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Keywords:

  • Akt;
  • antioxidants;
  • cyclic AMP-response element binding protein;
  • flavonoids;
  • mitogen-activated protein kinase;
  • neurodegeneration

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Emerging evidence suggests that the cellular actions of flavonoids relate not simply to their antioxidant potential but also to the modulation of protein kinase signalling pathways. We investigated in primary cortical neurons, the ability of the flavan-3-ol, (-)epicatechin, and its human metabolites at physiologically relevant concentrations, to stimulate phosphorylation of the transcription factor cAMP-response element binding protein (CREB), a regulator of neuronal viability and synaptic plasticity. (-)Epicatechin at 100–300 nmol/L stimulated a rapid, extracellular signal-regulated kinase (ERK)- and PI3K-dependent, increase in CREB phosphorylation. At micromolar concentrations, stimulation was no longer apparent and at the highest concentration tested (30 μmol/L) (-)epicatechin was inhibitory. (-)Epicatechin also stimulated ERK and Akt phosphorylation with similar bell-shaped concentration-response characteristics. The human metabolite 3′-O-methyl-(-)epicatechin was as effective as (-)epicatechin at stimulating ERK phosphorylation, but (-)epicatechin glucuronide was inactive. (-)Epicatechin and 3′-O-methyl-(-)epicatechin treatments (100 nmol/L) increased CRE-luciferase activity in cortical neurons in a partially ERK-dependent manner, suggesting the potential to increase CREB-mediated gene expression. mRNA levels of the glutamate receptor subunit GluR2 increased by 60%, measured 18 h after a 15 min exposure to (-)epicatechin and this translated into an increase in GluR2 protein. Thus, (-)epicatechin has the potential to increase CREB-regulated gene expression and increase GluR2 levels and thus modulate neurotransmission, plasticity and synaptogenesis.

Abbreviations used
AMPA

α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

BDNF

brain-derived neurotrophic factor

CRE

cAMP responsive element

CREB

cAMP-response element binding protein

ECL

enhanced chemiluminescence

EGCG

epigallocatechin gallate

ERK

extracellular signal-regulated kinase

HBM

HEPES-buffered incubation medium

MAPK

mitogen-activated protein kinase

MEK

MAP kinase kinase

PI

phosphatidylinositol

qPCR

quantitative RT-PCR

There is growing interest in the potential beneficial effects of flavonoids in the ageing and diseased brain. Dietary supplementation studies in humans and several animal models using flavonoid-rich plant or food extracts have shown improvements in cognition and learning (Youdim and Joseph 2001; Galli et al. 2002; Unno et al. 2004; Haque et al. 2006; Kuriyama et al. 2006; Wang et al. 2006) presumably by protecting vulnerable neurons, enhancing existing neuronal function or by stimulating neuronal regeneration. The cellular mechanisms underlying these actions of flavonoids are unknown but could potentially involve antioxidant and free radical scavenging activities, metal chelation or more likely the modulation of protein and lipid kinase signalling pathways (Mandel et al. 2004; Williams et al. 2004).

A wide-range of flavonoids protect cultured neurons against neurodegenerative disease-relevant insults and one of the most effective is (-)epicatechin (Schroeter et al. 2000). (-)Epicatechin, is a flavan-3-ol found in numerous fruits, green tea, red wine and cocoa products. Following oral ingestion flavanols undergo substantial phase I/II metabolism (Hackett and Griffiths 1983; Schroeter et al. 2006) but despite this, data on the effects of human flavanol metabolites in vitro are rare (Spencer et al. 2001, 2003). (-)Epicatechin is metabolised into glucuronidated, methylated and sulfated forms some of which have been detected in rat brain following dietary supplementation with (-)epicatechin (Abd El Mohsen et al. 2002), suggesting these flavonoids are able to cross the blood–brain barrier and thus have the potential to be active in vivo.

Emerging evidence suggests that the neuroprotective actions of (-)epicatechin and the green tea flavonoid epigallocatechin gallate (EGCG) relate not simply to their H-donating antioxidant potential but also to the modulation of protein kinase signalling cascades. For example, flavanols have been demonstrated to inhibits stress-activated extracellular signal-regulated kinase 1 and 2 (ERK1/2) and c-jun N-terminal kinase signalling pathways resulting in the protection of neurons from apoptosis (Schroeter et al. 2001). In contrast, the protective actions of EGCG against 6-hydroxy dopamine toxicity and serum deprivation involves the restoration of protein kinase C activity and ERK1/2 activities as well as the modulation of cell survival and cell cycle genes, respectively (Levites et al. 2002; Reznichenko et al. 2005).

