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Cell Calcium 45 (2009) 358–368

Contents lists available at ScienceDirect

Cell Calcium

journa l homepage: www.e lsev ier .com/ locate /ceca

olecular mechanisms of neuroprotection by two natural antioxidantolyphenols

aría Rosario Campos-Esparza, María Victoria Sánchez-Gómez, Carlos Matute ∗

epartamento de Neurociencias, Facultad de Medicina y Odontología. Universidad del País Vasco, and CIBERNED, 48940 Leioa, Spain

r t i c l e i n f o

rticle history:eceived 14 November 2008eceived in revised form 24 December 2008ccepted 31 December 2008vailable online 7 February 2009

eywords:

a b s t r a c t

Excessive activation of glutamate receptors, or excitotoxicity, contributes to acute and chronic neuro-logical disorders including stroke. We previously showed that two natural polyphenol antioxidants,mangiferin and morin, are neuroprotective in a model of ischemic brain damage. In this study, we analyzedthe molecular mechanisms underlying neuroprotection by mangiferin and morin in an in vitro model ofexcitotoxic neuronal death involving NMDA receptor overactivation. We observed that both polyphenolsreduce the formation of reactive oxygen species, activate the enzymatic antioxidant system, and restore

angiferinorin

xcitotoxicitypoptosisitochondriaxidative stress

the mitochondrial membrane potential. Moreover, both antioxidants inhibit glutamate-induced activa-tion of calpains, normalize the levels of phosphorylated Akt kinase and Erk1/2, as well as of cytosolicBax, inhibit AIF release from mitochondria, and regulate the nuclear translocation of NF-�B. Each of theseeffects contributes to the substantial reduction of apoptotic neuronal death induced by glutamate. Theseresults demonstrate that mangiferin and morin exhibit excellent antioxidant and antiapoptotic prop-

linica

erties, supporting their cneuronal death.

. Introduction

The overactivation of glutamate receptors provokes a massivenflux of calcium and alterations in the membrane potential of

itochondria and increments the production of reactive oxygenpecies (ROS). Both of these events are known to take place prior topoptosis-specific processes [1,2], suggesting that these events mayonstitute an initial trigger of apoptotic cellular death in diverseathologies such as cerebrovascular accident, epilepsy, stroke, brainrauma and spinal cord damage.

Increased levels of ROS induce Bax to permeabilize the externalitochondrial membrane [3], leading to the release of cytochromeand the activation of caspases, in addition to provoking the

elease of other proteins which are also found in the mito-hondrial intramembrane space. Excitotoxic events also induce

he inactivation of Akt kinase, either by activating protein phos-hatase 2A (PP2A) which can directly dephosphorylate Akt, ors a consequence of the entry of Ca2+ into the cytoplasm [4].he pathway of extracellular signal-regulated protein kinase (Erk),

Abbreviations: AIF, apoptosis inducing factor; Akt, protein kinase B; DMSO,imethyl sulfoxide; FBS, fetal bovine serum; HBSS, Hank’s buffered salt solution;AP kinase, mitogen-activated protein kinase; NF-�B, nuclear factor-�B; ROS, reac-

ive oxygen species; SOD, superoxide dismutase.∗ Corresponding author. Tel.: +34 94 601 3244; fax: +34 94 601 5055.

E-mail address: carlos.matute@ehu.es (C. Matute).

143-4160/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.oi:10.1016/j.ceca.2008.12.007

l application as trial neuroprotectors in pathologies involving excitotoxic

© 2009 Elsevier Ltd. All rights reserved.

which is essential for apoptotic neuronal death, can be stimu-lated and modulated by increases in the levels of intracellularcalcium, protein kinase A, diacylglycerol and cAMP [5]. Similarly,increased levels of Ca2+ and the generation of free radicals canactivate the NF-�B transcription factor [6,7] and growing evidencesuggests the involvement of NF-�B in both acute and chronicneurodegenerative disorders such as brain ischemia [8], spinalcord trauma [9], Parkinson’s disease [10] and Alzheimer’s disease[11].

Polyphenolic compounds, specifically found in fruit, vegetables,plant extracts, wine, tea and dry fruits, are natural antioxidantswhich have recently been proposed to be useful prophylactics forthe treatment of excitotoxic and oxidative neuronal death [12–14].The naturally occurring polyphenols, mangiferin and morin, areknown to be powerful antioxidants, but their neuroprotectivecapacity has not yet been characterized. Mangiferin is a xantonewhich is abundantly found in fruit and in the cortex of the stalkof Mangifera indica L (Mango) [15], whereas morin is abundantlyfound in the branches of Morus alba L (white mulberry) and redwine [16].

In the present study, we demonstrate using an in vitro modelthat both mangiferin and morin at submicromolar concentrations

exhibit a wide spectrum of antioxidant and anti-apoptotic activities,including the regulation of oxidative stress, mitochondrial mem-brane potential, the activation of protein kinases and the releaseof pro-apoptotic proteins and transcription factors. In conclusion,the results of this study indicate that these polyphenols are good

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andidate neuroprotectors for the clinical treatment of neurode-enerative diseases in which excitotoxicity plays a key role.

