Induction of the Protective Antioxidant Response Element Pathway by 6-Hydroxydopamine In Vivo and In Vitro

Induction of the Protective Antioxidant Response ElementPathway by 6-Hydroxydopamine In Vivo and In Vitro

Rebekah J. Jakel,*,†,‡ Jonathan T. Kern,* Delinda A. Johnson,* and Jeffrey A. Johnson*,‡,§,1

*Department of Pharmaceutical Sciences, School of Pharmacy, †Medical Scientist Training Program, Medical School, ‡Neuroscience Training Program,

§Center for Neuroscience, Molecular and Environmental Toxicology, and Waisman Center, University of Wisconsin-Madison, Madison, Wisconsin 53705

Received May 21, 2005; accepted June 20, 2005

Parkinson’s disease, a progressive neurodegenerative disorder, is

characterized by loss of midbrain dopaminergic neurons. The

etiology of sporadic Parkinson’s disease is unknown; however,

oxidative stress is thought to play a major role in disease patho-

genesis. Little is known regarding the transcriptional changes

that occur in Parkinson’s disease. The antioxidant response ele-

ment is a cis-acting enhancer sequence that is upstream of many

phase II detoxification and antioxidant genes. Here we show

that 6-hydroxydopamine, a mitochondrial inhibitor used to model

Parkinson’s disease, activates the antioxidant response element

both in cultured neurons and in the striatum and brainstem of

6-OHDA-lesioned mice. Pretreatment with antioxidants or NMDA

receptor antagonists reduced but did not abolish activation.

Further induction of this pathway with tert-butylhydroquinone

was able to significantly reduce cell death due to 6-OHDA in vitro.These observations indicate that 6-OHDA activates the antioxi-

dant response element through components of oxidative stress,

excitotoxicity, and potential structural factors. Further induction

of this endogenous defense mechanism may suggest a novel

therapeutic venue in Parkinson’s disease.

Key Words: 6-hydroxydopamine; Parkinson’s disease; oxidative

stress; antioxidant response element; tert-butylhydroquinone.

Parkinson’s disease (PD), the most common adult-onsetneurodegenerative movement disorder, is characterized by lossof the pigmented dopaminergic neurons in the substantia nigrapars compacta leading to a loss of striatal dopamine. Thehallmark features of PD include akinesia, tremor, rigidity, andpostural instability. Most cases of PD are sporadic, with aminority caused by known mutations. Although the etiology ofsporadic PD is unclear, oxidative stress, mitochondrial dys-function, and excitotoxicity likely play a role in pathogenesis(Jenner and Olanow, 1998). Indirect evidence of reactive

oxygen species (ROS) in PD has come from observationsof increased oxidized end-products such as 8-hydroxy-2-deoxyguanosine, 4-hydroxy-2-nonenol, and protein carbonylsin post mortem brain tissue from patients with Parkinson’sdisease (Alam et al., 1997a,b; Castellani et al., 2002; Dexteret al., 1986, 1989a, 1994; Jenner et al., 1992; Saggu et al.,1989; Schapira et al., 1990; Sian et al., 1994a,b).

There are several potential sources of ROS in PD. Impair-ment of the respiratory chain can cause oxidative stress throughsuperoxide production. There is evidence for complex I dys-function in post mortem human brain from PD patients(Schapira et al. 1989, 1990). Indeed, PD is modeled in vitroand in vivo using complex I inhibitors such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), rotenone, and6-hydroxydopamine (6-OHDA) (Betarbet et al., 2002). Mito-chondrial inhibition can also generate free radicals viaan excitotoxic mechanism (Albin and Greenamyre, 1992;Brouillet and Beal, 1993; Srivastava et al., 1993). Additionally,oxidative stress may be a consequence of high iron levelsnaturally present in the nigra, or due to changes in iron regu-lation observed in PD brains (Dexter et al., 1987, 1989b, 1990).

Another source of free radicals in PD may be intrinsic to thenigrostriatal dopaminergic system. Dopamine (DA), a catechol-aminergic neurotransmitter, is essential to normal basal gangliafunction; however, it can be oxidized to generate prooxidantspecies through autooxidation and enzymatic catabolism viamonoamine oxidase, prostaglandin H, or tyrosinase (Graham,1978; Graham et al., 1978; Hastings, 1995; Maker et al., 1981;Nappi and Vass, 2001; Tse et al., 1976). DA toxicity is most likelymediated by an oxidative stress mechanism (Hastings et al., 1996;Maker et al., 1981; Stokes et al., 2000). 6-OHDA, a hydroxylatedanalog of the DA used to model PD, is a catacholaminergicneurotoxin via mitochondrial complex I inhibition and oxidativestress (Adams et al., 1972; Soto-Otero et al., 2000), and may beformed via DA oxidation (Jellinger et al., 1995).

