**3. Peroxynitrite and apoptosis**

Unsurprisingly, peroxynitrite induces apoptosis or necrosis depending on the concentration of the oxidant (Bonfoco et al. 1995; Estévez et al. 1995), and it has become the accepted mechanism for the toxic effects of nitric oxide in biological systems (Dawson and Dawson 1996; Dawson and Dawson 1996; Dawson and Dawson 1996; Beckman and Koppenol 1996). Although growing evidence suggests that peroxynitrite induces apoptosis by interacting with specific cellular signaling pathways (Estévez et al. 1995; Shin et al. 1996; Spear, Estévez, Barbeito, et al. 1997; Spear, Estévez, Radi, et al. 1997; Shacka et al. 2006; Ye et al. 2007) (Fig 3), the cellular targets responsible for peroxynitrite-induced apoptosis remain unknown. In addition, most studies were performed using exogenously applied stock solutions of pure peroxynitrite or peroxynitrite donors (Bonfoco et al. 1995; Estévez et al. 1995).

Reactive Nitrogen Species in Motor Neuron Apoptosis 317

Fig. 4. Motor neuron apoptosis induced by trophic factor deprivation. Induction of motor neuron apoptosis by trophic factor deprivation is prevented by inhibition of JNK and p38 MAP kinases. The induction of neuronal NOS (nNOS) is regulated by the activation of p38 and responsible for the production of nitric oxide. Nitric oxide reacts with superoxide to form peroxynitrite. Inhbition of tyrosine nitration by peroxynitrite using tyrosine-

containing peptides is enough to prevent apoptosis induced by trophic factor deprivation. Caspase inhbitors also prevented apoptosis mediated by trophic factors deprivation. 1(Ricart et al., 2006); 2(Raoul et al., 1999b); 3(Raoul et al., 2002); 4(Estévez et al., 1998); 5(Estévez et al., 2000); 6(Estevez et al., 2006); 7(Estévez et al., 1999); 8(Peluffo et al., 2004); 9(Ye et al., 2007);

Apoptosis induced by trophic factor deprivation is preceded by the induction of Fas ligand expression and prevented, at least in part, by inhibition of Fas and caspase 8 (Raoul, Henderson, and Pettmann 1999). In the presence of trophic factors, Fas activates two parallel pathways leading to motor neuron apoptosis by a mechanism similar to trophic

10(Cassina et al., 2002); 11(Milligan et al., 1995); 12(Li et al., 1998); 13(Li et al., 2001)

Fig. 5. In motor neurons, the activation of the Fas pathway leads to the simultaneous activation of the FADD and DAXX components of the pathway. Downstream of DAXX, p38 induces the expression of nNOS leading to the formation of peroxynitrite while activation of FADD leads to activation of caspases. Upon activation of Fas, both pathways participate simoultaneously in the induction of cell death. Inhibition of JNK may be upstream of the Fas pathway through activation of the transcription factor FOXO3a and transcription of FasL.

factor deprivation (Raoul et al. 2002)(Fig 5).

Fig. 3. Cell death-pathway induced by peroxynitrite in PC12 cells.

