Superoxide Dismutase/Catalase Mimetics Are Neuroprotective against Selective Paraquat-mediated Dopaminergic Neuron Death in the Substantial Nigra

Exposure of mice to the herbicide paraquat has been demonstrated to result in the selective loss of dopaminergic neurons of the substantia nigra, pars compacta (SNpc) akin to what is observed in Parkinson disease (PD). In this study, we investigate the efficacy of two synthetic superoxide dismutase/catalase mimetics (EUK-134 and EUK-189) in protecting against paraquat-induced dopaminergic cell death in both the rat dopaminergic cell line 1RB3AN27 (N27) and primary mesencephalic cultures in vitro and in adult mice in vivo. Our data demonstrate that pretreatment with either EUK-134 or EUK-189 significantly attenuates paraquat-induced neurotoxicity in vitro in a concentration-dependent manner. Furthermore, systemic administration of EUK-189 decreases paraquat-mediated SNpc dopaminergic neuronal cell death in vivo. These findings support a role for oxidative stress in paraquat-induced neurotoxicity and suggest novel therapeutic approaches for neurodegenerative disorders associated with oxidative stress such as PD.


Exposure of mice to the herbicide paraquat has been demonstrated to result in the selective loss of dopaminergic neurons of the substantia nigra, pars compacta (SNpc) akin to what is observed in Parkinson disease (PD). In this study, we investigate the efficacy of two synthetic superoxide dismutase/catalase mimetics (EUK-134 and EUK-189) in protecting against paraquatinduced dopaminergic cell death in both the rat dopaminergic cell line 1RB 3 AN 27 (N27) and primary mesencephalic cultures in vitro and in adult mice in vivo. Our data demonstrate that pretreatment with either EUK-134 or EUK-189 significantly attenuates paraquat-induced neurotoxicity in vitro in a concentration-dependent manner. Furthermore, systemic administration of EUK-189 decreases paraquat-mediated SNpc dopaminergic neuronal cell death in vivo.
These findings support a role for oxidative stress in paraquat-induced neurotoxicity and suggest novel therapeutic approaches for neurodegenerative disorders associated with oxidative stress such as PD.
Parkinson disease (PD) 1 is a common age-related neurodegenerative disease that is pathologically characterized by the selective loss of nigrostriatal dopaminergic neurons in the substantia nigra pars compacta (SNpc) region of the ventral midbrain and the presence of ubiquinated protein deposits in residual neurons (Lewy bodies) (1,2). Epidemiological studies have suggested that exposure to agricultural chemicals is associated with an increased risk of developing PD (3)(4)(5)(6)(7)(8)(9). The herbicide 1,1Ј-dimethyl-4,4Ј-bipyridium (paraquat, PQ) may contribute to the pathogenesis of PD based on both epidemiological studies of parkinsonism correlated with exposure to the agent (3,7), and its structural similarity to the active metabolite of the parkinsonism-inducing agent 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, 1-methyl-4-phenylpyridium ion. Exposure of mice to either paraquat alone or in combination with the dithiocarbamate fungicide manganese ethylenebisdithiocarbamate results in preferential degeneration of dopaminergic nigrostriatal neurons, mimicking the cell loss observed in PD (10,11). Recently, we reported that paraquat administration triggers an apoptotic cell death program through oxidative stress-mediated activation of the JNK signaling pathway, suggesting a possible mechanism for selective dopaminergic neuron loss (12).
The molecular mechanisms that support the feasibility for the potential use of the salen manganese complexes as therapy for PD are far from being completely elucidated. The results of the current investigation demonstrate, for the first time, the capacity of these potentially therapeutic antioxidant compounds to neuroprotect against selective dopaminergic midbrain cell death not only in vitro but also in vivo in an animal model of the disease.
Cell Culture-The rat dopaminergic cell line 1RB 3 AN 27 (N27) was grown in RPMI 1640 medium supplemented with 10% fetal calf serum (Invitrogen), 100 units/ml penicillin, and 100 g/ml streptomycin. Cell * This work was supported by National Institutes of Health Grant U54 ES12077 (to J. K. A.). S. R. D. is Vice President, Research at Eukarion, Inc. and has been granted options to purchase stock in the company. EUK-189 is under development by Eukarion as a potential therapeutic product. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Primary Mesencephalic Cultures-Primary mesencephalic cell cultures were prepared from embryonic gestation day 14 -15 mouse embryos as described previously (12). Briefly, dissociated cells were seeded at 7 ϫ 10 5 cells per well onto poly-D-lysine-coated 24-well culture plates. Cultures were maintained at 37°C in a humidified atmosphere containing 95% air and 5% carbon dioxide, in Neurobasal medium (Invitrogen) containing 2% B27 supplement, 2 mM glutamate, 100 units/ml penicillin, and 100 g/ml streptomycin. After 4 days, one-half of the medium was replaced with fresh medium. Cells were grown an additional 2 days and then treated with 40 M paraquat for 18 or 24 h. The number of tyrosine hydroxylase (TH)-positive neurons in mesencephalic cultures was determined as described previously (12). The specificity of neurotoxicity was analyzed by double label immunostaining with the anti-TH antibody and antibodies against phospho-JNK, phospho-c-Jun, and cleaved caspase-3, respectively, as described previously (12). Experiments were repeated with cultures isolated from four independent dissections.
