Address correspondence and reprint requests to Maria Rosa Ciriolo, Department of Biology, University of Rome ‘Tor Vergata’, Via della Ricerca Scientifica 1, 00133 Rome, Italy. E-mail: email@example.com
Reactive oxygen and nitrogen species have emerged as predominant effectors of neurodegeneration. We demonstrated that expression of the fully active G93A Cu,Zn superoxide dismutase mutant in neuroblastoma cells is associated with an increased level of oxidatively modified proteins, in terms of carbonylated residues. A parallel increase in proteasome activity was detected and this was mandatory in order to assure cell viability. In fact, proteasome inhibition by lactacystin or MG132 resulted in programmed cell death. Nitrosative stress was not involved in the oxidative unbalance, as a decrease in neuronal nitric oxide production and down-regulation of neuronal nitric oxide synthase (nNOS) level were detected. The nNOS down-regulation was correlated to increased proteolytic degradation by proteasome, because comparable levels of nNOS were detected in G93A and parental cells upon treatment with lactacystin. The altered rate of proteolysis observed in G93A cells was specific for nNOS as Cu,Zn superoxide dismutase (Cu,Zn SOD) degradation by proteasome was influenced neither by its mutation nor by increased proteasome activity. Treatment with the antioxidant 5,5′-dimethyl-1-pyrroline N-oxide resulted in inhibition of protein oxidation and decrease in proteasome activity to the basal levels. Overall these results confirm the pro-oxidant activity of G93A Cu,Zn SOD mutant and, at the same time, suggest a cross-talk between reactive oxygen and nitrogen species via the proteasome pathway.
Reactive oxygen species (ROS), such as hydrogen peroxide and superoxide, are in large part generated as a result of the normal oxygen metabolism in mitochondria. ROS have recently been recognized as important signal transduction intermediates regulating gene expression, cell differentiation, immune activation and apoptosis by redox-mediated signalling (Finkel 2000; Herrlich and Bohmer 2000; Thannickal and Fanburg 2000). On the other hand, sustained oxidative insult is harmful to the cell, due to ROS high chemical reactivity with DNA, proteins, carbohydrates and lipids in a destructive manner leading to cell death (Halliwell and Gutteridge 1990). In biological systems, oxidative stress occurs when production of ROS exceeds the capability of the cellular natural defence system, consisting of low molecular weight antioxidants and cooperative redox enzymes (Fridovich 1999). Unbalanced ROS production is assumed to be involved in several pathological processes such as inflammation, cancer and neurodegenerative disorders (Halliwell and Gutteridge 1990). Among these the brain seems to be particularly vulnerable as evinced by the variety of diseases in the pathogenesis of which oxidative stress is implicated: Huntington's, Parkinson's, amyotrophic lateral sclerosis (ALS), Alzheimer's and retinal degenerative disorders (Floyd 1999). This is not surprising, as the central nervous system is highly aerobic and its antioxidant defences are relatively low, having almost no catalase (Aksenov et al. 1998) and very low levels of glutathione (Slivka et al. 1987).
Nitric oxide (NO), formed endogenously by the catalysis of different nitric oxide synthases (NOS), and its intermediates, reactive nitrogen species (RNS), have also emerged as predominant effectors of neurodegeneration (Leist and Nicotera 1998). However, NO have a variable degree of chemical reactivity and functions. These include regulation of the cardiovascular system, smooth muscle relaxation, neurotransmission, coagulation and immune regulation (Moncada et al. 1991). Moreover, NO has a controversial role in cell death because of its ability to both induce and protect against apoptosis (Kim et al. 1999).
Protein oxidation in cells is a natural consequence of ROS and RNS production as they can oxidize specific amino acids leading to the formation of carbonyls and nitrotyrosine (Chevion et al. 2000); these modifications have often been associated with a loss of the protein biological function. It has been proposed that oxidized proteins, like most partially denatured proteins, are rapidly degraded by proteases. Among these, the proteasome has emerged as the most important proteolytic system of the secondary defence against oxidative stress, as it selectively degrades oxidized and damaged proteins, thus preventing their accumulation. A growing list of proteasome substrates has been identified and contains numerous proteins of direct importance in neurophysiological and neuropathological processes. However, in spite of intensive research, demonstrating the involvement of proteasome in a diverse array of cellular activities, its role in central nervous system is beginning to be elucidated. Recent studies have demonstrated that proteasome inhibition occurs in several neurodegenerative conditions, previously indicated as oxidative stress-related diseases (Ding and Keller 2001), and is sufficient to induce neuronal death (Fenteany and Schreiber 1998; Lee et al. 2001).
