Inactivation of cytochrome c oxidase by mutant SOD1s in mouse motoneuronal NSC-34 cells is independent from copper availability but is because of nitric oxide


Address correspondence and reprint requests to Prof. Luisa Rossi, Department of Biology, ‘Tor Vergata’, University of Rome, Via della Ricerca Scientifica, Rome 00133, Italy. E-mail:


J. Neurochem. (2010) 112, 183–192.


The copper-enzyme cytochrome c oxidase (Cytox) has been indicated as a primary molecular target of mutant copper, zinc superoxide dismutase (SOD1) in familial amyotrophic lateral sclerosis (fALS); however, the mechanism underlying its inactivation is still unclear. As the toxicity of mutant SOD1s could arise from their selective recruitment to mitochondria, it is conceivable that they might compete with Cytox for the mitochondrial copper pool causing Cytox inactivation. To investigate this issue, we used mouse motoneuronal neuroblastoma × spinal cord cell line-34, stably transfected for the inducible expression of low amounts of wild-type or mutant (G93A, H46R, and H80R) human SOD1s and compared the effects observed on Cytox with those obtained by copper depletion. We demonstrated that all mutants analyzed induced cell death and decreased the Cytox activity, but not the protein content of the Cytox subunit II, at difference with copper depletion that also affected subunit II protein. Copper supplementation did not counteract mutant hSOD1s toxicity. Otherwise, the treatment of neuroblastoma × spinal cord cell line-34 expressing G93A, H46R, or H80R hSOD1 mutants, and showing constitutive expression of iNOS and nNOS, with either a NO scavenger, or NOS inhibitors prevented the inhibition of Cytox activity and rescued cell viability. These results support the involvement of NO in mutant SOD1s-induced Cytox damage, and mitochondrial toxicity.

Abbreviations used:

7-nitroindazole monosodium salt


amyotrophic lateral sclerosis




copper chaperone for superoxide dismutase




cytochrome c oxidase


familial ALS


human SOD1


inducible NOS


mouse SOD1


neuronal NOS


nitric oxide


NO synthase


neuroblastoma × spinal cord


phosphate-buffered saline




sporadic ALS


copper, zinc superoxide dismutase


manganese-containing superoxide dismutase


triethylene tetramine tetrahydrochloride

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease mainly affecting motor neurons in cortex, brainstem, and spinal cord. This pathology exists in two forms, idiopathic (sporadic ALS; sALS) or heritable (familial ALS; fALS). Interestingly, 20% of fALS cases are associated with point mutations in the gene coding for the main intracellular antioxidant enzyme, Cu, Zn superoxide dismutase (SOD1; EC (for review, see Cozzolino et al. 2008). This protein is active in form of homodimer, where the copper plays a catalytic role, while zinc has an essential structural role. The cause of motor neurons degeneration in ALS is still uncertain but the occurrence of similar features in most sALS and fALS cases suggests the existence of a common pathologic pathway responsible for the onset of the disease (Brown and Robberecht 2001); this makes the studies on fALS-associated SOD1 mutations particularly useful to identify the mechanism(s) underlying motor neuron damage in all ALS cases.

In the case of fALS associated with mutations of SOD1, the mutant protein seems to acquire a toxic property through misfolding or aberrant chemistry (Bruijn et al. 2004; Chattopadhyay and Valentine 2009). This hypothesis is sustained by the observation that mutations are distributed in all the exons coding for the protein, affecting or not its copper-binding affinity and catalytic activity; nonetheless, all mutants manifest toxicity to the motor neurons, and paradoxically, some of those retaining the dismutating activity (e.g. the G93A mutant) are even more toxic than others. Abnormal protein aggregation, excitotoxicity, and oxidative stress from reactive oxygen or nitrogen species have been suggested to concur to the motor neurons degeneration in fALS (Beckman et al. 2001; Julien 2001; Bendotti and Carri 2004).