. Materials and methods

.1. Reagents

l-glutamic acid, MK 801, mangiferin from Mangifera indicaeaves and morin hydrate were obtained from Sigma (St. Louis,

O, USA). Neurobasal medium, B-27 supplement Minus AO, B-27upplement, calcein-AM (calcein acetoxymethyl ester), 5-(and-)-chloromethyl-2ı,7ı-dichlorodihydrofluorescein diacetate acetylster (CM-H2DCFDA) and tetramethylrhodamine ethyl ester per-hlorate (TMRE) were purchased from Invitrogen. SOD-525 andatalase-520 assays kits were obtained from OxisResearch andalpain-Glo Protease Assay was from Promega. Bax antibody was

rom BD Pharmingen and polyclonal antibodies against phospho-kt (Ser473) and phospho-p44/42 Map Kinase (Thr202/Tyr204)ere purchased from Cell Signaling Technology (Beverley, MA,SA). Antibodies against AIF (H-300) and NF-�B p65 (C-20) werebtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA,SA). Secondary antibody Alexa fluor 488 goat anti-rabbit IgG andoechst 33258 were from Invitrogen.

.2. Cortical cell culture

Primary neuron cultures were obtained from the cortical lobes of18 Sprague-Dawley rat embryos according to previously describedrocedures [17]. The cells were resuspended in B27 Neurobasaledium plus 10% FBS and then seeded onto poly-l-ornithine-

oated glass coverslips (12 mm in diameter) or 6-well plates at× 104 cells per coverslip or 2 × 106 cells per well. One day later,

he medium was replaced by serum-free-, B27-supplemented Neu-obasal medium and after 4 days, by B27 Minus AO-supplementedeurobasal medium, which has no antioxidant. The cultures weressentially free of astrocytes and microglia; they were maintainedt 37 ◦C and 5% CO2. Cultures were used 8 days after plating.

.3. Toxicity assays

Cell toxicity assays were performed as described previously [18]ith modifications. Neurons were exposed to 50 �M glutamate plus

lycine (10 �M) in HBSS containing 2.6 mM CaCl2, 10 mM glucose,0 �M glycine, pH 7.4, for 10 min at 37 ◦C. When assayed, antag-nists were added 30 min before and during glutamate exposure.o evaluate the effects of mangiferin and morin hydrate on excito-oxicity, the polyphenols were added during and after glutamatexposure and cell viability was assessed 3 h later using calcein-M assay. Antioxidant stocks were dissolved in DMSO (final cultureoncentration 0.01%). All experiments were performed in quadru-licate, and the values provided are the average of at least three

ndependent experiments.

.4. Intracellular reactive oxygen species and mitochondrialotential measurements

Neuronal cultures (5 × 104 cells/well) were exposed to l-lutamate alone or with polyphenols (100 nM) as described. In dif-erent times, cells were loaded with 5-(and-6)-chloromethyl-2ı,7ı-ichlorohydrofluorescein diacetate, acetyl ester (CM-H2DCFDA;0 �M) to assay the levels of ROS or loaded with tetramethyl-

hodamine ethyl ester (TMRE; 20 nM) to quantify mitochondrialotential. Calcein-AM (1 �M) was used here to quantify the numberf cells within the reading field. Fluorescence was measured usingSynergy-HT fluorimeter (Bio-Tek Instruments Incl., Beverly, MA,SA), and excitation and emission wavelengths for CM-H2DCFDA,

alcium 45 (2009) 358–368 359

TMRE and calcein-AM were as suggested by the supplier. All exper-iments (n = 3) were performed at least in quadruplicate and plottedas mean ± S.E.M.

2.5. Antioxidant enzyme activity

In order to measure superoxide dismutase (SOD) and catalase,neuron cultures were exposed to glutamate (50 �M; 10 min) in theabsence or presence of polyphenol (100 nM) during and after theexcitotoxic stimulus. Cells were collected in 0.1 M phosphate buffer(pH 7.4) and homogenized [19]. The homogenates were centrifugedfor 30 min at 3000 g at 4 ◦C, and the supernatant was used to deter-mine the antioxidant enzymatic activity of the SOD and catalase,using the SOD-525 and OXIS catalase-520 tests.

2.6. Calpain activity assay

Calpain-Glo Protease Assay (Promega, Madrid, Spain) was usedto measure the enzyme activity of calpains on live cells, accord-ing to the manufacture’s instructions. For that purpose, neuronswere seeded onto 98-well plates (2 × 104 cells/well), and incubatedat day 8 in vitro with glutamate plus glycine in the presence orabsence of polyphenols, as described above. Freshly prepared dilu-tion of the Calpain-Glo Reagent stock solution was added to cellsat different times and luminescence was recorded with a plate-reading luminometer (Bio-Tek Instruments Incl., Beverly, MA, USA).Calpain inhibitor PD150606 or NMDA receptor antagonist MK-801was added for 30 min or 10 min, respectively, prior to exposure tothe agonist and the assay conducted in the presence of the inhibitor.All experiments were carried out in quadruplicate, and the valuesprovided calculated as the average of at least three independentexperiments.

2.7. Immunocytochemistry

For immunostaining with antibodies to phospho-Akt (1:100),phospho-p44/42 Map Kinase (1:100) and NF-�B p65 (2 �g/ml), pri-mary cortical neurons were exposed to 50 �M glutamate alone ortogether with polyphenol (100 nM) and processed as previouslydescribed in detail [20]. Cell nuclei were viewed using the Hoechst33258 nuclear stain (10 min; 5 �g/ml).