We previously demonstrated that human neuroblastoma cells (SH-SY5Y) transfected with wild-type Cu,Zn superoxide dismutase (Cu,Zn SOD) (WT cells) were protected from NO-induced apoptosis, whereas the same cells transfected with the mutant G93A Cu,Zn SOD (G93A cells) were highly susceptible to apoptosis (Ciriolo et al. 2000). These results point to an oxidative unbalance, occurring in SH-SY5Y cells as result of the expression of G93A Cu,Zn SOD, that was not detrimental to cell survival under resting conditions whereas it was a factor strengthening NO-mediated toxicity. Overall the results were in good agreement with the assumption that Cu,Zn SODs carrying mutations associated with the familial form of ALS, such as the G93A mutation, are pro-oxidant agents, although they retain full enzymatic SOD activity (Morrison and Morrison 1999; Roe et al. 2002).
Although precise mechanisms underlying mutant SOD toxicity in ALS remain unclear, among the major mechanisms underlying neurodegeneration, oxidative stress have been implicated in disease pathogenesis (Cluskey and Ramsden 2001). Thus, in the present study we further established the occurrence of oxidative unbalance in neuroblastoma cells carrying the G93A Cu,Zn SOD mutant and we dissected the molecular mechanisms activated by the cells to retain viability. We found that protein oxidation and proteasome activity were significantly augmented and that the increase in proteasome activity was mandatory to prevent cell death, as its inhibition by lactacystin or MG132 resulted in apoptosis. Moreover, by preventing ROS propagation, we evidenced an inhibition in carbonyls formation and a decrement in proteasome activity. A down-regulation of neuronal nitric oxide synthase (nNOS) in terms of protein content and NO production was also evidenced, that in G93A cell model might be regarded as a beneficial factor.
Materials and methods
HEPES, CHAPS [3-(cholamidopropyl)dimethylammonio-1-propanesulfonate], EGTA, Protease Inhibitor Cocktail, dithiothreitol, propidium iodide, NBT (nitro blue tetrazolium), monoclonal anti-catalase, monoclonal anti-actin were obtained from SIGMA Chemical Co. (St. Louis, MO, USA). Supersignal substrate chemiluminescence reagent was from Pierce (Rockford, IL, USA). NPA (Nω-propyl-l-arginine), suc-LLVY-AMC, Z-ARR-AMC, Z-VF-CHO, suc-LY-AMC, Lactacystin, MG132, DAF-2/DA (4,5-diaminofluorescein diacetate), Lipid Peroxidation Assay Kit were from Calbiochem-Novabiochem Corp. (La Jolla, CA, USA). Oxyblot was from Intergen (Purchase, NY, USA). Monoclonal anti-nNOS antibody was from Transduction Laboratories (Lexington, KY, USA). Polyclonal nNOS antibody was purchased from Biomol Research Laboratories (Plymouth Meeting, PA, USA). Polyclonal anti-Cu,Zn SOD was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Secondary antibodies were obtained from Bio-Rad Laboratory (Hercules, CA, USA). Anti-129S S2 subunit polyclonal antibody was from Alexis Biochemicals (Carlsbad, CA, USA). DMPO: 5,5′-dimethyl-1-pyrroline-N-oxide was obtained from Fluka-SIGMA-Aldrich (Milan, Italy). All other chemicals were obtained from Merck (Darmstadt, Germany).
Human neuroblastoma cells (SH-SY5Y) were purchased from the European Collection of Cell Culture and grown in Dulbecco's modified Eagle's/F12 medium supplemented with 15% fetal bovine serum, at 37°C in an atmosphere of 5% CO2 in air. Confluent monostrates were routinely trypsinized and plated at 2 × 105/mL. Experiments were performed on confluent monolayers unless otherwise stated. Monoclonal cell lines transfected with the mutated Cu,Zn SOD G93A (named G93A) and wild-type Cu,Zn SOD (named WT) were obtained as described previously (Carrìet al. 1997). Treatment with DMPO or 7-nitroindazole (7-NI) was performed with concentrations of 25 mm (Liu et al. 2002) and 0.1 mm, respectively, which under our experimental conditions do not result to be toxic. They were added 30 min before the addition of other reagents and maintained throughout the experiments.
Proteasome inhibition and viability assay
Cells were seeded at 2 × 105/mL and treated 24 h later with lactacystin (1 µm) or MG132 (1 µm) for additional 24 h. This concentration was selected on the basis of an adequate proteasome inhibition, ubiquitinated proteins accumulation and because it was not excessively toxic for the three cell lines used. The extent of apoptosis was determined by a cytofluorimetric analysis. Briefly, cells were collected and washed in phosphate-buffered saline and resuspended in solution containing 50 µg/mL of propidium iodide, 0.1% sodium citrate and 0.1% Triton X-100, incubated in the dark at 4°C for 1 h and analysed by a FACScan instrument (Becton and Dickinson, San Josè, CA, USA).
