Rotenone-Induced Apoptosis Is Mediated By p38 And JNK MAP Kinases In Human Dopaminergic SH-SY5Y Cells

Kathleen Newhouse*, Shih-Ling Hsuan*, Sandra H. Chang*, Beibei Cai*, Yupeng Wang* and Zhengui Xia*,{dagger},1

* Department of Environmental and Occupational Health Sciences and the {dagger} Department of Pharmacologyz, University of Washington, Seattle, Washington 98195–7234

Received September 20, 2003; accepted January 23, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rotenone is a naturally derived pesticide that has recently been shown to evoke the behavioral and pathological symptoms of Parkinson’s disease in animal models. Though rotenone is known to be an inhibitor of the mitochondrial complex I electron transport chain, little is known about downstream pathways leading to its toxicity. We used human dopaminergic SH-SY5Y cells to study mechanisms of rotenone-induced neuronal cell death. Our results suggest that rotenone, at nanomolar concentrations, induces apoptosis in SH-SY5Y cells that is caspase-dependent. Furthermore, rotenone treatment induces phosphorylation of c-Jun, the c-Jun N-terminal protein kinase (JNK), and the p38 mitogen activated protein (MAP) kinase, indicative of activation of the p38 and JNK pathways. Importantly, expression of dominant interfering constructs of the JNK or p38 pathways attenuated rotenone-induced apoptosis. These data suggest that rotenone induces apoptosis in the dopaminergic SH-SY5Y cells that requires activation of the JNK and p38 MAP kinases and caspases. These studies provide insights concerning the molecular mechanisms of rotenone-induced apoptosis in neuronal cells.

Key Words: rotenone; MAP kinase; p38; JNK; c-Jun; SH-SY5Y; apoptosis; pesticide; Parkinson’s disease.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Parkinson’s disease is the second most common neurodegenerative disorder, affecting about one million Americans (Olanow and Tatton, 1999Go). It is characterized by a selective and progressive degeneration of dopaminergic neurons and the presence of Lewy bodies in the neurons of the substantia nigra region of the brain. The death of greater than 80% of these dopamine-synthesizing neurons results in a scarcity of the catecholamine neurotransmitter dopamine and induces the characteristic motor symptoms of Parkinson’s disease. A growing body of evidence suggests that the neurodegeneration seen in several neurodegenerative disorders, including Parkinson’s disease, likely involves an increased level of apoptosis in sensitive neuronal populations (Blum et al., 2001Go; Olanow and Tatton, 1999Go; Tatton et al., 2003Go).

It has been hypothesized that environmental toxicants including pesticides may contribute to the development of Parkinson’s disease (Mouradian, 2002Go; Ramsden et al., 2001Go). For example, although genetic studies have linked several genes to genetic predisposition in Parkinson’s disease, most cases of Parkinson’s disease are sporadic, and their etiology remains largely undefined. Environmental factors and gene–environmental 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 Parkinson’s disease-like symptoms in humans provided the first evidence supporting this general hypothesis (Langston et al., 1983Go). Although still somewhat controversial, many epidemiological studies have established an association between increased risk for Parkinson’s disease and exposure to pesticides (Checkoway and Nelson, 1999Go; Di Monte et al., 2002Go; Le Couteur et al., 1999Go).

Several toxicant-induced model systems have been developed to study Parkinson’s disease in animals, including MPTP and 6-hydroxydopamine (Beal, 2001Go; Dauer and Przedborski, 2003Go). These models have been used for decades to evoke symptoms of Parkinson’s 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 Parkinson’s disease pathogenesis. MPP+, the metabolite of MPTP, has been the most widely used Parkinson’s disease model, contributing greatly to our current understanding of the pathogenesis of Parkinson’s disease (Blum et al., 2001Go). However, MPTP rarely induces formation of Lewy bodies, a hallmark of Parkinson’s disease (Beal, 2001Go).