One cellular defense mechanism to cope with oxidativestress is the antioxidant response element (ARE), a cis-actingenhancer element that is upstream of many phase II detox-ification and antioxidant enzymes such as heme oxygenase-Iand glutathione-S-transferases (Friling et al., 1990; Rushmore

Portions of this research were presented at the 44th annual meeting of the

Society of Toxicology, March 2005, New Orleans, LA, and at the 34th annual

meeting of the Society for Neuroscience, October 2004, San Diego, CA.1 To whom correspondence should be addressed at School of Pharmacy,

University of Wisconsin, 777 Highland Avenue, Madison WI 53705. Fax: (608)

262-5345. E-mail: [email protected].

� The Author 2005. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.For Permissions, please email: [email protected]

TOXICOLOGICAL SCIENCES 87(1), 176–186 (2005)

doi:10.1093/toxsci/kfi241

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et al., 1990, 1991; Rushmore and Pickett, 1990, 1991). TheARE is induced by xenobiotics, changes in the redox status, aswell as catechol and quinone structures (Nguyen et al., 2004).NF-E2 related factor (Nrf2), a basic leucine zipper transcrip-tion factor, is known to drive ARE-mediated gene expression(Nguyen et al., 2004). Following exposure to activators, Nrf2translocates to the nucleus where it binds the ARE and activatestranscription (reviewed by Jaiswal, 2004). Nrf2-knockout micedemonstrate decreased basal activity of some ARE regulatedgenes and normal expression of others; however, these animalsdo not display inducible ARE activity (Lee et al., 2003a).Because of the prominent role of oxidative stress in PD, wehypothesized that the ARE may be induced in response to thecellular dysfunction specific to this disease.

Previous research from our lab has shown that culturedneurons from Nrf2 knockout mice are more vulnerable to1-methyl-4-phenylpyridinium (MPPþ) and rotenone (Leeet al., 2003b). This suggests that the ARE system is criticalin mediating PD pathogenesis. ARE-inducers have been able toprotect against death due to DA and 6-OHDA in vitro (Duffyet al., 1998; Hara et al., 2003). Analysis of post mortemPD brains has revealed increased ARE-regulated enzymessuch as heme oxygenase-1 (HO-1) and NAD(P)H quinoneoxidoreductase-1 (NQO1) also suggesting the potential forcommon transcriptional regulation (Schipper et al., 1998; vanMuiswinkel et al., 2004; Yoo et al., 2003).

The current work tests the hypothesis that 6-OHDA induces theARE. Specifically, we evaluated (1) whether 6-OHDA activatesthe ARE in vivo and in vitro, (2) the roles of oxidative stress andexcitotoxicity on ARE activation in vitro, and (3) whether furtherinduction of the ARE with tert-butylhydroquinone (tBHQ) wouldprotect against 6-OHDA-mediated cytotoxicity in vitro.

MATERIALS AND METHODS

Animals. All animals were housed at the University of Wisconsin School

of Pharmacy Vivarium and treated in accordance with all IACUC regulations.

All mice were maintained under standard laboratory conditions with food and

water available ad libitum in a 12-h light/dark cycle. The transgenic ARE-

human Placental Alkaline Phosphatase (hPAP) animals were generated as

described previously (Johnson et al., 2002). The presence of the transgene was

confirmed by PCR amplification of a portion of the inserted gene. ARE-hPAP-

negative littermates were used as background controls for endogenous alkaline

phosphatase activity.

Chemicals and reagents. All chemicals were dissolved in neurobasal

media (as described below) and from Sigma unless specifically noted. 6-

Hydroxydopamine (RBI) was dissolved in 0.5% ascorbate in 0.9% sterile

saline. Apomorphine hydrochloride was dissolved in 0.15% ascorbate in saline.

Dizocilpine (MK801) was dissolved in 0.5% dimethylsulfoxide (DMSO). Tert-

butylhydroquinone and di-tert-butylhydroquinone (tBHQ and dtBHQ, Acros)

were dissolved in 0.1% DMSO, with appropriate DMSO vehicle controls.

