**2. Zn-deficient SOD1**

The first proposed mechanisms linking mutations of SOD1 with ALS were based on the loss of dismutase activity (Beckman et al., 1993; Deng et al., 1993a). However, the SOD1 mutants G37R and G93A remain fully active and were linked to familial ALS (Borchelt et al., 1994; Yim et al., 1996). In addition, the mouse knockout for SOD1 developed normally and did not show signs of motor neuron deficit, although the motor neurons were more susceptible to cell death upon axonal injury (Reaume et al., 1996). This evidence indicated that a gain-of-function rather than the loss of function was responsible for motor neuron degeneration in ALS, and that the gain-of-function could be related to the redox properties of SOD1.

The discovery that mutations on the gene for an antioxidant enzyme such as SOD1 were associated with a population of familial ALS patients led to speculate on the role of oxidative stress in the pathogenesis of ALS (Beckman et al., 1993; Deng et al., 1993b; Rosen et al., 1993). From this original discovery to the present the interest on oxidative stress in ALS has been a rollercoaster. Several different groups described the presence of a variety of markers for oxidative stress in human samples and animal models of ALS, including elevated protein carbonyl and nitrotyrosine levels as well as lipid and DNA oxidation. Oxidation of proteins, 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 still controversial.

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

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

The first proposed mechanisms linking mutations of SOD1 with ALS were based on the loss of dismutase activity (Beckman et al., 1993; Deng et al., 1993a). However, the SOD1 mutants G37R and G93A remain fully active and were linked to familial ALS (Borchelt et al., 1994; Yim et al., 1996). In addition, the mouse knockout for SOD1 developed normally and did not show signs of motor neuron deficit, although the motor neurons were more susceptible to cell death upon axonal injury (Reaume et al., 1996). This evidence indicated that a gain-of-function rather than the loss of function was responsible for motor neuron degeneration in ALS, and that the

The discovery that mutations on the gene for an antioxidant enzyme such as SOD1 were associated with a population of familial ALS patients led to speculate on the role of oxidative stress in the pathogenesis of ALS (Beckman et al., 1993; Deng et al., 1993b; Rosen et al., 1993). From this original discovery to the present the interest on oxidative stress in ALS has been a rollercoaster. Several different groups described the presence of a variety of markers for oxidative stress in human samples and animal models of ALS, including elevated protein carbonyl and nitrotyrosine levels as well as lipid and DNA oxidation. Oxidation of proteins,

gain-of-function could be related to the redox properties of SOD1.

copper/zinc superoxide dismutase.

142 Current Advances in Amyotrophic Lateral Sclerosis

**2. Zn-deficient SOD1**

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 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 copper-binding site still produced disease (Prudencio et al., 2012; Wang et al., 2003). In contrast, another study showed that the mutant enzymes A4V, G85R, and G93A had a higher affinity for copper than the wild type protein, and that this aberrant copper binding was mediated by cysteine 111 (Watanabe et al., 2007), implying that the enzyme binds copper in an alternate site (Figure 1A).

mutants induce activation of caspase 1 and promoted apoptosis in N2a cells and tissue expressing mutant SOD1 when exposed to hydrogen peroxide. In NSC34 cells, a motor neuron model, mutant SOD1 induces cell death upon exposure of the cells to hydrogen peroxide (Pasinelli et al., 1998; Wiedau-Pazos et al., 1996). These findings suggest that the ALS pheno‐ type may require both, the genetic background and an additional oxidative challenge.

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Nitric oxide alone is not toxic to normal motor neurons (Estévez et al., 1999), but when superoxide is also produced it can react with nitric oxide to form the powerful oxidant peroxynitrite, responsible for the induction of cell death. Overexpression of mutant SOD1 makes motor neurons vulnerable to exogenous and endogenous production of nitric oxide. The increased vulnerability is linked to the activation of the Fas death pathways (Raoul et al., 2002). More recently it was shown that motor neurons from mutant SOD1 transgenic animals have lower levels of a calcium-binding ER chaperone calreticulin. A decrease in the expression of this protein is necessary and sufficient to activate the Fas/NO pathways in motor neurons. Further evidence *in vivo* shows that this protein is decreased in the spinal motor neurons of SOD1G93A transgenic animals prior to muscle denervation (Bernard-Marissal et al., 2012). Therefore, motor neurons expressing mutant SOD1 may produce superoxide making them susceptible to the formation of peroxynitrite in the presence of nitric oxide. In the presence of reductants, Zn-deficient SOD1 is able to produce superoxide. For instance, ascorbate reduces