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 Parkinson’s disease (Alam and Schmidt, 2002Go; Betarbet et al., 2000Go; Sherer et al., 2003Go). These results have renewed interest in the link between exposure to pesticides and the development of Parkinson’s disease (Adam, 2000Go). Most importantly, the rotenone model not only evokes the behavioral symptoms of Parkinson’s 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., 2000Go; Sherer et al., 2003Go). Thus, rotenone treatment provides one of the best experimental models for Parkinson’s disease research (Beal, 2001Go; Dauer and Przedborski, 2003Go).

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, 2000Go). Our results indicate the importance of apoptosis and the stress-activated JNK and p38 MAP kinases in rotenone-induced neurotoxicity.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids.
The following plasmids have been described previously: the dominant negative MKK3 (pRc-RSV-Flag-MKK3 (Ala)), the dominant negative MKK4 (pcDNA3-MKK4 (Ala)) (Xia et al., 1995Go), and the dominant negative c-Jun (pCMV-TAM67) (Rapp et al., 1994Go).

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, 95–98% 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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FIG. 7. Inhibition of p38 signaling partially protects SH-SY5Y cells from rotenone-induced apoptosis. Cells were transiently transfected with 3 µg of plasmid DNA encoding a vector control pcDNA3 or a dominant negative (DN) MKK3, a p38 kinase. The cells were also cotransfected with 1 mg of green fluorescent protein (eGFP) DNA as a transfection marker. Two days after transfection (see Materials and Methods for details), cells were treated with vehicle control or 200 nM rotenone for 24 h, and apoptosis in transfected cells (GFP positive) was then quantified. Error bars are SE (n = 4). **p < 0.01.

 


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FIG. 9. Inhibition of the JNK signaling pathway attenuates rotenone-induced apoptosis. (A) Effect of expression of a dominant negative (DN) MKK4, a JNK kinase. (B) Effect of expression of a dominant negative (DN) c-Jun. Transfection and treatment were done as described in Figure 7. Results represent two independent experiments. Error bars represent SE. ***p < 0.005.

 


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FIG. 2. Rotenone induces apoptosis in SH-SY5Y cells in a dose-dependent manner. SH-SY5Y cells were treated with various concentrations of rotenone as indicated for 24 h (A and B) or 48 h (C and D), and apoptosis was scored (A and C). Because late-stage apoptotic cells detach from the culture dish, we also quantitated cell death by counting the number of remaining SH-SY5Y cells/field left on plates after 24- or 48-h treatment (B and D). Results are representative of three independent experiments. Error bars are standard deviation (SD).

 
Transient transfection of SH-SY5Y cells.
SH-SY5Y cells were transiently transfected by calcium phosphate coprecipitation method as previously described (Hetman et al., 1999Go; Namgung and Xia, 2000Go; Xia et al., 1996Go). Cells were split 24 h after transfection and replated onto replicate plates coated with poly-D-lysine for subsequent drug treatment. Cells were treated with rotenone (200 nM) in their regular serum-containing culture media 24 h after replating (i.e., 48 h after transfection). Cells were not treated in low-serum medium like the nontransfected cells, because they detach from the coverslips readily in low serum after the transfection procedure. Transfected cells were treated with 200 nM rotenone in order to induce sufficient level of apoptosis in the vector control transfected cell population without causing nonapoptotic cell death. This concentration of rotenone is higher than the 80 or 100 nM used in low-serum media, due to the presence of serum for reasons stated above under "drug treatment." Cells were fixed 24 h post rotenone treatment and stained with Hoechst dye 33258 to visualize nuclear morphology. All cells were cotransfected with an expression vector encoding enhanced green fluorescent protein (eGFP) as a marker for transfected cells. Transfected cells were identified by green fluorescence. Apoptosis in transfected cells was scored blind under a fluorescence microscope. The percentage of apoptotic cells in the total transfected cell population was quantitated.

