6-hydroxydopamine activates the mitochondrial apoptosis ... · 6-hydroxydopamine activates the...

6-Hydroxydopamine activates the mitochondrial apoptosispathway through p38 MAPK-mediated, p53-independentactivation of Bax and PUMA

Maria Gomez-Lazaro,* Maria F. Galindo,* Caoimhın G. Concannon,� Miguel F. Segura,�Francisco J. Fernandez-Gomez,* Nuria Llecha,§ Joan X. Comella,� Jochen H. M. Prehn� andJoaquin Jordan*,¶

*Grupo de Neurofarmacologıa, Departamento de Ciencias Medicas, Facultad de Medicina, Universidad Castilla-La Mancha,

Albacete, Spain

�Department of Physiology and RCSI Neuroscience Research Centre, Royal College of Surgeons in Ireland, Dublin, Ireland

�Cell Signaling and Apoptosis group, Departament de Ciencies Mediques Basiques, Univeristy of Lleida and Hospital Arnau de

Vilanova, Lleida, Spain

§Department of Pathology and Molecular Genetics, Hospital Universitari Arnau de Vilanova, Lleida, Spain

¶Centro Regional de Investigaciones Biomedicas, Albacete, Spain

Received June 4, 2007; revised manuscript received October 5, 2007;accepted October 22, 2007.

Address correspondence and reprint requests to Joaquin Jordan,Grupo de Neurofarmacologıa, Departamento de Ciencias Medicas,Facultad de Medicina, Universidad Castilla-La Mancha, Avda Almansa14, Albacete 02006, Spain. E-mail: [email protected] used: AFU, arbitrary fluorescent units; BH3, Bcl-2

homology domain 3; BIM, Bcl-2 interacting mediator of cell death;CHAPS, 3-[(3-cholamido propyl) dimethyl ammonio]-1-propanesulpho-nate; DMEM, Dulbecco’s modified Eagle’s medium; DTT, dithiothreitol;

E14, embryonic day 14; GFP, green fluorescent protein; GUSB,

beta glucuronidase; K-H, Krebs–HEPES buffer; MAPK, mitogen-

activated protein kinase; MEF, murine embryonic fibroblast; MKK,MAP kinase kinase; MOMP, mitochondrial outer membrane permeabi-lization; MPTP, mitochondrial permeability transitory pore; OHDA, 6-hydroxydopamine; PBS, phosphate-buffered saline; PD, Parkinson’sdisease; PMSF, phenylmethylsulfonyl fluoride; PTP, permeabilitytransition pore; PUMA, p53 upregulated modulater of apoptosis;zVAD-fmk, Z-Val-Ala-DL-Asp-fluoromethylketone.

Abstract

Mitochondrial alterations have been associated with the

cytotoxic effect of 6-hydroxydopamine (6-OHDA), a widely

used toxin to study Parkinson’s disease. In previous work, we

have demonstrated that 6-OHDA increases mitochondrial

membrane permeability leading to cytochrome c release, but

the precise mechanisms involved in this process remain

unknown. Herein we studied the mechanism of increased

mitochondrial permeability of SH-SY5Y neuroblastoma cells in

response to 6-OHDA. Cytochrome c release induced by

6-OHDA occurred, in both SH-SY5Y cells and primary cul-

tures, in the absence of mitochondrial swelling or a decrease

in mitochondrial calcein fluorescence, suggesting little

involvement of the mitochondrial permeability transition pore

in this process. In contrast, 6-OHDA-induced cell death was

associated with a significant translocation of the pro-apoptotic

Bax protein from the cytosol to mitochondria and with a sig-

nificant induction of the BH3-only protein PUMA. Experiments

in mouse embryonic fibroblasts deficient in Bax or PUMA

demonstrated a role for both proteins in 6-OHDA-induced

apoptosis. Although 6-OHDA elevated both total and nuclear

p53 protein levels, activation of p53 was not essential for

subsequent cell death. In contrast, we found that p38 mitogen-

activated protein kinase (MAPK) was activated early during

6-OHDA-induced apoptosis, and that treatment with the p38

MAPK inhibitor SKF86002 potently inhibited PUMA induction,

green fluorescent protein-Bax redistribution and apoptosis in

response to 6-OHDA. These data demonstrate a critical

involvement of p38 MAPK, PUMA, and Bax in 6-OHDA-

induced apoptosis.

Keywords: BH3 only proteins, cell death, mitochondrion,

mitochondrial outer membrane permeability, Parkinson’s

disease, permeability transition pore.

J. Neurochem. (2008) 104, 1599–1612.

d JOURNAL OF NEUROCHEMISTRY | 2008 | 104 | 1599–1612 doi: 10.1111/j.1471-4159.2007.05115.x

� 2007 The AuthorsJournal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2008) 104, 1599–1612 1599

6-Hydroxydopamine (6-OHDA) is a toxic oxidative metab-olite of dopamine that is detected in brains and the urine ofParkinson’s disease (PD) patients. Its administration intocertain brain areas produces selective destruction of cate-cholamine neurotransmitter neurons in animal models,making 6-OHDA a widely used tool for the study of PD.Different cell types have been described as susceptible to6-OHDA treatment including primary rat striatal neurons(Shinkai et al. 1997), bovine chromaffin cells (Galindo et al.2003b), human neuroblastoma cells (Simantov et al. 1996;Jordan et al. 2004), and PC12 cells (Walkinshaw and Waters1994; Biswas et al. 2005). However, the mechanismsresponsible for 6-OHDA-induced toxicity remain largelyunclear (for review, see Blum et al. 2001). Under physio-logical conditions 6-OHDA is rapidly and non-enzymaticallyoxidized by molecular oxygen to form 1,4-p-quinone and itsdegradation products, as well as reactive oxygen species(Saner and Thoenen 1971). Recently, we studied thecytotoxic effects of 6-OHDA on the human catecholamin-ergic SH-SY5Y neuroblastoma cells and have demonstratedthat 6-OHDA induces mitochondrial cytochrome c releaseand caspase 3 activation events that were blocked by over-expression of the anti-apoptotic Bcl-xL protein (Jordan et al.2004), and a role for p53 and p53 upregulated modulator ofapoptosis (PUMA) has been proposed (Biswas et al. 2005).

Increases in mitochondrial permeability appear to be acommon event in many forms of apoptotic and necrotic celldeath with evidence showing that both forms of cell deathmay play a role in PD (Greenamyre et al. 1999). The precisemechanisms regulating these events are a matter of currentdebate, and may involve the formation of specific pores,holes, or tears in the mitochondrial outer and innermembrane (Goldstein et al. 2000; Martinou et al. 2000).The formation of a voltage-dependent high conductancemultiproteic channel, referred to as mitochondrial perme-ability transitory pore (MPTP) has been reported (Bernardi1999; Baines et al. 2005). MPTP opening produces an innermitochondrial membrane permeabilization, resulting in mito-chondrial swelling, as a result of entrance of water thateventually may also break the outer membrane (Bernardi1999). Calcium, reactive oxygen species, and pro- and anti-apoptotic Bcl-2 family members have been reported toregulate this process (Wei et al. 2001; Letai et al. 2002;Galindo et al. 2003a). Moreover, the relevance of MPTP incell death process has been shown, as MPTP blockers protectagainst cytotoxic stimuli.

