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(The FASEB Journal. 2006;20:1683-1685.)
© 2006 FASEB

Mitochondrial damage due to SOD1 deficiency in SH-SY5Y neuroblastoma cells: a rationale for the redundancy of SOD1

Katia Aquilano, Paola Vigilanza, Giuseppe Rotilio and Maria Rosa Ciriolo1

Department of Biology, University of Rome "Tor Vergata," Rome, Italy

1Correspondence: Department of Biology, University of Rome "Tor Vergata," Via della Ricerca Scientifica, 1, Rome, 00133, Italy. E-mail: ciriolo{at}bio.uniroma2.it

ABSTRACT

Superoxide dismutases (SODs) represent the first line of defense against oxidative stress, which is considered an essential factor in several neurodegenerative diseases and aging. We investigated the role of the copper,zinc superoxide dismutase (SOD1) in the maintenance of intracellular redox homeostasis by analyzing the early effects of SOD1 down-regulation in SH-SY5Y neuroblastoma cells. Through the use of small interference RNA, SOD1 was efficiently down-regulated at 48 h after transfection without any significant effect on cell viability. The steady-state concentration of superoxide was significantly increased after 12 h, when SOD1 was only slightly decreased, and progressively returned to values close to those observed in control cells. The superoxide increase was buffered by the enhanced levels of antioxidant glutathione (GSH); however, GSH increase was not sufficient to avoid damage to proteins in terms of carbonyls. GSH-depleting agents, such as BSO or diamide, further increased protein damage and committed SOD1 deficient cells to death, confirming the pivotal role played by this antioxidant. Although SOD1 declined mostly in the cytosolic compartment, mitochondria were significantly affected with impairment of the mitochondrial transmembrane potential and a decrease in ATP production. Together with these effects carbonylation of mitochondrial proteins was detected and in particular a consistent carbonylation and decrease of the antiapoptotic protein Bcl-2. These conditions induced a high susceptibility of SOD1-depleted cells to treatment with the mitochondrial reactive oxygen species producing agent rotenone. Overall, the results demonstrate that loss of SOD1 leads to severe damage of mitochondria, suggesting an important biological role for this enzyme in the preservation of mitochondrial homeostasis. —Aquilano, K., Vigilanza, P., Rotilio, G., and Ciriolo, M. R. Mitochondrial damage due to SOD1 deficiency in SH-SY5Y neuroblastoma cells: a rationale for the redundancy of SOD1.


Key Words: oxidative stress • aging • Bcl-2 • neurodegeneration

SUPEROXIDE IS THE PRIMARY RADICAL among reactive oxygen species (ROS); the physiological value of its production is {approx}2% of the total oxygen consumed in the mitochondrial respiratory chain that represents the major cellular source of superoxide (1) . In the last years, it has become clear that this radical is not only a dangerous species but can be also part of the signaling network involved in the maintenance of cellular homeostasis (2) . For this reason, the production of superoxide has to be finely controlled, and its excess or deficit may negatively influence several physiological processes in the cell (2 , 3) .

Oxidative stress is a widely accepted concept that has been useful to explain, at least in part, several pathological processes, in particular in brain aging (4) and in a variety of neurodegenerative diseases including Parkinson’s, Alzheimer’s, and Huntington’s diseases, and amyotrophic lateral sclerosis (ALS; ref 4 5 6 7 ). Despite the growing support for the free radical theory of aging, direct evidence for it is still lacking, and it is unclear whether oxidative stress is the primary initiating event associated with neurodegeneration or a secondary effect related to other pathological pathways. However, mitochondrial dysfunction and inflammatory insults have emerged as major contributing factors in the aging process, while oxidative stress seems to be involved in the propagation of cellular injury leading to different damages observed in neurodegenerative diseases. A large body of recent work on oxidative stress-related pathologies points to mitochondrial impairment as a central causative factor. In fact, besides being a major source of ROS, mitochondria are also the main target of their harmful action. Decreased activity of specific complexes of the electron transport chain, increased oxidative damage, and altered activity of antioxidant defense enzymes have been shown in aging and neurodegenerative diseases (4) . Mitochondria actually possess an efficient and extensive defense system including two superoxide scavenging superoxide dismutases (SODs), namely the specific mitochondrial isoform, which contains manganese (SOD2) and is located at the matrix level, and a small fraction of the copper,zinc SOD (SOD1), which resides in the intermembrane space (8 , 9) .

Among SODs, SOD1 is the most abundant in the cell accounting for {approx}90% of the total SOD activity (10) . It is mainly present in the cytosol at high concentration (µM range) despite its very high catalytic efficiency (Kcat=109 M-1·s-1). The physiological role of this enzyme is still an open and debated question due to this apparent excess and to the evidence that its known substrate, superoxide, is present at very low concentration under unstressed conditions.

A huge amount of data demonstrate that SOD1 overexpression may protect against oxidative stress conditions and extends life span of some organisms (11 , 12) . However, higher levels of SOD1 are not always associated with beneficial effects. Indeed, an additional copy of sod1 gene, such as that found in Down’s syndrome, or overexpression of SOD1 in mice reduces life span and is associated with increased oxidative damage (13) . The controversial action of SOD1 has been also shown in studies carried out in cellular and animal models where a down-regulation of the enzyme was induced. In fact, chronic SOD1 depletion causes senescence in cultured fibroblasts and apoptotic cell death in HeLa cells (14) as well as in spinal neurons (15) , thus confirming the fundamental role played by this enzyme in ROS homeostasis. Although SOD2 knockout mice show a neonatal lethal oxidative stress phenotype (16) , SOD1 knockout mice display no remarkable somatic abnormalities in the development and the early adulthood even though they have a reduced life span (17) . Moreover, stress conditions after neuronal injury were detrimental for these animals (18) , and lack of SOD1 has been reported to lead to increased incidence of oxidative stress-related hepatocarcinogenesis (17) and increased apoptosis in liver during aging (19) and sterility (20) .