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., 2001Go). Regular culture medium was replaced with low-serum media (0.5% FBS) 1–2 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rotenone Induces Apoptosis in SH-SY5Y Cells
To define the mode of cell death induced by rotenone, we treated SH-SY5Y cells with rotenone and examined its effect on nuclear morphology. At concentrations equal to or lower than 150 nM in serum-free medium, or up to 250 nM in serum-containing medium, rotenone treatment caused condensation of the cell body, nuclear fragmentation, and condensation into discrete dense chromatin clumps (Fig. 1). These are hallmarks of morphological changes associated with apoptosis. However, at higher concentrations, rotenone treatment induced a mixture of apoptotic and nonapoptotic nuclear morphological changes (data not shown). The percentage of cells adherent to the culture dish that are undergoing apoptosis was quantitated as a function of the concentration and time after rotenone exposure (Fig. 2). Rotenone, at 50 nM, caused a 9-fold increase in apoptosis in SH-SY5Y cells after 24-h treatment (Fig. 2A). At 100 nM, rotenone induced apoptosis at 24 h in about 33% of the adherent cell population, a level 18 times higher than basal cell death (Fig. 2A). We also examined the effect on cell death 48 h after rotenone treatment. Surprisingly, there is no apparent increase in the percentage of apoptotic cells in the adherent cell population from 24-h to 48-h treatment (Fig. 2C). However, we noticed that there were much fewer cells left on plates after rotenone treatment for 48 h. Therefore, we counted the number of cells left in each field. Although only 150 nM rotenone caused some decrease in cell numbers after 24-h treatment (Fig. 2B), we found that after 48-h treatment, about 100 nM rotenone caused a 50% loss of total cell numbers (Fig. 2D). Therefore, the values in Figure 2C are an underestimate of the actual number of cells in the population that have undergone apoptosis upon rotenone treatment. This is most likely because cells that had progressed into later stages of apoptosis detached from the tissue culture dish and were not scored in the assay. The effect of rotenone on SH-SY5Y cell death was detectable at 25 nM after 24-h treatment (data not shown), and with a LD50 of 100 nM after 48-h treatment. We chose 100 nM concentration of rotenone in serum-free media or 200 nM rotenone in serum containing media for 24-h treatment for our subsequent studies because, at these concentrations, rotenone consistently induces significant levels of apoptosis without causing cells to detach from the culture dish or inducing nonapoptotic cell death.



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FIG. 1. Representative photomicrographs of nuclei morphology of SH-SY5Y cells treated with vehicle control (A) or 100 nM rotenone for 24 h (B). Nuclei morphology was visualized after Hoechst staining. Arrows identify cells with condensed or fragmented nuclei, characteristic of apoptosis. Scale bar: 20 µm.

 
Rotenone Sensitizes Cells to Exposure of Other Pesticides
Because multiple pesticides are often applied for agricultural use, we tested if coexposure of rotenone increased the sensitivity to other pesticides. We recently demonstrated that chlorpyrifos, an organophosphate and one of the most commonly used pesticides, induces neuronal apoptosis (Caughlan et al., 2004Go). In the absence of chlorpyrifos treatment, 50 nM rotenone alone caused a reduction in cell number after 48-h treatment (Fig. 3), consistent with results shown in Figure 2. Only 3 mM chlorpyrifos was needed to kill 50% of the cells when 50 nM rotenone was present (Fig. 3). The LD50 in the absence of rotenone was 17 µM, 5 to 6 times higher. These data suggest that coexposure of both chlorpyrifos and rotenone synergistically induces neuronal apoptosis.



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FIG. 3. Cotreatment with rotenone greatly increases the sensitivity of SH-SY5Y cells to exposure of another pesticide, chlorpyrifos. SH-SY5Y cells were treated with various concentrations of chlorpyrifos as indicated ±50 nM rotenone. The number of remaining SH-SY5Y cells/field left on plates was quantitated 48 h later. Error bars are standard error of the mean (SE).