During apoptosis, the release of pro-apoptotic intermem-brane proteins such as cytochrome c, Smac/DIABLO, andapoptosis-inducing factor occurs predominantly as a conse-quence of mitochondrial outer membrane permeabilization(MOMP) (Green and Kroemer 2004). The pro-apoptotic Bcl-2 family proteins Bax and Bak have been shown to mediatethis increase in permeability in many cell types. While therole of Bak in neuronal apoptosis is a matter of debate

(Fannjiang et al. 2003), there is a large body of evidenceshowing that Bax is critically involved in neuronal cell death(Blum et al. 1997; Mladenovic et al. 2004). Bax activationduring apoptosis involves a conformational change in theBax protein, translocation to mitochondria, and insertion intothe mitochondrial outer membrane (Goping et al. 1998).Here, Bax oligomers might form a specific release channel orregulate pre-existing channels (Jordan et al. 2004; Sharpeet al. 2004), inducing the release of cytochrome c and otherintermembrane proteins (Goping et al. 1998; Narita et al.1998). Postmortem studies indicate that the presence of Baxand its translocation to the outer mitochondrial membranemay contribute to the death of dopaminergic neurons in PD(Hartmann et al. 2001). Activation of Bax during apoptosisis thought to be triggered by the transcriptional or post-translational activation of Bcl-2 homology domain 3 (BH3)-only proteins (Ward et al. 2004). The p53 protein has alsobeen identified as a critical mediator of neuronal apoptosis(Jordan et al. 1997a, 2003; Tieu et al. 2001; Duan et al.2002). In most cases, p53-induced apoptosis proceedsthrough transcriptional induction of the BH3-only proteinsPUMA and Noxa, Bax activation, and mitochondrial releaseof cytochrome c (Schuler et al. 2000; Schuler and Green2001; Cregan et al. 2004; Wyttenbach and Tolkovsky 2006).Other upstream signals leading to BH3-only proteins andBax activation include the stress-activated kinases such asp38 MAPK. p38 MAPK is activated in response to variousstimuli including oxidative stress and is a potent effector ofneuronal apoptosis including apoptosis of dopaminergicneurons (Ferrer et al. 2001; Naderi et al. 2003; Choi et al.2004; Ouyang and Shen 2006).

The relative contribution of BH3-only proteins, Bax,p53, and p38 MAPK in 6-OHDA-induced apoptosis hasnot yet been studied in great detail. The current studydemonstrates a predominant role of p38 MAPK but notp53 in 6-OHDA-induced apoptosis in human SH-SY5Yneuroblastoma cells.

Materials and methods

Cell culture and drug treatment proceduresSH-SY5Y cultures were grown as previously described (Jordan

et al. 2004) [in Dulbecco’s modified Eagle’s medium (DMEM)

supplemented with 2 mmol/L L-glutamine, penicillin (20 U/mL),

streptomycin (5 lg/mL), and 15% (v/v) fetal bovine serum

(Gibco, Gaithersburg, MD, USA)]. Cells were grown in a

humidified cell incubator at 37�C under a 5% CO2 atmosphere.

For green fluorescent protein (GFP)-Bax translocation and

viability experiments, cells were plated on glass coverslip at

2.9 · 105 cells/cm2 and allowed to attach overnight. Immediately

before 6-OHDA addition, dilutions of 6-OHDA were made in

0.1% ascorbic acid and added to fresh cell culture medium to

achieve the required concentration. Murine embryonic fibroblast

(MEF) Bax)/) and p53)/) cells, a gift from Dr M. Serrano

Journal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2008) 104, 1599–1612� 2007 The Authors

1600 | M. Gomez-Lazaro et al.

(CNIO, Madrid, Spain), were grown in DMEM supplemented

with 2 mmol/L L-glutamine, penicillin (20 U/mL), streptomycin

(5 lg/mL), and 15% (v/v) fetal bovine serum. Primary MEFs

were isolated from PUMA+/+ and PUMA)/) littermates from

E14.5 day embryos either using standard methods and were

maintained in DMEM with 10% fetal bovine serum, 100 U/mL

penicillin, and 100 mg/mL streptomycin, in a humidified atmo-

sphere of 5% CO2 in air at 37�C.

TransfectionsCells were plated 24 h before transfection at a density of 5.3 · 104

cells/cm2, on poly D-lysine-coated glass slides. Transfection was

achieved using Lipofectamine� reagent (Invitrogen, Carlsbad, CA,

USA) according to the manufacturer’s protocol. Cells were

transfected with the following plasmids encoding caspase 9

dominant-negative mutant caspase 9 (C287A; Casp9DN, a gift

from H. F. Ding, Medical College of Ohio, Toledo, OH, USA), wild-

type Flag-tag MAP kinase kinase (MKK)-6 (MKK6-wt), or an

inactive kinase version (MKK6-kd) used as a negative control (Dr R

Sanchez-Prieto, CRIB, Albacete, Spain; Losa et al. 2003), bacul-oviral broad spectrum caspase inhibitor p35 (Dr J. Merino,

Universidad de Santander, Spain), GFP (pGFP-C1; Clontech

Laboratories Inc., Palo Alto, CA, USA), GFP-Bax (Poppe et al.2002). After 4 h incubation the transfection mixture was removed

and replaced with fresh complete medium. By 12 h post-transfec-

tion, the percentage of cells positive for GFP expression was > 60%

in every experiment.

Assessment of apoptotic cell deathSH-SY5Y cells were plated on poly D-lysine-coated glass slides. For

cell death assay, chromatin state was analyzed by staining cells with

the dye Hoechst 33342 (Molecular Probes Inc., Eugene, OR, USA).

Cultures were rinsed three times with phosphate-buffered saline

(PBS) and then incubated with 0.5 lg/mL of Hoechst 33342 for

5 min at 22–25�C. After two rinses with PBS, cell staining was

analyzed using a fluorescent microscope. Uniformly stained nuclei

were scored as healthy, viable cells. Condensed or fragmented nuclei

were scored as apoptotic.

Assay of caspase enzymatic activityAfter treatment with 100 lmol/L 6-OHDA, cells were collected in a

buffer with the following composition (in mmol/L): HEPES 25,

EDTA 5, EGTA 1, MgCl2 5, dithiothreitol (DTT) 5, phenylmeth-

ylsulfonyl fluoride (PMSF) 1, and 10 lg/mL each of pepstatin and

leupeptin, pH 7.5. The cellular material was left for 20 min on ice

and then sonicated. The lysate was centrifuged for 20 min at

10 000 g and the supernatant was quickly frozen in a methanol dry

ice bath and stored at )80�C. Lysates (30 lg protein) were

incubated at 37�C in a buffer containing 25 mmol/L HEPES (pH

7.5), 10% sucrose, 0.1 3-[(3-cholamido propyl) dimethyl ammonio]-

1-propanesulphonate (CHAPS), and 10 mmol/L DTT with the

fluorogenic substrate DEVD-AFC (15 lmol/L in dimethylsulfoxide;

Calbiochem System Products, San Diego, CA, USA) (Jordan et al.1997b). Substrate cleavage emitted a fluorescent signal that was

quantified in a fluorometer (luminescence-spectrophotometer

LS50B; Perkin Elmer, Buckinghamshire, England) (excitation

400 nm and emission 505 nm). Enzymatic activity is expressed as

arbitrary fluorescent units (AFU).

