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6Hydroxydopamine (6-OHDA) induces Drp1-dependent mitochondrial fragmentation in SH-SY5Y cells

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6Hydroxydopamine (6-OHDA) induces Drp1-dependent mitochondrial fragmentation in SH-SY5Y cells
  Original Contribution 6-Hydroxydopamine (6-OHDA) induces Drp1-dependent mitochondrialfragmentation in SH-SY5Y cells Maria Gomez-Lazaro a,b , Nina A. Bonekamp b , Maria F. Galindo a,c , Joaquin Jordán a, ⁎ , Michael Schrader b a Grupo de Neurofarmacología, Department Ciencias Médicas, Facultad de Medicina, Universidad de Castilla-La Mancha-Centro Regional de Investigaciones Biomédicas, Albacete, Spain b Centre for Cell Biology and Department of Biology, University of Aveiro, Aveiro, Portugal c Unidad P   fi  zer-Castilla-La Mancha de Neuropsicofarmacología Translacional, Complejo Hospitalario Universitario de Albacete, Spain a r t i c l e i n f o a b s t r a c t  Article history: Received 12 October 2007Revised 21 February 2008Accepted 11 March 2008Available online 20 March 2008 Mitochondrial alterations have been associated with the cytotoxic effect of 6-hydroxydopamine (6-OHDA), awidely used neurotoxin to study Parkinson's disease. Herein we studied the potential effects of 6-OHDA onmitochondrial morphology in SH-SY5Y neuroblastoma cells. By immuno fl uorescence and time-lapse fl uorescence microscopy we demonstrated that 6-OHDA induced profound mitochondrial fragmentation inSH-SY5Y cells, an event that was similar to mitochondrial  fi ssion induced by overexpression of Fis1p, amembrane adaptor for the dynamin-related protein 1 (DLP1/Drp1). 6-OHDA failed to induce any changes inperoxisome morphology. Biochemical experiments revealed that 6-OHDA-induced mitochondrialfragmentation is an early event preceding the collapse of the mitochondrial membrane potential andcytochrome  c   release in SH-SY5Y cells. Silencing of DLP1/Drp1, which is involved in mitochondrial andperoxisomal  fi ssion, prevented 6-OHDA-induced fragmentation of mitochondria. Furthermore, in cellssilenced for Drp1, 6-OHDA-induced cell death was reduced, indicating that a block in mitochondrial  fi ssionprotects SH-SY5Y cells against 6-OHDA toxicity. Experiments in mouse embryonic  fi broblasts de fi cient in Baxor p53 revealed that both proteins are not essential for 6-OHDA-induced mitochondrial fragmentation.Our data demonstrate for the  fi rst time an involvement of mitochondrial fragmentation and Drp1 function in6-OHDA-induced apoptosis.Published by Elsevier Inc. Keywords: DynaminMitochondriaPeroxisomesOrganelle dynamicsApoptosis6-HydroxydopamineParkinsonBaxp53 Introduction Accumulating evidence suggests that neuronal apoptosis contri-butes to the pathogenesis of acute and chronic neurodegenerativediseases in the adult brain following metabolic or neurotoxic insults[1,2]. 6-Hydroxydopamine (6-OHDA), an oxidative metabolite of dopamine, is a neurotoxin which has been broadly used to generateexperimental models of Parkinson's disease [3,4]. Although there is a consensus in the ability of 6-OHDA to induce cytotoxicity in differentcell types [5,6], the mechanism involved is still controversial [4]. Among the mechanisms discussed, the generation of reactive oxygenspecies (ROS) is the most accepted [6]. Thus, 6-OHDA can eitherundergo extracellular autooxidation or intracellular enzymatic oxida-tion through the monoamine oxidase type B, yielding ROS andquinolinic products [7]. The mechanisms downstream of these toxicproducts are complex and