Little is known about the sequence of events occurring in the early stages of SOD1 deficiency, which cannot be studied with transgenic mouse models. In this study, we investigated the role of SOD1 in maintaining neuronal cell integrity under physiological concentrations of oxy-radicals particularly focusing on mitochondria. The deciphering of events that mediate the occurrence of oxidative stress involves either increasing ROS production or decreasing the antioxidant defense system. Here, we studied the effects on human neuroblastoma SH-SY5Y cells after SOD1 down-regulation induced by RNAi. This powerful gene-silencing tool allowed detecting, time dependently, the early changes in cellular homeostasis due to SOD1 decrement. Our data demonstrate that the gradual loss of SOD1 is efficiently sensed by the GSH redox system that counteracts the increase of superoxide concentration and prevents cell commitment to death. However, GSH rise is not sufficient to protect mitochondria where protein oxidation occurs to a higher extent than in the cytosol and where a rapid decrease of Bcl-2 content and impairment of ATP production was detected.

MATERIALS AND METHODS

Materials
MTS assay kit "CellTiter 96 AQueous One Solution Cell Proliferation Assay" was purchased from Promega (Madison, WI). Xanthine, xanthine oxidase, diamide, rotenone, protease inhibitor cocktail, protein A Sepharose, monoclonal anti-p53, anti-catalase, anti-SOD1, anti-Hsp60, anti-Bcl-2, and ATP bioluminescence detection kit were from Sigma (St. Louis, MO, USA). Polyclonal anti-SOD1 and SOD2 were from Santa Cruz Biotecnology (Santa Cruz, CA, USA). IgG (H+L)-HRP-conjugated goat antimouse and anti-rabbit secondary antibodies were from Bio-Rad Laboratories (Hercules, CA, USA). Oligofectamine was from Invitrogen (Carlsbad, CA, USA). Tetramethyl rhodamine ethyl ester (TMRE), 2'7'-diclorofluorescein diacetate (DCF-DA), and dihydroethidium (DHE) were from Molecular Probes (Eugene, OR, USA). siRNAs against SOD1 and Bcl-2 and scramble siRNA were from Dharmacon (Lafayette, CO, USA). Oxyblot kit was from Intergen (Purchase, NY, USA). All other chemicals were obtained from Merck (Darmstadt, Germany).

Cell cultures
SH-SY5Y neuroblastoma cells were purchased from the European Collection of Cell Cultures. CHP100 neuroblastoma cells were kindly donated by Prof. Spinedi (University of Rome Tor Vergata). SH-SY5Y and CHP100 cells were grown in Dulbecco’s modified Eagle’s medium-F12 and RPMI medium, respectively (Sigma), supplemented with 15% FCS (GIBCO, Carlsbad, CA, USA) and maintained at 37°C in an atmosphere of 5% CO2 in air. Cell proliferation was assayed by an MTS test according to the manufacturer’s instructions. Alternatively, viable cells were determined using a hemocytometer by counting the number of cells that excluded trypan blue dye.

Treatments
For production of superoxide, 36 h after transfection, cells were incubated with xanthine oxidase at concentration of 0.1 U/ml and 250 µM of xanthine was added and maintained for the next 12 h. Diamide was added 24 h after transfection at concentration of 100 µM and maintained for the next 24 h. BSO was used at concentration of 1 mM and added immediately after transfection and maintained throughout the experiment. Rotenone was added 24 h after transfection at concentration of 0.5 µM and maintained for the next 24 h.

Transfections
Twenty-four hours after plating, 50% confluent cells were transfected, with a 21-nucleotide siRNA duplex directed against the following SOD1 and Bcl-2 mRNA target sequences: 5'-AAGGCCUGCAUGGAUUCCAUG-3' (for human SOD1); 5'-ACACCAGAAUCAAGUGUUCCG-3' (for human Bcl-2). These sequences were determined to be unique to the human SOD1 and Bcl-2 genes by basic local alignment search tool search of GenBank database. Control cells were transfected with a scramble siRNA duplex, which does not present homology with any other human mRNAs. Transfections were performed using Oligofectamine reagent according to the manufacturer’s instructions. For time-course experiments, cells were transfected by elecroporation using a Gene Pulser xcell system (Bio-Rad) according to the manufacturer’s instructions and immediately seeded into fresh medium. The initial point considered for time-course experiments was 12 h since only at this time cells were completely attached to the flask. Times longer than 48 h were not considered as a starting recovery of the protein was detected on 60 h. Transfection efficiency of siRNA into SH-SY5Y cells was estimated by cotransfecting siRNAs with nonspecific rhodamine-conjugated oligonucleotides and found to be >80%.

Western blotting
Cell pellets were resuspended in lysis buffer containing 50 mM Tris/HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, and protease inhibitor cocktail. After 30 min incubation on ice, cell lysates were centrifuged at 22,300 g for 15 min. Then protein extracts of the supernatant were electrophoresed on 12% SDS-polyacrylamide gels and blotted onto nitrocellulose. Membranes were stained with monoclonal primary antibodies against p53, catalase, Hsp60 and Bcl-2, and polyclonal primary antibodies against SOD1 and SOD2. After incubation with the appropriate HRP-conjugated secondary antibodies, protein bands were detected using a Fluorchem Imaging system (Alpha Innotech, San Leandro, CA, USA) after incubation with ChemiGlow chemiluminescence substrate (Alpha Innotech).