 
Rotenone-Induced Apoptosis Is Caspase-Dependent
Caspases are a family of cysteine-dependent, Asp-specific proteases (Budihardjo et al., 1999Go; Cryns and Yuan, 1998Go; Salvesen and Dixit, 1999Go). They cleave a number of cellular proteins including poly (ADP-ribose) polymerase-1 (PARP) by limited proteolysis, a process that plays a central role in the execution of many forms of apoptosis. To determine if caspases play a role in rotenone-induced apoptosis, we treated SH-SY5Y cells with zVAD, a broad-spectrum caspase inhibitor. Addition of 10 µM zVAD significantly attenuated rotenone-induced apoptosis in SH-SY5Y cells (Fig. 4A), suggesting a role for caspases in general in rotenone-induced neuronal apoptosis.



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FIG. 4. Rotenone-induced apoptosis is caspase-dependent. (A) Effect of the broad-spectrum caspase inhibitor zVAD on rotenone-induced apoptosis. SH-SY5Y cells were treated with 10 mM zVAD-FMK, 80 nM rotenone, or vehicle control (-) as indicated. Cell death was scored 24 h post treatment. Apoptosis was defined by nuclear fragmentation and condensation into discrete dense chromatin clumps. Cells that had shrunken nuclei but did not show chromatin condensation into discrete dense clumps were scored as nonapoptotic cell death. (B) Anti-PARP Western analysis. SH-SY5Y cells were treated with vehicle control, 80 nM rotenone, and various concentrations of DEVD or 10 mM zVAD-FMK as indicated. Cell lysates were prepared 24 h later for Western analysis. (C) Representative photomicrographs of nuclei morphology (Hoechst staining) of SH-SY5Y cells treated for 24 h with vehicle control (a), 80 nM rotenone (b), or 80 nM rotenone + 20 nM DEVD (c). Arrow in (B) identifies apoptotic cells, while arrow heads in (C) identify cells with shrunken nuclei that did not show chromatin condensation into discrete dense clumps (nonapoptotic cell death). Scale bar: 10 µm. (D). Quantitation of apoptotic and nonapoptotic cell death in SH-SY5Y cells treated with rotenone ± DEVD. Results are from two independent experiments. Error bars are SE.

 
To determine the specific caspase that is involved in rotenone-induced apoptosis, we first examined if rotenone induces cleavage of PARP, a substrate of caspase-3. PARP cleavage was assayed by Western analysis using an anti-PARP antibody that recognizes both the intact and cleaved PARP. Rotenone treatment caused PARP cleavage that was inhibited by coincubation with DEVD, a caspase-3 inhibitor (Fig. 4B). These data suggest that caspase-3 is activated by rotenone. In the presence of DEVD or zVAD, there is a reduction in the number of cells that showed classical hallmarks of apoptosis (Fig. 4D). However, many of the nuclei have a staining pattern distinct from that of control-treated, healthy cells (Fig. 4C). They are shrunken to variable degrees, but did not show chromatin condensation into discrete dense clumps. This type of nuclear morphology has been attributed to "caspase-independent," nonapoptotic cell death (Stefanis et al., 1999Go). Our observations suggest that rotenone-induced apoptosis is at least partially caspase-dependent. However, in the presence of caspases-3 inhibitor, rotenone will still lead to cell death, albeit in a caspase-independent form.

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 0–24 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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FIG. 5. Rotenone does not affect ERK1/2 phosphorylation. (A) SH-SY5Y cells were treated with 100 nM rotenone for indicated times. Cell lysates were prepared and 40 µg of total protein were submitted to Western analysis using an antibody recognizing phosphorylated and activated (p-) ERK1/2. Anti-ß-actin Western was used to normalize protein loading. Results are representative of three independent experiments. (B) Quantitation of the data shown in panel A. Error bars are SE.