might involve an increase in mitochondrialouter membrane permeability (MOMP), leading to the release of cytochrome  c   and other proapoptotic mitochondrial proteins thatactivate downstream effector proteins such as the effector caspase 3[8 – 10].Mitochondria form a highly dynamic semitubular network, themorphologyofwhichisregulatedbyfrequent fi ssionandfusioneventsas well as by movements along the cytoskeleton [11]. Although thephysiological signi fi cance of mitochondrial dynamics is not fully un-derstood, growing evidence indicates that maintaining correct mito-chondrial morphology by  fi ssion and fusion is critical for cell function[11 – 15].Thisnotionissupportedbyrecent fi ndingsthatlinkmutationsingenesencoding fi ssion/fusionproteinstohumandiseases[16,17,14]. Mitochondrial  fi ssion has also been implicated in synaptic and spineplasticity in neurons [18]. A remarkable episode occurring duringneuronal apoptosis is the disintegration of the semireticular mito-chondrial network into small punctiform organelles. Evidence hasbeen presented suggesting that mitochondrial fragmentation mightplay an active role in apoptotic cell death [19 – 21], although in somemodels mitochondrial  fi ssion is not a prerequisite for triggering the Free Radical Biology & Medicine 44 (2008) 1960 – 1969 ⁎  Corresponding author. Grupo de Neurofarmacología, Departamento de CienciasMedicas, Universidad Castilla-La Mancha-Centro Regional de Investigaciones Biomédi-cas, Avda Almansa 14, Albacete 02006, Spain. Fax: +34 967599327. E-mail address: (J. Jordán).  Abbreviations:  6-OHDA, 6-hydroxydopamine; ABC, ATP-binding cassette; DLP1/Drp1, dynamin-related protein 1; GDAP1, ganglioside-induced differentiation-associated protein 1; MARCH5, membrane-associated ring  fi nger (C3HC4) 5; MEF,mouse embryonic  fi broblast; Mfn2, mitofusin 2; MnSOD, manganese superoxidedismutase; MOMP, mitochondrial outer membrane permeability; PMP70, 70-kDaperoxisomalmembrane protein; R123, Rhodamine 123; ROS,reactive oxygen species;TEMPOL, 4-hydroxy-2,2,6,6-tetramethylpiperidine- N  -oxyl.0891-5849/$  –  see front matter. Published by Elsevier Inc.doi:10.1016/j.freeradbiomed.2008.03.009 Contents lists available at ScienceDirect Free Radical Biology & Medicine  journal homepage:  apoptoticprocess[22].Apoptosiscanbemediatedbyanincreaseinthe fi ssion of tubular mitochondria and an inhibition of fusion, suggestingan involvement of the components of the  “ classical ”  mitochondrial fi ssion machinery [19,23 – 25]. A reduction of the mitochondrialvolume had already been observed in pathological tissue [26 – 28],which was retrospectively found to undergo apoptosis [29 – 32].Furthermore, ultracondensation or fragmentation of mitochondriawas observed in a range of cell types undergoing apoptosis [33 – 35].Several components mediating mitochondrial  fi ssion in mammaliancells have already been identi fi ed, for example, dynamin-relatedprotein 1 (Drp1/DLP1), hFis1, MTP18 [36,37], GDAP1 [38,39], and MARCH5 [40]. Drp1 is a large cytosolic GTPase that translocates to themitochondria where it couples GTP hydrolysis with scission of themitochondrial tubule [41 – 43]. Its receptor at the mitochondrialmembrane is thought to be hFis1, a tail-anchored outer membraneproteinfacingthecytoplasm[44 – 47].Drp1andhFis1arealsoinvolvedin peroxisomal division and are shared by both mitochondria andperoxisomes [48].Bax is a proapoptotic member of the Bcl-2 family that migrates tomitochondria after apoptotic insults, and it is accepted that Bax andBak are essential for MOMP [49,50]. In most cases studied, dis- integrationof themitochondrialnetworkoccurswithinthesametimeframe as Bax activation and MOMP [51,52]. Indeed, Bax might play a critical role in the control of mitochondrial  fi ssion and morphology,since Bax colocalizes with Drp1 and Mfn2 in distinct foci, possiblyfuture scission sites, on the mitochondria after the induction of apop-tosis [53 – 55]. Furthermore, p53, a transcription factor that is up-regulated after DNA damage, and that translocates to the mitochon-dria during cell death [25], has been shown to increase after 6-OHDAtreatment [56].Here we show that 6-OHDA induces profound mitochondrial fi ssion in SH-SY5Y cell cultures, an event that precedes collapse of themitochondrial membrane potential and cytochrome  c   release. Weobserved that the lack of expression of Drp1, but not Bax or p53,prevented 6-OHDA-induced morphological mitochondrial changes.Furthermore, blocking mitochondrial  fi ssion by preventing Drp1expression alleviates cellular death induced by 6-OHDA. Materials and Methods cDNAs and antibodies The construct pmitoGFP for the labeling of the mitochondrialmatrix was kindly provided by R. Lill (University of Marburg, Ger-many). hFis1 fused to the Myc epitope tag (Myc-hFis1) was describedpreviously [45]. pGFP-C1 and mito-DsRed2 were obtained fromClontech Laboratories, Inc. (Mountain View, CA).Antibodies used were as follows: rabbit polyclonal antibodiesagainst PMP70, a peroxisomal membrane protein (kindly providedby A. Völkl, University of Heidelberg, Germany), anti-Cyt c (SantaCruz Biotechnology Inc., Santa Cruz), and DLP1/Drp1 (kindly providedby M.A. McNiven, Mayo Clinic, Rochester, MN). The followingmonoclonalantibodieswereused:anti-tubulinDM1 α  (Sigma-Aldrich,St. Louis, MO), anti-MnSOD (Axxora GmbH, Loerrach, Germany), andanti-myc (Santa Cruz Biotechnology Inc.). Species-speci fi c anti-IgGantibodies conjugated to HRP or to the  fl uorophores TRITC and Alexa488 were obtained from Bio-Rad (Hercules, CA) or Molecular ProbesEurope (Leiden, The Netherlands). Cell culture and drug treatment procedures SH-SY5Y cell cultures and mouse embryonic  fi broblasts (MEFs)(WT, Bax-/-, and p53-/-) (kindly provided by Dr. M. Serrano, CNIO,Spain; see [57]) were grown in Dulbecco's modi fi ed Eagle's medium(DMEM) and Ham's F-12 supplemented with 2 mM L-glutamine,penicillin (20 units/ml), streptomycin (5  μ g/ml), and 15% (v/v) fetalbovine serum (Invitrogen, Carlsbad, CA) as previously described [57].Cells were grown in a humidi fi ed cell incubator at 37 °C under a 5%CO 2  atmosphere.Immediately before 6-OHDA (Sigma) addition, stocks of 6-OHDA(50 mM, 1000X) were made in ascorbic acid (0.01%) and added tothe culture medium to achieve the required  fi nal concentration(50  μ M). In order to address an involvement of ROS in 6-OHDA-mediated mitochondrial fragmentation, we performed experimentswith  “ aged ”  (1 month) 6-OHDA stock solutions, from which ROShave been liberated by autooxidation [5]. Furthermore, dilutions of 6-OHDA in culture medium were pretreated for 1 h with the anti-oxidants catalase (Sigma) (10 U/ml), ascorbic acid (Sigma) (0.01%), orTEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine- N  -oxyl) (Calbio-chem) (0.2  μ M) before addition to the cells. RNA interference and transfection experiments To knock down the expression of Drp1 by RNA interference,21-nucleotide small interfering RNA (siRNA) was obtained fromDharmacon (Lafayette, CO) and annealed according to the ma-nufacturer's protocol. The siRNA sequence ef  fi ciently