Overexpression of mutated Cu,Zn-SOD in neuroblastoma cells results in cytoskeletal change

Rina Takamiya,1 Motoko Takahashi,1 Yong Seek Park,1 Yoshie Tawara,1 Noriko Fujiwara,2 Yasuhide Miyamoto,1 Jianguo Gu,1 Keiichiro Suzuki,2 and Naoyuki Taniguchi1

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


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Amyotrophic lateral sclerosis (ALS) involves the progressive degeneration of motor neurons in the spinal cord and the motor cortex. It has been shown that 15–20% of patients with familial ALS (FALS) have defects in the Sod1 gene, which encodes Cu,Zn-superoxide dismutase (SOD). To elucidate the pathological role of mutated Cu,Zn-SOD, we examined the issue of whether mutated Cu,Zn-SOD affects the cell cycle. Mouse neuroblastoma Neuro-2a cells were transfected with human wild-type or mutated (G37R, G93A) Cu,Zn-SOD. Mutated, Cu,Zn-SOD-transfected cells exhibited marked retardation in cell growth and G2/M arrest. They also displayed lower reactivity to phalloidin, indicating that the cytoskeleton was disrupted. Immunoprecipitation, two-dimensional gel electrophoresis, and Western blot analysis indicated that mutated Cu,Zn-SOD associates with actin. Similar results were obtained by in vitro incubation experiments with purified actin and mutated Cu,Zn-SOD (G93A). These results suggest that mutated Cu,Zn-SOD in FALS causes cytoskeletal changes by associating with actin, which subsequently causes G2/M arrest and growth retardation.

amyotrophic lateral sclerosis; copper; zinc superoxide dismutase; G2/M arrest; neurodegenerative disease


AMYOTROPHIC LATERAL SCLEROSIS (ALS) is a neurodegenerative disease characterized by the selective and progressive dysfunction of motor neurons initiated in middle-aged adults (1). The pathology of the disease results from the death of lower motor neurons in the brainstem and spinal cord and upper motor neurons in the cerebral cortex. Approximately 5–10% of ALS cases are familial ALS (FALS), and among FALS cases, 15–20% have been linked to autosomal dominant inheritance of mutations in Cu,Zn-superoxide dismutase (Cu,Zn-SOD) (10, 30). Cu,Zn-SOD functions as an antioxidative enzyme that catalyzes the conversion of O2· to hydrogen peroxide, which is further detoxified by other antioxidant enzymes such as catalase and glutathione peroxidase. More than 100 types of mutations in Cu,Zn-SOD, which comprises 153 amino acids, have been reported to be associated with the FALS (10). Although some FALS-related mutants show reduced enzymatic activities, many retain full activity (26). Furthermore, transgenic mice that have FALS-associated Sod1 mutations develop FALS-like symptoms despite elevated Cu,Zn-SOD activity, suggesting that the disease is not caused by the loss of normal enzymatic activity (15). Thus the motor neuron dysfunction observed in FALS is generally thought to be due to the newly acquired neurotoxicity of mutant Cu,Zn-SOD. Several hypotheses have been proposed to explain this toxic gain of function of mutated Cu,Zn-SOD in FALS (6, 18, 19, 29). For example, oxidative stress produced by aberrant catalysis (13, 36), abnormal Cu chemistry (26), decreased glutamate metabolism (6), and increased cytoplasmic aggregation (7, 11, 18, 20, 26, 29) have been studied in the setting of mutated Cu,Zn-SOD. However, the precise mechanism of pathogenesis is not understood.

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 Alzheimer’s 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture and transfection. Neuro-2a (N2a) cells, a mouse neuroblastoma cell line, were grown in DMEM (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum. Mutated Cu,Zn-SOD cDNA, designated G37R and G93A, were constructed using site-directed mutagenesis with a uracil template as described previously (14). The DNA fragments were ligated to a mammalian expression vector, pcDNA3.1/Zeo (Invitrogen, Carlsbad, CA), which was regulated by the cytomegalovirus promoter. The resulting plasmids were transfected into N2a cells using Lipofectamine reagent (Life Technologies/Invitrogen) according to the manufacturer’s instructions. Selection was performed in a medium that contained 1.0 mg/ml Zeocin (GIBCO-BRL/Invitrogen), and, after a 2-wk incubation, several stable colonies were isolated.

