* Department of Environmental and Occupational Health Sciences and the Department of Pharmacologyz, University of Washington, Seattle, Washington 981957234
Received September 20, 2003; accepted January 23, 2003
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ABSTRACT |
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Key Words: rotenone; MAP kinase; p38; JNK; c-Jun; SH-SY5Y; apoptosis; pesticide; Parkinsons disease.
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INTRODUCTION |
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It has been hypothesized that environmental toxicants including pesticides may contribute to the development of Parkinsons disease (Mouradian, 2002; Ramsden et al., 2001
). For example, although genetic studies have linked several genes to genetic predisposition in Parkinsons disease, most cases of Parkinsons disease are sporadic, and their etiology remains largely undefined. Environmental factors and geneenvironmental interactions may play a significant role in neurodegeneration. The discovery of a link between the neurotoxicant MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) and the development of Parkinsons disease-like symptoms in humans provided the first evidence supporting this general hypothesis (Langston et al., 1983
). Although still somewhat controversial, many epidemiological studies have established an association between increased risk for Parkinsons disease and exposure to pesticides (Checkoway and Nelson, 1999
; Di Monte et al., 2002
; Le Couteur et al., 1999
).
Several toxicant-induced model systems have been developed to study Parkinsons disease in animals, including MPTP and 6-hydroxydopamine (Beal, 2001; Dauer and Przedborski, 2003
). These models have been used for decades to evoke symptoms of Parkinsons disease in laboratory animals and, subsequently, to test clinical treatments and strategies. Accurate model systems aid in the study of molecular mechanisms involved and can give valuable insight into Parkinsons disease pathogenesis. MPP+, the metabolite of MPTP, has been the most widely used Parkinsons disease model, contributing greatly to our current understanding of the pathogenesis of Parkinsons disease (Blum et al., 2001
). However, MPTP rarely induces formation of Lewy bodies, a hallmark of Parkinsons disease (Beal, 2001
).
Rotenone is a naturally occurring plant compound and a common insecticide used in vegetable gardens. It is also used to kill or sample fish. Recently it has been demonstrated that rats administered subacute doses of rotenone develop biochemical, anatomical, and behavioral symptoms of Parkinsons disease (Alam and Schmidt, 2002; Betarbet et al., 2000
; Sherer et al., 2003
). These results have renewed interest in the link between exposure to pesticides and the development of Parkinsons disease (Adam, 2000
). Most importantly, the rotenone model not only evokes the behavioral symptoms of Parkinsons disease and causes degeneration of substantia nigra neurons, but also induces cytoplasmic inclusions in the substantia nigra neurons similar to Lewy bodies (Betarbet et al., 2000
; Sherer et al., 2003
). Thus, rotenone treatment provides one of the best experimental models for Parkinsons disease research (Beal, 2001
; Dauer and Przedborski, 2003
).
However, since the rotenone model is a recent discovery, the molecular mechanisms underlying rotenone-induced neurodegeneration are not well understood. In this study, we sought to define the mode of cell death induced by rotenone in a human dopaminergic cell line SH-SY5Y and to elucidate the underlying signal transduction pathways mediating rotenone-induced cell death. We were particularly interested in members of the mitogen activated protein (MAP) kinases. These include the extracellular signal-regulated protein kinase (ERK) 1/2, the c-Jun NH2-terminal protein kinase (JNK), and the p38 MAP kinase. ERK1/2 are preferentially activated by growth factors and neurotrophic factors, while JNK and p38 are preferentially activated by cell stress-inducing signals, such as oxidative stress, environmental stress, and toxic chemical insults (Davis, 2000). Our results indicate the importance of apoptosis and the stress-activated JNK and p38 MAP kinases in rotenone-induced neurotoxicity.
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MATERIALS AND METHODS |
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Cell culture.
Human SH-SY5Y neuroblastoma cells were maintained in DMEM/F12 (Gibco) supplemented with 10% fetal bovine serum (FBS) and 0.05 U/ml penicillin and 0.05 mg/ml streptomycin. Cells were plated on poly-D-lysine coated plates with (for immunostaining) or without (for biochemical analysis) coverslips.
Quantification of apoptosis.
To visualize nuclear morphology, cells were fixed in 4% paraformaldehyde/4% sucrose and stained with 2.5 µg/ml of the DNA dye Hoechst 33258 (bis-benzimide, Sigma, St. Louis, MO). Uniformly stained nuclei were scored as healthy, viable neurons. Condensed or fragmented nuclei were scored as apoptotic. In order to obtain unbiased counting, slides were coded and cells were scored blind without knowledge of their prior treatment.
