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Pyruvate protects cerebellar granular cells from 6-hydroxydopamine-induced cytotoxicity by activating the Akt signaling pathway and increasing glutathione peroxidase expression

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Pyruvate protects cerebellar granular cells from 6-hydroxydopamine-induced cytotoxicity by activating the Akt signaling pathway and increasing glutathione peroxidase expression
  Pyruvate protects cerebellar granular cells from6-hydroxydopamine-induced cytotoxicity by activating the Aktsignaling pathway and increasing glutathione peroxidase expression F.J. Fernandez-Gomez, a,e,f  M.D. Pastor, a,b,f  E.M. Garcia-Martinez, c,e R. Melero-Fernandez de Mera, a,e,f  M. Gou-Fabregas, d M. Gomez-Lazaro, a,e,f  S. Calvo, a,b,f  R.M. Soler, d M.F. Galindo, a,f  and J. Jordán a,e,f, ⁎ a   Departamento de Ciencias Médicas, Facultad de Medicina, Universidad Castilla-La Mancha, Spain  b Grupo Glia e Isquemia, Universidad Castilla-La Mancha, Spain c Servicio de Farmacia, Complejo Hospitalario Universitario de Albacete, Spain d  Departament de Ciències Mèdiques Bàsiques, Facultat de Medicin, Universitat de Lleida, Spain e Grupo Neurofarmacología, Universidad de Castilla-La Mancha, Spain f  Centro Regional de Investigaciones Biomédicas, Albacete, Spain Received 19 May 2006; revised 7 July 2006; accepted 16 July 2006Available online 15 September 2006 Parkinson disease (PD) is the second-most common age-relatedneurodegenerative disease and is characterized by the selectivedestruction of dopaminergic neurons. Increasing evidence indicatesthat oxidativestress plays a crucial rolein the pathogenesis of idiopathicPD. Anti-oxidant agents including catalase, manganese porphyrin andpyruvateconfercytoprotectiontodifferentcellcultureswhenchallengedwith 6-hydroxydopamine (6-OHDA). Herein we used rat cerebellargranular cell cultures to ascertain the plausible cellular pathwaysinvolved in pyruvate-induced cytoprotection against 0.1 mM 6-OHDA.Pyruvateprovidedcytoprotectioninaconcentration-dependentmanner(2 – 10 mM). Consistent with its well-established anti-oxidant capacity,pyruvate (10 mM) prevented 6-OHDA-induced lipid peroxidation byblocking the rise in intracellular peroxides and maintaining theintracellular reduced glutathione (GSH) levels. Further experimentsrevealed that pyruvate increased Akt, but not extracellular signal-regulated kinase phosphorylation. Moreover, phosphatidylinositol 3-kinase (PI3K) inhibitors attenuated pyruvate-induced cytoprotectionindicating that PI3K-mediated Akt activation is necessary for pyruvatetoinducecytoprotection.Ontheotherhand,pyruvatealsoup-regulatedglutathione peroxidase mRNA levels, but not those of the anti-oxidantenzymes superoxide dismutase-1 and -2, catalase or the anti-apoptoticoncogenes Bcl-2 or Bcl-x L . In summary, our results strongly suggestthat pyruvate, besides the anti-oxidant properties related to its struc-ture, exerts cytoprotective actions by activating different anti-apoptoticroutes that include gene regulation and Akt pathway activation.© 2006 Elsevier Inc. All rights reserved. Parkinson ’ s disease (PD) is a neurodegenerative disorder characterized by a progressive loss of dopaminergic neurons inthe substantia nigra pars compacta ( Noelker et al., 2005), with theresulting loss of nerve terminals accompanied by a dopamine(DA) deficiency in the striatum (Hornykiewicz, 1966). 