Ziram Causes Dopaminergic Cell Damage by Inhibiting E1 Ligase of the Proteasome
2008; Elsevier BV; Volume: 283; Issue: 50 Linguagem: Inglês
10.1074/jbc.m802210200
ISSN1083-351X
AutoresArthur P. Chou, Nigel T. Maidment, Rebecka Klintenberg, John E. Casida, Sharon Li, Arthur G. Fitzmaurice, Pierre‐Olivier Fernagut, Farzad Mortazavi, Marie‐Françoise Chesselet, Jeff M. Bronstein,
Tópico(s)Protein Degradation and Inhibitors
ResumoThe etiology of Parkinson disease (PD) is unclear but may involve environmental toxins such as pesticides leading to dysfunction of the ubiquitin proteasome system (UPS). Here, we measured the relative toxicity of ziram (a UPS inhibitor) and analogs to dopaminergic neurons and examined the mechanism of cell death. UPS (26 S) activity was measured in cell lines after exposure to ziram and related compounds. Dimethyl- and diethyldithiocarbamates including ziram were potent UPS inhibitors. Primary ventral mesencephalic cultures were exposed to ziram, and cell toxicity was assessed by staining for tyrosine hydroxylase (TH) and NeuN antigen. Ziram caused a preferential damage to TH+ neurons and elevated α-synuclein levels but did not increase aggregate formation. Mechanistically, ziram altered UPS function through interfering with the targeting of substrates by inhibiting ubiquitin E1 ligase. Sodium dimethyldithiocarbamate administered to mice for 2 weeks resulted in persistent motor deficits and a mild reduction in striatal TH staining but no nigral cell loss. These results demonstrate that ziram causes selective dopaminergic cell damage in vitro by inhibiting an important degradative pathway implicated in the etiology of PD. Chronic exposure to widely used dithiocarbamate fungicides may contribute to the development of PD, and elucidation of its mechanism would identify a new potential therapeutic target. The etiology of Parkinson disease (PD) is unclear but may involve environmental toxins such as pesticides leading to dysfunction of the ubiquitin proteasome system (UPS). Here, we measured the relative toxicity of ziram (a UPS inhibitor) and analogs to dopaminergic neurons and examined the mechanism of cell death. UPS (26 S) activity was measured in cell lines after exposure to ziram and related compounds. Dimethyl- and diethyldithiocarbamates including ziram were potent UPS inhibitors. Primary ventral mesencephalic cultures were exposed to ziram, and cell toxicity was assessed by staining for tyrosine hydroxylase (TH) and NeuN antigen. Ziram caused a preferential damage to TH+ neurons and elevated α-synuclein levels but did not increase aggregate formation. Mechanistically, ziram altered UPS function through interfering with the targeting of substrates by inhibiting ubiquitin E1 ligase. Sodium dimethyldithiocarbamate administered to mice for 2 weeks resulted in persistent motor deficits and a mild reduction in striatal TH staining but no nigral cell loss. These results demonstrate that ziram causes selective dopaminergic cell damage in vitro by inhibiting an important degradative pathway implicated in the etiology of PD. Chronic exposure to widely used dithiocarbamate fungicides may contribute to the development of PD, and elucidation of its mechanism would identify a new potential therapeutic target. Parkinson disease (PD) 2The abbreviations used are: PD, Parkinson disease; E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin ligase; UPS, ubiquitin proteasome system; VMC, ventral mesencephalic cultures; TH, tyrosine hydroxylase; DA, dopaminergic; DMDC, dimethyldithiocarbamate; NaDMDC, sodium DMDC; HEK, human embryonic kidney; GFP, green fluorescent protein; SNc, substantia nigra pars compacta; ANOVA, analysis of variance. is a common neurodegenerative disease characterized by relatively selective degeneration of dopaminergic (DA) neurons in the substantia nigra (nigrostriatal neurons). The etiology probably involves both environmental and genetic factors including pesticide exposure (1.Ascherio A. Chen H. Weisskopf M.G. O'Reilly E. McCullough M.L. Calle E.E. Schwarzschild M.A. Thun M.J. Ann. Neurol. 