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Keywords:

  • aggregates;
  • amyotrophic lateral sclerosis;
  • lactacystin;
  • proteasomal inhibition;
  • protein nitration;
  • SOD-1

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Cell culture
  5. DNA transfection
  6. Proteasomal activities
  7. Cell viability assays
  8. Hematoxylin and eosin immunostaining
  9. Fluorescence microscopy
  10. Protein nitration or ubiquitination
  11. Levels of NO2/NO3
  12. Data analysis
  13. Results
  14. Proteasomal enzyme activities
  15. Effect of lactacystin on cell viability and apoptosis
  16. Accumulation of ubiquitinated, tyrosine-nitrated or other proteins
  17. Formation of reactive nitrogen species
  18. Effects of l-NAME
  19. Discussion
  20. Acknowledgements
  21. References

Mutations in Cu,Zn-superoxide dismutase (SOD-1) are associated with some familial cases of amyotrophic lateral sclerosis (ALS), but it is not known how they result in cell death. We examined effects of overexpression of wild-type SOD-1 or the G37R or G85R mutations on the accumulation of ubiquitinated and nitrated proteins, and on loss of cell viability induced by the proteasome inhibitor, lactacystin. Wild-type SOD-1 had no effect on proteasomal activity, but the mutants decreased it somewhat. Treatment with lactacystin (1 µm) caused only limited cell viability loss, even though it induced a marked inhibition of proteasomal activities. However, viability loss due to apoptosis was substantial in response to lactacystin when cells were overexpressing a mutant SOD-1. The frequency of cells showing immunoreactivity against ubiquitinated- or nitrated-proteins was enhanced when wild-type and mutant SOD-1 s were overexpressed. Ubiquitinated or nitrated α-tubulin, SOD-1, α-synuclein and 68K neurofilaments were observed in the aggregates. Similar aggregates were observed in cells overexpressing mutant parkin (Del3–5, T240R and Q311′X). The nitric oxide synthase inhibitor, l-NAME, decreased viability loss and aggregation, suggesting that nitration of proteins may play an important role in aggregation and in the cell death accompanying it.

Abbreviations used
AD

Alzheimer's disease

ALS

amyotrophic lateral sclerosis

ChT-L

chymotrypsin-like

H&E

hematoxylin-eosin

IgG

immunoglobulin G

IgM

immunoglobulin M

Lac

lactacystin

MTT

3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide

l-NAME

NG-nitro-l-arginine methyl ester

NO

nitric oxide

NOS

nitric oxide synthase

PBS

phosphate-buffered saline

PD

Parkinson's disease

PGPH

peptidyl glutamyl peptide hydrolase

SOD-1

Cu,Zn-superoxide dismutase

T-L

trypsin-like

The ubiquitin/proteasome system plays an important role in degrading certain normal or damaged proteins following ubiquitination (Voges et al. 1999). Inhibition or malfunction of this system causes an accumulation of ubiquitinated proteins and such accumulations are often observed in neurodegenerative diseases. Indeed, defects in the 26S proteasome have been observed in the brains of patients with Alzheimer's disease (AD) (Keller et al. 2000), Parkinson's disease (PD) (McNaught et al. 2002) and patients with amyotrophic lateral sclerosis (ALS) (Hoffman et al. 1996; Johnston et al. 2000) and may contribute to neuronal cell death (Halliwell 2002).

Ubiquitinated proteins accumulate to produce protein aggregates in AD, PD and ALS (Mezey et al. 1998; Johnson 2000; Bence et al. 2001). In the case of ALS, the aggregates are thought to be composed of ubiquitin, SOD-1, some subunits of the proteasome and neurofilaments (Alves-Rodrigues et al. 1998; Johnson 2000). Accumulation of nitrated proteins is also found in the brains of patients with AD, PD and ALS or in mutant Cu,Zn-superoxide dismutase (SOD-1) or mutant α-synuclein transgenic animals (Johnson 2000). Nitrated proteins suggest that damage has occurred by peroxynitrite (ONOO) or other reactive nitrogen species (Eiserich et al. 1999; Halliwell et al. 1999; Greenacre and Ischiropoulos 2001; Przedborski et al. 2001). Some protein aggregates may be directly cytotoxic (Bence et al. 2001).

ALS is characterized by the progressive loss of motor neurons in the motor cortex, brain stem and medulla spinalis (Brown 1995). Some cases of familial ALS are associated with point mutations in the genes encoding SOD-1 (Rosen et al. 1993). Although the mechanism by which mutations in the SOD-1 genes cause neurodegeneration in this disease is far from clear, apoptotic cell death is associated with disease progression (Kostic et al. 1997). However, it is not known how the protein aggregates found in ALS relate to cell death.

The aim of the study was to investigate the effects of overexpression of abnormal mutant SOD-1 (and the wild-type SOD-1 for comparison) on the response of cells to the selective proteasomal inhibitor, lactacystin (Fenteany and Schreiber 1998). Since we have shown that proteasomal inhibition by a high level of lactacystin is associated with increased nitric oxide (NO) production and protein nitration, events which are involved in decreasing cell viability (Lee et al. 2001a), effects of the non-specific nitric oxide synthase (NOS) inhibitor, NG-nitro-l-arginine methyl ester (l-NAME), were also examined.

