Abstract
Exposure to pesticides is implicated in the etiopathogenesis of Parkinson's disease (PD). The organochlorine pesticide dieldrin is one of the environmental chemicals potentially linked to PD. Because recent evidence indicates that abnormal accumulation and aggregation of α-synuclein and ubiquitin-proteasome system dysfunction can contribute to the degenerative processes of PD, in the present study we examined whether the environmental pesticide dieldrin impairs proteasomal function and subsequently promotes apoptotic cell death in rat mesencephalic dopaminergic neuronal cells overexpressing human α-synuclein. Overexpression of wild-type α-synuclein significantly reduced the proteasomal activity. Dieldrin exposure dose-dependently (0–70 μM) decreased proteasomal activity, and 30 μM dieldrin inhibited activity by more than 60% in α-synuclein cells. Confocal microscopic analysis of dieldrin-treated α-synuclein cells revealed that α-synuclein-positive protein aggregates colocalized with ubiquitin protein. Further characterization of the aggregates with the autophagosomal marker mondansyl cadaverine and the lysosomal marker and dot-blot analysis revealed that these protein oligomeric aggregates were distinct from autophagosomes and lysosomes. The dieldrin-induced proteasomal dysfunction in α-synuclein cells was also confirmed by significant accumulation of ubiquitin protein conjugates in the detergent-insoluble fraction. We found that proteasomal inhibition preceded cell death after dieldrin treatment and that α-synuclein cells were more sensitive than vector cells to the toxicity. Furthermore, measurement of caspase-3 and DNA fragmentation confirmed the enhanced sensitivity of α-synuclein cells to dieldrin-induced apoptosis. Together, our results suggest that increased expression of α-synuclein predisposes dopaminergic cells to proteasomal dysfunction, which can be further exacerbated by environmental exposure to certain neurotoxic compounds, such as dieldrin.
Dieldrin, a long-lasting organochlorine pesticide, was widely used agriculturally before it was banned by the U.S. Environmental Protection Agency in 1974. The persistent accumulation of dieldrin in the environment and pesticide-contaminated food remains a major source of dieldrin exposure to humans even 30 years after its use was banned (Kanthasamy et al., 2005). Dieldrin is highly lipophilic and therefore accumulates in lipid-containing tissues, including the central nervous system, over a prolonged period, with a half-life of approximately 300 days. A recent investigation by the Centers for Disease Control and Prevention showed very high serum levels and a high dietary consumption level of dieldrin in farmers and their spouses in Iowa (Brock et al., 1998). Dieldrin exposures to the general population occur through various food sources such as meat, milk products, fruits, and fishes (Kanthasamy et al., 2005). A recent study reported a significant amount of dieldrin exposure through consumption of farm-raised salmon compared with North Atlantic salmon (Hites et al., 2004).
Parkinson's disease (PD), one of the most common neurodegenerative diseases, affects more than 1 million people in the United States, and the prevalence of the disease increases by approximately 70,000 individuals each year. Progressive and selective degeneration of dopaminergic neurons in the substantia nigra is characteristic of PD, which is accompanied by the formation of the cytoplasmic inclusions known as Lewy bodies (Dawson and Dawson, 2003). Although the causes and mechanisms underlying PD are not completely understood, accumulating evidence suggests that both environmental and genetic factors contribute to selective dopaminergic degeneration (Le Couteur et al., 2002). Among the genetic factors, at least 10 distinct loci are responsible for the familial forms of PD, including mutations in α-synuclein gene (A53T, A30P, and E46K), α-synuclein loci triplication, Parkin, ubiquitin C-terminal hydrolase-L1 (UCH-L1), DJ-1, phosphatase and tensin homolog deleted from chromosome 10-induced kinase 1 (PINK1), and leucine-rich repeat kinase 2 (LRRK2) (Dawson and Dawson, 2003; Moore DJ et al., 2005). Among the environmental factors, pesticides are one of the potential risk factors of PD, as revealed by recent epidemiological studies (Priyadarshi et al., 2000; Kanthasamy, 2005).
Dieldrin is implicated as one of the possible etiological factors for PD because of its detectable levels in the brains of some patients with PD but not in the brains of patients without PD (Fleming et al., 1994); significantly higher levels of dieldrin were detected in the caudate and substantia nigra of patients with PD compared with control subjects (Corrigan et al., 2000). Animal studies have shown that feeding dieldrin resulted in significantly decreased dopamine levels in brains of doves (Heinz et al., 1980). The relatively selective toxicity of dieldrin to dopaminergic neurons over GABAergic neurons has been reported in primary cultured neurons (Sanchez-Ramos et al., 1998). We have shown that dieldrin impairs mitochondrial function and induces oxidative stress and apoptotic cell death in dopaminergic cells (Kitazawa et al., 2001). We also showed that caspase-3-dependent proteolytic activation of the proapoptotic protein kinase Cδ contributes to apoptotic cell death (Kitazawa et al., 2003) and that the DNA repair enzyme poly(ADP-ribose) polymerase is inactivated by proteolytic cleavage in rat pheochromocytoma cells challenged by dieldrin (Kitazawa et al., 2004).