 
Rotenone Activates the p38 Pathway Which Contributes to Rotenone-Induced Apoptosis
To determine if p38 plays a role in rotenone-induced apoptosis, SH-SY5Y cells were treated with 100 nM rotenone (Fig. 6A) for 0–24 h in low-serum-containing medium. Activation of p38 was measured by Western analysis using an anti-phospho-p38 antibody that specifically recognizes dual-phosphorylated and activated p38. Phosphorylation of p38, indicative of p38 activation, was evident at 0.5- to 2-h treatment with rotenone. Because p38 can also be activated by serum deprivation in some cells (Xia et al., 1995Go), we treated SH-SY5Y cells with vehicle control ethanol. The control-treated cells did not exhibit an increase in p38 phosphorylation at 0.5 or 1 h (Fig. 6B), time points in which we observed dramatic p38 phosphorylation with rotenone treatment. These data suggest that rotenone activates the p38 signaling pathway.



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FIG. 6. Rotenone triggers p38 phosphorylation, indicative of p38 activation. SH-SY5Y cells were treated with 100 nM rotenone (A) or vehicle control ethanol (B) for indicated times (0–24 h). Cell lysates were prepared, and 40 µg of total protein was submitted to Western analysis using an antibody recognizing phosphorylated and activated (p-) p38. Anti-total p38 Western was used to normalize protein loading. Results are characteristic of two independent experiments.

 
Next, we transiently transfected SH-SY5Y cells with a dominant negative (DN) MKK3, a p38 kinase which phosphorylates and activates p38, in order to determine the role of p38 activation in rotenone-induced apoptosis (Fig. 7). The cloning vector was used as a control for transfection. Two days after transfection, cells were treated with 200 nM rotenone or vehicle control in regular serum-containing culture media (see Materials and Methods for details). Cells were not treated in low-serum medium like the nontransfected cells, because they detach from the coverslips readily in low serum after the transfection procedure. Apoptosis was scored in transfected cells after 24-h rotenone treatment. Transfected cells were identified by the green fluorescence of the cotransfected marker protein eGFP. The basal apoptosis in vehicle control-treated cells was approximately 10% for both vector or DN MKK3 transfected cells. This is higher than basal apoptosis without transfection. Because transfection is a form of stress that renders cells more susceptible to other insults, it is common to observe increased sensitivity to cell death after transfection (Hetman et al., 1999Go; Namgung and Xia, 2000Go). Rotenone treatment induced apoptosis in 28% of the vector control transfected cells, an almost 3-fold increase over baseline cell death of vehicle-treated cells. Expression of the DN MKK3 significantly reduced rotenone-induced apoptosis in transfected cells (p = 0.0053). Significantly, unlike in DEVD- or zVAD-treated cells, there was not any nonapoptotic nuclear morphology in cells transfected with DN MKK3 (data not shown). These results suggest that p38 is acting in a pro-apoptotic manner and contributing to apoptotic cell death resulting from rotenone exposure.

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 4–6 h after rotenone treatment but returned to baseline level at 12 h.



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FIG. 8. Rotenone treatment activates the JNK signaling pathway in SH-SY5Y cells. SH-SY5Y cells were treated with 100 nM rotenone (A, C) or vehicle control ethanol (B) for indicated times (0–24 h). Cell lysates were prepared, and 40 µg of total protein was submitted to Western analysis using antibodies recognizing phosphorylated and activated (p-) JNK or c-Jun. The blots were stripped and reprobed with antibodies against total JNK1 or ß-actin to normalize protein loading. Results are characteristic of 2 (panel C) or 3 (panels A and B) independent experiments.

 
c-Jun is a nuclear transcription factor and a known target of JNK. Its transcriptional activity is stimulated by JNK phosphorylation (Davis, 2000Go). There is a robust phosphorylation of c-Jun after rotenone treatment, indicative of c-Jun activation (Fig. 8C). Together, these data indicate that, in addition to p38, the JNK signaling pathway is activated in response to rotenone treatment.

