Induction of the Protective Antioxidant Response Element Pathway by 6-Hydroxydopamine In Vivo and In Vitro
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《毒物学科学杂志》
Department of Pharmaceutical Sciences, School of Pharmacy, Medical Scientist Training Program, Medical School, Neuroscience Training Program, Center for Neuroscience, Molecular and Environmental Toxicology
Waisman Center, University of Wisconsin-Madison, Madison, Wisconsin 53705
ABSTRACT
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 pathogenesis. Little is known regarding the transcriptional changes that occur in Parkinson's disease. The antioxidant response element 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 antioxidant 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.
INTRODUCTION
Parkinson's disease (PD), the most common adult-onset neurodegenerative movement disorder, is characterized by loss of the pigmented dopaminergic neurons in the substantia nigra pars compacta leading to a loss of striatal dopamine. The hallmark features of PD include akinesia, tremor, rigidity, and postural instability. Most cases of PD are sporadic, with a minority caused by known mutations. Although the etiology of sporadic PD is unclear, oxidative stress, mitochondrial dysfunction, and excitotoxicity likely play a role in pathogenesis (Jenner and Olanow, 1998). Indirect evidence of reactive oxygen species (ROS) in PD has come from observations of increased oxidized end-products such as 8-hydroxy-2-deoxyguanosine, 4-hydroxy-2-nonenol, and protein carbonyls in post mortem brain tissue from patients with Parkinson's disease (Alam et al., 1997a,b; Castellani et al., 2002; Dexter et 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. Impairment of the respiratory chain can cause oxidative stress through superoxide production. There is evidence for complex I dysfunction in post mortem human brain from PD patients (Schapira et al. 1989, 1990). Indeed, PD is modeled in vitro and in vivo using complex I inhibitors such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), rotenone, and 6-hydroxydopamine (6-OHDA) (Betarbet et al., 2002). Mitochondrial inhibition can also generate free radicals via an excitotoxic mechanism (Albin and Greenamyre, 1992; Brouillet and Beal, 1993; Srivastava et al., 1993). Additionally, oxidative stress may be a consequence of high iron levels naturally present in the nigra, or due to changes in iron regulation observed in PD brains (Dexter et al., 1987, 1989b, 1990).
Another source of free radicals in PD may be intrinsic to the nigrostriatal dopaminergic system. Dopamine (DA), a catecholaminergic neurotransmitter, is essential to normal basal ganglia function; however, it can be oxidized to generate prooxidant species through autooxidation and enzymatic catabolism via monoamine 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 likely mediated by an oxidative stress mechanism (Hastings et al., 1996; Maker et al., 1981; Stokes et al., 2000). 6-OHDA, a hydroxylated analog of the DA used to model PD, is a catacholaminergic neurotoxin via mitochondrial complex I inhibition and oxidative stress (Adams et al., 1972; Soto-Otero et al., 2000), and may be formed via DA oxidation (Jellinger et al., 1995).
One cellular defense mechanism to cope with oxidative stress is the antioxidant response element (ARE), a cis-acting enhancer element that is upstream of many phase II detoxification and antioxidant enzymes such as heme oxygenase-I and glutathione-S-transferases (Friling et al., 1990; Rushmore et al., 1990, 1991; Rushmore and Pickett, 1990, 1991). The ARE is induced by xenobiotics, changes in the redox status, as well as catechol and quinone structures (Nguyen et al., 2004). NF-E2 related factor (Nrf2), a basic leucine zipper transcription factor, is known to drive ARE-mediated gene expression (Nguyen et al., 2004). Following exposure to activators, Nrf2 translocates to the nucleus where it binds the ARE and activates transcription (reviewed by Jaiswal, 2004). Nrf2-knockout mice demonstrate decreased basal activity of some ARE regulated genes and normal expression of others; however, these animals do not display inducible ARE activity (Lee et al., 2003a). Because of the prominent role of oxidative stress in PD, we hypothesized that the ARE may be induced in response to the cellular dysfunction specific to this disease.