Immunoprecipitation
Cells were lysed by incubation for 5 min in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 12 mM deoxycolic acid) containing proteases inhibitor cocktail. Lysates were centrifuged at 3500 g for 20 min, and 400 µg of protein extracts (300 µl) were incubated overnight at 4°C with monoclonal anti-SOD1 or anti-Bcl-2 antibodies (1 µg/ml). Antibody (Ab) was then precipitated by incubation for 30 min with 10 µl of protein A agarose. Beads were washed three times with RIPA buffer and boiled for 5 min in Laemmli sample buffer. Immunoprecipitates were loaded on 12% SDS-PAGE, and SOD1/Bcl-2 heterocomplex or carbonylated Bcl-2 was detected by Western blot using an anti-Bcl-2 or anti-DNP Ab (Intergen).

Determination of ROS and mitochondrial-related function
Superoxide and ROS were detected by cytofluorimetric analysis after incubation for 30 min at 37°C with 5 µM DHE or 50 µM DCF-DA, respectively. Mitochondrial transmembrane potential was determined by cytofluorimetric analysis after staining with TMRE at concentration of 200 nM for 30 min at 37°C. For cytofluorimetric analysis, cells were scraped, washed, and resuspended in PBS. The fluorescence intensities of 10,000 cells from each sample were analyzed by FACScalibur instrument (Beckton-Dickinson, San Josè, CA). Data were analyzed using the WinMDI 2.8 software.

ATP content was determined luminometrically using ATP Bioluminescence assay kit (Sigma) according to the provided protocol. Cell extracts were assayed for ATP content using the ATP dependency of the light-emitting luciferase-catalyzed oxidation of luciferin. ATP concentrations were calculated according to a standard curve.

Preparation of cell fractions
Cell fractions were obtained by incubating in ice 9 x 106 cells in 0.2 ml of a hypotonic medium containing 10 mM Tris/HCl pH 7.5, 15 mM MgCl2, 10 mM KCl and protease inhibitor cocktail. After 10 min on ice, equal volumes of a "mitochondrial" buffer containing 400 mM sucrose, 10 mM TES, 0.1 mM EGTA, and 2 µM DTT (pH 7.2) were added and cells were disrupted by 20 strokes in a glass dounce. Supernatant obtained after centrifugation at 900 g for 10 min was further centrifuged at 10,000 g for 10 min to separate mitochondria (pellet) from cytosolic fraction (supernatant). For protein determination, mitochondria were lysed in RIPA buffer.

Determination of protein oxidation
Carbonylated proteins were detected using the Oxyblot kit (Intergen). Briefly, 20 µg of proteins were reacted with 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 Ab.

Determination of GSH
Intracellular GSH was assayed by HPLC on formation of S-carboxymethyl derivatives of free thiols with iodoacetic acid, followed by the conversion of free amino groups to 2,4-dinitrophenyl derivatives by the reaction with 1-fluoro-2,4-dinitrobenzene as described previously (21) . Data are expressed as nanomoles of GSH per milligrams of protein.

Proteins were assayed by the method of Lowry (22) .

Statistical analysis
Results are means ± SD; n = 6 unless otherwise stated. Statistical evaluation was made using ANOVA, followed by the post hoc Student-Newman-Keuls. Differences were considered to be significant at P < 0.05.

RESULTS

SOD1 knockdown does not affect cell growth and viability
SH-SY5Y cells were transiently transfected with a siRNA against SOD1 (siSOD) to induce SOD1 RNAi. As controls, cells were transfected with a scramble siRNA (siScr), a sequence having no homology with any other human mRNAs. Western blot reported in Fig. 1 A shows that siRNA against SOD1 significantly down-regulated the concentration of the protein. In particular, we evidenced the highest value of decrease at 48 h after transfection; densitometric analyses of Western blot indicated that the decrease was 40 ± 2% at 24 h and 80 ± 5% at 48 h (Fig. 1A , right panel). A parallel decrease in SOD1 activity was also found (data not shown). To test whether SOD1 RNAi could affect other intracellular antioxidants, we also determined the expression levels of catalase and SOD2. Figure 1A shows that, during the experimental period examined, the content of these enzymes was not changed. Cell growth and viability were determined by two independent methods: an assay based on the reaction of a tetrazolium salt (MTS) to a soluble formazan, which is a measure of the efficiency of intracellular dehydrogenases, and direct cell counting by trypan blue exclusion. Both analyses revealed that siSOD cells were not affected in cell growth and viability with respect to siScr cells (Fig. 1B, C ) and normal SH-SY5Y cells (data not shown). Moreover, a crucial biochemical marker of cell cycle regulation such as p53, the accumulation of which has been extensively demonstrated to be a marker of DNA damage and to cause cell growth arrest, did not show any significant change, confirming the unaltered cell cycle progression of siSOD cells (Fig. 1D ).


Figure 1
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  		Figure 1. SOD1 down-regulation does not affect cell viability under basal conditions. A) SH-SY5Y cells were transfected with siRNA against SOD1 (siSOD) or with scramble siRNA (siScr). After 24 and 48 h, total protein extracts were used for Western blot analysis: SOD1 (3 µg), SOD2 (30 µg) and catalase (3 µg). {alpha}-Tubulin was used as loading control. Right panel) Densitometric analysis of SOD1 was calculated using Quantitiy One Software (Bio-Rad), and data are shown as ratio of SOD1/{alpha}-tubulin (bars, mean±SD; n=10, *P<0.001). B) Cell proliferation of siSOD and siScr cells was assayed by the MTS test 24 and 48 h after transfection (bars, mean±SD; n=6). C) Viable cells number of siSOD and siScr cells was determined by trypan blue exclusion 48 h after transfection (bars, mean±SD; n=6). D) Total protein extracts were used for Western blot analysis of p53 (30 µg) 48 h after transfection. {alpha}-Tubulin was used as loading control. Immunoblot represents 3 with similar results. E) 36 h after transfection, siSOD and siScr cells were treated with xanthine/xanthine oxidase (X/XO) for 12 h as described in Materials and Methods. Cell viability was assessed by trypan blue exclusion (bars, mean±SD; n=5; *P<0.001).

