ToxSci Advance Access originally published online on March 6, 2007
Toxicological Sciences 2007 97(2):448-458; doi:10.1093/toxsci/kfm040
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Inhibition of Paraquat-Induced Autophagy Accelerates the Apoptotic Cell Death in Neuroblastoma SH-SY5Y Cells
* CIBER de Enfermedades Neurodegenerativas, Departamento de Bioquímica y Biología Molecular y Genética, E.U. Enfermería y T.O., Universidad de Extremadura, Avda. Universidad s/n 10071 Cáceres, Spain
Departamento de Química Orgánica
Departamento de Biología Celular
Departamento de Medicina y Sanidad Animal, Facultad de Veterinaria, Universidad de Extremadura, Cáceres, Spain
1 To whom correspondence should be addressed. Fax: +34-927-257-451. E-mail: rosapolo{at}unex.es.
Received January 12, 2007; accepted February 27, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Autophagy is a degradative mechanism involved in the recycling and turnover of cytoplasmic constituents from eukaryotic cells. This phenomenon of autophagy has been observed in neurons from patients with Parkinson's disease (PD), suggesting a functional role for autophagy in neuronal cell death. On the other hand, it has been demonstrated that exposure to pesticides can be a risk factor in the incidence of PD. In this sense, paraquat (PQ) (1,1'-dimethyl-4,4'-bipyridinium dichloride), a widely used herbicide that is structurally similar to the known dopaminergic neurotoxicant MPP+ (1-methyl-4-phenyl-pyridine), has been suggested as a potential etiologic factor for the development of PD. The current study shows, for the first time, that low concentrations of PQ induce several characteristics of autophagy in human neuroblastoma SH-SY5Y cells. In this way, PQ induced the accumulation of autophagic vacuoles (AVs) in the cytoplasm and the recruitment of a LC3-GFP fusion protein to AVs. Furthermore, the cells treated with PQ showed an increase of the long-lived protein degradation which is blocked in the presence of the autophagy inhibitor 3-methyladenine and regulated by the mammalian target of rapamycin (mTOR) signaling. Finally, the cells succumbed to cell death with hallmarks of apoptosis such as phosphatidylserine exposure, caspase activation, and chromatin condensation. While caspase inhibition retarded cell death, autophagy inhibition accelerated the apoptotic cell death induced by PQ. Altogether, these findings show the relationship between autophagy and apoptotic cell death in human neuroblastoma cells treated with PQ.
Key Words: paraquat; vacuoles; apoptosis; Parkinson.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Autophagy is an intracellular lysosome-mediated catabolic mechanism that is responsible for the bulk degradation and recycling of damaged or dysfunctional cytoplasmic components and intracellular organelles (Klionsky et al., 2000
). Autophagy is a cellular response to both extracellular (nutrient deprivation or hypoxia) and intracellular (accumulation of damaged organelles and cytoplasmic components) stress conditions. Autophagy, being an evolutionarily ancient cellular response to intra- and extracellular noxious stimuli, may precede or coexist with apoptosis, and it may be induced by apoptotic stimuli (Xue et al., 1999). Thus, autophagy and apoptosis may be interconnected (Bursch et al., 2000). Cellular autophagy is a physiological degradative process involved, like apoptosis, in embryonic growth and development, cellular remodeling, and the biogenesis of some subcellular organelles (Filonova et al., 2000; Hariri et al., 2000; Sattler and Mayer, 2000). Autophagic cell death involves accumulation of autophagic vacuoles (AVs) in the cytoplasm of dying cells as well as mitochondria dilation and enlargement of the endoplasmic reticulum and the Golgi apparatus. Autophagic cell death has been described during the normal nervous system development (Schweichel et al., 1973) and could be a consequence of a pathological process such as those associated with neurodegenerative diseases (Petersen et al., 2001). Of note, autophagy is highly enhanced in brain amyloidoses (Graeber et al., 2002), Alzheimer disease (Stadelmann et al., 1999), Huntington's disease (Landles et al., 2004), and Parkinson's disease (PD) (Anglade et al., 1997).
PD is a chronic progressive neurodegenerative disease, affecting at least 1% of the population over the age of 55. Genetic forms of the disease represent less than 5% of current cases, but the causes of the vast majority of sporadic cases of PD are still unknown. Accumulating evidence strongly points to environmental toxins as feasible triggers of neurodegeneration of nigrostriatal dopaminergic neurons, and the common use of pesticides in rural life has been correlated to parkinsonism in humans (Di Monte et al., 2000). In this sense, the herbicide 1,1'-dimethyl-4,4'-bipyridinium dichloride (paraquat [PQ]), widely used as a cationic nonselective bipyridyl herbicide to control weeds and grasses in many agricultural areas, has emerged as a putative risk factor on the basis of its structural homology to 1-methyl-4-phenyl-pyridine (MPP+), the active metabolite of 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine, a neurotoxicant that induces Parkinson's-like features in rodents, nonhuman primates, and humans (Langston and Ballard, 1984; Tanner and Ben Shlomo, 1999).