Impairment of ubiquitin-proteasome function and protein aggregation is an emerging area of investigation because genetic analysis of familial PD cases has elucidated mutation of key genes including α-synuclein, Parkin, and UCH-L1 (Le Couteur et al., 2002), some of which are important in protein processing and degradation. α-Synuclein has been identified as the major component of Lewy bodies in PD (Dawson and Dawson, 2003). Wild-type α-synuclein in monomeric and aggregated forms has been reported to interact with the S6′ subunit of 19S cap and inhibit proteasomal function (Snyder et al., 2003).
The recent discovery that an increased level of the α-synuclein gene resulting from the triplication of the α-synuclein locus causes PD in some individuals strongly suggests that overexpression of this gene could be a risk factor for PD (Singleton et al., 2003). The combination of α-synuclein overexpression and exposure to environmental pesticides is likely to contribute to increased vulnerability of nigral dopaminergic neurons. Therefore, in the present study, we investigated the effect of dieldrin, a pesticide with suspected involvement in PD pathogenesis, on proteasomal function and apoptotic cell death in the α-synuclein-overexpressing mesencephalic dopaminergic neuronal cell model (N27 cells).
Materials and Methods
Chemicals. Dieldrin, lactacystin, thioflavin S, monodansyl cadaverine (MDC), and Hoechst 33342 were purchased from Sigma Chemical Co. (St. Louis, MO). Methamphetamine was a gift from the National Institute on Drug Abuse (Bethesda, MD). The substrate used to measure proteasomal activity, Suc-Leu-Leu-Val-Try-7-amino-4-methylcoumarin (AMC), was purchased from Calbiochem (San Diego, CA). The caspase-3 substrate Ac-DEVD-AMC was obtained from Bachem Biosciences (King of Prussia, PA). An enhanced chemiluminescence (ECL) Western blot analysis kit was purchased from GE Healthcare (Little Chalfont, Buckinghamshire, UK). The Cell Death Detection enzyme-linked immunosorbent assay (ELISA) Plus Assay Kit was purchased from Roche Molecular Biochemicals (Indianapolis, IN). RPMI 1640 medium, fetal bovine serum, l-glutamine, penicillin/streptomycin, and hygromycin B were purchased from Invitrogen (Carlsbad, CA). Sytox green and Prolong antifade reagents were obtained from Invitrogen. The Bradford protein assay kit was purchased from Bio-Rad (Hercules, CA).
Cell Culture and Stable Expression of α-Synuclein. The immortalized rat mesencephalic dopaminergic cell line (referred to as N27 cells) was a gift from Dr. Kedar N. Prasad (University of Colorado Health Sciences Center, Denver, CO). N27 cells were grown in RPMI 1640 medium containing 10% fetal bovine serum, 2 mM l-glutamine, 50 units of penicillin, and 50 μg/ml of streptomycin in a humidified atmosphere of 5% CO2 at 37°C as described previously (Kaul et al., 2003). Dr. Eliezer Masliah (University of California, San Diego, CA) provided the pCEP4 expression vector containing the full-length human α-synuclein sequence, α-synuclein-pCEP4. α-Synuclein-pCEP4 and empty pCEP4 vector-conferring hygromycin resistance were transfected into N27 cells using Lipofectamine Plus reagent (Invitrogen) following the procedure recommended by the manufacturer and described recently (Kaul et al., 2005). For the stable transfection, N27 cells were selected in 400 μg/ml of hygromycin 48 h after transfection; supplementation of 200 μg/ml of hygromycin in the growth medium maintained the stable transfection.
Treatment Paradigm. Vector-expressing N27 cells and α-synuclein-expressing cells were treated with different concentrations of dieldrin or lactacystin dissolved in dimethyl sulfoxide (final concentration in the medium was no higher than 0.5%) for the duration of the experiments. After treatment, cells were collected by trypsinization, spun down at 200g for 5 min, and washed with ice-cold phosphate-buffered saline (PBS). The lysates from the cell pellets were used for various assays, including proteasome peptidase activity, caspase-3 activity, and measurement of DNA fragmentation.
Proteasomal Peptidase Activity Assay. The proteasome enzymatic assay was performed as described previously (Snyder et al., 2003) with slight modification. In brief, after treatment, cells were collected, washed, and lysed with hypotonic buffer (10 mM HEPES, 5 mM MgCl2, 10 mM KCl, 1% sucrose, and 0.1% CHAPS). The lysates were then incubated with fluorogenic Suc-LLVY-AMC (75 μM) in the assay buffer (50 mM Tris-HCl, 20 mM KCl, 5 mM MgOAc, and 10 mM dithiothreitol, pH 7.6) at 37°C for 30 min. The cleaved fluorescent product was measured at the excitation wavelength of 380 nm and emission wavelength of 460 nm using a fluorescence plate reader (Gemini Plate Reader; Molecular Devices, Sunnyvale, CA). The protein concentration was determined by the Bradford method. The enzymatic activity was normalized by protein concentration. Lysates from cells treated with 10 μM lactacystin for 12 h were used as the positive control for the assay.