2006; 60: 197-203Crossref PubMed Scopus (339) Google Scholar, 2.Kamel F. Tanner C. Umbach D. Hoppin J. Alavanja M. Blair A. Comyns K. Goldman S. Korell M. Langston J. Ross G. Sandler D. Am. J. Epidemiol. 2007; 165: 364-374Crossref PubMed Scopus (280) Google Scholar, 3.Brown T.P. Rumsby P.C. Capleton A.C. Rushton L. Levy L.S. Environ. Health Perspect. 2006; 114: 156-164Crossref PubMed Scopus (338) Google Scholar). Hundreds of pesticides are used alone or in combinations making it difficult to separate individual effects. Because no individual pesticide has been established by epidemiologic studies, we chose to perform an unbiased screen of potential toxicants for their ability to interfere with the ubiquitin-proteasome system (UPS), a biological pathway implicated in the etiology of PD. Impaired UPS activity has been reported in the brains of patients with PD, and mutations in two UPS genes, Parkin and UCHL-1, cause rare genetic forms of PD (4.McNaught K.S. Jenner P. Neurosci. Lett. 2001; 297: 191-194Crossref PubMed Scopus (566) Google Scholar). Although these results are not universally reproduced (5.Manning-Bog A.B. Reaney S.H. Chou V.P. Johnston L.C. McCormack A.L. Johnston J. Langston J.W. Di Monte D.A. Ann. Neurol. 2006; 60: 256-260Crossref PubMed Scopus (90) Google Scholar, 6.Kordower J.H. Kanaan N.M. Chu Y. Suresh Babu R. Stansell J. 3rd, Terpstra B.T. Sortwell C.E. Steece-Collier K. Collier T.J. Ann. Neurol. 2006; 60: 264-268Crossref PubMed Scopus (120) Google Scholar, 7.Bove J. Zhou C. Jackson-Lewis V. Taylor J. Chu Y. Rideout H.J. Wu D.C. Kordower J.H. Petrucelli L. Przedborski S. Ann. Neurol. 2006; 60: 260-264Crossref PubMed Scopus (126) Google Scholar), in some studies administration of UPS inhibitors to rodents recapitulates many of the clinical and pathological aspects of PD (8.McNaught K.S. Olanow C.W. Ann. Neurol. 2006; 60: 243-247Crossref PubMed Scopus (69) Google Scholar, 9.Schapira A.H. Cleeter M.W. Muddle J.R. Workman J.M. Cooper J.M. King R.H. Ann. Neurol. 2006; 60: 253-255Crossref PubMed Scopus (80) Google Scholar, 10.McNaught K.S. Perl D.P. Brownell A.L. Olanow C.W. Ann. Neurol. 2004; 56: 149-162Crossref PubMed Scopus (454) Google Scholar). We hypothesized that chronic pesticide exposure may increase the risk of developing PD by inhibiting the UPS. We screened several pesticides for their ability to inhibit the UPS and found a number of toxicants that can lower activity at relevant concentrations (11.Wang X.F. Li S. Chou A.P. Bronstein J.M. Neurobiol. Dis. 2006; 23: 198-205Crossref PubMed Scopus (117) Google Scholar). We then focused on dithiocarbamate fungicides because they were found to be one of the most potent UPS inhibitors and are widely used in crop protection. In the present study, zinc dimethyldithiocarbamate (ziram) was one of several dimethyl- and diethyldithiocarbamates found to inhibit the UPS at 0.15–1 μm. Furthermore, ziram increased α-synuclein expression in DA cells, induced relatively selective DA cell damage in vitro, and inhibited the UPS by interfering with ubiquitin E1 ligase activity. In vivo, systemic administration of the more soluble sodium dimethyldithiocarbamate (NaDMDC) in mice resulted in motor deficits and damage to the nigrostriatal pathway. These findings help explain how chronic pesticide exposure could increase the risk of developing PD. Chemicals—The test compounds (see Table 1) were from Chem Service (West