### **4. Motor neuron death and peroxynitrite** *in vivo*

More recently, cultured motor neurons have become one of the best-described models for apoptosis induced by endogenous peroxynitrite (Estévez, Spear, Manuel, Radi, et al. 1998; Estévez, Spear, Manuel, Barbeito, et al. 1998; Estévez et al. 2000; Sendtner et al. 2000; Raoul et al. 2002; Raoul, Pettmann, and Henderson 2000; Kaal et al. 2000; Bar 2000). Motor neurons are large neurons located in the ventral spinal cord and brain stem responsible for the stimulation of muscle contraction. Motor neuron survival is highly dependent on trophic factors (Oppenheim 1991; Oppenheim 1996; Sendtner et al. 2000). Chronic administration of trophic factors prevents avian and mammalian motor neurons death during the period of programmed cell death (Neff et al. 1993) and motor neuron apoptosis induced by axon injury in mammals (Yan, Elliott, and Snider 1992; Li et al. 1994; Novikov, Novikova, and Kellerth 1995; Pennica et al. 1996). Remarkably, motor neurons induce the expression of neuronal NOS and the p75 neurotrophin receptor after injury (Wu 1993). Trophic factors such as BDNF, and nerve grafts prevent the induction of neuronal NOS and motor neuron death (Wu et al. 1994; Novikov, Novikova, and Kellerth 1995). Furthermore, inhibition of NOS activity prevents motor neuron death induced by axonal injury (Wu and Li 1993; Casanovas et al. 1996), suggesting that induction of motor neuron death after axonal injury may result from trophic factor deprivation leading to the induction of neuronal NOS as well as nitric oxide and peroxynitrite production, evidenced by the increase levels of nitrotyrosine in motor neurons after axotomy (Martin, Kaiser, and Price 1999).

### **5. Motor neuron apoptosis** *in vitro*

Motor neuron survival in culture can be supported by a large number of trophic factors (Oppenheim 1996; Hughes, Sendtner, and Thoenen 1993), which also induce the extension of long and branched neurites. As many other cells in culture, trophic factor-deprived motor neurons undergo protein synthesis and caspase-dependent apoptosis (Milligan, Oppenheim, and Schwartz 1994; Milligan et al. 1995; Estévez, Spear, Manuel, Radi, et al. 1998; Estévez et al. 2000) (Fig 4).

More recently, cultured motor neurons have become one of the best-described models for apoptosis induced by endogenous peroxynitrite (Estévez, Spear, Manuel, Radi, et al. 1998; Estévez, Spear, Manuel, Barbeito, et al. 1998; Estévez et al. 2000; Sendtner et al. 2000; Raoul et al. 2002; Raoul, Pettmann, and Henderson 2000; Kaal et al. 2000; Bar 2000). Motor neurons are large neurons located in the ventral spinal cord and brain stem responsible for the stimulation of muscle contraction. Motor neuron survival is highly dependent on trophic factors (Oppenheim 1991; Oppenheim 1996; Sendtner et al. 2000). Chronic administration of trophic factors prevents avian and mammalian motor neurons death during the period of programmed cell death (Neff et al. 1993) and motor neuron apoptosis induced by axon injury in mammals (Yan, Elliott, and Snider 1992; Li et al. 1994; Novikov, Novikova, and Kellerth 1995; Pennica et al. 1996). Remarkably, motor neurons induce the expression of neuronal NOS and the p75 neurotrophin receptor after injury (Wu 1993). Trophic factors such as BDNF, and nerve grafts prevent the induction of neuronal NOS and motor neuron death (Wu et al. 1994; Novikov, Novikova, and Kellerth 1995). Furthermore, inhibition of NOS activity prevents motor neuron death induced by axonal injury (Wu and Li 1993; Casanovas et al. 1996), suggesting that induction of motor neuron death after axonal injury may result from trophic factor deprivation leading to the induction of neuronal NOS as well as nitric oxide and peroxynitrite production, evidenced by the increase levels of nitrotyrosine in motor neurons

Motor neuron survival in culture can be supported by a large number of trophic factors (Oppenheim 1996; Hughes, Sendtner, and Thoenen 1993), which also induce the extension of long and branched neurites. As many other cells in culture, trophic factor-deprived motor neurons undergo protein synthesis and caspase-dependent apoptosis (Milligan, Oppenheim, and Schwartz 1994; Milligan et al. 1995; Estévez, Spear, Manuel, Radi, et al.

Fig. 3. Cell death-pathway induced by peroxynitrite in PC12 cells.

**4. Motor neuron death and peroxynitrite** *in vivo*

after axotomy (Martin, Kaiser, and Price 1999).

**5. Motor neuron apoptosis** *in vitro*

1998; Estévez et al. 2000) (Fig 4).