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 {beta}-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 3–10, 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, 3–10). 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 manufacturer’s 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 Fisher’s multiple-comparison test. P < 0.05 was considered statistically significant.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Establishment of N2a clones stably expressing wild-type or mutant human Cu,Zn-SOD. N2a cells were stably transfected with wild-type or mutant Cu,Zn-SOD (G37R, G93A), and five Zeocin-resistant clones were selected for each cell type. As shown in Fig. 1, the expression levels of Cu,Zn-SOD were verified by Western blot analysis. The upper bands are human enzymes, and the lower bands are endogenous mouse enzymes, as judged by the molecular mass. Because we could not obtain high-expression clones for G93A, the data with indicated clones in Fig. 1 are shown in subsequent figures.



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 1. Characterization of human wild-type and mutated Cu,Zn-superoxide dismutase (SOD) in transfectants of Neuro-2a (N2a) cells, a mouse neuroblastoma cell line. Cell lysates (10 µg) were subjected to 15% SDS-PAGE and subsequent Western blot analysis using an anti-human Cu,Zn-SOD antibody. Mock, mock transfectant; WT, wild-type Cu,Zn-SOD transfectant; G37R, G37R-mutated Cu,Zn-SOD transfectant; G93A, G93A-mutated Cu,Zn-SOD transfectant.

 
Cell growth arrest in mutant Cu,Zn-SOD-transfected N2a cells. When cell growth was examined, marked inhibition in cell growth was observed in G37R and G93A Cu,Zn-SOD-transfected cells compared with wild-type Cu,Zn-SOD- and mock-transfected cells (Fig. 2). Wild-type Cu,Zn-SOD transfectants showed a growth curve that was similar to that of the mock transfectants. We further determined the cell cycle by conducting flow cytometric analysis. After subconfluent cells were incubated in medium with or without 10% FBS for 48 h, the cells were stained with propidium iodide and subjected to flow cytometric analysis. As shown in Fig. 3A, no significant differences were found among mock, wild-type, and mutant Cu,Zn-SOD transfectants in the G0/G1 and S phases when grown in a medium containing 10% serum. However, a higher percentage of G37R or G93A Cu,Zn-SOD transfectants was found in the G2/M phase than with the mock or wild-type Cu,Zn-SOD transfectants. When the cells were subjected to serum starvation, the difference became more obvious; a higher percentage of mock and wild-type Cu,Zn-SOD transfectants was found in the G0 phase compared with the G37R or G93A Cu,Zn-SOD transfectants, whereas a higher percentage of the mutant transfectants was found in the G2/M phase compared with mock or wild-type transfectants (Fig. 3B). These results suggest that G2/M arrest occurred with the G37R or G93A Cu,Zn-SOD transfectants.



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 2. Growth curves for wild-type or mutated Cu,Zn-SOD-transfected N2a cells. Equal numbers (n = 5 x 104 cells) of mock, wild-type, G37R, and G93A Cu,Zn-SOD-transfected cells were plated onto 6-well tissue culture dishes and incubated in DMEM containing 10% FBS. Cell numbers were determined by hemocytometric counting at 0, 1, 2, and 3 days. Each data point is the average of 4 independent experiments. *P < 0.05 vs. wild-type Cu,Zn-SOD transfectant.

 


View larger version (27K):
[in this window]
[in a new window]
 
Fig. 3. Cell cycle analysis of wild-type and mutated Cu,Zn-SOD-transfected N2a cells. Cells were incubated in DMEM containing 10% FBS for 1 day and then incubated with or without serum for an additional 48 h. Cell cycles were determined by staining with propidium iodide and by performing flow cytometric analysis. A: cells incubated with DMEM containing 10% FBS. B: cells were serum starved for 48 h, m, mock transfectant. *P < 0.05 vs. mock transfectant.