Converging evidence suggest that damage to mitochondria is an early event, critically involved in the pathogenesis of ALS. Mutant SOD1 transgenic models showed important structural mitochondrial defects (Wong et al. 1995; Kong and Xu 1998) and mitochondrial respiration was found to be impaired in G93A-SOD1 transgenic mice CNS (Higgins et al. 2002; Mattiazzi et al. 2002; Kirkinezos et al. 2005). A crucial molecular target for this effect seems to be cytochrome c oxidase (Cytox; EC, the Complex IV of the mitochondrial electron transport chain, a multimeric enzyme carrying a copper ion named CuB on subunit I and a binuclear copper center (CuA) on subunit II; the three copper ions, together with heme a and a3, are responsible for Cytox catalytic activity, and are also required for the correct assembly of the whole oligomer into the inner mitochondrial membrane (Rossi et al. 1998; Carr and Winge 2003). Indeed, Cytox impairment seems to be a general feature of both ALS and fALS: decreased activity of Complex IV was observed in biopsies of the spinal cord of sALS patients (Fujita et al. 1996; Borthwick et al. 1999) and in fALS mouse models Complex IV showed to be a relevant target of mutant SOD1 toxicity (Mattiazzi et al. 2002; Martin et al. 2007). However, the mechanism(s) leading to the enzyme failure is still debated. It has been proposed that a reduced association of cytochrome c to the inner mitochondrial membrane might be responsible for faulting delivery of electrons to Complex IV (Kirkinezos et al. 2005). Alternatively, nitric oxide (NO) production and nitrative damage may be responsible for inactivation of Complex IV (Ciriolo et al. 2000; Martin et al. 2007).

Another hypothesis on Cytox inactivation by mutant SOD1s may originate from the notions that (i) both SOD1 and Cytox are copper-dependent enzymes and (ii) physiologically, a small fraction of SOD1 associates with mitochondria (Okado-Matsumoto and Fridovich 2001; Sturtz et al. 2001; Field et al. 2003), when still partially or completely demetallated and receive copper from a specific copper-delivering chaperone, the protein copper chaperone for superoxide dismutase (CCS). It has been shown that mutant fALS-related SOD1s associate with mitochondria more easily then the wild-type enzyme (Liu et al. 2004; Ferri et al. 2006; Vande Velde et al. 2008), and this seems be specific for the motor neuron, as in transgenic animal models, mutant SOD1 has not been found in mitochondria from liver or brain (Liu et al. 2004). Therefore, it might compete for the copper mitochondrial pool with Cytox, thus impairing Complex IV assembly and activity. This idea is sustained by recent studies carried out by Son et al. (2007, 2008) who demonstrated in a very elegant in vivo model that the over-expression of CCS leads to increased mitochondrial localization of the mutant SOD1 and an isolated Cytox deficiency, without damage to other mitochondrial respiratory chain protein complexes, and that those transgenic mice manifest an acceleration in the disease course.

In this study, we sought to deeply investigate the effect of mutant SOD1s on Cytox, to provide further information about the mechanism of inhibition. Using motoneuronal mouse neuroblastoma × spinal cord cell line (NSC-34), the most accepted motor neuron-like cell model, stably transfected for the inducible expression of either wild-type or one of three mutant hSOD1s typical of fALS (G93A, H46R, and H80R) (Ferri et al. 2006) showing different chemicophysical properties, we demonstrate that the expression of all mutant-SOD1s investigated causes the irreversible inhibition of Cytox, that is not because of shortness of copper but of the formation of NO. This is supported by the recovery of the activity of Cytox upon treatment with either the NO scavenger carboxy-2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (C-PTIO), or by treatment with 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT) or 7-nitroindazole monosodium salt (7-NINA), two inhibitors of NO synthases (NOSs).

Materials and methods

Cell lines

Mouse motoneuronal cell line NSC-34 (originally, a gift of Dr N. R. Cashman, University of Toronto, Toronto, ON, Canada) is a hybrid NSC cell line that resembles motor neurons, displaying a multipolar neuron-like phenotype. Even when undifferentiated, it expresses choline acetyltransferase and neurofilament triplet proteins, induces twitching in co-cultured mouse myotubules, and generates action potentials (Cashman et al. 1992). Because of these properties and other properties expected of motor neurons, it is considered the best stable motoneuronal cell line model system available.

This line was stably transfected with the pTet-ON plasmid (Clontech, Palo Alto, CA, USA), coding for the reverse tetracycline-controlled transactivator, to obtain the line designed NSC-34-ON7, which displays a very low level of basal expression and high inducibility, and was used to construct inducible cell lines expressing the cDNAs encoding human wild-type-SOD1 or one of the three human fALS-typical mutSOD1s inserted in the pTRE2 plasmids (G93A, H64R, and H80R) as previously described (Ferri et al. 2006). To be able to compare the results obtained with each transfected hSOD1, we used cell clones expressing low and similar amounts of proteins upon induction (see Fig. 1). The cell lines used in this study were grown in Dulbecco’s modified Eagle’s/F-12 medium supplemented with 10% tetracycline-free FCS (Cambrex, East Rutherford, NJ, USA), at 37°C in an atmosphere of 5% CO2 in air. Induction of hSOD1 expression was obtained by adding 2 μg/mL doxycycline (BD Bioscience Clontech, Palo Alto, CA, USA) to the culture medium for the last 24 h of culture. For each experiment, cells were plated at the density of 2 × 10cell/mL of culture medium.