Primary cortical culture. Primary cortical neuronal cultures were derived

from E16-18 embryos pooled from litters resulting from crossing ARE-

hPAPþ/– males with ARE-hPAP–/– female mice as previously described

(Lee et al., 2003b). Briefly, following trypsin dissociation, cells were plated on

poly-D-lysine coated 96-well plates or on CC2-treated chamber slides

(LabTek) in media containing modified eagle media (MEM), fetal bovine

serum, horse serum, L-glutamine, and penicillin/streptamicin/fungicide (PSF)

for 24 h. Cells were then transferred to media containing neurobasal (Gibco

BRL), B27, PSF, and L-glutamine for the duration of the experiment. All toxin

exposures lasted 24 h. MK801 and antioxidant pretreatments (N-acetylcysteine

0.5 mM, catalase 100 units/ml, and reduced glutathione 0.5 mM) commenced

1 h prior to toxin exposure. All treatments were started on 3–7DIV with

exception of the cultures pretreated with tBHQ for 48 h starting on 2DIV prior

to toxin exposure.

Stereotaxic injections. 16–25 week old male and female mice were

anesthetized with isoflurane and received intrastriatal stereotactic injections

of 6-OHDA (6 lg, 1 ll) with contralateral vehicle control administration into the

following coordinates: 0.5 mm anterior to bregma, ± 2.0 mm lateral to midline,

and 3.1 mm ventral to dura. A Hamilton syringe was inserted and allowed to

equilibrate for 2 min followed by injection over 3 min. The syringe was allowed

to equilibrate again for 2 min, and then the syringe was withdrawn over 3 min.

Behavioral assessment. Mice in the 7-day time-point group for tissue

assays were administered 1mg/kg apomorphine HCl sc (0.15% ascorbate in

0.9% sterile saline). Mice were observed for turning behavior for 20 min during

the initial pretest 24–48 h prior to surgery. One week following surgery, animals

were again administered apomorphine and observed for 40 min for turning.

Animals not exhibiting contralateral turning stereotypy were excluded from

analysis (one animal).

Tissue collection and histology. All animals were euthanized with CO2

and transcardially perfused with PBS. Tissues collected for hPAP tissue enzyme

assay were first hemisected then dissected to remove cortex, brainstem, and

striatum, which were snap frozen and stored at �80�C until assayed. Tissues

collected for histology were post-fixed overnight with 4% paraformaldehyde

and cryoprotected with 30% sucrose. Brains were sectioned on a cryostat

(Leica, Deerfield, IL). Serial sections were taken as free-floating in PBS þazide (40 lm) or directly onto slides (20 lm). Free-floating and mounted

sections were stored at 4�C and �20�C, respectively until analysis.

Immunochemical staining. Free-floating sections were incubated in

100% methanol containing 1% H202 to abolish endogenous peroxidase activity.

Sections were blocked with PBS þ 0.3% Triton-X 100 (PBST) with 10%

normal goat serum. Sections were incubated in anti-tyrosine hydroxylase

(Chemicon, 1:800). Sections were then exposed to biotinylated goat anti-rabbit

IgG followed by the ABC and DAB reaction kits (Vector). All washes were

completed with PBST. Sections were mounted on glass slides, dried, and

cleared with xylenes before coverslipping.

Primary cultures were blocked with PBS containing 1% BSA, 10% NGS

and/or NHS, and 0.2% Triton-X 100. Slides were exposed to anti-beta-III-

tubulin (Promega, 1:200), anti-heme oxygenase-1 (Stressgen, 1:200) or anti-

Glial Fibrilary Acidic Protein (GFAP; Dako, 1:1000 and Chemicon, 1:200)

overnight. Secondary antibodies used include rabbit anti-goat conjugated to

Texas Red, goat anti-rabbit conjugated to Texas Red or fluorescein and horse

anti-mouse conjugated to Texas Red or fluorescein depending on whether the

samples were co-labeled with Vector Red or TUNEL as described. All

secondary antibodies came from Vector Labs. Cells were counterstained with

Hoescht 33258 to visualize nuclei. A Zeiss photomicroscope was used to

acquire all images, which were analyzed using Axiovision software.

Alkaline phosphatase assays. For alkaline phosphatase tissue activity,

tissues were homogenized in TMNC buffer (50 mM Tris, 5 mM MgCl2, 100

mM NaCl, 4% CHAPS) and refrozen. Samples were heat-inactivated at 65�C(to destroy endogenous phosphatase activity). HPAP activity was assayed in

a 96-well format using the chemiluminescent CSPD substrate (Tropix) with

Emerald (Tropix) enhancement in diethanolamine. Activity was measured in

a luminometer and calculated relative to protein concentration as was

determined by BCA kit (Pierce). Primary cortical cultures were also assayed

for activity using this method using known cell numbers.