 
Loss of actin polymerization in mutated, Cu,Zn-SOD-transfected cells. One of the characteristics of the cytoskeletal structure in the M phase is the formation of a contractile ring by actin filaments. The G2/M arrest observed in mutant transfectants prompted us to examine whether these cells could contain structurally modified actin. After 2-day serum starvation, polymerized actin fiber was stained with phalloidin and analyzed using laser scanning confocal microscopy. As seen in Fig. 4A, the G37R or G93A Cu,Zn-SOD transfectants displayed less reactivity to phalloidin than did the mock or wild-type transfectant controls. Such lower reactivity was evident not only in the dendritic processes but also in the cell surface (Fig. 4A). This evidence indicates that a decrease in actin polymerization occurred in the mutated Cu,Zn-SOD transfectants. Because tubulin governs the location of the cytoplasm, we next stained the cells with an anti-{alpha}-tubulin antibody. As shown in Fig. 4B, there were not clarified changes among mock, wild-type, and mutated Cu,Zn-SOD transfectants in {alpha}-tubulin immunoreactivities, but some cells with two nuclei were observed in the G93A and G37R Cu,Zn-SOD transfectants. The cells were stained with propidium iodide to determine the area of the nucleus. Fig. 5 indicates the size of the nucleus per cell in each of the transfectants. The mean values of the size of mutant transfectants were almost twice those of wild-type Cu,Zn-SOD or mock transfectants, suggesting that M-phase arrest can occur in mutant-transfected cells.



View larger version (24K):
[in this window]
[in a new window]
 
Fig. 4. Determination of cytoskeletal changes in wild-type and mutated Cu,Zn-SOD-transfected N2a cells. After cells were serum starved for 2 days, they were fixed with 2% paraformaldehyde. Florescent images are shown with tetramethylrhodamine isothiocyanate (TRITC)-labeled phalloidin and FITC-labeled anti-{alpha}-tubulin antibody. A: cells stained with TRITC-labeled phalloidin. B: cells stained with FITC-labeled {alpha}-tubulin antibody (green) and propidium iodide (red) to identify nuclei. mock, mock transfectant.

 


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 5. The size of nuclei in wild-type and mutated Cu,Zn-SOD-transfected N2a cells. After serum starvation for 2 days, the cells were stained with propidium iodide to identify the nucleus. After 100 cells from each culture were randomly collected, the size of the nucleus per cell was measured through the microscope and the image was processed digitally with an 8-bit image analyzer (NIH Image version 1.62). The line in each column indicates the mean value. mock, mock transfectant.

 
One of the mutated Cu,Zn-SOD-associated proteins is actin. To examine the changes in cellular protein in the mutant transfectants, 2-D gel electrophoresis was performed. As shown in Fig. 6A, significant differences were not found between the wild-type and mutant Cu,Zn-SOD transfectants when a whole cell lysate was examined. However, when the cell lysate was immunoprecipitated with an anti-Cu,Zn-SOD antibody and subjected to 2-D gel electrophoretic analysis, several protein spots with molecular mass of ~40 kDa were found only in mutated Cu,Zn-SOD transfectants as shown in Fig. 6B. Because the molecular mass of actin is ~43 kDa, we assumed that one of the spots might be actin. To identify the spots, similarly prepared gels were subjected to Western blot analysis using an anti-actin antibody. Actin-positive spots were observed in the G37R and G93A transfectants but not in the wild-type transfectants (Fig. 7A). The samples were next immunoprecipitated with an anti-actin antibody and subjected to Western blot analysis using an anti-Cu,Zn-SOD antibody, the reverse of the former experiment. As shown in Fig. 7B, stronger signals were detected in the G37R and G93A Cu,Zn-SOD transfectants than in the wild-type transfectants. These results suggest that mutated Cu,Zn-SOD has a greater tendency to associate with actin protein.



View larger version (94K):
[in this window]
[in a new window]
 
Fig. 6. Two-dimensional (2-D) gel electrophoretic pattern of wild-type and mutated Cu,Zn-SOD-transfected N2a cells. A: whole cell lysates (50 µg) of wild-type, G37R, and G93A Cu,Zn-SOD transfectants were subjected to 2-D gel electrophoresis and silver staining. B: samples were immunoprecipitated with an anti-Cu,Zn-SOD antibody and subjected to 2-D gel electrophoresis and silver staining.