Detection of mitochondrial permeability using calceinfluorescenceTo demonstrate induction of the MPTP, calcein fluorescence studies

were carried out following the method of Petronilli et al. (2001).This method allows one to directly visualize permeability changes in

mitochondria in situ. Calcein/acetoxymethyl ester enters the cells

and becomes fluorescent upon de-esterification. Co-loading of cells

with cobalt chloride quenches the fluorescence in the cell, except in

mitochondria, because cobalt is impermeable across mitochondrial

membranes. However, during induction of the MPTP, cobalt enters

mitochondria and is able to quench the mitochondrial calcein

fluorescence. Cultures were washed in Krebs–HEPES buffer (K–H)

with the following ionic composition (in mol/L): NaCl 140, KCl 5.9,

MgCl2 1.2, HEPES 15, glucose 10, and CaCl2 2.5, pH 7.4, and

incubated in fresh K–H containing calcein/acetoxymethyl ester

(1 lmol/L) and cobalt chloride (1 mmol/L) for 30 min at 22–25�C.Following cobalt quenching, cultures were washed with K–H, and

images were collected with a confocal microscopy within 5 min. We

have validated this method in cultured SH-SY5Y by the addition of

500 lmol/L CaCl2 along with the calcium ionophore A23187

(3 lmol/L) previously loaded with calcein and Co2+. The addition

of calcium caused a significant decrease in the mitochondrial calcein

fluorescence in a time-dependent manner (data not shown),

consistent with the earlier report by Petronilli et al. (2001).

Mitochondrial isolationMitochondria were isolated from the brains of adult Sprague–

Dawley rats. All the procedures followed in the present work were

in compliance with the European Community Council Directive of

24 November 1986 (86/609/EEC) and were approved by the Ethical

Committee of the University of Castilla-La Mancha. To exclude that

the observed effects were due to contaminating synaptosomes, we

isolated brain mitochondria using a Percoll gradient as previously

described (Sims 1990). Rats were killed by decapitation, forebrains

were rapidly removed, chopped, and homogenized in ice-cold

isolation buffer (225 mmol/L mannitol, 25 mmol/L sucrose,

10 mmol/L HEPES, and 1 mmol/L K2EDTA, pH 7.4 at 4�C). Thehomogenate was centrifuged at 1330 g for 3 min, and the pellet

obtained was resuspended and recentrifuged at 1330 g for 3 min.

The pooled supernatants were centrifuged at 21 300 g for 10 min.

The pellet was resuspended in 15% Percoll and layered on pre-

formed gradients (40% and 23%). The Percoll gradients were then

centrifuged at 31 700 g for 10 min. The mitochondrial fraction

located at the interface of the lower two layers was removed, diluted

with isolation buffer, and centrifuged at 16 700 g for 10 min. The

mitochondrial pellet was resuspended in solution III (215 mmol/L

mannitol, 71 mmol/L sucrose, 10 mmol/L succinate, and 10 mmol/

L HEPES, pH 7.4) and kept on ice for analysis.

Permeability transition pore activityPermeability transition pore opening was assayed spectrophoto-

metrically as previously described (Kristal et al. 2000). Specifically,mitochondria were suspended to reach a protein concentration of

1 mg/mL in 200 lL of solution containing 125 mmol/L KCl,

20 mmol/L HEPES, 2 mmol/L KH2PO4, 1 lmol/L EGTA, 1 mmol/

L MgCl2, 5 mmol/L malate, and 5 mmol/L glutamate with the pH

adjusted to 7.08 with KOH. Changes in absorbance at 540 nm (A540),

indicatingmitochondrial swelling as a result of permeability transition

� 2007 The AuthorsJournal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2008) 104, 1599–1612

Bax, PUMA, and p38MAPK participate in 6-OHDA cytotoxicity | 1601

pore (PTP) opening, were determined, after addition of different

compounds, using a microplate reader (BioRad, Hercules, CA, USA).

Initial A540 values were @ 0.8, and minor differences in the loading of

the wells were compensated by representing the data as the fraction of

the initial absorbance determination remaining at a given time.

Detection of mitochondrial cytochrome c releaseImmunoblot analysis was performed on cytosolic extracts from

control and 6-OHDA-treated cultures, as previously described

(Jordan et al. 2004). Cells were washed once with PBS and

collected by centrifugation (2000 g; 5 min). Cell pellets were

resuspended in 200 lL of extraction buffer containing (in mmol/L):

sucrose 250, Tris–HCl 50, EGTA 1, EDTA 1, DTT 1, and PMSF

0.1, pH 7.4, homogenized in a Teflon-glass homogenizer (five

strokes) and, after 15 min on ice, centrifuged (15 000 g; 15 min).

The supernatants, i.e. cytosolic fractions, were removed and stored

at )80�C until analyzed by gel electrophoresis using anti-cyto-

chrome c (1 : 1000 dilution of rabbit polyclonal IgG; Santa Cruz

Biotechnology Inc.) or with anti-cytochrome c oxidase subunit IV

(BD Biosciences, San Jose, CA, USA).

Reverse transcription – quantitative PCRcDNA was synthesized from 1 lg total RNA using TaqMan�Reverse Transcription Reagens (P/N N808-234) from Applied

Biosystems (Foster City, CA, USA) following the manufacturer’s

indications. The reaction was performed in a Thermal Cycler as

follows: 65�C for 5 min, 42�C for 60 min, and 70�C for 15 min.

Samples were then kept at )20�C until their utilization. PCR

amplifications were performed on the ABI Prism 7000 Sequence

Detection System (Applied Biosystems), using 96-well microtiter

plates. They were performed in a total volume of 25 lL, containing2 lL cDNA sample (equivalent to 100 ng); 1· of probe, forward

and reverse primers mix, and 1· Taqman Universal Master Mix

(Applied Biosystems). PCR amplifications were always performed

in duplicate wells, using the following temperature cycles: 2 min at

50�C, 10 min at 55�C followed by 40 cycles consisting of 15 s at

94�C and 1 min at 60�C. A sample without cDNA was used as

negative control. The quantification was performed by the compar-

ative Ct (cycle threshold) method (Livak and Schmittgen 2001),

using human beta glucuronidase (GUSB) as internal control. The

primers were designed and provided by Applied Biosystems and

were Hs 00560402-m1 for Noxa; Hs 248075-m1 for PUMA; Hs

00375807-m1 for Bcl2-interacting mediator of cell death (BIM), and

the Human GUSB (20·) (P/N 4326320E) for GUSB.

Preparation of cytosolic and nuclear extractsCells were washed with PBS and collected by centrifugation at

2000 g. Cell pellets were homogenized with 100 lL of buffer A

[10 mmol/L HEPES (pH 7.9), 1 mmol/L EDTA, 1 mmol/L EGTA,

100 mmol/L KCl, 1 mmol/L DTT, 0.5 mmol/L PMSF, 2 lg/mL

aprotinin, 10 lg/mL leupeptin, 2 lg/mL Na-p-tosyl-L-lysine chlo-

romethyl ketone, 5 mmol/L NaF, 1 mmol/L NaVO4, and 10 mmol/

L Na2MoO4]. After 10 min at 4�C, Nonidet P-40 was added to reacha 0.5% concentration. Nuclei were collected by centrifugation at

8000 g for 15 min (Gomez-Lazaro et al. 2005). The supernatants

were stored at )80�C (cytosolic extracts); the pellets were

resuspended in 50 lL of buffer A supplemented with 20%

glycerol–0.4 mol/L KCl. Nuclear protein extracts were obtained

by centrifugation at 13 000 g for 15 min, and the supernatant was

stored at )80�C. All cell fractionation steps were carried out at 4�C.