Immunofluorescence Staining of Protein Aggregation. Immunofluorescence staining was performed essentially as described previously (Lee et al., 2002a). In brief, after dieldrin treatment, α-synuclein-overexpressing N27 cells grown on coverslips precoated with poly-l-lysine were washed with PBS and fixed in 4% paraformaldehyde. Coverslips were then washed three times with PBS, permeabilized with 0.2% Triton X-100 in PBS, and then incubated with blocking buffer (5% bovine serum albumin, 5% goat serum in PBS) to block the nonspecific binding sites. Thioflavin S staining was performed by incubating the cells with 0.4% thioflavin S followed by washing with 80% alcohol before processing the cells for α-synuclein immunochemical analysis. For ubiquitin and α-synuclein double staining, cells were incubated overnight with antibodies against α-synuclein (mouse monoclonal antibody, 1:500; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and ubiquitin (rabbit polyclonal antibody, 1:100; DakoCytomation California Inc., Carpinteria, CA). α-Synuclein and ubiquitin were visualized with Cy3-conjugated goat anti-mouse and Alexa 488-conjugated goat anti-rabbit secondary antibodies, respectively. For visualization of α-synuclein aggregates and lysosomes (Wilson et al., 2004), polyclonal α-synuclein antibody (1:500; BIOMOL Research Laboratories, Plymouth Meeting, PA) and monoclonal lysosomal-associated membrane protein 1 (LAMP-1) antibody (1:500; Calbiochem) were used. Cy3-conjugated anti-rabbit and Alexa 488-conjugated anti-mouse antibodies were used for the visualization of α-synuclein and LAMP-1, respectively. Nuclei were counterstained with Hoechst 33342 for 3 min at a final concentration of 10 μg/ml. Finally, cells were washed once in PBS and mounted onto a slide with mounting medium containing Prolong antifade reagent. In cases of autophagosome staining, live cells were incubated with 50 μM MDC (Sigma Chemical Co.) for 10 min at 37°C before fixation and α-synuclein immunostaining (Larsen et al., 2002). The images were analyzed by either C1 confocal microscopy (model TE-2000U; Nikon, Tokyo, Japan) or by confocal microscopy (model TCS NT; Leica, Wetzlar, Germany). Areas of α-synuclein immunopositive aggregates were measured in 14 randomly chosen cells from each group using Metamorph 5.07 image analysis software (Molecular Devices).
Western Blot Analysis of Ubiquitin-Conjugated Proteins. Low detergent-soluble and -insoluble fractions were separated according to the procedure described previously with slight modification (Rideout and Stefanis, 2002). After exposure to dieldrin or lactacystin, cells were collected and washed once with ice-cold PBS. The cell pellets were resuspended in a low detergent lysis buffer (protease inhibitors and 0.5% Triton X-100 in PBS). The lysates were ultracentrifuged at 100,000g for 40 min. The detergent-soluble fraction was obtained by collecting the resulting supernatant. The detergent-insoluble pellets were washed once with the lysis buffer and resuspended in PBS containing protease inhibitors and 2% SDS and then sonicated for 20 s. Equal amounts of protein from the detergent-soluble and equal volumes of the suspension of the detergent-insoluble fractions were resolved on 8% SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membrane. Nonspecific binding to the membranes was blocked by 5% nonfat milk blocking solution, and the membranes then were probed with ubiquitin antibody (1:500; DakoCytomation California Inc.) overnight at 4°C. Incubation with horseradish peroxidase (HRP)-conjugated secondary anti-rabbit or anti-mouse IgG (1:2000; GE Healthcare) for an additional 1 h was followed by the detection of the antibody-bound proteins using the ECL detection kit. The membranes were reprobed with β-actin antibody (mouse monoclonal, 1:5000; Sigma Chemical Co.) to confirm equal protein loading. The densitometric analysis of ubiquitin conjugates was performed with one-dimensional image analysis software (Eastman Kodak, Rochester, NY).
Dot-Blot Analysis. Formation of protein oligomers was determined by dot-blot measurements using A11 antibody (rabbit polyclonal; BioSource International, Camarillo, CA), which recognizes oligomers of proteins independent of amino acid sequence (Kayed et al., 2003). Dot-blot analysis using this antibody has recently been used for identification of protein oligomers in various neurodegenerative models (Kayed et al., 2003; Glabe, 2004). The procedure for dot-blot analysis was followed as described by the manufacturer. In brief, vector and α-synuclein cells were harvested after dieldrin treatment, and cell lysates were prepared as described above for Western blots. Cell lysates containing equal protein (5–15 μg) were spotted on the nitrocellulose membrane and air dried for 30 min. Membranes were incubated with A11 anti-oligomer antibody (dilution 1:2000) for 1 h at room temperature and then treated with HRP-conjugated secondary anti-rabbit IgG for 1 h. Antibody-bound proteins were detected with an ECL detection kit, and densitometric analysis of dots representing the oligomeric protein aggregates was performed with one-dimensional image analysis software (Eastman Kodak).