Despite this evidence, few studies have investigated the direct signalling potential of physiological relevant concentrations of flavonoids or indeed their in vivo metabolites. Indeed most in vitro data on flavanol-mediated effects are obtained using concentrations that are much higher than can be achieved in vivo under normal physiological conditions. The concentrations used for studies in vitro are particularly important as numerous flavonoids exert anti-apoptotic actions at low concentrations but act as pro-apoptotic stimuli at higher concentrations. Indeed, we have previously shown that quercetin and its metabolites 3′-O-methyl quercetin and 4′-O-methyl quercetin inhibit protein kinase B/Akt (Akt) and ERK1/2 phosphorylation, an action underlying their potentially pro-apoptotic action in neurons. Interestingly, at lower concentrations quercetin stimulated phosphorylation of the transcription factor the cAMP-response element binding protein (CREB) (Spencer et al. 2003), which could reflect an anti-apoptotic protective response, as CREB-dependent up-regulation of gene expression is neuroprotective (Riccio et al. 1999). In addition to these pro-survival responses, CREB-mediated gene expression plays a role in memory formation (Bourtchuladze et al. 1994) through the up-regulation of receptors and growth factors that are involved in synaptic strengthening (Kandel 2001; Lonze and Ginty 2002). Thus, molecules that stimulate signalling pathways leading to the phosphorylation of CREB could act to modulate synaptic efficacy and potentially improve cognition not simply through neuroprotection but also via the up-regulation of genes involved in synaptic plasticity. Therefore, to determine if flavonoids have the potential to stimulate CREB-mediated gene expression, we have investigated whether nanomolar concentrations of (-)epicatechin and its major in vivo metabolites act on pathways leading to CREB phosphorylation and CRE (cAMP responsive element)-mediated gene expression in neurons.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Materials

(-)Epicatechin was purchased from Extrasynthase (Genay, France). The synthesis and purification of 3′-O-methyl-(-)epicatechin and (-)epicatechin glucuronide by semi-preparative HPLC was undertaken as previously described (Spencer et al. 2001). U0126, LY294002 and wortmannin were purchased from Calbiochem Corporation (La Jolla, CA, USA). Antibodies used were anti-ACTIVE MAPK (mitogen-activated protein kinase) (ERK1/2) (Promega, Madison WI, USA), total ERK2 and C14 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Akt, pAkt Ser473, CREB and pCREBSer133 (all from Cell Signaling Technologies, Danvers, MA, USA), GluR2 and GluR1 (Chemicon International, Temecula, CA, USA). Horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Sigma-Aldrich, Gillingham, UK), enhanced chemiluminescence (ECL) reagent and Hyperfilm-ECL were purchased from GE Healthcare (Little Chalfont, UK). pCRE-luc cis reporter plasmid (Stratagene, La Jolla, CA, USA) and Bright-Glo luciferase assay system (Promega) were used for this study. Oligonucleotides were obtained from Sigma-Genosys (Papiford, UK). All other reagents were obtained from Sigma or Merck (Poole, UK).

Neuronal cell culture

Primary cortical neurons were prepared from 15–16-day-old Swiss mouse embryos (NIH, Harlan, UK) as described previously (Crossthwaite et al. 2002). Briefly, dissociated cells were plated at 106 cells/mL into either 92 × 17 mm petri plates, 24-well plates or six-well plates, that had been coated previously overnight with 15 μg/mL poly-l-ornithine, in a serum-free medium composed of a mixture of Dulbecco’s modified Eagle’s medium and F-12 nutrient (1 : 1 v/v) (Invitrogen, Paisley, UK) supplemented with 33 mmol/L glucose, 2 mmol/L glutamine, 6.5 mmol/L sodium bicarbonate, 5 mmol/L HEPES buffer (pH 7.4), 100 μg/mL streptomycin and 60 μg/mL penicillin. A mixture of hormones and salts composed of insulin (25 μg/mL), transferrin (100 μg/mL), putrescine (60 μmol/L), progesterone (20 nmol/L) and sodium selenate (30 nmol/L) (all from Sigma) was also added to the culture medium. Cells were cultured at 37°C in a humidified atmosphere of 95% air and 5% CO2 and were used after 5–7 days in vitro when the majority of cells (>95%) were neuronal as determined by neurofilament and β-tubulin immunoreactivity and there were <5% glial elements as determined by glial fibrillary acidic protein immunoreactivity (not shown).