Measurement of Cu,Zn SOD activity
Cells were washed and collected by centrifugation, resuspended in phosphate-buffered saline and sonicated. Lysates were centrifuged at 22 300 g for 20 min at 4°C. The activity was measured on supernatants by a polarographic method (Rigo et al. 1975) with an AMEL (Milano, Italy) model 466 analyser, in tetraborate buffer at pH 9.6, conditions that did not allow detection of Mn SOD activity (Steinkuhler et al. 1991). Data were expressed as µg/mg protein with reference to purified human Cu,Zn SOD. Activity was also evidenced on non-denaturing 7.5% polyacrylamide gels, by loading 50 µg of supernatants proteins. After electrophoresis, the gel was incubated in NBT solution (2.5 mm) for 30 min in the dark with gentle shaking, followed by 30 min incubation with a solution containing 30 mm tetramethylenediamine and 10 µg/mL riboflavin. Cu,Zn SOD activity was detected as the achromatic band on the violet-coloured gel, obtained after light exposure (Beauchamp and Fridovich 1971).
Measurement of oxidative damage
Carbonylated proteins were detected using the Oxyblot Kit. Briefly, 20 µg of proteins were reacted with 2,4-dinitrophenylhydrazine (DNP) for 15 min at 25°C. Samples were resolved on 12% SDS-polyacrylamide gels and DNP-derivatized proteins were identified by immunoblot using an anti-DNP antibody. Non-derivatized samples were used as negative controls. Data are presented as a representative immunoblot of three giving similar results.
Levels of malondialdehyde (MDA) and 4-hydroxy-2(E)-nonenal (4-HNE) were measured by a colorimetric method using the Lipid Peroxidation Assay Kit according to the manufacturer's instructions. In brief, cells were lysated by cycles of freeze and thaw in distilled water, incubated with the appropriate reagents for 45 min and clarified by centrifugation. Lipid peroxidation was expressed as µmol MDA + 4-HNE/mg protein determined by standard curves obtained with known amounts of MDA and 4-HNE.
Measurement of NO production
Monostrates were washed and incubated for 5 h in Hank's balanced salt solution containing 0.3 mm arginine. The formation of nitrites and nitrates in culture medium was measured by the Griess reaction, according to Schmidt (Schmidt et al. 1996). Concentration of nitrites was determined by a standard curve obtained with known amount of sodium nitrite and expressed as µmol/mg protein. NO production was alternatively detected according to Lopez-Figueroa et al. (2001). Briefly, cells were incubated with 2.5 µm of cell permeant selective NO indicator DAF-2/DA for 30 min at 37°C. The cells were then washed in phosphate-buffered saline and prepared for fluorescent microscopic analyses. As negative controls, cells were incubated with the nNOS inhibitor NPA (1 mm) for 1 h prior DAF-2/DA staining. Images of cells were rapidly digitized with a Cool Snap video camera connected to Nikon Eclipse TE200 epifluorescence microscopy. All of the images were captured under constant exposure time, gain and offset.
Immunoblots, dot blots and immunofluorescence analyses
Protein extracts were obtained by disrupting cells with 30 min incubation on ice in 50 mm Tris-HCl pH 7.4, 1 mm EDTA, 1 mm EGTA, 0.1% Triton X-100 and protease inhibitors cocktail and by centrifugation at 22 300 g for 20 min. Proteins were electroforesed on sodium dodecyl sulfate–polyacrylamide gels and blotted onto nitrocellulose membrane. To detect nNOS 50 µg of proteins were loaded onto 7.5% polyacrylamide gels and a monoclonal antibody against C-terminal region of nNOS was used. Alternatively, upon lactacystin treatment, we used a polyclonal antibody against N-terminal and C-terminal regions of nNOS because it has been suggested that C-terminal nNOS epitopes may be masked by ubiquitination. Immunoblots were also performed to detect: nitrosylated proteins (50 µg of proteins, 12% sodium dodecyl sulfate–polyacrylamide gels), Cu,Zn SOD (50 µg of proteins, 12% sodium dodecyl sulfate–polyacrylamide gels), 19S S2 proteasome subunit (50 µg of proteins, 7.5% sodium dodecyl sulfate–polyacrylamide gels) with the respective polyclonal antibodies, catalase (50 µg of proteins, 12% sodium dodecyl sulfate–polyacrylamide gels), actin (50 µg of proteins, 12% sodium dodecyl sulfate–polyacrylamide gels) and ubiquitinated proteins (10 µg of proteins, 12% sodium dodecyl sulfate–polyacrylamide gels) with the respective monoclonal antibodies. After incubation with the appropriate horseradish peroxidase-conjugated secondary antibodies, proteins were detected by an enhanced chemiluminescence kit. Densitometric analyses of protein bands were performed by the Softwer Gel Pro Analyser and protein levels were normalized to the density of related actin bands. Alternatively, ubiquitinated proteins were determined by dot blot after filtering lysates to remove free ubiquitin (Centricon, MW cut-off 10 K) and nitrosylated proteins by immunofluorescence analysis on 4% paraformaldeyde-fixed cells after staining with anti-nitrotyrosine and rhodamine-conjugated antibodies. Data are presented as typical experiment out of four that gave similar results (five immunoblots were performed for nNOS).