Cell culture

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Cell culture
  5. DNA transfection
  6. Proteasomal activities
  7. Cell viability assays
  8. Hematoxylin and eosin immunostaining
  9. Fluorescence microscopy
  10. Protein nitration or ubiquitination
  11. Levels of NO2/NO3
  12. Data analysis
  13. Results
  14. Proteasomal enzyme activities
  15. Effect of lactacystin on cell viability and apoptosis
  16. Accumulation of ubiquitinated, tyrosine-nitrated or other proteins
  17. Formation of reactive nitrogen species
  18. Effects of l-NAME
  19. Discussion
  20. Acknowledgements
  21. References

NT-2 is a human teratocarcinoma cell line (Angulo et al. 1995) and SK-N-MC is a human neuroblastoma cell line, which has cholinergic characteristics (Biedler et al. 1978). These cell lines and their SOD-1 transfectants were maintained in 100 mm tissue culture plates (Greiner, Frickenhavsen, Germany) containing high glucose-Dulbecco's Modified Eagle's medium (Life Technologies, Paisley, UK), 1 mm sodium pyruvate (Sigma, Dorset, UK), 10% fetal bovine serum (Life Technologies) and 100 IU/mL penicillin and 100 µg/mL streptomycin (Life Technologies) under 5% CO2 and 95% air at 37°C.

DNA transfection

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Cell culture
  5. DNA transfection
  6. Proteasomal activities
  7. Cell viability assays
  8. Hematoxylin and eosin immunostaining
  9. Fluorescence microscopy
  10. Protein nitration or ubiquitination
  11. Levels of NO2/NO3
  12. Data analysis
  13. Results
  14. Proteasomal enzyme activities
  15. Effect of lactacystin on cell viability and apoptosis
  16. Accumulation of ubiquitinated, tyrosine-nitrated or other proteins
  17. Formation of reactive nitrogen species
  18. Effects of l-NAME
  19. Discussion
  20. Acknowledgements
  21. References

Wild-type and mutant SOD-1 constructs (pCB6-neo, pCB6-wild-type SOD-1, pCB6-G37R mutant SOD-1 and pCB6-G85R mutant SOD-1) were a gift from Professor Don Cleveland (The Ludwig Institute for Cancer Research, San Diego, CA, USA). Wild-type and mutant parkin constructs (pcDNA3.1(+)-myc-neo, pcDNA3.1(+)-myc-wild-type parkin, pcDNA3.1(+)-myc-Del3–5 mutant parkin, pcDNA3.1(+)-myc-T240R mutant parkin and pcDNA3.1(+)-myc-Q311X mutant parkin) were obtained from Professor Y. Mizuno (Juntendo University Medical School, Tokyo, Japan). DNA transfection was performed as described by Lee et al. (2001b). Transfectants were selected with 400 µg/mL G418 (neomycin analogue) (Life Technologies).

Cell viability assays

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Cell culture
  5. DNA transfection
  6. Proteasomal activities
  7. Cell viability assays
  8. Hematoxylin and eosin immunostaining
  9. Fluorescence microscopy
  10. Protein nitration or ubiquitination
  11. Levels of NO2/NO3
  12. Data analysis
  13. Results
  14. Proteasomal enzyme activities
  15. Effect of lactacystin on cell viability and apoptosis
  16. Accumulation of ubiquitinated, tyrosine-nitrated or other proteins
  17. Formation of reactive nitrogen species
  18. Effects of l-NAME
  19. Discussion
  20. Acknowledgements
  21. References

When cells reached confluence, the normal medium was changed to normal medium containing 1 µm lactacystin (Calbiochem), and the cells incubated for a maximum of 5 days. Viability assays (trypan blue exclusion and MTT tests) and assays for characteristics of apoptosis were performed with propidium iodide (Sigma) and Hoechst 33258 (Sigma) as described by Kelner et al. (2000) and Lee et al. (2001a).

Hematoxylin and eosin immunostaining

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Cell culture
  5. DNA transfection
  6. Proteasomal activities
  7. Cell viability assays
  8. Hematoxylin and eosin immunostaining
  9. Fluorescence microscopy
  10. Protein nitration or ubiquitination
  11. Levels of NO2/NO3
  12. Data analysis
  13. Results
  14. Proteasomal enzyme activities
  15. Effect of lactacystin on cell viability and apoptosis
  16. Accumulation of ubiquitinated, tyrosine-nitrated or other proteins
  17. Formation of reactive nitrogen species
  18. Effects of l-NAME
  19. Discussion
  20. Acknowledgements
  21. References

This procedure was carried out as described by Bruijn et al. (1998). Briefly, after treatment with lactacystin for 1 day, cells were re-plated on poly l-lysine-coated slides (BDH, Dorset, UK) and incubated at 37°C for 2 h. Cells were fixed with 4% paraformaldehyde for 1 h and permeabilized with 0.4% Triton X-100 for 30 min. The cells were stained with hematoxylin solution (Sigma) for 5 min at room temperature and then decolorized with 1% HCl in ethanol, and stained with eosin-Y (Sigma) for 3 min. The preparations were examined and photographed where necessary in a light microscope ( × 1000) (Zeiss, Göttingen, Germany). For immunostaining, the cells were incubated with a blocking solution, 3% BSA in phosphate-buffered saline (PBS, pH 7.4) for 1 h and washed with PBS. The cells were incubated with anti-ubiquitin antibody (IgG, 1 : 200 in 1% BSA/PBS) (Santa Cruz Technology, Santa Cruz, CA, USA), anti-SOD-1 antibody (IgG, 1 : 200 in 1% BSA/PBS) (Sigma), anti-3-nitrotyrosine antibody (IgG, 1/200 dilution in 1% BSA/PBS) (Zymed, South San Francisco, California, USA), anti-68K neurofilament antibody (IgG, 1 : 200 dilution in 1% BSA/PBS) (Sigma), anti-α-tubulin antibody (IgM, 1 : 200 in 1% BSA/PBS) (Santa Cruz Technology) or anti-c-myc antibody (IgG, 1 : 200 dilution in 1% BSA/PBS) (Sigma) overnight. The cells were incubated with horseradish peroxidase-conjugated anti-IgG or anti-IgM antibody (1 : 200 in 1% BSA/PBS) (Vector Laboratories, Burlingame, CA, USA) for 2 h. The colour reaction was performed by adding 0.5% diaminobenzidine (in 50 mm Tris-HCl, pH 7.4) and H2O2 (final concentration of 0.01%) for 3 min The same cells were examined and photographed using a light microscope (× 1000) (Zeiss).

