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Minocycline fails to protect cerebellar granular cell cultures against malonate-induced cell death

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Minocycline fails to protect cerebellar granular cell cultures against malonate-induced cell death
  Minocycline fails to protect cerebellar granular cell cultures againstmalonate-induced cell death F.J. Fernandez-Gomez, a  M. Gomez-Lazaro, a  D. Pastor, a  S. Calvo, a   N. Aguirre,  b M.F. Galindo, a  and J. Jorda´n a, * a   Departamento Ciencias Me´dicas, Facultad de Medicina, Universidad de Castilla-La Mancha, Avda, Almansa, s/n, 02006 Albacete, Spain  b  Departamento de Farmacologı´a, Universidad de Navarra, Facultad de Medicina, Pamplona, Spain Received 11 February 2005; revised 11 February 2005; accepted 25 March 2005Available online 3 May 2005 Experimental and clinical studies support the view that the semi-synthetic tetracycline minocycline exhibits neuroprotective roles inseveral models of neurodegenerative diseases, including ischemia,Huntington, Parkinson diseases, and amyotrophic lateral sclerosis.However, recent evidence indicates that minocycline does not alwayspresent beneficial actions. For instance, in an in vivo model of Huntington’s disease, it fails to afford protection after malonateintrastriatal injection. Moreover, it reverses the neuroprotective effectof creatine in nigrostriatal dopaminergic neurons. This apparentcontradiction prompted us to analyze the effect of this antibiotic onmalonate-induced cell death. We show that, in rat cerebellar granularcells, the succinate dehydrogenase inhibitor malonate induces cell deathin a concentration-dependent manner. By using DFCA, monochlorobi-mane and 10-  N  -nonyl-Acridin Orange to measure, respectively, H 2 O 2 -derived oxidant species and reduced forms of GSH and cardiolipin, weobserved that malonate induced reactive oxygen species (ROS)production to an extent that surpasses the antioxidant defense capacityof the cells, resulting in GSH depletion and cardiolipin oxidation. Thepre-treatment for 4 h with minocycline (10–100  M M) did not presentcytoprotective actions. Moreover, minocycline failed to block ROSproduction and to abrogate malonate-induced oxidation of GSH andcardiolipin. Additional experiments revealed that minocycline was alsounsuccessful to prevent the mitochondrial swelling induced by malo-nate. Furthermore, malonate did not induce the expression of the iNOS,caspase-3, -8, and -9 genes which have been shown to be up-regulated inseveral models where minocycline resulted cytoprotective. In addition,malonate-induced down-regulation of the antiapoptotic gene Bcl-2 wasnot prevented by minocycline, controversially the mechanism previ-ously proposed to explain minocycline protective action. These resultssuggest that the minocycline protection observed in several neuro-degenerative disease models is selective, since it is absent from culturedcerebellar granular cells challenged with malonate. D  2005 Elsevier Inc. All rights reserved.  Keywords:  Minocycline; Cytoprotection; Malonate; Mitochondria;Huntington; Neurodegeneration During the last decade, the effects on the central nervous systemof the highly lipophilic semisynthetic second-generation tetracy-cline, minocycline, have received much attention (Blum et al.,2004; Domercq and Matute, 2004; Yong et al., 2004). Minocy-cline, commonly used to treat acne and rheumatoid arthritis, iscapable of crossing the blood–brain barrier and exert neuro- protective actions. It has been proposed that such effects, which areindependent of its ability to inhibit bacterial protein synt hesis,result from its anti-inflammatory and antioxidant properties (Lin et al., 2003, Yrjanheikki et al., 1999). In this sense, tetracyclines caninhibit the matrix metalloproteases activity and superoxide production, and can also down-regulate the