 h. CT control cultures. Cells were treated with BDNF and inhibitors once, after 2–4 h in culture. The following inhibitors were used: 1 lM PP1 (Src); 4 lM U73122 (phospholipase C); 1.25 lM chelerithrine chloride (CC) (protein kinase C); 2 lM rottlerin (ROT) (PKCd). Data are reported as the mean ± SEM (n = 12). P \ 0.001, compared to the 48 h control * Significantly reverts the BDNF effect
Table 2 The trophic effect of 50 ng/mL BDNF on RGC survival is not mediated by an increase in intracellular calcium levels and EGF receptors activation Treatment
% of control CT 48 h
BDNF
-
SEM
?
SEM
-
SEM
?
SEM
AG1478 (2.5 lm)
59.70
9.70
55.45
9.80
103.74
3.35
100.07
4.50
BAPTA-AM (20 lm)
52.43
4.00
49.62
2.40
109.62
6.70
98.60
4.20
Cells were kept in culture for 48 h in the presence of complete culture medium (CT control cultures), or cells were treated with BDNF and inhibitors once, after 4 h in culture. The following inhibitors were used: 2.5 lM AG1478 (EGF receptor inhibitor) or 20 lM BAPTA-AM (an intracellular calcium chelator). Data are reported as the mean ± SEM (n = 12). P \ 0.001, compared to the 48 h control
inhibitor of PKC, abolished the trophic effect of BDNF (Table 1). To investigate the role of intracellular calcium levels in the BDNF effect, we used 20 lM BAPTA-AM (an intracellular calcium chelator). The results present in Table 2 indicate that an increase in intracellular calcium concentration is not necessary for the BDNF effect. Based on this last result and on the fact that the isoenzymes of the novel class of the PKC family are independent on calcium for their activation, we decided to analyze if PKCd is also involved on the BDNF effect. Our results show that 2 lM rottlerin (a PKCd inhibitor) completely blocked the BDNF effect (Table 1). Since 50 ng/mL BDNF and 3 nM ouabain isolated exert trophic effects on RGC maintained in culture for 48 h, we analyzed if the treatment with low doses of and BDNF (10 ng/mL) and ouabain (0.75 nM) could promote a synergistic action on the survival of this neuronal population. As can be seen in Fig. 1a, when both treatments were used alone, they are not able to increase RGC survival. However, when concomitantly used, they induce a significant increase in RGC survival. This result confirms that there is a synergism between ouabain and BDNF on RGC survival. Based on this result, we decided to investigate the intracellular pathways involved in the synergistic effect. We started by analyzing the role of TrkB receptors on it. The
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cultures were treated with 50 nM K252a, an inhibitor of Trk receptors, and we observed that K252a totally abolished the synergistic effect (Fig. 1b). Based on our previous results, we decided to analyze the pathways that are activated by 3 nM ouabain (Correˆa et al. 2005, 2010) and also by 50 ng/mL BDNF on retinal ganglion cells survival. To ascertain the involvement of Src, we used PP1 (1 lM) and we observed that this inhibitor blocked the synergistic effect (Fig. 2a). Analysis of the Src levels indicated the participation of this protein in the synergistic effect, confirming the results obtained previously. It can be observed in Fig. 2b that the individual treatments with 0.75 nM ouabain or 10 ng/mL BDNF do not affect the phosphorylation levels of this protein. However, the combined treatment, for 45 min, significantly elevates their levels (Fig. 2b, c). To evaluate the involvement of PLC in the synergistic effect, we used 4 lM U73122. As can be observed in Fig. 3a, U73122 completely abolished the synergistic trophic effect. We then analyzed the role of PKCd in the synergistic effect. Data present in Fig. 3b show that 2 lM rottlerin totally blocks the effect of ouabain and BDNF on retinal ganglion cell survival. We also studied a possible involvement of JNK in the synergistic effect using its specific inhibitor at the concentration of 1 lM. The results