Occupational PQ exposures have been associated with parkinsonism (Hertzman et al., 1990; Liou et al., 1997), although the mechanism of PQ toxicity is yet poorly understood. Several studies have suggested the involvement of reactive oxygen species (ROS) in its effect (Gonzalez-Polo et al., 2004; Mollace et al., 2003). Our group has demonstrated that free radicals play an active role in the PQ-induced apoptotic events that culminate with cell death (Gonzalez-Polo et al., 2004).
Here, we show for the first time that low concentrations of PQ induce several characteristics of autophagy in human neuroblastoma SH-SY5Y cells, which are commonly selected as a cellular PD model (Lai et al., 1997; Lee et al., 2000; Shavali et al., 2004). Finally, the cells succumbed to cell death with hallmarks of apoptosis. Thus, we investigated the relationship between autophagy and apoptosis in neurons treated with PQ to propose a new and exciting strategy to the knowledge of the PD mechanism and its possible treatment.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell line and culture condition.
Human neuroblastoma SH-SY5Y cells were grown in Dulbecco's modified Eagle medium (Gibco BRL, Paisley, U.K.) containing 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% fetal bovine serum (Hyclone, Brevieres, France). Cells were seeded at 5 x 105 in a 75-cm2 tissue culture flask (TPP, Trasadingen, Switzerland) and incubated at 37°C under a saturating humidity atmosphere of 5% CO2/95% air.
Reagents and cell death induction.
Confluent cells (
80%) in 75-cm2 tissue culture flasks were trypsinized and seeded in tissue culture dishes at a concentration of 5 x 104 cells/cm2. Twenty-four hours later, the medium was aspirated and replaced with fresh medium alone or containing the indicated concentrations of PQ (Niso-Santano et al., 2006). Serum and amino acid starvation of cells were performed using serum-free Earle's balanced salt solution medium (Sigma, St Louis, MO) (Boya et al., 2005). For caspase inhibiton, a sublethal dose of N-benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone (Z-VAD-fmk, 50µM, Bachem AG, Bubendorf, Switzerland) was added 1 h before PQ. The antioxidants N-acetylcysteine (NAC 1mM), vitamin E (100µM), glutathione (1mM), and the inihibitor of autophagy, 3-methyladenine (3-MA, 10mM), were added before the addition of PQ.
Flow cytometry.
To determine apoptosis-associated changes by cytofluorometry, we used 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3), 40nM) for the mitochondrial transmembrane potential quantification, propidium iodide (PI, 1 µg/ml) for the determination of cell viability (Boya et al., 2003; Gonzalez-Polo et al., 2005a), and Annexin-V labeled with fluorescein isothiocyanate (all of them from Molecular Probes, Eugene, OR) for the assessment of phosphatidylserine exposure. For the determination of superoxide anion generation, hydroethidine (HE, 10µM) was used. After different experimental conditions, cells were trypsinized and labeled with the fluorochromes at 37°C, followed by cytoflurometric analysis with a FACS Scan (Becton Dickinson, Frankin Lakes, NJ) (Gonzalez-Polo et al., 2005a,b).
Immunofluorescence and light microscopy.
Cells were cultured on coverslips. After the experimental conditions, the cells were stained with CMFDA (5-chloromethylfluorescein diacetate, 1µM) (Molecular Probes) for 30 min at 37°C to visualize the vacuoles. Cells were fixed with paraformaldehyde (4% wt:vol) for CMFDA staining and immunofluorescence assays (Gonzalez-Polo et al., 2005a,2005b). Cells were stained for the detection of activated caspase-3 with a polyclonal antibody from Cell Signaling Technology, Inc. (Danvers, MA) developed with an anti-rabbit immunoglobulin Alexa fluor conjugate (Molecular Probes) and counter-stained with Hoechst 33342, 2µM (Ho) (Sigma) before mounting. Fluorescence microscopy was analyzed with an Olympus IX51 equipped with a DC300F camera.
Western blot analysis.
Following experimental treatments, the cells (cultured in 60 mm dishes) were rinsed twice with cold phosphate-buffered saline (PBS) and removed by scrapping and then centrifuged at 900 x g for 5 min at 4°C. Cells were lysed in a buffer containing 50mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid pH 7.5, 100mM NaCl, 1% Triton X-100, 10% glycerol, 1mM ethyleneglycol-bis(aminoethylether)-tetraacetic acid, 5mM MgCl2, 25mM NaF, and Protease Inhibitor Cocktail (Sigma). Cells were centrifuged at 13,000 x g 5 min at 4°C. Supernatants were stored at – 80°C until analysis by Western blot. Protein concentration was measured according to Bradford (1976) using bovine serum albumin (BSA) as standard. Equal amounts of proteins (20 µg/condition) were resolved in 12–15% sodium dodecyl sulfate gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes according to conventional partially modified methods (Fuentes et al., 2000). Briefly, proteins were transferred (250 mA for 60 min) to PVDF membranes using Mini Trans-Blot Cell apparatus (Bio-Rad, Hercules, CA). The procedure for immunodetection includes the transfer and blocking of the membrane (60 min at room temperature) with Tween–Tris-buffered saline (TTBS) (10mM Tris/HCl pH 7.5, 150mM NaCl, and 0.2% Tween-20) containing 10% nonfat-dried milk. Membranes were then incubated overnight at 4°C with primary antibodies, p-mTORser2448, and cleaved caspase-3 from Cell Signalling (Beverly, MA) and anti-LC3 from MBL Medical and Biological Laboratories Co., Ltd (Nagoya, Japan), all of them diluted 1:1000 in TTBS + 10% nonfat-dried milk or 5% BSA. After washing (for two 5-min periods with TTBS), membranes were incubated (60 min at room temperature) with peroxidase-conjugated secondary antibodies (1:5000 in TTBS with 10% nonfat-dried milk). After washing (for two 5-min periods and one 10-min period), the detection of bound antibodies was visualized by chemiluminiscence using the ECL-plus reagent (GE Healthcare, Buckinghamshire, UK). Actin content was analyzed as a control by means of a rabbit polyclonal antibody (from Sigma).