Drug treatment.
Rotenone (Sigma, 9598% pure) was dissolved in ethanol. DEVD and zVAD (R & D System) were dissolved in dimethyl sulfoxide (DMSO). Final ethanol concentration in media did not exceed 0.025%. Rotenone was made fresh prior to each treatment. All treatments were one-time, single-dose exposures. Because rotenone is lipophilic and may bind to proteins present in the serum, cells were transferred into lower serum media (0.5% FBS) before rotenone treatment to prevent excessive retention of rotenone in the serum. This procedure was practiced for all experiments except those of transfected cells in Figures 7 and 9. This lower serum media did not noticeably increase basal apoptosis in the absence of rotenone for up to 48 h (Fig. 2). After transfection procedure, cells detach from culture dish readily in serum-free medium. Therefore, transfected cells were treated with 200 nM rotenone in serum-containing medium in order to induce sufficient level of apoptosis while minimizing cell detachment from culture dish.
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Statistical analysis.
Data are from or representative of at least two independent experiments, each of duplicate or triplicate determination (n 6). Statistical analysis of the data was performed using one-way analysis of variation (ANOVA). Error bars represent standard error of the mean (SE) or standard deviation (SD).
Western blot analysis.
This was done as described (Figueroa-Masot et al., 2001). Regular culture medium was replaced with low-serum media (0.5% FBS) 12 h prior to rotenone treatment in order to minimize background kinase activity. Anti-phospho-p38 antibody (Thr 180 and Tyr182), anti-phospho-JNK antibody (Thr183 and Tyr185), and anti-phospho-cJun (Ser73) were purchased from Cell Signaling Technology (Beverly, MA). Anti-phospho ERK1/2 antibody was from Promega, anti-ß-actin from Sigma, anti-total p38 from Santa Cruz, anti-total JNK1 from Pharmingen, and anti-ERK-2 from Upstate. The anti-PARP antibody that recognizes both the cleaved and uncleaved PARP was from R & D System. The intensity of the bands on Western blots was quantitated by densitometry analysis of the scanned blots using ImageQuant software. The relative phosphorylation was normalized to loading control.
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RESULTS |
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Rotenone Does Not Activate the ERK1/2 Pathway
In order to determine the effect of rotenone treatment on the ERK1/2 pathway, SH-SY5Y cells were treated with 100 nM rotenone for 024 h in low-serum-containing medium (see Materials and Methods for details). ERK1/2 activation was assayed by Western analysis using an anti-phospho-ERK1/2 antibody that specifically recognizes phosphorylated and activated (p) ERK1/2. p-ERK1/2 was stable over time in control-treated samples (data not shown). Rotenone treatment did not significantly change the level of ERK1/2 phosphorylation (Fig. 5), indicating that ERK1/2 activity is not affected in response to rotenone.
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Rotenone Treatment Activates the JNK Pathway
To evaluate a role of JNK in rotenone-induced apoptosis, SH-SY5Y cells were treated with vehicle control or 100 nM rotenone for the indicated times. JNK activity was assayed by Western analysis using an antibody that recognizes dual-phosphorylated and activated JNK (anti-p-JNK) (Fig. 8). JNK phosphorylation, indicative of JNK activation, was apparent at 0.5-, 1-, and 2-h treatment with rotenone (Fig. 8A), but not in vehicle control-treated cells (Fig. 8B). JNK phosphorylation was still noticeable at 46 h after rotenone treatment but returned to baseline level at 12 h.
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Inhibition of JNK Signaling Attenuates Rotenone-Induced Apoptosis
The importance of JNK pathway activation for induction of apoptosis was investigated by inhibition of JNK signaling (Fig. 9). This was achieved by transiently transfecting SH-SY5Y cells with a dominant negative (DN) MKK4, a JNK kinase that phosphorylates and activates JNK (Fig. 9A). The effect of DN MKK4 on rotenone-induced apoptosis was examined as described above for the DN MKK3. Expression of the DN MKK4 caused partial but statistically significant inhibition of rotenone-induced apoptosis (Fig. 9A; p = 0.0011). Similarly, expression of a dominant negative c-Jun reduced SH-SY5Y cell apoptosis after rotenone treatment (Fig. 9B, p = 0.0012). As with cells transfected with DN MKK3, there was no nonapoptotic nuclear morphology in cells transfected with DN MKK4 or DN c-Jun (data not shown). These data implicate a causative role of the JNK signaling pathway in rotenone-induced apoptosis.