 


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 7. Determination of Cu,Zn-SOD associated proteins. A: samples were immunoprecipitated with an anti-Cu,Zn-SOD antibody and subjected to 2-D gel electrophoresis (10%) and Western blot analysis using an anti-actin antibody. B: samples were immunoprecipitated with an anti-actin antibody and subjected to 2-D gel electrophoresis (15%) and Western blot analysis using an anti-Cu,Zn-SOD antibody.

 
Mutated Cu,Zn-SOD directly associate with actin in vitro. To determine whether mutated Cu,Zn-SOD directly suppressed actin polymerization, we purified wild-type and mutated Cu,Zn-SOD in a baculovirus insect cell system and performed an actin polymerization assay. In this assay, we found that both wild-type and mutated Cu,Zn-SOD did not prevent actin polymerization (data not shown). We then performed in vitro incubation of actin and Cu,Zn-SOD. Actin samples incubated with purified wild-type and mutated Cu,Zn-SOD were immunoprecipitated with anti-Cu,Zn-SOD antibody and subjected to Western blot analysis using an anti-actin antibody. As shown in Fig. 8, a band corresponding to the molecular mass of actin was observed in the mixture with the mutated Cu,Zn-SOD. These results suggest that mutated Cu,Zn-SOD directly associates with actin.



View larger version (28K):
[in this window]
[in a new window]
 
Fig. 8. In vitro association of G93A Cu,Zn-SOD with actin. After wild-type or G93A mutation, Cu,Zn-SOD (0.1 µg) was incubated with human platelet actin (0.1 µg) in actin polymerization buffer (in mM: 50 KCl, 2 MgCl2, and 1 ATP) for 1 h at 24°C, and the samples were immunoprecipitated with an anti-Cu,Zn-SOD antibody and subjected to Western blot analysis using anti-actin antibody. Lane 1: wild-type Cu,Zn-SOD incubated with actin; lane 2: G93A-mutated Cu,Zn-SOD incubated with actin.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell cycle changes in mouse neuroblastoma N2a cells transfected with FALS-associated mutants of Cu,Zn-SOD were examined. When G37R and G93A Cu,Zn-SOD mutants were transfected into N2a cells, G2/M arrest and significant cell growth retardation were observed. Although there were no significant changes in tubulin immunoreactivities, the mutated Cu,Zn-SOD transfectants displayed less reactivity to phalloidin than did the wild-type transfectants, indicating that actin disruption occurred in the mutant transfectants. Immunoprecipitation and 2-D gel electrophoresis followed by Western blot analysis indicated that the mutated Cu,Zn-SOD tended to associate with actin. The in vitro study in which purified Cu,Zn-SOD was incubated with actin indicated that the mutated Cu,Zn-SOD directly associated with actin. G2/M arrest also occurred in mutated Cu,Zn-SOD-transfected NIH-3T3 cells, although the extent was much less than observed in transfectants of N2a cells (data not shown). Therefore, these phenomena are not considered to be N2a cell specific.

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 {beta}-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 Alzheimer’s 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-{delta} (TRAP-{delta}) and lysyl-tRNA synthetase (KARS) using a yeast two-hybrid system. Although we were not able to identify TRAP-{delta} 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.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by a grant from the Amyotrophic Lateral Sclerosis Association, a grant on Specific Diseases (Itoyama) from Ministry of Health and Welfare, Japan, the 21st Century COE Program of the Ministry of Education, Science, Culture, Sports, and Technology, and a Grant-in-Aid for Scientific Research (C) No. 15500237.


    ACKNOWLEDGMENTS
 
We thank Dr. Yoshitaka Ikeda for technical support and critical discussion.


    FOOTNOTES
 

Address for reprint requests and other correspondence: N. Taniguchi, Dept. of Biochemistry, Osaka Univ. Graduate School of Medicine, B1, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan (E-mail: proftani{at}biochem.med.osaka-u.ac.jp)

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.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1. Andersen PM, Morita M, and Brown RH Jr. Genetics of Amyotrophic Lateral Sclerosis: An Overview. London: World Federation of Neurology, Committee on Motor Neuron Disease, 1999.

