**1. Introduction**

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140 Current Advances in Amyotrophic Lateral Sclerosis

Oxidative stress is defined as the imbalance between reactive species such as free radicals and oxidants and the antioxidant defenses. Free radicals are molecules with one or more unpaired electrons, while oxidants are molecules with a high potential for taking electrons from other molecules. The more recognized reactive species are the reactive oxygen species (ROS), which include oxygen and its reduction products superoxide, hydrogen peroxide and hydroxyl radical, and the reactive nitrogen species (RNS) such as the free radical nitric oxide and its byproducts, including the powerful oxidant peroxynitrite and the sub-product of peroxynitrite decomposition nitrogen dioxide.

As part of the antioxidant defense system, superoxide dismutase 1 (SOD1) is an abundant and highly conserved cytosolic enzyme responsible for the disproportionation of superoxide to molecular oxygen and hydrogen peroxide (McCord and Fridovich, 1969). SOD1 is a relatively small protein of 153 amino acids that works as a tight homodimer and requires a high stability for fast catalysis (Perry et al., 2010; Trumbull and Beckman, 2009). The stability is conferred by the quaternary structure of the protein, an eight-strand beta-barrel, as well as the binding of Cu and Zn, two metal ions with catalytic roles positioned in the active site channel (Perry et al., 2010; Trumbull and Beckman, 2009). The disproportionation of superoxide is a two-step oxidation-reduction reaction that involves the cycling of the copper atom in SOD1 from Cu2+ to Cu+ and back to Cu+2.

The zinc does not participate in this reaction but is essential for the structure of the active site. In addition, the formation of an intrasubunit disulfide bridge stabilizes the enzyme and plays an important role in preventing aggregation of metal-deficient SOD (Getzoff et al., 1989).

© 2013 Franco et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

lipids, and DNA was also found in transgenic mice and cell culture models (Barber and Shaw, 2010). On the other hand, other groups failed to find markers of oxidative damage in animal models of ALS, casting doubt on the relevance of oxidative stress in the pathogenesis of the disease (Barber and Shaw, 2010). Currently, a role for oxidative stress in ALS is generally accepted but whether oxidative stress is responsible for the mutant SOD1 gain-of-function is

Superoxide Dismutase and Oxidative Stress in Amyotrophic Lateral Sclerosis

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Mutant SOD1s have a tendency to aggregate when expressed in bacterial systems and transfected cells, and the presence of mutant and wild type SOD1-containing aggregates has been described in animal models of ALS (Bruijn and Cleveland, 1996; Watanabe et al., 2001). The formation of aggregates clogging the proteasome and containing other relevant proteins along with mutant SOD1 is one of the possible explanations for SOD1 toxic gain-of-function. However, in mice expressing the SOD1A4V mutant, the most common mutation linked to familial ALS in humans, the mutant is expressed at high levels and forms protein aggregates but does not cause disease (Gurney et al., 1994). Alternatively, other groups proposed a hypothesis in which the formation of aggregates is a protective mechanism rather than cause of toxicity. *In vitro* experiments showed that both wild type SOD1 and SOD1 with mutation of the cysteine residues involved in protein aggregation were able to stabilize the mutant SOD1 enzymes, increasing their toxicity (Clement et al., 2003; Fukada et al., 2001; Sahawneh et al., 2010; Witan et al., 2009). Additionally, it was recently described that overexpression of the deubiquitinating enzyme ataxin-3 stimulates the formation of SOD1-containing aggresomes by trimming K63-linked polyubiquitin chains. The knockdown of ataxin-3 decreases the formation of aggresomes and increases cell death induced by mutant SOD1 (Wang et al., 2012). These results suggest a toxic gain-of-function for the stabilized and soluble mutant SOD1, rather than toxicity due to aggregation. Indeed, by removing the toxic soluble mutant SOD1, the formation of aggregates has been proposed to be a protective mechanism (Trumbull and Beckman, 2009). Further support is provided by recent studies of crossbreeding showing an acceleration of the disease in mutant SOD1 transgenic mice overexpressing wild type SOD1, which was linked to the formation of disulfide bridges in the enzyme by oxidation of cysteine residues, increasing the formation of aggregates (Deng et al., 2006; Furukawa et al., 2006; Wang et al., 2009). Other investigations reproduced the acceleration of the disease in animals expressing both wild type and mutant SOD1 but failed to find a correlation between expression

still controversial.

**2.1. Mutant SOD1 aggregation and Zn-deficiency**

of wild type SOD1 and protein aggregation (Prudencio et al., 2009).

The link between the gain-of-function and the redox activity of soluble mutant SOD1 as a source of oxidative stress is based on the presence of the copper atom in the active site of the enzyme as well as the loss of zinc. The requirement for copper was challenged by genetic experiments in which the chaperone that delivers the copper metal to SOD1 was deleted. The ablation of the chaperone in the G93A, G85R, or G73R-SOD1 mutant mice decreased the activity of the enzyme but had no effect on the progression of the disease (Subramaniam et al., 2002), although it may be possible for SOD1 to acquire copper from an alternative source (Beckman et al., 2002). The transgenic expression of a SOD1 with mutations that eliminate the

Mutations in the gene codifying for SOD1 were linked to familial ALS almost 20 years ago. Currently, over 130 point mutations on more than 70 sites on SOD1 have been described, most of these being missense single residue mutations located in critical positions that affect the stability and folding of the enzyme (Beckman et al., 2001; Perry et al., 2010; Roberts et al., 2007). The goal of this chapter is to review recent advances in our understanding of the role of oxidative stress on the gain of a toxic function associated with mutations in the gene of the copper/zinc superoxide dismutase.