Fig. 4. Motor neuron apoptosis induced by trophic factor deprivation. Induction of motor neuron apoptosis by trophic factor deprivation is prevented by inhibition of JNK and p38 MAP kinases. The induction of neuronal NOS (nNOS) is regulated by the activation of p38 and responsible for the production of nitric oxide. Nitric oxide reacts with superoxide to form peroxynitrite. Inhbition of tyrosine nitration by peroxynitrite using tyrosinecontaining peptides is enough to prevent apoptosis induced by trophic factor deprivation. Caspase inhbitors also prevented apoptosis mediated by trophic factors deprivation. 1(Ricart et al., 2006); 2(Raoul et al., 1999b); 3(Raoul et al., 2002); 4(Estévez et al., 1998); 5(Estévez et al., 2000); 6(Estevez et al., 2006); 7(Estévez et al., 1999); 8(Peluffo et al., 2004); 9(Ye et al., 2007); 10(Cassina et al., 2002); 11(Milligan et al., 1995); 12(Li et al., 1998); 13(Li et al., 2001)

Apoptosis induced by trophic factor deprivation is preceded by the induction of Fas ligand expression and prevented, at least in part, by inhibition of Fas and caspase 8 (Raoul, Henderson, and Pettmann 1999). In the presence of trophic factors, Fas activates two parallel pathways leading to motor neuron apoptosis by a mechanism similar to trophic factor deprivation (Raoul et al. 2002)(Fig 5).

Fig. 5. In motor neurons, the activation of the Fas pathway leads to the simultaneous activation of the FADD and DAXX components of the pathway. Downstream of DAXX, p38 induces the expression of nNOS leading to the formation of peroxynitrite while activation of FADD leads to activation of caspases. Upon activation of Fas, both pathways participate simoultaneously in the induction of cell death. Inhibition of JNK may be upstream of the Fas pathway through activation of the transcription factor FOXO3a and transcription of FasL.

Reactive Nitrogen Species in Motor Neuron Apoptosis 319

On the other hand, although formation of nitrotyrosine can be catalyzed from nitrite and hydrogen peroxide by peroxidases and transition metals (van der Vliet et al. 1997; Eiserich et al. 1998; Schopfer, Baker, and Freeman 2003), incubation of motor neurons with micromolar concentrations of nitrite and/or hydrogen peroxide has no effect on the survival of motor neurons in culture (Estévez et al. 2000) or tyrosine nitration (Ye et al. 2007), further supporting peroxynitrite formation. Together these results suggest that peroxynitrite formation is necessary for the induction of motor neuron apoptosis by trophic factor deprivation and after Fas pathway activation. In addition, scavenging the nitrating radical products of peroxynitrite by tyrosine-containing peptides does not affect thiol oxidation but prevents nitrotyrosine formation and motor neuron death (Ye et al. 2007)(Fig 6). These results suggest that tyrosine nitration has a causal role in the induction of motor neurons apoptosis by peroxynitrite and it

Fig. 6. Peptides that scavenge the nitrating products derived from peroxynitrite decomposition prevent cell death induced by the pure oxidant or endogenously produced peroxynitrite.

The relevance of the Fas pathways in the regulation of motor neurons death *in vivo* was shown in studies on the effects of axonal injury in mice knockout for Fas and transgenic mice expressing a dominant negative form of FADD, where axotomy-induced motor neuron degeneration was blocked (Ugolini et al. 2003; Martin, Chen, and Liu 2005). Axonal injury is also associated with increased nitrotyrosine immunoreactivity (Martin, Kaiser, and Price 1999; Martin, Chen, and Liu 2005), reveling that in addition to the activation of the classical Fas pathway, peroxynitrite is also produced. These observations suggest that the atypical pathways involved in the induction of motor neuron apoptosis by Fas activation are also active *in vivo* and play a role in the degeneration of adult motor neurons. In addition, SOD deficiency increases motor neuron vulnerability to axotomy (Reaume et al. 1996), indicating that production of superoxide plays an important role in motor neuron degeneration i*n vivo*. Even when the source of superoxide for the formation of peroxynitrite *in vivo* and in cultured motor neurons remains unknown, evidence from other neuronal types suggest that the induction and activation of NADPH oxidase might be responsible for the production of

**6. Extrinsic apoptotic pathway and motor neuron apoptosis** *in vivo*

is not only a marker for the formation of reactive nitrogen species.