Determination of proteasome and calpain activity
Cells were washed with phosphate-buffered saline and lysates in 100 mm HEPES, 10% sucrose and 0.1% CHAPS. Then, 50 µL of lysates were incubated for 30 min at 37°C in 150 µL of 5 mm MgCl2, 50 mm Tris-HCl pH 7.8, 20 mm KCl, 5 mm MgOAc, 10 mm dithiothreitol. Specific proteasome substrates (50 µm), Suc-LLVY-AMC or Z-ARR-AMC, which detect, respectively, trypsin- and chymotrypsin-like activities of proteasome, were added. The reaction was stopped by addition of 200 µL of cold ethanol. Calpain I and II activity was detected using the specific fluorogenic substrate suc-LY-AMC. To assess the specificity of substrates, lactacystin (10 µm) or Z-VF-CHO (50 µm) were used to inhibit proteasome or calpain activity, respectively. The cleavage of substrates was monitored fluorimetrically using a Perkin-Elmer luminescence spectrometer LS-5 at 460 nm (excitation at 380 nm). Data were expressed as arbitrary units of fluorescence/mg proteins.
The results are presented as means ± SD. Statistical evaluation was conducted by anova, followed by the post-hoc Student–Newman–Keuls. Comparisons were considered to be significant at p < 0.05.
Oxidative damage to proteins in G93A cells
We previously reported that SH-SY5Y neuroblastoma cells transfected with G93A Cu,Zn SOD mutant (G93A cells) had an increased ROS production (Ciriolo et al. 2000) despite a fully active SOD activity (Fig. 1a). Here we further established the occurrence of oxidative unbalance by measuring some molecular targets of ROS. Under oxidative unbalance, proteins are susceptible to oxidation that can result in the formation of protein carbonyls. Monolayers of SH-SY5Y, G93A and WT cells were utilized as described under ‘Material and methods’; protein carbonyls were then derivatized by DNP and detected by immunoblotting with an anti-DNP polyclonal antibody. Consistent with our previous results, G93A cells showed evidence of increased oxidative damage in that they exhibit a higher content and a different pattern of oxidized proteins with respect to the other cell lines (Fig. 1b). The blot showed in Fig. 1(b) is from a typical experiment out of five, giving comparable results.
Cell membrane represents another target of ROS because of the presence of polyunsaturated phospholipids undergoing peroxidation. We determined the presence of MDA and 4-HNE that are end products of lipid peroxidation. These derivatives were analysed in SH-SY5Y, G93A and WT cells by a colorimetric lipid peroxidation assay kit. Very low levels of peroxides were found and no changes were observed in the different cell lines that gave an average value of 0.077 ± 0.012 µmol/mg protein of MDA + 4-HNE. This result was in agreement with G93A cells viability, assessed by Trypan blue exclusion, which did not differ from the other cell lines.
NO-mediated damage is not observed in G93A cells
RNS, derived from NO metabolism, may strengthen the ROS-mediated oxidative damage because they can interact with ROS, leading to highly reactive radical species (Beckman 1996). To address whether NO-mediated damage was also involved in this experimental model, we determined the levels of nitrite and nitrate that are considered stable oxidized products of NO. Culture medium were withdrawn and cells were incubated for 5 h with Hank's balanced salt solution in the presence of 0.3 mm arginine. During this incubation time, no changes were observed either in the morphology or in cell viability upon staining with the vital dye Trypan blue. Figure 2(a) shows that SH-SY5Y and WT cells had comparable levels of nitrite and nitrate. Surprisingly, we found a significant decrease of nitrite and nitrate levels in G93A cells (**p < 0.001, n = 8). These results were also confirmed by using a more sensitive method that directly evaluates in vivo production of NO (Brown et al. 1999). This assay was based on the usage of the cell permeant DAF-2/DA, which, by trapping NO, produces a green fluorescent signal. Following incubation of cells with DAF-2/DA the green-fluorescent signal that corresponds to basal production of NO was observed in the three cell types (Fig. 2b, upper panels). DAF-2/DA analysis revealed that NO production was significantly decreased in G93A cells, whereas no changes were observed in WT with respect to SH-SY5Y cells. The specificity of the reaction between NO and DAF-2 was demonstrated by almost undetectable fluorescence signal in cells incubated with 1 mm NPA, which is a strong inhibitor of nNOS activity (Fig. 2b, bottom panels). Moreover, nitrosylated proteins, which represent a biomarker of the attack of RNS upon proteins, were not detected under our experimental conditions, either by Western blot or by immunofluorescence analyses (data not shown).