Immunoblotting

Western blot analysis was performed as described previously (Spencer et al. 2003). Neurons cultured in six-well plates or 92 × 17 mm petri plates were placed in a water bath at 37°C and left for 5 min to equilibrate. After this period, the culture medium was removed and replaced with HEPES-buffered incubation medium (HBM; 20 mmol/L HEPES (pH 7.4), 140 mmol/L NaCl, 5 mmol/L KCl, 5 mmol/L NaHCO3, 5 mmol/L Na2HPO4, 1.2 mmol/L CaCl2 and 5.5 mmol/L glucose). Neurons were pre-incubated for 5 min with kinase inhibitors (MAP kinase kinase (MEK)1/2 inhibitor U0126 or phosphatidylinositol (PI)3-kinase inhibitor LY294002), or vehicle, prior to treatment with flavonoids. Following pre-incubation, neurons were stimulated for 15 min with (-)epicatechin or (-)epicatechin metabolites (30 nmol/L–30 μmol/L) in the further presence or absence of kinase inhibitors where appropriate. After 15 min, the HBM was removed and for the measurements of Akt, pAkt, CREB, pCREB ERK and pERK, the plates were quickly washed with ice-cold phosphate buffered saline (pH 7.4) (Ca2+-free with 200 μmol/L EGTA) and placed immediately on ice. For measurement of GluR2, GluR1 and ERK expression, the HBM was removed and replaced with neuronal conditioned medium and the cells returned to the CO2 incubator for a further 18 h. Crude lysates were prepared by scraping the cell monolayer in ice-cold 1% Triton X-100 lysis buffer (50 mmol/L Tris (pH 7.5), 150 mmol/L NaCl and 1% Triton X-100) containing a cocktail of inhibitors at a final concentration of 2 mmol/L EDTA, 2 mmol/L EGTA, 0.5 mmol/L phenylmethylsulfonyl fluoride, 10 μg/mL leupeptin, 10 μg/mL antipain, 1 μg/mL pepstatin A, 1 μg/mL chymostatin, 5 mmol/L sodium pyrophosphate, 1 mmol/L Na3VO4 and 50 mmol/L NaF, and left on ice for 45 min before centrifugation at 2000 × g at 4°C for 5 min. The protein concentration in the supernatants was determined using Bio-Rad protein assay reagent (Hemel-Hempstead, UK) and samples were then denatured by boiling for 5 min in gel loading buffer [62.5 mmol/L Tris (pH 6.8), 2% sodium dodecyl sulphate, 5% 2-mercaptoethanol, 10% glycerol and 0.0025% bromophenol blue]. Samples were run (20 μg/lane) on 9% sodium dodecyl sulphate–polyacrylamide gels and transferred to nitrocellulose membrane by electroblotting. Immunoblots were then blocked [20 mmol/L Tris (pH 7.5), 150 mmol/L NaCl] (TBS) containing 5% skimmed milk powder for 30 min washed for 10 min in TBS supplemented with 0.05% (v/v) Tween 20 (TTBS) and then incubated with primary antibodies overnight at 25°C in TTBS containing 1% skimmed milk powder at the following dilutions: anti-ACTIVE MAPK (1 : 5000), ERK1/2 (1 : 1000), phospho-CREB (1 : 2000), CREB (1 : 1000), phospho-Akt (1 : 2000), Akt (1 : 1000), GluR2 (1 : 1000) or GluR1 (1 : 1000). The immunoblots blots were washed twice in TTBS and then incubated in TTBS containing 1% skimmed milk powder with goat anti-rabbit IgG conjugated to horseradish peroxidase (1 : 1000 dilution of stock) for 1 h. Finally, immunoblots were washed in TTBS for 5 min, rinsed in TBS for 10 min and then treated with ECL reagent. Immunoblots were then exposed to Hyperfilm ECL for 1–2 min and developed. Relative bands intensities were obtained by densitometric analysis of films using Bioimage Intelligent Quantifier software (Ann Arbor, MI, USA). Molecular weights were calculated from comparison with pre-stained molecular weight markers (MW 29 000–205 000).

CRE-luciferase reporter assay

Cortical neurons cultured in 24-well plates (5 × 105/well) were transfected overnight with 0.5 μg of the pCRE-luc cis reporter plasmid (Stratagene) and 0.5 μg of the transfection efficiency Renilla luciferase phRL-TK plasmid (Promega) using 1 μL LipofectAMINE 2000 (Invitrogen) according to the manufacturer’s instructions. This resulted in a transfection efficiency of 5–10% as determined using a green fluorescent protein plasmid (not shown). Following transfection, the growth medium was removed and retained and neurons were pre-incubated for 5 min with U0126 (5 μmol/L) or vehicle in HBM. Following pre-incubation, neurons were stimulated for 15 min with (-)epicatechin or 3′-O-methyl-(-)epicatechin (100 nmol/L) in the presence or absence of U0126 as appropriate. After 15 min, the HBM was removed and replaced with the original conditioned medium and the cells returned to the CO2 incubator for a further 18 h. Luciferase assays were performed using a Dual-Glo luciferase assay system as described in the manufacturer’s instructions (Promega). Briefly, the growth medium was removed and Glo lysis buffer (40 μL/well) was added to the neuronal monolayer and left for 5 min. Lysates were then transferred to a 96-well luminometer plate (Greiner, Stonehouse, UK) and luciferase activities produced by the pCRE luciferase reporter plasmid was measured using a Veritas microplate luminometer (Turner Biosystems Inc., Sunnyvale, CA, USA). Renilla luciferase activities produced by the phRL-TK control plasmid were then assayed by adding an equal volume of Dual-Glo Stop and Glo substrate (comprising the stop solution for firefly luciferase and substrate for Renilla luciferase) and remeasuring in the luminometer. All treatments were performed in quadruplicate on five- to six-independent cultures. Firefly luciferase activities were standardised to the corresponding Renilla luciferase activities and statistical analyses were performed using one-way anova.

Reverse-transcriptase PCR and quantitative real-time PCR

Neurons in petri dishes were stimulated with (-)epicatechin (100 nmol/L in HBM for 15 min) or vehicle control. Eighteen hours after stimulation, RNA was extracted with RNABee (Biogenesis, Poole, UK) according to the manufacturer’s protocol and was resuspended with 10 mmol/L NaCl, 10 mmol/L Tris pH 8.0 and 1 mmol/L EDTA. Synthesis of cDNA was as previously described (Suchak et al. 2003) using oligo(dT)15 primer (Promega, Southampton, UK) and M-MLV RNase H minus reverse transcriptase (Promega). In the negative controls, reverse transcriptase was omitted from the reaction tubes. Primers were designed in house, except for the pair used for brain-derived neurotrophic factor (BDNF) (Fiore et al. 2003). The primer pairs used for PCR were as follows:

