Free Radical Biology and Medicine - Yonsei Universityweb.yonsei.ac.kr/neurolab/published/72.pdf · a Department of Systems Biology, Yonsei University College of Life Science and Biotechnology,

Free Radical Biology and Medicine 53 (2012) 936–950

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Free Radical Biology and Medicine

0891-58

http://d

Abbre

6-OHDA

5-(and-

PML, pr

signal;

HDAC, h

remoden Corr

E-m

journal homepage: www.elsevier.com/locate/freeradbiomed

Original Contribution

Nuclear translocation of DJ-1 during oxidative stress-induced neuronalcell death

Su-Jeong Kim a, Yun-Jong Park a, Ih-Yeon Hwang a, Moussa B.H. Youdim a, Kang-Sik Park b, Young J. Oh a,n

a Department of Systems Biology, Yonsei University College of Life Science and Biotechnology, Seoul 120-749, Koreab Department of Physiology, Kyung Hee University School of Medicine, Seoul 120-178, Korea

a r t i c l e i n f o

Article history:

Received 27 January 2012

Received in revised form

22 May 2012

Accepted 24 May 2012Available online 7 June 2012

Keywords:

PARK7/DJ-1

6-Hydroxydopamine

Reactive oxygen species

Dopaminergic neurodegeneration

Parkinson disease

Free radicals

49/$ - see front matter & 2012 Elsevier Inc. A

x.doi.org/10.1016/j.freeradbiomed.2012.05.03

viations: PD, Parkinson disease; DAergic, do

, 6-hydroxydopamine; ROS, reactive oxygen

6)-chloromethyl-20 ,70-dichlorodihydrofluores

omyelocytic leukemia; NAC, N-acetyl-L-cyste

STS, staurosporine; WT, wild type; TSA, trich

istone deacetylase; CBP, Creb binding protei

ling and deacetylase

esponding author. Fax: þ82 2 312 5657.

ail address: [email protected] (Y.J. Oh).

a b s t r a c t

Loss-of-function mutations in the PARK7/DJ-1 gene cause early onset autosomal-recessive Parkinson

disease. DJ-1 has been implicated in protection of neurons from oxidative stress and in regulation of

transcriptional activity. However, whether there is a relationship between the subcellular localization

of DJ-1 and its function remains unknown. Therefore, we examined the subcellular localization of DJ-1

during dopaminergic neurodegeneration induced by various insults. Immunoblotting and immunocy-

tochemistry showed that the nuclear pool of DJ-1 dramatically increased in both MN9D dopaminergic

neuronal cells and primary cultures of mesencephalic dopaminergic neurons after 6-hydroxydopamine

(6-OHDA) treatment. This was paralleled by a corresponding decrease in its cytosolic level, indicating

drug-induced nuclear translocation of DJ-1. The same phenomenon was detected in other cell death

paradigms induced by pro-oxidants including hydrogen peroxide and cupric chloride. Consequently,

cotreatment with the antioxidant N-acetyl-L-cysteine blocked the translocation of DJ-1 into the nucleus.

However, mutation at cysteine 106 had no effect on the translocation of DJ-1 into the nucleus,

suggesting that reactive oxygen species-mediated downstream signaling and/or modifications other

than oxidative modification are involved in its nuclear translocation. Ectopic expression of nucleus

localization signal (NLS)-tagged DJ-1 prevented cell death from 6-OHDA. We investigated whether

nuclear DJ-1 was involved in transcriptional regulation and found that DJ-1 was localized in

promyelocytic leukemia bodies, and this localization increased upon 6-OHDA treatment. We also

confirmed that binding of DJ-1 and promyelocytic leukemia bodies indeed increased after 6-OHDA

treatment. Consequently, expression levels of acetylated p53 and PUMA were downregulated in cells

overexpressing DJ-1 or NLS-tagged DJ-1. Taken together, our data suggest that nuclear translocation of

DJ-1 may protect neurons from cell death after oxidative stress.

& 2012 Elsevier Inc. All rights reserved.

Introduction

Parkinson disease (PD)1 is the second most common neuro-degenerative disorder of the central nervous system and ischaracterized by both motor and nonmotor symptoms [1,2].The motor symptoms of PD typically include resting tremors,rigidity, and bradykinesia, which result from a loss of dopami-nergic (DAergic) neurons in the substantia nigra pars compactaand a concomitant deprivation of dopamine (DA) in the striatal

ll rights reserved.

5

paminergic; DA, dopamine;

species; CM-H2DCFDA,

cein diacetate acetyl ester;

ine; NLS, nuclear localization

ostatin A; NA, nicotinamide;

n; NuRD, nucleosome

region [3]. Although most cases of PD are idiopathic, at least 5% ofPD cases are caused by mutations in specific genes. These genescode for a-synuclein, parkin, leucine-rich repeat kinase 2, PTEN-induced putative kinase 1, and DJ-1 [4]. The exact cause of DAneurodegeneration is unknown; however, several potential med-iators have been proposed based on studies using in vitro andin vivo experimental models of PD [3]. These include oxidativestress, mitochondrial dysfunction, failure in proteasomal and lyso-somal systems, endoplasmic reticulum stress, and inflammation.For example, studies using neurotoxins such as 6-hydroxydopa-mine (6-OHDA) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridinehave identified multiple molecular targets and signaling pathwaysthat impair one or several of these mechanisms. Recent studiesusing genetic models of PD indicate that gene mutations are alsoassociated with mitochondrial dysfunction, increases in reactiveoxygen species (ROS), and facilitation of protein aggregation [5,6],suggesting that PD is caused by an interaction of multiple geneticpredisposition and environmental factors [7].

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dx.doi.org/10.1016/j.freeradbiomed.2012.05.035



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S.-J. Kim et al. / Free Radical Biology and Medicine 53 (2012) 936–950 937

DJ-1, also known as PARK7, was initially mapped as the thirdlocus for the early onset autosomal-recessive inherited form ofparkinsonism [8]. Deletion or missense mutations of DJ-1 leading toloss of function were identified in Italian and other Europeanfamilies [9]. DJ-1 is a ubiquitous redox-responsive protein withdiverse functions such as transcriptional coregulator [10,11], mole-cular chaperone [12], and RNA-binding protein [13]. Biochemically,DJ-1 is easily oxidized in response to several oxidative stimuli, andthe oxidized, acidic isoforms of DJ-1 accumulate in the brains ofpatients with idiopathic PD and Alzheimer disease [14]. DJ-1 isproposed to be an atypical peroxiredoxin-like peroxidase thatreduces hydrogen peroxide through oxidation at C106 [15].However, the kinetics of the reaction is much slower than that ofhuman peroxiredoxin, suggesting that DJ-1 exerts its antioxidativeeffects mainly by regulating antioxidant gene expression ratherthan by simply scavenging hydrogen peroxide. Indeed, it has beenshown that DJ-1 seems to exert its antioxidative effects by directlyregulating gene expression. For example, previous studies showedthat during oxidative stress, DJ-1 may modulate the expression ofgenes such as glutamate–cysteine ligase, the rate-limiting enzymeof glutathione biosynthesis; extracellular superoxide dismutase(SOD3) [16,17]; or manganese superoxide dismutase (MnSOD)synergistic with peroxisome proliferator-activated receptor g coac-tivator 1a [18]. DJ-1 can also modulate expression of several othergenes including androgen receptor reporter activity by interactingwith PIASx, which is known as androgen receptor-interactingprotein-3 [19,20], as well as tyrosine hydroxylase [21] and Bax [22].