The decrease in NO production observed in G93A cells prompted us to evaluate the expression level of nNOS. Western blot analysis was performed as described under the ‘Material and methods’ section and nNOS was evidenced using a monoclonal antibody that recognizes its C-terminal region. As shown in Fig. 2(c), nNOS protein level was significantly decreased in G93A cells (− 68% ± 5); on the contrary, a slight increase in the protein content was detected in WT cells (+ 25% ± 6). The blot showed in Fig. 2(c) represents one of five separate experiments that gave comparable results.
Proteasome activity is fundamental for survival of G93A cells
In eukaryotic cells the ubiquitin–proteasome system represents both one of the mechanisms through which protein turnover is regulated and the first line of defence against protein oxidation. In fact, under mild oxidative conditions proteasome can be highly activated to selectively recognize and degrade the ubiquitinated damaged proteins (Strack et al. 1996; Reinheckel et al. 1998). To assess whether proteasome was involved in the degradation of oxidized proteins and consequently in the protection from oxidative stress in G93A cells, we measured its trypsin- and chymotrypsin-like activities following the cleavage of the fluorogenic peptides Z-ARR-AMC and suc-LLVY-AMC, respectively. As shown in Figs 3(a) and (b), we evidenced a significantly higher activity of the proteasome in G93A cells with respect to the other cell lines (**p < 0.001, n = 6). Moreover, WT cells showed a significant decrease in proteasome activity with respect to SH-SY5Y cells (trypsin-like activity, **p < 0.001; chymotrypsin-like activity, *p < 0.05). The specificity of fluorescent substrates for proteasome activity was determined using the selective proteasome inhibitor lactacystin. Addition of 10 µm lactacystin highly inhibited proteasome activities by almost 90% (Figs 3a and b). Moreover, a Western blot analysis was performed to detect the content of the regulatory 19S S2 subunit of the proteasome involved in the recognition of ubiquitinated proteins. Figure 3(c) shows that the levels of this protein did not change in the three different cell lines, suggesting comparable proteasome efficiency.
Calpains, together with the proteasome system, are involved in protein degradation and modulation of their turnover. They are a family of calcium-dependent thiol proteases and operate in a reduced environment. For this reason, these enzymes have been postulated to be inactivated by oxidative stress. We examined the activity of calpain I and II by a fluorimetric method following the proteolytic cleavage of the fluorescent substrate suc-LY-AMC. Figure 3(d) shows that the oxidative stress operative in G93A cells did not affect calpain activity. In fact, calpain activity in G93A cells was identical to that of SH-SY5Y cells, whereas a slightly increase was detected in WT cells (*p < 0.05 vs. SH-SY5Y cells). The specificity of the proteolytic attack was determined by performing the calpain activity in the presence of its inhibitor Z-VF-CHO (Fig. 3d).
In order to analyse further the role played by proteasome in G93A cells, we examined the effects of proteasome inhibition on neuroblastoma cell viability upon treatment with 1 µm lactacystin for 24 h. As markers of proteasome inhibition, we followed by dot blotting the accumulation of ubiquitinated proteins (free ubiquitin was removed prior to measurement using a 10-K MW cut-off filter). Ubiquitinated proteins were detectable in untreated cells, and treatment with lactacystin raised their levels to comparable extents in the three different cell lines (Fig. 4a). This was confirmed by Western blotting (a representative blot after treatment with lactacystin is reported in Fig. 4b). Furthermore, protein carbonyls were highly increased upon lactacystin treatment, both in SH-SY5Y and G93A cells (Fig. 4c), confirming the role played by proteasome in removing oxidized proteins. Despite the similar pattern and levels of ubiquitinated proteins, cells respond to lactacystin treatment in a very different manner. In fact, G93A cells were committed to programmed cell death, as demonstrated by cytofluorimetric analysis, that accounted for 63 ± 7% after 24 h of treatment [Fig. 5a(2)]. On the contrary, the viability of both SH-SY5Y and WT cells were almost unaffected [Fig. 5a(1 and 3)]. Higher concentration of lactacystin (10 µm) results in induction of apoptosis to a comparable extent in the three lines used (data not shown). In order to confirm that the effects of lactacystin involve the inactivation of proteasome, another inhibitor, MG132 was also used. As expected, treatment with 1 µm MG132 for 24 h resulted in accumulation of ubiquitinated proteins (Fig. 4a) and in apoptotic cell death especially for G93A cells (Fig. 5b). Moreover, the extent of apoptosis was comparable to that observed upon lactacystin treatment. As result of proteasome inhibition an increase in lipid peroxidation was detected in SH-SY5Y cells (n = 5, *p = 0.01) and at higher extent in G93A cells (n = 5, **p < 0.001) (Fig. 4d).