Fluorescence microscopy

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Cell culture
  5. DNA transfection
  6. Proteasomal activities
  7. Cell viability assays
  8. Hematoxylin and eosin immunostaining
  9. Fluorescence microscopy
  10. Protein nitration or ubiquitination
  11. Levels of NO2/NO3
  12. Data analysis
  13. Results
  14. Proteasomal enzyme activities
  15. Effect of lactacystin on cell viability and apoptosis
  16. Accumulation of ubiquitinated, tyrosine-nitrated or other proteins
  17. Formation of reactive nitrogen species
  18. Effects of l-NAME
  19. Discussion
  20. Acknowledgements
  21. References

This was performed with the same immunostaining procedure described above, except that fluorescein-conjugated anti-IgG antibody (1 : 200 dilution in 1% BSA/PBS) and Texas Red-conjugated anti-IgM antibody (1 : 200 dilution in 1% BSA/PBS) were used. The cells were examined and photographed in a light microscope (× 1000) (Zeiss) with UV filters (a filter BP 510–525 for green and a filter LP 570 for red fluorescence).

Protein nitration or ubiquitination

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Cell culture
  5. DNA transfection
  6. Proteasomal activities
  7. Cell viability assays
  8. Hematoxylin and eosin immunostaining
  9. Fluorescence microscopy
  10. Protein nitration or ubiquitination
  11. Levels of NO2/NO3
  12. Data analysis
  13. Results
  14. Proteasomal enzyme activities
  15. Effect of lactacystin on cell viability and apoptosis
  16. Accumulation of ubiquitinated, tyrosine-nitrated or other proteins
  17. Formation of reactive nitrogen species
  18. Effects of l-NAME
  19. Discussion
  20. Acknowledgements
  21. References

Protein nitration or ubiquitination was assessed using immunoprecipitation and immunoblotting as described by Shimura et al. (2000). Ubiquitinated or nitrated proteins were precipitated with protein G-coated bead-bound anti-ubiquitin antibody (IgG, 1 : 200 1% BSA/PBS, Santa Cruz Technology) or anti-3-nitrotyrosine antibody (IgG, 1 : 200 1% BSA/PBS) (Zymed), respectively. In order to get clear data, excess antibody was removed using filters (Centricon, molecular weight cut-off 100 K) (Millipore, Gloucestershire, UK). Immunoblotting was performed as described by Lee et al. (2001b), with anti-SOD-1 antibody (IgG, 1 : 200 in 1% BSA/PBS) (Sigma) or anti-68K neurofilament antibody (IgG, 1 : 200 in 1% BSA/PBS) (Sigma).

Levels of NO2/NO3

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Cell culture
  5. DNA transfection
  6. Proteasomal activities
  7. Cell viability assays
  8. Hematoxylin and eosin immunostaining
  9. Fluorescence microscopy
  10. Protein nitration or ubiquitination
  11. Levels of NO2/NO3
  12. Data analysis
  13. Results
  14. Proteasomal enzyme activities
  15. Effect of lactacystin on cell viability and apoptosis
  16. Accumulation of ubiquitinated, tyrosine-nitrated or other proteins
  17. Formation of reactive nitrogen species
  18. Effects of l-NAME
  19. Discussion
  20. Acknowledgements
  21. References

Levels of NO2/NO3 were measured using the Griess reaction with some modification (Guevara et al. 1998). Cells (105) were plated in 96-well plates and incubated under the experimental conditions stated. Aliquots (100 µL) of culture media were added to a reaction mixture containing final concentrations of 50 mm HEPES, 0.2 unit/mL nitrate reductase, 5 µm FAD and 100 µm NADPH in 500 µL. After the reduction described above, 900 µL methanol/diethyl ether was added to the sample to deproteinise followed by centrifugation (12 000 g, 10 min at 4°C). The supernatant was incubated at 37°C for 30 min and 100 µL 1 mm potassium ferricyanide and 200 µL deionized water then added to make a final volume of 800 µL. The mixture was incubated at room temperature for 20 min Griess reagent [1% N-(1-naphthyl)ethylene diamine (w/v) and 10% sulfanilamide (w/v) in 200 µL] was added and absorbance read at 543 nm for 3 min. A standard curve was constructed with sodium nitrite (Sigma) and sodium nitrate (Sigma). The levels of NO2/NO3 in medium alone (including FBS) were subtracted from the levels of NO2/NO3 in cells plus medium. The levels of NO2/NO3 in cells plus medium were 20–25 µm and in medium (plus FBS) alone 10–12 µm.