2.8. Western Blot

Cytosolic extracts from primary cortical neurons (2 × 106 cells)were performed as described previously [21]. Cultures were washedtwice with ice-cold phosphate buffered saline (PBS, 0.1 M) and har-vested in 100 �l of cell lysis buffer (10 mM HEPES, pH 7.9, 10 mM KCl,0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT and 1 mM phenylmethyl-sulfonylfluoride). Lysates were incubated on ice for 20 min, seven�l of 10% Nonidet P-40 was added for 2 min, and nuclei were col-lected by centrifugation at 10,000 g for 30 min at 4 ◦ C. The resultingsupernatant was stored as the cytosolic fraction at −80 ◦C.

For preparation of nuclear fraction, nuclei were collected by cen-trifugation and were suspended in 20 mM HEPES, pH 7.9, 400 mMNaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM phenylmethylsul-fonylfluoride, shaken for 20 min, centrifuged and the supernatantcontaining nuclear proteins was stored at −80 ◦C.

The supernatants were boiled for 5 min in urea buffer (2.5%SDS, 6% DTT, 15% urea) and 40 �g of protein was separatedby SDS-polyacrylamide gel electrophoresis (10%). After transfer

to a nitrocellulose membrane (Hybond ECL, Amersham Bio-sciences), proteins were detected with a specific primary antibodyand a horseradish peroxidase conjugated secondary antibodyusing enhanced chemiluminescence according to the manufac-turer’s instructions (Super Signal ULTRA, Pierce, Rockford, IL,

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presence of polyphenols, and ROS were quantified after 30, 60 and180 min. Glutamate induced a rapid increase in ROS levels, observedat 30 min, which were significantly diminished in the presence ofmangiferin but not morin, both at 100 nM (Fig. 1A). ROS production

60 M.R. Campos-Esparza et al.

SA). Primary antibodies were polyclonal antibodies specificor phospho-Akt (1:1000), phospho-p44/42 Map Kinase (Erk1/2;:1000), Bax (1:1000), AIF (0.2 �g/ml), NF-�B p65 (0.4 �g/ml), andctin (1:1000). Monoclonal antibody specific for tubulin was clon-5-1-2 (1:1000).

.9. Data analysis

All data are expressed as mean ± S.E.M. (n), where n refers tohe number of cultures assayed. Statistical analysis was carried outith the Student t test and significance was determined at p < 0.05.

. Results

.1. Mangiferin and morin protect cortical neurons fromxcitotoxic death

We have previously shown that in 8-day-old cultures of embry-nic cortical neurons, neuronal excitotoxicity is induced by thectivation of glutamate receptors and that mangiferin and morin atubmicromolar concentrations attenuate cell death in this model ofeuronal excitotoxicity [14]. Consistent with that finding, neuronaleath following excitotoxic insults mediated by NMDA receptorsglutamate 50 �M plus glycine 10 �M; 10 min) was attenuated byolyphenols (1–104 nM). The peak of protection by both polyphe-ols was at 100 nM, where cell death was attenuated up to5.1 ± 2.1% and 43.1 ± 1.7% respectively, compared with culturesxposed to glutamate in the absence of polyphenols (100% of celleath; Table 1). Lower and higher concentrations of these polyphe-ols did not further increase the viability of neurons after exposureo glutamate (Table 1).

.2. Mangiferin and morin reduce ROS levels and maintain theomeostasis of the enzymatic antioxidant system after excitotoxicvents

Previously, we have assayed the antioxidant properties ofangiferin and morin by measuring their ability to scavenge the

ree-radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) in an ethanol

olution [22] and both polyphenols scavenged DPPH in a signifi-ant way compared with control samples tested in their absence23].

Therefore, and in order to identify the mechanism(s) of protec-ive action of these polyphenols, we analyzed whether mangiferin

able 1angiferin and morin protect against neuronal death induced by the activation ofMDA type glutamate receptors.

olyphenol Concentration (nM) Cell death (% of control)

angiferin 1 76.4 ± 0.610 96.4 ± 3.4

100 45.2 ± 2.2***

1000 82.1 ± 1.610000 73.5 ± 1.3

orin 1 79.2 ± 1.910 68.5 ± 2.5

100 43.1 ± 1.7***

1000 54.1 ± 2.6**

10000 110.7 ± 3.6

ell cultures of cortical neurons were stimulated for 10 min with glutamate (50 �M)n the presence of glycine (10 �M) and treated with polyphenols (1–104 nM) duringnd for 3 h following this excitotoxic pulse. Neuronal viability was quantified usinghe calcein-AM method. Both polyphenols at a concentration of 100 nM substantiallyrotect against excitotoxic death (**p < 0.01; ***p < 0.001). Data were normalized withespect to cells subjected to excitotoxicity in the absence of polyphenol (29.7 ± 1.7%ell death, considered as 100% of death) and are expressed as mean neuronal death±S.E.M.; n = 3).

alcium 45 (2009) 358–368

and morin were capable of reducing oxidative stress levels causedby excitotoxic insults. Cortical neurons (5 × 104 cells per coverslip)were subjected to the excitotoxic pulse for 10 min, in the absence or