Chester, PA), Sigma-Aldrich, or other commercial sources as the highest available purity, except for 7 and 8, which were synthesized by Karl Fisher in the Casida laboratory.TABLE 1Structure-activity relationships for ziram-related compounds on 26 S UPS with 24-h treatment of SK-N-MC cells Open table in a new tab Measurement of 26 S Proteasome Activity and Cell Death in Cell Lines—26 and 20 S UPS activity and cell death were measured in human embryonic kidney (HEK) and neuroblastoma SK-N-MC cells by flow cytometry as previously described (12.Bence N.F. Sampat R.M. Kopito R.R. Science. 2001; 292: 1552-1555Crossref PubMed Scopus (1858) Google Scholar). Fluorescence of the green fluorescent protein degron fusion protein (GFP-U) was measured and expressed as a percentage of control. Rat Primary Ventral Mesencephalic Cultures (VMC)—VMC were prepared by using a protocol adapted from Rayport et al. (13.Rayport S. Sulzer D. Shi W.X. Sawasdikosol S. Monaco J. Batson D. Rajendran G. J. Neurosci. 1992; 12: 4264-4280Crossref PubMed Google Scholar). Briefly, the cultures were prepared in two stages. In the first stage, cortical glial feeder cells were established on polyornithine/laminin-coated coverslips, which formed the base of a 10-mm-diameter well cut into 35-mm culture dishes, until they reached confluency in ∼6 days. Fluorodeoxyuridine was then added to prevent additional glial proliferation. In the second stage, postnatal day 2 or 3 pups were anesthetized, and 1 mm3 mesencephalic blocks containing the ventral tegmental area were dissected from sagittal sections taken along the midline of the brain. The cells were dissociated and plated onto pre-established glial feeder cells at densities of 1 × 105/coverslip. The mixed cultures were grown in chemically defined media containing fluorodeoxyuridine for 10 days before treatment and analysis. Immunocytochemistry in VMCs and Cell Counts—At the conclusion of the experiment, the cultures were immediately washed and fixed in 4% paraformaldehyde for 30 min. The cultures were then incubated with anti-tyrosine hydroxylase (TH) antibodies (1:500; Calbiochem) and anti-NeuN antibodies (1:100; Chemicon) overnight at 4 °C. The cells were stained for 2 h with fluorescein isothiocyanate- and Cy3-conjugated secondary antibodies (Jackson ImmunoResearch). In some experiments, the cells were stained with anti-TH and anti-α-synuclein (1:500; Zymed Laboratories Inc.) antibodies. After staining and prior to counting, the coverslips were randomly assigned an identification number. TH- and NeuN-immunoreactive neuron counts were then determined manually by raters blinded to the experimental conditions. For TH, the entire coverslip was counted but for NeuN+ neurons, representative fields from each coverslip were counted and averaged because of their large number. Determination of Dopamine Content in VMCs—VMCs were homogenized in 0.1 m perchloric acid containing 0.1% EDTA. Insoluble debris was removed by centrifugation, and the supernatant was filtered through a Millipore MC cartridge. The filtrate was analyzed for dopamine by high pressure liquid chromatography with electrochemical detection (Antec Leyden, Leiden, The Netherlands) using a mobile phase consisting of sodium acetate (75 mm), sodium dodecane sulfonate (0.75 mm), EDTA (10 mm), triethylamine (0.01%), acetonitrile (12%), methanol (12%), and tetrahydrofuran (1%), pH 5.5, pumped at a rate of 200 μl/min (model LC-10AD; Shimadzu, Columbia, MD) through a 100 × 2 mm column (3 μm, Hypersil C18; Keystone Scientific, Bellefonte, PA). The data were collected and analyzed using ChromPerfect software (Justice Innovations, Mountain View, CA). Quantitation of α-Synuclein Expression—TH-positive cells in each coverslip were identified