Assessment of Cell Death by Sytox Green. Cell death was assessed with Sytox green, a membrane-impermeable DNA dye that enters dead cells as a result of altered membrane permeability and intercalates into the nucleic acid (Kaul et al., 2005). DNA-bound Sytox green can be detected at an excitation wavelength of 485 nm and an emission wavelength of 538 nm using a fluorescence microplate reader (Gemini Plate Reader; Molecular Devices). Cells grown in 24-well plates were incubated with 1 μM Sytox green for 20 min and then treated with 30 μM dieldrin or vehicle as a control. To quantify cell death, fluorescence intensity was monitored after the experiments were conducted and normalized by the time-matched control.
Caspase-3 Enzymatic Activity Assay. Caspase-3 activity was measured as described previously (Kitazawa et al., 2003). In brief, cell lysates were obtained by suspending the cells in 50 mM Tris-HCl lysis buffer containing 1 mM EDTA, 10 mM EGTA and 10 μM digitonin after cells were collected and washed with PBS. The supernatants from lysates collected after centrifugation at 14,000g were incubated with 50 μM Ac-DEVD-AMC at 37°C for 1 h, and caspase-3 activity was measured using a fluorescence plate reader (Molecular Devices) with excitation at 380 nm and emission at 460 nm. Protein concentration was determined by the Bradford protein assay.
DNA Fragmentation Assay. DNA fragmentation was measured using a Cell Death Detection ELISA Plus Assay Kit as described previously (Kaul et al., 2003). This method measures the amount of histone-associated low molecular weight DNA in the cytoplasm of cells and is more sensitive than DNA ladder analysis. Dieldrin-treated cells were washed with PBS, and the cell pellets were then resuspended with the lysis buffer provided in the assay kit. The lysate was spun down at 200g, and 20 μl of supernatant was incubated for 2 h with the mixture of HRP-conjugated antibody that recognizes histones and single- and double-stranded DNA. After washing away the unbound components, the final reaction product was measured colorimetrically with 2,2′-azino-di-[3-ethylbenz-thiazoline sulfonate] as an HRP substrate using a spectrophotometer at 405 nm and 490 nm. The difference in absorbance between 405 and 490 nm was used to determine the amount of DNA fragmentation in each sample.
Data Analysis. Data are presented as mean ± S.E.M., and the data analysis was performed with Prism 3.0 software (GraphPad Software Inc., San Diego, CA). The p values were determined by one-way analysis of variance (ANOVA), followed by either Dunnett's post-test to compare dieldrin treatment groups with the control group or by Bonferroni multiple comparison test to compare all the pairs of groups. Single comparisons were made using the Student's t test. A significant difference was accepted if p < 0.05.
Results
α-Synuclein Overexpression Impairs Proteasomal Activity in Dopaminergic Neuronal Cells. First we examined the effect of overexpression of human α-synuclein on proteasomal activity in mesencephalic rat dopaminergic neuronal cells (N27 cells). Stable expression of human α-synuclein in N27 cells was determined by Western blot analysis using an antibody that recognizes only the exogenously expressed human α-synuclein or an antibody that detects both exogenously expressed human α-synuclein and endogenous rat α-synuclein as described previously (Kaul et al., 2005). There was an 11-fold increase in stable α-synuclein expression compared with vector-expressing N27 cells (data not shown). Enzymatic activity of 20S/26S proteasome was evaluated in α-synuclein-overexpressing and vector-transfected N27 cells with the specific fluorogenic substrate Suc-LLVY-AMC. As shown in Fig. 1, α-synuclein overexpression significantly (p < 0.001) inhibited proteasomal activity compared with vector-expressing cells. An almost 50% reduction in proteasomal activity was observed in α-synuclein-overexpressing cells. The well-known proteasome inhibitor lactacystin (10 μM) was used as a positive control, which inhibited more than 90% of proteasomal activity in N27 cells.
Dieldrin Impairs Proteasomal Activity in a Dose- and Time-Dependent Manner. Next we examined the effect of dieldrin exposure on proteasomal activity in vector and α-synuclein-overexpressing N27 dopaminergic neuronal cells. Figure 2A shows a dose-dependent decrease in the proteasomal activity in both vector cells and α-synuclein-overexpressing cells after 0 to 70 μM dieldrin exposure for 24 h. The EC50 values for vector and α-synuclein-overexpressing cells were 50 and 32 μM, respectively. Exposure to 30 μM dieldrin for 12 and 24 h caused significant proteasomal dysfunction in both vector and α-synuclein-transfected cells, so this dose was used for further studies. We conducted a detailed time course analysis of proteasomal activity after 30 μM dieldrin exposure to determine the earliest time point at which dieldrin impairs proteasomal activity (Fig. 2B). Proteasomal activity significantly decreased within 12 h in vector-transfected and α-synuclein-overexpressing cells (p < 0.01) and remained reduced during the entire treatment period (Fig. 2B).