Previous research from our lab has shown that cultured neurons from Nrf2 knockout mice are more vulnerable to 1-methyl-4-phenylpyridinium (MPP+) and rotenone (Lee et al., 2003b). This suggests that the ARE system is critical in mediating PD pathogenesis. ARE-inducers have been able to protect against death due to DA and 6-OHDA in vitro (Duffy et al., 1998; Hara et al., 2003). Analysis of post mortem PD brains has revealed increased ARE-regulated enzymes such as heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase-1 (NQO1) also suggesting the potential for common transcriptional regulation (Schipper et al., 1998; van Muiswinkel et al., 2004; Yoo et al., 2003).
The current work tests the hypothesis that 6-OHDA induces the ARE. Specifically, we evaluated (1) whether 6-OHDA activates the ARE in vivo and in vitro, (2) the roles of oxidative stress and excitotoxicity on ARE activation in vitro, and (3) whether further induction of the ARE with tert-butylhydroquinone (tBHQ) would protect 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-hPAP+/– 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
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 + azide (40 μm) or directly onto slides (20 μm). 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 + 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 μm frozen sections were stored at –20°C until thawed and rehydrated in TMN (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 TUNEL+ or Hoescht+ 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 reporter transgene were exposed to 6-OHDA (1, 25, or 75 μM) for 24 h at three time-points in vitro and harvested for hPAP activity (Johnson et al., 2002). 6-OHDA induced ARE activation in a dose-dependent fashion at all three time-points At 7DIV, 75 μM 6-OHDA was sufficient to induce an over 50-fold increase in ARE-hPAP activity over vehicle control (Fig. 1A). As time in vitro progressed, the degree of ARE activation increased (Fig. 1A). Pretreatment with antioxidants (N-acetylcysteine, catalase, and reduced glutathione) significantly reduced ARE activation due to 75 μM 6-OHDA by approximately 50% (Fig. 1B).
ARE Induction by 6-OHDA Is Not Contingent upon Ability to Cause Neurotoxicity
6-OHDA and diethyl maleate (DEM), a known ARE activator through an oxidative stress mechanism, activate the ARE as compared to vehicle control. As shown in Figure 1C, pretreatment with antioxidants was sufficient to significantly reduce ARE activation by 6-OHDA and DEM. In contrast, 75 μM MPP+ and 75 mM glutamate, known oxidative stressors, fail to activate the ARE at relevant doses as compared to vehicle control, with or without antioxidants (Fig. 1C).
6-OHDA-Induced ARE Activation Is Reduced by NMDA Receptor Antagonism
At 3 and 7 DIV, primary hPAP+ neurons were exposed to 6-OHDA (75 μM) with or without pretreatment with MK801 (10 μM) and/or antioxidants. As shown in Figure 1D, at both time points, 6-OHDA exposure led to significantly increased ARE activation (fold change over vehicle control) which was reduced by pretreatment with antioxidants. At 3DIV, pretreatment 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-induced ARE activation was significantly reduced, but not to the level of ARE activity in the presence of MK801 alone (Fig. 1D).
At 7DIV, when primary cortical cells are vulnerable to excitotoxicity (Frandsen and Schousboe, 1990), pretreatment with MK801 significantly reduced ARE activation by approximately 50% (Fig. 1D). Pretreatment with MK801, however, did not fully abolish ARE activity due to 6-OHDA. Pretreatment with antioxidants in addition to MK801 did not further attenuate ARE activation (Fig. 1D). There was no significant difference between 6-OHDA + antioxidants and MK801 + 6-OHDA + antioxidants, suggesting that MK801 is blocking ROS due to excitotoxicity.