under 10×, and digital images were obtained using a 40× objective for TH and α-synuclein immunoreactivity. Exposure times were held constant between coverslips. After acquisition, the images were randomly assigned an identification number, and the experimental conditions were blinded. All of the images were stacked into a single sequence, a polygonal region of interest was manually drawn around the neuron, and the mean image intensity, total image intensity, and the area inside the region of interest were evaluated for each cell. Data analysis was performed with IPLab ver 3.x for Windows (Scanalytics Inc.) compensating for bleed-through between fluorescein isothiocyanate and Cy3 channels. To determine whether ziram treatment leads to high molecular weight α-synuclein species, we performed Western blots on the detergent-soluble fractions of culture lysates. VMCs treated with either 1 μm ziram or 5 μm lactocystin were lysed in buffer containing 1% Triton X-100 and 0.1% SDS. The lysates were sonicated, and insoluble debris was removed by centrifugation. The protein concentrations were determined, and 10 μg/lane were subjected to SDS-PAGE. The separated proteins were transferred to nitrocellulose membranes and incubated with an anti-α-synuclein antibody (BD Transduction Laboratories; 1:1000) followed by an anti-tubulin antibody for normalization. Evaluation of E1 Ligase Activity—The effects of ziram on E1 ligase activity were investigated using Western blot analysis of treated cellular extracts to determine E1/E1-ubiquitin ratios and using purified enzyme preparations. Ziram-treated SK-N-MC cells were washed with phosphate-buffered saline and then lysed in a thiol stabilizing buffer using the method of Jha et al. (14.Jha N. Kumar M.J. Boonplueang R. Andersen J.K. J. Neurochem. 2002; 80: 555-561Crossref PubMed Scopus (63) Google Scholar). The samples were sonicated for 10 s, centrifuged for 15 min at 13,000 × g, mixed with 2 parts thiol gel buffer (33 mm Tris-HCl, pH 6.8, 2.7 m urea, 2.5% sodium dodecyl sulfate, and 13% glycerol), and boiled for 2 min, and 10 μg of protein/lane was loaded on a 12% SDS-PAGE gel (15.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (212859) Google Scholar). The proteins were electrophoretically transblotted onto nitrocellulose paper, and immunoblots were performed as previously described (16.Chow E. Mottahedeh J. Prins M. Ridder W. Nusinowitz S. Bronstein J.M. Mol. Cell Neurosci. 2005; 29: 405-413Crossref PubMed Scopus (40) Google Scholar) using anti-E1 ligase antibody (BIOMOL, Plymouth Meeting, PA). Antigen-antibody interactions for immunoblots were visualized using horseradish peroxidase-conjugated secondary antibody and chemoluminesence substrate (Pierce). For purified enzyme assays, human recombinant ubiquitin-activating E1 enzyme and biotinylated ubiquitin (both from BIOMOL) were incubated for 5 or 10 min in thiolester buffer as per the manufacturer's protocol. The reactions were stopped using thiol-stabilizing buffer, and the proteins were subjected to SDS-PAGE and transblotted onto nitrocellulose paper. E1-ubiquitin conjugates were determined using streptavidin-horseradish peroxidase and an enhanced chemiluminescence kit. Band densities were measured using a scanning densitometer. Animal Studies—Male C57BL/6 mice were treated for 2 weeks by subcutaneous osmotic minipumps with NaDMDC 50 mg/kg/day) in phosphate-buffered saline or with phosphate-buffered saline only. The behavior of the mice was evaluated using the pole test as described by Fleming et al. (17.Fleming S.M. Salcedo J. Fernagut P.O. Rockenstein E. Masliah E. Levine M.S. Chesselet M.F. J. Neurosci. 