Address correspondence and reprint requests to Dr Luc Pellerin, Département de Physiologie, 7 Rue du Bugnon, 1005 Lausanne, Switzerland. E-mail: Luc.Pellerin@unil.ch
Monocarboxylate transporter 2 (MCT2) expression is up-regulated by noradrenaline (NA) in cultured cortical neurons via a putative but undetermined translational mechanism. Western blot analysis showed that p44/p42 mitogen-activated protein kinase (MAPK) was rapidly and strongly phosphorylated by NA treatment. NA also rapidly induced serine/threonine protein kinase from AKT virus (Akt) phosphorylation but to a lesser extent than p44/p42 MAPK. However, Akt activation persisted over a longer period. Similarly, NA induced a rapid and persistent phosphorylation of mammalian target of rapamycin (mTOR), a kinase implicated in the regulation of translation in the central nervous system. Consistent with activation of the mTOR/S6 kinase pathway, phosphorylation of the ribosomal S6 protein, a component of the translation machinery, could be observed upon treatment with NA. In parallel, it was found that the NA-induced increase in MCT2 protein was almost completely blocked by LY294002 (phosphoinositide 3-kinase inhibitor) as well as by rapamycin (mTOR inhibitor), while mitogen-activated protein kinase kinase and p38 MAPK inhibitors had much smaller effects. Taken together, these data reveal that NA induces an increase in neuronal MCT2 protein expression by a mechanism involving stimulation of phosphoinositide 3-kinase/Akt and translational activation via the mTOR/S6 kinase pathway. Moreover, considering the role of NA in synaptic plasticity, alterations in MCT2 expression as described in this study might represent an adaptation to face energy demands associated with enhanced synaptic transmission.
In both striated and cardiac muscles, changes in the expression of MCTs occur in relation with the level of activity. Thus, there is an important literature demonstrating that MCT1 and/or MCT4 are up-regulated upon various exercise training paradigms (Baker et al. 1998; Green et al. 2002; Coles et al. 2004). While denervation was shown to decrease MCT1 and MCT4 expression in striated muscle (Juel and Halestrap 1999), cross-reinnervation was shown to alter the expression patterns of these two transporters (Bergersen et al. 2006), further suggesting a direct relationship between their expression and the level of activity. In the brain, there is as yet few observations about modifications of MCT expression associated with synaptic activity. In cultured neurons, it was found that the level of MCT2 expression correlated with increased expression of synaptophysin (a pre-synaptic protein) along days in vitro, suggesting a relationship between synaptic development and energy substrate requirements (Debernardi et al. 2003). Furthermore, the neurotransmitter noradrenaline (NA) was shown to enhance the expression of MCT2 in cultured cortical neurons (Pierre et al. 2003). Interestingly, this effect appears to be regulated at the translational level as no change in the amount of MCT2 mRNA was detected following NA treatment. In this study, we have characterized the putative signal transduction mechanisms involved in the translational activation leading to enhanced MCT2 protein expression in neurons exposed to NA.