ARE Activation Due to 6-OHDA Is Primarily in Astrocytes
Cultured neurons exposed to various conditions were assayed for hPAP histochemistry using the fluorescing substrate Vector Red followed by immunostaining for either GFAP or beta-III-tubulin to discern astrocytes versus neurons, respectively (Fig. 2). Vehicle treated cells showed very little ARE-hPAP histochemistry (Figs. 2A and 2B). Treatment with 6-OHDA generated ARE-hPAP histochemistry primarily in astrocytes (Fig. 2C) as opposed to neurons (Fig. 2D). Treatment with MPP+ did not reveal hPAP histochemistry in either astrocytes (Fig. 2E) or neurons (Fig. 2F) confirming hPAP activity measures in Figure 1C.
To confirm that increased hPAP activity correlates with protein expression, we examined heme oxygenase-1 (HO-1). HO-1 expression is known be regulated in part by the ARE and has been shown previously to correspond to striatal injury due to 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-1 induction (Fig. 3C).
In order to determine if ARE activation was a component of a more general neurotoxic response to complex I inhibitors, we assayed for cell death using the TUNEL-labeling.
Both 6-OHDA (75 μM) and MPP+ (75 μM) caused significantly increased apoptotic cell death as revealed by TUNEL staining and observable pyknotic nuclei in Hoescht-stained images (Fig. 4). However, as demonstrated in Figures 1 and 2, MPP+ failed to activate the ARE. This suggests that the structural properties and/or the mechanism of cell death due to 6-OHDA may account for its induction of the ARE.
6-OHDA Activates the ARE In Vivo in the Striatum and Brainstem
Thirty-two adult ARE-hPAP transgenic reporter mice
Tissue hPAP activity assays did not demonstrate induction due to 6-OHDA in tissues collected at 24 h post-injection (Fig. 5A). However, by seven days post-injection, hPAP activity was significantly activated in the brainstem and striatum as compared to contralateral vehicle control hemisphere. The greatest fold change activation due to 6-OHDA lesions was found in the striatum, which demonstrated over 6-fold activation as compared to paired vehicle-treated contralateral hemisphere (Fig. 5B). There was no change in the cortex, a negative control region, due to 6-OHDA at 7 days (data not shown).
Increased ARE activity correlates with loss of tyrosine hydroxylase immunoreactivity (THir) as seen in sections from identically treated animals in a parallel study (Fig. 5C). At 24 h, there is no loss of THir; however, by one week, the 6-OHDA lesion 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 and counterstained with nuclear fast red. At 24 h, there were no hPAP+ cells present (data not shown). This agrees with data from tissue hPAP assays which did not reveal changes in ARE activity at 24 h (Fig. 5A). hPAP-negative tissue did not demonstrate any staining at any time point assayed (Figs. 6A and 6E). At 96 h post-injection, half of the animals assayed demonstrated hPAP+ 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 demonstrated hPAP+ cells encroaching into the core of the lesion (Figs. 6G and 6H), but not in the vehicle treated hemisphere (Fig. 6F). No visible increase in hPAP+ cells was seen in the brainstem (data not shown). The issue of specific cell type expressing hPAP in and around the lesion is discussed subsequently.
Induction of ARE Can Reduce Cell Death Due to 6-OHDA In Vitro
tBHQ (10 μM), a known ARE activator, can cause an over 30-fold induction in ARE activity, significantly more potent than 6-OHDA (75 μM; Fig. 7A). dtBHQ, a structural analog of tBHQ, does not activate the ARE and was used as a negative control (Fig. 7B). Treatment with both tBHQ, and 6-OHDA does not significantly increase ARE induction over tBHQ alone (Fig. 7A). This suggests tBHQ (10 μM) saturates the Nrf2-ARE induction cascade.
Primary cortical cells were exposed to 6-OHDA for 24 h following 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 increased viability as compared to vehicle pretreated cells (Fig. 7B).
Cells from the same culture were plated in chamber slides and exposed to 6-OHDA (75 μM) following pretreatment with vehicle or tBHQ. After 24 h, cells were fixed and assessed for apoptotic nuclei using the TUNEL assay and counterstained with Hoescht to indicate total cells in the field (Fig. 7C). 6-OHDA caused significantly increased TUNEL+ cells (Fig. 7C, middle panel and D) as compared to vehicle control (Fig. 7C, top panel). Pretreatment with tBHQ decreased the amount of TUNEL+ cells by approximately 35% indicating a reduction in apoptosis (Fig. 7C, bottom panel and D).