Paraquat Administration-Mice were intraperitoneally injected with either saline or 7 mg/kg paraquat (dissolved in saline) at 2-day intervals for a total of 10 doses. Animals were killed at day 7 or 8 after the last administration as previously described (12). Experimental protocols were in accordance with the National Institutes of Health Guidelines for Use of Live Animals and were approved by the Animal Care and Use Committee at the Buck Institute of Age Research.
Stereological SN TH-positive Neuron Counts-Littermates were fixed by perfusion as previously described (12). Cryostat-cut sections (40 m) were taken through the entire midbrain. TH-positive neurons were immunolabeled by incubating the tissue sections successively with a rabbit polyclonal anti-TH antibody (1:200) and biotinylated horse anti-rabbit IgG (1:200, Vector Laboratories) and following the staining procedure outlined by the manufacturers of Vectastain ABC kit (Vector Laboratories) in combination with 3,3Ј-diaminobenzidine (DAB) reagents. The total number of TH-positive neurons in the substantia nigra pars compacta was counted from four to five littermates per group by using the optical fractionator method, an unbiased stereological technique of cell counting (23), as previously described (12).
Western Blot Analysis-Total protein was isolated from brain tissue as described previously (12). Protein concentration of the supernatant was determined using a commercially available protein assay kit (Bio-Rad). Equal concentrations of protein extracts were electrophoretically resolved on SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. Primary antibodies for Western blot analysis were used at the following dilutions: phospho-JNK (1:1000), phosphoc-Jun (1:1000), caspase-3 (1:1000), and ␤-actin (1:5000). Detection was performed using horseradish peroxidase-conjugated secondary antibody and an ECL kit (Amersham Biosciences).
Statistical Analysis-All data are expressed as mean Ϯ S.E. for the number (n) of independent experiments performed. Differences among the means for all experiments described were analyzed using one-or two-way analysis of variance. Newman-Keuls post-hoc analysis was employed when differences were observed by analysis of variance testing (p Ͻ 0.05).

Protective Effects of EUK Compounds on Paraquat-induced Neurotoxicity in a Dopaminergic Midbrain-derived Rat Cell
Line-We used two separate salen manganese complexes, EUK-134 and EUK-189, in this study (Fig. 1). EUK-189 has both SOD and catalase activities equivalent to those of EUK-134 but has increased lipophilicity and neuroprotective activity (20). N27 is an immortalized dopaminergic neuronal cell line isolated from fetal rat mesencephalic cultures (24). The N27 cell line produces dopamine and expresses the dopamine-synthesizing enzyme tyrosine hydroxylase (TH) and the dopamine transport (DAT). Recently, we used this cell line as a model to study the potential role of paraquat on the JNK signaling pathway, because it relates to dopaminergic cell death associated with PD (12). As previously shown (12), treatment with 400 M paraquat for 18 -24 h increased caspase-3 activation, cell death, and DNA fragmentation compared with untreated control. However, when EUK-134 or EUK-189 (15 or 30 M) was added 1 h before addition of paraquat, caspase-3 activation, cell death, and DNA fragmentation were all significantly inhibited (Fig. 2). In the case of 30 M EUK-189, paraquatmediated increases in caspase-3 activation, cell death, and DNA fragmentation were inhibited by 60%, 85%, and 77%, respectively (Fig. 2). Neither EUK-134 nor EUK-189 at the concentrations used were stimulatory or inhibitory for neuronal survival without paraquat (data not shown).
Lactacystin is a selective proteasome inhibitor; even at high concentration and long treatment times, it does not significantly inhibit other proteases (25). To study whether the effects of the salen manganese complexes were specific for oxidative stress-induced cell death, N27 cells were treated with EUK-134 or EUK-189 (15 or 30 M) for 1 h prior to treatment with 5 M lactacystin. Cell death and DNA fragmentation were analyzed by MTT and TUNEL staining methods at 24 h, respectively. As seen in Fig. 3, there was no difference in lactacystin-induced apoptotic death in the absence or presence of EUK compounds.