6-Hydroxydopamine (6-OHDA), an endogenously generated meta- bolite of dopamine oxidation (Fornstedt et al., 1986), is extensively utilized in experimental models of PD because it causes cell death in different cell types, including humanneuroblastoma SK-N-SH (Shimizu et al., 2002), SH-SY5Y (Von Coelln et al., 2001; Jordan et al., 2004) and pheochromocytomaPC12 cells ( Nie et al., 2002), besides of being a specificneurotoxin of dopaminergic neurons in vivo ( Noelker et al., 2005;Blum et al., 2001). Indeed, if reactive oxygen species (ROS) arenot properly detoxified, they can damage cell lipids, proteins or DNA, impairing normal cell function. Uncoupling of mitochon-drial oxidative phosphorylation resulting in energy deprivationand by-products resulting from 6-OHDA auto-oxidation such asquinones and hydrogen peroxide (H 2 O 2 ) are involved in thesecytotoxic processes (Galindo et al., 2003; Thakar and Hassan,1988). Interestingly, both mitochondrial dysfunction and oxida-tive stress appear to play a central role in the pathogenesis of PD(Beal, 2003; Blum et al., 2001; Mazzio et al., 2004). In fact, anti-oxidant agents, such as catalase, vitamin E,  N  -acetyl cysteine,ascorbic acid and pyruvate are known to elicit cytoprotectionagainst 6-OHDA in experimental models (Jordan et al., 2004;Galindo et al., 2003; Lai and Yu, 1997).Alpha-ketoacid structures like pyruvate have been shown toconfer cytoprotection against different noxious stimuli includingtransient forebrain ischemia, hemorrhagic shock,  β -amyloid,exogenous H 2 O 2  and menadione (2-methyl-1,4-naphthoquinone) Neurobiology of Disease 24 (2006) 296 – 307 ⁎  Corresponding author. Grupo de Neurofarmacología, Facultad deMedicina, Universidad de Castilla-La Mancha-CRIB, Avda. Almansa, 14,02006 Albacete, Spain. Fax: +34 967599327.  E-mail address: (J. Jordan). Available online on ScienceDirect ( 0969-9961/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.nbd.2006.07.005  (Thor et al., 1982; Doroshow et al., 1990; Alvarez et al., 2003;Salahudeen et al., 1991; Slovin et al., 2001; Lee et al., 2001). Theunderlying mechanism of this cytoprotective effect is not completely understood. Pyruvate is considered a cytoprotectiveagent in experiments involving an attenuation of metabolicdysfunction and situations in which cellular glucose oxidation isrequired (Chung et al., 2004). In this manner, pyruvate seems to bethe critical component to explain the trophic activity of glia-conditioned media for central nervous system neurons (Selak et al.,1985). On the other hand, pyruvate might act as a ROS scavenger due to its anti-oxidant capacity. For example, pyruvate is able toundergo nonenzymatic decarboxylation in the presence of H 2 O 2 and thus prevent   • OH formation by the so-called Fenton reaction(Alvarez et al., 2003).Anti-oxidant systems and intracellular signaling pathways,including phosphatidylinositol 3-kinase (PI3K) and extracellular signal-regulated kinase (ERK), have been shown to be important in the regulation of cell survival. Superoxide dismutase, catalase,glutathione peroxidase and non-enzymatic molecules such asvitamin E, glutathione and alpha-ketoacid compounds are anti-oxidant systems that neutralize and eliminate ROS and confer cytoprotection versus 6-OHDA-induced cell death (Jordan et al.,2004; Love et al., 2002; Lai et al., 1997). The PI3K pathway has emerged as one of the critical factors in anti-apoptotical signaltransduction (Song et al., 2005; D ’ Mello et al., 1997; Franke et al., 2003). The activation of this pathway is known to protect cells from several apoptotical stimuli (Kennedy et al., 1997). TheERK (p42/p44 mitogen-activated protein kinase; MAPK) cascadeis a central pathway that transmits signals from manyextracellular agents to regulate cellular processes. It is thought to play a pivotal role in the integration and transmission of transmembrane signals required for growth and differentiation.ERK activation is essential for cell growth and plays a crucialrole in apoptosis inhibition (Yoon and Seger, 2006; Pages et al.,1993).Here we analyzed the possible mechanisms underlying thecytoprotective effects of pyruvate on 6-OHDA-induced toxicity ingranular cell cultures from rat cerebellum, a region wherestructural reorganization, alterations in neuronal activity andarchitecture were detectable following unilateral striatal 6-OHDAlesion (Perovic et al., 2005). We have found that pyruvate blocks6-OHDA-induced toxicity in a concentration-dependent manner without affecting the auto-oxidation process of 6-OHDA. Further experiments revealed that pyruvate prevented the 6-OHDA-induced, intracellular