DJ-1 is mainly localized in the cytosol and is also found inmitochondria and the nucleus [23,24]. Nevertheless, the relation-ship between the biochemical function and the subcellularlocalization of DJ-1 has not been clearly determined. Therefore,we investigated the potential role of nuclear DJ-1. We hypothe-sized that DJ-1 needs to be translocated into the nucleus toperform its function as a transcriptional coregulator. Second,based on previous studies indicating that DJ-1 regulates expres-sion of several genes during oxidative stress, we also hypothe-sized that ROS may regulate the nuclear translocation of DJ-1.Previously, our laboratory showed that ROS-mediated apoptoticsignaling is primarily responsible for 6-OHDA-induced neurode-generation in primary cultures of mesencephalic and corticalneurons, MN9D DAergic neuronal cells, and rat brain models ofPD [25–28]. In these 6-OHDA-induced cell death paradigms, ROSsuch as superoxide anion, hydrogen peroxide, and nitric oxide areamong several upstream triggers that are central to dopaminergicneurodegeneration. Therefore, we specifically asked whether6-OHDA triggers the nuclear translocation of DJ-1 in a ROS-dependent manner using MN9D cells and primary cultures ofmesencephalic neurons. Similarly, we examined a potential cor-relation between its nuclear localization and transcriptionalregulatory activity. Here, we provide evidence that DJ-1 istranslocated from the cytosol to the nucleus during oxidativestress-induced cell death. We also show that nuclear DJ-1 may beassociated with inhibition of apoptosis by decreasing p53 acet-ylation and PUMA expression.

Materials and methods

Cell culture and treatment

MN9D cells, a somatic fusion product between murine embryonicmesencephalic neurons and neuroblastoma N18TG [29], were cul-tured as previously defined in our laboratory [30]. Briefly, cells wereplated on 25 mg/ml poly-D-lysine (Sigma, St. Louis, MO, USA)-coatedculture plates or dishes and cultivated in Dulbecco’s modified Eagle’smedium (DMEM; Sigma) supplemented with 10% heat-inactivated

fetal bovine serum (Gibco, Grand Island, NY, USA) in an atmosphereof 10% CO2 for 2–3 day. For experiments, cells were switched toserum-free N2 supplement containing 100 mM 6-OHDA (Sigma),1 mM staurosporine (STS; Sigma), 1.5 mM hydrogen peroxide(Calbiochem, Darmstadt, Germany), or 150 mM cupric chloride(Sigma) in the presence or absence of 1 mM N-acetyl-L-cysteine(NAC; Sigma) for the time periods indicated. Primary cultures ofmesencephalic neurons were established using the ventral mesen-cephalon, which was removed from Sprague–Dawley rat embryos atgestation day 14.5 (Orient, Seongnam-si, Gyeonggi-do, Korea) aspreviously described by us [25]. Briefly, dissociated cells were seededon 100 mg/ml poly-D-lysine and 4 mg/ml laminin double-coated Aclarembedding film (Electron Microscopy Sciences, Hatfield, PA, USA).Primary cultures of mesencephalic neurons were maintained inminimal essential medium (MEM; Gibco) supplemented with 10%heat-inactivated fetal bovine serum, 6 mg/ml glucose (Sigma), and2 mM glutamine (Sigma). After 5 day of culture, cells were washedwith MEM and treated with 20 mM 6-OHDA in the presence orabsence of 500 mM NAC (Sigma) for 12 h. PC12 cells were culturedwith Ham’s F-12 medium (Lonza, Basel, Switzerland) supplementedwith 15% heat-inactivated horse serum (Gibco) and 2.5% heat-inactivated fetal bovine serum (Gibco) in an atmosphere of 5% CO2.After 2 day of culture, PC12 cells were cultivated in a mediumcontaining 50 ng/ml nerve growth factor (NGF; Invitrogen, Carlsbad,CA, USA) for at least 5 day. Cells were then treated with 100 mM6-OHDA in Ham’s F-12 medium for the indicated time periods.

Immunofluorescence microscopy

For immunocytochemistry, cells plated on Aclar embeddingfilms were fixed with 4% paraformaldehyde for 20 min at roomtemperature. After fixation, the cells were blocked in phosphate-buffered saline (PBS; Lonza) containing 0.2% Triton X-100 and5% normal goat serum for 1 h at room temperature. Cells werethen incubated with rabbit polyclonal anti-DJ-1 (Novus Biologi-cals, Littleton, CO, USA; 1:300), mouse monoclonal anti-c-Myc(Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:200), humananti-C106-oxidized DJ-1 (a generous gift from Dr. HiroyoshiAriga; 1:100), or mouse monoclonal anti-promyelocytic leukemia(PML; MBL International, Woburn, MA, USA; 1:200) in PBScontaining 0.2% Triton X-100 and 1% normal goat serum at 4 1Covernight. For double immunocytochemical localization of tyro-sine hydroxylase (TH; a rate-limiting enzyme of dopamine bio-synthesis and a marker of DAergic neurons) and DJ-1, primarycultures of mesencephalic neurons were incubated with mousemonoclonal anti-TH (Pel-Freez, Rogers, AR, USA; 1:200) and rabbitpolyclonal anti-DJ-1 in blocking solution at 4 1C overnight. Afterthree washes with PBS, the cells were further incubated at roomtemperature for 1 h with appropriate fluorescence-conjugatedsecondary antibodies in PBS containing 0.2% Triton X-100 and 1%normal goat serum. These included Alexa Fluor 488-conjugatedgoat anti-rabbit IgG (1:200), Alexa Fluor 568-conjugated goatanti-mouse IgG (1:200), Alexa Fluor 488-conjugated goat anti-mouse IgG (1:200), Alexa 488-conjugated goat anti-human IgG(1:200; all from Invitrogen), or appropriate combinations. Ifnecessary, nuclei were stained for 5 min at room temperature inPBS containing Hoechst 33258 (2 mg/ml; Invitrogen). Aclar filmswere mounted with Vectashield mounting medium (VectorLaboratories, Burlingame, CA, USA). Fluorescence was observedunder an LSM 510 META confocal laser scanning microscopeequipped with epifluorescence and an LSM digital image analyzer(Carl Zeiss, Jena, Germany). For quantitative analysis, Metamorph(Molecular Devices, Downington, PA, USA) was used for measur-ing fluorescence intensity of DJ-1 localized in the nuclei andcolocalized with PML.

