1Department of Biochemistry, Osaka University Graduate School of Medicine, Osaka, Japan; and 2Department of Biochemistry, Hyogo College of Medicine, Hyogo, Japan
Submitted 9 January 2004 ; accepted in final form 21 September 2004
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ABSTRACT |
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amyotrophic lateral sclerosis; copper; zinc superoxide dismutase; G2/M arrest; neurodegenerative disease
A pathological hallmark of both sporadic and familial ALS involves the abnormal accumulation of cytoskeletal proteins such as neurofilament, tubulin, and actin in the perikaryon and axon of motor neurons (12, 17, 19, 31). Recent studies have reported that these cytoskeletal changes induce slowing of axonal transport in mutated Cu,Zn-SOD transgenic mice (37).
Previous reports demonstrated that the abnormal regulation of mitotic proteins and an aberrant cell cycle have been observed in neurodegenerative diseases, including Alzheimers disease and ALS. In those studies, it was suggested that cell cycle signaling may affect the neuronal death pathway (25, 33). Thus we hypothesized that the cell cycle might be affected by mutated Cu,Zn-SOD in FALS.
In this study, mouse neuroblastoma cells were transfected with mutated Cu,Zn-SOD. The mutated Cu,Zn-SOD transfectants grew at a much slower rate than the wild-type transfectants or mock transfectants, and G2/M arrest was observed in the mutant transfectants. As evidenced by confocal microscopy, cytoskeletal destruction occurred in the mutant transfectants. The issue of how mutated Cu,Zn-SOD affected cytoskeletal components was examined.
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MATERIALS AND METHODS |
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Western blot analysis.
N2a cells were lysed in buffer [20 mM Tris·HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% (wt/vol) Nonidet P-40, 1% (wt/vol) Triton, 10% (wt/vol) glycerol, 5 mM sodium pyrophosphate, 10 mM NaF, 1 mM sodium orthovanadate, 10 mM -glycerophosphate, and 1 mM dithiothreitol (DTT)] with protease inhibitors. Protein (10 µg) was subjected to 15% SDS-PAGE. For Western blot analysis using an anti-Cu,Zn-SOD antibody, the proteins were transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, NH). The blots were blocked with 5% skim milk and 2% BSA and then probed with goat anti-human Cu,Zn-SOD (2). After incubation with a peroxidase-conjugated secondary antibody, immunoreactive bands were visualized using an enhanced chemiluminescence kit (Amersham Biosciences, Piscataway, NJ).
Cell growth curve. Equal numbers (5 x 104) of mock, wild-type, G37R, and G93A Cu,Zn-SOD transfected cells were plated onto six-well tissue culture dishes, and the cell number was determined by hemocytometric counting at 0, 1, 2, and 3 days after cell plating. Triplicate plates were used for each time point (22).
Determination of cell cycle by image analysis. The relative number of cells occupying a particular state of the cell cycle at a specific time was obtained by staining cells with the DNA intercalator propidium iodide (5 µg/ml). Stoichiometric binding resulted when the cells were fixed in cold 70% ethanol for at least 6 h, followed by two washes with PBS and incubation with 100 µg/ml RNase for 30 min at 37°C and two additional washes with PBS (22). Single-parameter histograms were generated by analyzing the cells on a FACStarPlus flow cytometer (BD Biosciences Immunocytometry Systems, San Jose, CA).
Confocal laser scanning microscopy. Cells grown on glass-bottom dishes were fixed with 2% paraformaldehyde-PBS for 10 min on ice. Cells were rinsed in PBS and then treated with 1% saponin for membrane permeabilization. The cells were incubated with 100 ng/ml of tetramethyl rhodamine isothiocyanate (TRITC)-labeled phalloidin (Sigma) or FITC-labeled anti-tubulin antibody (Sigma) for 2 h at room temperature. After washing with PBS with 0.05% Triton five times, the cells were analyzed using confocal laser microscopy. To identify the nuclei, cells were stained with propidium iodide, the size of the nuclei was measured through the microscope, and the image was processed digitally using an eight-bit image analyzer (NIH Image software, version 1.63).
Immunoprecipitation, two-dimensional gel electrophoresis and Western blotting. For the immunoprecipitation of Cu,Zn-SOD or actin, whole cell lysates were incubated with 4 µg of goat anti-human Cu,Zn-SOD antibody or mouse anti-actin antibody (Sigma) and 15 µl of protein G-Sepharose 4 Fast Flow (Amersham Biosciences) for 12 h at 4°C (32). For two-dimensional (2-D) gel electrophoretic analysis, immunoprecipitates were incubated with immobilized pH gradient (IPG) buffer {8 M urea, 2% (wt/vol) 3-([3-cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate, 0.5% (vol/vol) carrier ampholyte, pH 310, 10 mM DTT, and a trace amount of bromophenol blue} for 4 h. After the samples were centrifuged, the supernatants were subjected to isoelectric focusing using the IPGphor system (Amersham Biosciences) and a 7-cm IPG strip (pH range, 310). The IPG strip was then applied to an SDS-PAGE gel (10 or 15%). The gels were subjected to silver staining using the Silver Stain II kit (Daiichi Pure Chemicals, Tokyo, Japan). Western blotting was performed as described above using an anti-Cu,Zn-SOD antibody or an anti-actin antibody.