Proteasomal enzyme activities

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Cell culture
  5. DNA transfection
  6. Proteasomal activities
  7. Cell viability assays
  8. Hematoxylin and eosin immunostaining
  9. Fluorescence microscopy
  10. Protein nitration or ubiquitination
  11. Levels of NO2/NO3
  12. Data analysis
  13. Results
  14. Proteasomal enzyme activities
  15. Effect of lactacystin on cell viability and apoptosis
  16. Accumulation of ubiquitinated, tyrosine-nitrated or other proteins
  17. Formation of reactive nitrogen species
  18. Effects of l-NAME
  19. Discussion
  20. Acknowledgements
  21. References

Proteasome activity was measured after treatment with 1 µm lactacystin. Addition of lactacystin to the cell culture medium for 24 h inhibited all three enzyme activities, namely trypsin-like (T-L), chymotrypsin-like (ChT-L) and peptidyl glutamyl peptide hydrolase (PGPH) of the proteasome in both cell lines, by 70%, in agreement with previous results (Lee et al. 2001b) (Table 1). Enzymatic activity did not recover for 3–4 days (data not shown). Addition of l-NAME (1 mm) did not alter the degree of proteasomal inhibition caused by lactacystin, nor did it have any effect on the proteasomal activity in untreated cells.

Table 1.  Proteasomal activity
CellsProteolytic activityControlLactacystinl-NAMELactacystin + l-NAME
  1. When cells reached confluence, they were exposed to lactacystin (1 µm) and/or l-NAME (1 mm) for 24 h and extracted for measuring proteasome activities. Values are the means ±SEM (FU/min/mg protein), n = 6. Significance was examined by one-way anova. *p < 0.01 compared with the same cells under normal incubation conditions. This inhibitor affects both the 20S and the 26S proteasome (Fenteany and Schreiber 1998).

NT-2T-L15.1 ± 1.22.9 ± 0.3*17.3 ± 1.33.1 ± 0.3*
ChT-L4.1 ± 0.20.6 ± 0.1*4.3 ± 0.20.6 ± 0.1*
PGPH2.1 ± 0.10.4 ± 0.0*2.2 ± 0.10.4 ± 0.1*
SK-N-MCT-L13.9 ± 0.92.6 ± 0.2*14.2 ± 0.83.0 ± 0.2*
ChT-L3.7 ± 0.20.4 ± 0.1*3.8 ± 0.20.4 ± 0.1*
PGPH1.9 ± 0.10.3 ± 0.1*2.0 ± 0.10.3 ± 0.1*

Effect of lactacystin on cell viability and apoptosis

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Cell culture
  5. DNA transfection
  6. Proteasomal activities
  7. Cell viability assays
  8. Hematoxylin and eosin immunostaining
  9. Fluorescence microscopy
  10. Protein nitration or ubiquitination
  11. Levels of NO2/NO3
  12. Data analysis
  13. Results
  14. Proteasomal enzyme activities
  15. Effect of lactacystin on cell viability and apoptosis
  16. Accumulation of ubiquitinated, tyrosine-nitrated or other proteins
  17. Formation of reactive nitrogen species
  18. Effects of l-NAME
  19. Discussion
  20. Acknowledgements
  21. References

Treatment of both cell lines (non-transfectants or vector-only transfectants) with 1 µm lactacystin for up to 5 days did not cause a loss of viability, as assessed by trypan blue exclusion (Fig. 1a), but metabolic activity, as measured by the MTT test, was reduced by 15% at 12 h (p < 0.01), 17% at 24 h (Fig. 1b), and about 40% at 5 days (data not shown). Approximately 10–15% of NT-2 and SK-N-MC cells showed signs of early apoptosis at 24 h (p < 0.01, Fig. 2) and 47% at 5 days (data not shown). However, overexpression of either wild-type or mutant SOD-1 rendered cells more sensitive to lactacystin. Both wild-type and mutant SOD-1 proteins were expressed 2–2.5-fold more than in vector-only transfectants (data not shown), consistent with our previous report (Lee et al. 2001a). Overexpression of wild-type SOD-1 had no effect on proteasomal activities, whereas the mutant SOD-1 s decreased them by about 20%. For wild-type SOD-1 transfectants, 20% of lactacystin-treated cells failed to exclude trypan blue dye at 5 days and 20% of MTT reducing activity had been lost at 24 h (p < 0.01, Fig. 1a,b). About 30–40% of cells showed features of apoptosis (p < 0.01, Fig. 2). By contrast, mutant SOD-1 s greatly exacerbated the toxicity of lactacystin. Approximately 50–60% of cells lost viability or MTT reducing activity by 5 days or 24 h, respectively (p < 0.01). This was confirmed by the frequency of cells showing apoptotic features (Fig. 2). No effect of normal or mutant SOD-1 on cell viability was observed in the absence of lactacystin.

image

Figure 1. Effect of 1 µm lactacystin on the viability of NT-2 and SK-N-MC cell lines for 24 h. (a) Trypan exclusion (NT-2 cell line) and (b) MTT tests (SK-N-MC cell line). Cells were treated with lactacystin for 24 h □, Control; ▮, 1 µm lactacystin; ░, 1 µm lactacystin + l-NAME. Values are means ± SEM, n = 4. Two-way anova was carried out to test significance. Multiple comparisons were followed with post hoc Bonferroni t-tests where necessary. *(p < 0.01), Significant difference compared with the same cells under normal incubation conditions; †(p < 0.01), Significant difference compared with non- or vector-only transfectants under the same incubation conditions. Non, non-transfectant; Vector, vector-only transfectant; WT, wild-type SOD-1 transfectant, G37R, G37R mutant SOD-1 transfectant; G85R, G85R mutant SOD-1 transfectant.