Protective Effects of EUK Compounds on Paraquat-induced Dopaminergic Neurotoxicity in Primary Mesencephalic Cultures Is Due to Inhibition of JNK Pathway Activation-Our previous results suggested that paraquat-generated superoxide may lead to activation of the JNK signaling pathway resulting in subsequent dopaminergic neuronal apoptosis, because these could both be attenuated in vitro by either the SOD mimetic, manganese(III) tetrakis (4-benzoic acid) porphyrin (MnTBAP), or the JNK-specific inhibitor SP600125 (12). To assess the neuroprotective ability of the salen manganese complexes in relation to paraquat-induced cell death on a cellular level in primary dopamine midbrain neurons, we examined the effects of EUK-134 or EUK-189 administration on paraquattreated primary mesencephalic cultures via dual immunofluorescence with antibodies specific for TH and either phospho-JNK, phospho-c-Jun, or cleaved caspase-3, respectively, coupled with 4Ј,6-diamidino-2-phenylindole staining. Cultures were pretreated with EUK-134 (0.5 M) or EUK-189 (0.5 M) 1 h prior to treatment with 40 M paraquat. As shown in Fig.  4A, EUK-134 or EUK-189 both separately reduced the colocalization of phospho-JNK, phospho-c-Jun, and activated caspase-3 with TH-positive neurons after 18 h of paraquat treatment. Cells were next stained for TH at 24 h following paraquat treatment, and TH-positive neurons were counted. Pretreatment with EUK-134 or EUK-189 was found to protect TH-positive neurons from paraquat-induced cell death (Fig.  4B). These data, coupled with our previous findings (12), suggest that the salen manganese complexes attenuate paraquatinduced neuronal cell death via inhibition of JNK activation and c-Jun phosphorylation.  (12). A, neurons that were positive for phospho-JNK (black bar), phospho-c-Jun (white bar), and activated caspase-3 (striped bar) among TH-positive neurons were counted 18 h after paraquat treatment, respectively, as previously described (12). B, TH-positive neuron counts in paraquat-treated mesencephalic cultures 24 h after paraquat treatment. Mean Ϯ S.E., n ϭ 4. *, p Ͻ 0.01, significantly from paraquat.

EUK-189 Inhibits Paraquat-induced Reduction in TH-positive SNpc Cell Numbers in Vivo via Inhibition of JNK Pathway
Activation-To examine whether EUK-189 attenuates the selective loss of nigrostriatal dopamine neurons after paraquat administration in vivo, we implanted mice with pumps containing either 5% mannitol (as vehicle control) or 15 mM EUK-189 1 day prior to paraquat treatment. As previously demonstrated (12), exposure of mice to paraquat produced a substantial loss of nigral dopamine neurons when compared with unlesioned controls (Fig. 5A). However, subcutaneous administration of the EUK-189 significantly attenuated the loss of nigral dopamine neurons when examined at day 8 following the last paraquat treatment (Fig. 5A). In keeping with previous studies (11,12), stereological analysis revealed that paraquat administration resulted in an ϳ29% loss of substantia nigra pars compacta (SNpc) TH-positive neurons compared with salinetreated controls (Fig. 5B). In contrast, paraquat-treated mice that received EUK-189 displayed a significant reduction in the loss of SNpc dopaminergic neurons compared with animals treated paraquat alone (Fig. 5B). Indeed, the number of SNpc TH-positive neurons in EUK-189-treated animals did not significantly differ from saline-treated controls following paraquat administration.
To investigate whether inhibition of the JNK apoptotic cascade contributed to the neuroprotection conferred by EUK-189 following paraquat injection, the levels of phospho-JNK, phospho-c-Jun, and cleaved caspase-3 were detected by Western blot analysis of SNpc tissues. As previously demonstrated (12), the levels of phosphorylated JNK, phosphorylated c-Jun, and cleaved caspase-3 were all enhanced in SNpc tissues prepared from paraquat-treated mice compared with SNpc tissues prepared from mice in the saline treatment group (Fig. 6). However, pretreatment with EUK-189 completely suppressed the paraquat-induced increase in the levels of phosphorylation of JNK and c-Jun and caspase-3 cleavage (Fig. 6). DISCUSSION In the present study, we demonstrate that pretreatment of dopaminergic cultures in vitro or systemic treatment of mice in vivo with synthetic catalytic scavengers of reactive oxygen species, the salen manganese complexes EUK-134 and EUK-189, conferred neuroprotection against selective paraquat-mediated dopaminergic nigral cell death. In vitro and in vivo evidence from both our previous work (12) and this current study indicate that these compounds likely attenuate paraquat-induced neuron damage via inhibition of the activation of JNK-mediated apoptosis.