Material and methods
Neuronal cultures and pharmacological treatments
Primary cultures of mouse cortical neurons were prepared from embryonic day 17 OF1 mice (Charles Rivers, France). As previously described (Debernardi et al. 2003), cortices were mechanically dissociated in phosphate-buffered saline (PBS)/glucose (150 mmol/L NaCl, 3 mmol/L KCl, 1.5 mmol/L KH2PO4, 7.9 mmol/L Na2HPO4, 33 mmol/L glucose, 0.006 g/L penicillin, and 0.1 g/L streptomycin, pH 7.4). Cells were plated on poly-l-ornithine (15 mg/L) pre-coated dishes and cultured in neurobasal-B27 medium (Brewer et al. 1993) supplemented with 0.5 mmol/L l-glutamine. All experiments were performed on day 7 in vitro. At this stage, cultures contained approximately 1% of glial cells. Neuronal treatments with pharmacological agents were performed without changing the medium before or during the incubation time. NA (A9512; Sigma, Buchs, Switzerland) at a final concentration of 100 μmol/L was added directly into the culture medium and cells were incubated for various times. Rapamycin 20 ng/mL [mammalian target of rapamycin (mTOR) inhibitor], SB202190.HCl 10 μmol/L [p38 mitogen-activated protein kinase (MAPK) inhibitor], LY294002 10 μmol/L [phosphoinositide 3-kinase (PI3K) inhibitor], and PD98059 50 μmol/L [mitogen-activated protein kinase kinase (MEK) inhibitor] were added directly in the medium 30 min before NA. All these inhibitors were purchased from Alexis® biochemicals (Lausen, Switzerland) except LY294002 (L9908; Sigma). Concentrations used were selected on the basis of previous studies establishing the specificity and efficacy of each inhibitor (Davies et al. 2000).
Immunocytochemistry and related quantification
After removal of the culture medium, cells were carefully rinsed in PBS at 37°C and directly post-fixed in an ice-cold p-formaldehyde fixative (4% in PBS; 30 min at 20°C). Fixed cells were treated with casein (0.5% in PBS) for 1 h at 20°C to block non-specific sites. For immunostaining, cultures were incubated overnight at 4°C in 50 μL of freshly prepared MCT2 antibody solution (anti-MCT2 diluted 1 : 500 in PBS containing 0.25% bovine serum albumin). After careful rinsing in PBS, cultures were incubated in a solution containing goat fluorescein isothiocyanate-conjugated anti-rabbits Igs (diluted 1 : 500; 2 h at 20°C, protected from light). After rinsing in PBS twice and a final rinsing in water, coverslips were mounted with Vectashield (Reactolab SA; Sigma). Coverslips were examined and photographed with an Axioplan2 microscope (Axiocam, Zeiss, Germany) using epifluorescence with an appropriate filter. To quantitatively assess the influence of different treatments on MCT2 protein expression, a quantitative analysis on images obtained by epifluorescence with a 20× objective and acquired using a cooled CCD Camera (Zeiss) together with the 2.05 Axiovision software (Zeiss) was carried out. Three fields were chosen randomly in each untreated or treated cultures that contained at least 20 MCT2-labeled neurons per field. All pictures were acquired and presented as different levels of grey, with identical acquisition time for all. Pictures were then analyzed using the National Institute of Health software, version 1.62 (NIH, Bethesda, MD, USA). The fluorescence intensity of eight isolated cells taken randomly in each of the three captured areas was assessed. The mean fluorescence intensity representing neuronal MCT2 expression in a given condition was obtained by calculating the average of the 24 measurements. Measurements were obtained in a blind fashion with the investigator unaware of the culture treatments. Data were statistically analyzed with an analysis of variance (anova) followed by Bonferroni’s test.