Mouse GluR1 (accession no. NM008165): mGLUR1L5, 5′-TCCGCAAGATTGGTTACTGG-3′ and mGLUR1R5, 5′-CAGATCTCGTAGGCCAAAGG-3′; Mouse GluR2 (accession number NM013540): mGLUR2L5, 5′-AATAGAAAGGGCCCTCAAGC-3′ and mGLUR2R5, 5′-ATTCCAAGGCTCATGAATGG-3′; Mouse GluR3 (accession number NM016886): mGLUR3L1, 5′-GGGTGAGACTGGATGAAAGG-3′ and mGLUR3R1, 5′-CCTTCATAGCGCTCATTTCC-3′; Mouse GluR4 (accession number NM019691): mGLUR4L1, 5′-GAAGCACGTCAAAGGCTACC-3′ and mGLUR4R1, 5′-TTCCAATAGCCAACCTTTCG-3′; Mouse BDNF (accession number NM007540): mBDNFL-3, 5′-AGCTGAGCGTGTGTGACAGT-3′ and mBDNFR-3, 5′-TCCATAGTAAGGGCCCGAAC-3′; Mouse nR4a2 (accession number NM013613): mNR4a2L2, 5′-AGGTCGTTTACCCTCGAAG-3′ and mNR4a2R2 5′-ATTGCAACCTGTGCAAGACC-3′; Mouse Zfhx1a (accession number NM011546): mZfhx1aL4, 5′-ATGTGACAAGTGTGGCAAGC-3′ and mZfhx1aR4, 5′-GCTCCAACCTCCACTGTACC-3′; Beta-Actin (accession number NM031144): ACT-S, 5′-ATCGTGGGCCGCCCTAGGCAC-3′ and ACT-AS, 5′-TGGCCTTAGGGTTCAGAGGGGC-3′. PCR was carried out with 2.5 µL cDNA (equivalent to 0.125 μg total RNA) in a reaction volume of 25 µL containing 1.5 mmol/L MgCl2 (Promega), reaction buffer (Promega) comprising 45 mmol/L KCl, 9 mmol/L Tris-HCl (pH 9.0) and 0.09% Triton X-100, 0.05 mmol/L dNTP (GE Healthcare), 0.1 μmol/L primers and 0.625 U Taq polymerase (Promega). Amplification was performed in a GeneAmp 9700 thermal cycler (Applied Biosystems, Warrington, UK); the initial denaturation step was 5 min at 94°C, followed by 22–35 cycles of 30 s at 94°C, 30 s at 55°C, 30 s at 72°C and then a final step of 7 min at 72°C. Negative controls for each sample were also amplified to ensure that genomic DNA was not present. Semi-quantitative RT-PCR (qPCR) was carried out as previously described (Dolman et al. 2005) and intensities of ethidium-bromide stained PCR products were determined with a UVIpro gel documentation system and associated software (UVItec, Cambridge, UK). Ratios of intensity of bands for genes of interest compared with beta-actin internal controls were calculated for each sample. Data from six-independent pairs of cultures were used for each determination.

qPCR was carried out using the Rotor Gene 6 Machine (Corbett Research, Cambridge, UK), and 0.5 μL cDNA from each sample was amplified in a total volume of 25 μL containing a SYBR green amplification mixture (Applied Biosystems) and 0.1 μmol/L of each primer. Primers for qPCR were Mouse GluR1: RTGluR1L1, 5′-GTCCGCCCTGAGAAATCCAG-3′ and RTGluR1R1, 5′-CTCGCCCTTGTCGTACCAC-3′; Mouse GluR2: RTGluR2L2, 5′-TGTGTGGTGGTTCTTTACCCT-3′ and RTGluR2R2-3, 5′-AGTAGGCATACTTCCCTTTGGAT-3′; beta-actin, as described above. Gene expression levels per sample were obtained by fitting the fluorescence intensities to standard curves and levels of the genes of interest normalised to actin mRNA levels. Data from four-independent pairs of cultures were used for each determination.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

(-)Epicatechin stimulates ERK1/2-dependent CREB phosphorylation

There is considerable interest in identifying safe-effective agents that enhance the function of the transcription factor CREB, as these could have the potential to improve memory deficits associated with conditions of cognitive decline such as in Alzheimer’s disease (Carlezon et al. 2005). To investigate the potential for (-)epicatechin to signal to CREB, cortical neurons were stimulated for 15 min with a range of concentrations of (-)epicatechin (30 nmol/L–30 μmol/L) and cell lysates were immunoblotted with a phospho-CREB polyclonal antibody that detects the protein when it is phosphorylated at Ser133 within the kinase inducible domain. A 15 min treatment regime was chosen as other stimuli such as glutamate and hydrogen peroxide show a robust increase in phospho-CREB levels in cortical neurons at this time point (Crossthwaite et al. 2002; Perkinton et al. 1999). (-)Epicatechin treatments caused a robust phosphorylation of CREB with a maximum stimulation observed between 100 and 300 nmol/L (Figs 1a and b). At higher concentrations the levels of phosphorylation were much lower and returned to a level below basal at concentrations greater than 10 μmol/L (-)epicatechin. Increased CREB phosphorylation above basal was still evident 1 h after exposure to (-)epciatechin (not shown). Total protein levels of CREB were not significantly altered during the time of treatment and by any of the (-)epicatechin concentrations tested (Fig. 1a). A number of protein kinase signalling pathways regulate CREB phosphorylation, but in neurons key pathways involve ERK1/2 (Impey et al. 1998) and PI3-kinase (Perkinton et al. 1999). To test whether or not these pathways were involved in the transduction of information from (-)epicatechin to CREB, cortical neurons were treated with the MEK inhibitor U0126 (5 μmol/L), which blocks activation of ERK1/2, and/or the PI3-kinase inhibitor LY294002 (50 μmol/L), which blocks PI3-kinase-dependent signalling. U0126 treatments significantly reduced (-)epicatechin (100 nmol/L)-stimulated CREB phosphorylation by ∼55%, and the combined application of U0126 and LY294002 completely abolished (-)epicatechin-stimulated CREB phosphorylation (Fig. 2). Neither U0126 nor LY294002 significantly altered basal CREB phosphorylation (not shown). Together, this data demonstrates a causal involvement of both PI3-kinase- and ERK1/2-dependent signalling, in mediating neuronal response to (-)epicatechin.