To test whether SOD1 shortage could instead affect cell viability under an oxidative burst, we treated siSOD cells with externally produced superoxide. To this end, 36 after transfection, xanthine/xanthine oxidase were added to the culture medium as described in Materials and Methods and maintained for the next 12 h. Figure 1E shows that siSOD cells were extremely susceptible to death (74±6%) with respect to siScr cells (25±5%), confirming the pivotal role played by SOD1 against a pathological burst of superoxide.

SOD1 knockdown affects ROS production and induces a rise in GSH
SOD1 down-regulation could result in increased possibility for superoxide to accumulate intracellularly and/or increased superoxide-derived damage. To address these hypotheses, we measured the concentration of ROS production after SOD1 depletion, using cytofluorimetric analyses after incubation with either DHE or DCF-DA, the former being a more specific probe for superoxide, the latter being a widespread probe for ROS detection. Whereas DCF analysis did not evidence any variations in the concentration of ROS (data not shown), DHE staining indicated that 48 h after transfection, although a high value of SOD1 decrease was reached, the flux of intracellular superoxide only showed a slight (5.0±0.2%) but reproducible raise (Fig. 2 A) that was significantly different from siScr cells (n=10; P<0.05).


Figure 2
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  		Figure 2. SOD1 down-regulation affects superoxide production and increases GSH intracellular levels. A) SH-SY5Y cells were transfected with siSOD or with siScr. After 48 h, concentration of intracellular superoxide was determined by cytofluorimetric analyses after incubation with DHE. Histogram represents 10 that gave similar results. Increase of superoxide flux was equal to 5 ± 0.2%; n = 10; P < 0.05. B) 48 h after transfection, siSOD and siScr cells were prepared for GSH/GSSG assay by HPLC. Data are expressed as nmol of GSH or GSSG /mg total protein (bars, mean±SD; n=6; **P<0.001). SOD1 content was determined as reported in Fig. 1 A legend. C) CHP100 cells were transfected with siRNA against SOD1 (siSOD) or with a scramble siRNA (siScr). After 48 h, cells were prepared for GSH/GSSG assay by HPLC. Data are expressed as nmol of GSH or GSSG/mg total protein (bars, mean±SD; n=4; **P<0.001). SOD1 content was determined at 48 h by Western blot analysis on total protein extracts (3 µg). Immunoblot represents 3 with similar results. {alpha}-Tubulin was used as loading control.

We previously demonstrated that under impairment of SOD1 activity, GSH, the most important low molecular weight antioxidant, was significantly higher in neuroblastoma cells (23) . To test whether under SOD1 deficiency GSH could be induced to buffer superoxide production, we determined its intracellular concentration by HPLC analysis. As shown in Fig. 2B , 48 h after transfection we found a significant higher amount (47±2%) of the tripeptide in siSOD cells compared with siScr cells. To establish a role for GSH in counteracting superoxide flux and superoxide-mediated damage and to demonstrate that this phenomenon was not due to a mere consequence of a cell-type related effect, we analyzed GSH in another human neuroblastoma cell line (CHP100). As shown in Fig. 2C , in these cells GSH concentration also significantly increased (45±5%) during SOD1 down-regulation. In both cell lines, the increased concentration of GSH was not paralleled by a change in the levels of its oxidized form GSSG (Fig. 2B, C ).

To further confirm the protective role of GSH, we carried out experiments with two different GSH depleting agents. BSO, a specific inhibitor of {gamma}-glutamyl cysteine synthetase, the rate-limiting enzyme of GSH synthesis, was added after transfection to culture medium at concentration of 1 mM and maintained throughout the experiment. Diamide, a cell-permeable compound, able to efficiently oxidize GSH, was added to culture medium 24 h after transfection at a concentration of 100 µM and maintained throughout the experiment. Under our experimental conditions, BSO did not affect cell viability (data not shown), whereas diamide was significantly cytotoxic. Figure 3 A shows that more siSOD cells were killed (63±4%) than siScr cells (15±4%), demonstrating that rapid oxidation of available GSH is detrimental to cells deficient in SOD1. However, with the use of BSO treatment, we showed, by cytofluorimetric analyses, after staining with DCF-DA, a significant increase of ROS (Fig. 3B ), confirming the pivotal role played by GSH in scavenging ROS under physiological conditions. Moreover, siSOD displayed a larger increase in the concentration of ROS that could be ascribed to the lack of both antioxidants (Fig. 3B ).


Figure 3
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  		Figure 3. GSH depletion results in increased oxidative stress. A) SH-SY5Y cells were transfected with siSOD or with siScr; 24 h after transfection, diamide was added at concentration of 100 µM and maintained for 24 h. Viable cells number was determined by trypan blue exclusion (bars, mean±SD; n=6; *P<0.05, **P<0.001). B) After cell transfection, BSO was added at concentration of 1 mM and maintained for 48 h. ROS concentration was assayed by cytofluorimetric analysis after staining with DCF-DA. Histogram represents 3 with similar results. C) After cell transfection, BSO was added at concentration of 1 mM and maintained for 48 h. Protein extracts (20 µg) were then used for protein carbonyls analysis on derivatization with DNP followed by Western blot using an anti-DNP Ab. A representative immunoblot of 5 with similar results is shown. {alpha}-Tubulin was used as loading control. Right panel) Densitometric analysis of each lane was calculated using Quantity One Software (Bio-Rad), and data are shown as ratio of protein carbonyls/{alpha}-tubulin (bars, mean±SD; n=5; **P<0.001; ##P<0.001).