S.-J. Kim et al. / Free Radical Biology and Medicine 53 (2012) 936–950938

Cellular fractionation and immunoblot analysis

After drug treatment for the indicated time periods, cytosolicand nuclear fractions of MN9D cells were obtained as previouslydescribed with some modifications [31]. Briefly, MN9D cell pelletsharvested by centrifugation at 100g were washed in ice-cold PBSonce and resuspended in PB buffer (250 mM sucrose, 10 mMMgCl2, 2 mM potassium phosphate, 240 mM EGTA, pH 8.0, 20 mMHepes, pH 7.2) containing 0.008% w/v digitonin (Sigma) andincubated for 5 min at 4 1C. The resuspended homogenate wascentrifuged at 500g for 5 min at 4 1C. The resulting supernatantswere considered the cytosolic fraction. To remove the remainingmembrane fraction, the resulting pellets were resuspended andhomogenized in PB buffer containing 0.1% Triton X-100 andcentrifuged at 5000g for 10 min at 4 1C. These pellets were thenresuspended in a nuclear lysis buffer (5 M urea, 2 M thiourea, 2%Chaps, 0.25% Tween 20, 100 mM dithiothreitol) and centrifuged at15,000g for 10 min at 4 1C. The resulting supernatants wereconsidered the nuclear fraction. For collecting total cellularlysates, cells were lysed with RIPA buffer (50 mM Tris–HCl, pH7.4, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mMEDTA) containing 0.1% SDS plus complete protease inhibitorcocktail (Roche, Basel, Switzerland). The lysates were homoge-nized using a 1-ml syringe with a 26-gauge needle. The super-natant was collected by centrifugation at 15,000g for 10 min at4 1C. Protein amounts in the cellular lysates were quantified usingthe Bradford protein assay reagent (Bio-Rad, Hercules, CA, USA).Predetermined amounts of proteins (5–20 mg) were separated on12.5% SDS–PAGE gels and blotted onto prewetted polyvinylidenedifluoride membranes (Pall Corp., Port Washington, NY, USA).Membranes were incubated with one of the following antibodiesat 4 1C overnight: goat anti-DJ-1 (Abcam, Cambridge, MA, USA;1:2000), rabbit anti-cleaved caspase-3 (Cell Signaling, Beverly,MA, USA; 1:3000), anti-c-Myc-HRP (Santa Cruz Biotechnology;1:2000), rabbit anti-PUMA (Cell Signaling; 1:1000), or rabbit anti-Bax (Cell Signaling; 1:1000). Goat anti-superoxide dismutase 1(SOD1; Santa Cruz Biotechnology; 1:1000), rabbit anti-nucleolin(Santa Cruz Biotechnology; 1:3000), mouse anti-cytochrome c

oxidase subunit IV (COX IV; Molecular Probes, Eugene, OR, USA;1:2000), and mouse anti-GAPDH antibody (Millipore, Billerica,MA, USA; 1:3000) were used as loading controls for the cytosolicfraction, nuclear fraction, mitochondrial fraction, and total cellu-lar lysates, respectively. After several washes with Tris-bufferedsaline containing 0.1% Tween 20, membranes were incubated atroom temperature for 1 h with goat anti-rabbit horseradishperoxidase (HRP)-conjugated antibody (Santa Cruz; 1:5000), goatanti-mouse HRP-conjugated antibody (Santa Cruz; 1:5000), ordonkey anti-goat HRP-conjugated IgG (Abcam; 1:20,000).Enhanced chemiluminescence (PerkinElmer, Waltham, MA, USA)was used to detect specific bands. If necessary, relative intensitiesof the DJ-1 band in each condition were measured using ImageJ,free software provided by the National Institutes of Health(Bethesda, MD, USA). As previously described by us [28], nuclear(50 mg) fractions and whole-cell lysates (50 mg) obtained fromMN9D cells were processed for two-dimensional electrophoresis(2-DE) using 7-cm Immobiline DryStrip (pH 3–10NL) for iso-electric focusing and subjected to immunoblot analysis using goatanti-DJ-1 antibody.

DNA constructs and transfection

Wild-type (WT) human DJ-1 was generated by polymerasechain reaction (PCR) from SHSY5Y cell lysates with the followingpair of primers: forward, 50–GCGAATTCATGGCTTCCAAAAGAGC-TCTGG-30, and reverse, 50–CGCTCGAGGTCTTTAAGAACAAGTGGAG-CC-30. These primers contained an EcoRI or XhoI site, respectively.

The PCR product was cloned into a pcDNA3.1/V5-His A vector(Invitrogen). Myc-tagged WT DJ-1 was generated by cloning thePCR product for human DJ-1 into the pCI-neo/myc mammalianexpression vector (Promega, Madison, WI, USA). The C106A humanDJ-1 mutant was generated with a site-directed mutagenesis kit(Agilent Technologies, Santa Clara, CA, USA). Myc-tagged nuclearlocalization signal (NLS) WT DJ-1 was generated by cloning the PCRproduct for human DJ-1 into a pEF/myc/nuc vector (Invitrogen). ThecDNA sequence that corresponds to human p53 or PML wasamplified and inserted into the pCI-Neo/Flag mammalian expressionvector (Promega). For transient transfection, MN9D cells werefirst plated at a density of 4.3�104 or 6.5�105 cells on 25 mg/mlpoly-D-lysine-coated four-well culture dishes or P-100 culture plates,respectively. After 36 h, transient transfection was performed withLipofectamine 2000 as recommended by the supplier (Invitrogen).Unless otherwise described, 0.8 mg plasmid DNA was used forfour-well culture dishes and 8 mg plasmid DNA was used for P-100culture plates. For transient transfection of PC12 cells, cells wereplated at a density of 2�105 cells on 25 mg/ml poly-D-lysine-coated35-mm culture plates, cultivated in a medium containing 50 ng/mlNGF for 5 day, and transfected using 2.6 mg plasmid DNA withFuGENE HD as recommended by the supplier (Roche). After transfec-tion, the cells were incubated for 18 h, switched into a completeculture medium, and further incubated for an additional 36 h.

Immunoprecipitation

MN9D cells grown in a P-100 culture plate were transfectedwith a total of 5 mg DNA using Lipofectamine 2000. Plasmids usedfor transfection included pcDNA 3.1/V5-His, pcDNA3.1/V5-His/DJ-1, and pCI-neo/Flag/PML. Twenty-four hours after transfection,the cells were treated with 100 mM 6-OHDA for 12 h. After drugtreatment, the cells were washed in PBS and lysed in 0.5 ml RIPAbuffer containing complete protease inhibitor cocktail and incu-bated on ice for 30 min. Lysates were then collected by centrifu-gation at 15,000g for 15 min at 4 1C. Protein amounts in thesupernatant were quantified using the Bradford protein assayreagent. One milligram of protein from each sample was incu-bated with 10 ml anti-Flag M2 affinity gel (Sigma) in RIPA buffer at4 1C overnight. The conjugates were collected by centrifugation at3000g for 2 min at 4 1C, and the resulting pellets were washed fivetimes with RIPA buffer. Proteins were eluted from the beads byadding 25 ml 1� protein sample buffer, further denatured byboiling, separated on 9 or 12% SDS–PAGE gels, and subjected toimmunoblot analysis using anti-V5-HRP (Invitrogen; 1:5000) andanti-Flag M2-HRP (Sigma). To detect acetylation of p53, MN9Dcells grown in a P-100 culture plate were transfected with a totalof 6 mg DNA using Lipofectamine 2000. Plasmids used fortransfection included pcDNA 3.1/V5-His, pcDNA3.1/V5-His/DJ-1,pCI-neo/myc-His/DJ-1, and pCI-neo/Flag/p53. Twenty-four hoursafter transfection, the cells were treated with 1 mM trichostatin A(TSA; Sigma) and 5 mM nicotinamide (NA; Sigma) for 5 h. Thecells were lysed in 0.5 ml RIPA buffer containing 2 mM TSA,10 mM NA, and complete protease inhibitor cocktail and sub-jected to immunoprecipitation as described above. Briefly, 1 mgprotein from each sample was incubated with 10 ml anti-Flag M2affinity gel (Sigma) in RIPA buffer at 4 1C overnight. After fivewashes with lysis buffer, proteins were eluted from the beads byadding 25 ml 1� protein sample buffer, further denatured byboiling, separated on 9% SDS–PAGE gels, and subjected to immu-noblot analysis using anti-Flag M2-HRP (Sigma) and rabbit anti-acetylated lysine antibody (Cell Signaling; 1:3000) followed bygoat anti-rabbit HRP-conjugated antibody (Santa Cruz Biotech-nology; 1:5000).