Proteasome inhibition affects nNOS and Cu,Zn SOD turnover
Recent studies suggest that turnover of NOS can be regulated by the proteasome system (Bender et al. 2000; Noguchi et al. 2000; Musial and Eissa 2001). To assess whether the decrease in nNOS protein level was related to the higher proteasome activity in G93A cells, we performed Western blot analyses of cell lysates after proteasome inhibition with 1 µm lactacystin. Figure 6(a) shows the amount of nNOS protein after lactacystin treatment. In particular, in both SH-SY5Y and G93A cells, bands at higher molecular weights than 150 kDa, which are typical of ubiquitinated-nNOS accumulation, appeared. Moreover, we found that nNOS levels were affected differently in the three cell lines used (SH-SY5Y 2.71 ± 0.25 fold; G93A 4.34 ± 0.35 fold; WT 0.88 ± 0.09 fold, n = 4), with G93A having the higher rate of accumulation and WT nNOS being unaffected. Proteasome inhibition resulted in Cu,Zn SOD accumulation (Fig. 6a), indicating that Cu,Zn SOD turnover also may be regulated by the proteasome, as suggested by Lee et al. (2001). In addition, the Cu,Zn SOD increase was proportional to the basal level of the protein in the three different cell lines (SH-SY5Y 1.53 ± 0.15 fold; G93A 1.30 ± 0.12 fold; WT 1.50 ± 0.14 fold, n = 4), indicating a similar Cu,Zn SOD degradation by the proteasome. The specificity of the proteasome system for particular substrates was also investigated by additional experiments demonstrating that the levels of another antioxidant enzyme, such as catalase, were not altered upon treatment with lactacystin (Fig. 6a).
In order to investigate whether the observed rise in both nNOS and Cu,Zn SOD protein levels upon lactacystin treatment corresponds to a parallel increase in their activities, we measured nitrite and nitrate contents and the rate of superoxide dismutation. As shown in Fig. 6(b), the activity of Cu,Zn SOD was unchanged after lactacystin treatment, whereas an increase in the content of nitrite and nitrate was observed only in G93A cells (n = 8, **p < 0.001) (Fig. 6c). This increase in NO production may be responsible, at least in part, for the observed susceptibility of G93A cells to proteasome inhibition. Thus, we followed the effect of lactacystin treatment on cell death upon nNOS inhibition. Figure 6(d) shows that apoptotic cell death was significantly decreased in the presence of 0.1 mm 7-NI, a specific nNOS inhibitor (n = 4, **p < 0.001).
The spin-trap DMPO reduces G93A Cu,Zn SOD-mediated oxidative damage
It has been suggested that increased oxygen free-radical production by mutant Cu,Zn SOD contributes significantly to the motor neurone death in ALS (Cluskey and Ramsden 2001). Therefore, spin trapping molecules could prevent mutant Cu,Zn SOD-mediated oxidative damage because these compounds react efficiently with oxygen free radicals, preventing their propagation. We hence tested whether DMPO, a highly effective spin trapping compound, could prevent protein carbonylation and the increase in proteasome activity in G93A cell culture system. Figure 7(a and b) shows that both protein carbonylation and chymotrypsin-like proteasome activity (n = 6, **p < 0.001) were significantly reduced upon treatment with 25 mm DMPO. This effect results in a considerable decrease of cell death upon incubation of G93A cells with 1 µm lactacystin (n = 4, **p < 0.001) (Fig. 7c).
There is growing evidence for a potential role of ROS in acute neurological events and chronic neurodegenerative diseases. In particular, considerable experimental evidence implicates oxidative damage as a relatively early event in patients affected by ALS, a neurodegenerative disorder where a percentage of the familial forms is associated with mutations in Cu,Zn SOD gene. Although, the precise molecular pathways that ultimately lead to motorneurones degeneration and death in ALS remain unknown, research studies point to abnormal Cu,Zn SOD protein aggregation, enhancement of protein nitrosylation by mutant Cu,Zn SOD, enhanced peroxidase activity, exposure of the toxic copper at the active site, disorganization of intermediate neurofilaments, glutamate-mediated excitotoxicity and abnormal regulation of intracellular calcium (Morrison and Morrison 1999). In particular, it is widely accepted that mutated Cu,Zn SODs exert their noxious effects by a gain of novel toxic properties, involving unbalance of ROS (Wiedau-Pazos et al. 1996; Yim et al. 1996; Roe et al. 2002).