Analysis of protein degradation.
Human neuroblastoma SH-SY5Y cells were incubated with 0.2 mCi/ml. L-[14C]valine (GE Healthcare) in complete medium (CM) for 24 h at 37°C. Unincorporated radioisotope was removed by washing the cells three times with PBS (pH 7.4). Cells were then incubated in Earle's balanced salt solution buffer plus 0.1% BSA (nutrient-free [NF] medium) in the presence of 10mM cold valine, for 1 h (prechase period). After this time, the medium was replaced by the appropriate fresh medium (NF or CM) plus cold valine 10mM in the presence or absence of 10mM 3-MA and 10µM PQ for 4 h (chase period). Cells and radiolabeled proteins from the medium were precipitated in trichloroacetic acid at the final concentration of 10% (vol/vol). The precipitated proteins were separated from the soluble radioactivity by centrifugation at 600 x g for 20 min and then dissolved in 1 ml of 0.2 N NaOH. The rate of protein degradation was calculated by determining the ratio of acid-soluble radioactivity recovered from both cells and medium to the ratio of radioactivity in trichloroacetic acid-precipitated proteins obtained from both cells and medium (Pattingre et al., 2003).
Electron microscopy.
Scraped cells were spinned and immersed in a mixture of 2% glutaraldehyde and 2% paraformaldehyde in phosphate buffer (pH 7.4) for 1 h at 4°C. Pellets were then rinsed in phosphate buffer, postfixed in 1% osmium tetroxide for 90 min, rinsed, dehydrated in a graded series of acetone, and embedded in Araldite resin. Ultrathin 90-nm–thick sections were obtained using a Leica Reichert Ultracut ultramicrotome equipped with a glass knife. Sections were mounted on copper grids, stained with uranyl acetate and lead citrate, examined, and photographed with a Jeol JEM-100 CX II transmission electron microscope.
Transient transfections.
SH-SY5Y cells were transiently transfected with the Fugene procedure, with 1–2 µg DNA per 35-mm plate, according to the manufacturer's protocol (Roche Diagnostics, GmbH, Mannheim, Germany) to label AVs with LC3-GFP plasmid (Kabeya et al., 2000).
Statistical analysis.
Data are representative of at least three independent experiments each in triplicate determination. Statistical analysis of the data was performed using one-way ANOVA. Significance (*) was defined at p < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PQ Induces Apoptotic Death in Neuroblastoma Cells
As previously described (Chun et al., 2001
; Gonzalez-Polo et al., 2004; Niso-Santano et al., 2006), several cell lines, including human neuroblastoma SH-SY5Y cells, are sensitive to the PQ induced toxicity. We investigated, for the first time, the cell death type in neuroblastoma cells exposed to the bipiridinic pesticide PQ. As we show in Figure 1, the PQ treatment caused phosphatidylserine exposure on the outer leaflet of the plasma membrane, which can manifest before plasma membrane permeabilization. This phenomenon was increased in combination with PQ and starvation conditions. The cells treated with 10–25µM of PQ exhibited a range of 25–50% loss of cell viability in CM and NF medium. In parallel experiments, we analyzed the involvement of caspases in the apoptotic death induced by 10µM of PQ (Fig. 2). The maximum level of caspase-3 activation was evident after 24 h of PQ incubation, shown by Western blot detection of a 17-kDa fragment (active form) and caspase-3 total, both in CM (Fig. 2A) and NF medium (Fig. 2B). Morphological analysis of cells subjected to the combined treatment of PQ 10µM and starvation indicate a typical apoptotic chromatin condensation (as detectable with the nuclear stain Hoechst 33342) associated with caspase-3 activation (Figs. 2C and 2D). Therefore, we wondered whether inhibition of caspases with the broad-spectrum caspase inhibitor Z-VAD-fmk would prevent cell death in this model. As shown in Figure 2E, Z-VAD-fmk retarded cell death of PQ-treated, SH-SY5Y cells, both in CM and NF medium.