Alkaline phosphatase tissue histochemistry was assayed as follows: 20 lm

frozen sections were stored at �20�C until thawed and rehydrated in TMN

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(50 mM Tris, 5 mM MgCl2, 100 mM NaCl). Sections were heat-inactivated in

TMN (65�C) and treated with BCIP/NBT (Promega) at 37�C until color

reaction product developed. Vector red alkaline phosphatase substrate (Vector

Labs) was used on fixed primary cells as follows. Cells cultured on CC2-treated

Lab-Tek chamber slides were incubated in TMN and heat inactivated as above,

followed by incubation with Vector Red kit as per manufacturer’s instructions.

Cytotoxicity measurements. Terminal deoxynucleotidyltransferase-

mediated dUTP nick end labeling (TUNEL; Roche) staining for primary

cortical cells was completed as per manufacturer’s instructions. Cells were

further counterstained with Hoescht. Five fields from each condition were

quantified for number of either TUNELþ or Hoeschtþ cells by a non-biased

observer who was blinded to the conditions of the experiment. The MTS assay

[3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-

2H-tetrazolium salt; Promega] was also used as per instructions.

Statistical analysis. All data reported as averages ± SEM, using p < 0.05

as the cutoff for significance. For primary culture data, all data points were

collected in triplicate and analyzed with unpaired, two-tailed Student t-tests.

For tissue assays, paired, two-tailed Student t-tests were used to analyze the

data. Actual p values are reported in figure legends.

RESULTS

6-OHDA Activates the ARE In Vitro

Primary cortical neurons containing an ARE-driven reportertransgene were exposed to 6-OHDA (1, 25, or 75 lM) for 24 hat three time-points in vitro and harvested for hPAP activity(Johnson et al., 2002). 6-OHDA induced ARE activation in adose-dependent fashion at all three time-points At 7DIV, 75 lM6-OHDA was sufficient to induce an over 50-fold increase inARE-hPAP activity over vehicle control (Fig. 1A). As timein vitro progressed, the degree of ARE activation increased(Fig. 1A). Pretreatment with antioxidants (N-acetylcysteine,catalase, and reduced glutathione) significantly reduced AREactivation due to 75 lM 6-OHDA by approximately 50%(Fig. 1B).

ARE Induction by 6-OHDA Is Not Contingent uponAbility to Cause Neurotoxicity

6-OHDA and diethyl maleate (DEM), a known AREactivator through an oxidative stress mechanism, activate theARE as compared to vehicle control. As shown in Figure 1C,pretreatment with antioxidants was sufficient to significantlyreduce ARE activation by 6-OHDA and DEM. In contrast,75 lM MPPþ and 75 mM glutamate, known oxidativestressors, fail to activate the ARE at relevant doses as comparedto vehicle control, with or without antioxidants (Fig. 1C).

6-OHDA-Induced ARE Activation Is Reduced by NMDAReceptor Antagonism

At 3 and 7 DIV, primary hPAPþ neurons were exposed to6-OHDA (75 lM) with or without pretreatment with MK801(10 lM) and/or antioxidants. As shown in Figure 1D, at bothtime points, 6-OHDA exposure led to significantly increased

ARE activation (fold change over vehicle control) which wasreduced by pretreatment with antioxidants. At 3DIV, pre-treatment with MK801 did not have any significant effect on 6-OHDA-induced ARE-activation in the absence of antioxidants;however, in the presence of antioxidants, 6-OHDA-inducedARE activation was significantly reduced, but not to the levelof ARE activity in the presence of MK801 alone (Fig. 1D).

At 7DIV, when primary cortical cells are vulnerable toexcitotoxicity (Frandsen and Schousboe, 1990), pretreatmentwith MK801 significantly reduced ARE activation by approx-imately 50% (Fig. 1D). Pretreatment with MK801, however,did not fully abolish ARE activity due to 6-OHDA. Pre-treatment with antioxidants in addition to MK801 did notfurther attenuate ARE activation (Fig. 1D). There was nosignificant difference between 6-OHDA þ antioxidants andMK801 þ 6-OHDA þ antioxidants, suggesting that MK801 isblocking ROS due to excitotoxicity.