To determine whether the effects of dieldrin and α-synuclein on proteasome activity are additive or synergistic, we exposed vector cells to 300 nM lactacystin for 3 h to reduce the baseline ubiquitin proteasome system (UPS) activity to the level of α-synuclein-expressing cells; these cells then were incubated with 30 μM dieldrin for an additional 24 h, and UPS activity was measured. As shown in Fig. 2C, exposure of vector cells to 300 nM lactacystin decreased the UPS activity by 53%, which approximated the baseline UPS activity in α-synuclein-expressing cells. A 24-h exposure to 30 μM dieldrin further decreased UPS activity by 18% and 13% in vector cells and in lactacystin-treated vector cells, respectively. Because the extent of UPS inhibition by dieldrin is similar in vector and α-synuclein cells, these results suggest that the effects of α-synuclein and dieldrin are additive and not synergistic.
Dieldrin Exposure Induces the Formation of Intracellular Inclusions Containing α-Synuclein. The biological consequences of impaired proteasomal function are accumulation of proteins, formation of intracellular protein inclusions, and up-regulation of ubiquitin-conjugated proteins. Accumulation of α-synuclein in the form of intracellular inclusions and increased levels of ubiquitinated protein are typical pathological changes associated with PD. In this study, we examined whether the inhibition of proteasomal function by dieldrin exposure promotes the formation of intracellular protein inclusions and ubiquitinated protein accumulation. Confocal analysis revealed the formation of α-synuclein-positive aggregation in a time-dependent manner, with small aggregates appearing as early as 12 h and progressively increasing over 24 h in dieldrin-treated cells (Fig. 3A). Vehicle only-treated cells for a 24-h period did not show any significant formation of α-synuclein aggregates. However, as anticipated, the proteasome inhibitor lactacystin induced a profound aggregation in α-synuclein-overexpressing cells (data not shown). Quantitative analysis of α-synuclein-positive aggregates using Metamorph image analysis software is shown in Fig. 3B. The results show that dieldrin exposure for 12 and 24 h significantly increased the number of intracellular inclusion bodies (Fig. 3B). In this experiment, we observed that the protein aggregates were negative for thioflavin S staining. Furthermore, we observed no protein inclusions in either dieldrin-treated vector cells or untreated α-synuclein cells. We attribute this finding to recent reports that suggest thioflavin S stains large perinuclear inclusions but not the small oligomeric aggregates (Lee et al., 2002a).
Dieldrin Induces the Formation of Soluble Oligomer Proteins in α-Synuclein Cells. Neurodegenerative diseases are associated with the accumulation of misfolded proteins in the form of fibrillar protein aggregates (Kayed et al., 2003; Glabe, 2004). The formation of oligomers from misfolded monomeric proteins has been suggested to precede fibrillar formation. Recent development of an antibody A11, which recognizes amino acid sequence-independent oligomers of proteins including β-amyloid, α-synuclein, polyglutamine proteins, and prion peptide 106 to126, has enabled the determination of protein oligomers in many experimental neurodegenerative models (Kayed et al., 2003; Glabe, 2004). Thus, to further verify dieldrin-induced protein aggregation, we used the A11 antibody in dot-blot experiments. Vector and α-synuclein cells were exposed to 30 μM dieldrin for 12 and 24 h. As shown in Fig. 4A, dieldrin treatment for 12 and 24 h significantly increased the protein oligomer levels in α-synuclein cells. The vector-treated cells showed only a background staining. Densitometric analysis of the dot-blot revealed 170% and 217% increases in oligomeric protein staining at 12 and 24 h, respectively, in dieldrin-treated α-synuclein-expressing cells (Fig. 4B) compared with 91% and 115% increases in dieldrin-treated vector cells. Together, these results with confocal experiments indicate that dieldrin induces protein aggregates in α-synuclein-expressing cells.