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PQ Induces Reactive Oxygen Species Production in SH-SY5Y Neuroblastoma Cells
The mechanism of action of PQ is as yet poorly understood. However, it is well known that PQ acts as a reactive oxygen species (ROS) producer (Bus et al., 1984
; Suntres 2002). These ROS interact with unsaturated lipids of membranes (lipid peroxidation) and destroy organelles subsequently leading to cell death (Dodge, 1971). In this sense, several studies have suggested the involvement of ROS in the toxicity induced by PQ, including studies in vitro in several cell lines as in human lung epithelial cells (Cappelletti et al., 1998) or in cerebellar granule cells (Bus et al., 1984; Gonzalez-Polo et al., 2004), as well as studies in vivo (Mollace et al., 2003). In our study, we showed that exposure to low concentrations of PQ (10µM) for 24 h caused an increase of intracellular levels of ROS in human neuroblastoma SH-SY5Y cells (as measured by the oxidation of the nonfluorescent dye HE into its fluorescent product ethidium [Eth]) (Fig. 3). This production was enhanced when the cells were treated with a combination of nutrient depletion conditions and PQ. The ROS generation was abolished in the presence of several antioxidants as N-acetylcysteine as well as the ROS scavenger vitamin E (
-tocopherol) (data not shown).
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The Treatment of SH-SY5Y Neuroblastoma Cells with Low Concentrations of PQ Produces Characteristics of Autophagy
It has been described that autophagy is a cellular response to extracellular (nutrient deprivation, hypoxia) and intracellular stress conditions (accumulation of damaged organelles and cytoplasmic components). This phenomenon is characterized morphologically by the accumulation of AVs in the cytoplasm. We have tested in our model that, in NF medium, that is, in the absence of serum and amino acids, as well as in CM, control cells did not develop any discernible vacuoles, while cells treated with PQ 10µM exhibited an increase in cytoplasmic vacuolization visible as "holes" in CMFDA-stained cells of a time-dependent way (Figs. 4A and 4B). Moreover, these vacuoles induced in the presence of PQ showed, clearly, the characteristic double membranes of AVs as visible by transmission electron microscopy (Fig. 4C). Figures 4A and 4C show images in CM conditions. Microphotographs in NF conditions are equivalents (data not shown).
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Similarly, after treatment of serum and amino acid starvation, 10µM of PQ induced the recruitment of a LC3-GFP fusion protein to AV in discrete foci (Kabeya et al., 2000
; Mizushima 2004) (Figs. 5A and 5B). In agreement with the microscopic observations, we detected by Western blotting the accumulation of the autophagosome marker LC3-II (Boya et al., 2005; Kabeya et al., 2000) both in CM (Fig. 5C) and under the conditions of starvation (data not shown) in SH-SY5Y treated with PQ 10µM. Furthermore, as compared to the prototypic autophagy inhibitor 3-MA, PQ (10µM) induced the autophagy (as determined by quantifying the turnover of proteins in starved cells) as shown in Figure 6A. In this sense, exposure of human neuroblastoma cells with 10µM of PQ produced a marked decrease in the phosphorylation (Ser2448) status of mTOR (Figs. 6B and 6C), protein involved in the autophagy signaling, with a maximal effect after 30 min of herbicide exposure in CM (Fig. 6B). In starved cells incubated with PQ, the activation of phosphorylated mTOR was invaluable to all the tested times (Fig. 6C).
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The Inhibition of PQ-Induced Autophagy Accelerates the Apoptotic Cell Death in SH-SY5Y Cells
We investigated the possible connection between the accumulation of autophagic phenomenon and the apoptotic cell death induced by PQ in SH-SY5Y cells. The morphological analysis (Figure 7A) showed that the prototypic autophagy inhibitor 3-MA inhibited the PQ-induced autophagic vacuolization as well as protein degradation, already previously mentioned in Figure 6A. Moreover, cytofluorometric quantification revealed that a significant fraction of cells treated with 3-MA and/or PQ lost the capacity to retain the dye DiOC6(3) and hence dissipated the mitochondrial transmembrane potential (
m). Among this m low population, a fraction of cells incorporated the vital dye PI and thus lost the barrier function of their plasma membrane (Fig. 7B). This event was more evident in combination of 3-MA and the herbicide PQ. In the same way, the caspase-3 activation, as measured by Western blot detection of 17-kDa fragment (Fig. 7C), was more increased with PQ and 3-MA together that each one separately. Therefore, the combination of these results clearly suggests that the inhibition of PQ-induced autophagy accelerates the apoptotic cell death in SH-SY5Y cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several studies have shown the possibility that environmental neurotoxicants such as pesticides may be related to the development of nongenetic PD (Cory-Slechta et al., 2005
; Landrigan et al., 2005; Norris et al., 2004; Ritz et al., 2000; Sherer et al., 2001). PQ is one of the possible pesticides involved in PD genesis because of the similar chemical structure to the dopaminergic neurotoxicant MPP+ and the strong correlation between the incidence of the disease and the amount of PQ used (Ritz et al., 2000). In the present study we report, for the first time, that low concentrations of PQ induce molecular events compatible with autophagy in neuroblastoma SH-SY5Y cells. We also show that the inhibition of autophagy exacerbates PQ-induced apoptosis.