DISCUSSION
In the current study, we have shown that 6-OHDA, a catecholaminergic neurotoxin used to model PD, activates the ARE both in vivo and in vitro. Oxidative stress is a critical factor in PD pathogenesis and consequently, we hypothesized that the cellular injury in PD may lead to activation of the ARE. Although known ARE-regulated genes such as HO-1 and NQO1 are increased in the PD brain (Schipper et al., 1998; van Muiswinkel et al., 2004), the nature of the regulation of these changes on a transcriptional level has not been elucidated. The ARE is an enhancer sequence found in the promoter of many cytoprotective genes. Oxidative stress and xenobiotic exposures can lead to Nrf2 translocation to the nucleus and subsequent ARE-regulated transcription. In this way, the ARE can coordinate the upregulation of a multitude of protective genes with a single insult.
In primary neuronal cultures from reporter mice, 6-OHDA activated the ARE in a dose-dependent fashion over a seven-day period (Fig. 1A). ARE-driven hPAP activity was observed primarily in astrocytes rather than in neurons (Figs. 2C and 2D). This agrees with previous work that ARE-mediated activity is primarily induced in astrocytes in vitro (Eftekharpour et al., 2000; Kraft et al., 2004; Shih et al., 2003). ARE activation due to 6-OHDA (75 μM) was reduced but not eliminated in the presence of antioxidants (Figs. 1B–1D). At 7DIV, pretreatment with MK801, an NMDA antagonist, also reduced, but did not eliminate ARE activity (Fig. 1D). Antioxidants in combination with MK801 did not further reduce the ARE activation. Therefore, 6-OHDA activates the ARE by a combination of factors including oxidative stress generated in part through an excitotoxic mechanism. In addition, 6-OHDA may activate the ARE due to its catecholamine structure that is independent of ROS formation. The latter mechanism of activation is probably the same used by tBHQ.
DA and its metabolites share structural similarities to tBHQ and hydroquinone. tBHQ activates the ARE without producing ROS, suggesting that its mode of induction is purely structural. MPP+, another chemical used to model of PD, does not induce the ARE (Figs. 1B, 2E, and 2F) in cell culture and lacks structural similarities to known ARE activators. Experiments designed to determine the effect of MPTP administration in vivo are currently underway. The pro-oxidant nature of the quinones and catecholamines suggests that DA breakdown may be a contributing factor to PD pathogenesis. However, these chemicals, by virtue of their structure, may induce the ARE. If 6-OHDA and DA behave like tBHQ in the ARE induction cascade, it is possible that they alter the redox status of Keap1 and stabilize Nrf2 protein, allowing for enhanced binding to the ARE (Dinkova-Kostova et al., 2002; Nguyen et al., 2003). Further studies are needed to confirm the mechanism of direct activation of the ARE by catecholamines like 6-OHDA.
Direct intrastriatal administration of 6-OHDA in vivo lesions the nigrostiatal dopaminergic pathway modeling PD pathology in the live animal. 6-OHDA induces ARE activation in ARE reporter mice at one week, but not 24 h post-injection (Fig. 5). 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 underlying 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 Nrf2 is important for determining the sensitivity of primary neurons to complex I inhibitors (Lee et al., 2003b). Although the ARE is induced by 6-OHDA, it is clear that this host response is insufficient to quell pathogenesis (Fig. 5C). However, further induction of the ARE may protect against cell death. Preliminary in vitro data shown herein imply that pre-activation with tBHQ can protect against 6-OHDA-induced cell death. We have also shown that Nrf2-mediated protection is efficacious in the malonate model of Huntington's disease (Calkins et al., 2005). We are currently exploring the potential for using ARE inducers in vivo in the Parkinson's disease animal models. Successful translation of this work into animal models of PD could lead to new approaches for the treatment of PD via activation of the Nrf2-ARE pathway.