levels of expressionof interleukin 1 beta, inducible nitric oxide synthase (iNOS),caspase-3 and -1 (Chen et al., 2000). Perhaps the reports that more widely opened an important expectative for the neuroprotectiveeffect of this drug were those showing that minocycline, but not other tetracyclines, blocks the opening of the mitochondrial highconductance permeability transition pore (PTP) (Zhu et al., 2002). This blockade, in turn, inhibits the release of the proapoptoticfactors cytochrome  c , SMAC (DIABLO) and apoptosis-inducingfactor from the mitochondria (Wang et al., 2003; Zhu et al., 2002). The neuroprotective action of minocycline in animal models,including global and focal ischemia (Arvin et al., 2002; Yrjanheikkiet al., 1998, 1999), amyotr ophic lateral sclerosis (Zhang et al., 2003), Parkinson’s disease (Thomas et al., 2004; Wu et al., 2002), and Huntington disease (HD) (Gordon et al., 2004; HuntingtonStudy Group, 2004), has prompted the use of minocycline in several clinical trials (Gordon et al., 2004; Thomas and Le, 2004). However, it has been recently reported that minocycline may havevariable or even deleterious effects in different species and modelsdepending on the mode of administration and the dose (Diguet et al., 2004). Thus, minocycline presents deleterious effects in two phenotypic models of PD and HD (Diguet et al., 2003, 2004). Similarly, it exacerbates MPTP-induced dopamine damage, andreverses the neuroprotective effect of creatine in nigrostriataldopaminergic neurons (Yang et al., 2003). Further, it does not   present any beneficial action in an in vivo model of Huntington’sdisease (Bantubungi et al., 2005; Smith et al., 2003), and fails to 0969-9961/$ - see front matter   D  2005 Elsevier Inc. All rights reserved.doi:10.1016/j.nbd.2005.03.019* Corresponding author. Fax: +34 967 599327.  E-mail address: (J. Jorda´n). Available online on ScienceDirect ( Neurobiology of Disease 20 (2005) 384 – 391  afford any protection against intrastriatal injections of malonate(Cornet et al., 2004; Gon˜i-Allo et al., 2005).The main obstacle for the development of new neuroprotectivetherapies relies on the limited understanding of the key molecular events involved in neurodegeneration. Huntington’s disease is aninherited neurodegenerative disease in which there is a pro-gressive motor and cognitive deterioration. Mitochondrial toxinslike 3-nitropropionic acid (3-NP) or malonate, functioning asinhibitors of the complex II of the mitochondrial respiratorychain, have been shown to effectively induce specific behavioralchanges and striatal lesions in rats and non-human primateswhich resemble some of the histopat hologic hallmarks in HD(Beal et al., 1993; Greene et al., 1993). In this study, we have used rat cerebellar granular cell culturesto study the effects of minocycline on the cellular and molecular mechanisms responsible for the cytotoxic effects of malonate. Theaddition of this reversible succinate dehydrogenase inhibitor to cellcultures induced ROS production that resulted in the depletion of glutathione and cardiolipin in their reduced form and a significant reduction of cell viability. Minocycline did not modify any of theabovementioned deleterious effects of malonate nor preventedmalonate-induced swelling of brain mitochondria. Moreover,although no modifications in the expression of the mRNA’sencoding for iNOS, caspase-3, -8, and -9 after malonate additionswere found, we observed a significant reduction of blc-2 mRNAexpression which was not reverted by minocycline. Materials and methods  Materials Minocycline, malonic acid (Sigma); 2  V ,7  V -dichlorodihydrofluor-escein diacetate, monochlorobimane, 10-  N  -nonyl-Acridin Orangewere purchased from Molecular Probes (Eugene, OR, USA). Cell culture Primary cultures of cerebellar granule neurons were obtainedfrom dissociated cerebella of 7- to 8-day-old rats (Scorziello et al.,2001). Dissection