ARE Activation Due to 6-OHDA Is Primarily in Astrocytes

Cultured neurons exposed to various conditions wereassayed for hPAP histochemistry using the fluorescing sub-strate Vector Red followed by immunostaining for either GFAPor beta-III-tubulin to discern astrocytes versus neurons, re-spectively (Fig. 2). Vehicle treated cells showed very littleARE-hPAP histochemistry (Figs. 2A and 2B). Treatment with6-OHDA generated ARE-hPAP histochemistry primarily inastrocytes (Fig. 2C) as opposed to neurons (Fig. 2D). Treatmentwith MPPþ did not reveal hPAP histochemistry in eitherastrocytes (Fig. 2E) or neurons (Fig. 2F) confirming hPAPactivity measures in Figure 1C.

To confirm that increased hPAP activity correlates withprotein expression, we examined heme oxygenase-1 (HO-1).HO-1 expression is known be regulated in part by the ARE andhas been shown previously to correspond to striatal injury dueto 6-OHDA (Munoz et al., 2005; Prestera et al., 1995).Increased HO-1 is seen in 6-OHDA-treated cultures (Fig. 3).tBHQ treatment is a positive control for heme oxygenase-1induction (Fig. 3C).

In order to determine if ARE activation was a component ofa more general neurotoxic response to complex I inhibitors, weassayed for cell death using the TUNEL-labeling.

Both 6-OHDA (75 lM) and MPPþ (75 lM) causedsignificantly increased apoptotic cell death as revealed byTUNEL staining and observable pyknotic nuclei in Hoescht-stained images (Fig. 4). However, as demonstrated in Figures 1and 2, MPPþ failed to activate the ARE. This suggests that thestructural properties and/or the mechanism of cell death due to6-OHDA may account for its induction of the ARE.

6-OHDA Activates the ARE In Vivo in the Striatumand Brainstem

Thirty-two adult ARE-hPAP transgenic reporter mice re-ceived midstriatal 6-OHDA lesions with contralateral vehicle

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control injections. For tissue hPAP activity, animals weresacrificed either at 24 h or seven days post-injection. To testfor the efficacy of the lesions, animals were administeredapomorphine (1 mg/kg) and observed for turning behaviorseven days post-injection (Ungerstedt and Arbuthnott, 1970).All but one 7-day animal demonstrated a significant increase inturning contralateral to the hemisphere of the lesion (data notshown). The animal that did not show a contralateral turningpreference was omitted from the study. 24-hour animals werenot tested for rotational turning as this behavior is not present atearly time points.

Tissue hPAP activity assays did not demonstrate inductiondue to 6-OHDA in tissues collected at 24 h post-injection(Fig. 5A). However, by seven days post-injection, hPAPactivity was significantly activated in the brainstem andstriatum as compared to contralateral vehicle control hemi-sphere. The greatest fold change activation due to 6-OHDAlesions was found in the striatum, which demonstrated over 6-fold activation as compared to paired vehicle-treated contra-lateral hemisphere (Fig. 5B). There was no change in thecortex, a negative control region, due to 6-OHDA at 7 days(data not shown).

FIG. 1. 6-OHDA activates the antioxidant response element in vitro. 6-OHDA activates the ARE in a dose-dependent fashion at three time-points [3DIV

(vehicle vs. 1 lM, p ¼ 0.03; 25 lM, p ¼ 0.04; 75 lM, p ¼ 0.0003); 5DIV (vehicle vs. 1 lM, p > 0.05; 25 lM, p ¼ 0.01; 75 lM, p ¼ 0.0006); 7DIV (vehicle vs.

1 lM, p¼ 0.008; 25 lM, p¼ 0.008; 75 lM, p¼ 0.001); A]. 6-OHDA-induced ARE activation can be partially reduced by pretreatment with antioxidants (p¼ 0.02;

B). 6-OHDA and DEM activate the ARE in the absence of antioxidants (p ¼ 0.00001 and p ¼ 0.002, respectively), and in the presence of antioxidants (6-OHDA,

p¼ 0.001; DEM, p¼ 0.03; C). However, MPPþ and glutamate fail to activate the ARE in vitro (C). Like B, pretreatment with antioxidants was sufficient to reduce

ARE activation by 6-OHDA (p¼ 0.002) and DEM (p¼ 0.003) but had no effect on MPPþ (p > 0.05). 6-OHDA activated the ARE compared to vehicle control in

the absence of antioxidants (3DIV, p ¼ 0.01; 7DIV, p ¼ 0.005) and the presence of antioxidants (3DIV, p ¼ 0.002; 7DIV, p ¼ 0.001). ARE activation was reduced

by antioxidants (3DIV, p¼ 0.02; 7DIV, p¼ 0.01). At 3DIV, in the presence of antioxidants, 6-OHDA-induced ARE activation was significantly reduced by MK801

(p ¼ 0.05), but not back to level of ARE activity due to the presence of MK801 alone (p ¼ 0.04; D). At 7DIV, MK801 significantly reduced ARE activation (p ¼0.01; D). Pretreatment with MK801 did not fully abolish ARE activity due to 6-OHDA (p ¼ 0.003). Unless noted, data shown as relative light units (RLU).