targetinghuman Drp1 (Accession No. NM_012062) corresponded to thecoding region 783 – 803 relative to the  fi rst nucleotide of thestart codon (siRNA, sense strand: 5 ′ -UCCGUGAUGAGUAUGCUU-UdTdT-3 ′ ). Transfection of siRNA was carried out by electropora-tion. Cells grown to 90% con fl uence on an area of 75 cm 2 (1×10 7 cells) were harvested by trypsination, washed in 10 ml of HBSsolution (21 mM Hepes, 137 mM NaCl, 5 mM KCl, 0.7 mM Na 2 HPO 4 ,6 mM dextrose), resuspended in 0.5 ml HBS, and transferred to a0.4-cm-gap, sterile electroporation cuvette containing 10 µg of DNA. Electroporation was performed in an Easyject Plus electro-porator (Peqlab, Erlangen, Germany) at 230 V, 150 0 µF, 25 – 30 msduration. After electroporation, cells were immediately resus-pended in complete medium and plated on coverslips. As a control,cells were transfected with siRNA duplexes targeting luciferase(Dharmacon). Cells were transfected with siRNA duplexes 24 hafter seeding and treated with 6-OHDA (50  μ M) 72 h after elec-troporation, when tubular peroxisomes reached their maximum[58]. Successful Drp1 silencing was assayed by immuno fl uores-cence and immunoblotting. For transfection with plasmids, cellswere plated at a density of 5.3×10 4 cells/cm 2 on poly-L-lysine-coated glass coverslips. After 24 h cells were transfected withLipofectamine reagent (Invitrogen) according to the manufacturer'sprotocol. After 4 h, the transfection mixture was replaced by freshcomplete medium, and the cells were treated with 6-OHDA. Fluorescence microscopy Cells grown on glass coverslips were washed twice with PBS,pH 7.35,  fi xed with 4% paraformaldehyde in PBS, pH 7.35, for 20 minat RT and then washed three times with PBS. Next, cellular mem-branes were permeabilized by incubation in 0.2% Triton-X100 for10minatRT.CellswerewashedthreetimesinPBSandthenincubatedfor 10 min with 1% BSA. After washing once with PBS, cells wereincubated for 1 h with primary antibodies diluted in PBS. After ex-tensive washing in PBS, cells were incubated with  fl uorescentlylabeled secondary antibodies diluted in PBS (Alexa-488 or TRITC),washed an additional 3 times, and mounted in Mowiol 4-88 contain-ing n -propylgalateasdescribed[59].SampleswereexaminedusinganOlympus BX-61 fl uorescence microscope (Olympus Optical Co. GmbH,Hamburg, Germany) equipped with the appropriate  fi lter combina-tions and a 100× objective (Olympus Plan-Neo fl uar; numerical aper-ture, 1.35). Fluorescence images were acquired with a F-view II CCDcamera (Soft Imaging System GmbH, Münster, Germany) driven bySoft imaging software. Digital images were optimized for contrast andbrightness using Adobe Photoshop software. 1961 M. Gomez-Lazaro et al. / Free Radical Biology & Medicine 44 (2008) 1960 – 1969  Mitochondrial morphology To examine mitochondrial morphology we used Mitotracker Red-CMX-ROS staining (Invitrogen). Cells grown on 24-mm-diameterpoly-L-lysine-coated glass coverslips were stained with 300 nMMitoTracker Red CMX-ROS for 30 min in Hepes buffer with thefollowing ionic composition (in mM): NaCl 140, KCl 5.9, MgCl 2  1.2,Hepes15,glucose10,CaCl 2 2.5,pH7.4.CellswerethenwashedinfreshHepes buffer and mounted in a chamber for confocal microscopy. Theexcitation/emission wavelengths for MitoTracker Red CMX-ROS were578/599 nm. Images were captured with a Leica DMIRE2 confocalmicroscope, using a 63× 1.4 NA objective. Digital images wereoptimized for contrast and brightness using Adobe Photoshop soft-ware. To quantify the occurrence of mitochondrial fi ssion, we countedthe number of