NOTES
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.
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 Distinguished Rath Fellowship. The authors wish to thank Marcus Calkins and Andrew Kraft for helpful discussions.
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Waisman Center, University of Wisconsin-Madison, Madison, Wisconsin 53705
ABSTRACT
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 pathogenesis. Little is known regarding the transcriptional changes that occur in Parkinson's disease. The antioxidant response element 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 antioxidant 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.
INTRODUCTION
Parkinson's disease (PD), the most common adult-onset neurodegenerative movement disorder, is characterized by loss of the pigmented dopaminergic neurons in the substantia nigra pars compacta leading to a loss of striatal dopamine. The hallmark features of PD include akinesia, tremor, rigidity, and postural instability. Most cases of PD are sporadic, with a minority caused by known mutations. Although the etiology of sporadic PD is unclear, oxidative stress, mitochondrial dysfunction, and excitotoxicity likely play a role in pathogenesis (Jenner and Olanow, 1998). Indirect evidence of reactive oxygen species (ROS) in PD has come from observations of increased oxidized end-products such as 8-hydroxy-2-deoxyguanosine, 4-hydroxy-2-nonenol, and protein carbonyls in post mortem brain tissue from patients with Parkinson's disease (Alam et al., 1997a,b; Castellani et al., 2002; Dexter et 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. Impairment of the respiratory chain can cause oxidative stress through superoxide production. There is evidence for complex I dysfunction in post mortem human brain from PD patients (Schapira et al. 1989, 1990). Indeed, PD is modeled in vitro and in vivo using complex I inhibitors such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), rotenone, and 6-hydroxydopamine (6-OHDA) (Betarbet et al., 2002). Mitochondrial inhibition can also generate free radicals via an excitotoxic mechanism (Albin and Greenamyre, 1992; Brouillet and Beal, 1993; Srivastava et al., 1993). Additionally, oxidative stress may be a consequence of high iron levels naturally present in the nigra, or due to changes in iron regulation observed in PD brains (Dexter et al., 1987, 1989b, 1990).
Another source of free radicals in PD may be intrinsic to the nigrostriatal dopaminergic system. Dopamine (DA), a catecholaminergic neurotransmitter, is essential to normal basal ganglia function; however, it can be oxidized to generate prooxidant species through autooxidation and enzymatic catabolism via monoamine 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 likely mediated by an oxidative stress mechanism (Hastings et al., 1996; Maker et al., 1981; Stokes et al., 2000). 6-OHDA, a hydroxylated analog of the DA used to model PD, is a catacholaminergic neurotoxin via mitochondrial complex I inhibition and oxidative stress (Adams et al., 1972; Soto-Otero et al., 2000), and may be formed via DA oxidation (Jellinger et al., 1995).
One cellular defense mechanism to cope with oxidative stress is the antioxidant response element (ARE), a cis-acting enhancer element that is upstream of many phase II detoxification and antioxidant enzymes such as heme oxygenase-I and glutathione-S-transferases (Friling et al., 1990; Rushmore et al., 1990, 1991; Rushmore and Pickett, 1990, 1991). The ARE is induced by xenobiotics, changes in the redox status, as well as catechol and quinone structures (Nguyen et al., 2004). NF-E2 related factor (Nrf2), a basic leucine zipper transcription factor, is known to drive ARE-mediated gene expression (Nguyen et al., 2004). Following exposure to activators, Nrf2 translocates to the nucleus where it binds the ARE and activates transcription (reviewed by Jaiswal, 2004). Nrf2-knockout mice demonstrate decreased basal activity of some ARE regulated genes and normal expression of others; however, these animals do not display inducible ARE activity (Lee et al., 2003a). Because of the prominent role of oxidative stress in PD, we hypothesized that the ARE may be induced in response to the cellular dysfunction specific to this disease.