*Significantly different from corresponding vehicle-treated control sample (p < 0.05). †Significantly different from corresponding sample in the absence of

antioxidants (p < 0.05). #Significantly different from corresponding sample in the absence of MK801 (p < 0.05). ‡Significantly different from corresponding

sample in the absence of 6-OHDA (p < 0.05).

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Increased ARE activity correlates with loss of tyrosinehydroxylase immunoreactivity (THir) as seen in sections fromidentically treated animals in a parallel study (Fig. 5C). At 24 h,there is no loss of THir; however, by one week, the 6-OHDAlesion was nearly complete (Fig. 5C).

Sections were taken from 6-OHDA-injected brains at 24 h,96 h, and one week post-lesion for hPAP histochemisty andcounterstained with nuclear fast red. At 24 h, there were nohPAPþ cells present (data not shown). This agrees with datafrom tissue hPAP assays which did not reveal changes in AREactivity at 24 h (Fig. 5A). hPAP-negative tissue did notdemonstrate any staining at any time point assayed (Figs. 6Aand 6E). At 96 h post-injection, half of the animals assayeddemonstrated hPAPþ cells at the penumbra of the lesion (Figs.6C and 6D), but not in the vehicle control-treated hemisphere

(Fig. 6B). At one week, all animals assayed demonstratedhPAPþ cells encroaching into the core of the lesion (Figs. 6Gand 6H), but not in the vehicle treated hemisphere (Fig. 6F). Novisible increase in hPAPþ cells was seen in the brainstem (datanot shown). The issue of specific cell type expressing hPAP inand around the lesion is discussed subsequently.

Induction of ARE Can Reduce Cell Death Due to6-OHDA In Vitro

tBHQ (10 lM), a known ARE activator, can cause an over30-fold induction in ARE activity, significantly more potentthan 6-OHDA (75 lM; Fig. 7A). dtBHQ, a structural analog oftBHQ, does not activate the ARE and was used as a negativecontrol (Fig. 7B). Treatment with both tBHQ, and 6-OHDA

FIG. 2. ARE-hPAP activation in vitro. Primary cultures derived from ARE-hPAP mice were stained for hPAP histochemistry (red), Hoescht (blue), and either

anti-GFAP or anti-beta III tubulin (green) in order to assess the identity of ARE-active cells (403). Vehicle treated cells demonstrate little ARE-activity (A and B).

6-OHDA-treated cultures demonstrate induction primarily in astrocytes (C) as compared to neurons (D). Cells treated with MPPþ demonstrate little ARE

activation (E and F).

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does not significantly increase ARE induction over tBHQ alone(Fig. 7A). This suggests tBHQ (10 lM) saturates the Nrf2-AREinduction cascade.

Primary cortical cells were exposed to 6-OHDA for 24 hfollowing 48 h of pretreatment with tBHQ or vehicle. 6-OHDA

led to loss of cellular viability in a dose-dependent fashion(Fig. 7B). Pretreatment with tBHQ significantly increasedviability as compared to vehicle pretreated cells (Fig. 7B).

Cells from the same culture were plated in chamber slidesand exposed to 6-OHDA (75 lM) following pretreatment with

FIG. 3. Increased HO-1 expression in 6-OHDA- and tBHQ-treated cultures (403). HO-1, an ARE-regulated enzyme is upregulated in cells treated with either

6-OHDA (B) or tBHQ (C) as compared to vehicle controls (A). *Indicates HO-1 immunoreactive cells.

FIG. 4. Apoptotic cell death as demonstrated by TUNEL-labeling (203). As compared to vehicle control cells (A), 6-OHDA (B) and MPPþ (C) both lead to

apoptotic cell death, with differential activation of the ARE. A, B, and C shown as merged pseudo-colored overlay images (Hoescht, blue; TUNEL, green).