Dieldrin-Induced α-Synuclein Inclusions Colocalize with Ubiquitin but Not with Autophagosomes or Lysosomes. Because dieldrin treatment impairs UPS activity, we also determined whether ubiquitin colocalizes with α-synuclein inclusions. Confocal microscopic analysis of antiubiquitin immunohistochemical images revealed that the α-synuclein-positive aggregates tended to be ubiquitin-immunoreactive, as seen by the colocalization of ubiquitin with α-synuclein-positive aggregates (Fig. 5A). To further distinguish the α-synuclein inclusions from autophagosomes, we performed autophagosome marker MDC staining. As shown in Fig. 5B, dieldrin-treated cells showed a moderate increase in autophagic vacuoles, but these vacuoles did not colocalize with α-synuclein aggregates, indicating that the protein aggregates observed after dieldrin treatments are not autophagosomes. Methamphetamine was used as a positive control because it induced autophagy in mouse primary dopaminergic neurons (Larsen et al., 2002) and in the N27 dopaminergic cell model used in this study (Ar. Kanthasamy and An. Kanthasamy, unpublished observations). MDC labeling revealed large autophagic vacuoles in methamphetamine-treated cells (Fig. 5B), and these vacuoles clearly excluded the α-synuclein staining. To further distinguish the α-synuclein aggregates from enlarged lysosomes (Wilson et al., 2004), we performed lysosomal marker LAMP-1 immunohistochemical analysis. As shown in Fig. 5C, confocal microscopic analysis of anti-LAMP-1 immunocytochemical images revealed that dieldrin-induced α-synuclein-positive aggregates did not colocalize with LAMP-1 immunoreactivity, as shown by the distinct staining patterns of LAMP-1 and α-synuclein aggregates.
Accumulation of High Molecular Weight Ubiquitin-Conjugated Proteins during Dieldrin Exposure. Impairment of the proteasomal machinery leads to accumulation of ubiquitinated protein in the cytosol caused by the reduced clearance of proteins by the proteasome (Rideout and Stefanis, 2002). High molecular weight (HMW) ubiquitin-conjugated proteins accumulated dramatically in the low detergent-insoluble fraction from both vector and α-synuclein-overexpressing cells after 24 h of dieldrin exposure (Fig. 6A). However, the accumulation of insoluble HMW ubiquitin conjugates in α-synuclein-overexpressing cells was much higher than in vector-transfected cells. Densitometric analysis of the HMW bands from Western blots revealed 136% and 121% of the proteins in vector cells compared with 172% and 301% in α-synuclein cells after 30 μM dieldrin treatment for 12 and 24 h, respectively (Fig. 6B). The slight decrease in HMW ubiquitin conjugates in vector cells at 24 h measured by Western blot analysis was not statistically significant. In addition to the HMW ubiquitin conjugates in insoluble fractions, dieldrin treatment also produced a significant increase in the level of HMW ubiquitin conjugates in the soluble fraction of both α-synuclein cells and vector-transfected cells (Fig. 6C). However, the level of ubiquitin conjugates did not differ significantly between α-synuclein and vector cells (Fig. 6D). These results suggest that dieldrin induces a time-dependent increase in insoluble HMW ubiquitin conjugates in vector and α-synuclein cells. Treatment with 10 μM lactacystin for 12 h dramatically increased the level of the HMW ubiquitin conjugates in both soluble and insoluble fractions and was used as a positive control (Fig. 6, A and B).
Overexpression of α-Synuclein Increases the Sensitivity of N27 Cells to Dieldrin-Induced Neurotoxicity. To determine whether the formation of insoluble protein aggregates and accumulation of ubiquitin proteins during dieldrin treatments enhances the neurodegenerative processes in α-synuclein-overexpressing cells, we investigated the temporal dieldrin cytotoxicity in vector and α-synuclein-overexpressing cells. Cell death was assessed using the membrane-impermeable Sytox green fluorescence dye at various time intervals after 24 h of dieldrin treatment. As shown in Fig. 7, α-synuclein-overexpressing cells showed enhanced cytotoxicity at 24 h, whereas cytotoxicity was not significantly increased in vector-transfected cells up to 24 h. We also noted a consistent decrease in dieldrin-induced cytotoxicity in α-synuclein-overexpressing cells compared with vector cells up to the 12-h time point.
Dieldrin Induced Caspase-3 Activation and DNA Fragmentation in Vector and α-Synuclein-Overexpressing Cells. To determine the functional consequence of dieldrin-induced proteasomal inhibition and protein aggregation on cell survival, we determined caspase-3 activation and DNA fragmentation after dieldrin exposure. As shown in Fig. 8A, measurement of caspase-3 activity using Ac-DEVD-AMC as a substrate revealed that dieldrin activated caspase-3 to 205.1% at 12 h and to 311.5% at 24 h in α-synuclein-overexpressing N27 cells, whereas only a minimal increase of 138.1% and 168.5% was observed in vector cells at 12 and 24 h after treatment, respectively. In addition, the classic proteasome inhibitor lactacystin dramatically increased caspase-3 activation (Fig. 8A).
Furthermore, to determine the extent of apoptotic cell death in dopaminergic neuronal cells after dieldrin exposure, we measured DNA fragmentation using the ELISA method. As shown in Fig. 8B, dieldrin significantly (*, p < 0.05; ***, p < 0.001) increased DNA fragmentation in both vector and α-synuclein-overexpressing cells, but dieldrin-induced DNA fragmentation was more pronounced in α-synuclein-overexpressing cells than in vector cells (##, p < 0.01), indicating that α-synuclein-overexpressing cells are more sensitive to apoptotic cell death.