Fig. 1. Mangiferin and morin reduce ROS levels and restore SOD and catalase activ-ity following excitotoxic stimuli. (A) Levels of reactive oxygen species produced byactivation of NMDA type glutamate receptors are reduced by mangiferin and morin.Cortical neurons were stimulated with glutamate (50 �M) in the presence of glycine(10 �M) and treated with polyphenols (100 nM) during and following excitotoxicstimulation at different times. ROS levels were quantified using the CM-H2DCFDAprobe (30 �M) and data represent means ± S.E.M. of the % of CM-H2DCFDA/calcein-AM (n = 3). (B) SOD and (C) catalase activity 1 h post-excitotoxic stimulus in theabsence or presence of 100 nM polyphenol. Data were normalized with respect tocells treated without agonist in the absence or presence of polyphenol and repre-sented as a percentage of U/mg protein with respect to its control (100%). In allinstances, the mean ± S.E.M. (n = 3) is shown. #p < 0.05; ###p < 0.001 with respect tocells without treatment; *p < 0.05; **p < 0.01, ***p < 0.001 with respect to cells withagonist alone, without polyphenols.

/ Cell Calcium 45 (2009) 358–368 361

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Fig. 2. Mangiferin and morin attenuate the depolarization of the mitochondrialmembrane induced by the activation of glutamate receptors. (A) Chronological pro-file of the mitochondrial membrane potential induced by glutamate (50 �M) in thepresence of glycine (10 �M) for 10 min in cortical neurons. Data were calculatedwith respect to neurons control, vehicle-treated cultures (*p < 0.05, ***p < 0.001). (B)Cortical neurons were treated with mangiferin or morin (both at 100 nM) duringthe duration of the excitotoxic pulse (10 min) and for 30 min thereafter, and valueswere represented as a percentage with respect to the corresponding control. Data

M.R. Campos-Esparza et al.

ad similar values after 60 min (196.8 ± 18.9% respect to control),ut in this time the differences between mangiferin and morin dis-ppeared and both were similar effective reducing the amount ofOS caused by exposure to glutamate (about 25%). In all cases, dif-

erent concentrations of polyphenols were assayed but a significanteduction of ROS was only observed at 100 nM for mangiferin andorin (data not shown).In addition, we next analyzed if mangiferin and morin, besides

educing the ROS production, can modify the equilibrium of thendogenous antioxidant system. For this, cortical neurons (2 × 106

ells per well) were subjected to the excitotoxic stimulus in thebsence or presence of polyphenols and, one hour later, cellsere collected, homogenized and the supernatant was obtained

o quantify the activity of the SOD and catalase enzymes, usinghe corresponding enzymatic tests. Glutamate reduced the activityf SOD and catalase (79.5 ± 4.1% and 50.3 ± 4.8% respect to controlells, respectively) and this reduction was blocked in the presencef mangiferin or morin, both at 100 nM (Fig. 1B,C). These results sug-est that these polyphenols act at early stages of excitotoxic damagend maintain the equilibrium of the endogenous enzymatic antiox-dant system, thus helping to avoid the formation of ROS and toequester oxidative species which had been generated during therocess.

.3. Effect of mangiferin and morin on mitochondrial membraneotential after excitotoxic events

Mitochondrial dysfunction associated with the loss of cal-ium homeostasis and the increase in cellular oxidative stresslays a very important role in cell damage of excitotoxic origin,

eading to apoptotic-type neuronal death [1,24]. We evaluated ifangiferin or morin was capable of modulating the mitochondrialembrane potential following excitotoxic stimuli. Cortical neu-

ons were subjected to the excitotoxic pulse and mitochondrialembrane potential was subsequently determined at different

tages. This pulse induced an immediate transitory depolariza-ion of the mitochondrial membrane, reaching minimum values of7.7 ± 2.4% after 30 min, with respect to control (100%, untreatedells), (Fig. 2A). These values were restored in the presence ofangiferin (82.4 ± 1.3%) and morin (93.4 ± 3.5%), both at 100 nM

Fig. 2B). We can thus conclude that these polyphenols are capablef attenuating the depolarization of the mitochondrial membranenduced by the activation of glutamate receptors, and thus, of reduc-ng mitochondria-dependent apoptotic neuronal death at an earlytage.

.4. Glutamate-induced calpain activity is inhibited byangiferin and morin

Excitotoxic insult mediates by glutamate receptors cause anlevation in the concentration of cytosolic Ca2+ which in turnctivate intracellular enzymes, like calcium-activated protease cal-ains. We observed previously that NMDA receptor activation inortical neurons caused a significant cytosolic Ca2+ overload thatas attenuated by mangiferin and morin [14]. Because of that,e examined whether these polyphenols would diminish calpain

ctivity under these experimental conditions. To that end, we ana-yzed by luminescence assays the time course activity of calpainsn neurons exposed to glutamate in the absence or presence ofolyphenols. Exposure to glutamate plus glycine increased calpainctivity with respect to untreated cells (100% control; Fig. 3A).

levated activity was monitored after 10 min of exposure to ago-ists and reached its peak at 20 min (144.2 ± 8.7%) and 30 min139.6 ± 0.8%) (Fig. 3A). In turn, we found that calpain activity wasignificantly reduced by co-application of agonist together with00 nM mangiferin (83.7 ± 10.4% at 20 min; 86.6 ± 4.0% at 30 min)

are means ± S.E.M., n = 3 (###p < 0.001 with respect to cells vehicle-treated, withoutagonist, and *p < 0.05; ***p < 0.001 with respect to cells with agonist alone, withoutpolyphenols. In all cases, mitochondrial membrane potential was determined usingthe TMRE (20 nM) and calcein-AM (1 �M) probes.