Measurement of ROS

MN9D cells were incubated with or without the indicated drugsfor 6 h. The cells were then incubated with DMEM containing 3 mM5-(and-6)-chloromethyl-20,70-dichlorodihydrofluorescein diacetateacetyl ester (CM-H2DCFDA; Invitrogen) for 30 min at 37 1C. Forflow cytometric analysis, cells were harvested using trypsin,washed twice with PBS, and resuspended in PBS. Intracellular DCFfluorescence was measured using FACSCalibur (BD Biosciences,Franklin Lakes, NJ, USA) with excitation at 488 nm and emissionat 530 nm. Approximately 20,000 cells from each sample were

Fig. 1. Nuclear translocation of DJ-1 after 6-OHDA treatment in MN9D dopaminergic ne

(Images a–d) Cells were then processed for immunostaining of DJ-1 (green). Nuclei we

were magnified with additional 3� digital zoom. (Graphs i, j) Fluorescence intensity of D

intensities of DJ-1 (green) and nuclear (blue) signals from the cross-sectional area mark

(15 mg) lysates were subjected to immunoblot analysis of DJ-1. Anti-SOD1 and anti

densitometrically measured and normalized against SOD1 and nucleolin for the cytoso

ANOVA and Tukey’s post hoc test (n¼3). **po0.001. (C) Total cell lysates were immunob

control. Significance was determined with one-way ANOVA and Tukey’s post hoc test (n¼

transfected with DJ-1-GFP were performed after 100 mM 6-OHDA treatment for 12 h. Bo

acquired, and histograms were analyzed with the software programCellQuest (BD Biosciences).

Apoptosis in MN9D cells

MN9D cells transiently transfected with Myc-tagged NLS/WTDJ-1 or control Myc-tagged NLS/GFP vector were treated with100 mM 6-OHDA for the indicated time periods. The cells werestained with mouse monoclonal anti-c-Myc (1:200), incubatedwith Alexa Fluor 488-conjugated goat anti-mouse IgG, and

uronal cells. (A) MN9D cells were treated with or without 100 mM 6-OHDA for 12 h.

re stained with Hoechst 33258 (blue). Scale bar, 10 mm. (Images e–h) Stained cells

J-1 in cells was analyzed with an LSM digital image analyzer. Graphs represent the

ed with a white arrow. (B) After drug treatment, both cytosolic (5 mg) and nuclear

-nucleolin were used as the fraction markers. Relative intensities of DJ-1 were

lic and nuclear fractions, respectively. Significance was determined with one-way

lotted using anti-DJ-1 or anti-cleaved caspase-3. Anti-GAPDH was used as a loading

3). ns, not significant. (D) Live cell image analyses of GFP in MN9D cells transiently

ttom row: enlarged images marked with white dotted boxes. Scale bar, 10 mm.


counterstained with Hoechst 33258 (2 mg/ml). Cells with frag-mented or shrunken nuclei or both were scored as apoptotic cells.Approximately 600 green fluorescent protein-positive cells wereassessed for each condition, and the percentage of apoptotic cellswas subsequently determined.

Statistics

Data are presented as the means7standard deviation. Sig-nificant differences were determined with Student’s t tests orone-way ANOVA and Tukey’s post hoc test using GraphPadPrism 5. Values of *po0.05 or **po0.001 were considered statis-tically significant.

Fig. 2. Effect of staurosporine on the localization of DJ-1 in MN9D cells. (A) After

treatment with or without 1 mM STS for 12 h, immunocytochemistry for DJ-1 was

performed as described for Fig. 1A. Insets boxed at the lower left corner are the

enlarged view. Scale bar, 10 mm. (B) Total cell lysates were immunoblotted using

anti-DJ-1 or anti-cleaved caspase-3. Quantitative analysis of relative intensity of

DJ-1 to GAPDH was performed. Significance was determined with student’s t tests

(n ¼3). ns, not significant. (C) Cytosolic or nuclear extracts were immunoblotted

with anti-DJ-1.

Results

In this study, we specifically explored the subcellular localiza-tion of DJ-1 during oxidative stress-induced DAergic neuronal celldeath. For this purpose, both MN9D DAergic neuronal cells andprimary cultures of mesencephalic neurons were treated with ahydroxylated derivative of DA, 6-OHDA, which has been used toestablish an experimental model of PD associated with DAergicneuronal death [32]. Previous studies including ones from ourlaboratory showed that 6-OHDA, once accumulated withinDAergic neurons, is involved in the generation of ROS, includingautoxidation, leading to typical ROS-mediated apoptosis [33,34].Therefore, we first examined and compared the cellular localiza-tion of DJ-1 in MN9D cells before and after 6-OHDA treatment. Asshown in Fig. 1A, both confocal photomicrographs and digitalimage analyses revealed that DJ-1 was mainly localized in thecytoplasm under normal conditions. In contrast, increased levelsof DJ-1 in the nuclei were detected after 6-OHDA treatment.Consistent with these immunocytochemical localization analyses,immunoblot analyses using fractionated cellular extracts indi-cated that 6-OHDA treatment indeed led to a time-dependentincrease in DJ-1 levels in the nuclear fraction (Fig. 1B). This wasparalleled by a corresponding decrease in the cytosolic level,showing drug-induced translocation of DJ-1 into the nuclei.Quantitative analyses indicated that slight but detectableamounts of DJ-1 were translocated into the nuclei 9 h after6-OHDA treatment (2.7370.67-fold increase over the untreatedcontrol), and substantial amounts of translocated DJ-1 were foundin the nuclear fraction thereafter (10.8874.22-fold increase at 12h; 13.4571.54-fold increase at 18 h; n¼3). Because the predictedmolecular weight of DJ-1 is approximately 20 kDa, it may pas-sively diffuse from the cytosol into the nucleus upon 6-OHDAtreatment via the nuclear pore complex [35]. To exclude thispossibility, we first examined the expression levels of DJ-1 in cellsbefore and after 6-OHDA treatment. As shown in Fig. 1C, immu-noblot analysis using total cellular lysates indicated that 6-OHDAtreatment did not affect total expression levels of DJ-1 in MN9Dcells. We then transiently transfected cells with a eukaryoticexpression vector containing green fluorescent protein (GFP)conjugated with DJ-1 sequences. Because the estimated molecularweight of this fusion protein is approximately 46 kDa, it cannotpassively diffuse into the nucleus. As shown in Fig. 1D, DJ-1–GFPfusion protein was mainly localized in the cytosol in the absenceof 6-OHDA, whereas increased levels of DJ-1–GFP were detectedin the nuclei after 6-OHDA treatment. Taken together, theseresults indicate that the increased level of DJ-1 in the nuclei after6-OHDA treatment was a consequence of drug-induced nucleartranslocation of DJ-1.

We previously demonstrated that 6-OHDA induces caspase-dependent apoptosis in both MN9D neuronal cells and primarycultures of cortical and mesencephalic neuronal cells [25,27].

Thus, we examined if drug-induced nuclear translocation of DJ-1is a common event in other caspase-dependent cell death para-digms. Therefore, we treated MN9D cells with or without 1 mMSTS, a prototypic apoptotic stimulus, and then compared thecellular localization of DJ-1. As shown in Fig. 2A, confocal micro-scopic examination revealed that DJ-1 was mainly localized in thecytosol regardless of STS treatment. Immunoblot analyses usingtotal cellular lysates or fractionated cellular lysates indicated thatSTS did cause obvious activation of caspase but did not lead to anysignificant changes in total DJ-1 levels or nuclear levels of DJ-1 inMN9D cells (Figs. 2B and 2C). Quantitative evaluation of thenuclear-to-cytoplasmic ratios of DJ-1 confirmed the absence ofnuclear translocation of DJ-1 in STS-treated MN9D cells (data notshown). Exposure of MN9D cells to STS for longer than 12 h or toetoposide (another prototypic apoptotic insult) also did not causeany detectable levels of nuclear translocation of DJ-1 (data notshown), indicating that nuclear translocation of DJ-1 is a drug-specific event.