The human dopaminergic cell line SH-SY5Y, which has many qualities of nervous system neurons, was used as target neuronal cells. First, we confirmed that PQ induced neurotoxicity in SH-SY5Y cells in a dose-dependent manner both in CM and in starvation conditions (Fig. 1). This result was similar to the results reported by Gonzalez-Polo et al. (2004) and Yang et al. (2005). Our group found that PQ induced dose-dependent cell death and apoptosis in SH-SY5Y cells within a period of 24 h. PQ is a well-known oxidative-stress inducer (Gonzalez-Polo et al., 2004; Mollace et al., 2003), so most of the reports about it are focused on ROS production. In this sense, we show in our study that low concentrations of PQ (10µM) induce a robust ROS generation (Fig. 3), which is more evident in NF conditions. However, the major finding of this study is that low PQ concentrations produce characteristics of autophagy.
Autophagy was described for the first time by Schweichel et al. (1973). It is characterized by the appearance of numerous cytoplasmic AVs of lysosomal origin, followed by mitochondrial dilation and enlargement of the endoplasmic reticulum and the Golgi apparatus. Dysfunction of cellular degradation has been implicated in PD pathogenesis. Macroautophagy is the primary mechanism of degrading long-lived proteins and damaged organelles. The autophagic process begins with the remodeling of subcellular membranes into double-membrane vesicles that sequester cytoplasm and organelles in AVs. These vacuoles are fused with the lysosome to form the autophagolysosome. The content of the autophagolysosome are ultimately degraded by lysosomal degradative enzymes (Klionsky et al., 2000). The electron microscopy images in Figure 4 show that PQ induces the accumulation of double-membrane AVs in the cytoplasm. This event is accompanied by other autophagic characteristics as accumulation of the autophagosome marker LC3-II (Fig. 5) or protein degradation regulated negatively by the mTOR kinase signaling (Fig. 6). In this sense, it has been demonstrated that the inactivation of mTOR in yeast or mammalian cells leads to induction of autophagy, even in nutrient-rich medium, indicating that mTOR negatively controls starvation-induced autophagy (Blommaart et al., 1995). So, under unfavorable conditions, mTOR is inactive, leading to a reduction in protein synthesis and an upregulation of protein degradation (Dennis et al., 1999). Thus, we report here that PQ is involved in the control of autophagy in neuroblastoma SH-SY5Y cells. So, one possibility is that the cell treated with PQ triggers autophagy as a defense mechanism to degrade the oxidized proteins by ROS and damaged or dysfunctional cytoplasmic components.
The pesticide induced, in the first place, the morphological appearance of autophagy (Figs. 4–6). But finally the cells succumbed in morphological and biochemical changes that are typical of apoptotic cell death, as phosphatidylserine exposure (Fig. 1), m dissipation (Fig. 7), caspase-3 activation (Fig. 2), and chromatin condensation (Fig. 2). Interestingly, inhibition of caspases retarded cell death (Fig. 2), yet had no influence on autophagic vacuolization (data not shown). In this sense, there is some controversy in the bibliography regarding the precise role that autophagy might play in programed cell death. There are many reports describing situations in which autophagy activation accompanies apoptosis, but there are also cases in which autophagy enhancement leads to cell death in the absence of caspase activation (Bursch et al., 1996; Butler et al., 2000; Chi et al., 1999; Xiang et al., 1996). Moreover, autophagic cell death has been described in association with several neurodegenerative diseases, including PD (Anglade et al., 1997; Marino et al., 2004; Petersen et al., 2001). Increased levels of autophagy were observed in neuronal cell lines expressing mutant proteins associated with such diseases. Expression of PD-associated A53T, but not wild-type, -synuclein in PC12 cells induced massive AV formation (Stefanis et al., 2001). In addition, dopaminergic neurons with AVs have been observed in the substantia nigra of PD patients, along with neurons displaying apoptotic features (Anglade et al., 1997). However, the functional consequence of increased autophagic activity for neurodegeneration is not clear but has been suggested that excessive autophagy may directly lead to neuronal cell death (Yuan et al., 2003).
In the literature, concentrations of high micromolar or millimolar range of PQ are normally used to study its neurotoxicity and apoptosis induction (Gonzalez-Polo et al., 2004; Kim et al., 2004; Peng et al., 2004). The evidence that PQ triggers apoptotic cell death has been demonstrated in other cellular models, as in human lung epithelial cells (Cappelletti et al., 1998) as well as in PC12 cells (Li et al., 1999). Our experiments show that PQ induces apoptosis in SH-SY5Y cells at low concentrations (10µM) at the same concentrations that produces autophagic events. These low concentrations are not normally used in the PQ toxicity study, and nobody in the present has described evidences that relate PQ-induced apoptosis and autophagy.
In our study, we have shown for the first time that PQ initially induces a morphological appearance of autophagy but it finally succumbed to a typical apoptotic cell death caspase dependent. Hereby, we demonstrated that, with the prototypic autophagy inhibitor 3-MA 10mM, not only the vacuoles formation and the protein degradation induced by PQ are inhibited but also the apoptotic cell death was accelerated and the caspase-3 activation increased. In this regard, recent studies (Boya et al., 2005; Gonzalez-Polo et al., 2005a) already have demonstrated that the inhibition of autophagy triggers apoptosis, so in this case autophagy would be considered as a defense mechanism and not as a type of cell death; moreover, recent evidence (Kiffin et al., 2006) supports a protective role of the lysosomal system, which can eliminate altered intracellular components through autophagy, at least in the first stages of oxidative injury. So, we suggest that the early activation of autophagy induced by low concentrations of PQ in neuroblastoma SH-SY5Y can be attributed to the cell response like a defense mechanism against the rapid oxidative stress induced after PQ exposure. Our results indicated that the systems of defense of the cell seemed not to be sufficient because the cell finally succumbed to the apoptotic cell death. In addition, we have seen for the first time in our study that when we inhibited PQ-induced autophagy in presence of 3-MA, we accelerated the apoptotic death process.