the electrons from ascorbate to oxygen to produce superoxide slowly but significantly over a period of minutes. Indeed, Zn-deficient SOD1 is able to oxidize ascorbate 3000-fold faster than mutant or wild type Cu,Zn-SOD1 *in vitro* (Estévez et al., 1999). In the cells, ascorbate and other cellular antioxidants such as glutathione, urate, and cysteine could have a similar effect. Normally, superoxide would be removed by the dismutase activity of the remaining and fully active Cu,Zn-SOD1. However, if nitric oxide is also produced it can effectively compete with Cu,Zn-SOD1 for superoxide to produce peroxynitrite. Because nitric oxide is a small molecule able to diffuse 10-fold faster than a small size protein, the reaction of nitric oxide with super‐ oxide occurs 10 times faster than that with SOD1 (Beckman et al., 2001; Estévez et al., 1999; Franco and Estévez, 2011; Nauser and Koppenol, 2002) (Figure 1B). Wild type Cu,Zn-SOD1 can also produce peroxynitrite by a similar mechanism but requires superoxide in the initial

Cu,Zn-SOD1 is not only responsible for the production of peroxynitrite but it can also catalyze tyrosine nitration *in vitro* (Beckman et al., 1993; Crow et al., 1997b; Ischiropoulos et al., 1992). The mechanism for tyrosine nitration depends on the copper atom in SOD1 that reacts with peroxynitrite. The loss of zinc from Cu,Zn-SOD1 increases by 2-fold the efficiency of the enzyme to catalyze tyrosine nitration (Crow et al., 1997a) (Figure 1B). Moreover, SOD1 is not inactivated by peroxynitrite and can catalyze tyrosine nitration indefinitely. Indeed, reactivity for nitrotyrosine was found *in vivo* in the SOD1G93A mouse model and in patients with ALS

. In turn, Zn-deficient SOD1 can transfer

**2.3. Production of peroxynitrite**

the copper on Zn-deficient SOD1 from Cu2+ to Cu+

step to be efficiently reduced (Beckman et al., 2001).

**2.4. Catalysis of tyrosine nitration**

Some SOD1 mutants bind copper and zinc and are fully active (Borchelt et al., 1994; Marklund et al., 1997) but many mutations affect the binding of zinc while copper remains tightly bound, thus favoring the formation of Zn-deficient SOD. In the SOD1G93A mouse model of ALS, the dietary depletion of zinc accelerates the progression of the disease while moderate supplement of zinc provides protection (Ermilova et al., 2005). Indeed, a peak corresponding to one-metal SOD1 was detected *in vivo* in spinal cords from the SOD1G93A rat model using the recently developed methodology of electrospray mass spectrometry. The one-metal peak was 2-fold larger in the disease-affected ventral spinal cord compare to that of the dorsal spinal cord (Rhoads et al., 2011), suggesting that Zn-deficient SOD1 may be present *in vivo* in the affected tissue.

ALS-linked mutant SOD1s have 5-50 fold less affinity for zinc than the wild type protein (Crow et al., 1997a; Lyons et al., 1996). The loss of zinc disorganizes the structure of the active site leaving the copper metal more expose and accessible to substrates other than superoxide, decreasing the normal activity of the enzyme. When replete with zinc, SOD1 mutants can generally fulfill the antioxidant activity of wild type SOD (Crow et al., 1997a). Early studies showed that mutant SOD1 has an aberrant chemistry and is reduced abnormally fast which allows the reaction with oxidants such as hydrogen peroxide and peroxynitrite (Crow et al., 1997a; Crow et al., 1997b; Lyons et al., 1996; Wiedau-Pazos et al., 1996), thus turning the antioxidant enzyme into a catalyst for oxidation. The conversion of SOD1 from antioxidant to pro-oxidant due to the loss of zinc is a simple explanation for the gain-of-function attributed to the ALS-linked SOD mutants, but is still highly controversial.