cells with fragmented mitochondria of each coversliptotalling ∼ 150cells/coverslip. Thepercentage ofcellswithfragmentedmitochondria was determined on three coverslips for each conditionand normalized to parallel controls. Each independent coverslip wastreatedasasingleobservation.Theaveragerelativepercentageofcellswith fragmented mitochondria from at least three separate cultureswas determined. Results are expressed as mean±SE values, andsigni fi cancewas determinedbyStudent's t   test. Statisticalsigni fi cancewas considered at the  P  b 0.05 level.  Assessment of apoptotic cell death To assess apoptotic cell death, SH-SY5Y cells were plated on poly-L-lysine-coated glass coverslips, and were treated with 6-OHDA(50  μ M) for 1, 3, 6, and 24 h. Nuclei were stained with 0.5 µg/ml of Hoechst 33342 in PBS for 5 min. Cells were examined with an UV illumination microscope (Leica DMRXA). Uniformly stained nucleiwere scored as healthy, viable cells. Condensed or fragmented nucleiwere scored as apoptotic. Viable and dead cells were counted onadjacent  fi elds of each coverslip totaling  ∼ 350 – 450 cells. Thepercentage of dying cells was determined on four coverslips foreach condition and normalized to parallel controls. Each independentcoverslip was treated as a single observation, and at least fourcoverslips were used in each experiment. The average relativepercentage of apoptotic cells from at least three separate cultureswas determined. Results are expressed as mean±SE values, andsigni fi cance was determined by Student's  t   test. Statistical signi fi -cance was considered at the  P  b 0.05 level. Fig.1.  Effects of hFis1 overexpression and 6-OHDA on the mitochondrial fi ssionpattern. SH-SY5Y cell cultures were fi xed with 4% paraformaldehyde and mitochondrial morphologywas assessed by immuno fl uorescence microscopy using antibodies against the mitochondrial matrix protein MnSOD. (A) Control cultures exhibit mainly mitochondria with a fi lamentous morphology. (B,C) SH-SY5Y cells overexpressing hFis1-myc for 24 h (B) or treated with 6-OHDA (6 h, 50 μ M) presented dramatic fragmentation of mitochondria (C). Theimages shown are representative of results obtained in four separate experiments, each performed in triplicate. Fig. 2.  Time course of 6-OHDA-induced mitochondrial  fi ssion. SH-SY5Y cells were transfected with DsRed2-mito and after 24 h changes in mitochondrial morphology weremonitored byconfocallaser scanning microscopy.Zoomed confocaltime-lapse imageswere acquiredevery15 min for1 hfromcells culturedunderdifferentconditions: Control(A),and fresh(B) or “ aged ”  (C) 50 μ M 6-OHDA.Onlyfresh 6-OHDA(B,D) was able toinduce changes in mitochondrial morphology. (D) ROS participatein 6-OHDA-induced mitochondrialfragmentation. 6-OHDA(50 μ M) was exposed tothe antioxidants catalase (10 U/ml), ascorbic acid (0.01%), andTEMPOL (0.2 μ M) for 1 hbeforethe addition to cultured SH-SY5Y cells.Cellsexhibitingfragmented mitochondriawere counted3 hafterthe addition of 6-OHDA.Results aregiven as percentage of total cells (mean±SE). Experiments arerepresentativeof at least  fi ve experiments, each performed in triplicate.1962  M. Gomez-Lazaro et al. / Free Radical Biology & Medicine 44 (2008) 1960 – 1969  1963 M. Gomez-Lazaro et al. / Free Radical Biology & Medicine 44 (2008) 1960 – 1969  Cytochrome c determination Cytosolic extracts from nontreated and 6-OHDA-treated cultureswere obtained as previously described [60]. Cells were washed oncewith PBS and collected by centrifugation (2000 ×  g  ; 5 min). The