Previously we demonstrated that SH-SY5Y neuroblastoma cells transfected with the G93A Cu,Zn SOD mutant, associated with severe form of familial ALS, were highly susceptible to NO-mediated apoptosis. The increased NO-toxicity was mediated by a persistent higher rate of ROS production and modulated by metal chelators (Ciriolo et al. 2000). In the present study we have extended our previous findings by examining the targets of oxidative stress in G93A cells under resting conditions. The data presented here demonstrate that the observed ROS unbalance results in significantly higher levels of protein carbonyls that are considered markers of oxidative damage to proteins, whilst other putative targets such as polyunsaturated lipids were not affected. Oxidatively damaged proteins are mainly cleared in eukaryotes via the proteasome, a large multicatalytic protease, recently involved in the regulation of crucial cellular processes as well as in a wide array of neurodegenerative disorders (Davies 2001; Ding and Keller 2001). The vast majority of known protein substrates of the proteasome must be modified by the covalent attachment of a polyubiquitin chain, which serves as a substrate-targeting and recognition signal for the proteasome. We propose that in G93A cells the increase in chymotrypsin- and trypsin-like activities of the proteasome is strictly related to oxidative stress in order to remove oxidatively damaged proteins. This assumption was nicely demonstrated by the experiments carried out in the presence of DMPO, a well-established spin-trapping agent, that by impeding free radicals-mediated protein carbonylation was able to decrease the activity of proteasome in G93A cells to values comparable to that observed in untransfected cells.
The proteasome system function can be regulated by altering levels of proteasome, proteasome regulatory proteins or proteins of the ubiquitin conjugation system (Fruh et al. 1994; De Martino and Slaughter 1999). Under our experimental conditions, the 19S S2 proteasome subunit was similarly expressed among the three different cell lines used, suggesting comparable proteasome efficiency in recognizing ubiquitinated proteins. Moreover, the ubiquitin conjugation system was not altered in G93A cells with respect to the other cell lines used, as demonstrated by Western and dot blot analyses of ubiquitinated proteins, either under basal conditions or in the presence of lactacystin.
Moreover, under our conditions, we did not evidence changes in calpain activity of G93A cells with respect to SH-SY5Y cells, whereas a small significant increase was observed in WT cells, which in turn possessed the lowest proteasome activity. Calpains are proteolytic enzymes that, as well as proteasome, have been postulated to play a role in many physiological processes (Lynch and Baudry 1987; Pontremoli and Melloni 1986) and disease states (Nixon et al. 1994; Blomgren et al. 1995; Gafni and Ellerby 2002). Interestingly, calpains can be modulated by redox state, because their activity decreases, in vitro, in the presence of oxidants (Guttmann et al. 1997). The reason for this relies on the presence of a cysteine residue in the catalytic site that has to be kept in the reduced state for proper calpain activity. In this context, the lack of sensitivity of calpain in G93A cells reinforces the role played by proteasome in cleaning up oxidatively damaged proteins. It has to be pointed out that in WT cells, although other various mechanisms have been left unexamined, the previously demonstrated low levels of ROS (Ciriolo et al. 2001) may be, at least in part, responsible for the regulation of the two proteolytic systems.
Recent studies have demonstrated that proteasome impairment may occur in several neurodegenerative diseases (Keller et al. 2000a; Lopez Salon et al. 2000; Ding and Keller 2001; McNaught and Jenner 2001) previously related to oxidative damage as well as during the ageing of the CNS (Keller et al. 2000b). Moreover, chemical inhibition of proteasome was sufficient to commit neuronal cells to death (Lee et al. 2001). Among the molecular mechanisms involved in proteasome activity modulation, oxidative stress plays a dual role: sustained oxidative insult is reported to inactivate proteasome by direct oxidation or by the formation of protein cross-linking and aggregates (Halliwell 2002); but on the other hand exposure to mild oxidative injury has been demonstrated to stimulate proteasome activity, presumably through favouring conformation changes in the 20S proteasome complex (Strack et al. 1996; Reinheckel et al. 1998). In our model, inhibition of proteasome by either lactacystin or MG132 results in the induction of programmed cell death, the extent of which was much higher in G93A cells than in the other two cell lines. It has to be noticed that induction of apoptosis was highly significant with 1 µm lactacystin; other authors report that the death process was detectable only upon treatment with 10 µm lactacystin (Lee et al. 2001; Noguchi et al. 2000). This reinforces the pivotal role played by proteasome in assuring cell viability of G93A cells under the mild oxidative unbalance. The proteasome inhibition also resulted in a significant increase in lipid peroxidation, which was higher in G93A cells; this could be regarded as the consequence of the excess of oxidatively modified proteins that overwhelms the cellular protein-buffer.