#### **2.2. Formation of hydroxyl radical from hydrogen peroxide**

In normal conditions SOD1 catalyzes the disproportionation of superoxide to hydrogen peroxide, but due to changes in mutant SOD1 conformation, the mutant enzyme can catalyze the production of hydroxyl radical from hydrogen peroxide *in vitro* (Yim et al., 1990) (Figure 1B). The G93A-SOD1 mutant has enhanced free-radical production compare to the wild type enzyme due to a more open active site, decreasing the *Km* for hydrogen peroxide (Yim et al., 1996). Accordingly, an increase in the levels of hydrogen peroxide and hydroxyl radical was reported *in vivo* in the spinal cord from mice expressing the G93A mutant (Liu et al., 1999).

The aberrant chemistry of mutant SOD1 was shown to inactivate the glutamate transporter EAAT2 by oxidative reactions catalyzed by the A4V and I113T-SOD1 mutants and triggered by hydrogen peroxide (Trotti et al., 1999; Trotti et al., 1996). The function of this transporter is down regulated in human patients and animal models of ALS and its inactivation results in neuronal degeneration (Rothstein et al., 2005; Tanaka et al., 1997). Moreover, the aberrant SOD1 chemistry increases the vulnerability of a variety of cells in culture to hydrogen peroxide, with an increased susceptibility to inhibition by copper chelators. The G37R, G41D, and G85R-SOD1 mutants induce activation of caspase 1 and promoted apoptosis in N2a cells and tissue expressing mutant SOD1 when exposed to hydrogen peroxide. In NSC34 cells, a motor neuron model, mutant SOD1 induces cell death upon exposure of the cells to hydrogen peroxide (Pasinelli et al., 1998; Wiedau-Pazos et al., 1996). These findings suggest that the ALS pheno‐ type may require both, the genetic background and an additional oxidative challenge.

#### **2.3. Production of peroxynitrite**

copper-binding site still produced disease (Prudencio et al., 2012; Wang et al., 2003). In contrast, another study showed that the mutant enzymes A4V, G85R, and G93A had a higher affinity for copper than the wild type protein, and that this aberrant copper binding was mediated by cysteine 111 (Watanabe et al., 2007), implying that the enzyme binds copper in an alternate site

Some SOD1 mutants bind copper and zinc and are fully active (Borchelt et al., 1994; Marklund et al., 1997) but many mutations affect the binding of zinc while copper remains tightly bound, thus favoring the formation of Zn-deficient SOD. In the SOD1G93A mouse model of ALS, the dietary depletion of zinc accelerates the progression of the disease while moderate supplement of zinc provides protection (Ermilova et al., 2005). Indeed, a peak corresponding to one-metal SOD1 was detected *in vivo* in spinal cords from the SOD1G93A rat model using the recently developed methodology of electrospray mass spectrometry. The one-metal peak was 2-fold larger in the disease-affected ventral spinal cord compare to that of the dorsal spinal cord (Rhoads et al., 2011), suggesting that Zn-deficient SOD1 may be present *in vivo* in the affected

ALS-linked mutant SOD1s have 5-50 fold less affinity for zinc than the wild type protein (Crow et al., 1997a; Lyons et al., 1996). The loss of zinc disorganizes the structure of the active site leaving the copper metal more expose and accessible to substrates other than superoxide, decreasing the normal activity of the enzyme. When replete with zinc, SOD1 mutants can generally fulfill the antioxidant activity of wild type SOD (Crow et al., 1997a). Early studies showed that mutant SOD1 has an aberrant chemistry and is reduced abnormally fast which allows the reaction with oxidants such as hydrogen peroxide and peroxynitrite (Crow et al., 1997a; Crow et al., 1997b; Lyons et al., 1996; Wiedau-Pazos et al., 1996), thus turning the antioxidant enzyme into a catalyst for oxidation. The conversion of SOD1 from antioxidant to pro-oxidant due to the loss of zinc is a simple explanation for the gain-of-function attributed