image

Figure 1.  (-)Epicatechin stimulates cAMP-response element binding protein (CREB) phosphorylation in a concentration-dependent manner. Cortical neurons were exposed to increasing concentrations of (-)epicatechin (30 nmol/L–30 μmol/L) for 15 min and the levels of phosphorylated CREB (Ser133) and total CREB was measured by immunoblotting. (a) Representative immunoblots from a single experiment showing phosphorylated CREB and total CREB in the same cell lysates. (b) Band intensities were determined by densitometric analysis using Bioimage Intelligent Quantifier Software. (-)Epicatechin exhibited bell-shaped concentration response characteristics and stimulated a significant increase in CREB phosphorylation at 100–300 nmol/L (a upper panel and b) compared with the untreated basal, without altering the total levels of CREB (a lower panel). **p < 0.01 one-way anova, followed by Dunnett’s post hoc test, n = 3.

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image

Figure 2.  (-)Epicatechin stimulates cAMP-response element binding protein (CREB) phosphorylation through an extracellular signal-regulated kinase (ERK)- and PI3K-dependent pathway. Cortical neurons were exposed to 100 nmol/L epicatechin for 15 min following a 5 min pre-treatment with or without the MEK inhibitor U0126 (5 μmol/L) and/or the PI3K inhibitor LY294002 (50 μmol/L) and the levels of phosphorylated CREB (Ser133) measured by immunoblotting. (a) Representative immunoblot from a single experiment showing phosphorylated CREB. (b) Band intensities were determined by densitometric analysis using Bioimage Intelligent Quantifier Software. U0126 alone, or in combination with LY294002, significantly reduced (-)epicatechin stimulated CREB phosphorylation. *p < 0.05; **p < 0.01 one-way anova followed by Dunnett’s post hoc test, n = 3.

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(-)Epicatechin stimulates ERK and Akt phosphorylation in neurons

The sensitivity of CREB phosphorylation to U0126 and LY294002 strongly suggested that (-)epicatechin was able to recruit and to stimulate ERK1/2 and PI3-kinase pathways in neurons Therefore, the ability of (-)epicatechin to directly stimulate ERK1/2 and PI3-kinase-sensitive signalling pathways was tested. As demonstrated for CREB above, low concentrations of (-)epicatechin also stimulated a robust phosphorylation of ERK1/2 (Fig. 3a). In addition, (-)epicatechin-mediated activation of PI3-kinase culminated in the subsequent phosphorylation of Akt (Fig. 4a) with maximum effects at (-)epicatechin concentrations between 100 and 300 nmol/L (Figs 3b and 4b). At higher (-)epicatechin concentrations, the extent of phosphorylation was much lower, returning to basal at levels greater than 1 μmol/L. The total protein levels of ERK1/2 and Akt were not significantly altered by any of the treatments tested (Figs 3a and 4a). Thus, acute exposure to (-)epicatechin stimulates a concentration-dependent increase in ERK1/2 and Akt phosphorylation, which mediates signalling to CREB phosphorylation in neurons.

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Figure 3.  (-)Epicatechin stimulates extracellular signal-regulated kinase (ERK) phosphorylation in a concentration-dependent manner. Cortical neurons were exposed to increasing concentrations of (-)epicatechin (100 nmol/L–10 μmol/L) for 15 min and the levels of dually phosphorylated ERK1/2 (pTEpY) and total ERK1/2 was measured by immunoblotting (a). Representative immunoblots from a single experiment showing phosphorylated ERK1/2 and total ERK1/2 in the same cell lysates. Band intensities were determined by densitometric analysis using Bioimage Intelligent Quantifier Software (b). (-)Epicatechin exhibited bell-shaped concentration response characteristics with a maximum stimulation of ERK1/2 phosphorylation between 100 and 300 nmol/L (n = 3 for 100 nmol/L–1 μmol/L; n = 2 for 3–10 μmol/L).

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Figure 4.  (-)Epicatechin stimulates Akt phosphorylation in a concentration-dependent manner. Cortical neurons were exposed to increasing concentrations of (-)epicatechin (100 nmol/L–10 μmol/L) for 15 min and the levels of phosphorylated Akt (Ser473) and total Akt was measured by immunoblotting (a). Representative immunoblots from a single experiment showing phosphorylated Akt and total Akt in the same cell lysates. Band intensities were determined by densitometric analysis using Bioimage Intelligent Quantifier Software (B). (-)Epicatechin exhibited bell-shaped concentration response characteristics with a maximum stimulation of Akt phosphorylation observed at 100–300 nmol/L (n = 3 for 100 nmol/L–1 μmol/L; n = 2 for 3–10 μmol/L).