Motor neuron apoptosis induced by trophic factor deprivation is also dependent on the expression of neuronal NOS and the production of nitric oxide (Estévez, Spear, Manuel, Radi, et al. 1998; Estévez, Spear, Manuel, Barbeito, et al. 1998; Estévez et al. 2000; Raoul et al. 2002; Raoul et al. 2005). Either inhibition of nitric oxide production or scavenging of superoxide with Cu,Zn SOD prevents motor neuron apoptosis induced by trophic factor deprivation up to seven days after plating (Estévez et al. 2000). The protective effects of NOS inhibition are reverted by steady state concentrations of exogenous nitric oxide as low as 80 nM (Estévez et al. 2000). Remarkably, 7 days old motor neuron cultures undergo apoptosis when deprived of trophic factors by a mechanism indistinguishably from the cell death induced by plating motor neurons in the absence of trophic factors (Estévez et al. 2000). These results reveal that production of nitric oxide or superoxide alone is not sufficient for the induction of motor neuron apoptosis by trophic factor deprivation (Estévez et al. 2000; Estévez, Spear, Manuel, Radi, et al. 1998; Raoul et al. 2002; Raoul et al. 2005). In addition, an increase in nitrotyrosine immunoreactivity is detected in motor neurons deprived of trophic factors suggesting peroxynitrite formation (Estévez, Spear, Manuel, Radi, et al. 1998; Raoul et al. 2002)(Fig 4).

Inhibition of the JNK MAP kinase activity blocks trophic factor deprivation-induced apoptosis, but has not effect on motor neuron apoptosis induced by Fas activation (Raoul et al. 2002; Ricart et al. 2006; Li, Oppenheim, and Milligan 2001; Newbern et al. 2007). Activation of JNK leads to the phosphorylation of transcription factors and the induction of protein synthesis and might induce the expression of Fas ligand (Le-Niculescu et al. 1999; Morishima et al. 2001), suggesting that JNK activation may be upstream of Fas activation (Barthelemy, Henderson, and Pettmann 2004). JNK phosphorylation of 14-3-3 proteins can stimulate the translocation of Bad to the mitochondria and the activation of FOXO3a (Sunayama et al. 2005; Vogt, Jiang, and Aoki 2005). In turn, FOXO3a regulates the expression of Fas ligand in motor neurons (Barthelemy, Henderson, and Pettmann 2004), which suggests a pathway integrating the dependence of both JNK and FOXO3a in the induction of motor neuron apoptosis.

Inhibition of p38 MAP kinase prevents apoptosis induced by Fas pathway activation but has no effect on trophic factor deprivation-induced apoptosis, further suggesting the activation of more than one apoptotic pathway by trophic factor deprivation. In fact, motor neuron apoptosis induced by Fas activation occurs by an atypical mechanism involving the two parallel pathways (Raoul et al. 2002; Raoul et al. 2006)(Fig 5). One of the pathways is the classical caspase 8-mediated mitochondrial apoptotic pathway. The other pathway is responsible for the induction of neuronal NOS by a mechanism involving sequential activation of DAXX, ASK1 and p38 MAP kinase (Raoul et al. 2002). Activation of either pathway seems to be able to induce apoptosis by itself, but when activated together the process occurs faster. The original discussion on a possible mechanism for peroxynitrite and nitric oxide to enhance the caspase 8-mitochondria apoptotic pathway was based in the literature indicating that both peroxynitrite and nitric oxide affect the mitochondrial function. On the other hand, activation of caspase 8 by Fas occurs by means of the DISC complex recruitment (Medema et al. 1997). Another possible explanation is that peroxynitrite may be able to induce the activation of caspase 8 by interacting with some of the components of the DISC, which also could make the Fas activation of this complex easier, resulting in a faster induction of motor neuron apoptosis.