In normal conditions SOD1 catalyzes the disproportionation of superoxide to hydrogen peroxide, but due to changes in mutant SOD1 conformation, the mutant enzyme can catalyze the production of hydroxyl radical from hydrogen peroxide *in vitro* (Yim et al., 1990) (Figure 1B). The G93A-SOD1 mutant has enhanced free-radical production compare to the wild type enzyme due to a more open active site, decreasing the *Km* for hydrogen peroxide (Yim et al., 1996). Accordingly, an increase in the levels of hydrogen peroxide and hydroxyl radical was reported *in vivo* in the spinal cord from mice expressing the G93A mutant (Liu et al., 1999). The aberrant chemistry of mutant SOD1 was shown to inactivate the glutamate transporter EAAT2 by oxidative reactions catalyzed by the A4V and I113T-SOD1 mutants and triggered by hydrogen peroxide (Trotti et al., 1999; Trotti et al., 1996). The function of this transporter is down regulated in human patients and animal models of ALS and its inactivation results in neuronal degeneration (Rothstein et al., 2005; Tanaka et al., 1997). Moreover, the aberrant SOD1 chemistry increases the vulnerability of a variety of cells in culture to hydrogen peroxide, with an increased susceptibility to inhibition by copper chelators. The G37R, G41D, and G85R-SOD1

to the ALS-linked SOD mutants, but is still highly controversial.

**2.2. Formation of hydroxyl radical from hydrogen peroxide**

(Figure 1A).

144 Current Advances in Amyotrophic Lateral Sclerosis

tissue.

Nitric oxide alone is not toxic to normal motor neurons (Estévez et al., 1999), but when superoxide is also produced it can react with nitric oxide to form the powerful oxidant peroxynitrite, responsible for the induction of cell death. Overexpression of mutant SOD1 makes motor neurons vulnerable to exogenous and endogenous production of nitric oxide. The increased vulnerability is linked to the activation of the Fas death pathways (Raoul et al., 2002). More recently it was shown that motor neurons from mutant SOD1 transgenic animals have lower levels of a calcium-binding ER chaperone calreticulin. A decrease in the expression of this protein is necessary and sufficient to activate the Fas/NO pathways in motor neurons. Further evidence *in vivo* shows that this protein is decreased in the spinal motor neurons of SOD1G93A transgenic animals prior to muscle denervation (Bernard-Marissal et al., 2012). Therefore, motor neurons expressing mutant SOD1 may produce superoxide making them susceptible to the formation of peroxynitrite in the presence of nitric oxide. In the presence of reductants, Zn-deficient SOD1 is able to produce superoxide. For instance, ascorbate reduces the copper on Zn-deficient SOD1 from Cu2+ to Cu+ . In turn, Zn-deficient SOD1 can transfer the electrons from ascorbate to oxygen to produce superoxide slowly but significantly over a period of minutes. Indeed, Zn-deficient SOD1 is able to oxidize ascorbate 3000-fold faster than mutant or wild type Cu,Zn-SOD1 *in vitro* (Estévez et al., 1999). In the cells, ascorbate and other cellular antioxidants such as glutathione, urate, and cysteine could have a similar effect. Normally, superoxide would be removed by the dismutase activity of the remaining and fully active Cu,Zn-SOD1. However, if nitric oxide is also produced it can effectively compete with Cu,Zn-SOD1 for superoxide to produce peroxynitrite. Because nitric oxide is a small molecule able to diffuse 10-fold faster than a small size protein, the reaction of nitric oxide with super‐ oxide occurs 10 times faster than that with SOD1 (Beckman et al., 2001; Estévez et al., 1999; Franco and Estévez, 2011; Nauser and Koppenol, 2002) (Figure 1B). Wild type Cu,Zn-SOD1 can also produce peroxynitrite by a similar mechanism but requires superoxide in the initial step to be efficiently reduced (Beckman et al., 2001).