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image

Figure 2. Frequency of cells showing apoptotic features after exposure to 1 µm lactacystin for 24 h. (a) Cell morphology change (NT-2 cell line) and (b) chromatin condensation (SK-N-MC cell line). Cells were treated with lactacystin for 24 h and counted in a fluorescence microscope. □: Control; ▮: 1 µm lactacystin. Values are the means ± SEM, n = 10. Significance was examined by one-way anova. Multiple comparisons were followed with post hoc Bonferroni t-tests where necessary. *(p < 0.01), Significant difference compared with non- or vector-only transfectants under normal incubation conditions; †(p < 0.01), Significant difference compared with the same cells under normal condition; ‡(p < 0.01), Significant difference compared with non- or vector-only transfectants under the same incubation conditions. Non, non-transfectant; Vector, vector-only transfectant; WT, wild-type SOD-1 transfectant, G37R, G37R mutant SOD-1 transfectant; G85R, G85R mutant SOD-1 transfectant.

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Accumulation of ubiquitinated, tyrosine-nitrated or other proteins

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Cell culture
  5. DNA transfection
  6. Proteasomal activities
  7. Cell viability assays
  8. Hematoxylin and eosin immunostaining
  9. Fluorescence microscopy
  10. Protein nitration or ubiquitination
  11. Levels of NO2/NO3
  12. Data analysis
  13. Results
  14. Proteasomal enzyme activities
  15. Effect of lactacystin on cell viability and apoptosis
  16. Accumulation of ubiquitinated, tyrosine-nitrated or other proteins
  17. Formation of reactive nitrogen species
  18. Effects of l-NAME
  19. Discussion
  20. Acknowledgements
  21. References

Accumulation of ubiquitinated, nitrated or other proteins was investigated after addition of lactacystin for 24 h. Under normal culture conditions, only a few cells were stained by antibodies against ubiquitin or 3-nitrotyrosine (Fig. 3a and 4a and Table 2). The addition of lactacystin to the culture medium produced an accumulation of ubiquitinated or nitrated proteins, especially clustered near the cell membrane (Fig. 3). Immunostaining against SOD-1, α-synuclein or 68K neurofilaments was also observed following lactacystin treatment (Fig. 3). A similar effect of lactacystin was observed in cells transfected with mutant parkins (Del3–5, T240R and Q311X) (Fig. 4).

image

Figure 3. Protein aggregates observed in SOD-1 transfectants after treatment with 1 µm lactacystin for 24 h. (a) Control (NT-2 cells, H&E staining); (b) control (NT-2 cells, immunostaining with anti-ubiquitin mAb); (c) lactacystin-treatment (NT-2 cells, H&E staining); (d) lactacystin-treatment (NT-2 cells, immunostaining with anti-ubiquitin mAb); (e) control (SK-N-MC cells, H&E staining); (f) control (SK-N-MC cells, immunostaining with anti-nitrotyrosine mAb; (g) lactacystin-treatment (SK-N-MC cells, H&E staining); (h) lactacystin-treatment (NT-2 cells, immunostaining with anti-nitrotyrosine mAb; (i) control (NT-2 cells, H&E staining); (j) control (NT-2 cells, immunostaining with anti-SOD-1 mAb; (k) Lactacystin-treatment (NT-2 cells, H&E staining); (l) lactacystin-treatment (NT-2 cells, immunostaining with anti-SOD-1 mAb; (m) control (SK-N-MC cells, H&E staining); (n) control (SK-N-MC cells, immunostaining with anti-α-synuclein mAb; (o) lactacystin-treatment (SK-N-MC cells, H&E staining); (p) Lactacystin-treatment (SK-N-MC cells, immunostaining with anti-α-synuclein mAb; (q) control (NT-2 cells, H&E staining); (r) control (NT-2 cells, immunostaining with anti-68K neurofilament mAb; (s) lactacystin-treatment (NT-2 cells, H&E staining); (t) lactacystin-treatment (NT-2 cells, immunostaining with anti-68K neurofilament mAb). Arrows indicate protein aggregates. Scale bars: 10 µm.

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image

Figure 4. Protein aggregates observed in cells transfected with mutant parkins following treatment with 1 µm lactacystin for 24 h. (a) Control (NT-2 cells, immunostaining with anti-ubiquitin mAb); (b) control (SK-N-MC cells, immunostaining with anti-nitrotyrosine mAb; (c) lactacystin-treatment (NT-2 cells, immunostaining with anti-ubiquitin mAb); (d) lactacystin-treatment (SK-N-MC cells, immunostaining with anti-ubiquitin mAb); (e) lactacystin-treatment (NT-2 cells, immunostaining with anti-nitrotyrosine mAb); (f) lactacystin-treatment (SK-N-MC cells, immunostaining with anti-nitrotyrosine mAb); (g) Lactacystin-treatment (NT-2 cells, immunostaining with anti-c-myc mAb); (h) Lactacystin-treatment (SK-N-MC cells, immunostaining with anti-c-myc mAb); (i) lactacystin-treatment (NT-2 cells, immunostaining with anti-SOD-1 mAb; (j) lactacystin-treatment (SK-N-MC cells, immunostaining with anti-SOD-1 mAb; (k) lactacystin-treatment (NT-2 cells, immunostaining with anti-α-synuclein mAb; (l) lactacystin-treatment (SK-N-MC cells, immunostaining with anti-α-synuclein mAb; (m) lactacystin-treatment (NT-2 cells, immunostaining with anti-α-tubulin mAb; (n) lactacystin-treatment (SK-N-MC cells, immunostaining with anti-α-tubulin mAb; (o) lactacystin-treatment (NT-2 cells, immunostaining with anti-68K neurofilament mAb; (p) lactacystin-treatment (SK-N-MC cells, immunostaining with anti-68K neurofilament mAb). Arrows indicate protein aggregates. Scale bars: 10 µm.