SOD1 knockdown is associated with increased protein carbonylation
Protein oxidation in cells is usually increased as a consequence of ROS production, as they can oxidize specific amino acids leading to the formation of oxidative modifications. Therefore, we tested the concentration of protein carbonyls, one of the most frequent oxidative modifications, of both untreated and BSO treated cells where a high increase in ROS production was determined. Forty-eight hours after transfection, cells were lysed and protein extracts were derivatized with DNP that specifically reacts with carbonyl groups. The immunoelectroforesis carried out with an anti-DNP Ab revealed that siSOD cells under basal conditions already possessed higher levels of protein carbonyls with respect to siScr cells (Fig. 3C ), suggesting that SOD1 deficiency affected protein integrity even in the presence of augmented intracellular levels of GSH. Moreover, BSO treatment gave rise to a further increase in protein carbonyls, especially in siSOD cells (Fig. 3C ). These results indicate the occurrence of an oxidative stress under SOD1 deficiency and, at the same time, reinforce the fundamental role played by GSH in buffering the superoxide-mediated damage.

SOD1 down-regulation induces mitochondrial impairment
Since SOD1 is present also in the mitochondria, we evaluated the concentration of SOD1 in both cytosolic and mitochondrial fractions after SOD1 RNAi. The immunoblot in Fig. 4 A shows that SOD1 was easily detectable in mitochondria extracts and that transfection with siSOD induced a decrease of SOD1 in both cytosol and mitochondria, although to a different extent. Densitometric analyses demonstrated that SOD1 was decreased 55 ± 6% in mitochondria with respect to 79 ± 5% in cytosol indicating a different turnover of SOD1 in the two compartments. Figure 4A also shows the levels of Hsp60 and Bcl-2 proteins. Although Hsp60 protein, a chaperone that is important for folding proteins after import into the mitochondria (24) , did not change in siSOD cells, the content of the antiapoptotic protein Bcl-2 in mitochondria was significantly reduced (60±4%).


Figure 4
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  		Figure 4. SOD1 deficiency induces Bcl-2 down-regulation and preferential mitochondrial protein carbonylation. A) SH-SY5Y cells were transfected with siSOD or with siScr; 48 h after transfection, mitochondrial and cytosolic extracts were used for Western blot analysis of Bcl-2 (20 µg) and SOD1 (3 µg). Hsp60 was used as loading control. A representative immunoblot of 5 with similar results is shown. Bottom panels) Densitometric analyses of SOD1 and Bcl-2 were calculated using Quantity One Software (Bio-Rad) and data are shown as arbitrary units (bars, mean±SD; n=5; *P<0.001). B) 48 h after transfection, mitochondrial (20 µg) and cytosolic extracts (20 µg) were prepared and used for protein carbonyls analysis on derivatization with DNP followed by Western blot using an anti-DNP Ab. Representative immunoblots of 3 with similar results are shown.

Mitochondria, being the major source of ROS, are also the preferential target of oxidative stress. We determined the effects of SOD1 down-regulation on mitochondrial fraction by evaluating the contribution of oxidized mitochondrial proteins to the observed increased concentration of carbonyls. Figure 4B shows that the carbonyl contents of mitochondria from siSOD cells were significantly increased 48 h after transfection. Moreover, our data showed that no changes in oxidized proteins could be detected in the cytosolic fraction (Fig. 4B ), indicating that oxidatively modified mitochondrial proteins could give a significant contribution to the increment of carbonyls observed in total proteins extracts.

It is well established that overexpression of Bcl-2 protects against induction of apoptosis mainly through a mechanism that, by enhancing H+ efflux, protects against collapse of mitochondrial transmembrane potential ({Delta}{psi}) under apoptotic stimuli (25) . As we detected a significant decrease in the amount of Bcl-2 protein under SOD1 deficiency, we inquired into the possible consequence on {Delta}{psi} by performing cytofluorimetric analyses after incubation of the cells with the fluorescent probe TMRE. As usually observed, TMRE staining allowed us identifying two distinct cell populations. Figure 5 A shows that a significant percentage of siSOD cell population shifted toward the lower levels of {Delta}{psi}, indicating that mitochondrial potential was altered in these cells. Since {Delta}{psi} is fundamental to phosphorylate ADP via the F1-F0 ATP synthase, we measured the ATP production by a luminometric method. Figure 5B shows that in concomitance with {Delta}{psi} impairment, we observed a significant decrease in ATP production 48 h after transfection (20±1%; n=10, *P=0.002).


Figure 5
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  		Figure 5. SOD1 down-regulation affects mitochondrial homeostasis. A) SH-SY5Y cells were transfected with siSOD or with siScr. After 48 h, cells were stained with 200 nM TMRE and used for cytofluorimetric analyses as described in Materials and Methods. Histograms represent 6 with similar results. B) 48 h after transfection siSOD and siScr cells were used for ATP measurement by a bioluminescence detection kit as described in Materials and Methods. Data are expressed as nmol/mg prot (bars, mean±SD; n=10; *P=0.002). C) 24 h after transfection, siSOD and siScr cells were treated with 0.5 µM rotenone for the next 24 h. Viable cells number was determined by trypan blue exclusion (bars, mean±SD; n=5; **P<0.001)

To confirm the occurrence of mitochondrial impairment under SOD1 deficiency, we treated cells with low dose of rotenone, a specific inhibitor of complex I of the electron transport chain. Twenty four hours after transfection, rotenone was added in culture medium at concentration of 0.5 µM and maintained for the next 24 h. As shown in Fig. 5C , the low dose of rotenone dramatically decreased cell viability only in siSOD cells (52±5%), which shows the importance of high intracellular concentration of SOD1 to preserve both mitochondrial integrity and cell viability.