2004; 24: 9434-9440Crossref PubMed Scopus (392) Google Scholar) except that a cut-off time of 60 s was used. One week after the last behavioral testing, the mice were perfused with fixative, and their brains were sectioned for TH staining. Fiber density was measured with a computer-assisted image analysis system as previously described (18.Bordelon Y.M. Chesselet M.F. Neuroscience. 1999; 93: 843-853Crossref PubMed Scopus (23) Google Scholar). For stereological analysis in the substantia nigra pars compacta (SNc), the neurons were counted using the optical fractionator method with the Stereo Investigator software (Microbrightfield, Colchester, VT) coupled to a Leica DM-LB microscope with a Ludl XYZ motorized stage and z axis microcator (MT12; Heidenheim, Traunreut, Germany). The SNc was delineated at 5× objective using previously reported criteria (19.McCormack A.L. Thiruchelvam M. Manning-Bog A.B. Thiffault C. Langston J.W. Cory-Slechta D.A. Di Monte D.A. Neurobiol. Dis. 2002; 10: 119-127Crossref PubMed Scopus (652) Google Scholar, 20.West M.J. Slomianka L. Gundersen H.J. Anat. Rec. 1991; 231: 482-497Crossref PubMed Scopus (2651) Google Scholar). After delineation at low magnification, every fourth section throughout the SNc was counted at 100× magnification. Statistical Analysis—Statistical analysis was performed with GraphPad Prism software. Statistical significance was determined using one-way ANOVA with Dunnett's multiple comparison post-test and Bonferroni's multiple comparison post-test as appropriate. For histochemistry and behavior, statistical analysis was performed with GB-Stat software (Dynamic Microsystems, Inc., Silver Spring, MD, 2000) for Macintosh. A 2 × 3 mixed design ANOVA was followed by post hoc analysis with Fisher's least significant difference. To maintain homogeneity of variance, an inverse transform was calculated (21.Cohen J. Cohen P. Applied Multiple Regression/Correlation Analysis for the Behavioral Sciences. 2nd Ed. Lawrence Erlbaum Associates, Hillsdale, NJ1983Google Scholar) for each score in the pole test. The level of significance was set at p < 0.05. Ziram Inhibits 26 S Proteasome Activity in HEK Cells—We reported earlier that ziram inhibits 26 S UPS in SK-N-MC cells expressing a GFP-degron reporter system (11.Wang X.F. Li S. Chou A.P. Bronstein J.M. Neurobiol. Dis. 2006; 23: 198-205Crossref PubMed Scopus (117) Google Scholar). To determine whether ziram causes a similar inhibition in a non-neuronal cell line, HEK cells were exposed, and GFP accumulation was measured using flow cytometry. We found that HEK cells were similarly sensitive to the 26 S inhibitory effects of ziram with an IC50 of 161 nm (Fig. 1). Structure-Activity Relationships of Ziram-related Compounds for UPS Inhibition—Ziram and 14 related compounds were tested at 1 and 10 μm for their ability to inhibit 26 S UPS activity in GFP-U-transfected SK-N-MC cells (Table 1). Ziram (1) and the other DMDC derivatives (sodium DMDC (2), free acid (3), and bis-disulfide (4)) were similarly very active. Two diethyldithiocarbamates (5 and 6) were also very potent. Thiram (4) and disulfiram (6) are cleaved into DMDC (3) and diethyldithiocarbamate (5), respectively. Ziram in aqueous solution dissociates to zinc ion (7) and forms dithio acid (3), which breaks down into carbon disulfide (8) and dimethylamine (9), all of which were inactive. The disulfide functionality of etem (10) (an ethylenebis(dithiocarbamate) fungicide oxidation product) can cleave to a dithiocarbamic acid possibly associated with its activity, whereas that of the phenyl analog (11) cannot give a dithiocarbamate. The carbon disulfide progenitor enzone (12) was also inactive. Environmental degradation converts ethylenebis(dithiocarbamate) fungicides to ethylenethiourea (13) and sodium methyldithiocarbamate (14) to methyl isothiocyanate (15), all