and dissociation were carried out in Ca 2+ /Mg 2+ - phosphate buffered saline (PBS). Tissues were incubated withtrypsin for 20 min at 37 - C and dissociated by trituration in amedium containing DNase, bovine serum albumin, and trypsininhibitor. Cells were plated on 96 plastic well dishes or on 60-mm plastic Petri dishes precoated with poly- l -lysine (10 g/ml) at aconcentration of 8    10 6 cells/ml in basal Medium Eagle (BME;Life Technology) containing 25 mM KCl, 10% de-complementedfetal calf serum (FCS; Life Technology), glutamine, and anti- biotics. Cytosine- d -Arabino-furanoside (Ara-C) (10 M) was addedat days in vitro (DIV) 3 of plating to prevent the growth of non-neuronal cells. At DIV 7, neurons were treated with variousconcentrations of malonic acid that was added to the culturemedium from a fresh 50 mM stock in PBS. For minocyclineexperiments, the drug was prepared in 50-fold stock in mediumculture and added to the cultures as indicate in the experiments. Cell survival assay Viable cerebellar granular cells were quantified by measuringfluorescein fluorescence resulted from fluorescein diacetate stain-ing living cells. After treatments, cells were washed twice withPBS, and then incubated with 0.2 ml of standard PBS 36  A Mfluorescein diacetate (FDA, SIGMA) for 5 min in dark conditionsat room temperature. After washing twice, the stained cells’fluorescence intensities were examined immediately at 530 nmafter excitation at 485 nm in a Spectra Max Gemini XS (Molecular Devices). Values were expressed as a % control cultures for eachexperiment and the data are represented as the mean  T  standarderror. Six wells were used for each treatment.  Intracellular generation of ROS  We used the oxidation-sensitive fluorescent dye 2  V ,7  V -dichlor-odihydrofluorescein diacetate (DCFH-DA) to measure the produc-tion of reactive oxygen species, mainly hydrogen peroxide andhydroxyl radicals. DCFH-DA is deacetylated by esterases todichlorofluorescein (DCFH). This nonfluorescent product is thenconverted by reactive species into DCF, which can easily bevisualized by strong fluorescence at 530 nm when excited at 485nm. Cells seeded in 96-well culture plates were incubated withDCFC-DA (10  A g/ml); after 15 min, vehicle or minocycline wasadded. By 30 min, malonate or vehicle was added. Fluorescenceintensity was measured in a Spectra Max Gemini XS (Molecular Devices) every 2 min for 4 h. Six wells were used for eachtreatment. The average relative percent ROS production from at least three separate cultures was determined. Results are expressedas mean  T  SE values, and significance was determined by Student’s t   test. Statistical significance was considered at the  P   < 0.05 level.  Measurement of glutathione levels Levels of glutathione were determined by using monochlor-obimane (mBCl) fluorescence. GSH is specifically conjugated withmBCl to form a fluorescent bimane–GSH adduct, in a reactioncatalyzed by glutathione  S  -transferases (Shrieve et al., 1988). The concentration of the bimane–GSH adducts increases during theinitial 10- to 12-min period of this reaction with first-order kinetics, before leveling off (Young et al., 1994). Culture medium was removed and cells were washed twice with 0.2 ml PBS andincubated for 30 min at 37 - C in 0.2 ml fresh PBS containing 160 A M mBCl. After incubation, cells were washed twice with PBSand fluorescence was measured at an excitation wavelength of 340nm and emission wavelength of 460 nm in a Spectra Max GeminiXS (Molecular Devices). The average relative percent reducedGSH levels from at least three separate cultures were determined.Results are expressed as mean  T  SE values, and significance wasdetermined by Student’s  t   test. Statistical significance wasconsidered at the  P   < 0.05 level.  