JNK and p38 Function Cooperatively in Rotenone-Induced Apoptosis
Because all dominant negative constructs were partially effective at reversing rotenone-induced toxicity, we examined whether there is an additive effect by inhibiting both JNK and p38 pathways. Coexpression of DN c-Jun and DN MKK3 did not offer more protection than either construct alone (Fig. 10), suggesting that the JNK and p38 pathways function cooperatively, rather than independently, to mediate rotenone-induced apoptosis. Unlike in DEVD- or zVAD-treated cells, there was not any nonapoptotic nuclear morphology in cells transfected with both constructs. The incomplete inhibition of apoptosis also suggests that in addition of JNK and p38, other signaling pathways such as GSK 3b may also be at play in rotenone-induced apoptosis (Hetman et al., 2000, 2002
; King et al., 2001
).
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DISCUSSION |
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In the literature, concentrations of rotenone ranging from 30 to 100 nM are normally used to study its neurotoxicity, although rotenone at concentrations up to 1 mM have also been used in several different cell types (Betarbet et al., 2000; Kitamura et al., 2002
; Lee et al., 2002
; Pei et al., 2003
; Sherer et al., 2001
; Wang et al., 2002
). In our experiments, rotenone induced apoptosis in SH-SY5Y cells at concentrations as low as 25 nM. This concentration is fairly consistent with the calculated concentration of rotenone (2030 nM) found in rat brain tissue in the in vivo study by Betarbet et al. (2000)
. At 100 nM, rotenone killed 50% of SH-SY5Y cells after 48-h exposure. Therefore, the dopaminergic SH-SY5Y cells are very sensitive to rotenone and offer a good model system to study rotenone neurotoxicity in vitro.
Although epidemiological studies have suggested a correlation between general pesticide exposure and increased risk for Parkinsons disease, it has been difficult to convincingly identify risk factors associated with exposure to any single pesticide. This could be due to the practical difficulty in identifying a large population that is only exposed to one particular pesticide. Alternatively, exposure to multiple pesticides, rather than to any single pesticide, may be more critical in the pathogenesis of Parkinsons disease. Interestingly, coexposure to both chlorpyrifos and rotenone synergistically induced apoptosis in SH-SY5Y cells. Cotreatment with 50 nM rotenone greatly increased the sensitivity of SH-SY5Y cells to chlorpyrifos. Only 3 mM chlorpyrifos was needed to kill 50% of the cells when 50 nM rotenone was present. The LD50 of chlorpyrifos in the absence of rotenone was 17 µM, 5 to 6 times higher. Even if humans are not exposed to both pesticides at the same time, preexposure to one pesticide may predispose the subject to subsequent injury following exposure to another pesticide. Furthermore, this type of synergism may also occur upon coexposure of chlorpyrifos or rotenone with other pesticides. In addition, the synergistic effect of chlorpyrifos and rotenone on cell death suggests that the two pesticides may operate in a common or related pathway, rather than by totally independent mechanisms, which would lead to additive effects. This is consistent with the observation that both pesticides stimulate JNK activity and require JNK for apoptosis (data shown here and Caughlan et al., in press).
Treatment with zVAD or DEVD partially inhibited rotenone-induced apoptosis in SH-SY5Y cells, suggesting a general role for caspases in rotenone-induced apoptosis. This is consistent with others reports (Kitamura et al., 2002; Pei et al., 2003
; Wang et al., 2002
). Interestingly, although zVAD and DEVD blocked PARP cleavage and reduced the number of classical apoptotic cell deaths after rotenone treatment, many of the cells now have shrunken nuclei without chromatin fragmentation or condensation into discrete dense clumps. Our observations suggest that rotenone-induced apoptosis is at least partially caspase-dependent. Because rotenone is a complex I inhibitor, it can inhibit mitochondria function independent of the downstream caspase activation. Therefore, in the presence of caspases-3 inhibitor, rotenone will eventually lead to a form of caspase-independent, nonapoptotic cell death when mitochondria function is severely impaired.