Motor neuron apoptosis induced by trophic factor deprivation is also dependent on the expression of neuronal NOS and the production of nitric oxide (Estévez, Spear, Manuel, Radi, et al. 1998; Estévez, Spear, Manuel, Barbeito, et al. 1998; Estévez et al. 2000; Raoul et al. 2002; Raoul et al. 2005). Either inhibition of nitric oxide production or scavenging of superoxide with Cu,Zn SOD prevents motor neuron apoptosis induced by trophic factor deprivation up to seven days after plating (Estévez et al. 2000). The protective effects of NOS inhibition are reverted by steady state concentrations of exogenous nitric oxide as low as 80 nM (Estévez et al. 2000). Remarkably, 7 days old motor neuron cultures undergo apoptosis when deprived of trophic factors by a mechanism indistinguishably from the cell death induced by plating motor neurons in the absence of trophic factors (Estévez et al. 2000). These results reveal that production of nitric oxide or superoxide alone is not sufficient for the induction of motor neuron apoptosis by trophic factor deprivation (Estévez et al. 2000; Estévez, Spear, Manuel, Radi, et al. 1998; Raoul et al. 2002; Raoul et al. 2005). In addition, an increase in nitrotyrosine immunoreactivity is detected in motor neurons deprived of trophic factors suggesting peroxynitrite formation (Estévez, Spear, Manuel,

Inhibition of the JNK MAP kinase activity blocks trophic factor deprivation-induced apoptosis, but has not effect on motor neuron apoptosis induced by Fas activation (Raoul et al. 2002; Ricart et al. 2006; Li, Oppenheim, and Milligan 2001; Newbern et al. 2007). Activation of JNK leads to the phosphorylation of transcription factors and the induction of protein synthesis and might induce the expression of Fas ligand (Le-Niculescu et al. 1999; Morishima et al. 2001), suggesting that JNK activation may be upstream of Fas activation (Barthelemy, Henderson, and Pettmann 2004). JNK phosphorylation of 14-3-3 proteins can stimulate the translocation of Bad to the mitochondria and the activation of FOXO3a (Sunayama et al. 2005; Vogt, Jiang, and Aoki 2005). In turn, FOXO3a regulates the expression of Fas ligand in motor neurons (Barthelemy, Henderson, and Pettmann 2004), which suggests a pathway integrating the dependence of both JNK and FOXO3a in the

Inhibition of p38 MAP kinase prevents apoptosis induced by Fas pathway activation but has no effect on trophic factor deprivation-induced apoptosis, further suggesting the activation of more than one apoptotic pathway by trophic factor deprivation. In fact, motor neuron apoptosis induced by Fas activation occurs by an atypical mechanism involving the two parallel pathways (Raoul et al. 2002; Raoul et al. 2006)(Fig 5). One of the pathways is the classical caspase 8-mediated mitochondrial apoptotic pathway. The other pathway is responsible for the induction of neuronal NOS by a mechanism involving sequential activation of DAXX, ASK1 and p38 MAP kinase (Raoul et al. 2002). Activation of either pathway seems to be able to induce apoptosis by itself, but when activated together the process occurs faster. The original discussion on a possible mechanism for peroxynitrite and nitric oxide to enhance the caspase 8-mitochondria apoptotic pathway was based in the literature indicating that both peroxynitrite and nitric oxide affect the mitochondrial function. On the other hand, activation of caspase 8 by Fas occurs by means of the DISC complex recruitment (Medema et al. 1997). Another possible explanation is that peroxynitrite may be able to induce the activation of caspase 8 by interacting with some of the components of the DISC, which also could make the Fas activation of this complex easier, resulting in a faster induction of motor neuron

Radi, et al. 1998; Raoul et al. 2002)(Fig 4).

induction of motor neuron apoptosis.

apoptosis.