of which were inactive. Thus, the dialkyldithiocarbamate moiety is required to alter UPS activity. Furthermore, preincubation of ziram with the reducing agents GSH, dithiothreitol, or N-acetylcysteine completely abolished its inhibitory activity (data not shown). Ziram Causes Dopaminergic Cell Damage in Primary VMC—Because proteasome inhibition has been implicated in the etiology of PD and ziram causes UPS dysfunction, dopaminergic cells might be selectively vulnerable to the toxicity of ziram. Primary VMC were exposed to ziram for 48 h, and then cellular toxicity was measured by counting TH immuno-positive (TH+) cells and NeuN immuno-positive (NeuN+) cells (a nonspecific neuronal marker). Ziram at 5 and 10 μm was highly toxic to all cells, including the glial bed, causing the cell layers to lift off the coverslip after treatment or during immunostaining (data not shown). At lower doses Ziram exposure had a significant overall effect on TH+ cell survival (F4,110 = 4.52, p < 0.005) reducing TH+ cell number at 0.5 and 1 μm (p < 0.05, Dunnet's post-hoc test versus control; Fig. 2). The small, nonsignificant decrease in total NeuN+ cells measured after ziram treatment therefore represents primarily the loss of the TH+ subset of the entire NeuN+ pool. The loss of TH+ cells is likely reflective of cell death and not simply the loss of expression because TH levels were increased in the surviving positive cells (data not shown). The 20 S proteasome inhibitor lactacystin is reported to cause selective DA cell death in VMC, but this has not been a universal finding (28.Rideout H.J. Lang-Rollin I.C. Savalle M. Stefanis L. J. Neurochem. 2005; 93: 1304-1313Crossref PubMed Scopus (76) Google Scholar, 29.Kikuchi S. Shinpo K. Tsuji S. Takeuchi M. Yamagishi S. Makita Z. Niino M. Yabe I. Tashiro K. Brain Res. 2003; 964: 228-236Crossref PubMed Scopus (53) Google Scholar, 30.Hoglinger G.U. Carrard G. Michel P.P. Medja F. Lombes A. Ruberg M. Friguet B. Hirsch E.C. J. Neurochem. 2003; 86: 1297-1307Crossref PubMed Scopus (237) Google Scholar, 31.McNaught K.S. Mytilineou C. Jnobaptiste R. Yabut J. Shashidharan P. Jennert P. Olanow C.W. J. Neurochem. 2002; 81: 301-306Crossref PubMed Scopus (275) Google Scholar, 32.Petrucelli L. O'Farrell C. Lockhart P.J. Baptista M. Kehoe K. Vink L. Choi P. Wolozin B. Farrer M. Hardy J. Cookson M.R. Neuron. 2002; 36: 1007-1019Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar). In the present study lactacystin was toxic to NeuN+ neurons (F4,110 = 13.12, p < 0.0001) and to the TH+ subset of such neurons (F4,110 = 4.26, p < 0.003), but the TH+/NeuN+ ratios revealed that the effects of lactacystin were not specific to dopaminergic neurons (F4,110 = 0.90, p > 0.05; Fig. 2). Because ziram caused preferential loss of TH+ neurons and lactacystin did not, they appear to act via different mechanisms, despite the fact that they are both UPS inhibitors. Ziram Toxicity Is Not Dopamine-dependent—One possible mechanism for the relative selective effect to TH+ neurons is that ziram interacts with dopamine metabolism to produce preferential toxicity. To test this hypothesis, ziram toxicity was measured in the presence of α-methyl l-tyrosine, an inhibitor of dopamine synthesis. Treatment of VMCs with α-methyl l-tyrosine (250 μm) for 48 h resulted in a decrease in dopamine content of 62 ± 6% compared with controls (p ≤ 0.05) but did not significantly alter the number of TH+ cells. Reducing dopamine content with α-methyl l-tyrosine was ineffective in attenuating the toxicity of ziram to TH+ neurons (Fig. 3). α-Synuclein Expression in VMCs—Several converging lines of evidence support a role for α-synuclein in the pathogenesis of PD. Mutations in the α-synuclein gene or increased expression of wild-type α-synuclein cause PD in familial PD, and α-synuclein is a major component of Lewy bodies in sporadic PD (22.Singleton A.B. Trends Neurosci. 