Western blot and related quantification
Neuronal cultures were homogenized in 50 μL/dish of a buffer containing 20 mmol/L Tris–HCl pH 6.8, 0.27mol/L sucrose, 1 mmol/L EGTA, 1 mmol/L EDTA, 50 mmol/L NaF, 1% Triton X-100, 10 mmol/L β-glycerophosphate, 10 mmol/L dithiothreitol, 10 mmol/L 4-nitrophenylphosphate, and a mixture of protease inhibitors (Complete 11257000; Roche, Rotkreuz, Germany). Each condition was carried out in duplicate and the contents of two Petri dishes were pooled. Protein samples were sonicated and heated at 95°C for 5 min in half the final volume of SDS-PAGE sample buffer (62.5 mmol/L Tris–HCl, 50 mmol/L dithiothreitol, 2% SDS, 10% glycerol, and 0.1% bromophenol blue). Samples were loaded onto polyacrylamide gels composed of a 10% or 6% acrylamide/bisacrylamide running gel and a 4.5% acrylamide/bisacrylamide stacking gel. After electrophoresis, proteins were transferred onto nitrocellulose membranes (Trans-Blot® Transfer Medium 162-0115, BIO-RAD, Reinach, Switzerland) using a Transblot SD Semi Dry Transfer cell (BIO-RAD). For protein detection, membranes were incubated in a blocking solution of TBST (50 mmol/L Tris–HCl pH 7.5, 150 mmol/L NaCl, and 0.1% Tween-20) containing 5% non-fat milk for 1 h at 20°C. Membranes were incubated overnight at 4°C with the anti-P-serine/threonine protein kinase from AKT virus (Akt) -Ser473 (1 : 700), anti-P-p44/p42 MAPK-Thr202/Thr204 (1 : 1000), anti-P-mTOR-Ser2448 (1 : 1000), anti-mTOR (1 : 1000), anti-P-S6-Ser235/236 ribosomal protein (1 : 1000), and anti-β-actin (1 : 10 000). All primary antibodies were purchased from Cell Signaling (Bioconcept, Allschwil, Switzerland) except the anti-β-actin (A5441; Sigma). After three washes in TBST, membranes were incubated with the secondary antibodies, Alexa Fluor 680 goat anti-rabbit IgG (Juro, Lucerne, Switzerland) and IRDye™ 800 anti-mouse IgG (Rockland, Gilbertsville, PA, USA), diluted at 1 : 5000 in TBST containing 1% non-fat milk, 2 h at 20°C, and protected from light. After three washes in TBST, membranes were scanned using the ODYSSEY® Infrared Imaging System (LI-COR® Biosciences, Lincoln, NE, USA) which permits to detect and quantify proteins of interest. β-actin, revealed in green, was used for normalization and the proteins of interest were revealed in red. As P-mTOR has a very high molecular weight (289 kDa), actin was not visible on the same gel as P-mTOR. In order to normalize western blots for P-mTOR, samples were thus loaded in duplicate on a 6% running gel and proteins were transferred on a nitrocellulose membrane using a 10% methanol transfer buffer. The membrane was then cut in two identical pieces and probed either with the P-mTOR antibody (1 : 1000) or the mTOR antibody (1 : 1000). Thus, quantifications were performed on samples resolved on the same gel and transferred on the same membrane, and normalization was performed against mTOR (instead of β-actin). Please note that the remaining of the procedure was unchanged.
Data were statistically analyzed with an analysis of variance (anova) followed by Bonferroni’s test.
It was previously shown that NA treatment increases MCT2 protein expression in primary cultures of mouse cortical neurons (Pierre et al. 2003). On the basis of unchanged mRNA levels as determined by quantitative RT-PCR measurements, it has been proposed that the increase in protein expression is mediated by a translational activation rather than a transcriptional mechanism. To further characterize the mechanism leading to this effect, we first investigated the activation/phosphorylation of a few steps in some signaling pathways putatively involved in translation regulation in primary cultures of mouse cortical neurons. Then, we verified the involvement of these distinct pathways in NA-mediated enhancement in MCT2 protein expression.
NA activates the p44/p42 MAPK and PI3K/Akt signaling pathways in cultured mouse cortical neurons
Cultured cortical neurons were treated with NA 100 μmol/L for three time periods (5 min, 30 min, and 1 h) and the phosphorylation level of p44/p42 MAPK [or extracellular-regulated kinase (ERK)] on Thr202/Thr204 was determined by western blot. Figure 1a shows that NA strongly induced the phosphorylation of p44/p42 MAPK after 5 min of treatment. The level of phosphorylation was increased by 219 ± 50% and 312 ± 62% (for p44 and p42, respectively) above control condition (set at 100%) after 5 min of treatment. Phosphorylation of p44/p42 MAPK decreased sharply after 30 min and returned almost to control level after 1 h of treatment with NA. In order to assess the specificity of the effect of NA on the p44/p42 MAPK signaling pathway, we pre-treated cortical neuron cultures with PD98059 50 μmol/L (a potent and selective inhibitor of MEK) during 30 min prior to adding NA 100 μmol/L during 5 min. Figure 1b shows that PD98059 pre-treatment completely prevented the phosphorylation of p44/p42 MAPK induced by NA in cultured cortical neurons.