Western blot analysisSH-SY5Y cell cultures were washed with ice-cold PBS twice and

then collected by mechanical scraping with 1 mL of PBS per tissue

culture dish. The suspension was centrifuged at 13 000–14 000 gfor 5 min. The supernatant was discarded, and the pellet was

brought up in 150 lL of sample buffer. The protein from each

condition was quantified spectrophotometrically (Micro BCA

Protein Reagent Kit; Pierce, Rockford, IL, USA), and an equal

amount of protein (30 lg) was loaded onto 10% sodium dodecyl

sulfate–polyacrylamide gel electrophoresis gels. After electrophore-

sis, proteins were transferred to Immobilon PVDF membranes (Life

Sciences, Pall Corporation, Pensacola, FL, USA). Non-specific

protein binding was blocked with Blotto [4% w/v non-fat dried

milk, 4% bovine serum albumin (Sigma, St Louis, MO, USA), and

0.1% Tween-20 (Sigma)] in PBS for 1 h. The membranes were

incubated with anti-p53 [1 : 1000 dilution of anti-mouse monoclo-

nal (Pab240) sc-99; Santa Cruz], anti-pan p38 [(C-20) sc-535; Santa

Cruz], and anti-phospho-p38 MAPK (Thr180/Tyr182) (3D7)

(1 : 1000 dilution of rabbit monoclonal antibody) overnight at

4�C. Anti-alpha tubulin (1 : 40 000) and anti-histone 3 (1 : 500)

were purchased from Sigma. Anti-PUMA (1 : 1000) and anti-Bax

(1 : 3000) were obtained from Cell Signaling (Beverly, MA, USA).

Anti-BIM (1 : 1000) was from Stressgen (San Diego, MI, USA).

After washing with Blotto, the membranes were incubated with a

secondary antibody (1 : 5000 dilution of peroxidase-labeled anti-

mouse; Promega, Madison, WI, USA) in Blotto. The signal was

detected using an enhanced chemiluminescence detection kit

(Amersham ECL RPN 2106 Kit, Amersham Pharmacia Biotech,

QC, Canada). Immunoblots were developed by exposure to X-ray

film (Eastman-Kodak, Rochester, NY, USA).

ImmunofluorescenceFor the analysis of activation of endogenous Bax in primary cortical

neurons were prepared from day 17–19 (E17–E19) as previously

described (Kushnareva et al. 2005). Neurons were treated with

0.1 mmol/L 6-OHDA on 5–7 day in vitro. Following treatments,

neuronal cultures were fixed in 2% p-formaldehyde for 15 min at

22–25�C and permeabilized with 0.2% CHAPS/PBS for 5 min.

Following permeabilization, slides were blocked for 1 h in PBS/1%

bovine serum albumin/3% serum. Bax conformational change was

detected with an anti-Bax 6A7 antibody (Upstate Biotechnologies,

Millipore Ireland BV, Carrigtwohill, Co. Cork, Ireland), which only

recognizes active Bax. Primary antibody was diluted 1 : 500 in

blocking buffer was added and incubated overnight at 4�C. Cellswere washed three times with PBS, and incubated with a 1 : 250

dilution of FITC-conjugated goat anti-mouse secondary antibody

(Jackson Immuno Research Europe, Suffolk, UK) for 2 h at 22–

25�C. Following washing, slides were mounted in VectaShield

(Vector Laboratories, Burlingame, CA, USA) and imaged by

confocal microscopy.

StatisticsThe results were expressed at the mean ± SD of at least three

independent experiments. Student’s two-tailed, unpaired t-test wasused, and values of p < 0.05 were considered to be significant.



When comparing more than two conditions statistically significant

differences between groups were determined by ANOVA followed by

a Newman–Keuls post hoc analysis. The level of statistical

significance was set at p < 0.05.

Results

Consistently with our previous observation (Jordan et al.2004), 0.1 mmol/L 6-OHDA induced cell death in SH-SY5Y

cell cultures through a mechanism that involved caspaseactivation. Figure 1a illustrates that the addition of0.1 mmol/L 6-OHDA resulted in an increase in the numberof nuclei exhibiting nuclear pyknosis when assayed atdifferent times points following addition of 6-OHDA, usingHoechst 33342 as a chromatin stain. The increase inapoptotic cells correlated with a time-dependent increase inDEVD-like activity, which was evident after 6 h (Fig. 1b).To confirm that caspases participated in 6-OHDA-induced

0 5 10 15 20 25 30 35 40

0.6

0.7

0.8

0.9

1.0

1.1

KO2

6-OHDA

Control

A54

0

Time (s)

0 5 10 15 20 25 30 35 40 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5

Cal

cein

flu

ore

scen

ce

Time (min)

Control 6-OHDA

GFP

GFP

Casp9DN p35

0

20

40

60

* *

0.1 mmol/L 6-OHDA

Ap

op

toti

c n

ucl

ei (

%)

0 6 12 18 24

250

300

350

400

450

500

550

***

*** ***

DE

VD

-lik

e ca

spas

e ac

tivi

ty

(AF

U)

Time (h) 0 6 12 18 24

0 10 20 30 40 50 60 70

Ap

op

toti

c n

ucl

ei

(%)

Time (h)

15 min 0 min 35 min

(a) (b) (c)

(g) (f) (e) (d)

(h) (i)

C CsA C CsA

6-OHDA

COX-IV

Cyt c

Fig. 1 6-OHDA induces a caspase-dependent apoptotic cell death in

SH-SY5Y neuroblastoma cells independent of the PTP. (a) Time course

plot of cell viability after 0.1 mmol/L 6-OHDA of SH-SY5Y cell cultures as

assessed by Hoechst 33342 staining. (b) DEVD-like caspases are

activated after 6-OHDA treatment. Caspase activity was determined by

measuring DEVD-AFC hydrolysis in lysates from SH-SY5Y cells chal-

lenged with 6-OHDA for 1, 3, 9, 12, 18, and 24 h. (c) SH-SY5Y cell

cultures were co-transfected with GFP and Casp9DN or p35 24 h before

treatment with 0.1 mmol/L 6-OHDA. Cell viability was assessed by

Hoechst 33342 staining in GFP-positive cells. Each column represents

the average obtained from four independent experiments;

Results ± SEM. ***p < 0.001 versus control conditions. (d–g). Detec-

tion of mitochondrial calcein fluorescence intensities. SH-SY5Y cells

were co-loaded with calcein-AM and CoCl2 as described under Materi-

als and Methods and images were collected at 5 min intervals [0 (d), 15

(e), and 35 min (f) are shown]. (g) The initial fluorescence intensities

were normalized for comparative purposes, and values on the ordinate

report the mean ± SD of four independent experiments. (h) 6-OHDA

failed to induced mitochondrial swelling. Brain mitochondrial suspen-

sions were exposed to 0.1 mmol/L 6-OHDA and A540 was recorded. KO2

was used as a positive control for brain mitochondrial swelling. Cyclo-

sporine A failed to block 6-OHDA-induced cytochrome c release. (i)