We then specifically examined whether the nuclear transloca-tion of DJ-1 may be a consequence of drug-induced buildup of ROS-mediated signaling. To do so, we investigated whether ROS gen-eration is correlated with the nuclear localization of DJ-1. Usinga hydrogen peroxide-sensitive fluorescent probe, CM-H2DCFDA,we measured ROS levels in MN9D cells after 6-OHDA or STStreatment. As shown in Fig. 3A, FACS analyses indicated that theintensity of the DCF-sensitive fluorescent signals increased after

Fig. 3. Nuclear translocation of DJ-1 in response to various pro-oxidants in MN9D cells. (A, B) Cells were treated for 6 h with or without (A) 100 mM 6-OHDA, 1 mM STS, or

(B) 150 mM cupric chloride (CuCl2), 1.5 mM hydrogen peroxide (H2O2). DCF-sensitive intracellular ROS levels were measured with flow cytometry. Numerical values

represent the percentage of cells in the M1 fraction. Significant differences were determined with Student’s t tests (n¼3). **po0.001, *po0.05; ns, not significant.

(C) Immunocytochemistry for DJ-1 was performed in MN9D cells after 150 mM cupric chloride or 1.5 mM hydrogen peroxide for 12 h. Insets boxed at the lower left corner

are the enlarged view. Scale bar, 10 mm.


6-OHDA but not STS treatment (arbitrary units, 59.173.9 in theuntreated control cells; 84.172.5 in 6-OHDA-treated cells;

51.671.1 in STS-treated cells; n¼3). These results suggest thatan increase in ROS inside MN9D cells may be one cause that led tonuclear translocation of DJ-1. To directly test this possibility, wetreated MN9D cells with other ROS-inducing drugs, includinghydrogen peroxide and cupric chloride. Indeed, FACS analysesdemonstrated that the intensity of DCF-sensitive fluorescent signalsincreased in MN9D cells treated with hydrogen peroxide or cupricchloride (arbitrary units, 58.274.6 in the untreated control;

82.571.4 in hydrogen peroxide-treated cells; 97.970.3 in cupricchloride-treated cells; Fig. 3B, n¼3). After drug treatment, we usedimmunocytochemistry to examine the localization of DJ-1 in MN9Dcells. As expected, DJ-1 increased in the nuclei of cells treated withthese ROS-inducing drugs (Fig. 3C). These results suggest that theincrease in DJ-1 in the nucleus is primarily controlled by ROS. Tofurther confirm whether ROS were involved in DJ-1 translocation,we assessed the nuclear translocation of DJ-1 after 6-OHDA treat-ment in the presence or absence of NAC, a widely used antioxidantthat scavenges free radicals including hydrogen peroxide and


hydroxyl radical [36]. As shown in Fig. 4A, confocal microscopicanalyses showed that NAC inhibited the increase in 6-OHDA-mediated nuclear translocation of DJ-1. Immunoblot and quantita-tive analyses using fractionated cellular extracts indicated that6-OHDA-mediated nuclear translocation of DJ-1 was substantiallyinhibited in the presence of NAC (3.4970.73-fold increase overthe untreated control for nuclear DJ-1 after 6-OHDA treatment;0.8170.36 in 6-OHDA plus NAC treatment; Fig. 4B, n¼3).We previously demonstrated that 6-OHDA induces ROS generationand ROS-mediated cell death signaling in primary cultures ofmesencephalic neurons, and therefore, cotreatment with NAClargely blocks ROS-mediated cell death signaling [25,27,28]. There-fore, we further examined whether DJ-1 is also localized in thenucleus after 6-OHDA treatment and whether this phenomenon isinhibited in the presence of NAC using primary cultures of mesen-cephalic neurons. As in MN9D cells, DJ-1 was largely present in thecytoplasm of TH-positive DAergic neurons before drug treatment(Fig. 5A). In contrast, both confocal photomicrographs and imageanalyses indicated that levels of DJ-1 in the nucleus increased after6-OHDA treatment in TH-positive DAergic neurons, and thisphenomenon was inhibited in the presence of NAC (Fig. 5A).To quantitate the levels of nuclear DJ-1 in the three treatment groups,we measured the fluorescence intensity in the nucleus marked bydotted white lines in each cell and compared the intensity (Fig. 5B).As shown in Fig. 5C, quantitative analyses using 20 randomly selectedTH-positive cells revealed that the average intensity of DJ-1 in thenuclei increased after 6-OHDA treatment, and cotreatment with NACinhibited this phenomenon (average intensity per area 22.274.41in untreated control TH-positive cells; 64.63713.83 in 6-OHDA-treated TH-positive cells; 23.7374.16 in 6-OHDA plus NAC-treated

Fig. 4. Inhibition of nuclear translocation of DJ-1 by the antioxidant N-acetyl-L-

cysteine. (A) MN9D cells were incubated for 12 h with or without 100 mM 6-OHDA

in the presence or absence of 1 mM NAC. Immunocytochemistry for DJ-1 was

performed. Scale bar, 10 mm. (B) After drug treatment for 18 h, immunoblot

analyses of the cytosolic and nuclear extracts were performed, and densitometry

was performed. Relative intensities of each fraction were normalized to SOD1 and

nucleolin for the cytosolic and nuclear fractions, respectively. Significant differ-

ences were determined with Student’s t tests (n¼3). *po0.05.

TH-positive cells; n¼3), implying that 6-OHDA causes ROS-mediatednuclear translocation of DJ-1 in both MN9D DAergic neuronal cellsand primary cultured of DAergic neurons.

We then examined the localization of DJ-1 using a human anti-C106-oxidized DJ-1 antibody, which recognizes DJ-1 that isoxidized at cysteine 106. We observed that the oxidized formsof DJ-1 increased in the nuclei when MN9D cells were treatedwith 6-OHDA (Fig. 6A). Therefore, we hypothesized that oxidationof cysteine 106 rather than a typical NLS is critical for targetingDJ-1 to the nucleus. By mutating cysteine 106 to alanine, wegenerated a GFP-tagged DJ-1 mutant that cannot be oxidized.Contrary to our expectation, image analysis of live cells showedthat levels of both WT and mutant DJ-1 increased in the nucleiafter 6-OHDA treatment (Fig. 6B), indicating that direct oxidativemodification of the cysteine residue C106 of DJ-1 itself is not aprerequisite for its nuclear translocation. Therefore, other ROS-mediated modifications of DJ-1 alone or in combination withoxidation are likely to be involved.

To examine which isoforms of DJ-1 are found in total lysatesand nuclear fractions, we performed a mini-2-DE followed byimmunoblot analysis using anti-DJ-1 antibody. From total lysatesobtained from cells treated with or without 6-OHDA, we observedthe presence of at least four distinct isoforms of DJ-1 monomersthat have the same apparent molecular mass of approximately20 kDa but different isoelectric points (Fig. 6C). Upon exposure to6-OHDA, the more acidic isoforms of DJ-1 (indicated as 2, 3, and 4)increased in the total lysates. Next, we separated the isoforms ofDJ-1 from the nuclear fractions through mini-2-DE followedby immunoblot analysis with the same antibody (Fig. 6D).As determined by immunoblot analysis using fraction markers,we confirmed that the nuclear fractions prepared for 2-DE werefree of cytosolic and mitochondrial contaminants (Fig. 6D, right).At 12 h, we observed that very slight but detectable levels of threeisoforms of DJ-1 (designated as 2, 3, and 4) were present in thenuclear fraction of untreated control cells. In contrast, 6-OHDAtreatment dramatically increased the levels of all three acidicisoforms in the nuclear fraction, and this phenomenon wasblocked in the presence of NAC. Although we failed to identifythe nature of these acidic forms of DJ-1, our data suggest thatROS-mediated acidic modifications of DJ-1 may be critical for itsnuclear translocation.