Figure 1.

 The expression of controlled levels of mutant hSODs affect cell viability and mitochondrial network of motoneuronal NSC-34 cells. NSC-34 cells were induced to express hSOD1 by 24 h treatment with 2 μg/mL doxycycline. (a) Immunoreactive SOD1s were detected by western blot, using a polyclonal rabbit antibody; 10 μg of total protein from whole cell extracts were loaded on each lane to detect SOD1. Glyceraldeyde-3-phosphate dehydrogenase (GAPDH) was detected for the loading control. h, human transfected SOD1; m, mouse endogenous SOD1. The blot shown is representative of at least five different experiments, giving comparable results. (b) After 24 h incubation with doxycycline, cells were detached and observed under a light microscope in the presence of the vital stain Trypan Blue (0.2%). Cells excluding the stain were considered viable. Data shown are the average of five experiments, ± SD; **p < 0.005. (c) After 24 h incubation with doxycycline, to observe mitochondrial network, NSC-34 cells were permeabilized and incubated with a rabbit polyclonal antibody against SOD2, followed by an anti-rabbit secondary antibody, tetramethyl rodhamine isothiocyanate-conjugated, and observed under a confocal microscope, equipped with a 60× objective (red). Nuclei were stained with Hoechst 3342 (1 μg/mL) (blue).


Where indicated, cells were treated for 72 h with the specific copper chelator triethylene tetramine tetrahydrochloride (Trien; 125 μmol/L, final concentration), added 24 h after plating. The NO scavenger C-PTIO (Alexis, Lausen, Switzerland), 50 mmol/L in phosphate-buffered saline (PBS; phosphate buffer 10 mmol/L, KCl 2.7 mmol/L, and NaCl 137 mmol/L, pH 7.4), was added at 20 or 50 μmol/L final concentration in the last 24 h of cell culture. The NOS inhibitors AMT (Sigma, St Louis, MO, USA) or 7-NINA (Sigma) were also added at the last 24 h of cell culture at the concentrations 20 or 125 μmol/L, respectively.

Cell number and viability assay

Cells in PBS were counted in a hemocytometric chamber under a phase contrast optical microscope. Cell viability was evaluated by their impermeability to Trypan Blue (0.2% wt/vol; Sigma).

Preparation of cell lysates

After the treatments, the cells were detached from the monolayer by a plastic scraper, extensively washed in PBS, resuspended in hypotonic PBS (1 : 2; vol/vol), and sonicated for 10 s. Whole cell lysates were used for copper content estimation and Cytox enzymatic assay.

For western blot analyses, detached and washed cells were treated with a lysis buffer [Tris–HCl 10 mmol/L, 150 mmol/L NaCl, 1% (vol/vol) Triton X-100, 10% (v/v) proteases inhibitor cocktail (Sigma)] for 30 min on ice, followed by centrifugation (23 000 g, 30 min). Supernatants were stored at −80°C until used.

Mitochondria isolation

NSC-34-hSOD1s expressing cells (5 × 109) were homogenized in mitochondrial buffer (0.2 mmol/L EDTA, 0.25 mol/L sucrose, and 10 mmol/L Tris/HCl, pH 7.4) and proteases inhibitor cocktail (Sigma) in a Potter homogenizer with a Teflon pestle. Centrifugation of the supernatant at 12 000 g for 15 min yielded the crude mitochondrial fraction. The crude mitochondrial pellet was then resuspended in 0.8 mol/L sucrose, loaded on a discontinuous sucrose gradient (1, 1.3, 1.6, and 2 mol/L), and ultracentrifuged for 2 h at 80 000 g. The material at the 1.3–1.6 mol/L interface was collected, washed in PBS, and designated as purified mitochondria.

Total protein content

Protein content was assayed according to Lowry et al. (1951).

Copper content measurement

Copper levels in the samples were measured by atomic absorption spectrophotometry using an AAnalyst 300 Perkin Elmer instrument, equipped with a graphite furnace with platform (HGA800) and an AS-72 autosampler (PerkinElmer, Waltham, MA, USA). Before analysis, an equal volume of 65% nitric acid was added to whole cell lysates, and the samples digested at 25°C for at least 1 week.

