**4. SOD1 catalyzes increased hydroperoxide production in IMS**

The growing body of evidence provides support to the concept that superoxide dismutation in IMS may cause an increased hydroperoxide production with toxic consequences. Mitochondria are the major intracellular source of superoxide, the primary ROS, where superoxide anion radical is generated by one electron reduction of oxygen.

The two major pathways of superoxide production in mitochondria are autooxidation or complex III catalyzed oxidation of ubisemiquinone (Muller et al., 2004) and complex I catalyzed reduction of oxygen through reversed electron flow in the respiratory chain (Fig. 2.) (Liu et al., 2002). The produced superoxide anion radical has ability to actively react with a number of cellular targets leading to the loss of their proper function. The main detoxifying mechanism for superoxide instead of reverse oxidation of superoxide to oxygen, includes dismutation to hydroperoxide and oxygen.

Besides SOD1, there are other dedicated enzymes catalyzing this dismutation reaction. In mitochondria Mn-superoxide dismutase (SOD2), which is found in the mitochondrial matrix, scavenges superoxide in this compartment. Extracellular superoxide dismutase (SOD3) is secreted into the extracellular space and protects tissues against excess of superoxide (Zelko et al., 2002). In the IMS superoxide is produced presumably by complex III (Fig. 2.) (Muller et al., 2004). Unlike hydroperoxide, which freely diffuses through the membranes, superoxide cannot cross the mitochondrial inner membrane. In the matrix SOD2 converts superoxide to hydroperoxide, which in turn is reduced to water by the matrix glutathione peroxidase (Inoue et al., 2003). Homozygous SOD2 knockout mice are neonatally lethal (Li et al., 1995), whereas deletion of SOD1 gene does not have apparent motor neuron disease phenotype (Maier & Chan, 2002).

 In IMS the fate of superoxide is determined by SOD1 and cytochrome c, which is present there in millimolar concentrations (Forman & Azzi, 1997; van Beek-Harmsen & van der Laarse, 2005). Cytochrome c is a heme containing protein, which functions as an electron carrier between complex III and cytochrome oxidase in the respiratory chain. Cytocrome c can also efficiently oxidize superoxide to oxygen. In this respect, cytochrome c can function

Mutant Cu/Zn-Superoxide Dismutase Induced

oxidize a number of vital biological targets (V).

causes paradoxically augmented ROS production.

increased cytochrome c-catalyzed oxidation in the IMS.

Mitochondrial Dysfunction in Amyotrophic Lateral Sclerosis 255

Fig. 3. Deleterious role of superoxide dismutase in the mitochondrial intermembrane space. Superoxide (O2.-) is released in IMS by one electron reduction of oxygen at a site in the inner membrane (I). Cu/Zn Superoxide dismutase (SOD1) in IMS is activated by oxidation of cysteine thiols, leading to formation of intramolecular S=S bounds (II). SOD1 produces hydroperoxide (H2O2) by dismutating superoxide (III). Hydroperoxide oxydizes cytochrome c (CytC) to oxoferryl-CytC (CytC(Fe4+=O)), an exceptionally strong oxidant (IV), able to

We have recently proposed a model, where upon mitochondrial stress SOD1 may compete with cytochrome c for superoxide in the IMS and generate hydroperoxide, which then could react with cytochrome c and form peroxidase compound I-type intermediate, eventually leading to a deleterious increase in ROS production and cellular injury (Fig. 2) (Goldsteins et al., 2008). According to this model the SOD1-catalyzed superoxide dismutation in the IMS

The data obtained demonstrate that inhibition of electron transfer at the level of complex III leads to SOD1 activation in the IMS, resulting in increased hydroperoxide production and, consequently, cytochrome c-catalyze peroxidation (Goldsteins et al., 2008). This could trigger a vicious circle where oxidative damage to mitochondrial respiratory components leads to further ROS production and peroxidation. Indeed, we have demonstrated that inhibition of mitochondrial respiration at the level of complex III causes SOD1-dependent ROS production and apoptotic death of isolated blood lymphocytes. In contrast, mitochondria isolated from SOD1 knockout mice do not show increased ROS production upon mitochondrial stress. Moreover, accumulation of mutant human G93A-SOD1 in the IMS that is observed in the tg animal models of ALS, leads to elevated SOD1 activity and