Specificity and time dependency of the events occurring under SOD1 deficiency
To establish the sequence of events occurring under SOD1 down-regulation, we analyzed the previously reported effects at different time points after transfection. We first followed the decrease of SOD1 protein by Western blot analysis and found that it decreased gradually starting from 12 h (Fig. 6 A). Then, we analyzed superoxide production citofluorimetrically by DHE staining. The histograms reported in Fig. 6B show that a massive increase of superoxide flux was already detectable at 12 h, with a gradual decrease in the following time points (Fig. 6B ). We therefore evaluated the concentration of Bcl-2 protein content in total cell extracts. Figure 6A shows that Bcl-2 follows the same trend as SOD1 decrement: it was already decreased after 12 h and reached the highest decrement (60±4%) 48 h after transfection (Fig. 6A ). The measurement of GSH by HPLC analysis showed that its concentration followed the superoxide concentration, as a considerable raise was observed at 12 h with a progressive decrease up to 48 h (Fig. 6C ). These data are in agreement with a role for GSH as principal sensor of ROS production under SOD1 deficiency and at the same time could explain the low levels of superoxide observed at 48 h. As reported in Fig. 6C , {Delta}{Psi} loss was significant only after 36 h reaching the maximum value at 48 h. Figure 6C graphically summarizes all the effects induced by the time-dependent loss of SOD1 decrement.


Figure 6
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  		Figure 6. Time-dependent effects of SOD1 down-regulation. A) SH-SY5Y cells were transfected with siSOD or with siScr. After transfection, total protein extracts were used for Western blot analysis: SOD1 (3 µg) and Bcl-2 (20 µg). {alpha}-Tubulin was used as loading control. Lane 1: siScr at 12 h; lane 2: siSOD at 12 h, lane 3: siSOD at 24 h; lane 4: siSOD at 36 h; lane 5: siSOD at 48 h; lane 6: siScr at 48 h. Immunoblot represents 6 with similar results. B) After transfection of siRNA against SOD1, concentration of intracellular superoxide was determined by cytofluorimetric analyses after incubation with DHE. Histograms represent 6 with similar results. C) After transfection of siRNA against SOD1, the time-dependent changes of SOD1, Bcl-2, {Delta}{Psi}, GSH, superoxide were evaluated and expressed as percent of controls. SOD1 and Bcl-2 protein content were determined by Western blot and quantified by densitometric analysis using {alpha}-tubulin as loading control; GSH was measured by HPLC analysis; superoxide concentration and {Delta}{Psi} were determined cytofluorimetrically after staining with DHE and TMRE, respectively. All data reported were statistically significant, except values of {Delta}{Psi} at 12 and 24 h (n=6).

To discriminate which of the events analyzed were directly linked to SOD1 deficiency and/or to Bcl-2 decrease, SH-SY5Y cells were transiently transfected with a siRNA against Bcl-2 (siBcl2). Forty-eight hours after transfection, a decrement of Bcl-2 was observed (46±3%) without any effect on SOD1 protein content (Fig. 7 A). Cytofluorimetric analyses after TMRE staining showed a marked and significant loss of {Delta}{Psi} concomitant with Bcl-2 decrement (Fig. 7B ), confirming the importance of Bcl-2 in maintaining transmembrane potential. Moreover, in siBcl2 cells DHE staining showed no rise in superoxide flux as well as in carbonylated proteins (data not shown) confirming the specificity of SOD1 in mediating these effects.


Figure 7
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  		Figure 7. Relationship between SOD1 and Bcl-2 in SH-SY5Y cells. A) SH-SY5Y cells were transfected with siBcl2 or with siScr. After 48 h, total protein extracts were used for Western blot analysis: SOD1 (3 µg), Bcl-2 (20 µg). {alpha}-Tubulin was used as loading control. Immunoblots represent 3 with similar results. Right panel: densitometric analysis of each lane was calculated using Quantity One Software (Bio-Rad) and data are shown as ratio of Bcl-2/{alpha}-tubulin (bars, mean ±SD; n=3; *P <0.01). B) 48 h after transfection of siRNA against Bcl-2, cells were stained with 200 nM TMRE and used for cytofluorimetric analyses as described in Materials and Methods. Histograms represent 3 experiments with similar results. C) SH-SY5Y cells were lysed and protein extracts were immunoprecipitated using a monoclonal anti-SOD1 Ab as described in Materials and Methods. Bcl-2 and SOD1 were determined by Western blot analysis using a monoclonal or polyclonal antibody, respectively. Immunoblots represent experiments with similar results. D) SH-SY5Y cells were transfected with siSOD or with siScr; 48 h after transfection, total protein extracts were immunoprecipitated with an anti-Bcl-2 monoclonal antibody. Immunoprecipitates were then used for Bcl-2 carbonylation analysis on derivatization with DNP followed by Western blot using an anti-DNP Ab. A representative immunoblot of 3 with similar results is shown.