In conclusion, our results help to suggest PQ neurotoxicity as an emerging PD model system. This study also provides new molecular bases for the knowledge of the relationship between autophagy and the cell response to stress and apoptosis cell death. However, further studies are required to elucidate the exact mechanism involved in PQ-induced autophagy and its relation with neuronal cell death.
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ACKNOWLEDGMENTS |
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Supported by grants 2PR04B002, 2PR06B124, and 3PR05C015 (Junta de Extremadura, Spain) and PI040828 (Fondo de Investigación Sanitaria, Ministerio de Sanidad y Consumo, Spain). R.A.G.P. was supported by the Junta de Extremadura re-incorporation fellowship. M.N.S. was supported by the Valhondo-Calaff Foundation fellowship. M.A.O. was supported by the Junta of Extremadura predoctoral fellowship. The authors would like to thank P. Delgado, R. Tarazona, and V. Llorente Vera for their invaluable technical assistance. We also thank Dr Guido Kroemer (Institute Gustave Roussy, Villejuif, France) and Tamotsu Yoshimori (National Institute of Genetics, Mishima, Japan) for regeants.
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REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Anglade P, Vyas S, Javoy-Agid F, Herrero MT, Michel PP, Marquez J, Mouatt-Prigent A, Ruberg M, Hirsch EC, Agid Y. Apoptosis and autophagy in nigral neurons of patients with Parkinson's disease. Histol. Histopathol. (1997) 12:25–31.[Web of Science][Medline]
Blommaart EF, Luiken JJ, Blommaart PJ, van Woerkom GM, Meijer AJ. Phosphorylation of ribosomal protein S6 is inhibitory for autophagy in isolated rat hepatocytes. J. Biol. Chem. (1995) 270:2320–2326.
Boya P, Gonzalez-Polo RA, Casares N, Perfettini JL, Dessen P, Larochette N, Metivier D, Meley D, Souquere S, Yoshimori T, et al. Inhibition of macroautophagy triggers apoptosis. Mol. Cell Biol. (2005) 25:1025–1040.
Boya P, Gonzalez-Polo RA, Poncet D, Andreau K, Vieira HL, Roumier T, Perfettini JL, Kroemer G. Mitochondrial membrane permeabilization is a critical step of lysosome-initiated apoptosis induced by hydroxychloroquine. Oncogene (2003) 22:3927–3936.[CrossRef][Web of Science][Medline]
Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. (1976) 72:248–254.[CrossRef][Web of Science][Medline]
Bursch W, Ellinger A, Kienzl H, Torok L, Pandey S, Sikorska M, Walker R, Hermann RS. Active cell death induced by the anti-estrogens tamoxifen and ICI 164 384 in human mammary carcinoma cells (MCF-7) in culture: The role of autophagy. Carcinogenesis (1996) 17:1595–1607.
Bursch W, Hochegger K, Torok L, Marian B, Ellinger A, Hermann RS. Autophagic and apoptotic types of programmed cell death exhibit different fates of cytoskeletal filaments. J. Cell Sci. (2000) 113(Pt 7):1189–1198.[Abstract]
Bus JS, Gibson JE. Paraquat: Model for oxidant-initiated toxicity. Environ. Health Perspect. (1984) 55:37–46.[Web of Science][Medline]
Butler R, Mitchell SH, Tindall DJ, Young CY. Nonapoptotic cell death associated with S-phase arrest of prostate cancer cells via the peroxisome proliferator-activated receptor gamma ligand, 15-deoxy-delta12,14-prostaglandin J2. Cell Growth Differ. (2000) 11:49–61.