2. Arai K, Maguchi S, Fujii S, Ishibashi H, Oikawa K, and Taniguchi N. Glycation and inactivation of human Cu-Zn-superoxide dismutase: identification of the in vitro glycated sites. J Biol Chem 262: 16969–16972, 1987.[Abstract/Free Full Text]

3. Banan A, Fitzpatrick L, Zhang Y, and Keshavarzian A. OPC-compounds prevent oxidant-induced carbonylation and depolymerization of the F-actin cytoskeleton and intestinal barrier hyperpermeability. Free Radic Biol Med 30: 287–298, 2001.[CrossRef][ISI][Medline]

4. Banan A, Zhang Y, Losurdo J, and Keshavarzian A. Carbonylation and disassembly of the F-actin cytoskeleton in oxidant induced barrier dysfunction and its prevention by epidermal growth factor and transforming growth factor alpha in a human colonic cell line. Gut 46: 830–837, 2000.[Abstract/Free Full Text]

5. Beckman JS, Carson M, Smith CD, and Koppenol WH. ALS, SOD and peroxynitrite. Nature 364: 584, 1993.[CrossRef][ISI][Medline]

6. Bruijn LI, Becher MW, Lee MK, Anderson KL, Jenkins NA, Copeland NG, Sisodia SS, Rothstein JD, Borchelt DR, Price DL, and Cleveland DW. ALS-linked SOD1 mutant G85R mediates damage to astrocytes and promotes rapidly progressive disease with SOD1-containing inclusions. Neuron 18: 327–338, 1997.[ISI][Medline]

7. Bruijn LI, Houseweart MK, Kato S, Anderson KL, Anderson SD, Ohama E, Reaume AG, Scott RW, and Cleveland DW. Aggregation and motor neuron toxicity of an ALS-linked SOD1 mutant independent from wild-type SOD1. Science 281: 1851–1854, 1998.[Abstract/Free Full Text]

8. Bucciantini M, Giannoni E, Chiti F, Baroni F, Formigli L, Zurdo J, Taddei N, Ramponi G, Dobson CM, and Stefani M. Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases. Nature 416: 507–511, 2002.[CrossRef][ISI][Medline]

9. Burke JR, Enghild JJ, Martin ME, Jou YS, Myers RM, Roses AD, Vance JM, and Strittmatter WJ. Huntingtin and DRPLA proteins selectively interact with the enzyme GAPDH. Nat Med 2: 347–350, 1996.[ISI][Medline]

10. Cleveland DW and Rothstein JD. From Charcot to Lou Gehrig: deciphering selective motor neuron death in ALS. Nat Rev Neurosci 2: 806–819, 2001.[CrossRef][ISI][Medline]

11. Durham HD, Roy J, Dong L, and Figlewicz DA. Aggregation of mutant Cu/Zn superoxide dismutase proteins in a culture model of ALS. J Neuropathol Exp Neurol 56: 523–530, 1997.[ISI][Medline]

12. Farah CA, Nguyen MD, Julien JP, and Leclerc N. Altered levels and distribution of microtubule-associated proteins before disease onset in a mouse model of amyotrophic lateral sclerosis. J Neurochem 84: 77–86, 2003.[CrossRef][ISI][Medline]

13. Ferrante RJ, Browne SE, Shinobu LA, Bowling AC, Baik MJ, MacGarvey U, Kowall NW, Brown RH Jr, and Beal MF. Evidence of increased oxidative damage in both sporadic and familial amyotrophic lateral sclerosis. J Neurochem 69: 2064–2074, 1997.[ISI][Medline]

14. Fujii J, Myint T, Seo HG, Kayanoki Y, Ikeda Y, and Taniguchi N. Characterization of wild-type and amyotrophic lateral sclerosis-related mutant Cu,Zn-superoxide dismutases overproduced in baculovirus-infected insect cells. J Neurochem 64: 1456–1461, 1995.[ISI][Medline]

15. Gurney ME, Pu H, Chiu AY, Dal Canto MC, Polchow CY, Alexander DD, Caliendo J, Hentati A, Kwon YW, Deng HX, Chen W, Zhai P, Sufit RL, and Siddique T. Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation. Science 264: 1772–1775, 1994.[ISI][Medline]