Cytochrome c oxidase assay

Cytochrome c oxidase (Cytox) was assayed spectrophotometrically in 30 mmol/L phosphate buffer, pH 7.4, 25°C by following the oxidation of reduced cytochrome c (from horse heart; Sigma) (0.02 mmol/L) at 550 nm, as previously described (Lombardo et al. 2003). A Beckman-Coulter DU800 spectrophotometer (Beckman-Coulter, Fullerton, CA, USA) was used. Activity was expressed as units (μmol cytochrome oxidized/min)/mg proteins.

Western blot analyses

Immunoelectrophoresis was performed using 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and by overnight blotting on Protran nitrocellulose transfer membranes (Schleicher and Schuell, Dassel, Germany). The protein load is reported in the figure legends. Mitochondrial proteins were recognized by monoclonal antibodies obtained from Molecular Probes (Eugene, OR, USA), except for Cytox subunit II that was detected with a polyclonal antibody from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and for the manganese-containing superoxide dismutase (SOD2) that was detected by a rabbit polyclonal antibody obtained from Upstate Biotechnology (Billerica, MA, USA). Immunoreactive SOD1 was detected by a rabbit polyclonal anti-SOD1 antibody (Stressgen, San Diego, CA, USA). Glyceraldehyde-3-phosphate dehydrogenase (EC was recognized by mouse monoclonal antibodies obtained from Santa Cruz Biotechnology. Protein–antibody complexes were identified with the Super Signal Chemiluminescent Substrate (Pierce, Rockford, IL, USA). The inducible isoform of NOS (iNOS) was detected by rabbit polyclonal antibody (Santa Cruz Biotechnology), while neuronal NOS (nNOS) was identified by a monoclonal antibody (Santa Cruz Biotechnology).


Cells cultured in 35 mm Petri dishes and incubated for 24 h with 2 μg/mL doxycycline were washed in PBS and fixed with 4%p-formaldehyde in PBS for 15 min. After permeabilization with 0.2% Triton X-100 in PBS for 5 min, cells were blocked in 2% horse serum in PBS (blocking buffer) and incubated for 1 h at 37°C with primary rabbit polyclonal antibody against SOD2 (Upstate Biotechnology). Cells were then washed in blocking buffer and incubated for 1 h with labeled anti-rabbit secondary antibody, tetramethyl rodhamine isothiocyanate-conjugated (Molecular Probes). After rinsing in blocking buffer, cells were examined under a Zeiss LSM 510 Confocal Microscope equipped with a 60× objective (Carl Zeiss MicroImaging, Inc., Thornwood, NY, USA). Fluorescence images were adjusted for brightness, contrast, and color balance by using Adobe Photoshop 7.0 (Adobe Systems Inc., San Jose, CA, USA).

Statistical analysis

The results are presented as mean ± SD. Data were analyzed by the Student’s t-test. A value of p < 0.05 was accepted as the level of significance. Western blots and fluorescence microscopy shown are representative of at least three analyses performed on samples from at least three separate experiments.


Figure 1a shows western blot analysis of SOD1s expressed by NSC-34 cells, a mouse cell line. The bands corresponding to mouse (mSOD1) or to human SOD1s (hSOD1) are visible; as already described, hSOD1s show lower mobility than the endogenous mSOD1. The clones of the transfected cells and the protocol employed to induce hSOD1s expression (2 μg/mL doxycycline for only 24 h) were selected to accomplish low expression level of all the transfected hSOD1s, which is always lower than that of the endogenous SOD1. This in order to prevent possible criticisms raised against the models of SOD1-related fALS (both transgenic mice or transfected cells), which, when expressing huge amounts of the human proteins, are considered to be too far from the physiological conditions of fALS patients and possibly generating artifacts (Bergemalm et al. 2006).

Cell viability of the transfected cells was assessed by the capacity of the cells to exclude the Trypan blue staining (Fig. 1b). At difference with wild-type SOD1, after 24 h from the induction of the expression with doxycycline all mutant hSOD1s examined produced cell toxicity (18%, 25%, and 28% for H46R, G93A, and H80R hSOD1, respectively).