Determination of cardiolipin peroxidation levels 10-  N  -nonyl-Acridin Orange (NAO), which binds to mitochon-dria-specific cardiolipin, was used. Decreases in the fluorescence of  NAO in apoptotic cells have been reported to reflect the peroxidation of intracellular cardiolipin ( Nomura et al., 2000) because the fluorochrome loses its affinity for peroxidisedcardiolipin. Cells were seeded at a final concentration of 8    10 5 cells/well in 96-well tissue culture plates, treated with 5  A M NAOfor 30 min. After washing twice, fluorescence emitted bycardiolipin-bounded NAO was measured at 530 nm after excitationat 485 nm in a Spectra Max Gemini XS (Molecular Devices). The  F.J. Fernandez-Gomez et al. / Neurobiology of Disease 20 (2005) 384–391  385  average relative percent cardiolipin levels from at least threeseparate cultures were determined. Results are expressed as mean  T SE values, and significance was determined by Student’s  t   test.Statistical significance was considered at the  P   < 0.05 level.  Mitochondrial isolation Mitochondria were isolated from the brains of adult Sprague– Dawley rats. All the procedures followed in the present work werein compliance with the European Community Council Directive of 24 November 1986 (86/609/EEC) and were approved by the EthicalCommittee of the University of Castilla-La Mancha. To exclude that the observed effects were due to contaminating synaptosomes, weisolated br ain mitochondria using a Percoll gradient as previouslydescribed (Sims, 1990). Rats were killed by decapitation; forebrains were rapidly removed, chopped, and homogenized in ice-coldisolation buffer (225 mM mannitol, 25 mM sucrose, 10 mMHEPES, 1 mM K  2 EDTA, pH 7.4 at 4 - C). The homogenate wascentrifuged at 1330   g   for 3 min, and the pellet obtained was re-suspended and re-centrifuged at 1330    g   for 3 min. The pooledsupernatants were centrifuged at 21,300   g   for 10 min. The pellet was re-suspended in 15% Percoll and layered on pre-formedgradients (40 and 23%). ThePercoll gradientswere then centrifugedat 31,700   g   for 10 min. The mitochondrial fraction located at theinterface of the two lower layers was removed, diluted withisolation buffer, and centrifuged at 16,700    g   for 10 min. Themitochondrial pellet was suspended in a solution containing 125mM KCl, 20 mM HEPES, 2 mM KH 2 PO 4 , 1  A M EGTA, 1 mMMgCl 2 , 5 mM malate, and 5 mM glutamate with the pH adjusted to7.4 with KOH.  Permeability transition pore activity Permeability transition pore opening was assayed s pectrophoto-metrically as previously described (Kristal et al., 2000). Changes in absorbance at 540 nm (  A 540 ), indicating mitochondrial swellingdue to PTP opening, were determined, after the addition of different compounds, using a microplate reader (BioRad, Hercules,CA). Initial  A 540  values were  ;  0.8, and minor differences in theloading of the wells were compensated by representing the data asthe fraction of the initial absorbance determination remaining at agiven time.  RNA isolation Total RNA was obtained with Trizol \ Reagent (Invitrogen)following the manufacturer’s indications. Ten million cells wereused per milliliter of Trizol. The isolated RNA was subsequentlytreated with DNase (Promega) to remove any genomic DNAcontamination. The integrity of RNA was always checked byrunning an aliquot in an agarose gel.  Real-time RT-PCR cDNA was synthesized from 10  A g total RNA in 100  A lvolume containing 1  RT Buffer (Applied BioSystems), 500  A MdNTPs, 2.5  A M random hexamers, and 1.25 U/  A l MultiScribeReverse Transcriptase. Reaction was performed in a thermalCycler at 48 - C for 30 min. Samples were then kept at    20 - Cuntil their utilization. PCR amplification was performed on theABI Prism 7000 Sequence Detection System (Applied Biosys-tems), using the SYBR Green PCR Master Mix (AppliedBiosystems). 