16. Hart PJ, Liu H, Pellegrini M, Nersissian AM, Gralla EB, Valentine JS, and Eisenberg D. Subunit asymmetry in the three-dimensional structure of a human CuZnSOD mutant found in familial amyotrophic lateral sclerosis. Protein Sci 7: 545–555, 1998.[Abstract/Free Full Text]

17. Hirano A, Nakano I, Kurland LT, Mulder DW, Holley PW, and Saccomanno G. Fine structural study of neurofibrillary changes in a family with amyotrophic lateral sclerosis. J Neuropathol Exp Neurol 43: 471–480, 1984.[ISI][Medline]

18. Johnston JA, Dalton MJ, Gurney ME, and Kopito RR. Formation of high molecular weight complexes of mutant Cu, Zn-superoxide dismutase in a mouse model for familial amyotrophic lateral sclerosis. Proc Natl Acad Sci USA 97: 12571–12576, 2000.[Abstract/Free Full Text]

19. Julien JP. Amyotrophic lateral sclerosis: unfolding the toxicity of the misfolded. Cell 104: 581–591, 2001.[ISI][Medline]

20. Koide T, Igarashi S, Kikugawa K, Nakano R, Inuzuka T, Yamada M, Takahashi H, and Tsuji S. Formation of granular cytoplasmic aggregates in COS7 cells expressing mutant Cu/Zn superoxide dismutase associated with familial amyotrophic lateral sclerosis. Neurosci Lett 257: 29–32, 1998.[CrossRef][ISI][Medline]

21. Kunst CB, Mezey E, Brownstein MJ, and Patterson D. Mutations in SOD1 associated with amyotrophic lateral sclerosis cause novel protein interactions. Nat Genet 15: 91–94, 1997.[ISI][Medline]

22. Lee PJ, Alam J, Wiegand GW, and Choi AM. Overexpression of heme oxygenase-1 in human pulmonary epithelial cells results in cell growth arrest and increased resistance to hyperoxia. Proc Natl Acad Sci USA 93: 10393–10398, 1996.[Abstract/Free Full Text]

23. Li XJ, Li SH, Sharp AH, and Nucifora FC Jr, Schilling G, Lanahan A, Worley P, Snyder SH, and Ross CA. A huntingtin-associated protein enriched in brain with implications for pathology. Nature 378: 398–402, 1995.[CrossRef][ISI][Medline]

24. Mourelatos Z, Gonatas NK, Stieber A, Gurney ME, and Dal Canto MC. The Golgi apparatus of spinal cord motor neurons in transgenic mice expressing mutant Cu,Zn superoxide dismutase becomes fragmented in early, preclinical stages of the disease. Proc Natl Acad Sci USA 93: 5472–5477, 1996.[Abstract/Free Full Text]

25. Nguyen MD, Boudreau M, Kriz J, Couillard-Després S, Kaplan DR, and Julien JP. Cell cycle regulators in the neuronal death pathway of amyotrophic lateral sclerosis caused by mutant superoxide dismutase 1. J Neurosci 23: 2131–2140, 2003.[Abstract/Free Full Text]

26. Okado-Matsumoto A, Myint T, Fujii J, and Taniguchi N. Gain in functions of mutant Cu,Zn-superoxide dismutases as a causative factor in familial amyotrophic lateral sclerosis: less reactive oxidant formation but high spontaneous aggregation and precipitation. Free Radic Res 33: 65–73, 2000.[ISI][Medline]

27. Ookawara T, Kawamura N, Kitagawa Y, and Taniguchi N. Site-specific and random fragmentation of Cu,Zn-superoxide dismutase by glycation reaction: Implication of reactive oxygen species. J Biol Chem 267: 18505–18510, 1992.[Abstract/Free Full Text]

28. Pasinelli P, Belford ME, Lennon N, Bacskai BJ, Hyman BT, Trotti D, and Brown RH Jr. Amyotrophic lateral sclerosis-associated SOD1 mutant proteins bind and aggregate with Bcl-2 in spinal cord mitochondria. Neuron 43: 19–30, 2004.[CrossRef][ISI][Medline]