Cultured cortical neurons were treated with NA 100 μmol/L for three time intervals (5 min, 30 min, and 1 h) and the phosphorylation level of Akt on Ser473 was determined by western blot. Figure 2 shows that NA induced the phosphorylation of Akt after 5 min of treatment. Akt phosphorylation was increased by 90 ± 35% above control level (set at 100%). Akt phosphorylation remained elevated at 30 min (+80 ± 33%) and started to decrease after 1 h of NA treatment. In order to assess the specificity of the effect of NA on the PI3K/Akt signaling pathway, we pre-treated the cortical neuron cultures with LY294002 10 μmol/L (a potent and selective inhibitor of PI3K) during 30 min prior to adding NA 100 μmol/L during 5 min. Results in Fig. 2 show that LY294002 pre-treatment completely prevented the phosphorylation of Akt induced by NA in cultured cortical neurons.
NA activates the mTOR/S6K signaling pathway in cultured mouse cortical neurons
Cultured cortical neurons were treated with NA 100 μmol/L for three time intervals (5 min, 30 min, and 1 h) and the phosphorylation level of mTOR on Ser2448 was determined by western blot. Figure 3 shows that NA induced the phosphorylation of mTOR as soon as 5 min after the beginning of the treatment. Phosphorylation of mTOR was increased by 114 ± 36% above control level (set at 100%). This increased phosphorylation state reached a maximum after 30 min (+134 ± 37%) and remained significant after 1 h (+113 ± 35%) of treatment with NA. In order to assess the specificity of the effect of NA on the mTOR signaling pathway, we pre-treated the cortical neuron cultures with rapamycin 20 ng/mL (a potent and selective inhibitor of mTOR) during 30 min prior to adding NA 100 μmol/L during 30 min. As shown in Fig. 3, rapamycin pre-treatment prevented the enhanced phosphorylation of mTOR induced by NA in cultured cortical neurons.
Cultured cortical neurons were treated with NA 100 μmol/L for three time intervals (5 min, 30 min, and 1 h) and the phosphorylation level of the S6 ribosomal protein on Ser235/236 was determined by western blot. Figure 4 shows that NA significantly induced the phosphorylation of S6 as soon as 5 min after the beginning of the treatment (+109 ± 37% above control level set at 100%). This effect reached its maximum at 30 min of treatment with an increase of 225 ± 50% above control level. This phosphorylation state decreased after 1 h of treatment to approach control level. As S6 protein is known to be a downstream effector of the mTOR signaling pathway, we pre-treated the cortical neuron cultures with rapamycin 20 ng/mL (inhibitor of mTOR) during 30 min prior to adding NA 100 μmol/L during 30 min. Rapamycin pre-treatment completely prevented the phosphorylation of S6 ribosomal protein induced by NA in cultured cortical neurons (Fig. 4).