Representative immunoblot showing levels of cytochrome c in cytosolic

fractions from SH-SY5Y cells cultures. Cells were pre-treated for 3 h

with 10 lmol/L cyclosporine A (CsA), and challenged with 0.1 mmol/L 6-

OHDA for 12 h. Cytochrome c oxidase subunit IV (COX-IV) protein

levels were used as an index of mitochondrial contamination. Similar

results were found in four different experiments. Data are mean ± SEM

values from n = 5–6 cultures. Different from respective controls:

***p < 0.001 (one-way ANOVA followed by Tukey’s test).



cell death, we co-transfected SH-SY5Y cell cultures withGFP and the p35 expression plasmids. By 24 h, cells werechallenged with 0.1 mmol/L 6-OHDA and nuclear apoptosisof GFP positive cells were assayed. Consistent with theconcept that caspase pathway participates in 6-OHDA-induced cell death, the cultures over-expressing Casp9DNor p35 were more resistant to 6-OHDA (Fig. 1c). Thenumber of apoptotic cells over-expressing caspase 9 domi-nant negative or p35 are not statistically different, suggestingthat the mitochondrial apoptotic pathway is pivotal in 6-OHDA induced cell death.

We then employed the calcein fluorescence method tostudy a possible involvement of MPTP activation in6-OHDA-induced cell death. This method allows the directvisualization of permeability changes of the mitochondrialinner membrane in situ, and a decrease in calceinfluorescence can be interpreted as MPTP formation. Asillustrated in the histogram of Fig. 1g no change in calceinfluorescence was detected upon addition of 0.1 mmol/L6-OHDA.

Mitochondrial swelling associated with MPTP formationis a colloid osmotic process which can be measured as adecrease in A540 (Bernardi et al. 1994). We next analyzed apotential effect of 6-OHDA on mitochondrial swelling. Asshown in Fig. 1h, the addition of 0.1 mmol/L 6-OHDA tobrain mitochondrial suspensions failed to induce significantmitochondrial swelling. As a positive control (Jordan et al.2002), addition of the MPTP activator KO2 induced a rapiddecrease in A540.

In a third approach, cells were pre-treated for 3 h with10 lmol/L cyclosporine A, an inhibitor of the MPTP, andwere then challenged with 0.1 mmol/L 6-OHDA. Consis-tent with our previous observations (Jordan et al. 2004),cytochrome c was released from the mitochondria to thecytosol 12 h after 6-OHDA addition, an event which wasinsensitive to inhibition by cyclosporine A pre-treatment(Fig. 1i).

As these experiments provided little evidence for theinvolvement of the MPTP in 6-OHDA-induced cell death,we were next interested in determining the role of Bcl-2family proteins in this process. In response to apoptoticstimuli, Bax undergoes specific conformational changes thatallow its targeting/insertion into mitochondrial outer mem-brane (Cory and Adams 2002). To investigate whether6-OHDA regulates Bax cellular distribution we used a GFP-Bax fusion protein. As illustrated in Fig. 2, confocalmicroscopy studies revealed that in untreated SH-SY5Ycells GFP-Bax distribution was cytosolic, as evident from thediffuse GFP fluorescence distribution pattern observed incells expressing GFP-Bax. This was evidenced by a lowstandard deviation of the GFP fluorescence (51 ± 6 AFU).Only around 12.4% of the GFP-transfected SH-SY5Y cellsexhibited evidence of GFP-Bax fluorescence clustering(Fig. 2a and c, n = 267 cells). However, 0.1 mmol/L 6-

OHDA induced, in a time-dependent manner, a markedredistribution of GFP-Bax (Fig. 2c and d). By 3 h after 6-OHDA treatment no significant difference were found eitherin GFP-Bax fluorescence standard deviation or in the numberof cells with changes in GFP-Bax distribution. However, by6 h an increase in GFP-Bax fluorescence standard deviationwas noted (69 ± 9 AFU; n = 109; p < 0.05; Fig. 2c); andafter 12 h 45.4% of the GFP-Bax expressing cells presentedwith a clustered GFP-Bax fluorescence (n = 359; p < 0.01).No clustered of GFP fluorescence was noted in 6-OHDA-treated cells that were transfected with a GFP plasmid alone(Fig. 2b). Incubation of 6-OHDA treated cells with themitochondrion-selective dye red Mito-tracker Red demon-strated that the GFP-Bax fluorescence was clustered at ornearby mitochondria (Fig. 2a).

To gain insight in the significance of Bax in 6-OHDA-induced cell death, we exposed mouse embryonic fibroblastsfrom wild-type and Bax knockout animals (Bax)/) MEFs)to 6-OHDA. As Fig. 2e illustrates, the lack of Bax affordedsignificant cytoprotection to MEF cultures exposed to0.1 mmol/L 6-OHDA.

Caspase family members participate in the late phases ofapoptotic cell death. To determine whether caspases partic-ipate in the translocation of GFP-Bax we pre-treated cellswith the broad spectrum caspase inhibitor Z-Val-Ala-DL-Asp-fluoromethylketone (zVAD-fmk). As depicted inFig. 2f, GFP-Bax redistribution was not inhibited in SH-SY5Y cells treated with 100 lmol/L zVAD-fmk for 12 h andexposed to 0.1 mmol/L 6-OHDA for a further 12 h.

In order to corroborate the results of the SH-SY5Y cells inprimary neuronal cultures we treated primary corticalneurons with 6-OHDA and monitored the formation ofMPTP using calcein fluorescence. As demonstrated inFig. 3a, 6-OHDA failed to induce mitochondrial swelling.Despite the absence of MPTP formation, activation ofendogenous Bax as assessed by immunostaining with anantibody which specifically recognizes the confromationallyactive Bax (6A7) was clearly evident in 6-OHDA treatedcultures (Fig. 3b). Interestingly, those neurons with activeBax clusters displayed alterations in their nuclear chromatin.

Several studies suggest that p53 expression may correlatewith neuronal death in neurodegenerative diseases (Steffanet al. 2000; Gomez-Lazaro et al. 2004) including PD (Duanet al. 2002; Mandir et al. 2002). To elucidate the participa-tion of p53 in 6-OHDA-induced cell death we tested whethertotal p53 protein levels changed in response to 6-OHDAtreatment in SH-SY5Y cells. As shown in Fig. 4a, exposingcultures to 0.1 mmol/L 6-OHDA had a marked effect on p53protein levels and significantly elevated levels were alreadyapparent by 2 h of 6-OHDA treatment. We also assayed thesubcellular localization of p53 in SH-SY5Y cells treated with6-OHDA for 3 h. In parallel with the enhanced p53 proteinlevel increase, 6-OHDA enhanced the nuclear accumulationof p53 (Fig. 4a, lower panel).