Corresponding Hoescht photos displayed below demonstrate increased pyknotic neuclei, indicative of apoptosis. Figure D shows quantification of TUNEL

counts. Approximately 10% of vehicle cells demonstrated apoptotic nuclei, whereas 6-OHDA and MPPþ treatment caused approximately 41% and 31% TUNELþcells, respectively. 6-OHDA and MPPþ induced significantly more apoptotic cells than vehicle control cultures (p ¼ 0.004; p ¼ 0.001), but were not significantly

different from each other the specific dose shown (p > 0.05).

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vehicle or tBHQ. After 24 h, cells were fixed and assessed forapoptotic nuclei using the TUNEL assay and counterstainedwith Hoescht to indicate total cells in the field (Fig. 7C). 6-OHDA caused significantly increased TUNELþ cells (Fig. 7C,middle panel and D) as compared to vehicle control (Fig. 7C,top panel). Pretreatment with tBHQ decreased the amount ofTUNELþ cells by approximately 35% indicating a reductionin apoptosis (Fig. 7C, bottom panel and D).

DISCUSSION

In the current study, we have shown that 6-OHDA, acatecholaminergic neurotoxin used to model PD, activatesthe ARE both in vivo and in vitro. Oxidative stress is a criticalfactor in PD pathogenesis and consequently, we hypothesizedthat the cellular injury in PD may lead to activation of the ARE.Although known ARE-regulated genes such as HO-1 andNQO1 are increased in the PD brain (Schipper et al., 1998; vanMuiswinkel et al., 2004), the nature of the regulation of thesechanges on a transcriptional level has not been elucidated. TheARE is an enhancer sequence found in the promoter of manycytoprotective genes. Oxidative stress and xenobiotic expo-sures can lead to Nrf2 translocation to the nucleus andsubsequent ARE-regulated transcription. In this way, theARE can coordinate the upregulation of a multitude ofprotective genes with a single insult.

In primary neuronal cultures from reporter mice, 6-OHDAactivated the ARE in a dose-dependent fashion over a seven-

day period (Fig. 1A). ARE-driven hPAP activity was observedprimarily in astrocytes rather than in neurons (Figs. 2C and2D). This agrees with previous work that ARE-mediatedactivity is primarily induced in astrocytes in vitro (Eftekharpouret al., 2000; Kraft et al., 2004; Shih et al., 2003). AREactivation due to 6-OHDA (75 lM) was reduced but noteliminated in the presence of antioxidants (Figs. 1B–1D). At7DIV, pretreatment with MK801, an NMDA antagonist, alsoreduced, but did not eliminate ARE activity (Fig. 1D).Antioxidants in combination with MK801 did not furtherreduce the ARE activation. Therefore, 6-OHDA activates theARE by a combination of factors including oxidative stressgenerated in part through an excitotoxic mechanism. In addi-tion, 6-OHDA may activate the ARE due to its catecholaminestructure that is independent of ROS formation. The lattermechanism of activation is probably the same used by tBHQ.

DA and its metabolites share structural similarities to tBHQand hydroquinone. tBHQ activates the ARE without producingROS, suggesting that its mode of induction is purely structural.MPPþ, another chemical used to model of PD, does not inducethe ARE (Figs. 1B, 2E, and 2F) in cell culture and lacksstructural similarities to known ARE activators. Experimentsdesigned to determine the effect of MPTP administrationin vivo are currently underway. The pro-oxidant nature of thequinones and catecholamines suggests that DA breakdown maybe a contributing factor to PD pathogenesis. However, thesechemicals, by virtue of their structure, may induce the ARE. If6-OHDA and DA behave like tBHQ in the ARE inductioncascade, it is possible that they alter the redox status of Keap1

FIG. 5. 6-OHDA activates the ARE in vivo. At 24 h and 1 week post-lesion, individual brains were hemisected and dissected for regions of interest. 6-OHDA

injections did not lead to increased ARE activation at 24 hours post-lesion (N¼ 5; A). By 1 week, tissue ARE assays demonstrated significantly increased activity

in the striatum (p ¼ 0.02) and brainstem (p ¼ 0.02) as compared to vehicle control hemisphere (N ¼ 7; B). Data shown as relative light units (RLU) per tissue

weight. Sections taken from unilateral (right) 6-OHDA-lesioned animals with contralateral vehicle controls (left) were immunostained for TH. At 24 h, there was

little loss of THþ cells in the striatum (13); however, by 1 week, there is a nearly complete lesion (C).