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Stimulation of ERK1/2 phosphorylation by an in vivo metabolite of (-)epicatechin

Few in vitro investigations using flavonoids consider that under physiological in vivo conditions flavonoids undergo extensive phase I and II metabolism, which potentially alters their physical and chemical properties and ultimately their tissue distribution and bioactivity. Thus, to determine whether or not human metabolites of (-)epicatechin could also stimulate protein kinase signalling pathways in cells originating from the CNS, neurons were exposed to 3′-O-methyl-(-)epicatechin and (-)epicatechin-5′-O-β-d-glucuronide, and the levels of phosphorylated ERK1/2 were measured by immunoblotting. Treatments with 100 nmol/L of 3′-O-methyl-(-)epicatechin stimulated ERK1/2 phosphorylation to a degree comparable to that observed using 100 nmol/L (-)epicatechin (Fig. 5). In contrast at a concentration of 100 nmol/L, the O-glucuronide of (-)epicatechin did not stimulate ERK1/2 phosphorylation above basal levels.

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Figure 5.  Bioactivity of in vivo metabolites of (-)epicatechin. Cortical neurons were exposed to 100 nmol/L of (-)epicatechin (EC), 3′-O-methyl-(-)epicatechin (Me-EC) or (-)epicatechin-5-O-β-d-glucuronide (Gluc-EC) (structures shown) for 15 min and the levels of dually phosphorylated extracellular signal-regulated kinase 1/2 (ERK) (pTEpY) determined by immunoblotting. Band intensities were determined by densitometric analysis using Bioimage Intelligent Quantifier Software. (-)Epicatechin and Me-EC but not Gluc-EC caused a significant increase in the levels of phosphorylated ERK1/2. **p < 0.01, one-way anova, followed by Dunnett’s post hoc test, n = 3.

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Submicromolar (-)epicatechin stimulates a CRE-reporter in neurons

CREB family members bind to DNA via a conserved basic region/leucine zipper motif that recognises the CRE (Conkright et al. 2003). Upon exposure of neurons to BDNF, CREB becomes rapidly bound to DNA coincident with phosphorylation at Ser133. This inducible CREB-DNA binding is however, not simply dependent on CREB Ser133 phosphorylation or on either the ERK or PI3-kinase signalling pathways (Riccio et al. 2006). Therefore, to determine whether (-)epicatechin signalling to CREB phosphorylation via ERK and PI3-kinase has the potential to drive CRE-mediated gene expression, experiments were performed using a CRE-luciferase reporter assay. Primary cortical neurons were transfected overnight with a CRE-luciferase reporter and then treated for 15 min with either (-)epicatechin (100 nmol/L) or the human metabolite 3′-O-methyl-(-)epicatechin (100 nmol/L) and luciferase activities were measured 18 h later. Exposure to 100 nmol/L (-)epicatechin or 3′-O-methyl-(-)epicatechin stimulated a moderate, but statistically significant, increase in luciferase activity by approximately 30% as compared with basal (Fig. 6a). This suggests that (-)epicatechin and one of its principal metabolites have the potential to stimulate CRE-mediated gene expression in neurons. (-)Epicatechin stimulation of CREB phosphorylation was significantly reduced following inhibition of ERK1/2. Therefore, in order to better link CREB phosphorylation and the observed increase in CRE-luciferase expression, experiments were performed in presence and absence of the MEK inhibitor U0126. (-)Epicatechin stimulation of CRE-luciferase expression was reduced in the presence of U0126 (Fig. 6b), suggesting that ERK1/2-dependent phosphorylation of CREB results in increased CRE activity in neurons.

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Figure 6.  (-)Epicatechin stimulates extracellular signal-regulated kinase-dependent CRE activity. (a) Cortical neurons transfected overnight with a CRE-luciferase reporter plasmid and a Renilla luciferase phRL-TK control plasmid were exposed for 15 min to either (-)epicatechin (EC 100 nmol/L) or the in vivo metabolite 3′-O-methyl-(-)epicatechin (Me-EC 100 nmol/L) and luciferase activity measured 18 h later. Data is expressed as firefly luciferase luminescence relative to Renilla luciferase luminescence. (-)Epicatechin and 3′-O-methyl-(-)epicatechin stimulated CRE-luciferase expression relative to vehicle-treated control, *p < 0.05 one-way anova, followed by Dunnett’s post hoc test (n = 6). (b) Transfected neurons (as above) were pre-treated for 5 min with or without the MEK inhibitor U0126 (5 μmol/L) and then exposed for 15 min to (-)epicatechin (EC 100 nmol/L) in the continued presence (black bars) or absence (white bars) of U0126 and luciferase activity measured 18 h later. Exposure to 100 nmol/L (-)epicatechin stimulated a modest increase in luciferase activity only in the absence of U0126. *p < 0.05 paired t-test, n = 5.