The morphology of mitochondria was observed by fluorescence microscopy, by labeling SOD2, a mitochondrial matrix enzyme, with a tetramethyl rodhamine isothiocyanate-conjugated secondary antibody. The mitochondrial network of NSC-34 expressing wild-type hSOD1 for 24 h (Fig. 1c, upper panel) is well defined and well distributed. Conversely, upon G93A-hSOD1 expression, swollen mitochondria were detectable and the organelles network was almost barely visible (Fig. 1c, lower panel).

The copper-enzyme Cytox is considered to be a putative target for the toxic action to mitochondria of mutant hSOD1 (Kirkinezos et al. 2005; Son et al. 2007). We have evaluated Cytox activity in total cell extracts by a spectrophotometric assay, following the oxidation of reduced cytochrome c at 550 nm; 24 h after the induction of hSOD1s expression, all mutant hSOD1s produced a significant decrease of Cytox activity (30% for H46R- and H80R- and 40% for G93A-hSOD1) (Fig. 2a); conversely, wild-type hSOD1 expression did not affect Cytox activity. As shown in Fig. 2b, the expression of hSOD1 mutants has no effect on Cytox subunit II levels evaluated in isolated mitochondria. Thus, Cytox inactivation seems to be a general consequence of expression of all mutant SOD1s, despite their different chemical–physical behavior, and it is not ascribable to effects on Cytox protein subunit level.

Figure 2.

 Familial ALS mutant hSOD1s expression in NSC-34 cells is accompanied by the decrease of cytochrome c oxidase (Cytox) activity. Cells were incubated for 24 h with 2 μg/mL doxycycline. (a) Cytox activity was measured in fresh whole cell homogenate by a spectrophotometric assay, by following the oxidation of reduced cytochrome c at 550 nm; 100% activity was about 80 mU/mg protein. The experiment was repeated five times, and the data shown are the mean ± SD; **p < 0.005. (b) Western blot analysis of Cytox, in extracts from isolated mitochondria; 10 μg protein was applied to each lane. Subunit II of Cytox was detected by a polyclonal antibody. Porin was used as loading control. Blot shown is representative of analysis performed on samples from five different experiments, showing comparable results.

It is widely accepted that a small fraction of SOD1, which is primarily a cytosolic enzyme, associates with mitochondria (Okado-Matsumoto and Fridovich 2001; Sturtz et al. 2001), and that this fraction is higher as far as the mutant SOD1s are concerned (Ferri et al. 2006). On this basis, one can infer that the inactivation of Cytox might be because of competition between over-expressed mutant SOD1s and Cytox for the mitochondrial copper pool (Cobine et al. 2006). To clarify this issue, experiments were carried out by treating NSC-34 cells over-expressing hSOD1s for 72 h with 125 μmol/L Trien, a well-known copper chelator, which depletes copper levels in neuronal cells (Rossi et al. 2001). Upon Trien treatment, copper content of transfected NSC-34 cells, expressing or not wild-type or mutant hSOD1s, was lowered by at least 50% (Fig. 3a). Correspondingly, Trien treatment of NSC-34 cells induced the decrease of Cytox activity (Fig. 3b). However, Trien treatment also decreased the subunit II protein levels in mitochondria isolated from NSC-34 cells expressing hSOD1, either wild-type or G93A, as demonstrated by western blot analysis performed (Fig. 3c), at difference with the unchanged subunit II level observed in isolated mitochondria upon expression of mutant-SOD1s (Fig. 2b). The measurement of the levels of the mitochondrial protein of the α-subunit of the F0-F1 ATP synthase, of SOD2 and of the VDAC (voltage-dependent anion channel, porin), which are not affected by copper deprivation (Fig. 3c), demonstrate that the change observed is specific for the subunit II of Cytox.

Figure 3.

 The decrease of cytochrome c oxidase (Cytox) activity following expression of fALS mutant hSOD1s in NSC-34 cells is not because of shortness of copper. Where indicated, cells were incubated for 72 h with the copper chelator Trien and for the last 24 h with doxycycline or with doxycycline plus copper sulfate for 24 h. (a) Copper content was assayed in whole cells samples digested by nitric acid by atomic absorption spectophotometry; 100% copper of uninduced cells = 16 ng/mg protein; n = 5, **p < 0.005 (vs. NSC-34 not treated with doxycycline). (b) Cytox activity was measured in fresh whole cell homogenate; 100% activity was about 80 mU/mg protein. The experiment was repeated five times, and the data shown are the mean ± SD; **p < 0.005. (c) Western blot analyses were performed on extracts of isolated mitochondria, and 10 μg protein were loaded on each lane. The results shown are representative of three different experiments. (d) Where indicated, cells were incubated with doxycycline alone, or plus copper sulfate for 24 h. At the end of the incubation, Cytox activity was assayed. The experiment was repeated five times, and the data shown are the mean ± SD; **p < 0.005.