peroxide rise and blocked the depletion of GSH levels. The activation of the Akt pathway and the increase inthe expression of glutathione peroxidase also participated in thecytoprotective mechanism activated by this alpha-ketoacid.Finally, the metabolic product, malate, did not protect cellcultures against 6-OHDA toxicity and failed to activate theabovementioned cytoprotective pathways. Materials and methods Cell culture Primary cultures of cerebellar granular neurons were obtainedfrom dissociated cerebella of 7- to 8-day-old rats (Fernandez-Gomez et al., 2005a). Dissection and dissociation were carried out in Basal Medium Eagle (BME; Life Technology). Tissues wereincubated with trypsin for 20 min at 37°C and dissociated bytrituration in a medium containing DNase and trypsin. Cells were plated on 96 plastic well dishes or on 60-mm plastic Petri dishes pre-coated with poly- L -lysine (10 g/ml) at a concentration of 8×10 6 cells/ml in BME containing 25 mM KCl, 10% de-complemented fetal calf serum (FCS; Life Technology), glutamineand antibiotics. Cytosine- β - D -arabino-furanoside (Ara-C) (10  μ M)was added at 3 days  in vitro  (DIV) to prevent the growth of non-neuronal cells. All experiments were carried out after 7 days inculture.  Intracellular generation of reactive oxygen species We used the oxidation-sensitive fluorescent dye 2 ′ ,7 ′ -dichlor-odihydrofluorescein diacetate (DCFH-DA) to measure the produc-tion of reactive oxygen species (ROS), mainly hydrogen peroxideand hydroxyl radicals. DCFH-DA is deacetylated by esterases todichlorofluorescein (DCFH). This non-fluorescent product is thenconverted by reactive species into DCF, which can easily bevisualized by strong fluorescence at 530 nm when excited at 485 nm. Cells seeded in 96-well culture plates were incubated withDCFC-DA (10  μ g/ml) for 5 min, treated with or without 10 mM pyruvate before adding either 0.1 mM 6-OHDA or vehicle and 4 hlater fluorescence intensity was measured in a Spectra Max GeminiXS (Molecular Devices). The average relative percent ROS production from four wells of at least three separate cultures wasdetermined. Results are expressed as mean±SD values, andsignificance was determined by Student  ’ s  t   test. Statisticalsignificance was considered at the  p <0.05 level.  Measurement of glutathione levels Levels of glutathione were determined by using monochlor-obimane (mBCl) fluorescence. Glutathione (GSH) is specificallyconjugated with mBCl to form a fluorescent bimane-GSHadduct in a reaction catalyzed by glutathione  S  -transferases(Shrieve et al., 1988). The concentration of the bimane-GSHadduct increases during the initial 10 to 12 min period of thisreaction with first order kinetics, before leveling off (Young et al., 1994). Culture medium was removed and cells were washedtwice with 0.2 ml Krebs and incubated for 1 h at 37°C in0.2 ml fresh Krebs containing 160  μ M mBCl. After incubation,cells were washed twice with Krebs and fluorescence was Table 1Sequences of the oligonucleotide primer pairs used for real-time PCR SOD1-F 5 ′ -TGCAGGGCGTCATTCACTT-3 ′ SOD1-R 5 ′ -CACAACTGGTTCACCGCTT-3 ′ SOD2-F 5 ′ -TTAACGCGCAGATCATGCA-3 ′ SOD2-R 5 ′ -GTAGGTCGCGTGGTGCTT-3 ′ CATALASE-F 5 ′ -CACACCTACGTACAGGCCG-3 ′ CATALASE-R 5 ′ -TTAGCTTTTCCCTTGGCAGC-3 ′ Gpx1-F 5 ′ -GCAGATACACCAGGCGCTTT-3 ′ Gpx1-R 5 ′ -GGCTTCTATATCGGGTTCGA-3 ′ GAPDH-F 5 ′ -CCAGCCTCGTCTCATAGACA-3 ′ GAPDH-R 5 ′ -GTAACCAGGCGTCCGATACG-3 ′ RBCL-XL-F 5 ′ -CTGCCTTGTTGGTGGGAC-3 ′ RBCL-XL-R 5 ′ -AAAGCATTCCCGAGAGGCT-3 ′ BCL2-F 5 ′ -ACGGTGGTGGAGGAACTCTT-3 ′ BCL2-R 5 ′ -CACAATCCTCCCCCAGTTC-3 ′ 297  F.J. Fernandez-Gomez et al. / Neurobiology of Disease 24 (2006) 296   –  307   measured at an excitation wavelength of 340 nm and emissionwavelength of 460 nm in a Spectra Max Gemini XS (Molecular Devices). The average relative percent reduced GSH levels fromat least three separate cultures was determined. Results areexpressed as mean±SD values, and significance was determined by Student  ’ s  t   test. Statistical significance was considered at the  p  < 0.05 level.  