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Table 2.  Frequency of immunostaining-positive cells
CellsControl (%)Lactacystin (%)l-NAME (%)Lactacystin + l-NAME (%)
  1. When cells reached confluence, they were exposed to lactacystin or HNE for 24 h and extracted for measuring frequency of immunostaining-positive cells. Values are the means ±SEM, n = 10. Significance was examined by one-way anova. Multiple comparisons were followed with post hoc Bonferroni t-tests where necessary. *p < 0.01, significant difference compared with-non-or vector-only transfectants under normal incubation conditions; p < 0.01, significant difference compared with the same cells under normal incubation conditions; p < 0.01, significant difference compared with non- or vector-only transfectants under the same incubation conditions; §p < 0.01, significant difference compared with same cells under culture conditions (control or lactacystin-treatment) without l-NAME. Non, Non-transfectant; Vector, vector-only transfectant; WT, wild-type SOD-1 transfectant, G37R, G37R mutant SOD-1 transfectant; G85R, G85R mutant SOD-1 transfectant.

Ubiquitinated proteins (NT-2)
 Non3 ± 09 ± 11 ± 0§6 ± 1§
 Vector3 ± 110 ± 11 ± 0§7 ± 1§
 WT13 ± 2*26 ± 28 ± 1§16 ± 2§
 G37R19 ± 2*37 ± 311 ± 2§18 ± 2§
 G85R17 ± 2*38 ± 310 ± 1§20 ± 3§
Tyrosine-nitrated proteins (SK-N-MC)
 Non4 ± 111 ± 21 ± 0§7 ± 1§
 Vector4 ± 110 ± 22 ± 1§8 ± 1§
 WT12 ± 2*38 ± 37 ± 1§19 ± 2§
 G37R17 ± 2*49 ± 312 ± 1§25 ± 2§
 G85R18 ± 2*49 ± 313 ± 1§28 ± 3§
SOD-1 (NT-2)
 Non3 ± 112 ± 21 ± 0§6 ± 1§
 Vector4 ± 112 ± 11 ± 0§6 ± 1§
 WT12 ± 3*39 ± 27 ± 1§16 ± 2§
 G37R19 ± 3*57 ± 310 ± 1§18 ± 2§
 G85R20 ± 2*54 ± 310 ± 2§19 ± 2§
α-Synuclein (SK-N-MC)
 Non3 ± 111 ± 11 ± 0§5 ± 1§
 Vector4 ± 112 ± 11 ± 0§6 ± 1§
 WT14 ± 1*24 ± 37 ± 1§12 ± 3§
 G37R23 ± 2*42 ± 311 ± 2§17 ± 3§
 G85R22 ± 2*46 ± 39 ± 1§18 ± 3§
68K neurofilament (NT-2)
 Non4 ± 111 ± 22 ± 0§8 ± 1§
 Vector4 ± 112 ± 22 ± 0§8 ± 1§
 WT12 ± 2*29 ± 28 ± 1§18 ± 2§
 G37R19 ± 2*40 ± 313 ± 2§23 ± 3§
 G85R20 ± 2*40 ± 313 ± 2§24 ± 3§

In order to investigate how the expression of wild-type or mutant SOD-1 s affects accumulation of ubiquitinated or nitrated proteins, the frequency of cells immunostained by monoclonal antibodies against ubiquitin, 3-nitrotyrosine, SOD-1, α-synuclein or 68K neurofilaments was determined (Table 2). Under normal incubation conditions, approximately 15–25% of cells transfected with wild-type or mutant SOD-1 s were identified as immunostaining-positive with all antibodies used (p < 0.01), whereas non-transfectants and vector-only transfectants showed few aggregates. Exposure of cells to lactacystin raised the frequency of aggregates in normal cells (p < 0.01), and to a greater extent in cells overexpressing normal or mutant SOD-1 s (p < 0.01) or mutant parkins (p < 0.01) (Fig. 4).

In order to investigate which proteins might be colocalized in the aggregates, double staining was employed using fluorescence-conjugated antibodies (Fig. 5). Since α-tubulin was found in protein inclusions in a previous study with microglial cells (Gai et al. 1999), we used anti-α-tubulin IgM in a double staining technique (Fig. 5). Ubiquitinated proteins, tyrosine-nitrated proteins, SOD-1, α-synuclein and 68K neurofilaments appeared to be colocalized in the protein aggregates in lactacystin-treated cells.

image

Figure 5. Double staining with fluorescein-conjugated secondary antibody (anti-IgG antibody) or Texas red-conjugated secondary antibody (anti-IgM antibody) after treatment with 1 µm lactacystin for 24 h. (a) Control (NT-2 cells, fluorescein-conjugated anti-ubiquitin mAb); (b) control (NT-2 cells, Texas red-conjugated anti-α-tubulin mAb); (c) lactacystin-treatment (SK-N-MC cells, fluorescein-conjugated anti-ubiquitin mAb); (d) lactacystin-treatment (SK-N-MC cells, Texas-conjugated anti-α-tubulin mAb); (e) lactacystin-treatment (NT-2 cells, fluorescein-conjugated anti-SOD-1 mAb); (f) lactacystin-treatment (NT-2 cells, Texas red-conjugated anti-α-tubulin mAb); (g) lactacystin-treatment (SK-N-MC cells, fluorescein-conjugated anti-α-synuclein mAb); (h) lactacystin-treatment (SK-N-MC cells, Texas red-conjugated anti-α-tubulin mAb); (i) lactacystin-treatment (NT-2 cells, fluorescein-conjugated anti-68K neurofilament mAb); (j) lactacystin-treatment (NT-2 cells, Texas red-conjugated anti-α-tubulin mAb); (k) lactacystin-treatment (SK-N-MC cells, fluorescein-conjugated anti-nitrotyrosine mAb); (l) lactacystin-treatment (SK-N-MC cells, Texas red-conjugated anti-α-tubulin mAb). Arrows indicate protein aggregates. Scale bars: 10 µm.