Our proposed model provides also an explanation for observations in other neurodegenerative disorders that elevated SOD1 activity worsens the pathology instead of the expected protective effect. For instance, immature mouse brains overexpressing SOD1 show an increased propensity for injury and accumulate more hydroperoxide after hypoxiaischemia than wt mouse brains (Fullerton et al., 1998). Also, elevation of SOD1 increases acoustic trauma from noise exposure in some models (Endo et al., 2005). Impotantly, mice

as an efficient antioxidant, scavenging superoxide without production of secondary ROS (Fig. 2. reaction II), in contrast to SOD1, which produces hydroperoxide (Fig. 2, reaction III) (Pereverzev et al., 2003). However, cytochrome c has also a potential to catalyze oxidation by hydroperoxide. Upon this reaction, hydroperoxide oxidizes the prosthetic heme in the cytochrome c molecule to oxoferryl heme, forming so-called peroxidase compound I-type intermediate, a highly reactive oxidant that is able to react with a number of intracellular targets including proteins, nucleic acids and lipids, causing cell damage (Fig. 3) (Lawrence et al., 2003). Cytochrome c peroxidase activity is controlled by the coordination state of heme iron, particularly by the sulphur ligand of methionine-80 (Met-80), which can be easily displaced by hydroperoxide (Barr et al., 1996; Qian et al., 2002). The peroxidase activity of cytochrome c may increase by unfolding and post-translational modifications, such as proteolytic cleavage, nitration and oxidation (Diederix et al., 2002; Everse & Coates, 2005; Jang & Han, 2006).

Fig. 2. Mitochondrial production and clearance of superoxide. Upon respiration superoxide is inevitably generated predominantly at respiratory complexes CI and CIII. The superoxide released to the matrix is dismutated by mitochondrial SOD2 (I) and the hydroperoxide produced is cleared by glutathione peroxidase and peroxyredoxins. Most of the superoxide released in IMS is generated at respiratory complex CIII. Oxidized form of cytochrome c, present in the IMS at high concentration can exercise clean clearance of superoxide by its oxidation to oxygen (II). An alternative dismutation catalyzed by SOD1 results in increased hydroperoxide generation in the IMS (III).

as an efficient antioxidant, scavenging superoxide without production of secondary ROS (Fig. 2. reaction II), in contrast to SOD1, which produces hydroperoxide (Fig. 2, reaction III) (Pereverzev et al., 2003). However, cytochrome c has also a potential to catalyze oxidation by hydroperoxide. Upon this reaction, hydroperoxide oxidizes the prosthetic heme in the cytochrome c molecule to oxoferryl heme, forming so-called peroxidase compound I-type intermediate, a highly reactive oxidant that is able to react with a number of intracellular targets including proteins, nucleic acids and lipids, causing cell damage (Fig. 3) (Lawrence et al., 2003). Cytochrome c peroxidase activity is controlled by the coordination state of heme iron, particularly by the sulphur ligand of methionine-80 (Met-80), which can be easily displaced by hydroperoxide (Barr et al., 1996; Qian et al., 2002). The peroxidase activity of cytochrome c may increase by unfolding and post-translational modifications, such as proteolytic cleavage, nitration and oxidation (Diederix et al., 2002; Everse & Coates, 2005;

Fig. 2. Mitochondrial production and clearance of superoxide. Upon respiration superoxide is inevitably generated predominantly at respiratory complexes CI and CIII. The superoxide released to the matrix is dismutated by mitochondrial SOD2 (I) and the hydroperoxide produced is cleared by glutathione peroxidase and peroxyredoxins. Most of the superoxide released in IMS is generated at respiratory complex CIII. Oxidized form of cytochrome c, present in the IMS at high concentration can exercise clean clearance of superoxide by its oxidation to oxygen (II). An alternative dismutation catalyzed by SOD1 results in increased

hydroperoxide generation in the IMS (III).

Jang & Han, 2006).

Fig. 3. Deleterious role of superoxide dismutase in the mitochondrial intermembrane space. Superoxide (O2.-) is released in IMS by one electron reduction of oxygen at a site in the inner membrane (I). Cu/Zn Superoxide dismutase (SOD1) in IMS is activated by oxidation of cysteine thiols, leading to formation of intramolecular S=S bounds (II). SOD1 produces hydroperoxide (H2O2) by dismutating superoxide (III). Hydroperoxide oxydizes cytochrome c (CytC) to oxoferryl-CytC (CytC(Fe4+=O)), an exceptionally strong oxidant (IV), able to oxidize a number of vital biological targets (V).

We have recently proposed a model, where upon mitochondrial stress SOD1 may compete with cytochrome c for superoxide in the IMS and generate hydroperoxide, which then could react with cytochrome c and form peroxidase compound I-type intermediate, eventually leading to a deleterious increase in ROS production and cellular injury (Fig. 2) (Goldsteins et al., 2008). According to this model the SOD1-catalyzed superoxide dismutation in the IMS causes paradoxically augmented ROS production.