Bcl-2 decrement under SOD1 deficiency is associated with its carbonylation
The existence of a strict relationship between SOD1 and Bcl-2 has recently been demonstrated and seems to be exclusive for neuronal cells where they interact at the mitochondrial level (26) ; therefore, it was reasonable to postulate a crosstalk between these two proteins under our experimental conditions. To evaluate this hypothesis, we investigated on the possible direct interaction between SOD1 and Bcl-2 by immunoprecipitation analysis. Total protein extracts from SH-SY5Y cells were immunoprecipitated with an anti-SOD1 Ab and subsequently used for immunoelectrophoresis using an anti-Bcl-2 Ab. This analysis demonstrated that Bcl-2 and SOD1 form a stable heterocomplex in SH-SY5Y cells (Fig. 7C ). Moreover, it was recently shown that Bcl-2 under oxidative stress conditions can undergo carbonylation and that this damage precedes its down-regulation (27) . Since high levels of carbonylated mitochondrial proteins were detected in our model, we explored the possibility that Bcl-2 down-regulation could be due to oxidative modification of this protein. To this end, protein extracts were incubated with a monoclonal Bcl-2 Ab and the immunoprecipitates were derivatized with DNP. Western blot analysis carried out with a polyclonal anti-DNP Ab revealed an evident increase of carbonylated Bcl-2 protein in siSOD cells (Fig. 7D ), indicating that the decrease of Bcl-2, under our experimental conditions, could be due to increased degradation after carbonylation.

DISCUSSION

In recent years, there has been a growing interest in the role of mitochondria malfunction in the pathogenesis of neurodegenerative disorders and during aging (4) , not only because of bioenergetics failure but also because of their pivotal function in the intrinsic apoptotic processes (28) . Moreover, oxidative stress has been directly or indirectly implicated in such pathologies as important contributing factors (29) . Our data showed that despite the fact that RNAi caused a drasticdecrease of SOD1 ({approx}80% at 48 h), cell viability as well as cell cycle progression was not altered as demonstrated by the lack of variations in the number of viable cells and in the expression and activation level of p53, a transcription factor that efficiently senses early DNA damage. Therefore, SOD1 depletion allowed us to study the consequence of chronic decrement in dismutating activity without the accompanying apoptosis that usually occurs under more cytotoxic conditions (14 , 15) . As result of the gradual SOD1 decrement, we expected to observe a parallel increase in the steady-state concentration of superoxide and/or in its damaged targets. Intriguingly, superoxide increase occurred rapidly when the rate of SOD1 decrement was not still at the maximum level, but it was efficiently buffered at longer time points when SOD1 protein was massively reduced. These results indicated that 1) high levels of intracellular SOD1 are necessary for inhibiting the raise of superoxide; and 2) other antioxidant systems subsequently counteract superoxide increase. Taking into account that neither catalase nor SOD2 was changed under SOD1 depletion, we suggest that the observed increase in GSH content could represent the alternative buffering system for superoxide under physiological conditions. Moreover, the rise in GSH under SOD1 deficiency was not specific for SH-SY5Y cells but may represent a more general process to counteract superoxide increase. In fact, we demonstrated that another human neuroblastoma cell line (CHP100 cells), transfected with siRNA against SOD1, also displayed increased concentration of the tripeptide. The importance of GSH in compensating for SOD1 loss was clearly demonstrated by the experiments carried out in the presence of compounds able to modulate its intracellular concentration. In particular, diamide, a thiol oxidizing agent that promptly reacts with GSH, killed siSOD cells, while BSO, which inhibits GSH neosynthesis, caused a significant increase of ROS, thus resulting in protein carbonylation at higher degree in siSOD cells. These results point to an efficient capacity of GSH to scavenge ROS under SOD1 deficiency in neuronal cells. They are also in good agreement with our previous work where we demonstrated that stable expression of an inactive SOD1 mutant, in neuroblastoma cells, induced an increase in the concentration of intracellular GSH and that this event was critical for cell survival (23) . Therefore, we suggest that the absence of pathological phenotype in the early childhood of sod1–/– mice may depend on the occurrence of compensatory increases of antioxidants capable to scavenge the superoxide physiologically produced. In this context, it has recently been demonstrated that redox systems related to GSH are up-regulated in neurons that are less susceptible to death induced by SOD1 mutants typical of familial ALS (FALS; ref 30 ) and in liver of sod1–/– mice (31) . However, our results show that under SOD1 deficiency the rise in GSH was not sufficient to avoid superoxide-mediated damage to proteins. These data are further evidence for the unique role played by SOD1 in 1) avoiding side-chain reactions with proteins as consequence of the short diffusion distance of superoxide, and 2) buffering cytotoxic high flux of superoxide, such as that generated by the xanthine/xanthine oxidase system that could mimic some pathological conditions (inflammation, ischemia/reperfusion). Indeed, it has been demonstrated that in sod1–/– mice the lack of SOD1 was not detrimental unless stressful conditions, such as ischemia/reperfusion, were induced (18) .

Previous studies on the subcellular localization of SOD1 have provided contradictory results depending on the tissues studied (32) . Some authors claimed that it is localized in the intermembrane space (32 33 34) , while others reported the presence of this enzyme also in the mitochondrial matrix (35) . Liu et al. (36) reported that only FALS-linked SOD1 but not wild-type SOD1 is associated with mitochondria from spinal cord and brain in mice. The results obtained in the present report are in agreement with an association of SOD1 with mitochondria in cells of neuronal origin, since we found a significant amount of the protein in the mitochondrial fraction. Moreover, our data support the concept that SOD1 contributes to counteract oxidative-mediated damage to mitochondrial proteins. Under basal conditions, SH-SY5Y mitochondria already showed a high rate of protein oxidation consistent with their susceptibility to oxidative injury (37 , 38) . After SOD1 depletion, mitochondria underwent a further increase in protein oxidation that roughly accounted for the observed raise of protein carbonyls in total cell extracts. The importance of the presence of SOD1 in mitochondria is confirmed by the fact that, even though in these organelles SOD1 was decreased less with respect to the cytosolic counterpart, its deficiency affects mitochondrial rather than cytosolic proteins. Taking into account the cytosolic origin of mitochondrial SOD1, the diverse degree of its decrement may reflect a different turnover of SOD1 between the two compartments in terms of degradation. An interesting result is that the oxidative damage to mitochondria is not accompanied by an alteration of SOD2 content, thus reinforcing the hypothesis of the need for double site protection (matrix and intermembrane space) against superoxide in the mitochondrial compartment. It has been suggested that SOD1 located in the mitochondrial intermembrane space could play an important role also in protecting matrix proteins from superoxide-mediated damage by impeding the formation of protonated superoxide in the intermembrane space and its diffusion toward the matrix (39) . Moreover, O’Brien et al. (40) suggested that SOD2 and mitochondrial SOD1 have both unique and overlapping functions in protecting mitochondrial proteins from oxidative damage. The protective role of SOD1 against mitochondrial dysfunction has further been reinforced by Vijayvergiva et al. (35) , who demonstrated that SOD1 is located also in the matrix of brain mitochondria. Work is in progress in our laboratory to characterize the submitochondrial localization of SOD1 and the pattern of mitochondrial proteins that are susceptible to oxidation in the absence of SOD1.