Previous research from our lab has shown that cultured neurons from Nrf2 knockout mice are more vulnerable to 1-methyl-4-phenylpyridinium (MPP+) and rotenone (Lee et al., 2003b). This suggests that the ARE system is critical in mediating PD pathogenesis. ARE-inducers have been able to protect against death due to DA and 6-OHDA in vitro (Duffy et al., 1998; Hara et al., 2003). Analysis of post mortem PD brains has revealed increased ARE-regulated enzymes such as heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase-1 (NQO1) also suggesting the potential for common transcriptional regulation (Schipper et al., 1998; van Muiswinkel et al., 2004; Yoo et al., 2003).
The current work tests the hypothesis that 6-OHDA induces the ARE. Specifically, we evaluated (1) whether 6-OHDA activates the ARE in vivo and in vitro, (2) the roles of oxidative stress and excitotoxicity on ARE activation in vitro, and (3) whether further induction of the ARE with tert-butylhydroquinone (tBHQ) would protect 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-hPAP+/– 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
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 + azide (40 μm) or directly onto slides (20 μm). 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 + 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 μm frozen sections were stored at –20°C until thawed and rehydrated in TMN (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 TUNEL+ or Hoescht+ 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 reporter transgene were exposed to 6-OHDA (1, 25, or 75 μM) for 24 h at three time-points in vitro and harvested for hPAP activity (Johnson et al., 2002). 6-OHDA induced ARE activation in a dose-dependent fashion at all three time-points At 7DIV, 75 μM 6-OHDA was sufficient to induce an over 50-fold increase in ARE-hPAP activity over vehicle control (Fig. 1A). As time in vitro progressed, the degree of ARE activation increased (Fig. 1A). Pretreatment with antioxidants (N-acetylcysteine, catalase, and reduced glutathione) significantly reduced ARE activation due to 75 μM 6-OHDA by approximately 50% (Fig. 1B).
ARE Induction by 6-OHDA Is Not Contingent upon Ability to Cause Neurotoxicity
6-OHDA and diethyl maleate (DEM), a known ARE activator through an oxidative stress mechanism, activate the ARE as compared to vehicle control. As shown in Figure 1C, pretreatment with antioxidants was sufficient to significantly reduce ARE activation by 6-OHDA and DEM. In contrast, 75 μM MPP+ and 75 mM glutamate, known oxidative stressors, fail to activate the ARE at relevant doses as compared to vehicle control, with or without antioxidants (Fig. 1C).
6-OHDA-Induced ARE Activation Is Reduced by NMDA Receptor Antagonism
At 3 and 7 DIV, primary hPAP+ neurons were exposed to 6-OHDA (75 μM) with or without pretreatment with MK801 (10 μM) and/or antioxidants. As shown in Figure 1D, at both time points, 6-OHDA exposure led to significantly increased ARE activation (fold change over vehicle control) which was reduced by pretreatment with antioxidants. At 3DIV, pretreatment 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-induced ARE activation was significantly reduced, but not to the level of ARE activity in the presence of MK801 alone (Fig. 1D).
At 7DIV, when primary cortical cells are vulnerable to excitotoxicity (Frandsen and Schousboe, 1990), pretreatment with MK801 significantly reduced ARE activation by approximately 50% (Fig. 1D). Pretreatment with MK801, however, did not fully abolish ARE activity due to 6-OHDA. Pretreatment with antioxidants in addition to MK801 did not further attenuate ARE activation (Fig. 1D). There was no significant difference between 6-OHDA + antioxidants and MK801 + 6-OHDA + antioxidants, suggesting that MK801 is blocking ROS due to excitotoxicity.
ARE Activation Due to 6-OHDA Is Primarily in Astrocytes
Cultured neurons exposed to various conditions were assayed for hPAP histochemistry using the fluorescing substrate Vector Red followed by immunostaining for either GFAP or beta-III-tubulin to discern astrocytes versus neurons, respectively (Fig. 2). Vehicle treated cells showed very little ARE-hPAP histochemistry (Figs. 2A and 2B). Treatment with 6-OHDA generated ARE-hPAP histochemistry primarily in astrocytes (Fig. 2C) as opposed to neurons (Fig. 2D). Treatment with MPP+ did not reveal hPAP histochemistry in either astrocytes (Fig. 2E) or neurons (Fig. 2F) confirming hPAP activity measures in Figure 1C.