NA-induced increase in MCT2 protein expression predominantly depends on activation of PI3K/Akt/mTOR/S6K cascade
As it was shown that p44/p42 MAPK, PI3K/Akt and mTOR signaling pathways were indeed activated by NA in cultured mouse cortical neurons, we tested the effects of some specific inhibitors of these pathways on MCT2 protein expression following treatment with NA 100 μmol/L. It was observed that NA 100 μmol/L applied during 6 h on cultured mouse cortical neurons induced a significant increase of MCT2 immunoreactivity (IR) (Fig. 5a). This increase reached 100 ± 20% above the control level (Fig. 5b). It was also found that a 30 min pre-treatment with LY294002 10 μmol/L or with rapamycin 20 ng/mL blocked the NA-induced increase of MCT2 IR after 6 h of treatment (Fig. 5a). The quantification of MCT2 IR shows that LY294002 (PI3K inhibitor) and rapamycin (mTOR inhibitor) significantly reduced by more than 75% MCT2 protein expression compared with NA-stimulated condition (Fig. 5b). Although to a lesser extent, PD98059 50 μmol/L (MEK inhibitor) and SB202190 10 μmol/L (p38 MAPK inhibitor) also significantly reduced MCT2 protein expression compared with NA-stimulated condition (by 40% and 30%, respectively). In contrast, when tested alone, none of the four inhibitors used was found to alter either MCT2 IR levels or MCT2 mRNA levels (data not shown), thus excluding a non-specific effect of the inhibitors on transcription or translation.
It has been previously shown that NA activates ERK (or p44/p42 MAPK) via its phosphorylation in primary cultures of cortical or hippocampal neurons (Tolbert et al. 2003; Lenz and Avruch 2005; Chen et al. 2006). Our results obtained from mouse cortical neurons are fully consistent with this observation. Moreover, the p44/p42 MAPK phosphorylation upon treatment with NA was entirely prevented by the MEK inhibitor PD98058, thus confirming the activation of the MAPK cascade. Although the PI3K can be one of the upstream elements implicated in this activation (Tolbert et al. 2003), it is also involved in the canonical PI3K/Akt signaling pathway (Brunet et al. 2001). In our cultured neurons, we could demonstrate for the first time that NA causes a phosphorylation of Akt that is dependent on PI3K. Interestingly, both of these signal transduction pathways (MAPK and PI3K/Akt cascades) have been implicated in the regulation of translation in neurons and might even exert a synergistic effect (Klann et al. 2004; Shahbazian et al. 2006). A key protein responsible for the activation of translation is the protein kinase mTOR, which is presumably activated by the PI3K/Akt pathway (Gingras et al. 2001; Raught et al. 2001; Hay and Sonenberg 2004). In this study, we show that NA promotes the phosphorylation of mTOR leading to its activation. One main target of mTOR kinase activity is the S6 kinase (S6K), which subsequently itself phosphorylates the ribosomal S6 protein (Inoki and Guan 2006). This protein is part of the components of the translation machinery responsible for the regulation of translation, especially for a group of mRNAs bearing a 5′-terminal oligopyrimidine tract in their 5′ untranslated region (5′UTR) (Klann et al. 2004). Our data reveals that NA increases the phosphorylation of the ribosomal S6 protein, an effect prevented by the mTOR inhibitor rapamycin. A similar effect of NA on S6 phosphorylation has been reported recently (Lenz and Avruch 2005). Altogether, our results suggest that NA could promote translation of specific target proteins in neurons via at least two cascades: one involving the MAPK pathway and the other via the PI3K/Akt/mTOR/S6K pathway (see Fig. 6 for a scheme illustrating the different pathways investigated).