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and stabilize Nrf2 protein, allowing for enhanced binding to theARE (Dinkova-Kostova et al., 2002; Nguyen et al., 2003).Further studies are needed to confirm the mechanism of directactivation of the ARE by catecholamines like 6-OHDA.

Direct intrastriatal administration of 6-OHDA in vivo lesionsthe nigrostiatal dopaminergic pathway modeling PD pathologyin the live animal. 6-OHDA induces ARE activation in AREreporter mice at one week, but not 24 h post-injection (Fig. 5).

FIG. 6. In vivo ARE-hPAP staining at 96 h (A–D) and 1 week (E–H). Sections were taken at 24 h, 96 h, and 1 week post-injection for hPAP histochemistry

(103, scale bar ¼ 100 lm). At 24 h, there were no hPAP þ cells (data not shown). Figures 6A and E show sections from an ARE-hPAP (-) mouse, processed for

hPAP histochemistry, and counterstained with nuclear fast red at 96 h and one week, respectively. The contralateral striatum receiving vehicle did not demonstrate

hPAPþ cells (B and F). However, by 96 h, there was increased ARE activity circumscribing the lesion in half of the animals assayed (C), which was present at one

week in all animals (G). Inlays D and H show same sections as C and G prior to nuclear fast red processing.

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The loss of THir, indicating loss of nigrostriatal terminals, is

observable at 96 h and nearly complete by one week. This

suggests that ARE induction follows a time course similar to

retrograde degeneration. ARE induction, as measured by

a tissue assay, occurs primarily in the brainstem and striatum.

In the striatum, ARE activation appears at the penumbra of

the lesion at 96 h (Figs. 6C and 6D). Previous work in a

Huntington’s disease model suggests that these cells may be

reactive astrocytes (Calkins et al., 2005). It is possible that

a small number of surviving nigral neurons of the lesioned

hemisphere may also be differentially active, as there is

observable basal hPAP expression in this region of the brain

(data not shown). This could explain the mechanism un-

derlying the expression of NQO1 observed in nigral neurons

of human PD brains (van Muiswinkel et al., 2004).The importance of ARE induction in PD pathogenesis is

currently being explored. Previously we have shown that Nrf2is important for determining the sensitivity of primary neurons

to complex I inhibitors (Lee et al., 2003b). Although the AREis induced by 6-OHDA, it is clear that this host response isinsufficient to quell pathogenesis (Fig. 5C). However, furtherinduction of the ARE may protect against cell death. Pre-liminary in vitro data shown herein imply that pre-activationwith tBHQ can protect against 6-OHDA-induced cell death.We have also shown that Nrf2-mediated protection is effica-cious in the malonate model of Huntington’s disease (Calkinset al., 2005). We are currently exploring the potential for usingARE inducers in vivo in the Parkinson’s disease animal models.Successful translation of this work into animal models of PDcould lead to new approaches for the treatment of PD viaactivation of the Nrf2-ARE pathway.

ACKNOWLEDGMENTS

This work was sponsored by grants ES08089 and ES10042 from NIEHS.

The authors disclose no conflicts of interest. R.J.J. is supported by a Wisconsin

FIG. 7. tBHQ protects against 6-OHDA-induced cell death. tBHQ (10 lM) is a known ARE activator (p ¼ 0.00001) that is significantly more potent than

6-OHDA (75 lM; p ¼ 0.01; A). A structural analog of tBHQ, dtBHQ, fails to activate the ARE (A). Data shown as relative light units (RLU). Pretreatment with

tBHQ for 48 h lead to increased viability following 6-OHDA treatment as demonstrated by the MTS assay at two different doses (75 lM 6-OHDA, p ¼ 0.04;

125 lM 6-OHDA, p¼ 0.0006; B). C shows condensed TUNELþ nuclei (right) and Hoescht-stained nuclei (left). As compared to vehicle control (C, top panel), 75 lM

6-OHDA generated significantly more TUNELþ cells (C, middle panel). Pretreatment with tBHQ reduced the amount of TUNELþ cells (C, bottom panel).

Images taken at 20X. TUNELþ cells were quantified (D). Treatment with 6-OHDA significantly increases the number of TUNELþ cells (p ¼ 0.000001). tBHQ

pretreatment significantly reduced TUNELþ cells by 37% (p ¼ 0.01). *Significantly different from corresponding vehicle-treated control sample (p < 0.05).

#Significantly different from corresponding sample in the absence of tBHQ (p < 0.05).

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Distinguished Rath Fellowship. The authors wish to thank Marcus Calkins and

Andrew Kraft for helpful discussions.

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