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(-)Epicatechin stimulates GluR2 mRNA and protein expression in neurons

There are many genes whose expression has been reported to be altered following CREB activation. These include transcription factors such as Zhfx1a and N4ra2 (Conkright et al. 2003; Zhang et al. 2005; Riccio et al. 2006) and key proteins involved in neuronal plasticity, including the neurotrophin BDNF (Tao et al. 1998) and ionotropic glutamate receptor subunits (Borges and Dingledine 2001). Reverse-transcriptase PCR was used to evaluate whether a 15 min incubation of primary cortical neurons with 100 nmol/L (-)epicatechin would cause an elevation of various selected mRNAs as measured at 18 h after treatment. The glutamate receptor subunit GluR2 showed a modest and highly variable increase in mRNA level of 60% (Figs 7a and b), although other genes reported to be rapidly induced by CREB were not altered at this time-point, these included BDNF, Zhfx1a and N4ra2 (Fig. 7a). A number of other CREB target genes were elevated at 2 h post-epicatechin treatment but not at 18 h showing that the kinetics of activation has a bearing on the profile of up-regulated genes detected (data not shown). The elevation of GluR2 mRNA was confirmed by real time PCR with GluR2 levels following (-)epicatechin treatment 171% of untreated controls (Fig. 7c). The levels of GluR1 mRNA were ∼120%. As the mRNA levels were highly varaible, it was important to determine if elevated GluR2 mRNA was translated into increased GluR2 protein. In order to do this, cortical neurons were treated with 100 nmol/L (-)epicatechin for 15 min and the levels of GluR2 in crude cell lysates were determined 18 h later by immunoblotting and compared with the levels of GluR1 and ERK1/2. The anti-GluR2 antibody detected major bands at ∼105 kDa in crude lysates, corresponding to the predicted molecular weight of the receptor subunit and an additional lower molecular weight band. Neurons treated with (-)epicatechin showed increased levels of GluR2 and GluR1 immunoreactivity but no alterations in ERK1/2 (Fig. 8a). Densitometric analysis of immunoblots from multiple experiments showed that the levels of GluR2 increased significantly by ∼250% following exposure to (-)epicatechin, compared with a modest non-significant increase of ∼125% for GluR1 (Fig. 8b). The levels of total ERK1/2 were unchanged indicating that alterations in GluR2 were not due to protein loading.

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Figure 7.  (-)Epicatechin stimulates GluR2 mRNA expression in neurons. Cortical neurons were exposed to 100 nmol/L (-)epicatechin for 15 min and 18 h later the levels of brain-derived neurotrophic factor (BDNF), Zhfx1a, n4ra2, GluR1, GluR2 and GluR4 mRNA were measured by RT-PCR (a) and GluR2 and GluR1 mRNA by real time PCR (c). The data shown in (a) and (c) are mRNA levels following (-)epicatechin treatment relative to non-treated controls (100% hatched lines) means ± SEM n = 6 independent cultures of primary neurons. (b) Representative gel from RT-PCR showing GluR2 mRNA levels relative to actin mRNA from untreated (−) and epicatechin-treated (+) cortical neurons.

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Figure 8.  (-)Epicatechin stimulates GluR2 protein expression in neurons. Cortical neurons were exposed to 100 nmol/L (-)epicatechin for 15 min, and 18 h later the levels GluR2, GluR1 and extracellular signal-regulated kinase (ERK) protein were measured by immunoblotting (a). Immunoblots of GluR2, GluR1 and ERK expression in neuronal cell lysates following exposure to vehicle (control) or (-)epicatechin (EC) at 100 nmol/L. (b) Data shows relative band intensity obtained by densitometric analysis of immunoblots. (-)Epicatechin (100 nmol/L) treatment increased the levels of an immunoreactive band at 105 kDa corresponding to the expected molecular weight of GluR2, *p < 0.05 paired t-test, n = 6. The levels of GluR1 and ERK following (-)epicatechin treatment were not significantly different from control (n = 6 and n = 3, respectively).

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Dietary flavonoids are emerging as promising compounds for conferring protection to the nervous system against neurodegeneration (Mandel and Youdim 2004). Although the beneficial bioactivities of flavanoids are often claimed to be based on their antioxidant properties, it is becoming increasingly evident that this is unlikely to be the case in vivo. It is true that flavonoids exert significant antioxidant effects, including hydrogen-donation and metal-chelating actions, in the test tube, and in certain cell culture systems. However, considering that (i) almost all flavonoids are subject to extensive metabolism, (ii) O-methylation, O-glucuronidation and O-sulfation will decrease antioxidant properties by blocking essential hydroxyl groups and (iii) the maximal concentrations of flavonoids in the circulation, and even more so in the CNS, is much lower as compared with the levels of many other antioxidants [i.e. urate, ascorbate, albumin, glutathione, catalase, superoxide oxide dismutase, metal chelating proteins], then antioxidant properties alone are unlikely to figure greatly as the basis for any bioactivity of flavonoids in vivo. Indeed, recent evidence has shown that the green tea flavanol EGCG reduces cerebral amyloidosis by increasing α-secretase activity in a mouse model of Alzheimer’s disease (Rezai-Zadeh et al. 2005), an action mediated by an up-regulation of active a-disintegrin-and-metalloprotease 10 (Obregon et al. 2006). Although various flavonoids, especially flavanols, can modulate neuronal function and survival under conditions of stress, data on the direct effects of flavonoids or their metabolites, in the absence of added insults, particularly at physiologically relevant concentrations are only just starting to accumulate. In this study, (-)epicatechin was found for the first time to rapidly and potently stimulate MAPK and PI3-kinase signalling pathways, CREB phosphorylation and ERK-dependent CRE activity at nanomolar concentrations in primary cortical neurons. Moreover, stimulation of these signalling pathways was also observed with a major circulating human metabolite 3′-O-methyl-(-)epicatechin, but not with (-)epicatechin-5′-O-β-d-glucuronide. This lack of activity is consistent with our previous findings demonstrating that the O-glucuronidation of the native flavanol renders the compound inactive against stress-induced neurotoxicity and can be attributed to a lack of cell or membrane permeability (Spencer et al. 2001).