Lipid peroxidation Lipid peroxidation was measured by determining malondialde-hyde (MDA) levels. Each sample (8×10 6 cells) was collected in100  μ L of ice-cold 20 mM BTris  –  HCl buffer, pH 7.4, andsonicated. Amounts of MDA were determined in the cellular extracts using a Lipid Peroxidation Assay Kit from Calbiochem Fig. 1. Protective effects of pyruvate against 6-OHDA-induced cell death. (A – C) Phase contrast. Control cultures (A) or cells challenged during 24 h with0.1 mM 6-OHDA pre-treated (C) or not (B) for 1 h with 10 mM pyruvate. (D) Cell viability was assayed 24 h after 6-OHDA addition by using the MTT test.Pyruvate(0.5 – 10mM)was added1 hbefore6-OHDA(0.1mM)andmaintaineduntilthe endof the experiment. (E)Pyruvate(10 mM)additionswereperformed1 h before( − 1), at the time (0) or 0.5, 1, 3 or 6 h after 6-OHDA treatment.Cell viability was measured 24 h after 6-OHDAaddition. Data represent the mean±SDof three independent experiments. ***  p  <0.001 vs. control conditions (0 pyruvate),  t   test significantly different from 6-OHDA alone.298  F.J. Fernandez-Gomez et al. / Neurobiology of Disease 24 (2006) 296   –  307   (No. 437634) based on the condensation reaction of thechromogene 1-methyl-2-phenylindole with MDA. The stablechromophores were determined using a VERSAmax Lunablemicroplate reader (Molecular Devices) with absorbance at 586 nm.Results are expressed as micromole MDA per mg protein.  Formation of quinoidal products by 6-OHDA auto-oxidation The formation of quinoidal products by 6-OHDA auto-oxidation was determined using a cell-free system. Briefly, 6-OHDA (0.1 mM) was incubated at room temperature (22  –  25°C) Fig. 2. Pyruvatepreventsintracellular6-OHDA-inducedH 2 O 2 -likespeciesgenerationbutnotauto-oxidationprocesses.(A)Cerebellargranularcellsweretreatedwith 6-OHDA (0.1 mM) 1 h after the addition of different concentrations of pyruvate. Four hours later DCF fluorescence was measured in a Spectra Max GeminiXS microplate reader.(B) LevelsofMDA incellscultures weredetermined by 24h after0.1 mM 6-OHDAadditionincontrolandcultures pre-treatedornot with10 mM pyruvate. Data are mean±SD, of at least 3 different cultures were used. **  p  <0.05; ***  p  <0.001 vs. vehicle conditions. Treatments with the samelowercase letters are not significantly different among them (  p >0.05). (C – F) Pyruvate did not interfere with 6-OHDA auto-oxidation. (C – D) Pyruvate did not  block the formation of quinoal oxidation products from 6-OHDA. Absorbance at 490 nm (A 490 ) was determined for 6-OHDA dissolved in fresh culture mediumwithout or with pyruvate (0.5, 2, 10 mM); ascorbic acid (AA, 0.01%) was used as a 6-OHDA auto-oxidation inhibitor control. (E – F) Hydrogen peroxidegenerationby6-OHDAauto-oxidationwasmeasuredbythescopoletinmethodinthepresenceorabsenceofpyruvate(0.5 – 10mM).Similarresultswereobtainedin three separate experiments. Ascorbic acid (AA, 0.01%) was used to block 6-OHDA auto-oxidation. Lines (C, E) represent mean values of a representativeexperiment performed by triplicate. Histogram (D, F) represents mean values±SEM of percent of control A 490  at 1800 s from at least five different experiments.299  F.J. Fernandez-Gomez et al. / Neurobiology of Disease 24 (2006) 296   –  307   for 15 min in culture medium with or without pyruvate, and theformation of quinoidal products of 6-OHDA auto-oxidation wasmonitored by absorbance at 490 nm as previously described(Tiffany-Castiglioni et al., 1982) using a VERSAmax Lunablemicroplate reader (Molecular Devices).  