Mitochondria plays a crucial role in apoptosis due to the presence in these organelles of several factors modulating the death process among which Bcl-2 represents the most important antiapoptotic protein. Previous studies demonstrated that Bcl-2 overexpression was able to counteract the apoptosis induced by SOD1 deprivation in PC12 cells (41) . Moreover, a relationship between SOD1 and Bcl-2 has emerged in studies carried out with cellular (42) and animal (43) models of FALS, where a protection by Bcl-2 against the toxicity of SOD1 mutants was demonstrated. A down-regulation of both SOD1 and Bcl-2 has also been demonstrated in lymphocytes from patients affected by sporadic ALS (44) . A direct interaction between these two proteins has been recently demonstrated in mitochondria where SOD1 binds to the cytosolic loop domain of Bcl-2. This interaction is enhanced with FALS-linked mutations of SOD1 where it results in aggregation with Bcl-2, thus inhibiting its antiapoptotic function leading the authors to suggest that SOD1 could be envisaged also as a genuine antiapoptotic factor besides being an antioxidant enzyme (26) . The interaction between Bcl-2 and SOD1 was also operative in our experimental model as demonstrated by the isolation of a stable SOD1/Bcl-2 heterocomplex and by the parallel decrease in the amount of Bcl-2 on SOD1 depletion. It has been reported that Bcl-2 under oxidative stress conditions can undergo carbonylation and that this phenomenon precedes its down-regulation (27) . On the basis of our results demonstrating a significantly augmented carbonylation of Bcl-2, we can suggest that the decrease in SOD1 and the corresponding increase in ROS flux could favor Bcl-2 oxidation thus leading to its degradation. This aspect is very intriguing and opens new perspectives on the role of SOD1 in preserving mitochondrial function both by preventing mitochondrial oxidative damage and by inhibiting Bcl-2 down-regulation. However, it requires further investigations, which are in progress in our laboratory.

The fact that Bcl-2 is localized in the mitochondrial membrane has generated considerable studies on the role played by mitochondria in apoptosis and on the ability of Bcl-2 to block apoptosis at the mitochondrial level (25) . It has been demonstrated that overexpression of Bcl-2 in neurons prevents the collapse of {Delta}{Psi}, which is an event underlying mitochondrial mediated-apoptosis (25) . Our data show that SOD1 decrement leads to a loss of mitochondrial {Delta}{Psi} and, concomitantly, reduces ATP production. Moreover, the data obtained after Bcl-2 knockdown by RNAi indicated that, among the events analyzed, only the loss of {Delta}{Psi} was concomitant to Bcl-2 decrement and not strictly related to SOD1 shortage. These results, while confirming a relationship between Bcl-2 down-regulation and impairment of {Delta}{Psi}, are not sufficient to explain the lack of induction of apoptosis in our experimental system. We also demonstrated that by increasing the mitochondrial superoxide production by rotenone, at the respiratory chain level, we observed cell death only in siSOD cells thus confirming the importance of SOD1 in protecting the cell against mitochondrial superoxide-mediated damage. At the same time, these data indicate the weakness of mitochondria under SOD1 depletion, which could represent one of the mechanisms in the processes leading to aging and/or to neurodegeneration.

Overall, the results indicate that under SOD1 deficiency, the increase in the superoxide level gives rise to an induction of GSH, which efficiently scavenges it allowing cell viability. However, SOD1 is fundamental to counteract the damaging effects of superoxide toward mitochondrial proteins and to preserve mitochondrial function through maintenance of Bcl-2 stability. The events observed in our experimental system correlate with the phenomena observed in studies carried out with animal or cellular models expressing mutant forms of SOD1 associated with familial FALS. In fact, it has been demonstrated that even small amounts of mutant SOD1 put motor neurons in a condition of oxidative stress and mitochondrial damage that cause cell vulnerability and death (45 46 47) . Therefore, the mitochondrial damage reported in this study let us to speculate that SOD1 deficiency or the "gain of function" typical of FALS mutants may result in the same detrimental effects. Our data open a new avenue of investigation into the role of SOD1 in the preservation of mitochondrial integrity taking into account its crosstalk with Bcl-2, not only in the diseases strictly related to SOD1 imbalance but also in other pathologies where mitochondrial impairment and/or oxidative stress are implicated.

ACKNOWLEDGMENTS

We thank Prof. J. Z. Pedersen for stimulating discussions and helpful advice. This work was partially supported by grants from FIRB, MIUR, and Ministero della Sanità "Progetto di Ricerca Finalizzata."

Received for publication October 14, 2005. Accepted for publication March 31, 2006.

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