To demonstrate that the p53 apoptosis pathway is func-tional in SH-SY5Y cells, we tested whether p53 expressionresulted in an increase of cell death in SH-SY5Y cellcultures. Cellular cultures were co-transfected with a plasmidencoding GFP and either wild-type p53 (wtp53) or aninactivating mutation (R273 fi H) in the p53 core domain(mp53) one of the most common mutations found in humancancers. As shown in Fig. 4b, by 24 h after transfection theover-expression of wtp53, but not mp53, resulted in asignificant increase of apoptotic nuclei when we analyzed thestate of chromatin condensation of GFP-positive cells byusing Hoechst 33342.

The next set of experiments was addressed to determinewhether p53 over-expression is sufficient to induce cellularGFP-Bax redistribution. GFP-Bax plasmid was co-transfect-

ed either with wtp53 or mp53 plasmids in SH-SY5Y cellsand by 24 h the cell were fixed. As shown in Fig. 4c,confocal microscopy studies revealed that in SH-SY5Y cellsco-transfected with wtp53 the percentage of cells showing apunctate/clustered GFP-Bax fluorescence was significantincreased, being evident in 38 ± 4% of the transfected cells(p < 0.01; n = 168 cells; standard deviation of the GFP-Baxfluorescence: 62 ± 6 AFU). However, mp53 expression didnot induce a significant GFP-Bax redistribution, with only14% of the GFP-Bax transfected SH-SY5Y cells exhibitingGFP-Bax fluorescence clusters (NS; n = 267 cells; standarddeviation of the GFP-Bax fluorescence: 42 ± 7 AFU).

To gain insight into the relevance of p53 in 6-OHDA-induced cell death, we used mouse embryonic fibroblast fromp53 knockout animals (p53)/) MEF). As Fig. 4e illustrates,

Control 3 6 1240

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l)

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Control

6-OHDA

GFP(a) (b)

(c) (d) (e) (f)

Fig. 2 6-OHDA-induced apoptosis requires Bax. (a and b) SH-SY5Y

cells were transfected with GFP-Bax, incubated for 24 h to allow for

sufficient GFP-Bax expression and treated with 0.1 mmol/L 6-OHDA.

After 12 h exposure the cells were fixed in 4% p-formaldehyde and

confocal images were captured using a 63· oil immersion lens. GFP-

Bax demonstrated primarily diffuse staining in control (a), while by

12 h after 6-OHDA treatments a punctate pattern is evident. The

images shown are representative of results obtained in four separate

experiments, each performed in triplicate. (b) No clustered of GFP

fluorescence was noted in 6-OHDA-treated cells that were transfected

with a GFP plasmid alone. (c) The translocation of Bax was assessed

by measuring the standard deviation of the GFP-Bax fluorescence

following treatment with 0.1 mmol/L 6-OHDA at various time periods.

(d) The number of cells with punctuate Bax distribution were counted

and expressed as a percentage of total number of cells. (e) MEFs from

Bax+/+ and Bax )/) cells were treated with 0.1 mmol/L 6-OHDA for

12 h and cell viability assessed by Hoechst 33342 staining. (f) Bax

translocation is caspase independent. Cell were pre-treating with

100 lmol/L zVAD-fmk for 12 h prior addition of 0.1 mmol/L 6-OHDA;

12 h after 6-OHDA treatment Bax pattern was assayed. Results are

presented as mean ± SD; they are representative of at least three

experiments, each performed in triplicate. Data are mean ± SEM

values. Different from respective controls: *p < 0.05, **p < 0.01 (one-

way ANOVA post hoc Tukey).



the lack of p53 did not afford cytoprotection to MEF cultureswhen compared with wild-type MEFs. Furthermore, 6-OHDAwas able to induce significant GFP-Bax redistributionin p53)/) MEF (Fig. 4d). Treatment of MEFs with0.1 mmol/L 6-OHDA also caused a time-dependent caspase3 activation in both wild-type and p53)/) MEFs (Fig. 4e).Taken together our data revealed that although 6-OHDAinduced p53 protein levels, p53 was not required for theneurotoxin to induce Bax redistribution and cell death.

Finally, we used the p53 inhibitor Pifithrin-a to determinethe role of p53 in 6-OHDA-induced apoptosis of humanSH-SY5Y cells. A pre-treatment for 3 h with 100 nmol/LPifithrin-a failed to protect the cells against 6-OHDA-induced toxicity (Fig. 4e).

p38 MAPK has been shown to contribute to several celldeath modes by mediating Bax redistribution (Gomez-Lazaroet al. 2007). To demonstrate the participation of p38 MAPKin 6-OHDA-induced apoptosis, SH-SY5Y cell cultures werechallenged with 0.1 mmol/L 6-OHDA and the levels ofactive, phopho-p38 MAPK were determined at different timepoints. Protein extracts from SH-SY5Y cell cultures chal-lenged with 0.1 mmol/L 6-OHDA presented with a marked

increase in activation-specific phosphorylation at Thr180 andThr182 of p38 MAPK. Maximal increases in p38MAPKphosphorylation occurred after 1 h, after which phosphory-lation levels slowly declined towards basal levels.

Next, we used the p38 MAPK inhibitor SKF86002 toaddress the significance of p38 MAPK activation in 6-OHDA-induced Bax redistribution and cell death. Cells pre-treated with SKF86002 (10 lmol/L) 12 h before 6-OHDAaddition showed strongly reduced levels of active phospho-p38 MAPK (Fig. 5a). Administration of SKF86002 to GFP-Bax transfected SH-SY5Y cells revealed that p38 MAPKinhibition resulted in a marked decrease in GFP-Baxredistribution (Fig. 5b). Inhibition of p38 MAPK alsoprevented 6-OHDA induced cell death as judged by thereduced number of nuclei with the characteristic apoptoticmorphology, compared with control cells (Fig. 5c).

To further ascertain whether an activation of p38 MAPK issufficient to activate Bax, we transfected SH-SY5Y cellswith its upstream activator MKK6. Activation of p38 MAPKpathway through MKK6-wt expression induced a markedGFP-Bax distribution, while the MKK6-kd mutant did notmodify GFP-Bax distribution (Fig. 5d).

Control

6-OHDA

Hoechst 33342 BAX 6A7-FITC(b)

(a)

0 min 35 min 0 5 10 15 20 25 30 35 400.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

Cal

cein

flu

ore

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ce

Time (min)

Control 6-OHDA

Fig. 3 6-OHDA effects on primary neuron cultures. (a) 6-OHDA failed

to decrease mitochondrial calcein fluorescence intensities. Neurons

were co-loaded with calcein-AM and CoCl2 as described under

Materials and Methods and images were collected at 5 min intervals

(0 and 35 min are shown). Histogram shows normalized calcein fluo-

rescence intensities, and values on the ordinate report the mean ± SD

of three independent experiments. (b) Cortical neurons were treated

with 0.1 mmol/L 6-OHDA or vehicle for 12 h. following treatment the

neurons were fixed and immunostained with an anti-Bax 6A7 antibody

which recognizes the activate form of Bax. Neurons were counter-

stained with Hoechst 33342 to visualize the nuclear structure. Similar

results were found in at least three different experiments.



We were next interested to directly compare the effects ofp38 MAPK activation and Bax deficiency on 6-OHDA-induced caspase activation in MEF cells. As shown inFig. 5e, 0.1 mmol/L 6-OHDA induced a time-dependentDEVDase (caspase) activity in the MEF cells. In line with theabove described findings, 6-OHDA failed to induce anycaspase activity in MEF that were pre-treated withSKF86002 (10 lmol/L, 12 h) or that lacked Bax (MEFBax)/)), while p53-deficient MEFs were not protected(MEF p53)/)).