In our experimental conditions, the oxidative damage observed was not related to an increase in NO synthesis, as stated by either direct measurement of intracellular NO production or determination of extracellular nitrite and nitrate release. Moreover, in G93A cells the diminished production of NO finely correlates with the results obtained from nNOS analysis that evidenced a significant decrease in the expression levels of this protein. The observed changes represent a rare phenomenon for a constitutive enzyme such as nNOS. In fact, its modulation does not usually require alterations in the protein levels as described for the inducible form of NOS (iNOS) (Dawson and Snyder 1994). It has been reported that suicide inactivation of nNOS enhances the proteolytic degradation of the enzyme, in part due to the proteasome. Bender et al. (2000) also demonstrated that inhibition of the proteasome with lactacystin leads to the accumulation of nNOS and its conjugated forms with one or few ubiquitins attached. Ubiquitination of nNOS, as demonstrated for the iNOS isoform (Musial and Eissa 2001), has probably been involved in the regulated removal of non-functional enzyme. These findings are in agreement with our results that showed lower levels of nNOS protein in association with increased proteasome activity, which may be responsible for the rapid proteolytic turnover of nNOS. This hypothesis is supported by the experiments carried out in the presence of lactacystin that revealed a significantly stronger accumulation of nNOS in G93A cells. On the contrary, nNOS did not accumulate in WT cells, where the lowest proteasome activity was detected. Since it has been suggested that increased flux of ROS profoundly affects NOS activity in vitro (Kotsonis et al. 1999), we can speculate that the increased nNOS proteolysis observed in G93A cells may be the results of a twin mechanism: increased proteasome activation and nNOS inhibition, as consequences of ROS increase. This assumption is in line with the results obtained with WT cells that were characterized by low levels of ROS (Ciriolo et al. 2001), low proteasome activity and higher nNOS, which in turn seems to be unaffected by lactacystin treatment. It is worth noting that calpain activity was higher in WT cells compared with the other cell lines and that nNOS is a calpain-sensitive protein (Lainé and Ortiz de Montellano 1998); thus, we suggest that in these cells the lack of oxidative stress could switch the mechanism through which nNOS is proteolysed, although the details remain to be determined. Furthermore, G93A cells were found to be deficient in the activity of calcineurin due to direct oxidation by the G93A Cu,Zn SOD enzyme (Ferri et al. 2001). Calcineurin is a serine/threonine phosphatase involved in a wide range of cellular responses to calcium mobilizing signals, including NO signalling. It has been suggested that NOS is a calcineurin substrate, with calcineurin dephosphorylating NOS and increasing its catalytic activity (Dawson et al. 1993). Therefore, under our experimental conditions, calcineurin activity impairment may play a role in the process leading to nNOS inactivation and subsequent degradation.
Lactacystin treatment causes also Cu,Zn SOD accumulation, whereas no changes were observed in the levels of the antioxidant enzyme catalase according to the results obtained by Lee and coworkers (Lee et al. 2001). Under our experimental conditions, the increase in Cu,Zn SOD levels was proportional to the basal content of the enzyme present in the three cell lines, suggesting that its proteolytic turnover was neither influenced by the mutated protein nor by the increased proteasome activity.
The observed increase in nNOS and Cu,Zn SOD protein levels upon lactacystin treatment in G93A cells is reflected differently in their activities; in fact, although an increase was determined in the nitrite and nitrate levels, no changes were observed in the dismutase activity, as previously reported by Lee and coworkers (Lee et al. 2001). The increased flux of NO observed upon lactacystin treatment could be responsible, at least in part, for the observed increased susceptibility of G93A to undergo apoptosis under these conditions and for the partial recovery from apoptosis in the presence of DMPO. This assumption is validated by the experiment carried out in the presence of 7-NI, a specific inhibitor of nNOS, where we obtained a significant inhibition of the lactacystin-induced apoptotic cell death.
Finally, in the present report we demonstrated that the over-expression of fully active Cu,Zn SOD G93A mutant, which represents one of more than 90 different autosomal dominant mutations in Cu,Zn SOD of familial ALS, induces an oxidative stress which results in carbonylated proteins, a concomitant raise in proteasome activity and nNOS down-regulation. This condition ultimately leads to less synthesis of NO, which is necessary, especially in neurones, at physiological concentration in order to elicit or suppress apoptosis. We suggest that down-regulation of nNOS allows cells to survive in a condition of increased flux of ROS. Although the cascade of deleterious effects initiated by mutant SOD in our experimental model may not be completely representative of the situation in motor neurones, the alterations of these key molecular factors may underlie the pathogenesis of ALS and other neurodegenerative diseases in which oxidative stress is involved.
This work was partially supported by MURST and by CNR Special Projects.