Next, we evaluated whether nuclear localization of DJ-1 mayaffect cell viability. We targeted WT DJ-1 expression to thenucleus by fusing Myc-tagged DJ-1 with the NLS. Regardless of6-OHDA treatment, expression levels of both ectopic NLS-taggedDJ-1 and endogenous DJ-1 were quite similar (Fig. 7A, left).Immunocytochemistry data confirmed that the NLS-tagged DJ-1was exclusively localized in the nucleus (Fig. 7A, right). Afternuclear staining with Hoechst dye, we counted the number ofgreen fluorescence-positive apoptotic cells with nuclear conden-sation and/or nuclear fragmentation in MN9D cells overexpres-sing either an empty vector (GFP/NLS) or a vector expressing NLS-tagged DJ-1 cDNA (Fig. 7B). We found a lower rate of apoptoticcell death in MN9D cells overexpressing NLS-tagged DJ-1(9.5971.34% in GFP/NLS-overexpressing cells vs 4.2070.30% inNLS-tagged DJ-1-overexpressing cells at 10 h; 13.8471.20% vs8.671.73% at 15 h), suggesting that nuclear DJ-1 may play aprotective role in 6-OHDA-induced apoptosis in MN9D cells.Nuclear DJ-1 has been previously linked to expression of neuro-protective antioxidant proteins [16–18]. Therefore, we performedimmunoblot analyses to measure expression levels of MnSOD andthioredoxin 1 in NGF-differentiated PC12 cells overexpressing anempty vector or NLS-tagged DJ-1. As shown in SupplementaryFig. 1, their expression levels remained unchanged in both celltypes regardless of 6-OHDA treatment, indicating that a protec-tive mechanism other than upregulation of antioxidant protein

Fig. 5. Nuclear translocation of DJ-1 during 6-OHDA-induced cell death in primary cultures of mesencephalic DAergic neurons. (A–C) Primary cultured neurons were

treated for 12 h with or without 20 mM 6-OHDA in the presence or absence of 500 mM NAC. (A) Cells were processed for double immunolocalization of DJ-1 and TH. Nuclei

were stained with Hoechst 33258. The fluorescent signals observed by confocal microscopy were analyzed with an LSM digital image analyzer. Graphs on the right

represent the intensities of DJ-1 (green), TH (brown), and nuclear (blue) signals from the cross-sectional area marked by the white arrow. Scale bar, 10 mm. (B and C) Using

Metamorph, arbitrary fluorescence intensity was determined by dividing the total fluorescence intensity of DJ-1 from the nucleus by the nuclear area marked by the white

dotted line. Each dot on the bottom graph represents the calculated value from one cell. (C) Twenty randomly selected cells per experiment were analyzed in three

independent experiments. Significant differences were determined with Student’s t tests (n¼3). **po0.001; ns, not significant.


itself is involved. Furthermore, we examined the possibility thatDJ-1 can directly bind to DNA to modulate the transcription oftarget genes. A random nucleotide binding assay showed that DJ-1 was detected only in the outflow and wash steps, suggestingthat DJ-1 cannot bind to either double- or single-strandednucleotides (Supplementary Fig. 2A). To evaluate a binding cap-ability of the oxidized forms of DJ-1, we treated the purified DJ-1proteins with 10 mM or 1 mM hydrogen peroxide to induce DJ-1oxidization in vitro and performed a random nucleotide bindingassay. We found that neither intact nor oxidized forms of DJ-1were bound to double-stranded nucleotides (SupplementaryFig. 2B). This was quite contrary to results obtained from apositive control using MYB23 (Supplementary Fig. 2C), suggestingthat DJ-1 may modulate transcription of targets by binding a

particular transcription factor or cofactor rather than by directinteraction with DNA. Second, we tried to correlate the sub-nuclear localization of DJ-1 with its potential protective role. Thenucleus is compartmentalized and contains numerous subnuclearbodies in addition to nucleoplasm containing chromosomes [37].These compartments include nucleoli, Cajal bodies, nuclear speck-les, and PML bodies, which are involved in regulating andenhancing the efficiency of specific nuclear processes includingtranscriptional regulation [37]. Therefore, we further attempted todetermine whether nuclear DJ-1 is localized in the nucleoplasmor in any of these subnuclear bodies. Although we did not see anyobvious signs of localization of DJ-1 in the nucleoli and nuclearspeckles, DJ-1 was found within PML bodies probed with anti-PML, and this colocalization increased after 6-OHDA treatment

Fig. 6. Translocation of the acidic isoforms of DJ-1 into the nuclei after 6-OHDA treatment. (A) MN9D cells treated with or without 100 mM 6-OHDA for 12 h were

immunostained with anti-C106-oxidized DJ-1 antibody (Ox-DJ-1). Nuclei were stained with Hoechst 33258. Scale bar, 10 mm. (B) MN9D cells were transiently transfected

with a eukaryotic expression vector containing either GFP-tagged wild-type DJ-1 (WT/DJ-1/GFP) or GFP-tagged mutant DJ-1 (C106A/DJ-1/GFP) and then treated with or

without 100 mM 6-OHDA for 12 h. Cells were then subjected to confocal microscopic examination of live cells. Scale bar, 10 mm. (C) Total cell lysate (50 mg) or (D, left)

nuclear fractions (50 mg) obtained from MN9D cells treated with or without 100 mM 6-OHDA for 12 h in the presence or the absence of 1 mM NAC were subjected to 2-DE

immunoblot analysis of DJ-1. The arrows indicate isoforms of DJ-1 and are serially numbered from basic to acidic isoforms. Numeric values above the blot indicate

approximate pI values of the blot. (D, right) The nuclear extracts were blotted with anti-nucleolin, anti-SOD1, and anti-COX IV to demonstrate the nuclear fraction free of

the cytosolic and mitochondrial proteins.


(Fig. 8A). To evaluate the binding of DJ-1 and PML in the nucleus,we performed a coimmunoprecipitation assay using MN9D cellstransiently expressing Flag-tagged PML or V5-tagged DJ-1 andtreated cells with or without 6-OHDA for 12 h. As shown inFig. 8B, DJ-1 bound to PML, and their interaction increased after6-OHDA treatment. Based on the previous studies demonstratingthat PML bodies regulate apoptotic processes via acetylation ofp53 [38], we then asked whether DJ-1 exerted an antiapoptoticfunction by regulating the extent of p53 acetylation. To examinethis possibility, MN9D cells were first transfected with Flag-tagged p53 alone and in combination with Myc-tagged or V5-tagged DJ-1 and incubated with or without 1 mM TSA and 5 mMNA, which are pan-histone deacetylase (HDAC) inhibitors. Cellularlysates were subsequently subjected to immunoprecipitationusing Flag-conjugated beads followed by immunoblot analysisusing an antibody that recognizes acetylated lysines. As shown inFig. 8C, we found that levels of acetylated p53 increased in thepresence of TSA/NA and were downregulated in cells

overexpressing either of the tagged forms of DJ-1, suggesting thatDJ-1 may play a protective role by regulating transcriptionalactivity of p53 in PML bodies in MN9D cells. Next, we examinedwhether DJ-1 subsequently decreased the expression of p53target genes. Because of a high basal expression level of PUMAin MN9D cells, it was difficult practically to reveal slight butsignificant changes in its expression in DJ-1-overexpressing cellsbefore and after 6-OHDA treatment. p53 has been demonstratedto increase expression of proapoptotic proteins including Bax andPUMA during 6-OHDA-induced cell death in NGF-differentiatedcatecholaminergic PC12 cells [39,40]. Therefore, we first exam-ined whether DJ-1 is translocated into the nucleus after 6-OHDAtreatment in NGF-differentiated PC12 cells. As shown in Fig. 9A,levels of DJ-1 increased in the nucleus after 6-OHDA treatment for8 h. The increase in DJ-1 in the nucleus was detected as early as4 h after 6-OHDA treatment (data not shown). Next, we trans-fected the NGF-differentiated PC12 cells with NLS-tagged DJ-1. Asshown in Fig. 9B, overexpression of DJ-1 decreased the expression