Purification of Cu,Zn-SOD produced from Sf21 cells. Recombinant Cu,Zn-SOD was isolated from Spodoptera frugiperda 21 (Sf21) cells as described previously (34). Briefly, wild-type and mutated Cu,Zn-SOD expression vectors were transfected into Sf21 cells to produce a recombinant virus. For the production of active Cu,Zn-SOD, aqueous solutions of CuCl2 and ZnCl2 were added directly to the medium until reaching a concentration of 1 mM after viral infection. Infected cells (n = 4 x 108) were lysed at 4°C in a hypotonic buffer containing 2.5 mM potassium phosphate, pH 7.4, 1 mM benzamidine, and 0.1 mM p-amidinophenylmethanesulfonyl fluoride (Wako Pure Chemical Industries, Osaka, Japan). After homogenization and centrifugation at 100,000 g for 1 h, the supernatant was subjected to a DE52 ion exchange column chromatography (Whatman, Brentford, UK) in 2.5 mM K+-phosphate, pH 7.4. Bound proteins were eluted with a linear gradient of K+-phosphate from 2.5 to 200 mM. To further purify the enzyme, column chromatography was performed on a hydroxyapatite type I column (Bio-Rad, Hercules, CA) with a liner gradient of K+-phosphate from 10 to 500 mM using Akta Explorer 10s (Amersham Biosciences).
Assay of actin polymerization. An actin polymerization assay was performed in the presence and absence of Cu,Zn-SOD using an Actin Polymerization Biochem kit (Cytoskeleton, Denver, CO) according to the manufacturers instructions. The samples also were examined by performing immunoprecipitation with anti Cu,Zn-SOD antibody followed by Western blotting using anti-actin antibody.
Statistical evaluation. Differences among mean values were determined by performing one-way ANOVA with Fishers multiple-comparison test. P < 0.05 was considered statistically significant.
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RESULTS |
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DISCUSSION |
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Mutated Cu,Zn-SOD have several enhanced activities that could be capable of selectively damaging interacting proteins or their associated organelles (5, 24, 36, 38). Investigators at our laboratory previously reported that Cu-binding affinities are decreased in mutated Cu,Zn-SOD (26) and mutated Cu,Zn-SOD are highly susceptible to nonenzymatic glycosylation, i.e., glycation (34). It is possible that actin-bound mutated Cu,Zn-SOD is glycated in vivo and reactive oxygen species could be produced via the Fenton reaction involving free Cu ions released from Cu,Zn-SOD (27). Recent reports indicate that hydrogen peroxide causes an increase in F- and G-actin oxidation and a decrease in the F-actin fraction (3, 4). The association of mutated Cu,Zn-SOD with actin might be unexpectedly toxic to the actin cytoskeleton.
Cytoskeletal changes and subsequent G2/M arrest are commonly seen in apoptotic cells. However, when we performed the apoptotic assay by examining the DNA ladder, no significant apoptotic evidence was seen in mutated Cu,Zn-SOD-transfected N2a cells after 2-day serum starvation (data not shown).
The cytoskeletal changes observed in the mutated Cu,Zn-SOD in this study could be related to neuronal cell death observed in patients with FALS. In recent studies, researchers have reported that cytoskeletal proteins are significantly altered in ALS spinal motor neurons. For example, neurofilament aggregation is known to be an early pathological hallmark of the disease process. Furthermore, Vukosavic et al. (35) reported that the level of -actin decreases in the mutated Cu,Zn-SOD transgenic mice. The association of mutated Cu,Zn-SOD with actin reported in the present study could be an another important factor in cytoskeletal disruption and the apoptosis of neuronal cells in FALS.
The association of mutant Cu,Zn-SOD with actin could also be involved in the formation of aggregates that are observed in ALS. An increasing number of studies have stressed the role of mutant Cu,Zn-SOD-derived aggregation in the pathogenesis of ALS. Intracellular Cu,Zn-SOD aggregates are found in cultured motor neurons after the microinjection of mutant Sod1 cDNA (11). Aggregates containing Cu,Zn-SOD were also detected in motor neurons and astrocytes of transgenic mice that expressed mutant Sod1 (7). Mutant Cu,Zn-SOD aggregation into insoluble protein complexes is considered to be an early event in the pathogenic mechanism of FALS (18). Transfection studies indicated that mutant but not wild-type Cu,Zn-SOD forms cytoplasmic aggregates (18, 20). Such aggregates might interact inappropriately with other cellular components to impair cellular function. Pasinelli et al. (28) recently reported that mutant Cu,Zn-SOD-containing aggregates binds to Bcl-2 in spinal cord mitochondria, suggesting possible mechanisms of neurotoxicity of mutant Cu,Zn-SOD. Aggregates observed in ALS are also likely substrates for dynein-mediated transport and cause the disruption of microtubule-dependent axonal transport of other substrates. In addition, they are considered to stimulate neurodegeneration by overwhelming the capacity of the protein-folding chaperones and normal proteasome function. Thus the toxicity of mutant Cu,Zn-SOD could result from their propensity to aggregate. Similar mechanisms have been proposed for other degenerative diseases such as Alzheimers disease, light-chain amyloidosis, and the spongiform encephalopathies (8). What, then, is the cause of the formation of such toxic aggregations? One possible mechanism is aberrant protein-protein interactions as demonstrated in mutant proteins in some neurodegenerative and/or neuromuscular disorders (9, 23). Kunst et al. (21) reported that G85R and G93A Cu,Zn-SOD bind to translocon-associated protein- (TRAP-
) and lysyl-tRNA synthetase (KARS) using a yeast two-hybrid system. Although we were not able to identify TRAP-
and KARS as proteins that bind to mutated Cu,Zn-SOD, it is possible that they were also present in the 2-D gels shown in Fig. 6. The mechanisms by which the mutated Cu,Zn-SOD associated with actin are currently under investigation. We assume that the structural changes or instability of Cu,Zn-SOD caused by the mutation suggested by the crystal structures (16) might be involved in this phenomenon. Further analysis of mutated Cu,Zn-SOD proteins will provide clues to their aberrant interactions with other proteins, including actin.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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