or 100 nM morin (89.5 ± 12.1% at 20 min; 92.5 ± 6.5% at 30 min).Finally, and in order to verify that this enzymatic activity was cal-pain specific and was induced by NMDA receptor activation, wecarried out similar experiments in the presence of calpain inhibitorPD150606 or MK-801, an antagonist of NMDA receptor, both at50 �M (Fig. 3B). Calpain activity was measured at 20 and 30 minafter the excitotoxic stimulus and, in both cases, values were sig-nificantly reduced and similar to those in controls (96.6 ± 6.5% and96.1 ± 1.3% for PD150606 and 77.5 ± 11.0% and 79.6 14.5% for MK-801). Overall, these results indicate that glutamate, through itsNMDA receptor, causes activation of calpains in neurons and thatpolyphenols mangiferin and morin inhibit calpain activity increasesinduced by that excitotoxic insult.

3.5. Mangiferin and morin regulate the release of pro-apoptoticproteins implicated in caspase-dependent and -independentapoptotic neuronal death

NMDA receptor mediated excitotoxicity induces early eventsin mitochondria, such as the accumulation of calcium and the

subsequent transitory permeability of the membrane, provokingthe release of molecules which activate caspase-dependent and -independent apoptotic death pathways. In order to determine ifmangiferin and morin modulate the release of Bax and AIF, corticalneurons were subjected to excitotoxicity as before in the presence

362 M.R. Campos-Esparza et al. / Cell Calcium 45 (2009) 358–368

Fig. 3. Mangiferin and morin diminish calpain activation induced by excitotoxic insults. Calpain activity was assessed in intact neurons in vitro using the cell-permeable,luminescent calpain substrate Suc-LLVY-aminoluciferin (A) Neurons were stimulated with glutamate (50 �M) plus glycine (10 �M) in the absence or presence of polyphenol(100 nM) and calpain activity was monitorized at different times. Both polyphenols significantly reduced calpain activity at 20 and 30 min post-stimulus. (B) Neurons wereincubated for 30 min with calpain inhibitor PD150606 or for 10 min with antagonist MK-801 before addition of agonists, and calpain activity was measured at given times.Both molecules significantly inhibited calpain activity. Values are illustrated as a percentage with respect to the corresponding control, untreated cells (100%), and data aremeans ± S.E.M., n = 3. In all instances, significance is given as *p < 0.05; **p < 0.01 with respect to cells with agonist alone.

Fig. 4. The release of Bax and AIF into the cytosol following excitotoxic stimulus is modulated by mangiferin and morin. Neurons were stimulated with glutamate (50 �M)in the presence of glycine (10 �M), in the presence or absence of polyphenol (100 nM) during and after the excitotoxic stimulus. (A and C) Cytosolic proteins were extractedat 15 min and 60 min after excitotoxicity and using Western blot, we quantified the expression of Bax and AIF. (B) Quantitative representation of Bax, which is expressed asa percentage of the optical density of the Western blot bands Bax/actin with respect to 100% of the corresponding control. (D) AIF expression, represented as a percentageof the optical density of the Western blot bands AIF/actin with respect to 100% of the corresponding control. In all cases, bars represent the mean ± S.E.M. (n = 4). ##p < 0.01;###p < 0.001 with respect to cells without agonist. *p < 0.05; **p < 0.01 with respect to cells with agonist, without polyphenol.

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r absence of polyphenol and subsequently incubated in the pres-nce or absence of polyphenol for 15 min and 60 min. After thisime, neurons were lysed to obtain cytosol proteins free of mito-hondria and nuclei.

We found that in cytosol extracts at 15 min post-excitotoxictimulus, Bax is upregulated (221.3 ± 67.3%), but the presence ofK 801 (20 �M; 84.4 ± 22.8%), mangiferin (100 nM; 51.5 ± 11%) andorin (100 nM; 102.3 ± 12.5%) modulated Bax upregulation. In con-

rast, at 60 min post-stimulation, Bax expression in the cytosolas significantly reduced (77.2 ± 1.8%), while treatment with MK01, mangiferin or morin managed to restore Bax to control lev-ls (Fig. 4A,B). The presence of polyphenols regulates Bax levelsnd maintains these near control levels at all stages. This couldvoid the alteration of the mitochondrial membrane and as a con-equence the release of pro-apoptotic factors and the activation ofaspases. Thus, it is possible that both polyphenols exert their neu-oprotective effect via an inhibition of caspase-dependent apoptoticeath [14] which in turn depends on the pro-apoptotic proteinax.