Fig. 7. Enhancement of cell survival by nuclear-targeted DJ-1 in response to 6-OHDA treatment. (A) To examine the total expression levels and expression pattern of

nuclear-targeted DJ-1, immunoblot analyses (left) and immunocytochemistry (right) were performed. Cellular lysates obtained from MN9D cells transiently transfected

with a blank vector (CON) or a vector containing the Myc-tagged nuclear-targeted DJ-1 sequence (DJ-1/NLS) were immunoblotted with anti-DJ-1– and anti-c-Myc–HRP.

Immunocytochemical localization of nuclear-targeted DJ-1 was carried out using anti-c-Myc. For the positive control, MN9D cells transiently transfected with NLS-

tagged GFP (GFP/NLS) were subjected to live cell fluorescence image analysis. Nuclei were stained with Hoechst 33258. Scale bar, 10 mm. (B) MN9D cells transiently

expressing nuclear-targeted GFP or DJ-1 were treated with 100 mM 6-OHDA and stained with Hoechst 33258. Using randomly selected fluorescence photomicrographs

taken from four independent experiments, green fluorescence-positive cells with fragmented and/or condensed nuclei (indicated by white arrows) were counted as

apoptotic cells. Each bar represents the average of approximately 600 cells counted. Scale bar, 10 mm. Significant differences were determined with Student’s t tests.

**po0.001, *po0.05.


level of PUMA in PC12 cells 6 h after 6-OHDA treatment (77.9%compared to the empty-vector-transfected cells (100%); n¼2).Decreased expression level of PUMA was greater in cells over-expressing NLS-tagged DJ-1 after 6-OHDA treatment (65.3 or49.1% compared to the control cells at 6 or 8 h, respectively;n¼2). Interestingly, the expression level of Bax was not affectedin these transfected cells. Taken together, nuclearly localized DJ-1decreased the expression of a p53 target gene, PUMA, which maybe the result of the decrease of p53 acetylation.

Discussion

DJ-1 is a multifunctional protein primarily expressed in astro-cytes and neurons in the human and mouse brain [41]. Fromprevious studies using DJ-1 knockout mice, DJ-1 knockdown, andDJ-1 overexpression paradigms, DJ-1 is thought to play a protectiverole by decreasing oxidative stress, reducing protein misfolding, andstabilizing mitochondrial potential and integrity [42–47]. At sub-cellular levels, DJ-1 is mainly localized in the cytosol and is alsopresent in the mitochondria and nucleus. Although the relationshipbetween its subcellular compartmentalization and its cytoprotectivefunction is not fully understood, several studies have suggested thatthere is a certain degree of correlation between its mitochondriallocalization and maintaining mitochondrial integrity. For example,

DJ-1 is mainly present in the cytosol under normal conditions andis preferentially partitioned to the mitochondrial matrix and intra-membranous space to mediate its protective role in oxidant-challenged neuroblastoma [48]. Oxidant-mediated modificationof DJ-1 at the conserved cysteine residue 106 is essential forits mitochondrial relocalization, and therefore, its cytoprotectiverole is abrogated in C106A mutants after exposure to paraquator 1-methyl-4-phenylpyridinium [48,49]. More recently, loss ofDJ-1 has been shown to result in loss of mitochondrial polariza-tion, fragmentation of mitochondria, and impaired mitochondrialdynamics in the presence of oxidative environments [46,50].Similarly, DJ-1 double-knockout flies have a shortened life span,and mitochondrial respiration and ATP production are defective inthese mutant flies [51,52]. In our present study, we also attemptedto provide insight into the molecular link between the subcellularlocalization of DJ-1 and its functional role during oxidative stress-induced neuronal cell death. More specifically, we examinedwhether DJ-1 is translocated into the nucleus in several oxidativestress-induced cell death paradigms. We also investigated at whichnuclear subdomain DJ-1 is preferentially localized and how nuclearDJ-1 consequently exerts its protective role in pro-oxidant-inducedneurodegeneration. We found that 6-OHDA caused nucleartranslocation of DJ-1 in both MN9D DAergic neuronal cells andprimary cultures of DAergic neurons and this phenomenon wassubstantially blocked in the presence of a ROS scavenger, indicating

Fig. 8. Decrease in p53 acetylation by nuclear DJ-1. (A) MN9D cells were treated with or without 100 mM 6-OHDA for 12 h. The cells were then processed for double

immunofluorescence localization of DJ-1 and PML. After staining with Hoechst 33258, colocalization of DJ-1 and PML in the nuclei was analyzed with Metamorph. Typical

cells with the apparent colocalized dot signals in the nuclei are shown on the right. Scale bar, 10 mm. (B) Coimmunoprecipitation analysis of cellular lysates was performed

after transient transfection with the indicated vectors before and after 6-OHDA treatment for 12 h. Briefly, cellular lysates were immunoprecipitated with anti-Flag M2

affinity gel and blotted with anti-V5–HRP or anti-Flag–HRP antibody. The lower two blots indicate immunoblotted input signals for the same preparations. (C) The level of

p53 acetylation was determined with immunoprecipitation analysis of MN9D cells transfected with p53 alone or in combination with Myc-tagged DJ-1 or V5-tagged DJ-1

in the absence or presence of 1 mM trichostatin A (TSA) and 5 mM nicotinamide (NA). Briefly, cellular lysates were immunoprecipitated with anti-Flag M2 affinity gel and

immunoblotted with rabbit anti-acetylated lysine antibody followed by goat anti-rabbit HRP-conjugated antibody.


that ROS are a key factor in the drug-mediated nuclear transloca-tion of DJ-1. Our data also showed that DJ-1 was localized in andbound to PML bodies in the nucleus, which are associated with adecrease in the acetylation level of p53. Overexpression of DJ-1 andNLS-tagged DJ-1 caused decreased expression of PUMA. Although itremains to be thoroughly determined, we hypothesize that thesesequential events may comprise one of the mechanisms underlyinghow nuclear DJ-1 plays a cytoprotective role in oxidative stress-induced neurodegeneration.