#### **2.4. Catalysis of tyrosine nitration**

Cu,Zn-SOD1 is not only responsible for the production of peroxynitrite but it can also catalyze tyrosine nitration *in vitro* (Beckman et al., 1993; Crow et al., 1997b; Ischiropoulos et al., 1992). The mechanism for tyrosine nitration depends on the copper atom in SOD1 that reacts with peroxynitrite. The loss of zinc from Cu,Zn-SOD1 increases by 2-fold the efficiency of the enzyme to catalyze tyrosine nitration (Crow et al., 1997a) (Figure 1B). Moreover, SOD1 is not inactivated by peroxynitrite and can catalyze tyrosine nitration indefinitely. Indeed, reactivity for nitrotyrosine was found *in vivo* in the SOD1G93A mouse model and in patients with ALS (Beal et al., 1997; Ferrante et al., 1997). In spite of the indirect evidence of mass spectrometry showing a peak corresponding to a one-metal SOD1 in a rat model of ALS (Rhoads et al., 2011), whether Zn-deficient SOD1 is present *in vivo* and catalyzes tyrosine nitration is still source of debate and remains to be determined.

mice (Mattiazzi et al., 2002; Panov et al., 2011) support the mutant SOD1 aberrant catalytic gain-of-function. Indeed, it was shown that metal-deficient SOD1s are prone to mitochondrial translocation and are found in the mitochondrial intermembrane space (Okado-Matsumoto and Fridovich, 2002). The mitochondria contain the majority of the cellular copper because is required by the oxygen-consuming proteins. The insertion of copper into the translocated metal-deficient SOD would result in the formation of Zn-deficient SOD inside the mitochon‐ dria (Figure 1A). This could explain why the mitochondria are affected early in the onset of the disease (Beckman et al., 2002). The ROS-linked toxic gain-of-function of mutant SOD1 would produce hydroxyl radical from H2O2 as well as peroxynitrite in the mitochondria. The mutant enzyme could then catalyze the nitration of mitochondrial proteins such as cyclophilin D and the adenine nucleotide translocator (Martin, 2010). Due to these toxic effects of mutant SOD1 on mitochondria, it has been proposed that the abnormal activity of the mitochondria in ALS may account for the initiation and progression of the disease. However, whether the mitochondrial localization of mutant SOD1 is cause or a consequence of pathology needs to

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**5. Expression of mutant SOD1 in motor neurons and neighboring cells**

macrophages, and astrocytes would play a role in the late disease progression.

**5.1. Expression of mutant SOD1 in motor neurons**

A new mechanism integrating the autonomous and non-autonomous induction of motor neuron death in ALS is emerging. In this scenario, the role of motor neurons and surrounding cells in the onset and progression of ALS is temporally determined. Several studies were conducted where mutant SOD1 was selectively expressed *in vivo* either in motor neurons or microglia of chimeric mice, or in culture in embryonic primary or stem cell-based models, allowing the study of the role of individual population of cells in the onset and progression of ALS. The cell-autonomous degeneration of motor neurons expressing mutant SOD1 seems to be more relevant for the onset and early progression of the disease, while microglia, peripheral

ALS is a motor neuron disease characterized by the gradual and selective loss of both, upper and lower motor neurons. Expression of mutant SOD1 in spinal motor neurons and inter‐ neurons of chimeric mice is enough to induce neuronal degeneration (Boillee et al., 2006; Wang et al., 2008). The mice do not develop clinical ALS but the motor neurons expressing mutant SOD1 exhibit pathological and immunohistochemical abnormalities, while motor neurons negative for mutant SOD1 expression do not. These observations indicate that in the chimeric mice the degeneration of motor neurons can be cell-autonomous. The fact that only some of the motor neurons express mutant SOD1 in this model may explain why the animals do not develop the disease (Wang et al., 2008). Indeed, normal motor neurons can prevent or delay the degeneration of mutant SOD1-expressing motor neurons (Clement et al., 2003). In addition, decreased expression of mutant SOD1 in motor neurons has a modest effect on the duration of the disease but significantly delay the onset and early phase of the disease progression (Wang et al., 2008). Similar results were observed in culture, where primary spinal motor

be established.