1  A l cDNA was used for each reaction. PCR amplifications were always performed in triplicate wells, using 40two-temperature cycles (15 s at 94 - C and 1 min at 60 - C). Thequantification was performed by the comparative Ct (Cyclethreshold) method (Livak and Schmittgen, 2001), using GAPDHas internal control, once demonstrated that the efficiency for thedifferent primer com binations was similar. Primers for all target sequences (Table 1) were designed using the computer Primer  Express software program specially provided with the 7000 SDS(Applied Biosystems). Statistics The results were expressed as the mean  T  SD of at least threeindependent experiments. Student’s two-tailed, unpaired  t   test wasused, and values of   P   < 0.05 were considered to be significant. Results  Minocycline does not protect against malonate-induced cell death Malonate has been reported to induce cell death in different models (Beal et al., 1993; Cornet et al., 2004; Greene andGreenamyre, 1995; Greene et al., 1993). The results shown hereconfirm and expand these observations. We used the fluoresceindiacetate staining method to analyze the effects of malonate oncerebellar granular cell culture viability. As shown in Fig. 1,malonate (1 mM to 50 mM) induced a marked reduction in cellviability measured 24 h after treatment, in a concentration-depend-ent manner (Fig. 1). The lower concentration of malonate tested did not compromise cell viability, while higher concentration (50 mM)resulted in a marked cell death (Fig. 1). To analyze the possible  protective effects of minocycline on malonate-induced cytotoxicity,7 DIV cerebellar granular cultures were pre-treated with minocy-cline (10–100  A M) for 4 h before the addition of malonate (1–50mM), a dose range that  has been shown to afford cytoprotectionagainst several stimuli (Wang et al., 2003; Zhu et al., 2002). As shown in Fig. 1, minocycline did not prevent the cell death caused  by 24 h exposure to malonate (10–50 mM).  Minocycline has no antioxidant action on cerebellar granular cells Tetracyclines have been reported to have the ability to inhibit ROS production in various cell types (Gabler et al., 1992; Table 1Sequences of the oligonucleotide primer pairs used for real-time PCR Bcl-2-F 5  V -CGGTGGTGGAGGAACTCTT-3  V Bcl-2-R 5  V -ACAATCCTCCCCCAGTTCA-3  V iNOS-F 5  V -ACATCAGGTCGGCCATTACT-3  V iNOS-R 5  V -TAGCCAGCGTACCGGATGA-3  V Caspase 3-F 5  V -ACGGGTCATGGTTCATCCA-3  V Caspase 3-R 5  V -TGCGCGTACAGTTTCAGCAT-3  V Caspase 9-F 5  V -TGTCCCGTGAAGCAAGGATT-3  V Caspase 9-R 5  V -TCCCACGTCTCCTCCAACC-3  V Caspase 8-F 5  V -GGAGGACATACCCAAACTC-3  V Caspase 8-R 5  V -TGCTGTGCAATCACTGAAGG-3  V GAPDH-F 5  V -CCAGCCTCGTCTCATAGACA-3  V GAPDH-R 5  V -GTAACCAGGCGTCCGATACG-3  V  F.J. Fernandez-Gomez et al. / Neurobiology of Disease 20 (2005) 384–391 386  Miyachi et al., 1986). The oxidation of DCFH, a nonfluorescent  probe, to a fluorescent dichlorofluorescein (DCF) was used tomeasure intracellular H 2 O 2 -derived oxidants. In the first set of experiments, we determined whether minocycline treatment modulates the endogenous production of  H 2 O 2 -derived oxidantsin cerebellar granular cells. As shown in Fig. 2A, 4 h after theaddition of minocycline (10–100  A M), cultures presented areduction in the basal peroxide levels. Secondly, and in agree-ment with previous observations (Ferna´ndez-Go´mez et al., 2005;Maragos et al., 2004), malonate increased ROS production in cerebellar granular cell cultures (Fig. 2B). Cerebellar granular cells were incubated with malonate (1–50 mM) and H 2 O 2 -derived oxidant production was monitored during 4 h. By 4 hafter malonate addition, peroxides production significantlyincreased at a malonate concentration of 10 mM and above(Fig. 2B). To analyze the effect of minocycline on this paradigm,we pre-treated cellular cultures for 30 min before the addition of malonate. Under these conditions, minocycline failed to prevent malonate-induced increase in H 2 O 2 -derived oxidants in cerebellar granular cell cultures (Fig. 2B).The antioxidant tripeptide