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., 2000Go, 2002Go; King et al., 2001Go).



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FIG. 10. Blocking both JNK and p38 signaling pathways did not offer additive protection against rotenone-induced apoptosis. SH-SY5Y cells were transiently transfected with a vector control, a DN MKK3, a DN c-Jun, or both DN MKK3 and DN c-Jun as indicated. Cells were treated and apoptosis scored as described in Figure 7. Results represent two independent experiments. Error bars represent SE. ***p < 0.001 comparing to vector transfected cells that have been treated with rotenone. However, there is no statistically significant difference (n. s.) in rotenone-induced apoptosis between cells transfected with DN MKK3 together with DN c-Jun and cells transfected with either construct alone.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The objectives of this study were to determine if rotenone induces apoptosis in human dopaminergic SH-SY5Y cells and to elucidate the underlying molecular mechanisms. Our results indicate that rotenone induces apoptosis in these cells, which is inhibited by a broad-spectrum caspase inhibitor zVAD or a caspase-3 specific inhibitor DEVD. Furthermore, rotenone activated the p38 and JNK but not ERK 1/2 signaling pathways. Blocking p38 or JNK signaling inhibited rotenone-induced apoptosis, supporting a pro-apoptotic role for these pathways in rotenone-induced apoptosis.

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., 2000Go; Kitamura et al., 2002Go; Lee et al., 2002Go; Pei et al., 2003Go; Sherer et al., 2001Go; Wang et al., 2002Go). 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 (20–30 nM) found in rat brain tissue in the in vivo study by Betarbet et al. (2000)Go. 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 Parkinson’s 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 Parkinson’s 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 pressGo).

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., 2002Go; Pei et al., 2003Go; Wang et al., 2002Go). 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., 1999Go). Furthermore, ERK1/2 is activated by camptothecin treatment itself as a compensatory response to counteract camptothecin-induced apoptosis (Hetman et al., 1999Go). In contrast, chlorpyrifos activates ERK1/2 signaling as part of the apoptotic mechanism (Caughlan et al, 2004Go). 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, 2000Go; Namgung and Xia, 2000Go; Xia et al., 1995Go), although in some cases p38 acts as a compensatory response or a pro-survival mechanism (Caughlan et al, 2004Go; Mao et al., 1999Go). 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, 2000Go; Namgung and Xia, 2000Go; Xia et al., 1995Go). 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 Parkinson’s 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., 1999Go). Furthermore, MPTP increases the levels of phosphorylated JNK and the JNK kinase MKK4 in the nigrostriatal system (Saporito et al., 2000Go), indicating activation of these kinases. This activation is inhibited by CEP-1347 at a dose that attenuates MPTP-induced dopaminergic loss (Saporito et al., 2000Go).

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, 2001Go). Fetal cell transplantation therapies are being developed for the treatment of Parkinson’s 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., 2001Go).

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 Parkinson’s 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 Parkinson’s 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 Parkinson’s disease. Thus these pathways may be viable drug targets for slowing down the disease progression of Parkinson’s 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 Parkinson’s disease (NLM, 2003Go).

In conclusion, our results help to better characterize rotenone as an emerging Parkinson’s disease model system. Accurate Parkinson’s disease models are invaluable to the study of the disease and in the testing of new potential therapies and clinical strategies (Betarbet et al., 2002Go; Eberhardt and Schulz, 2003Go). 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 Parkinson’s disease.


    ACKNOWLEDGMENTS
 
This work was supported by APP#3010 from Burroughs Wellcome Fund for New Investigator Award in Toxicology (ZX), the UW NIEHS sponsored Center for Ecogenetics and Environmental Health (NIEHS P30ES07033), NIH grant NS44069, and by the NIH Postdoctoral Training grant Environmental Pathology/Toxicology NIHNIEHS 5T32ES07032 (SHC).


    NOTES
 

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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