To reinforce the finding that Cytox activity decrease is not because of competition of mutant hSOD1 for the copper pool, experiments were carried out to guarantee complete availability of the metal during SOD1s expression in NSC-34 cells. Cells transfected with wild-type or mutant hSOD1(G93A) were grown in the presence of 50 μmol/L copper sulfate, added to the cell culture medium together with doxycycline (24 h before analysis). The decrease of Cytox activity produced by the expression of G93A mutant of hSOD1 was not affected by the presence of extra-copper in the culture media, thus suggesting that the amount of copper physiologically present in the cells is already sufficient to allow the refill of Cytox. From the whole of the results shown in Fig. 3, it can be concluded that mutant hSOD1s effect on Cytox is because of inactivation of the mature enzyme rather than of the decreased expression or increased turnover of the complex subunits because of copper shortness. Therefore another, copper-independent, mechanism should be responsible for Cytox inactivation in the presence of mutant hSOD1.

Several reports demonstrate that NO regulates/inhibits Cytox in in vitro or in vivo experimental models (Zhang et al. 2005; Brunori et al. 2006; Martin et al. 2007), and increased nitrative stress has been reported both in sALS and fALS patients and in the mouse model as well (Beal et al. 1997; Sasaki et al. 2001). To investigate possible correlation between the formation of NO, triggered by mutant hSOD1, and Cytox inhibition, we have incubated NSC-34 cells, transfected for the expression of wild-type, H46R, H80R or G93A hSOD1s for 24 h with doxycycline (2 μg/mL) plus C-PTIO (final concentration 20 μmol/L or 50 μmol/L), a well-known NO scavenger (Vicente et al. 2006). As illustrated in Fig. 4a, C-PTIO treatment did not affect the expression of hSOD1s (both wild-type or mutant) at both concentrations tested, but it rescued Cytox activity, in all cell lines expressing mutant hSOD1s (Fig. 4b), the 50 μmol/L concentration showing a better effect for H46R and H80R cells. Therefore, NO seems to be the actual mediator of Cytox inactivation by mutant hSOD1s. Furthermore, the NO scavenger was also able to protect cells from the toxic effect of G93A and H80R hSOD1s, leading to an increment of cell survival (Fig. 4c).

Figure 4.

 The treatment with the NO scavenger carboxy-PTIO (C-PTIO) rescues cell viability and cytochrome c oxidase (Cytox) activity in NSC-34 cells expressing hSOD1s but does not affect the hSOD1s protein level. (a) Western blot on h-SOD1s NSC-34 expressing cells upon 24 h treatment with C-PTIO (20 or 50 μmol/L). Glyceraldeyde-3-phosphate dehydrogenase (GAPDH) was assayed as loading control; 10 μg of total protein from whole cell extracts were loaded on each lane to detect SOD1s and GAPDH. Images shown are representative of three different experiments. (b) Trypan blue exclusion test. (c) Spectrophotometric evaluation of Cytox activity was performed after the C-PTIO (20 or 50 μmol/L) treatment. For both assays, n = 3 and ##p < 0.005 versus not treated and not expressing hSOD1 cells; *0.05 < p < 0.005 and **p < 0.005 versus hSOD1s expressing NSC-34 cells.

To understand the involvement of the enzymes devoted to NO formation in NSC-34 cells expressing mutant-hSOD1s, western blot analysis was performed to detect the presence and the amount of the nNOS or iNOS. Figure 5 shows that both isoforms are present constitutively in NSC-34 cells and that their amount does not change upon expression of either wild-type or mutant hSOD1s. However, the activity of NOSs seems to be crucial to the inactivation of Cytox by mutant hSOD1s expression. This was demonstrated by treatment with either 20 μmol/L AMT, or with 125 μmol/L 7-NINA, two routinely used inhibitors of NOSs (Roy et al. 2008) added at the same time as doxycycline. Cells were analyzed 24 h later. Figure 6a shows that both inhibitors were not toxic for NSC-34 cells at the concentrations used and protected the cells from the toxicity of mutant hSOD1. Furthermore, they rescued Cytox activity to levels similar to that of wild-type hSOD1 expressing cells (Fig. 6b). This effect was not mediated by a decrease in the expression of mutant SOD1s (Fig. 6c).