We also analyzed the expression of AIF after excitotoxictimulus and found that the release of AIF does not occur imme-

iately after the activation of NMDA receptors. Thus, at 1 host-excitotoxicity, AIF levels were increased in the cytosolic frac-ion up to 215.3 ± 37.8% compared with non-treated controls, butot in the presence of MK 801 (97.7 ± 2.2%). In addition, mangiferinnd morin treatment regulated AIF release, which presented values

ig. 5. The decrease of phosphorylated Akt kinase levels following stimulation of glutamatith glutamate (50 �M) in the presence of glycine (10 �M) for 10 min and treated witholyphenols avoid the inactivation of phosphorylated Akt induced by the overactivationprimary antibody which specifically recognizes the phosphorylated form of Akt (greenroteins were extracted 15 and 60 min post-excitotoxic stimulus and using Western blot, lf the bands normalized to those of actin. (C) The reduction of phosphorylated Akt induceean ± S.E.M. (n = 4). ##p < 0.01; ###p < 0.001 with respect to cells without agonist. *p < 0.0

olyphenol.

alcium 45 (2009) 358–368 363

of 96.4 ± 0.7% and 80.5 ± 0.2% respectively, with respect to the 100%control value (Fig. 4 C,D). Polyphenol treatment avoids the release ofAIF from mitochondria to the cytosol following excitotoxic stimula-tion, suggesting that these polyphenols exert their neuroprotectiveeffects via the inhibition of caspase-independent apoptosis, a routein which AIF is a key player.

3.6. Mangiferin and morin modulate the activity of the Akt andErk1/2 kinases after excitotoxic events

We next analyzed if the observed neuroprotective effects wereassociated with the activation of protein kinases, since recent stud-ies have shown that the excitotoxicity induced by the overactivationof glutamate receptors activates different signaling cascades inwhich different molecules such as the Akt [25] and MAP/Erk [5]kinases participate.

Using indirect immunofluorescence and confocal microscopy,we observed that the excitotoxic glutamate stimulus provoked aninactivation of phosphorylated Akt in the cytosol of cortical neu-rons. This event was reverted in the presence of either mangiferinor morin (100 nM) (Fig. 5A). We also quantified the expression of

phosphorylated Akt in extracts of cytosolic protein using Westernblot (Fig. 5B) and we found that mangiferin and morin attenuate thereduction of phosphorylated Akt in the cytosol at 15 min and 60 minpost-excitotoxic stimulus (Fig. 5B,C). These polyphenols thus pre-vent the inactivation of Akt, maintaining levels of phosphorylated

e receptors is regulated by mangiferin and morin. Cortical neurons were stimulatedor without polyphenol (100 nM) during and after the excitotoxic stimulus. (A) Theof glutamate receptors. Neurons were fixed 60 min post-stimulus and labeled with). Nuclei were stained with Hoechst 33258 (blue). Scale bar, 30 �m. (B) Cytosolic

evels of phosphorylated Akt (Ser473) were quantified in terms of the optical densityd by glutamate is modulated by mangiferin and morin (100 nM). Data represent the5; **p < 0.01; ***p < 0.001 with respect to cells with agonist alone, in the absence of

364 M.R. Campos-Esparza et al. / Cell Calcium 45 (2009) 358–368

Fig. 6. The increase of the levels of phosphorylated Erk1/2 kinase in response to excitotoxicity is rapidly regulated by polyphenols. Neurons were stimulated with glutamate(50 �M) in the presence of glycine (10 �M) for 10 min and treated with or without polyphenol (100 nM) during and after the excitotoxic stimulus for varying time periods.(A) Neurons were fixed 15 min post-stimulus and labeled with an antibody to p44/42 phosphorylated MAP kinase (Erk1/2; green). Nuclei were stained with Hoechst 33258( ost-sta ssed at cellso

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blue). Scale bar, 30 �m. (B) Cytosolic proteins were extracted 15 min and 60 min pnd alpha tubulin. (C) Quantitative representation of phosphorylated Erk1/2 expreubulin. Data represent means ± S.E.M. (n = 4). ##p < 0.01; ###p < 0.001 with respect tof polyphenol.

kt after excitotoxic stimuli, and probably contributing in an impor-ant way to neuronal survival under these circumstances.

In a similar manner, we determined that excitotoxic stimuli pro-oke the overactivation of phosphorylated Erk1/2 kinase in theytosol. This event is reverted by treatment with mangiferin ororin (Fig. 6A). In addition, in extracts of cytosolic protein, the pres-

nce of mangiferin or morin was found to maintain the basal levelsf Erk1/2 15 min after excitotoxic stimulus, without any significanthanges being apparent 60 min post-stimulus (Fig. 6B,C).

.7. Mangiferin and morin prevent the activation of nuclearactor �B and its subsequent translocation to the nucleus

The activation of NF-�B can be caused by activation of ionotropiclutamate receptors and of l type, voltage-dependent calciumhannels [7]. It seems that the activation of this transcription factors due to the degradation of I�B-� via a mechanism which is caspasedependent [26] and by calcium-dependent calpains [27].

Here, we analyzed if the polyphenols inhibit the activation of NF-B in the cytoplasm and/or inhibit its translocation to the nucleus

n response to excitotoxic events in cultured cortical neurons. Fol-owing excitotoxic stimulus, NF-�B was found to be distributed inoth the cytosol and nucleus (Fig. 7A). However, in the presence

imulus and using Western blot were detected p44/42 phosphorylated MAP kinases optical density of Western blot bands of phosphorylated Erk1/2 with respect towithout agonist. *p < 0.05; **p < 0.01 with respect to cells with agonist in the absence

of 100 nM mangiferin or morin, NF-�B was found to be presentprincipally in the cytosol.