With increased understanding both of the mechanisms of oxidative stress and the role of antioxidants, it has become apparent that antioxidant defense systems normally exist in a balance with endogenous reactive oxygen species. Disruption of this balance appears to be one of the major factors involved in the selective neuropathogenesis associated with PD (26). Studies have shown, for example, that the levels of the thiol-reducing agent glutathione decrease within the SN of patients with Parkinson disease (27,28), whereas the concentrations of iron, which can act as a catalyst for detrimental oxidative reactions, are elevated (29,30). Furthermore, the levels of the DNA oxidation by-product 8-hydroxy-2Ј-deoxyguanosine (31,32), lipid peroxidation (33), and the protein oxidation by-product 4-hydroxy-2-nonenal (34) are all elevated in the SN of PD patients versus age-matched controls, suggesting an involvement of oxidation stress in the ensuing midbrain dopaminergic cell loss.
Some epidemiological investigations have suggested that increased exposure to agricultural chemicals via living in a rural environment, drinking well water, or occupational exposure may be a potential environmental risk factor for the disease (3)(4)(5)(6)(7)(8)(9). The widely used herbicide paraquat has been demonstrated to selectively damage the nigrostriatal dopaminergic system (11) and exposure to this specific agricultural chemical has been postulated to be a prime risk factor for PD (3,7). Experimental paraquat exposure in vivo causes brain ␣-synuclein aggregation and formation of Lewy body-like neuronal occlusions (35). The mechanisms of neurotoxicity associated with exposure to paraquat are most likely mediated via oxidative stress. Mutations in the DJ-1 gene are linked with autosomal recessive early onset familial form of PD (36), and paraquat has been shown to oxidize the DJ-1 protein (37).
Superoxide anion radicals can be generated by paraquat through both redox cycling via reaction with molecular oxygen and electron transfer reactions with NADH-dependent oxidoreductases (38 -40). Superoxide anion radicals may then be converted to H 2 6. EUK-189 attenuates JNK signaling pathway activation within the SNpc. Western blot analysis for phospho-JNK, phospho-c-Jun, and cleaved caspase-3 in the SNpc at day 7 after the last paraquat administration as previously described (12). ␤-Actin was used as a loading control (C). presence of iron, subsequently undergo conversion to highly reactive and harmful hydroxyl radicals via the Fenton reaction. Catalase or glutathione peroxidase normally catalyzes the breakdown of H 2 O 2 to O 2 and H 2 O. EUK-134 and EUK-189 are mimetics of both superoxide dismutase and catalase and can therefore act to detoxify hydroxyl radicals completely (13)(14)(15). In this study, the administration of EUK-134 or EUK-189 prevented paraquat-induced dopaminergic neuron death, suggesting that reactive oxygen species acts upstream in the cell death pathway following exposure to paraquat.
Our results clearly indicate that oxidative stress is involved in the selective neuronal cell death of dopaminergic neurons following paraquat treatment both in vitro and in vivo. Activation of the JNK signaling pathway is considered as part of an oxidative stress response in a number of cell types (41,42). We previously demonstrate that activation of JNK and c-Jun is increased within the SNpc of paraquat-mediated adult animals and that MnTBAP administration prevents paraquat-induced JNK activation and subsequent dopaminergic apoptosis in vitro (12). However, MnTBAP does not cross the blood-brain barrier (43) and therefore was not a viable option for assessing the role of oxidative stress in paraquat-induced dopaminergic cell death in vivo. Pretreatment of mice with the bioavailable compound EUK-189 was found to inhibit the phosphorylation of JNK and c-Jun and caspase-3 activation in SNpc dopaminergic neurons in agreement with our previous in vitro experiment data, suggesting that EUK-189 can cross the blood-brain barrier and attenuate dopaminergic nigral cell death. Chronic systemic administration of EUK-189 was previously found to significantly decrease in lipid peroxidation and protein oxidation in the aging brain and to reverse age-related learning impairment (21). EUKs have previously proven to be effective against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-mediated toxicity at least in vitro, and we now demonstrate that they are protective in vivo in a second model of the disease, paraquat intoxication. This suggests a common mechanism of action, i.e. oxidative stress, in selective dopaminergic SN neurodegeneration associated with two separate models of the disease.
Together, our data provide direct evidence that systemic administration of bioavailable synthetic SOD/catalase mimetics protects against selective paraquat-mediated dopaminergic neuronal cell death through mechanisms involving the elimination of oxidative damage and the modulation of signal transduction pathways. Given the potential for exposure to this herbicide to increase the risk for the development of PD and the recent establishment of its systemic administration as a viable model for the disease, this has obvious therapeutic implications.