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Fig. 1 Synergistic effect of ouabain and BDNF on RGC survival. Cells were kept in culture for 48 h in the presence of complete culture medium. a The simultaneous treatment with 10 ng/mL BDNF and 0.75 nM ouabain significantly increased the RGC survival. However, the treatment with 10 ng/mL BDNF or 0.75 nM ouabain did not
increase the survival of RGC. b The synergistic effect of ouabain and BDNF was totally abolished by the presence of 50 nM K252a (an inhibitor of Trk receptors). CT control, OUA ouabain. Data are reported as the mean ± SEM (n = 12). P \ 0.001, compared to the 48 h control
Fig. 2 The synergistic effect of 0.75 nM ouabain and 10 ng/mL BDNF is mediated by Src. Cells were kept in culture in the presence of complete culture medium. a The inhibition of Src (1 lM PP1) abolished the synergistic effect on RGC survival after 48 h in culture. b The graph shows the levels of p-Src in cultures treated with OUA, BDNF and BDNF ? OUA for 45 min. c Representative blot of samples. CT control, OUA ouabain. Data are reported as the mean ± SEM (n = 12). P \ 0.001 compared to the 45 min (blot) or 48 h (survival) control
represented in Fig. 3b show that the inhibition of JNK blocks the synergistic effect and Fig. 3c show that the combined treatment induces a decrease in the levels of phospho-JNK (Fig. 3c, d). To further investigate the signaling pathways, we decided to assess which transcription factor might be involved on the effects of BDNF 50 ng/mL and ouabain 3 nM. We analyzed the role of NFj-B using a selective inhibitor of this protein (PDTC 20 nM). Figure 4a clearly shows that the NFj-B is involved in the effect of ouabain 3 nM on retinal ganglion
cell survival. However, the survival pathways activated by BDNF do not involve the activation of this transcription factor (Fig. 4b). Figure 4c and d shows the levels of phospho-NFj-B after ouabain, BDNF, and the combined treatment. We did not observe changes in its levels in any of these three conditions. Figure 4e shows that NFj-B does not participate in the trophic effect promoted by the treatment with ouabain and BDNF in low doses. We next investigated a possible role of the transcription factor CREB, analyzing the change in the levels of
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Cell Mol Neurobiol Fig. 3 The synergistic effect of 0.75 nM ouabain and 10 ng/mL BDNF is mediated by PLC, PKCd, and JNK. Cells were kept in culture in the presence of complete culture medium. a The inhibition of phospholipase C (4 lM U73122) abolished the synergistic effect after 48 h in culture. b The blockade of PKCd (2 lM ROT) or JNK (1 lM iJNK) also inhibited the synergistic effect after 48 h in culture. c The graph shows the levels of p-JNK in different treatments. Both BDNF and BDNF ? OUA treatments for 45 min induced a decrease in the p-JNK levels. d Representative blot of samples. CT control, OUA ouabain, U73122 phospholipase C selective inhibitor, ROT rottlerin, iJNK JNK inhibitor. Data are reported as the mean ± SEM (n = 12). P \ 0.001 compared to the 45 min (blot) or 48 h (survival) control
phospho-CREB in retinal cells cultures treated for 45 min. Figure 5 shows that BDNF (10 ng/mL) treatment alone decreases phospho-CREB levels, while ouabain treatment (0.75 nM) increases these levels. Accordingly, the combined treatment (OUA ? BDNF) did not significantly affect the phosphor-CREB levels.
Discussion The literature strongly supports the trophic effect of BDNF on retinal ganglion cells (von Bartheld 1998). For this reason we studied, in the present work, if ouabain could potentiate the effect of this classical trophic factor on retinal ganglion cells. Our data show that simultaneous treatment with BDNF 10 ng/mL and ouabain 0.75 nM increases the survival of retinal ganglion cells kept in mixed retinal cell cultures for 48 h. In fact this treatment is able to maintain all retinal ganglion cells initially plated suggesting that this protocol induces a trophic effect on axotomized neurons in vitro. As the isolated treatments with ouabain or BDNF, in such low concentrations, do not increase the survival of ganglion cells, we conclude that the effect occurs by a synergistic action of these two molecules.