The data obtained demonstrate that inhibition of electron transfer at the level of complex III leads to SOD1 activation in the IMS, resulting in increased hydroperoxide production and, consequently, cytochrome c-catalyze peroxidation (Goldsteins et al., 2008). This could trigger a vicious circle where oxidative damage to mitochondrial respiratory components leads to further ROS production and peroxidation. Indeed, we have demonstrated that inhibition of mitochondrial respiration at the level of complex III causes SOD1-dependent ROS production and apoptotic death of isolated blood lymphocytes. In contrast, mitochondria isolated from SOD1 knockout mice do not show increased ROS production upon mitochondrial stress. Moreover, accumulation of mutant human G93A-SOD1 in the IMS that is observed in the tg animal models of ALS, leads to elevated SOD1 activity and increased cytochrome c-catalyzed oxidation in the IMS.

Our proposed model provides also an explanation for observations in other neurodegenerative disorders that elevated SOD1 activity worsens the pathology instead of the expected protective effect. For instance, immature mouse brains overexpressing SOD1 show an increased propensity for injury and accumulate more hydroperoxide after hypoxiaischemia than wt mouse brains (Fullerton et al., 1998). Also, elevation of SOD1 increases acoustic trauma from noise exposure in some models (Endo et al., 2005). Impotantly, mice

Mutant Cu/Zn-Superoxide Dismutase Induced

of general mechanism in neurodegeneration.

diseases involving mitochondrial pathogenesis.

**6. Acknowledgment** 

Diagrams were used.

485.

**7. References** 

Mitochondrial Dysfunction in Amyotrophic Lateral Sclerosis 257

fuel the cytochrome c-catalyzed peroxidation and play a key role in oxidation of biological targets in the IMS. Thus, SOD1 activity and factors leading to its increase in this compartment can be regarded as deleterious mechanisms to the mitochondria and the cell. Increased SOD1 activity causing elevated hydroperoxide production in the IMS may be one

At the moment it is not clear how mutations in SOD1 directly affect hydroperoxide production in IMS. One possibility may be the already demonstrated increased mitochondrial import for mutant molecules in neurons of ALS models. Another possible mechanism is linked to less strict dismutase activity control. Altogether, we hypothesize that the mutant SOD1 may gain toxic features because the proper control mechanism for its dismutase activity in mitochondrial IMS may be lost. In conclusion, we suggest that SOD1 activity in the IMS is a relevant therapeutic target for ALS and other neurodegenerative

For the construction of figures, elements from www.motifolio.com Free PowerPoint

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Arnesano, F., Banci, L., Bertini, I., Martinelli, M., Furukawa, Y. & O'Halloran, T.V. (2004).

Atsumi, T. (1981). The ultrastructure of intramuscular nerves in amyotrophic lateral

Barr, D.P., Gunther, M.R., Deterding, L.J., Tomer, K.B. & Mason, R.P. (1996). ESR spin-

Beckman, J.S., Chen, J., Crow, J.P. & Ye, Y.Z. (1994). Reactions of nitric oxide, superoxide

van Beek-Harmsen, B.J. & van der Laarse, W.J. (2005). Immunohistochemical determination

hydrogen peroxide. *J Biol Chem*, Vol.271, No.26, (1996), pp. 15498-503. Basova, L.V., Kurnikov, I.V., Wang, L., Ritov, V.B., Belikova, N.A., Vlasova, I., Pacheco,

peroxynitrite. *Nature*, Vol.364, No.6438, (August 1993), pp. 584.

sclerosis. *Acta Neuropathol*, Vol.55, No.3, (1981), pp. 193-198.

Mutant SOD1 from spinal cord of G93A rats is destabilized and binds to inner mitochondrial membrane. *Neurobiol Dis*, Vol.32, No.3, (December 2008), pp. 479-

The unusually stable quaternary structure of human Cu,Zn-superoxide dismutase 1 is controlled by both metal occupancy and disulfide status. *J Biol Chem*, Vol.279,

trapping of a protein-derived tyrosyl radical from the reaction of cytochrome c with

A.A., Winnica, D.E., Peterson, J., Bayir, H., Waldeck, D.H. & Kagan, V.E. (2007). Cardiolipin switch in mitochondria: shutting off the reduction of cytochrome c and turning on the peroxidase activity. *Biochemistry*, Vol.46, No.11, (2007), pp. 3423-34. Beckman, J.S., Carson, M., Smith, C.D. & Koppenol, W.H. (1993). ALS, SOD and

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deficient in SOD1 have been reported to be resistant to acetaminophen toxicity (Lei et al., 2006). Even though SOD1 as a cytosolic antioxidant protects against mitochondrial dysfunction in a mouse model of transient focal cerebral ischemia (Fujimura et al., 2000), SOD1 deficiency, rather than overexpression, is associated with enhanced recovery and attenuated activation of NF-kappaB after brain trauma in mice (Beni et al., 2006).