Discussion
Our study clearly shows that dieldrin impairs ubiquitin-proteasome function additively with α-synuclein and triggers apoptosis in dopaminergic neuronal cells. The direct consequence of the proteasome inhibition is the abnormal accumulation of ubiquitinated protein and the formation of intracellular protein aggregates (Rideout and Stefanis, 2002). In this study, we showed that dieldrin exposure promotes the formation of the α-synuclein-positive intracellular inclusions and accumulation of HMW ubiquitin-conjugated proteins. Overall, our data suggest that the combination of environmental exposure to neurotoxic chemicals like pesticides and α-synuclein overexpression may enhance the susceptibility to apoptotic cell death by impairing the ubiquitin-proteasome system.
Epidemiological studies increasingly implicate pesticides as an important risk factor of PD (Priyadarshi et al., 2000) and suggest that exposure to the organochlorine class of pesticide may be of particular concern in the development of PD (Corrigan et al., 2000; Kanthasamy et al., 2005). Epidemiological studies and case control findings in PD brains (Fleming et al., 1994; Corrigan et al., 2000) and experimental studies in cell culture (Sanchez-Ramos et al., 1998; Kitazawa et al., 2001, 2003, 2004) and animal models (Heinz et al., 1980; Kanthasamy et al., 2005) have implicated the specific organochlorine pesticide dieldrin in PD. Dieldrin is a highly lipophilic compound and accumulates significantly in the central nervous system (Fleming et al., 1994; Corrigan et al., 2000). The concentration of dieldrin detected in postmortem PD brain tissue was approximately 50 ppm (Fleming et al., 1994), and the blood dieldrin level was up to 250 ng/ml in workers manufacturing or using aldrin/dieldrin (Nair et al., 1992). A high level of dieldrin accumulates in the body over a lifetime because of the extremely low clearance of this lipophilic neurotoxic compound from the body. Based on reports (MacIntosh et al., 1996; Doong et al., 1999; Campoy et al., 2001), we calculated the cumulative lifetime exposure to dieldrin of approximately 30 μM. We deduced this based on a daily dietary intake of 1.3 μg of dieldrin over a 50-year exposure period, resulting in intake of 41 μM. The 30 μM concentrations used in the present study are lower than concentrations used in previous neurotoxicological studies in the rat pheochromocytoma cell culture model (Kitazawa et al., 2001, 2003, 2004). The EC50 of 12 μM dieldrin for primary dopaminergic neurons and 85 μM for nondopaminergic neurons (Sanchez-Ramos et al., 1998) indicates an increased susceptibility of the dopaminergic system to the neurotoxic effect of dieldrin.
We show that exposure to a subtoxic concentration of dieldrin (30 μM dieldrin, 11.2 μg/ml) resulted in decreased proteasomal activity and the subsequent accumulation of ubiquitin-conjugated proteins and formation of α-synuclein/ubiquitin-immunopositive inclusions in α-synuclein-overexpressing cells. We also show that dieldrin induces formation of oligomeric aggregates in a time-dependent manner in α-synuclein-expressing cells. Although our results show that dieldrin-induced α-synuclein-positive aggregates do not colocalize with either autophagosomes or lysosomes for up to 24 h of dieldrin treatment, prolonged accumulation of protein aggregates after long-term exposure of dieldrin may be degraded via the lysosomal pathway. In addition, our results are in agreement with a recent study showing that a prolonged exposure to dieldrin can change the conformation of α-synuclein to produce protein fibrils in a cell-free system (Uversky et al., 2001). In addition, an interaction between filamentous α-synuclein and subunits of the 20S proteasome core has been shown to decrease its proteolytic activity (Lindersson et al., 2004). Our study clearly suggests that α-synuclein contributes to the dysfunction of the UPS because accumulated insoluble HMW ubiquitin-conjugated proteins were predominantly observed only in α-synuclein-overexpressing N27 cells but not in vector N27 cells during dieldrin treatment. Previous studies have shown that the overexpression of α-synuclein A53T mutant impairs UPS in cultured cells (Lee et al., 2002b). We showed recently that overexpression of A53T α-synuclein mutant potentiates MPP+-induced apoptotic cell death in N27 cells (Kaul et al., 2005). Therefore, it is likely that overexpression of A53T mutant may result in greater dieldrin-induced UPS dysfunction than impairment in wild-type α-synuclein-expressing cells.