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In order to investigate whether SOD-1, α-synuclein and/or 68K neurofilaments were ubiquitinated or nitrated, and how treatment with lactacystin modulates levels of them, immunoprecipitation and western blotting assays were performed. Under normal culture conditions, ubiquitinated SOD-1 and 68K neurofilaments were detectable only to a limited extent. Overexpression of wild-type and mutant SOD-1 s increased the levels of ubiquitinated proteins (1.2–1.4-fold in the wild-type SOD-1 transfectants and 1.6–1.8-fold in the mutant transfectants). Exposure of cells to lactacystin increased the levels of the ubiquitinated proteins by 2–2.5-fold (2.1 ±0.5-fold, n = 4); this was enhanced when wild-type and mutant SOD-1 s were overexpressed (4.5 ± 0.4-fold, n = 4, Fig. 6g).

image

Figure 6. Frequency of ubiquitinated or tyrosine-nitrated SOD-1, α-synuclein and 68K neurofilaments using immunoprecipitation and immunoblotting. Cells were incubated with 1 µm lactacystin for 24 h, and extracted for immunoprecipitation with anti-ubiquitin antibody (a and c) or anti-3-nitrotyrosine antibody (b), and western blotting were performed with anti-SOD-1 antibody (a), anti-α-synuclein antibody (b) or anti-68K neurofilament antibody (c). 50 µg protein was incubated with appropriated monoclonal antibody and protein G. Lane 1: control; lane 2: 1 µm lactacystin-treatment. (a) Frequency of ubiquitinated SOD-1 (SK-N-MC cells); (b) frequency of nitrated α-synuclein (NT-2 cells); and (c) frequency of ubiquitinated 68K neurofilament (SK-N-MC cells). □: Control; ▮: 1 µm lactacystin. Values are the means ± SEM, n = 6. Significance was examined by one-way anova. Multiple comparisons were followed with post hoc Bonferroni t-tests where necessary. *(p < 0.01), Significant difference compared with non- or vector-only transfectants under normal incubation conditions; †(p < 0.01), Significant difference compared with the same cells under normal incubation conditions; ‡(p < 0.01), Significant difference compared with non-or vector-only transfectants under the same incubation conditions.

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Tyrosine-nitrated proteins were also observed within aggregates. Expression of wild-type and mutant SOD-1 s increased levels of the nitrated proteins (1.5-fold in the wild-type SOD-1 transfectant and 1.8-fold in the mutant transfectant) (Fig. 6). Addition of lactacystin also elevated the nitrated proteins by 2–3-fold and this was greater in wild-type and mutant SOD-1 transfectants (3.9–4.2-fold in the wild-type SOD-1 transfectant and 6–7-fold in the mutant transfectant). However, tyrosine-nitrated SOD-1 was not detected under normal incubation conditions or in the presence of lactacystin (data not shown), as expected since human SOD-1 does not undergo tyrosine nitration.

Formation of reactive nitrogen species

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Cell culture
  5. DNA transfection
  6. Proteasomal activities
  7. Cell viability assays
  8. Hematoxylin and eosin immunostaining
  9. Fluorescence microscopy
  10. Protein nitration or ubiquitination
  11. Levels of NO2/NO3
  12. Data analysis
  13. Results
  14. Proteasomal enzyme activities
  15. Effect of lactacystin on cell viability and apoptosis
  16. Accumulation of ubiquitinated, tyrosine-nitrated or other proteins
  17. Formation of reactive nitrogen species
  18. Effects of l-NAME
  19. Discussion
  20. Acknowledgements
  21. References

Levels of NO2/NO3, as indicators of NO production, were measured. Exposure of cells to lactacystin elevated levels of NO2/NO3, which were further increased when wild-type and mutant SOD-1 were overexpressed (Fig. 7).

image

Figure 7. The levels of reactive nitrogen species after addition of lactacystin. (a) NO2 and (b) NO3. When cells were grown to 80% confluence in 96 well plates, they were subjected to lactacystin for 24 h and then were extracted. □: Control; ▮: 1 µm lactacystin. Values are the means ± SEM, n = 6. Significance was examined by one-way anova. Multiple comparisons were followed with post hoc Bonferroni t-tests where necessary. *(p < 0.01), Significant difference compared with non- or vector-only transfectants under normal incubation conditions; †(p < 0.01), Significant difference compared with the same cells under normal incubation conditions; ‡(p < 0.01), Significant difference compared with non- or vector-only transfectants under the same incubation conditions.

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Effects of l-NAME

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Cell culture
  5. DNA transfection
  6. Proteasomal activities
  7. Cell viability assays
  8. Hematoxylin and eosin immunostaining
  9. Fluorescence microscopy
  10. Protein nitration or ubiquitination
  11. Levels of NO2/NO3
  12. Data analysis
  13. Results
  14. Proteasomal enzyme activities
  15. Effect of lactacystin on cell viability and apoptosis
  16. Accumulation of ubiquitinated, tyrosine-nitrated or other proteins
  17. Formation of reactive nitrogen species
  18. Effects of l-NAME
  19. Discussion
  20. Acknowledgements
  21. References

Treatment with l-NAME (10 µm-10 mm) decreased the cell viability loss induced by lactacystin (Fig. 1). This effect was concentration-dependent (p < 0.01 from 1 to 3 days, data not shown). Based on these studies, a concentration of 200 µm was chosen for further experiments. l-NAME also attenuated the loss of cell viability in wild-type SOD-1 transfectants (p < 0.01, Figs 1a,b), and decreased the frequency of cells showing aggregates with immunoreactivity against ubiquitin, 3-nitrotyrosine, SOD-1 or 68K neurofilaments under normal condition or following treatment with lactacystin (p < 0.01, Table 2). l-NAME did not prevent inhibition of proteasomal activity by lactacystin (Table 1).