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Data from the literature show that ouabain binding to Na?/K?-ATPase induces the activation of intracellular pathways involving protein–protein interactions, promoting responses such as cell proliferation and differentiation (Xie and Askari 2002). This mechanism of signal transduction is triggered by low concentrations of ouabain and does not appear to alter the cell ionic concentration (Scheiner-Bobis and Schoner 2001). Corroborating Correˆa and co-workers, using ouabain in a quite low concentration (3 nM) demonstrated that this treatment induced a trophic effect on retinal ganglion cell population (Correˆa et al. 2005). These results are in agreement with data reported by Dvela, in 2012, showing that the endogenous concentration of ouabain corresponds to nanomolar levels (Dvela et al. 2012). It is interesting to note that the low concentration used by Correˆa et al. is equivalent to the physiological concentration of endogenous ouabain, therefore, in the nanomolar level. From the analysis of the signaling pathways of BDNF in our experimental model, we found some of them in common with those trigged by ouabain and previously described: Src, PLC, and PKCd. According to these data, we decided to check whether these pathways are actually used to elicit the synergism between BDNF and ouabain (Correˆa et al. 2005, 2010). In the present paper, we demonstrate
Cell Mol Neurobiol Fig. 4 The involvement of NFjB in the trophic effect elicited by OUA in RGC survival. a The treatment with 20 nM PDTC (an inhibitor of NFjB) abolished the trophic effect of 3 nM OUA in RGC. b However, the trophic effect of 50 ng/mL BDNF and OUA ? BDNF (e) is not mediated by NFjB. c The graph shows the levels of p-NFjB in different experimental conditions. It was not observed any difference between the conditions analyzed. d Representative blot of samples. e NFj-B does not participate in trophic effect promoted by treatment with ouabain and BDNF in low doses. CT, control; 0.75 nM OUA, ouabain; 20 nM PDTC, NFjB selective inhibitor. Data are reported as the mean ± SEM (n = 12). P \ 0.001 compared to the 45 min (blot) or 48 h (survival) control
that Src is also participating in BDNF effect on retinal ganglion cell survival as well as in the synergistic effect of ouabain and BDNF. Our data concerning the involvement of Src in the BDNF effect are in agreement with previous results of Vries and colleagues. This group demonstrated in 2010 that following BDNF binding to its receptor, Src activation occurs (Vries et al. 2010). Also in 2010, You and colleagues demonstrated in the brain of adult mice the involvement of the Src protein subtype D (SrcD) in signal transduction of TrkB receptors, via SH3 and PTB domains present on these receptors (You et al. 2010). Our previous data show that EGFR was involved in the trophic effect of ouabain on retinal ganglion cells (Correˆa et al. 2010). However, after investigating the possible involvement of EGFR in the BDNF effect, we did not observe any correlation. Based on this result, we can suggest that the EGF receptor does not mediate the effect of BDNF on retinal ganglion cell survival.
Zhang and colleagues recently demonstrated the involvement of PLC in the effect promoted by BDNF on glutamate release in cortical neurons (Zhang et al. 2013). Bagayogo and Dreyfus, in 2009, indicated the participation of the PLC pathway in the effect of BDNF on oligodendrocytes (Bagayogo and Dreyfus 2009). Our previous data showed the involvement of PLC in the signaling pathway activated by ouabain treatment in neonatal rats RGC (Correˆa et al. 2010), and in the present work, we observe that this enzyme is also activated in BDNF effect and in the synergism between BDNF and ouabain. Protein kinase C family plays an important role controlling proliferation, differentiation, survival, neuritogenesis, and apoptosis in the nervous system (Zhao et al. 2012; Pinzon-Guzman et al. 2011; Santos and Araujo 2000). In 2008, Tomimatsu and Arakawa demonstrated that BDNF induces a decrease in the damage induced by glutamatergic excitotoxicity in motoneurons via PKC activation (Tomimatsu and
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Cell Mol Neurobiol
Fig. 5 The dual effect of 0.75 nM OUA and 10 ng/mL BDNF in p-CREB levels. a Levels of p-CREB levels are increased in cultures treated for 45 min with OUA and decreased in cultures treated with BDNF. However, the combined treatment with OUA ? BDNF did not significantly influence the levels of p-CREB. b Representative blot of samples. CT control, OUA ouabain. Data are reported as the mean ± SEM (n = 4). P \ 0.001 compared to the 45 min control