The ERK1/2 pathway is often stimulated by growth factors, though stressors can also stimulate it. For example, the brain-derived neurotrophic factor (BDNF) activates ERK1/2 in order to protect cortical neurons against DNA damage after camptothecin treatment (Hetman et al., 1999). Furthermore, ERK1/2 is activated by camptothecin treatment itself as a compensatory response to counteract camptothecin-induced apoptosis (Hetman et al., 1999
). In contrast, chlorpyrifos activates ERK1/2 signaling as part of the apoptotic mechanism (Caughlan et al, 2004
). Here we show that rotenone treatment does not cause any appreciable changes of ERK1/2 phosphorylation, ruling against an involvement of this pathway in rotenone-induced apoptosis.
p38 activation often leads to a pro-apoptotic response (Davis, 2000; Namgung and Xia, 2000
; Xia et al., 1995
), although in some cases p38 acts as a compensatory response or a pro-survival mechanism (Caughlan et al, 2004
; Mao et al., 1999
). Inhibition of p38 in this study suppressed rotenone-induced apoptosis. JNK is another stress-activated MAP kinase and has been implicated in many forms of neuronal apoptosis (Davis, 2000
; Namgung and Xia, 2000
; Xia et al., 1995
). We show here that JNK is also activated by rotenone and contributes to rotenone-induced apoptosis in SH-SY5Y cells. Because c-Jun phosphorylation is induced by rotenone and expression of a dominant negative c-Jun blocks rotenone-induced apoptosis, JNK-induced gene expression is likely critical for induction of apoptosis upon rotenone treatment.
Prior to the discovery of the rotenone model, several other models including MPTP, 6-hydroxydopamine, and dopamine have been used extensively in Parkinsons disease research both in vitro and in vivo. Interestingly, activation of the JNK signaling pathways has been implicated in neurodegeneration in these model systems. For example, CEP-1347, an inhibitor of JNK activation, attenuates MPTP-mediated nigrostriatal dopaminergic loss, indicating that the JNK signaling may be activated by MPTP administration in vivo (Saporito et al., 1999). Furthermore, MPTP increases the levels of phosphorylated JNK and the JNK kinase MKK4 in the nigrostriatal system (Saporito et al., 2000
), indicating activation of these kinases. This activation is inhibited by CEP-1347 at a dose that attenuates MPTP-induced dopaminergic loss (Saporito et al., 2000
).
There is also existing evidence implicating p38 in dopaminergic neuron cell death. For instance, dopamine induces apoptosis in SH-SY5Y cells that is dependent on p38 activation (Junn and Mouradian, 2001). Fetal cell transplantation therapies are being developed for the treatment of Parkinsons disease; however, massive apoptotic cell death is a major limiting factor for the success of neurotransplantation. Interestingly, inhibitors of p38 MAP kinase increase the survival of rat dopamine neurons in vitro upon trophic withdrawal and in vivo after transplantation into hemiparkinsonian rats (Zawada et al., 2001
).
Together with evidence presented in this study, it appears that JNK and p38 MAP kinases are activated and play a role in cell death in multiple in vitro and in vivo model systems relevant for Parkinsons disease. Although each individual model system has its own advantages and limitations and may not represent what is truly going on in the brain of Parkinsons disease patients, the involvement of JNK and p38 pathways in various model systems strongly suggests that these pathways may be involved in toxicant-induced nigrostriatal dopaminergic death in the degenerative process of Parkinsons disease. Thus these pathways may be viable drug targets for slowing down the disease progression of Parkinsons disease by preserving dopamine synthesizing neurons that have not yet been lost to the disease. Importantly, unlike caspase inhibitors zVAD or DEVD that did not prevent cells from eventually dying in a caspase-independent manner (Fig. 4), inhibition of JNK or/and p38 did not induce the appearance of nonapoptotic nuclear morphology. Thus, inhibition of earlier signaling events like the JNK and p38 signaling pathways may be more advantageous than inhibition of downstream caspases, and may provide functional and long term rescue of cells. In fact, a pharmacological inhibitor of the JNK pathway, CEP1347, is currently undergoing phase II and III clinical trials for the treatment of Parkinsons disease (NLM, 2003).
In conclusion, our results help to better characterize rotenone as an emerging Parkinsons disease model system. Accurate Parkinsons disease models are invaluable to the study of the disease and in the testing of new potential therapies and clinical strategies (Betarbet et al., 2002; Eberhardt and Schulz, 2003
). Our studies of rotenone signaling mechanisms support the hypothesis that JNK and p38 signaling pathways may be involved in the degeneration of dopaminergic neurons in idiopathic Parkinsons disease.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at Department of Environmental Health Sciences, Box 357234, University of Washington, Health Science Building, Rm. F561C, Seattle, WA 98195-7234. E-mail: zxia{at}u.washington.edu
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