GSH plays an important role indetoxifying oxygen radicals and its relevance in malonate-induceddamagehasbeenreported(Klivenyietal.,2000).Thefactthatdrugs known to increase GSH levels confer cytoprotection to cellular cultures (Jordan et al., 2004) prompted us to measure the cellular tripeptide glutathione levels after minocycline treatment. Levels of reduced GSH were determined using monochlorobimane, whichspecifically conjugates with GSH to form a fluorescent bimane– GSH adduct. No modifications in basal levels of r educed GSH werefound in minocycline-treated cultures for 24 h (Fig. 3). However,  parallel cell cultures treated with 1–50 mM malonate for 24 h presented a severe depletion of around 50% at 50 mM (Fig. 3), an effect not prevented by minocycline (Fig. 3).Oxidation of proteins and lipids commonly occurs as aconsequence of increased ROS production and depletion of theantioxidant capacity. Cardiolipin is a phospholipid located in theinner mitochondrial membrane that becomes oxidized under stressconditions. Therefore, we next measured reduced cardiolipin le-vels by using NAO staining. NAO is a fluorochrome with a highaffinity for reduced cardiolipin form. As shown in Fig. 4, 10–50mM malonate-treated cerebellar granular cell cultures present lower fluorescence intensity than control cultures, as a result of the increase in peroxidised cardiolipin form. Once more, pre-treatment with minocycline (1–100  A M) for 4 h failed to block theoxidation of this phospholipid (Fig. 4).  Effects of minocycline on malonate-induced mitochondrial swelling  Under some circumstances, mitochondria respond to cellular stress by opening the PTP resulting in the swelling of the organelle.This morphological alteration can be monitored by measuring thechanges in 540 nm absorbance of a purified mitochondrialsuspension (Kristal et al., 2000). In this sense, it has been shown t hat minocycline has the capacity to block mitocondrial swelling(Zhu et al., 2002). Because malonate induces a delayed mitochon- Fig. 2. Minocycline did not abrogate malonate-induced H 2 O 2 -like speciesgeneration. (A) Four hours after the addition of minocycline (10–100  A M),cerebellar granular cell cultures presented a reduction in the basal peroxidelevels. (B) Cerebellar granular cells were treated with various concen-trations of malonate (1–50 mM) once pre-treated for 30 min withminocycline (1–100  A M). After 4 h of treatments, the green fluorescencecharacteristic of DCF was measured in a Spectra Max Gemini XSmicroplate reader. The data represent mean  T  SD of three independent experiments. *  P   < 0.05, ***  P   < 0.001 for control conditions versus controlconditions (0 minocycline).Fig. 1. Minocycline fails to protect malonate-induced cell death. Cerebellar granular cells were treated with various concentrations of malonate (1–50mM). Minocycline (1–100  A M) was added 4 h before malonate andmaintained until the end of the experiment. By 24 h after malonateadditions, the medium was aspirated, and the cells were washed twice withPBS and subsequently incubated with 10  A g/ml fluorescein-AM for 5 min.After washing the cells with PBS, the green fluorescence characteristic of fluorescein was measured in a Spectra Max Gemini XS microplate reader.The data represent mean  T  SD of three independent experiments. *  P   < 0.05,***  P   < 0.001 for control conditions (0 minocycline).  