Cappelletti G, Maggioni MG, Maci R. Apoptosis in human lung epithelial cells: Triggering by paraquat and modulation by antioxidants. Cell Biol. Int. (1998) 22:671–678.[CrossRef][Web of Science][Medline]
Chi S, Kitanaka C, Noguchi K, Mochizuki T, Nagashima Y, Shirouzu M, Fujita H, Yoshida M, Chen W, Asai A, et al. Oncogenic Ras triggers cell suicide through the activation of a caspase-independent cell death program in human cancer cells. Oncogene (1999) 18:2281–2290.[CrossRef][Web of Science][Medline]
Chun HS, Gibson GE, DeGiorgio LA, Zhang H, Kidd VJ, Son JH. Dopaminergic cell death induced by MPP(+), oxidant and specific neurotoxicants shares the common molecular mechanism. J. Neurochem. (2001) 76:1010–1021.[CrossRef][Web of Science][Medline]
Cory-Slechta DA, Thiruchelvam M, Barlow BK, Richfield EK. Developmental pesticide models of the Parkinson disease phenotype. Environ. Health Perspect. (2005) 113:1263–1270.[Web of Science][Medline]
Dennis PB, Fumagalli S, Thomas G. Target of rapamycin (TOR): Balancing the opposing forces of protein synthesis and degradation. Curr. Opin. Genet. Dev. (1999) 9:49–54.[CrossRef][Web of Science][Medline]
Di Monte DA, McCormack A, Petzinger G, Janson AM, Quik M, Langston WJ. Relationship among nigrostriatal denervation, parkinsonism, and dyskinesias in the MPTP primate model. Mov. Disord. (2000) 15:459–466.[CrossRef][Web of Science][Medline]
Dodge AD. The mode of action of the bipyridylium herbicides, paraquat and diquat. Endeavour (1971) 30:130–135.[CrossRef][Medline]
Filonova LH, Bozhkov PV, Brukhin VB, Daniel G, Zhivotovsky B, von Arnold S. Two waves of programmed cell death occur during formation and development of somatic embryos in the gymnosperm, Norway spruce. J. Cell Sci. (2000) 113(Pt 24):4399–4411.[Abstract]
Fuentes JM, Lompre AM, Moller JV, Falson P, le Maire M. Clean Western blots of membrane proteins after yeast heterologous expression following a shortened version of the method of Perini et al. Anal. Biochem. (2000) 285:276–278.[CrossRef][Web of Science][Medline]
Gonzalez-Polo RA, Boya P, Pauleau AL, Jalil A, Larochette N, Souquere S, Eskelinen EL, Pierron G, Saftig P, Kroemer G. The apoptosis/autophagy paradox: Autophagic vacuolization before apoptotic death. J. Cell Sci. (2005a) 118:3091–3102.
Gonzalez-Polo RA, Carvalho G, Braun T, Decaudin D, Fabre C, Larochette N, Perfettini JL, Djavaheri-Mergny M, Youlyouz-Marfak I, Codogno P, et al. PK11195 potently sensitizes to apoptosis induction independently from the peripheral benzodiazepin receptor. Oncogene (2005b) 24:7503–7513.[CrossRef][Web of Science][Medline]
Gonzalez-Polo RA, Rodriguez-Martin A, Moran JM, Niso M, Soler G, Fuentes JM. Paraquat-induced apoptotic cell death in cerebellar granule cells. Brain Res. (2004) 1011:170–176.[CrossRef][Web of Science][Medline]
Graeber MB, Moran LB. Mechanisms of cell death in neurodegenerative diseases: Fashion, fiction, and facts. Brain Pathol. (2002) 12:385–390.[Web of Science][Medline]
Hariri M, Millane G, Guimond MP, Guay G, Dennis JW, Nabi IR. Biogenesis of multilamellar bodies via autophagy. Mol. Biol. Cell (2000) 11:255–268.
Hertzman C, Wiens M, Bowering D, Snow B, Calne D. Parkinson's disease: A case-control study of occupational and environmental risk factors. Am. J. Ind. Med. (1990) 17:349–355.[Web of Science][Medline]
Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, Kominami E, Ohsumi Y, Yoshimori T. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J. (2000) 19:5720–5728.[CrossRef][Web of Science][Medline]
Kiffin R, Bandyopadhyay U, Cuervo AM. Oxidative stress and autophagy. Antioxid. Redox Signal. (2006) 8:152–162.[CrossRef][Web of Science][Medline]
Kim SJ, Kim JE, Moon IS. Paraquat induces apoptosis of cultured rat cortical cells. Mol. Cells (2004) 17:102–107.[Web of Science][Medline]
Klionsky DJ, Emr SD. Autophagy as a regulated pathway of cellular degradation. Science (2000) 290:1717–1721.
Lai CT, Yu PH. Dopamine- and L-beta-3,4-dihydroxyphenylalanine hydrochloride (L-Dopa)-induced cytotoxicity towards catecholaminergic neuroblastoma SH-SY5Y cells. Effects of oxidative stress and antioxidative factors. Biochem. Pharmacol. (1997) 53:363–372.[CrossRef][Web of Science][Medline]
Landles C, Bates GP. Huntingtin and the molecular pathogenesis of Huntington's disease. Fourth in molecular medicine review series. EMBO Rep. (2004) 5:958–963.[CrossRef][Web of Science][Medline]
Landrigan PJ, Sonawane B, Butler RN, Trasande L, Callan R, Droller D. Early environmental origins of neurodegenerative disease in later life. Environ. Health Perspect. (2005) 113:1230–1233.[Web of Science][Medline]
Langston JW, Ballard P. Parkinsonism induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP): Implications for treatment and the pathogenesis of Parkinson's disease. Can. J. Neurol. Sci. (1984) 11:160–165.[Web of Science][Medline]
Lee HS, Park CW, Kim YS. MPP(+) increases the vulnerability to oxidative stress rather than directly mediating oxidative damage in human neuroblastoma cells. Exp. Neurol. (2000) 165:164–171.[CrossRef][Web of Science][Medline]
Li X, Sun AY. Paraquat induced activation of transcription factor AP-1 and apoptosis in PC12 cells. J. Neural Transm. (1999) 106:1–21.[CrossRef][Web of Science][Medline]
Liou HH, Tsai MC, Chen CJ, Jeng JS, Chang YC, Chen SY, Chen RC. Environmental risk factors and Parkinson's disease: A case-control study in Taiwan. Neurology (1997) 48:1583–1588.