Subsequently, we sought to determine the involvement of each of these pathways in the increase of MCT2 expression observed following NA treatment using different inhibitors for key elements in these signal transduction cascades. First, results indicate that the MAPK p38 and p44/p42 (ERK) may play a small but significant role in this effect. Possible downstream effectors are the translation initiation factors eIF4B and eIF4E, which have been shown to be phosphorylated upon activation of the MAPK cascade and are critical components of the translation machinery (Gingras et al. 1999; Raught and Gingras 1999; Shahbazian et al. 2006). Quite remarkably, however, it was found that the PI3K/Akt/mTOR/S6K cascade most likely plays a major role in the increased MCT2 protein expression observed upon NA treatment in neurons, an effect which was shown previously to be regulated at the translational level rather than at the transcriptional level (Pierre et al. 2003). Apart from phosphorylation of the ribosomal S6 protein mentioned above that participates in the translation initiation complex, the PI3K/Akt/mTOR/S6K pathway might also activate translation via phosphorylation of two eIF4E-binding proteins called 4E-BP1 and 4E-BP2. These two proteins act as repressors of translation by inhibiting interactions between the translation initiation factors eIF4E and eIF4G, while their phosphorylation by S6K relieves this inhibition (Raught and Gingras 1999). Another possible and concomitant mechanism could be the activation of the translation initiation factor eIF4B, which was shown recently to be activated via its phosphorylation both by the MAPK and the PI3K/Akt/mTOR/S6K cascades (Shahbazian et al. 2006). It is important also to note that in parallel with these activated cascades, specific elements found in the 5′UTR and 3′UTR of mRNAs are responsible for the selective translational regulation of certain classes of mRNAs (Pesole et al. 2001; Wilkie et al. 2003). In the case of MCT2, it remains to be determined which of these 5′ and/or 3′UTR elements present in the MCT2 mRNA sequence can explain the observed NA-induced translational activation.
Noradrenaline is known to be involved in memory formation and synaptic plasticity, notably by enhancing the early and late phase of long-term potentiation (Kobayashi and Yasoshima 2001). Such events involve not only a transcriptional activation to induce the expression of memory-associated genes but increasing evidence indicate that induction of translation is a key mechanism in synaptic plasticity (Kelleher et al. 2004a; Klann and Dever 2004; Klann et al. 2004), including for the effects of NA (Gelinas and Nguyen 2005). Furthermore, it was shown that activation of both the MAPK cascade (Kelleher et al. 2004b) and the PI3K/Akt/mTOR/S6K pathway (Tsokas et al. 2005; Horwood et al. 2006) leading to enhanced protein synthesis are involved in synaptic plasticity and memory formation. A number of proteins involved in synaptic plasticity and shown to be translationally regulated have been identified so far including α Ca2+-calmodulin-dependent protein kinase II (Wu et al. 1998; Scheetz et al. 2000), α-amino-3-hydroxy-5-methyl-4-isoxazolpropionate receptor subunits (Narisawa-Saito et al. 1999), and postsynaptic density-95 (Todd et al. 2003). Our results suggest that MCT2 might represent an additional target protein to be regulated at the translational level in conditions leading to or modulating synaptic plasticity. Such an observation may seem surprising as MCT2 is involved in cerebral energy metabolism rather than being a component of synaptic transmission. However, MCT2 was recently shown to be located in dendrites of glutamatergic neurons (Bergersen et al. 2001; Pierre et al. 2002). More specifically, it was found in the post-synaptic density of glutamatergic synapses as well as on vesicle-like structures within the dendritic spine head, forming a reserve pool (Bergersen et al. 2005). Such a distribution suggests that it might participate in the post-synaptic changes occurring as part of synaptic plasticity. The regulation of MCT2 protein expression described in the present work might represent an additional level of control to ensure a sufficient and steady supply of energy substrates to potentiated synapses. The concept that energy metabolism is coupled to synaptic plasticity has emerged recently (Vaynman and Gomez-Pinilla 2006; Vaynman et al. 2006). Indeed, it is a common finding that expression of several proteins involved in energy metabolism are altered in paradigms inducing synaptic changes (Ding et al. 2006; McNair et al. 2006). Our observation that MCT2 can be up-regulated by NA, a neurotransmitter involved in learning and memory processes, is consistent with the possibility of a coupling between energy metabolism and synaptic plasticity. Therefore, providing evidence for an essential role of MCT2 in synaptic plasticity should attract attention and will now require further investigation.
The authors would like to thank Ms. Annabelle Parent for expert technical assistance and Dr Karin Pierre for carefully reading the manuscript as well as for her advice on many aspects of this work. This study was supported by the Swiss Fonds National de la Recherche Scientifique grant N° 3100A0-112119 to LP.