F.J. Fernandez-Gomez et al. / Neurobiology of Disease 20 (2005) 384–391  387  drial swelling (Ferna´ndez-Go´mez et al., 2005), in the next set of  experiments, we were interested in ascertain whether minocyclinewould block malonate-induced brain mitochondria swelling. Theresults, shown in Fig. 5, demonstrate that minocycline (50  A M)failed to block 50 mM malonate-induced brain mitochondrialswelling. iNOS, caspase-3, -8, and -9 mRNA expression levels are not modulated by malonate The regulation of antiapoptotic and proapoptotic genes has been suggested among the mechanisms invoked to explain theneuroprotective effects of minocycline. Among them, the down-regulation of caspases and iNOS and the up-regulation of Bcl-2have been documented recently (Wang et al., 2004). In order to check whether these pathways were involved in malonate-induced granular cell death, we first analyzed the pattern of expression of iNOS, caspase-3, -8, and -9 and the antiapopt oticgene Bcl-2 in malonate-treated cells. The results, shown in Fig.6A, revealed that malonate at the concentration which causes asignificant decrease in cell viability does not modify theexpression of the iNOS, caspase-3, -8, and -9 genes. However,50 mM malonate caused a significant down-regulation of theantiapoptotic gene Bcl-2 (Fig. 6B). Interestingly, the up-regulation of this gene has been pointed as a protectivemechanism for minocycline in an HD experimental model.Therefore, we studied whether minocycline would prevent thedown-regulation of Bcl-2 gene expression induced by malonate.As depicted in Fig. 6B, minocycline, consistently with its lack of  protective effect, failed to block the down-regulation of Bcl-2caused by malonate. Discussion The main finding of this study is that minocycline does not exert any cytoprotective effect on rat cerebellar granular cellsexposed to malonate, a pharmacological tool widely used tostudy the neurodegenerative events caused by mitochondrialinhibition. Here, we report that minocycline fails to abrogate anyof the effects caused by malonate, including increased ROS production and the subsequent depletion of GSH and cardiolipin.Indeed, the presence of the antibiotic does not block malonate-induced brain mitochondrial swelling. Additionally, althoughmalonate treatment fails to induce the mRNA expression of caspase-3, -8, and -9 as well as iNOS, it decreases the mRNA of the antiapoptotic protein Bcl-2, an effect that was not abrogated by minocycline.Reactive oxygen species can function either as physiologicalintermediates of cellular responses or as inducers of toxic oxidativestress depending on their intracellular concentration (e.g., Ha et al.,2003).Forthisreason,webeganmeasuringROSlevelsincerebellar granular cells treated with malonate, minocycline, or the combina- Fig. 5. Minocycline did not inhibit malonate-induced mitochondrialswelling. Changes in absorbance at 540 nm (  A 540nm ), indicating mitochon-drial swelling, were followed, after addition of malonate (50 mM) toisolated brain mitochondria. The effect of 50  A M minocycline on PTP wasalso measured. Data are mean values obtained from one experiment  performed by triplicate. Similar data were found in at least five different mitochondria preparations.Fig. 4. Minocycline failed to block the oxidation of cardiolipin by malonate.Cerebellar granular cells were pre-treated with minocycline (1–100  A M) for 4 h before the addition of malonate for 24 h. After the treatments, themedium was aspirated, and the cells were washed twice with PBS andsubsequently incubated with 5  A M NAO for 30 min. The cells were thenwashed with PBS. The NAO fluorescence was measured in a Spectra MaxGemini XS microplate reader. The data represent mean  T  SD of threeindependent experiments. *  P   < 0.05, ***  P   < 0.001 for control conditionsversus control conditions (0 minocycline).Fig. 3. Minocyclineunsuccessfulto blockmalonate-inducedGSH depletion.Cerebellar granular cells were pre-treated with minocycline (1–100  A M) for 4 h before the addition of malonate (1–50 mM) for 24 h, and mBcl stainingwas carried out as described under Materials and methods. The datarepresent mean  T  SD of three independent experiments. *  P   < 0.05, ***  P   <0.001 for control conditions versus control conditions (0 minocycline).  F.J. Fernandez-Gomez et al. / Neurobiology of Disease 20 (2005) 384–391 388
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