2005; 28: 416-421Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). To determine whether ziram alters the levels of α-synuclein in VMCs, the neurons were stained for both TH and α-synuclein, and relative fluorescence was measured. TH+ neurons showed robust and punctate α-synuclein staining, suggesting that they are mature and terminally differentiated (23.Quilty M.C. Gai W.P. Pountney D.L. West A.K. Vickers J.C. Exp. Neurol. 2003; 182: 195-207Crossref PubMed Scopus (37) Google Scholar, 24.Lesuisse C. Martin L.J. J. Neurobiol. 2002; 51: 9-23Crossref PubMed Scopus (199) Google Scholar). Ziram at 0.5 μm resulted in increased α-synuclein expression, whereas lactacystin at 5 μm decreased expression (Fig. 4). The sizes of the regions of interest were the same among all of the conditions varied in all of the experiments (data not shown). An important pathological marker in PD is the formation of α-synuclein inclusions or aggregates. TH+ cells from ziram- and lactacystin-treated VMCs were determined to be either positive or negative for nuclear, perinuclear, or cytoplasmic α-synuclein inclusions by blinded raters. Aggregates were relatively common in both treated and untreated cells, but surprisingly no significant differences were found with ziram compared with controls. Interestingly, a decrease in nuclear and perinuclear aggregates was seen in the lactacystin-treated cultures (Table 2).TABLE 2Ziram does not induce the formation of α-synuclein aggregatesCells with inclusionsNuclearPerinuclearCytoplasmic%Control32 ± 522 ± 44.3 ± 2.1Ziram 0.5 μm38 ± 624 ± 56.6 ± 2.9 1.0 μm19 ± 914 ± 84.8 ± 4.8Lactacystin (5 μm)19 ± 4ap = 0.04.8.6 ± 3.1bp = 0.01.7.4 ± 2.9a p = 0.04.b p = 0.01. Open table in a new tab To determine whether ziram treatment results in increased formation of detergent-soluble α-synuclein oligomer formation, we subjected VMCs lysates to Western blot analysis. Both monomeric and oligomeric forms of α-synuclein were apparent in detergent-soluble fractions as previously described (25.Rideout H.J. Dietrich P. Wang Q. Dauer W.T. Stefanis L. J. Biol. Chem. 2004; 279: 46915-46920Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Ziram treatment resulted in a nonsignificant trend for an increase in oligomeric forms of α-synuclein compared with controls (170 ± 120% optical density units of controls, n = 8 for ziram and n = 5 for controls, p = 0.18). Oligomeric α-synuclein was unchanged in lactacystin-treated VMCs, and monomeric α-synuclein levels were similar in all three conditions (data not shown). Ziram Inhibits E1 Ligase Activity—Ziram was found to inhibit the UPS using an assay that requires the substrate (i.e. degron) to be ubiquitinylated via ubiquitin ligases and recognized by the 26 S proteasome before it can be degraded by 20 S proteases (12.Bence N.F. Sampat R.M. Kopito R.R. Science. 2001; 292: 1552-1555Crossref PubMed Scopus (1858) Google Scholar). Disruption of any of these steps would be detected in the screen. It has been suggested that another dithiocarbamate fungicide, maneb, inhibits the 20 S component of the UPS (26.Zhou Y. Shie F.S. Piccardo P. Montine T.J. Zhang J. Neuroscience. 