The best studied system for the transport of proteins betweenthe cytosol and the nucleus is the classical nuclear import path-way. Typically, nuclear-localized proteins have a so-called NLS,which targets the corresponding proteins to the nucleus via aninteraction with importin on the nuclear pore [53]. In addition,posttranslational modifications such as oxidation/reduction,phosphorylation, and sumoylation have also been shown toregulate nuclear translocation of proteins [54–56]. Because DJ-1does not contain one (monopartite) or two (bipartite) NLS motifs,we hypothesize that one or more of these posttranslationalmodifications may be involved in the nuclear translocation of

DJ-1 after oxidative stress. Oxidation or phosphorylation pro-cesses usually affect the isoelectric point (pI) of the intact protein.When proteins have such modifications, an acidic shift can beobserved with 2-DE. Using a combination of 2-DE and massspectrometry, two independent groups indeed identified severalDJ-1 isoforms [57,58]. The acidic isoforms of DJ-1 accumulate inthe brain tissues of patients with idiopathic PD and Alzheimerdisease compared with age-matched control brains. We analyzedthe nuclear extract of MN9D cells using 2-DE analyses and foundthat relatively more acidic isoforms of DJ-1 were localized in thenucleus, and their levels were significantly increased after6-OHDA treatment, supporting the possibility that posttransla-tional modifications including oxidation or phosphorylation mayfacilitate the nuclear translocation of DJ-1. Based on previousstudies indicating that a conserved cysteine residue at position106 in DJ-1 is functionally essential and is subjected to prefer-ential oxidation under oxidative stress [48,59–62], we mainlyfocused on this possibility by using two approaches in this study:immunocytochemical localization of the oxidized form of endo-genous DJ-1 and an ectopically expressed mutant form of

Fig. 9. Decreased level of PUMA expression in cells overexpressing DJ-1 after 6-OHDA treatment. (A) NGF-differentiated PC12 cells were treated with or without 100 mM

6-OHDA for 8 h and processed for immunostaining of DJ-1. Nuclei were stained with Hoechst 33258. Fluorescence intensity of DJ-1 in cells was analyzed with an LSM

digital image analyzer. Graphs represent the intensities of DJ-1 (green) and nuclear (blue) signals from the cross-sectional area marked with a white arrow (bottom).

(B) NGF-treated PC12 cells transiently transfected with empty vector (control), DJ-1, or NLS-tagged DJ-1 were treated with 100 mM 6-OHDA. Total cell lysates were

immunoblotted with anti-PUMA or anti-Bax. Anti-V5 and anti-Myc were used as transfection controls.


DJ-1 (C106A) in the nucleus after 6-OHDA treatment. AlthoughDJ-1 that was oxidized at C106 was found in the nucleus using aspecific antibody that recognizes the C106-oxidized form ofDJ-1 [63], mutation of the cysteine residue (C106A) had nodetectable impact on the translocation of DJ-1 to the nucleus. Aprior study also reported that mutating cysteine 106 had no impacton the translocation of DJ-1 to the mitochondria [23]. This findingraises the possibility that in addition to oxidation of cysteine, DJ-1undergoes several other types of oxidative modifications, which

may be critical for its nuclear translocation after oxidative stress.These modifications may include oxidation of methionine residues,glutathionylation of cysteine residues, carbonylation, and nitration[64]. Although no direct evidence has been shown so far, ROS-induced phosphorylation of DJ-1 was previously indicated [65]. Ifthis is the case, ROS-driven phosphorylation of DJ-1 may also beinvolved in its nuclear translocation. Based on our unpublisheddata, we are tempted to propose that phosphorylation of tyrosineas well as serine/threonine residues of DJ-1 is likely to be involved


in its nuclear translocation upon 6-OHDA treatment. Althoughthere still exists the possibility that translocation is not directlydependent on posttranslational modification of DJ-1 itself butother related downstream events, careful examination and identi-fication of the nature of posttranslational modifications remain tobe done.

To assess the functional significance of the increase in DJ-1 inthe nucleus, we adapted two independent approaches: examinationof the effect of NLS-tagged DJ-1 in response to 6-OHDA-induced celldeath and its subnuclear localization. First, we demonstrated thatectopic expression of NLS-tagged DJ-1 in MN9D cells inhibited celldeath in response to 6-OHDA. Second, we found that DJ-1 waslocalized in PML bodies after 6-OHDA treatment. The mammaliannucleus consists of subnuclear compartments as well as chromo-some territory for maintenance and expression of genetic materials.Subnuclear compartments include nucleoli, nuclear speckles,Cajal bodies, and PML bodies that are not compartmentalizedwith membranous structures [37]. They are present in the steadystate and dynamically respond to both basal physiological pro-cesses and various forms of stress [66]. Several proteins that areclosely related to nuclear body functions are recruited into thenuclear bodies [37]. PML bodies consist of quite diverse proteinsthat regulate apoptosis induction through dynamic regulation ofproapoptotic transcriptional events. For example, the histoneacetyltransferase Creb-binding protein (CBP) is colocalized withPML [67] and recruited to PML bodies with p53 during apoptoticstimuli [68]. The formation of a complex of PML–p53–CBPinduces acetylation of p53, which in turn increases its DNA-binding ability [68]. Here, we found in MN9D cells that increasedlevels of DJ-1 were localized in PML bodies and bound to PMLafter 6-OHDA treatment. Similarly, we observed that 6-OHDAcaused upregulation of p53 and its binding to DJ-1 as determinedby immunoblot analysis, immunocytochemistry, and immuno-precipitation assays (Supplementary Fig. 3). In a paradigm usingectopic expression of p53 and DJ-1, we also found that levels ofacetylated p53 decrease in a DJ-1-dependent manner. Althoughthe mechanism of how DJ-1 possibly downregulates p53 acetyla-tion remains to be explored, we hypothesize three possible rolesfor DJ-1 in modulating p53 acetylation. First, binding betweenDJ-1 and p53 may destabilize the p53–CBP complex in PMLbodies. Because PML has no known intrinsic acetyltransferaseactivity [69], PML may directly interact with CBP for p53acetylation. Therefore, the formation of the PML–CBP–p53 com-plex may be essential for p53 acetylation. ROS-mediated nucleartranslocation of DJ-1 may interrupt its formation. Second, DJ-1may directly modulate the activity of CBP. The activity of CBP isregulated by posttranslational modification, including sumoyla-tion [70]. Because DJ-1 may inhibit protein sumoylation [18],sumoylation of p300/CBP may be inhibited by nuclear DJ-1.Third, DJ-1 may play a role in decreased acetylation of p53 as acomponent of the deacetylase complex. Deacetylation of p53 ismediated by an HDAC1-containing complex that includes Mi-2/nucleosome remodeling and deacetylase (NuRD) complex [71].Opsahl et al. [72] reported that DJ-1 is a core component of theNuRD complex. They suggested that downregulation of DJ-1facilitates p53-dependent cell death in response to apoptoticstimuli. Therefore, DJ-1 may decrease p53 acetylation by inter-acting with the NuRD complex. In any case, our data indicate thatthe acetylation status of p53 eventually affects expression levelsof proapoptotic proteins including PUMA during 6-OHDA-induced neurodegeneration. This is largely consistent with pre-vious studies indicating that acetylation of the p53 regulatesapoptotic induction [22,73,74].

In summary, our data support the previous study demonstrat-ing that DJ-1 protects against 6-OHDA-induced degenerationof nigral DAergic neurons in a PD rat brain model [75]. We extend

the previous findings by showing that DJ-1 is increased inthe nucleus in response to several oxidative stressors and bysuggesting that nuclear DJ-1 plays a role in protecting cellsfrom death after oxidative stress. Further studies examining themolecular mechanisms of DJ-1 in the nucleus, especially PMLbodies in more detail, will provide a better understanding of thepathogenesis of PD.

Acknowledgments

This work was supported by grants from the Ministry ofScience and Technology through BRC, KOSEF (SRC, R11-2008-036),Mid-Career Research Program, and WCU (R33-10014 to Y.J.O.).We thank Dr. H. Ariga for providing the human anti-C106-oxidized DJ-1 antibody.

Appendix A. Supporting information

Supplementary data associated with this article can be foundin the online version at http://dx.doi.org/10.1016/j.freeradbiomed.2012.05.035.

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