In cytosol extracts, we found that NF-�B levels are reduced15 min post-excitotoxicity, while the presence of either polyphenolsmanaged to avoid the reduction of NF-�B levels in cytosol (Fig. 7B,C).Polyphenol treatment may regulate calcium entry and caspase acti-vation [14], thus avoiding the phosphorylation of I�B-� complexand as a consequence the activation of NF-�B and its transloca-tion to the nucleus. Finally, we analyzed the presence of NF-�B inthe nucleus by using Western blot with nuclear extracts obtainedfrom neuronal cultures treated with excitotoxic stimulus in absenceor presence of polyphenols (Fig. 7D). Control cells with glutamatestimulation exhibited high levels of NF-�B in the nucleus com-pared with neuron untreated (130.2 ± 20.1%), while the presenceof mangiferin or morin significantly reduced the detection of NF-�B in the nucleus (42.5 ± 0.8%; 69.2 ± 3.5◦1%). Thus, polyphenolsmangiferin and morin inhibited glutamate-induced NF-�B activityby blockade of NF-�B translocation to the nucleus.

4. Discussion

Overactivation of glutamate receptors is known to lead tooxidative stress and activation of apoptotic molecules involved

M.R. Campos-Esparza et al. / Cell Calcium 45 (2009) 358–368 365

Fig. 7. The activation of NF-�B in response to the excitotoxic stimulus is regulated by polyphenols. Neurons were subjected to the excitotoxic stimulus in the presence/absenceof 100 nM polyphenol. (A) The translocation of NF-�B p65 (green) to the nucleus in response to the excitotoxic stimulus was examined. Cells were fixed 15 min post-stimulusand labeled with anti-NF-�B p65 (green) and Hoechst 33258 (blue). Scale bar, 10 �m. Cytosolic proteins were extracted at 15 min and 60 min post-excitotoxic stimulus (B)a ressioo 65 (Cw of po

iTkrnbpi

nd nuclear proteins were extracted at 15 min after stimulus (D) and NF-�B p65 expr nuclear NF-�B expressed as the optical density of Western blot bands of NF-�B pithout agonist. *p < 0.05; **p < 0.01 with respect to cells with agonist in the absence

n neuronal death in a variety of neurodegenerative pathologies.he naturally occurring polyphenols mangiferin and morin arenown to have excellent antioxidant properties and can thus act

apidly in the face of oxidative stress. Moreover, since they areon-polar polyphenols, they can efficiently cross the blood–brainarrier. However, the possible neuroprotective action of theseolyphenols had not been characterized before the present study,

n which we demonstrate that both mangiferin and morin pro-

n was quantified by Western blot. (C and E) Quantitative representation of cytosolic-20). Each bar represents the mean ± S.E.M. n = 4. ###p < 0.001 with respect to cellslyphenol.

tect against excitotoxic neuronal death, by modulating molecularmechanisms which are known to participate in apoptotic-like neu-ronal death. Interestingly, these agents are most effective at very

low concentrations, within the nanomolar range, a concentra-tion that could be achieved in the CNS in potential future clinicaltrials.

Sustained overactivation of glutamate receptors has beenreported to induce the partial depolarization of the mitochondrial

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embrane, due to the entry of calcium into the cytoplasm andts uptake by mitochondria [24,28] leading to apoptotic neuronaleath [1]. Here we show that mangiferin and morin attenuatehis drop in mitochondrial membrane potential. This finding maye related to the capacity of these polyphenols to reduce cal-ium entry via glutamate receptors [14]. These results corroboratehose obtained in liver mitochondria [29], demonstrating that

angiferin was capable of protecting against loss of mitochon-rial membrane potential and mitochondrial swelling induced by

ron. Moreover, studies using SH-SY5Y cells stimulated with 6-ydroxydopamine, a pre-parkinsonian neurotoxin, showed thatolyphenols from green tea prevent the reduction in mitochon-rial membrane potential and inhibit the increase in intracellularalcium levels [30].

The accumulation of calcium induced by excitotoxic stimulinduces the formation of ROS via the activation of phospholi-ases, xanthine oxidase, nitric oxide synthetase and the alterationf normal mitochondrial functioning (revised in [31]). Our find-ngs indicate that treatment with mangiferin or morin is capablef inhibiting intracellular ROS and maintains in equilibrium theomeostasis of the enzymatic antioxidant system, reducing oxida-ive stress and avoiding lipid peroxidation. These in vitro resultsomplement those in vivo carried out by Gottlieb et al. in 200614], in which using ischemic rats, it was shown that early post-reatment with mangiferin or morin reduces ROS production in theyramidal layer of the CA1 region of the hippocampus. Our resultslso corroborate those of other studies which employed a varietyf cellular models, and showed that mangiferin [32] and morin33] have a high capacity to sequester ROS and to reduce oxida-ive species in non-neural cells. Mangiferin is capable of chelatingron, avoiding its participation in the Fenton reaction and preventsipid peroxidation induced by iron better than that induced by per-xide [29]. In contrast, morin can inhibit xanthine oxidase, reducinghe production of ROS [16] and radicals derived from nitrogen34].

In addition, increases in cytosolic Ca2+ concentration causectivation of various proteolytic enzymes, as calpains, which areeleterious to neurons. Calpains are pivotal mediators of bothecrotic cell death and apoptosis [35] following acute hypoxia,raumatic brain injury and chronic neurodegeneration [36–38]. Inarticular, calpains are linked to excitotoxicity [39–42] and an �-pectrin cleavage product of calpains appears in the CA1 region ofhe hippocampus within 20 min of an ischemic insult, supportingn early involvement of calpains in mediating excitotoxic inducedell death [43]. The finding that polyphenols mangiferin and morinan inhibit the glutamate-induced calpain activity in a model ofxcitotoxicity in vitro supports their neuroprotective potential.