To confirm that increased hPAP activity correlates with protein expression, we examined heme oxygenase-1 (HO-1). HO-1 expression is known be regulated in part by the ARE and has been shown previously to correspond to striatal injury due to 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-1 induction (Fig. 3C).
In order to determine if ARE activation was a component of a more general neurotoxic response to complex I inhibitors, we assayed for cell death using the TUNEL-labeling.
Both 6-OHDA (75 μM) and MPP+ (75 μM) caused significantly increased apoptotic cell death as revealed by TUNEL staining and observable pyknotic nuclei in Hoescht-stained images (Fig. 4). However, as demonstrated in Figures 1 and 2, MPP+ failed to activate the ARE. This suggests that the structural properties and/or the mechanism of cell death due to 6-OHDA may account for its induction of the ARE.
6-OHDA Activates the ARE In Vivo in the Striatum and Brainstem
Thirty-two adult ARE-hPAP transgenic reporter mice
Tissue hPAP activity assays did not demonstrate induction due to 6-OHDA in tissues collected at 24 h post-injection (Fig. 5A). However, by seven days post-injection, hPAP activity was significantly activated in the brainstem and striatum as compared to contralateral vehicle control hemisphere. The greatest fold change activation due to 6-OHDA lesions was found in the striatum, which demonstrated over 6-fold activation as compared to paired vehicle-treated contralateral hemisphere (Fig. 5B). There was no change in the cortex, a negative control region, due to 6-OHDA at 7 days (data not shown).
Increased ARE activity correlates with loss of tyrosine hydroxylase immunoreactivity (THir) as seen in sections from identically treated animals in a parallel study (Fig. 5C). At 24 h, there is no loss of THir; however, by one week, the 6-OHDA lesion 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 and counterstained with nuclear fast red. At 24 h, there were no hPAP+ cells present (data not shown). This agrees with data from tissue hPAP assays which did not reveal changes in ARE activity at 24 h (Fig. 5A). hPAP-negative tissue did not demonstrate any staining at any time point assayed (Figs. 6A and 6E). At 96 h post-injection, half of the animals assayed demonstrated hPAP+ 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 demonstrated hPAP+ cells encroaching into the core of the lesion (Figs. 6G and 6H), but not in the vehicle treated hemisphere (Fig. 6F). No visible increase in hPAP+ cells was seen in the brainstem (data not shown). The issue of specific cell type expressing hPAP in and around the lesion is discussed subsequently.
Induction of ARE Can Reduce Cell Death Due to 6-OHDA In Vitro
tBHQ (10 μM), a known ARE activator, can cause an over 30-fold induction in ARE activity, significantly more potent than 6-OHDA (75 μM; Fig. 7A). dtBHQ, a structural analog of tBHQ, does not activate the ARE and was used as a negative control (Fig. 7B). Treatment with both tBHQ, and 6-OHDA does not significantly increase ARE induction over tBHQ alone (Fig. 7A). This suggests tBHQ (10 μM) saturates the Nrf2-ARE induction cascade.
Primary cortical cells were exposed to 6-OHDA for 24 h following 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 increased viability as compared to vehicle pretreated cells (Fig. 7B).
Cells from the same culture were plated in chamber slides and exposed to 6-OHDA (75 μM) following pretreatment with vehicle or tBHQ. After 24 h, cells were fixed and assessed for apoptotic nuclei using the TUNEL assay and counterstained with Hoescht to indicate total cells in the field (Fig. 7C). 6-OHDA caused significantly increased TUNEL+ cells (Fig. 7C, middle panel and D) as compared to vehicle control (Fig. 7C, top panel). Pretreatment with tBHQ decreased the amount of TUNEL+ cells by approximately 35% indicating a reduction in apoptosis (Fig. 7C, bottom panel and D).