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Cell culture
  5. DNA transfection
  6. Proteasomal activities
  7. Cell viability assays
  8. Hematoxylin and eosin immunostaining
  9. Fluorescence microscopy
  10. Protein nitration or ubiquitination
  11. Levels of NO2/NO3
  12. Data analysis
  13. Results
  14. Proteasomal enzyme activities
  15. Effect of lactacystin on cell viability and apoptosis
  16. Accumulation of ubiquitinated, tyrosine-nitrated or other proteins
  17. Formation of reactive nitrogen species
  18. Effects of l-NAME
  19. Discussion
  20. Acknowledgements
  21. References

The pathology of ALS results from the loss of specific sets of neurons, accompanied by increased levels of oxidative or nitrative damage to proteins (Brown 1995; Bruijn et al. 1997; Pedersen et al. 1998; Shibata et al. 2000; McNaught et al. 2001). Such damaged proteins are normally cleared by the proteasome after ubiquitination (Ciechanover et al. 2000). Ubiquitinated or nitrated proteins rarely accumulate in normal cells, but are frequently seen in neurons from patients with various neurodegenerative diseases such as ALS or PD (Eiserich et al. 1999; Giasson et al. 2000; Greenacre and Ischiropoulos 2001). This is most probably due, at least in part, to malfunction or inhibition of the ubiquitin/proteasome system (Halliwell and Jenner 1998; McNaught et al. 2001; Halliwell 2002). Consistent with this view, a decrease in the three enzymatic activities in the proteasome has been reported in patients with AD, PD or ALS (Chou et al. 1998; Keller et al. 2000; McNaught et al. 2002).

We investigated effects of overexpression of wild-type and mutant SOD-1 s on the accumulation of ubiquitinated or nitrated proteins, and viability change in cells in which proteasomes were inhibited by lactacystin. We have already shown that overexpression of wild-type SOD-1 has no direct effect on proteasomal activities, whereas the mutant SOD-1s decrease them slightly, by about 20% (Lee et al. 2001b; Hyun et al. 2002). Treatment of both cell lines with lactacystin to inhibit the proteasome to a much greater extent (Table 1) caused limited loss of viability, but it increased levels of protein damage products such as protein carbonyls, lipid peroxidation or 3-nitrotyrosine (Lee et al. 2001b). Some cells showed evidence of apoptosis (Fig. 2). Increased frequency of aggregates, which contained SOD-1, α-synuclein, α-tubulin, ubiquitinated or nitrated proteins, or 68K neurofilaments was also observed. Rideout et al. (2001) observed that proteasomal inhibition caused formation of ubiquitin/α-synuclein immunoreactive inclusions in PC12 cells.

Overexpression of wild-type and mutant SOD-1 s increased viability loss and the prevalence of protein aggregates induced by lactacystin. This may be a general effect because it was also seen in cells overexpressing mutant parkins (Del3–5, T240R and Q311X). Mice overexpressing the α1B-adrenergic receptor also accumulate aggregates containing ubiquitin, α-synuclein and nitrated protein (Papay et al. 2002). It may be that protein aggregates are the cause of the loss of cell viability, as suggested by Bence et al. (2001). An important issue is whether proteins aggregate and then are nitrated as aggregated proteins, or whether the nitration occurs first and facilitates aggregation. This is difficult to study using immunostaining techniques, which are semiquantitative. Consistent with the view that nitration plays a key role is our observation that lactacystin increased NO production, whereas and l-NAME reduced loss of viability in wild-type and mutant transfectants and had parallel effects on nitration and on the prevalence of the aggregates. NO was not responsible for the proteasomal inhibition induced by lactacystin, as this was not reversed by l-NAME. We therefore propose that NO contributes to aggregate formation, most likely by increasing levels of nitrated proteins. Indeed, it was shown that ONOO formation in HEK293 cells leads to α-synuclein aggregation (Paxinou et al. 2001). Tyrosine nitration of α-synuclein is involved in its dimerization after exposure to ONOO (Takahashi et al. 2002). SOD-1 is known to catalyze ONOO dependent nitration of other proteins (Crow et al. 1997a) and ALS-associated mutant SOD-1 s may be a more effective catalyst of this reaction (Crow et al. 1997b).

There appears to be no qualitative difference in components of the protein aggregates between control transfectants and SOD-1 (G37R and G85R) or parkin (Del3–5, T240R and Q311X) transfectants, but the number of aggregates was elevated in wild-type and mutant transfectants. Ubiquitinated or nitrated α-tubulin, or 68K neurofilaments were found in all the aggregates, suggestive of precipitation of a wide range of proteins, by a mechanism that involves protein nitration. This proteasomal inhibition and protein nitration may be key mechanisms contributing to neurodegeneration and accompanying protein aggregate formation.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Cell culture
  5. DNA transfection
  6. Proteasomal activities
  7. Cell viability assays
  8. Hematoxylin and eosin immunostaining
  9. Fluorescence microscopy
  10. Protein nitration or ubiquitination
  11. Levels of NO2/NO3
  12. Data analysis
  13. Results
  14. Proteasomal enzyme activities
  15. Effect of lactacystin on cell viability and apoptosis
  16. Accumulation of ubiquitinated, tyrosine-nitrated or other proteins
  17. Formation of reactive nitrogen species
  18. Effects of l-NAME
  19. Discussion
  20. Acknowledgements
  21. References
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