At higher micromolar concentrations, the (-)epicatechin-mediated activation of protein kinase pathways was lost and there was an inhibition of CREB phosphorylation. These concentration-dependent effects of (-)epicatechin on kinase signalling cascades may result from high affinity receptor agonist-like actions at low concentrations and direct-enzyme inhibition at higher concentrations (Agullo et al. 1997; Walker et al. 2000). However, it is equally possible that the loss of ERK, Akt and CREB phosphorylation at higher concentrations resulted from enhanced phosphatase activity or receptor desensitisation. The identity of the primary (-)epicatechin interacting site in neurons is unknown and could be either at the cell surface or intracellular, although the ERK and PI3-kinase dependence to CREB phosphorylation is reminiscent of ionotropic receptor signalling (Perkinton et al. 1999). Receptors reported to act as flavonoid binding sites that are present in cortical neurons are adenosine (Jacobson et al. 2002) and GABAA receptors (Johnstone 2005; Adachi et al. 2006). However, a specific plasma membrane binding site for polyphenols has been recently described in rat brain (Han et al. 2006) raising the possibility that (-)epicatechin might be acting through a steroid-like receptor in neurons to modulate CREB-mediated gene expression. An alternative explanation is that (-)epicatechin is mediating its signalling actions through the generation of hydrogen peroxide, which we have previously shown to be a rapid activator of ERK-dependent CREB phosphorylation in neurons (Samanta et al. 1998; Crossthwaite et al. 2002). It is known that long-term exposure to high concentrations of (-)epicatechin and other flavonoids can produce micromolar hydrogen peroxide in certain cell culture media (Long et al. 2000), although it is very unlikely that this accounts for the rapid signalling actions observed at nanomolar concentrations of (-)epicatechin in the present study.

CREB-dependent gene expression is neuroprotective (Riccio et al. 1999) and through the up-regulation of receptors and growth factors modulates the responsiveness of synapses to excitatory neurotransmission (Lonze and Ginty 2002). (-)Epicatechin-induced increases in the levels of glutamate GluR2 receptor subunits, which are regulated by CREB, could therefore have important implications both for neuronal viability and for synaptic plasticity in vivo. For example, abnormal influx of Ca2+ through α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors has been strongly implicated in neuronal death associated with a number of brain disorders through activation of Ca2+-dependent proteases, phospholipases and stress-activated kinases (Williams and Glowinski 1996; Tanaka et al. 2000). It is the presence of the GluR2 subunit that renders heteromeric AMPA receptor assemblies Ca2+-impermeable, thus (-)epicatechin could potentially confer some protection to neurons by increasing GluR2 levels in the membrane. This is not unprecedented as EGCG has been shown to have neuroprotective effects against AMPA through inhibition of AMPA-induced Ca2+ increases, albeit at micromolar rather than nanomolar concentrations (Bae et al. 2002). Ca2+-permeable AMPA receptors are also critical determinants of cerebellar long-term depression (Gardner et al. 2005), nociceptive plasticity and inflammatory pain (Hartmann et al. 2004). Indeed a reduction in the number of Ca2+-permeable AMPA receptors in spinal neurons could result in a reduction in acute inflammatory hyperalgesia. Whether dietary-derived flavonoids could exert any such effects in vivo is currently unknown although orally delivered (-)epicatechin is bioactive in the vasculature in humans (Schroeter et al. 2006). In order to be active within the nervous system, (-)epicatechin would also need to penetrate the blood–brain barrier. There is evidence to support this as (-)epicatechin metabolites have been identified in rat brain following oral administration of (-)epicatechin (Abd El Mohsen et al. 2002), although it is presently difficult to predict whether the concentrations achievable are sufficient to activate the signalling pathways observed in the present study. However, despite this, the potential clearly exists for dietary-derived (-)epicatechin to exert beneficial actions within the mammalian CNS.

In summary, we report for the first time that (-)epicatechin at nanomolar concentrations acts as a rapid stimulator of ERK-dependent CREB phosphorylation in neurons leading to an up-regulation of GluR2 containing AMPA receptors. This bioactivity is in addition to the previously described actions of (-)epicatechin as an inhibitor of oxidative stress-induced protein kinase phosphorylation at higher micromolar concentrations (Schroeter et al. 2001). Thus, (-)epicatechin not only protects neurons against oxidative insults but also has the potential to modulate synaptic function via the up-regulation of genes involved in synaptic plasticity and synaptogenesis.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This work was supported by a project grant from the Biotechnology and Biological Sciences Research Council (D20463). The authors thank Dr L Moon for his advice and assistance with qPCR.

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  6. Acknowledgements
  7. References
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