Oxidative stress and nitrative stress have been shown to contribute to various neurodegenerative diseases, including PD (Ischiropoulos, 2003). In support of this view, studies have shown that formation of reactive oxygen species and reactive nitrogen species can promote α-synuclein aggregation in in vitro models (Paxinou et al., 2001; Ischiropoulos, 2003). Because reactive oxygen species generation and oxidative stress have also been linked to proteasome inhibition (Okada et al., 1999), dieldrin-induced oxidative stress may play a role in the impairment of proteasomal function (Kitazawa et al., 2001). However, the antioxidant Trolox and the superoxide dismutase mimetic MnTBAP did not block dieldrin-induced UPS impairment in preliminary studies (F. Sun, V. Anantharam, Ar. Kanthasamy, and An. Kanthasamy, unpublished observations). Administration of paraquat, a pesticide known to generate superoxide radicals, induced α-synuclein aggregation in transgenic mice models (Manning-Bog et al., 2002). ATP depletion caused by the mitochondrial complex I inhibitor rotenone reduced proteasomal activity and formation of intracellular inclusions, which could be prevented by promoting ATP production (Hoglinger et al., 2003). Dieldrin is a mitochondrial electron transport inhibitor, which inevitably impairs oxidative ATP production (Kanthasamy et al., 2005). Therefore, a reduction in energy production may also contribute to proteasomal dysfunction during dieldrin exposure. However, further mechanistic studies are needed to establish the exact cellular mechanism underlying dieldrin-induced proteasomal dysfunction in dopaminergic neurons.
The impairment of proteasome function and protein aggregation during dieldrin treatment contributes to cell death. A time course analysis revealed that the proteasomal dysfunction and protein aggregation precedes the cellular toxicity. The α-synuclein aggregation starts to occur as early as 6 h after dieldrin treatment, whereas significant toxicity is noted only after 12 h of dieldrin treatment. In addition, dieldrin-induced cell death occurs only in α-synuclein-overexpressing N27 cells but not in vector N27 cells, suggesting that α-synuclein aggregation plays a causal role in the cytotoxic response. The time course analysis of the apoptotic marker caspase-3 further indicates that dieldrin-induced protein aggregation precedes capase-3 activation. In addition, we showed that the selective proteasome inhibitor lactacystin activates caspase-3. Measurement of DNA fragmentation by ELISA after dieldrin treatment in α-synuclein-overexpressing dopaminergic cells indicates that dieldrin-induced protein aggregation promotes apoptotic cell death. We also noted an increase in autophagosomes after dieldrin treatment, but detailed studies are needed regarding the individual contributions of apoptosis and autophagy during dieldrin-induced cell death.
The exact proapoptotic mechanisms downstream of the proteasome inhibition remain unclear in neuronal cells, although various signaling molecules involved in the regulation of apoptotic cell death have been identified as substrates of UPS, including p53, IκB, Smac, and the BclII family of proteins (Jesenberger and Jentsch, 2002). The UPS is the major cellular proteolytic machinery for the degradation of intracellular proteins. Identification of the mutant Parkin and ubiquitin C-terminal hydrolase (UCH-L1) genes in familial PD, as well as the impaired function and altered component levels of proteasome in the substantia nigra region of patients with sporadic PD, together suggest a critical role of UPS dysfunction in PD. Dopaminergic neurons are particularly susceptible to proteasome inhibition, and α-synuclein fibrillar inclusion is a characteristic pathological feature of PD (Dawson and Dawson, 2003). A recent study in transgenic flies revealed that overexpression of one of the Parkin substrates, Pael-R, caused selective degeneration of dopaminergic neurons, which could be suppressed by the coexpression of Parkin, which has E3 ligase activity (Yang et al., 2003). Administration of the proteasome inhibitor epoxomicin in rats has recently been shown to induce delayed symptoms and pathology similar to PD (McNaught et al., 2004). It is noteworthy that impaired proteasome function and formation of Lewy bodies were observed in this model, indicating enhanced vulnerability of the dopaminergic system to impairment of UPS. The recent interesting discovery of multiple copies of the α-synuclein gene in some patients with PD (Singleton et al., 2003) suggests that overexpression of α-synuclein can increase the risk of dopaminergic generation, and it was shown that in wild-type human α-synuclein transgenic mice, the loss of dopaminergic terminals was accompanied by the formation of intracellular inclusions (Masliah et al., 2000). Our data show that overexpression of human α-synuclein can dramatically inhibit proteasomal activity in dopaminergic neuronal cells. It is noteworthy that our results also suggest that exposure to environmental chemicals in individuals with increased copies of α-synuclein may enhance their vulnerability to PD.
In summary, we show for the first time that dieldrin and α-synuclein cumulatively induce impairment of ubiquitin-proteasome function to promote apoptotic cell death in dopaminergic neuronal cells. This study also reveals a close interaction between environmental factors and genetic defects in the promotion of dopaminergic degeneration involved in PD.
Footnotes
-
This study was supported by National Institutes of Health Grants NS45133, ES10586, and NS38644.
-
doi:10.1124/jpet.105.084632.
-
ABBREVIATIONS: PD, Parkinson's disease; MDC, monodansyl cadaverine; AMC, 7-amino-4-methylcoumarin; ECL, enhanced chemiluminescence; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; LAMP-1, lysosomal associated membrane protein 1; HRP, horseradish peroxidase; ANOVA, analysis of variance; UPS, ubiquitin proteasome system; HMW, high molecular weight; Trolox, 6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid; Suc, N-succinyl.
- Received February 5, 2005.
- Accepted June 27, 2005.
- The American Society for Pharmacology and Experimental Therapeutics