Fluorescence measurements of H  2 O 2  production from 6-OHDAauto-oxidation Hydrogen peroxide generation was measured fluorimetricallyas previously described (Votyakova and Reynolds, 2001). Briefly,6-OHDAwas added, at room temperature, to a standard incubation buffer that contained: 125 mM KCl, 2 mM K  2 HPO 4 , 5 mM MgCl 2 ,10 mM HEPES (pH adjusted to pH 7.0 with KOH), 10  μ M EGTAand scopoletin (2  μ M) in the presence of 1 U/ml horseradish peroxidase. Scopoletin fluorescence was monitored at excitation/ emission wavelengths of 365 nm/460 nm in a Spectra Max GeminiXS (Molecular Devices). Cell viability The cells were exposed to 6-OHDA (0.1 mM) and cell viabilitywas measured 24 h later by the ability to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) tothe blue formazan product. After removal of culture medium, cellswere incubated with 1 mg/ml MTT in regular BME for 2 h at 37°C. BME was then aspirated and the formazan dissolved in200  μ L DMSO. Absorbance at 570 nm was measured in aVERSAmax Lunable microplate reader (Molecular Devices), andthe absorbance of control conditions was used as 100% cell cultureviability. Western blot analysis For immunoblotting of phosphorylated forms of Akt or ERK in total cell lysates, 30  μ g of total protein was resolved in SDS  –   polyacrylamide gels and transferred onto polyvinylidene difluor-ide Immobilon-P transfer membrane filters (Millipore, Hucoa-Erlos, Barcelona) using an Amersham Biosciences semidry Trans-Blot (Amersham Biosciences, Barcelona). Membranes were blotted with an anti-phospho-Akt-specific antibody: anti-P-Ser473, or an anti-phospho-p44/42 MAPK antibody: anti-P-ERK (New England Biolabs, Servicios Hospitalarios, Barcelona)following the instructions of the provider. To control the contentsof the specific protein in each lane, membranes were re-probedwith a monoclonal anti- α -tubulin antibody (Sigma) or with anti-Akt antibody (C-20) (Santa Cruz Biotechnology, Quimigranel,Barcelona). Blots were developed using the ECL AdvanceWestern Blotting Detection Kit chemiluminescent substrate(Amersham Biosciences). Band intensity was estimated densito-metrically on a GS-800 calibrated densitometer (Bio-RadQuantity One).  RNA isolation Total RNA was obtained with Trizol® Reagent (Invitrogen)following the manufacturer  ’ s indications. Eight million cells wereused per milliliter of Trizol. The isolated RNA was thensubsequently treated with DNase (Promega) to remove anygenomic DNA contamination. The integrity of RNA was alwayschecked by running an aliquot in an agarose gel.  Real-time-PCR cDNAwas synthesized from 10  μ g total RNA in 100  μ l volumecontaining 1× RT Buffer (Applied BioSystems), 500  μ M dNTPs,2.5  μ M random hexamers and 1.25 U/  μ l MultiScribe ReverseTranscriptase. Reaction was performed in a thermal Cycler at 48°Cfor 30 min. Samples were then kept at   − 20°C until their utilization.PCR amplification was performed on the ABI Prism 7000Sequence Detection System (Applied Biosystems), using theSYBR Green PCR Master Mix (Applied Biosystems). Onemicroliter cDNA was used for each reaction. PCR amplificationswere always performed in triplicate wells, using 40 two-temperature cycles (15 s at 94°C and 1 min at 60°C). Oncedemonstrated that the efficiency for the different primer combina-tions was similar, the quantification was performed by thecomparative cycle threshold method (Livak and Schmittgen,2001), using GAPDH as internal control. Primers for all target sequences (Table 1) were designed using the computer Primer  Fig. 3. (A) Pyruvate blocks 6-OHDA-induced intracellular GSH depletion.Cerebellar granular cells were pre-treated with pyruvate (0.5 – 10 mM) for 1 h before the addition of 6-OHDA (0.1 mM). Measurement of reducedGSH levels was performed 24 h later by analyzing mBcl fluorescence.Data represent the mean ± SD of at least five independent experiments inthe presence (black column) or absence (white column) of pyruvate.***  p <0.001 vs. vehicle conditions, Student's  t   test. (B) BSO pre-treatment blocks pyruvate-induced cytoprotection. Throughout theseexperiments, pyruvate was used at a concentration of 10 mM, whereas6-OHDA was used at 0.1 mM. Cell cultures were pre-treated for 12 hwith 100  μ M BSO. Data are mean±SD of at least 3 different cultures.Treatments with the same lowercase letters are not significantly different among them (  p >0.05).300  F.J. Fernandez-Gomez et al. / Neurobiology of Disease 24 (2006) 296   –  307 
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