Arakawa 2008). In accordance, our results show that both BDNF effect and the synergistic effect of ouabain and BDNF depend on PKC activation, specifically of PKCd. It was previously demonstrated by our group that PKCd was involved in the survival of retinal ganglion cells stimulated either by ouabain or by phorbol 12-myristate 13-acetate (PMA), an activator of the conventional and novel isoforms of PKC (Correˆa et al. 2010). Literature shows the involvement of JNK in progressive phenomena of nervous system development such as neuritogenesis and cell survival. NGF induces neuritogenesis in PC12 cells via activation of JNK (Waetzig et al. 2008), and in HepG2 cells, the JNK activation mediates their survival (Granado-Serrano et al. 2009). JNK can be activated by PKC and calcium-calmodulin, and is involved in the activation of the transcription factor AP-1. This factor regulates cellular survival, proliferation, differentiation, and gene expression (Schoner and Scheiner-Bobis 2007). Sanna and co-workers in 2002 demonstrated that JNK regulates the survival of human embryonic kidney cells via the activation of the proteins that inhibits the apoptosis (IAP) (Sanna et al. 2002). Our data are in accordance with these previous results since
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JNK is involved in the synergistic effect of BDNF and ouabain. We have demonstrated the role of the transcription factor NFj-B on the ouabain effect. However, this transcription factor does not mediate the trophic effect on retinal ganglion cells of BDNF alone neither that of ouabain and BDNF in low doses. Literature shows that CREB is an important transcription factor involved in the BDNF synthesis in neurons (Nibuya et al. 1996). Data from our group already demonstrated that a chronic treatment of retinal cell cultures with BDNF, for 48 h, decreases the levels of BDNF, phospho-TrkB, and phospho-CREB (Perı´golo-Vicente et al. 2013). Our results presented here are in agreement with this previous one and also show that the treatment with ouabain induces an increase in the phospho-CREB levels. Future work will be performed in order to elucidate the intracellular pathways involved in the increase of phospho-CREB levels following ouabain treatment. In this work, we showed for the first time that ouabain can potentiate the trophic effect of BDNF in axotomized neuronal cells. We also described that either BDNF or ouabain treatments induce the activation of some intracellular proteins. The mechanisms involved in the crosstalk between ouabain and BDNF are presently not completely known. As this is the first evidence of the crosstalk, data from the literature cannot conveniently support further discussions on the mechanism involved in this effect. Further experiments will be addressed to further investigate which molecules are mediating this crosstalk. The results presented in this study demonstrate a classic synergistic effect of ouabain and BDNF treatments in increasing the survival of retinal ganglion cells maintained in cultures for 48 h, after axotomy, through the activation of the Src, PLC, PKC, and JNK. As ouabain was already used clinically in the treatment of congestive heart failure and in our work its presence potentiates the trophic effect of a classical trophic factor for retinal ganglion cells, ouabain could be used in order to increase neuronal survival following neuronal injuries. Indeed, it was demonstrated that ouabain has a neuroprotective effect after traumatic brain injury (Dvela-Levitt et al. 2014). Treatment with low concentrations of ouabain, in combination with low concentrations of trophic factor, could increase neuronal survival and decrease the side effects observed when drugs were used in higher concentrations. In the future, our results can contribute to the establishment of new therapeutic protocols able to decrease retinal ganglion cells damage following several insults. Acknowledgments We would like to thank Alexandre Jose´ Fernandes, Bernardino Matheus dos Santos and Arnaldo de Sa´ for technical assistance. We also thank Arnaldo Paes de Andrade for
Cell Mol Neurobiol critical reading of our manuscript. Gustavo de Rezende Correˆa, Vinı´cius Henrique Pedrosa Soares and Leandro de Araujo Martins received fellowships from CAPES. This work was supported by Grants from CAPES, PRONEX-MCT, PROPPi and FAPERJ. Conflict of interest interests.
None of the authors declares competing
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