In cultures of cortical and hippocampal neurons stimulated withMDA or kainate, reduced levels of phosphorylated Akt are associ-ted with both caspase-independent and -dependent death [4,44].hese findings suggest that the inactivation of PI3K kinase, or alter-atively, the activation of the PP2A phosphatase, which can directlyephosphorylate Akt [4], may be responsible for the lower lev-ls of phosphorylated Akt. We found that mangiferin and morinct rapidly following the excitotoxic stimulus, avoiding the inacti-ation of phosphorylated Akt. Consistently, neuroprotection withstradiol after ischemia prevents the drop in the levels of phospho-ylated Akt and phosphorylated glycogen synthase kinase [44]. Thisuggests that the modulation of phosphorylated Akt by polyphe-ols is a key event in neuroprotection, since Akt can inhibit deathia the following mechanisms: (1) maintenance of mitochondrial

embrane potential, (2) phosphorylation of Bad and inhibition of

onformational changes of Bax, thus blocking its translocation toitochondria, (3) inhibition of the transcriptional activity of p53

nd (4) lowering of ROS levels and increasing the levels of SODrotein [45,46].

alcium 45 (2009) 358–368

We have also shown that mangiferin and morin inhibit theoveractivation of the phosphorylated Erk1/2 kinases which arecorrelated with apoptotic death [5]. It thus appears that bothpolyphenols modulate the Erk1/2 kinase pathway by regulating cal-cium entry into the cytoplasm, and in this line, we have previouslyobserved that the presence of mangiferin or morin diminishedthe influx of calcium induced by glutamate receptors activation[14].

We have shown that these polyphenols, following excitotoxicstimulus, impede the upregulation of Bax in the cytosol, thus inter-cepting apoptotic death which depends on caspases and/or AIFsince a Bax-induced pro-oxidant state is critical for cytochromec release during programmed neuronal death [3]. Our results areconsistent with those obtained in a model of myocardial stroke,in which Elsholtzia blanda flavones prevent apoptosis by regulatingthe expression of members of the Bcl-2 family, i.e. increasing theexpression of Bcl-2 and reducing the expression of Bax [47]. Alsoin line with our findings, recent studies described that Silymarin,a natural polyphenol, reduces apoptosis induced by ultravioletradiation, by activating the Akt kinase pathway, reducing theexpression of Bax and increasing the expression of the Bcl-2 yBcl-xL proteins [48]. In turn, the results reported here indicatethat mangiferin and morin prevent the release of AIF from mito-chondria and its subsequent translocation to the nucleus followingan excitotoxic stimulus. This may be due to the inhibition of cal-cium entry by polyphenols following excitoxic insults [14] andthe ensuing attenuation of calpain activation which is responsi-ble for the activation of AIF, provoking its release to the cytosol[49].

Finally, we have shown that the activation of NF-�B and itstranslocation to the nucleus following excitotoxic events is pre-vented by mangiferin and morin. Glutamate is known to induce thephosphorylation of I�B-� and the release of NF-�B, which is translo-cated to the nucleus where it activates diverse pro-apoptotic genes[6,27]. Here, we have observed that both polyphenols reduce thenuclear translocation of NF-�B and elevate its cytosolic levels, andit is likely that these effects are due to the capacity of mangiferin andmorin to act on molecules which have been implicated in the degra-dation of I�B-� such as Ca2+ [27] and the Erk and Akt protein kinases[50]. Moreover, these results are consistent with those in mousemacrophages showing that mangiferin modulates the expressionof diverse genes related to the NF-�B signaling pathway [51]. Sim-ilarly, other polyphenols such as curcumine and epigallocatechininhibit the activation of NF-�B in epithelial alveolar cells and inmyocardial ischemia respectively [52,53].

The results reported here are relevant to major chronic neu-rodegenerative diseases. Altered neuronal Ca2+ homeostasis isimplicated in age-related cognitive impairment and Alzheimer’sdisease [54]. In this disorder, neurons undergo increased oxida-tive stress and impaired energy metabolism, which compromisethe function of proteins that control membrane excitability andsubcellular Ca2+ handling. The capacity of morin and mangiferinto attenuate Ca2+ overload [14,23] and oxidative stress in experi-mental paradigms employing excitotoxic insults, as reported here,can help re-establishing Ca2+ dynamics and thus favour neuropro-tection in Alzheimer’s disease. In turn, increased oxidative stressappears to be a common causative aspect involved in the prefer-ential loss of dopaminergic neurons in the substantia nigra, thehallmark of Parkinson’s disease [55]. Dietary antioxidant polyphe-nols such as morin and mangiferin may aid this vulnerable region toameliorate the antioxidant cellular defence at the onset of disease

and thus slow its progression.

Overall, the present results demonstrate that both mangiferinand morin have a wide spectrum of antioxidant and anti-apoptoticproperties, which can reduce neuronal damage associated with thesustained overactivation of glutamate receptors. These polyphenols

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ay represent useful components in therapies for diseases whichnvolve excitotoxic neuronal damage.

cknowledgments

This work was supported by CIBERNED, University of País VascoGobierno Vasco. R.C.-E. was a recipient of fellowships from Fun-ación Carolina y CONCYTEA.

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