The activation of BH3-only proteins is believed to beresponsible for the activation of Bax and subsequent

apoptosis (Ward et al. 2004). We next determined whether6-OHDA might increase mRNA levels of two BH3-onlyproteins which have previously been implicated in stress-induced neuronal apoptosis, BIM and PUMA (Wong et al.2005). To this end, we used quantitative real-time PCR andwestern blot analysis to determine both mRNA and proteinlevels in SH-SY5Y cells in response to 0.1 mmol/L6-OHDA. The results, shown in Fig. 6a, b, and c, demon-strated a potent induction of the BH3-only protein PUMA inresponse to 6-OHDA at both the mRNA and protein level. Incontrast neither the BH3-only protein BIM, nor Bax or Bakdirectly were induced in response to 6-OHDA.

mp53 p5305

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Vehicle 6-OHDA

6-OHDA

– –+ + 6-OHDACytosolic Nuclear

p53

p53

C 3 6 12 24 h

α-tubulin

α-tubulin

p53

H3

(a) (b)

(c)

(e)

(d)

Fig. 4 p53 is activated but not required for 6-OHDA-induced GFP-Bax

redistribution. (a) Time-dependent induction of p53 protein levels in

SH-SY5Y cell exposed to 0.1 mmol/L 6-OHDA. Whole-cell extracts

(upper panel) and nuclear extracts (3 h, lower panel) from SH-SY5Y

treated with or without 0.1 mmol/L 6-OHDA were subjected to western

blotting and probed with anti-p53 antibody. a-Tubulin and Histone 3

(H3) were used to show equal loading in cytoplasmic and nuclear

fractions. (b) p53 over-expression induces cell death in SH-SY5Y cell

cultures. SH-SY5Y cells cultures were co-transfected with expression

plasmids encoding wild-type p53 (p53) or its mutated form (mp53)

together with GFP; 24 h after transfection cells were fixed and stained

with Hoechst 33342. GFP was used as a marker to indicate trans-

fected cells that were subsequently scored for viability by chromatin

morphology. (c) SH-SY5Y cell cultures were co-transfected with GFP-

Bax and either plasmid coding for a wild-type p53 or its mutated form

used as a negative control, and 24 h after cell cultures were fixed in

4% p-formaldehyde and GFP-Bax distribution patterns were analyzed.

(d and e) MEF p53+/+ and p53)/) cells were transfected with GFP-

Bax and were incubated for 24 h and treated with 0.1 mmol/L

6-OHDA. Following 12 h 6-OHDA treatment cell cultures were fixed in

4% p-formaldehyde. GFP-Bax distribution patterns (d) or the per-

centage of apoptotic nuclei (e) were analyzed and also assayed the

effect of Pifithrin alpha (PFT-a). Results are presented as mean ± SD

and are representative of at least three experiments, each performed

in triplicate. **p < 0.01 and ***p < 0.001 (one-way ANOVA post hoc

Tukey).



We then examined whether treatment with the p38MAPK inhibitor SKF86002 could reduce PUMA levels inresponse to 6-OHDA. Treatment of SH-SY5Y cells withSKF86002 (10 lmol/L, 12 h) potently inhibited the6-OHDA induced mRNA increase (Fig. 6d). Treatmentwith SKF86002 likewise reduced PUMA protein levels in6-OHDA-treated SH-SY5Y cells (Fig. 6e). Finally, wedetermined whether the induction of PUMA was involvedin 6-OHDA-induced cell death. Indeed, MEFs derived fromPUMA-deficient mice exhibited a significant reduction inapoptosis compared with wild-type MEFs in response to6-OHDA (Fig. 6f).

Discussion

The present data provide insights into the cellular pathwaysinvolved in 6-OHDA induced cytotoxicity, a processbelieved to be involved in neurodegeneration in PD andtoxin used as a model for this disorder. Our data demonstratea lack of involvement of the MPTP in 6-OHDA-induced celldeath and define a role for the p38 MAPK, PUMA and, Baxpathway in mediating apoptosis triggered by 6-OHDA.

Our study provides evidence for the activation of themitochondrial apoptosis pathway in response to 6-OHDA,with activation of caspase 3 and the release of cytochrome c

01020304050607080

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Control0

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Control SKF86002

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MKK6-kdMKK6-wtMock

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210186420

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Time (h)

twFEM–/––/–

35pFEMxaBFEM

20068FKS

p38

p-p38

p38

SKFVC

6-OHDA

6-OHDA

Control 6-OHDA

Fig. 5 p38 MAPK is activated and required for 6-OHDA-induced GFP-

Bax redistribution. (a) Upper panel: Immunoblot analysis of phospho-

p38 MAKP protein levels in cell extract from control and 0.1 mmol/L

6-OHDA-challenged SH-SY5Y cells at the indicated time points. Lower

panel: SKF86002 blocked 6-OHDA-induced increase in phospho-p38.

SKF82002 was added 12 h before 6-OHDA addition and maintained

until the end of experiments, cells from control (C), vehicle (V), and

SKF82006 (SKF) were collected 1 h after 6-OHDA addition. Similar

results were achieved in at least three independent experiments. (b)

SH-SY5Y cells were transfected with GFP-Bax and were incubated for

24 h to allow for sufficient GFP-Bax expression and treated with

0.1 mmol/L 6-OHDA. Cells were pre-treated with SKF86002 (10 lmol/

L) for 12 h prior to the addition of 6-OHDA for an additional 12 h.

Following treatments cells were fixed in 4% p-formaldehyde and GFP-

Bax distribution patterns analyzed. (c) Cells were treated as described

above and cell viability assessed by Hoechst 33342 staining. (d) SH-

SY5Y cell cultures were co-transfected with GFP-Bax and a plasmid

coding either for a wild-type Flag-tag MKK6 (MKK6-wt) or an inactive

kinase version (MKK6-kd) used as a negative control, and GFP-Bax

distribution analyzed after 24 h. (e) Caspase activity was determined

by measuring DEVD-AFC hydrolysis. Enzymatic determinations were

performed in lysates from MEF wt, p53)/), and Bax)/) cell cultures

challenged with 0.1 mmol/L 6-OHDA for 3, 6, and 12 h. For p38

inhibition MEF wt cell were treated with 10 lmol/L SKF86002 12 h

before 6-OHDA insult. Results are presented as mean ± SD; they are

representative of at least three experiments, each performed in tripli-

cate. *p < 0.05 and ***p < 0.001 versus control conditions (one-way

ANOVA post hoc Tukey).



clearly evident in SH-SY5Y cells challenged with 6-OHDA.This event occurs indirectly via the redistribution of Baxrather than via MPTP formation or direct mitochondrialdamage. This assertion comes from several lines of exper-imental evidence. First we did not find a decrease in thelevels of mitochondrial calcein fluorescence when SH-SY5Ycells were challenged with 0.1 mmol/L 6-OHDA. Second,the 6-OHDA-induced mitochondrial cytochrome c releasewas not inhibited in cell cultures pre-treated for 3 h with theMPTP inhibitor cyclosporine A. Furthermore, a brainisolated mitochondria suspension did not readily undergoswelling upon exposure to 0.1 mmol/L 6-OHDA. Theseresults strongly suggest that 6-OHDA-induced cytochrome crelease takes place throughout a MPTP formation indepen-dent process. Furthermore, SH-SY5Y cells challenged with0.1 mmol/L 6-OHDA for 12 h did not present with mor-phological changes indicative of MPTP formation asevidenced by electron microscopy: abundant mitochondriawith spherical profiles along with normal elongated mito-chondria were observed in untreated cultures as well as incultures treated with 0.1 mmol/L 6-OHDA, all of them

possessing two clear mitochondrial membranes and thincristae (authors’ unpublished data). These results are in linewith our previous report that 6-OHDA failed to inducemitochondrial swelling in mitochondria isolated from thebovine adrenal medulla (Galindo et al. 2003b), and inagreement with a previous study demonstrating that theParkinsonian toxin MPP+ but not 6-OHDA induced MPTPformation in PC12 cells (Lee et al. 2006).