This apparent discrepancy concerning the role of SOD1 in cellular injury can be explained by the model introduced, showing that increased SOD1 activity in the IMS paradoxically produces peroxides which are converted to highly toxic ROS. This view is further supported by an observation in mouse model of genetic disorder ataxia-telangiectasia, where elevated levels of SOD1 exacerbate the phenotype of neurodegeneration (Peter et al., 2001). It is also of interest that SOD1 overexpression and high tissue dismutase activity may potentiate atherogenesis in fat-fed atherosclerosis-susceptible mice (Tribble et al., 1997). The evidence about deleterious role of increased SOD1 expression has been most recently complemented by studies demonstrating that overexpression of SOD1 in retina leads to increased hydroperoxide levels and accelerated damage of cone cells (Usui et al., n.d.).

The key component for the SOD1-derived hydroperoxide toxicity in IMS is cytochrome c. Previous studies, including electron paramagnetic resonance (EPR) studies (Barr et al., 1996; Svistunenko, 2005; Belikova et al., 2006; Basova et al., 2007) have demonstrated that the reaction of cytochrome c with hydroperoxide results in formation of oxoferryl cytochrome c (peroxidase compound I-type intermediate) and corresponding protein-derived tyrosyl radical, which is highly reactive and has a potential to oxidize proteins, DNA, and lipids, as well as endogenous antioxidants such as glutathione, NADH, and ascorbate (Lawrence et al., 2003) (Fig. 3). In particular, oxidation of cardiolipin, a phospholipid which is in complex with cytochrome c on the surface of the inner mitochondrial membrane, causes the release of proapoptotic factors from mitochondria (Kagan et al., 2005; Belikova et al., 2006). This leads to a scenario where the hydroperoxide produced by increased SOD1 activity in the IMS, would thus serve as a substrate for cardiolipin-bound cytochrome c and consequently switch on very early proapoptotic processes, inducing consecutive programmed cell death. Additionally, upon increased hydroperoxide levels cytochrome c peroxidase activity may cause NADH oxidation producing a radical, which in turn donates an electron to oxygen augmenting superoxide formation (Velayutham et al., 2011).

The toxicity based on the dismutase activity of mutant SOD1 in the IMS might also be true even for dismutase inactive mutant SOD1 proteins. In human FALS SOD1 mutations are dominantly inherited resulting in the presence of both wild type and mutant SOD1 subunits in each cell. Thus, dismutase activity lacking G85R-SOD1 can form active heterodimers with wt SOD1 molecules. In mice the co-expression of human mutant and wt SOD1 accelerated disease (Jaarsma et al., 2000; Fukada et al., 2001; Deng et al., 2006). Importantly, unaffected A4V-SOD1 mutant mice developed the disease only when mated with human wt SOD1 overexpressing mice (Deng et al., 2006). It was also shown recently that the toxicity of mutant SOD1 dimers is not correlated with their capacity to form protein aggregates but rather with their dismutase activity (Witan et al., 2008).

### **5. Conclusion**

Until now, several pathological mechanisms have been demonstrated how mutant SOD1 induces mitochondrial dysfunction in FALS models. Among them, the emerging evidence indicates that the SOD1-dependant hydroperoxide production in mitochondrial IMS may fuel the cytochrome c-catalyzed peroxidation and play a key role in oxidation of biological targets in the IMS. Thus, SOD1 activity and factors leading to its increase in this compartment can be regarded as deleterious mechanisms to the mitochondria and the cell. Increased SOD1 activity causing elevated hydroperoxide production in the IMS may be one of general mechanism in neurodegeneration.

At the moment it is not clear how mutations in SOD1 directly affect hydroperoxide production in IMS. One possibility may be the already demonstrated increased mitochondrial import for mutant molecules in neurons of ALS models. Another possible mechanism is linked to less strict dismutase activity control. Altogether, we hypothesize that the mutant SOD1 may gain toxic features because the proper control mechanism for its dismutase activity in mitochondrial IMS may be lost. In conclusion, we suggest that SOD1 activity in the IMS is a relevant therapeutic target for ALS and other neurodegenerative diseases involving mitochondrial pathogenesis.