Marino G, Lopez-Otin C. Autophagy: Molecular mechanisms, physiological functions and relevance in human pathology. Cell Mol. Life Sci. (2004) 61:1439–1454.[Web of Science][Medline]
Mizushima N. Methods for monitoring autophagy. Int. J. Biochem. Cell Biol. (2004) 36:2491–2502.[CrossRef][Web of Science][Medline]
Mollace V, Iannone M, Muscoli C, Palma E, Granato T, Rispoli V, Nistico R, Rotiroti D, Salvemini D. The role of oxidative stress in paraquat-induced neurotoxicity in rats: Protection by non peptidyl superoxide dismutase mimetic. Neurosci. Lett. (2003) 335:163–166.[CrossRef][Web of Science][Medline]
Niso-Santano M, Moran JM, Garcia-Rubio L, Gomez-Martin A, Gonzalez-Polo RA, Soler G, Fuentes JM. Low concentrations of paraquat induces early activation of extracellular signal-regulated kinase 1/2, protein kinase B, and c-jun N-terminal kinase 1/2 pathways: Role of c-jun N-terminal kinase in paraquat-induced cell death. Toxicol. Sci. (2006) 92:507–515.
Norris EH, Giasson BI, Lee VM. Alpha-synuclein: Normal function and role in neurodegenerative diseases. Curr. Top. Dev. Biol. (2004) 60:17–54.[Web of Science][Medline]
Pattingre S, Bauvy C, Codogno P. Amino acids interfere with the ERK1/2-dependent control of macroautophagy by controlling the activation of Raf-1 in human colon cancer HT-29 cells. J. Biol. Chem. (2003) 278:16667–16674.
Peng J, Mao XO, Stevenson FF, Hsu M, Andersen JK. The herbicide paraquat induces dopaminergic nigral apoptosis through sustained activation of the JNK pathway. J. Biol. Chem. (2004) 279:32626–32632.
Petersen A, Larsen KE, Behr GG, Romero N, Przedborski S, Brundin P, Sulzer D. Expanded CAG repeats in exon 1 of the Huntington's disease gene stimulate dopamine-mediated striatal neuron autophagy and degeneration. Hum. Mol. Genet. (2001) 10:1243–1254.
Ritz B, Yu F. Parkinson's disease mortality and pesticide exposure in California 1984–1994. Int. J. Epidemiol. (2000) 29:323–329.
Sattler T, Mayer A. Cell-free reconstitution of microautophagic vacuole invagination and vesicle formation. J. Cell Biol. (2000) 151:529–538.
Schweichel JU, Merker HJ. The morphology of various types of cell death in prenatal tissues. Teratology (1973) 7:253–266.[CrossRef][Web of Science][Medline]
Shavali S, Carlson EC, Swinscoe JC, Ebadi M. 1-Benzyl-1,2,3,4-tetrahydroisoquinoline, a Parkinsonism-inducing endogenous toxin, increases alpha-synuclein expression and causes nuclear damage in human dopaminergic cells. J. Neurosci. Res. (2004) 76:563–571.[CrossRef][Web of Science][Medline]
Sherer TB, Betarbet R, Greenamyre JT. Pathogenesis of Parkinson's disease. Curr. Opin. Investig. Drugs (2001) 2:657–662.[Medline]
Stadelmann C, Deckwerth TL, Srinivasan A, Bancher C, Bruck W, Jellinger K, Lassmann H. Activation of caspase-3 in single neurons and autophagic granules of granulovacuolar degeneration in Alzheimer's disease. Evidence for apoptotic cell death. Am. J. Pathol. (1999) 155:1459–1466.
Stefanis L, Larsen KE, Rideout HJ, Sulzer D, Greene LA. Expression of A53T mutant but not wild-type alpha-synuclein in PC12 cells induces alterations of the ubiquitin-dependent degradation system, loss of dopamine release, and autophagic cell death. J. Neurosci. (2001) 21:9549–9560.
Suntres ZE. Role of antioxidants in paraquat toxicity. Toxicology (2002) 180:65–77.[CrossRef][Web of Science][Medline]
Tanner CM, Ben Shlomo Y. Epidemiology of Parkinson's disease. Adv. Neurol. (1999) 80:153–159.[Medline]
Xiang J, Chao DT, Korsmeyer SJ. BAX-induced cell death may not require interleukin 1 beta-converting enzyme-like proteases. Proc. Natl. Acad. Sci. U.S.A (1996) 93:14559–14563.
Xue L, Fletcher GC, Tolkovsky AM. Autophagy is activated by apoptotic signalling in sympathetic neurons: An alternative mechanism of death execution. Mol. Cell Neurosci. (1999) 14:180–198.[CrossRef][Web of Science][Medline]
Yuan J, Lipinski M, Degterev A. Diversity in the mechanisms of neuronal cell death. Neuron (2003) 40:401–413.[CrossRef][Web of Science][Medline]
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