DISCUSSION
In the current study, we have shown that 6-OHDA, a catecholaminergic neurotoxin used to model PD, activates the ARE both in vivo and in vitro. Oxidative stress is a critical factor in PD pathogenesis and consequently, we hypothesized that the cellular injury in PD may lead to activation of the ARE. Although known ARE-regulated genes such as HO-1 and NQO1 are increased in the PD brain (Schipper et al., 1998; van Muiswinkel et al., 2004), the nature of the regulation of these changes on a transcriptional level has not been elucidated. The ARE is an enhancer sequence found in the promoter of many cytoprotective genes. Oxidative stress and xenobiotic exposures can lead to Nrf2 translocation to the nucleus and subsequent ARE-regulated transcription. In this way, the ARE can coordinate the upregulation of a multitude of protective genes with a single insult.
In primary neuronal cultures from reporter mice, 6-OHDA activated the ARE in a dose-dependent fashion over a seven-day period (Fig. 1A). ARE-driven hPAP activity was observed primarily in astrocytes rather than in neurons (Figs. 2C and 2D). This agrees with previous work that ARE-mediated activity is primarily induced in astrocytes in vitro (Eftekharpour et al., 2000; Kraft et al., 2004; Shih et al., 2003). ARE activation due to 6-OHDA (75 μM) was reduced but not eliminated in the presence of antioxidants (Figs. 1B–1D). At 7DIV, pretreatment with MK801, an NMDA antagonist, also reduced, but did not eliminate ARE activity (Fig. 1D). Antioxidants in combination with MK801 did not further reduce the ARE activation. Therefore, 6-OHDA activates the ARE by a combination of factors including oxidative stress generated in part through an excitotoxic mechanism. In addition, 6-OHDA may activate the ARE due to its catecholamine structure that is independent of ROS formation. The latter mechanism of activation is probably the same used by tBHQ.
DA and its metabolites share structural similarities to tBHQ and hydroquinone. tBHQ activates the ARE without producing ROS, suggesting that its mode of induction is purely structural. MPP+, another chemical used to model of PD, does not induce the ARE (Figs. 1B, 2E, and 2F) in cell culture and lacks structural similarities to known ARE activators. Experiments designed to determine the effect of MPTP administration in vivo are currently underway. The pro-oxidant nature of the quinones and catecholamines suggests that DA breakdown may be a contributing factor to PD pathogenesis. However, these chemicals, by virtue of their structure, may induce the ARE. If 6-OHDA and DA behave like tBHQ in the ARE induction cascade, it is possible that they alter the redox status of Keap1 and stabilize Nrf2 protein, allowing for enhanced binding to the ARE (Dinkova-Kostova et al., 2002; Nguyen et al., 2003). Further studies are needed to confirm the mechanism of direct activation of the ARE by catecholamines like 6-OHDA.
Direct intrastriatal administration of 6-OHDA in vivo lesions the nigrostiatal dopaminergic pathway modeling PD pathology in the live animal. 6-OHDA induces ARE activation in ARE reporter mice at one week, but not 24 h post-injection (Fig. 5). 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 underlying 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 Nrf2 is important for determining the sensitivity of primary neurons to complex I inhibitors (Lee et al., 2003b). Although the ARE is induced by 6-OHDA, it is clear that this host response is insufficient to quell pathogenesis (Fig. 5C). However, further induction of the ARE may protect against cell death. Preliminary in vitro data shown herein imply that pre-activation with tBHQ can protect against 6-OHDA-induced cell death. We have also shown that Nrf2-mediated protection is efficacious in the malonate model of Huntington's disease (Calkins et al., 2005). We are currently exploring the potential for using ARE inducers in vivo in the Parkinson's disease animal models. Successful translation of this work into animal models of PD could lead to new approaches for the treatment of PD via activation of the Nrf2-ARE pathway.
NOTES
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.
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 Distinguished Rath Fellowship. The authors wish to thank Marcus Calkins and Andrew Kraft for helpful discussions.
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