2004; 128: 281-291Crossref PubMed Scopus (111) Google Scholar). To determine whether ziram acted in this manner, HEK cells were treated with ziram (1 and 10 μm) for 24 h, but there was no change in 20 S chymotryptic activity (data not shown). The amount of α-subunit of the 20 S proteasome was also measured using Western blot analysis, and no differences were found (data not shown). Furthermore, our earlier study showed that ziram had no effect on 20 S UPS activity when added directly to cell lysate (11.Wang X.F. Li S. Chou A.P. Bronstein J.M. Neurobiol. Dis. 2006; 23: 198-205Crossref PubMed Scopus (117) Google Scholar). Because 20 S proteolytic activity was not altered by ziram, Western blot analysis on lysates was performed from ziram-treated cells to determine whether ubiquitinated proteins accumulated. As expected, inhibition of the 20 S UPS by lactacystin and rotenone resulted in the accumulation of high molecular weight ubiquitin conjugates (Fig. 5). Conversely, ziram treatment resulted in a significant decrease in these proteins, suggesting that it inhibits the UPS by interfering with ubiquitin ligation. Polyubiquitin is added to proteins targeted for UPS degradation by a series of ligases (E1, E2, and E3 ligases). Dithiocarbamates can lead to GSH oxidation (27.Burkitt M.J. Bishop H.S. Milne L. Tsang S.Y. Provan G.J. Nobel C.S. Orrenius S. Slater A.F. Arch. Biochem. Biophys. 1998; 353: 73-84Crossref PubMed Scopus (118) Google Scholar), and GSH depletion results in the loss of E1 ligase activity (14.Jha N. Kumar M.J. Boonplueang R. Andersen J.K. J. Neurochem. 2002; 80: 555-561Crossref PubMed Scopus (63) Google Scholar). Ziram does not appear to act by lowering GSH, because depleting cellular GSH using buthionine sulfoximine (1 mm) had no effect on UPS activity, and ziram did not alter the amount of reduced GSH in the cells at concentrations up to 50 μm (data not shown). Ubiquitin is activated by E1 ligase in an ATP-dependent manner forming an E1-ubiquitin adduct before the ubiquitin is transferred to E2 ligase. Because activated ubiquitin binds covalently to a cysteine of E1, the thiol group of ziram might interfere with this reaction. Indeed, ziram markedly reduced E1 ligase activity as measured by a reduction of endogenous E1-ubiquitin conjugates and the reduced formation of E1-ubiquitin conjugates in a purified preparation (Fig. 6).FIGURE 6Effect of ziram on E1-ubiquitin conjugates and E1 ligase activity. A, endogenous E1-ubiquitin conjugates in lysates of ziram-treated HEK cells. Ziram resulted in a dose-dependent reduction in the E1-Ub/E1 ratio compared with untreated controls. The Western blot is shown in the inset. B, E1 ligase activity was inhibited by ziram in purified preparations after 5 and 10 min of incubation (n = 4, p ≤ 0.05).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Motor Deficits in NaDMDC-treated Mice—NaDMDC was used for in vivo experiments because of its greater solubility than the zinc DMDC salt, ziram, with a similar effect on proteasomal function (Table 1). The treatment of male C57BL/6 mice for 2 weeks with NaDMDC resulted in increased time to turn in the pole test, a deficit also seen in mice with dysfunction of the nigrostriatal pathway (Fig. 7). The effect persisted 8 weeks after cessation of drug treatment, indicating that it was not because of an acute drug effect. Striatal TH fiber density was nonsignificantly reduced across striatal regions at 2 weeks, with a significant reduction in the ventrolateral quadrant 9 weeks after drug treatment in the NaDMDC-treated animals (Table 3). Stereological TH immunoreactive neuron counts in the SNc of treated mice were not changed (Table 3). Thus, 2 weeks of treatment of mice with NaDMDC, an E1-activating enzyme inhibitor, resulted in a motor deficit with a minor loss of nigrostriatal dopaminergic fibers.TABLE 3Effect of subcutaneous treatment with NaDMDC on TH immunoreactivity (IR) fiber density in striatal subregions and stereological estimates of TH-IR neurons in the SNcRegionMean ± S.E. (n = 5)2W2 + 9WControlTreatedControlTreatedTH-IR fiber density DL striatum1515 ± 2071456 ± 2211934 ± 2991951 ± 269 DM striatum1
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