Figure 5.

 Constitutive expression of nitric oxide synthases is not altered by hSOD1 mutants in NSC-34 cells. Western blot analysis of inducible nitric oxide synthase (iNOS) and neuronal nitric oxide synthase (nNOS) were performed on NSC-34 cells after 24 h induction of Wt, or G93A, or H46R, or H80R hSOD1s. Glyceraldeyde-3-phosphate dehydrogenase (GAPDH) was assayed as loading control; 20 μg protein was applied to each lane. Blot shown is representative of five different experiments, showing comparable results.

Figure 6.

 Inhibition of nitric oxide synthase (NOS) restores cytochrome c oxidase (Cytox) activity and cell viability in NSC-34 cells expressing mutant hSOD1s. Cells were incubated for 24 h with 2 μg/mL doxycycline and, where indicated, NOS inhibitors AMT or 7-NINA were also added, at concentrations 20 or 125 μmol/L, respectively. (a) Trypan Blue exclusion test, in the presence or in the absence of AMT or 7-NINA; n = 3, *p < 0.05 versus untreated cells and #p < 0.05 versus doxycycline treated cells. (b) Spectrophotometric evaluation of Cytox activity; n = 3, *p < 0.05 versus doxycycline treated wild-type cells; #p < 0.05 versus AMT or 7-NINA untreated cells. (c) hSOD1 levels in NSC-34 cells, upon treatment with doxycycline, and AMT or 7-NINA, evaluated by western blot analysis; 10 μg of total protein were loaded on each lane.


It is a widely shared opinion that motoneuronal mitochondria and Cytox are specific and early targets in ALS (Martin et al. 2007; Son et al. 2008). We confirm here the occurrence of mitochondrial damage induced by mutant SOD1s in NSC-34 cells by a change in the mitochondrial morphology, and by a decrease of the activity of Cytox (Figs 1 and 2).

Our study was performed on the model system represented by the mouse cell line NSC-34, which is a hybrid cell line (neuroblastoma × spinal cord-34), considered to be the best motor neuron-like cell available, that was transfected with human wild-type or mutant hSOD1s, under the control of the inducible pTet-On promoter. Indeed, some concern has been expressed on the possibility that the use of cell and animal models of fALS expressing excessive, over-physiological, amounts of hSOD1 can lead to misleading results; for instance, the system might have not enough copper and/or CCS to supply the protein with the metal (Bergemalm et al. 2006). Under these conditions, large amounts of copper-free SOD1 may thus be present in the cells, being more easily picked up by the mitochondria (Okado-Matsumoto and Fridovich 2001; Field et al.2003). In this study, care was therefore taken to adjust the degree of expression of the transfected hSOD1s, to avoid extreme over-expression, by properly selecting conditions for the induction (Fig. 1). In addition, the possibility that a large amount of apo-SOD1 was present in the expression system was ruled out by checking that no increase of mouse or human wild-type SOD1s activity occurred upon incubation with exogenous copper sulfate of either whole cells or cell extracts (data not shown). Furthermore, owing to the low level of expression of the three mutant SOD1s, their effect on cell viability was not devastating, thus allowing the observation of very early events concerning mitochondria. In this study, we investigated three different mutant SOD1s (namely G93A, H46R, and H80R), which show quite different physicochemical properties in vitro and are associated with different severity of the disease. However, all the three mutants selected caused the decrease of the Cytox activity in NSC-34 cells and a similar cell toxicity, thus confirming that the affection of Cytox is a general mechanism in fALS.

It has been proposed that the increased affinity for mitochondria of the mutant hSOD1s with respect to the wild-type enzyme may be relevant to the mitochondrial damage (Higgins et al. 2002; Mattiazzi et al. 2002; Ferri et al. 2006; Vande Velde et al. 2008). In yeast, wild-type hSOD1 carrying a signal peptide to mitochondria was shown to compete with Cytox for the mitochondrial copper pool, because a mitochondrial matrix copper-ligand complex is the likely source of copper for both Cytox and SOD1 (Cobine et al. 2006). However, this does not seem the case in our model, because no change in Cytox activity was observed in copper-deficient cells expressing wild-type-hSOD1. Furthermore, a comparable decrease of Cytox activity was observed in cells expressing either H46R- or H80R-hSOD1 mutants, that are defective in copper binding because they lack a metal-binding histidine residue (Valentine et al. 2005). In addition, supplementation of the culture media with copper sulfate during hSOD1s expression did not change the effect of the mutant SOD1s on Cytox activity.