29. Rakhit R, Cunningham P, Furtos-Matei A, Dahan S, Qi XF, Crow JP, Cashman NR, Kondejewski LH, and Chakrabartty A. Oxidation-induced misfolding and aggregation of superoxide dismutase and its implications for amyotrophic lateral sclerosis. J Biol Chem 277: 47551–47556, 2002.[Abstract/Free Full Text]

30. Rosen DR, Siddique T, Patterson D, Figlewicz DA, Sapp P, Hentati A, Donaldson D, Goto J, O’Regan JP, Deng HX, Rahmani Z, Krizus A, McKenna-Yasek D, Cayabyab A, Gaston SM, Berger R, Tanzi RE, Halperin JJ, Herzfeldt B, van den Bergh R, Hung WY, Bird T, Deng G, Mulder DW, Smyth C, Laing NG, Soriano E, Pericak-Vance MA, Haine J, Rouleau GA, Gusella JS, Horritz HR, and Brown RH Jr. Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature 362: 59–62, 1993.[CrossRef][ISI][Medline]

31. Rouleau GA, Clark AW, Rooke K, Pramatarova A, Krizus A, Suchowersky O, Julien JP, and Figlewicz D. SOD1 mutation is associated with accumulation of neurofilaments in amyotrophic lateral sclerosis. Ann Neurol 39: 128–131, 1996.[ISI][Medline]

32. Sato Y, Takahashi M, Shibukawa Y, Jain SK, Hamaoka R, Miyagawa J, Yaginuma Y, Honke K, Ishikawa M, and Taniguchi N. Overexpression of N-acetylglucosaminyltransferase III enhances the epidermal growth factor-induced phosphorylation of ERK in HeLaS3 cells by up-regulation of the internalization rate of the receptors. J Biol Chem 276: 11956–11962, 2001.[Abstract/Free Full Text]

33. Suzuki T, Oishi M, Marshak DR, Czernik AJ, Nairn AC, and Greengard P. Cell cycle-dependent regulation of the phosphorylation and metabolism of the Alzheimer amyloid precursor protein. EMBO J 13: 1114–1122, 1994.[Abstract]

34. Takamiya R, Takahashi M, Myint T, Park YS, Miyazawa N, Endo T, Fujiwara N, Sakiyama H, Misonou Y, Miyamoto Y, Fujii J, and Taniguchi N. Glycation proceeds faster in mutated Cu, Zn-superoxide dismutases related to familial amyotrophic lateral sclerosis. FASEB J 17: 938–940, 2003.[Abstract/Free Full Text]

35. Vukosavic S, Stefanis L, Jackson-Lewis V, Guégan C, Romero N, Chen C, Dubois-Dauphin M, and Przedborski S. Delaying caspase activation by Bcl-2: A clue to disease retardation in a transgenic mouse model of amyotrophic lateral sclerosis. J Neurosci 20: 9119–9125, 2000.[Abstract/Free Full Text]

36. Wiedau-Pazos M, Goto JJ, Rabizadeh S, Gralla EB, Roe JA, Lee MK, Valentine JS, and Bredesen DE. Altered reactivity of superoxide dismutase in familial amyotrophic lateral sclerosis. Science 271: 515–518, 1996.[Abstract]

37. Williamson TL and Cleveland DW. Slowing of axonal transport is a very early event in the toxicity of ALS-linked SOD1 mutants to motor neurons. Nat Neurosci 2: 50–56, 1999.[CrossRef][ISI][Medline]

38. Yim MB, Kang JH, Yim HS, Kwak HS, Chock PB, and Stadtman ER. A gain-of-function of an amyotrophic lateral sclerosis-associated Cu,Zn-superoxide dismutase mutant: An enhancement of free radical formation due to a decrease in Km for hydrogen peroxide. Proc Natl Acad Sci USA 93: 5709–5714, 1996.[Abstract/Free Full Text]





This Article
Abstract
Full Text (PDF)
A corrigendum has been published
All Versions of this Article:
288/2/C253    most recent
00014.2004v1
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Search for citing articles in:
ISI Web of Science (2)
Google Scholar
Articles by Takamiya, R.
Articles by Taniguchi, N.
Articles citing this Article
PubMed
PubMed Citation
Articles by Takamiya, R.
Articles by Taniguchi, N.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2005 by the American Physiological Society.