cellpellet was resuspended in 200  μ l of extraction buffer containing(mM): sucrose 250, Tris – HCl 50, EGTA 1, EDTA 1, DTT 1, PMSF 0.1, pH7.4. Cells were homogenized in a Te fl on-glass homogenizer ( fi vestrokes) and, after 15 min on ice, the suspension was centrifuged(15,000 ×  g  ; 15 min). The cytoplasmic supernatant was removed andstored at - 80 °C until analysis by gel electrophoresis. The amount of 15  μ g of protein from control and treated samples was run on 15%SDS – polyacrylamide gels and transferred to a PVDF membrane, thatwas incubated with anti-cytochrome  c   (1:1000 dilution of rabbitpolyclonal IgG, Santa Cruz Biotechnology Inc.). Mitochondrial con-taminationwas addressed with an anti-cytochrome  c   oxidase subunitIV antibody (COX-IV; BD Biosciences). The signal was detected usingan enhanced chemiluminescence detection kit (Amersham ECL RPN2106 Kit, GE Healthcare, Waukesha, WI). Mitochondrial potential For the determination of mitochondrial membrane potential,SH-SY5Y cellsweregentlyresuspendedinPBS,collectedbycentrifuga-tion at 300 ×  g  . Control and 6-OHDA-treated cells were resuspended inPBS(0.5×10 6 cells/ml)andincubatedfor30minat37°CwithRhodamine123(0.004 μ g/ml) (MolecularProbes, Inc.).AftertwowashesinPBS, the fl uorescencewas measured in anEPICS fl owcytometer(BDLSR,, BectonDickinson), and results were analyzed in real time using the BDCellQuestProprogramanddisplayedastwo-parameterhistograms:cellnumber versus  fl uorescence. Gel electrophoresis and immunoblotting  Protein samples were separated by SDS-PAGE, transferred tonitrocellulose or PVDF membranes using a semidry apparatus andanalyzed by immunoblotting. Immunoblots were processed usingHRP-conjugated secondary antibodies and enhanced chemilumines-cence reagents (Amersham Corp., Arlington Heights, IL). Results 6-OHDA induces mitochondrial fragmentation in SH-SY5Y cells In order to study the potential effects of 6-hydroxydopamine onmitochondria in SH-SY5Y cells, we  fi rst characterized the normalmitochondrial morphology in these cells. SH-SY5Y cells were pre-pared for indirect immuno fl uorescence and stained with speci fi cantibodies to MnSOD, a mitochondrial matrix protein. As shown inFig.1A, mitochondriain control cells exhibit an elongated-tubularand fi lamentous morphology and show an even distribution within thecytoplasm and along cellular processes. In these cultures less than 5%of the cells present mitochondrial fragmentation hallmarks. To de-monstrate that mitochondrial fi ssion can be induced in SH-SY5Y cells,and to analyze the typical  fi ssion pattern in these cells, we over-expressed Myc-tagged hFis1, a known inducer of mitochondrial di-vision [45]. At 24 h after hFis1 transfection SH-SY5Y cells presentedmitochondria with striking  fi ssion hallmarks including small and Fig. 3.  6-OHDA failed to induce changes in peroxisome morphology. SH-SY5Y cells were treated with 50  μ M 6-OHDA for 3 h and  fi xed with 4% paraformaldehyde.Immuno fl uorescencemicroscopyusing antibodies toPMP70,aperoxisomal membraneprotein, revealednoapparentmorphological changesofperoxisomes (1and3) whileMnSODimmuno fl uorescence revealed changes in mitochondrial morphology (4). Expression of Pex11p β -myc induced peroxisomal elongation (5), constriction, and division into smallspherical peroxisomes (6), indicating that SH-SY5Y cells are capable of multiplying their peroxisomes by growth and division.1964  M. Gomez-Lazaro et al. / Free Radical Biology & Medicine 44 (2008) 1960 – 1969
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