It is known that copper shortness or unavailability affects Cytox both by decreasing the enzyme activity and by affecting Cytox assembly and increasing protein subunits turnover (Rossi et al. 1998, 2001; Lombardo et al. 2003). A similar pattern is also observed in copper-depleted NSC-34 cells expressing wild-type or G93A hSOD1; treatment with the specific copper chelator Trien, beside producing similar canonical decline of intracellular copper levels and SOD1s activity, leads to decrease of Cytox activity as well as of the protein level of subunit II. This effect is different from that observed by the sole expression of mutant hSOD1s, which do not alter subunit II protein level. This clearly demonstrates that the effect on Cytox consequent to mutant hSOD1s expression is not because of competition between SOD1 and Cytox for copper, but rather to an inhibition process.

Reactive nitrogen species, up-regulation of iNOS and nitrative stress have been implicated in motor neurons death induced by fALS-linked SOD1 mutants, in isolated cells (Ciriolo et al. 2000; Raoul et al. 2002), in transgenic mice (Almer et al.1999; Casoni et al. 2005; Martin et al. 2007), and also in both sALS and fALS patients (Beckman et al. 1993; Beal et al. 1997). Disruption in the SOD1 structure by mutations reduces the affinity for zinc and it has been demonstrated that zinc-deficient SOD1 catalyzes the nitration of motoneuronal proteins such as neurofilaments (Crow et al. 1997; Trumbull and Beckman 2009). Furthermore, a nitration event might cause an irreversible inhibition of Cytox caused by NO (Cooper et al. 2003); indeed, nitrated and aggregated Cytox subunit I was found in association with G93A-hSOD1 expression in pre-symptomatic transgenic mice, together with NO and NOO production (Martin et al. 2007). In this study, the inhibition of Cytox is detected after cell disruption by a spectrophotometric test, indicating irreversible, persistent, possibly covalent inhibition. As Cytox inactivation is rescued by both C-PTIO, a renowned NO scavenger, and by the treatment with two routinely used NOS inhibitors (AMT and 7-NINA), it is conceivable that, under these experimental conditions, NO is responsible for impairment of Cytox. As these effects occur not only upon G93A hSOD1 expression but also upon H80R and H46R mutants, this process may represent a common acquired toxic property of all mutant SOD1s.

Cytox inhibition by NO in our cell model occurs under mild mutants hSOD1 expression, accompanied by a low extent of cell death, thus confirming the conclusion by Martin et al. that Cytox damage by NO is an early event in mutant SOD1 toxicity. Furthermore, the elimination of NO rescues cell viability of NSC-34 cells expressing mutant hSOD1s, thus demonstrating that Cytox inhibition via NO is a mechanism that can be relevant to the progression of the pathology.

As shown in Fig. 5, NSC-34 cells express a constitutive level of both nNOS and iNOS, which is not sensibly affected by wild-type or mutant SOD1s expression; thus we can speculate that the constitutive expression of iNOS may make the cells particularly vulnerable to SOD1s mutations. Furthermore, as iNOS was also reported to localize in mitochondria (Martin et al. 2007), its strict proximity with a physiological NO target, such as Cytox, is quite appealing. Indeed, the amount of mitochondrial iNOS increased in G93A transgenic mice and the deletion of iNOS gene significantly extended the life span of the G93A transgenic mice (Martin et al. 2007). The mechanism through which mutant SOD1 causes increase in NOS activity will certainly deserve further investigation.

In conclusion, this study demonstrates, in a motor neuron-like cell line, that a moderate expression of three different mutant hSOD1s affects the function of the Complex IV of the respiratory chain, at difference with the expression of comparable amounts of the wild-type enzyme. This effect is not because of competition of the mitochondrial fraction of the mutant hSODs with Cytox for the mitochondrial pool of copper, but rather to increased production of NO which irreversibly binds to the enzyme. The Cytox impairment by NO-induced stress could represent a realistic unifying mechanism between fALS and sALS. An increase in NO may occur in non-SOD1 linked ALS following different stress, thus causing the progressive failure of mitochondrial electron transport chain, decreased production of ATP and oxidative damage, leading to degeneration of motor neurons, all common features of all the forms of this fatal disease.


A grant from the Italian Ministry of Health (PF ‘Meccanismi molecolari e cellulari delle malattie neurodegenerative del sistema motorio’ to MTC) partially supported this work.