Confocal microscopy demonstrated a significant transloca-tion of GFP-Bax to mitochondria during 6-OHDA inducedcell death. Bax is a multidomain pro-apoptotic member ofthe Bcl-2 family of proteins which along with another Bcl-2protein, Bak, mediates the process of MOMP and the releaseof caspase activating molecules such as cytochrome c.Importantly, Bax deficient cells were resistant to 6-OHDAtreatment suggesting an absolute requirement for Bax expres-sion with Bak expression unable to mediate MOMP alone.

Interestingly, our study points to a central role for p38MAPK activation in mediating 6-OHDA toxicity. Inhibitionof p38 MAPK was sufficient to inhibit mitochondrialclustering of Bax and the activation of cell death. Moreover,

MEF PUMA–/– MEF wt 0

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MA

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h 12 6 3 0

Fig. 6 6-OHDA induces up-regulation of PUMA expression. (a) Effect

of 0.1 mmol/L 6-OHDA on mRNA levels of BH3-only proteins. SH-SY5Y

cell cultures were challenged with 6-OHDA for 12 h and the levels of

NOXA, BIM, and PUMA mRNA were assessed by real-time quantitative

PCR. Results are expressed relative to expression levels of control

cultures and are presented as mean ± SD; they are representative of

two experiments, each performed in triplicate. (b) SH-SY5Y cells were

challenged with 0.1 mmol/L 6-OHDA for varying time periods (0–12 h).

Expression levels of Bim, PUMA, Bax, and Bak were analyzed by

western blotting. Similar results were found in three separate experi-

ments. (c) SH-SY5Y cells were treated with 0.1 mmol/L 6-OHDA for 0–

12 h. Bim, PUMA, Bax, and Bak mRNA levels were determined by real-

time qPCR. Results are shown as mean ± SD from sat least two dif-

ferent experiments. (d) p38 MAPK inhibition abrogated 6-OHDA-in-

duced PUMA mRNA expression; 12 h before insults cells were treated

with SKF86002 (10 lmol/L) and cells subsequently treated with

0.1 mmol/L 6-OHDA for 12 h. PUMA mRNA levels were determined by

real-time qPCR. Results are expressed as fold increase and represent

the mean ± SD of at least two different experiments using two different

samples of RNA. (e) Cells were treated as described in (d). PUMA

protein expression was examined using western blotting. (f) PUMA

protein is required for 6-OHDA induced cell death. MEF wt and

PUMA)/) cells were treated with 0.1 mmol/L 6-OHDA for 12 h. Cell

viability was assessed using Hoechst 33342. *p < 0.05, **p < 0.01.



we demonstrate that transcriptional activation of the BH3-only protein PUMA also had an important role in inducingcell death. BH3 proteins couple cellular stress to theactivation of Bax by directly activating it or by antagonizingthe function of anti-apoptotic members of the Bcl-2 family.Previously, p38 MAPK has been implicated in the regulationof the BH3-only proteins, Bim and Bmf (Ramjaun et al.2007). However, we could find no evidence for transcrip-tional up-regulation of Bim in our model, although inhibitionof p38 MAPK did significantly inhibit the transcriptionalincrease in PUMA expression. These results suggest that p38MAPK is critically involved in the induction of various BH3-only proteins in response to cellular stress. However, thesefindings also suggest that the induction of specific BH3-onlyproteins by p38 MAPK is subject to secondary controlprocesses, such as stress- and tissue-specific expression andactivation of co-transcription factors/co-activators.

PUMA is thought to mediate the majority of the pro-apoptotic effects of p53 activity. Although originally iden-tified as a p53-dependent target, in response to certain stimuliincluding growth factor withdrawal (Villunger et al. 2003)and tunicamycin-induced endoplasmic reticulum stress(Reimertz et al. 2003) its expression is regulated in a p53-independent manner. However, PUMA induction in ourmodel appeared to occur in a p53-independent manner andloss of p53 expression did not affect responses to 6-OHDAtreatment. The lack of an effect in the p53 deficient cellssuggests that the activation of p53 plays a redundant role in6-OHDA-mediated toxicity. Interestingly, the increasedexpression of PUMA was found to be predominantly p38kinase dependent, although a role for p53 in regulating theresidual expression of PUMA cannot be excluded. Impor-tantly, loss of PUMA expression only partially protectedfrom 6-OHDA, while Bax deficient cells were completelyprotected, suggesting the potential involvement of otherBH3-only proteins which may compensate for the loss ofPUMA expression. One such BH3 protein is Noxa which hasbeen demonstrated to functionally compensate for loss ofPUMA expression (Villunger et al. 2003), although at leastin human cells Noxa is not a potent inducer of apoptosis(Kim et al. 2006; Shibue et al. 2006). Alternatively, p38MAPK may activate BH3-only proteins post-translationallythrough phosphorylation (Cai et al. 2006). It has also beenreported that p38 MAPK may directly activate Bax throughphosphorylation (Kim et al. 2006). The absolute requirementfor Bax expression suggests that mitochondria-independentpathways of cell death such as autophagy or lysosomalmediated cell death do not appear to play a predominant rolein 6-OHDA-mediated toxicity.

In summary, this study identifies new elements in thesignaling cascade that leads to mitochondrial membranepermeabilization in 6-OHDA-challeneged SH-SY5Y cellsand suggests that p38 MAPK and PUMA might signal toBax to engage the mitochondrial pathway of apoptosis.

Acknowledgements

We are grateful to Remedios Sanchis for technical assistance and to

Eva M. Garcıa-Martınez and RM Melero-Fernandez de Mera for

invaluable discussions. We thank Drs Andreas Strasser (WEHI,

Australia) and A. Villunger (Innsbruck, Austria) for PUMA deficient

mice. This work was funded by SAF2002-04721 and SAF2005-

07919-C02-01 from CICYT and 04005-00 Consejerıa de Sanidad

from Junta de Comunidades de Castilla La Mancha to JJ; Ministerio

de Sanidad y Consumo (contract number PI020051 and Redes

Tematicas de Investigacion Cooperativa), Fundacio La Caixa (02/

055-00) and Generalitat de Catalunya (Suport als Grups de Recerca

Consolidats) to JXC and by a grant from Science Foundation Ireland

(03/RP1/B344) to JHMP. MG-L and FJF-G are fellows from JCCM.

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