On the other hand, although formation of nitrotyrosine can be catalyzed from nitrite and hydrogen peroxide by peroxidases and transition metals (van der Vliet et al. 1997; Eiserich et al. 1998; Schopfer, Baker, and Freeman 2003), incubation of motor neurons with micromolar concentrations of nitrite and/or hydrogen peroxide has no effect on the survival of motor neurons in culture (Estévez et al. 2000) or tyrosine nitration (Ye et al. 2007), further supporting peroxynitrite formation. Together these results suggest that peroxynitrite formation is necessary for the induction of motor neuron apoptosis by trophic factor deprivation and after Fas pathway activation. In addition, scavenging the nitrating radical products of peroxynitrite by tyrosine-containing peptides does not affect thiol oxidation but prevents nitrotyrosine formation and motor neuron death (Ye et al. 2007)(Fig 6). These results suggest that tyrosine nitration has a causal role in the induction of motor neurons apoptosis by peroxynitrite and it is not only a marker for the formation of reactive nitrogen species.

Fig. 6. Peptides that scavenge the nitrating products derived from peroxynitrite decomposition prevent cell death induced by the pure oxidant or endogenously produced peroxynitrite.

### **6. Extrinsic apoptotic pathway and motor neuron apoptosis** *in vivo*

The relevance of the Fas pathways in the regulation of motor neurons death *in vivo* was shown in studies on the effects of axonal injury in mice knockout for Fas and transgenic mice expressing a dominant negative form of FADD, where axotomy-induced motor neuron degeneration was blocked (Ugolini et al. 2003; Martin, Chen, and Liu 2005). Axonal injury is also associated with increased nitrotyrosine immunoreactivity (Martin, Kaiser, and Price 1999; Martin, Chen, and Liu 2005), reveling that in addition to the activation of the classical Fas pathway, peroxynitrite is also produced. These observations suggest that the atypical pathways involved in the induction of motor neuron apoptosis by Fas activation are also active *in vivo* and play a role in the degeneration of adult motor neurons. In addition, SOD deficiency increases motor neuron vulnerability to axotomy (Reaume et al. 1996), indicating that production of superoxide plays an important role in motor neuron degeneration i*n vivo*. Even when the source of superoxide for the formation of peroxynitrite *in vivo* and in cultured motor neurons remains unknown, evidence from other neuronal types suggest that the induction and activation of NADPH oxidase might be responsible for the production of

Reactive Nitrogen Species in Motor Neuron Apoptosis 321

Further evidence for the participation of apoptosis in ALS was provided by the use of transgenic models of the disease (Shibata 2001). Histological analysis of motor neurons from transgenic mice carrying G93A SOD mutant shows decreased expression of the antiapoptotic proteins Bcl-2 and Bcl-xl and increased expression of the pro-apoptotic Bcl-2 family members Bax and Bad, which expression was attenuated following over-expression of Bcl-2 (Vukosavic et al. 1999; Vukosavic et al. 2000). Genetic deletion of Bax in transgenic mice for the G93A ALS-linked mutant SOD delays onset and extends the lifespan of the animals, but it does not prevent the disease in spite of preventing motor neuron death (Gould et al. 2006). These results are a clear testimony to the complexity of the disease process. Although motor neurons are protected against apoptosis, other abnormalities such as neuromuscular denervation and mitochondrial vacuolization are still occurring (Gould et al. 2006). Other studies suggest that alterations of the neuromuscular junction are between the first symptoms of the disease both in humans and animals models of the disease (Fischer et al. 2004). However, it is important to remember that in the human disease motor neurons do die. The artificial deletion of a gene that regulates apoptosis may just inhibit the final step in the death process, without affecting upstream pathways responsible for the death of the cells. In addition, these results suggest that reactive oxygen and nitrogen species formed during the disease process are not final effectors of cell death, but rather upstream triggers

of the activation of signaling pathways resulting in motor neuron death.

apoptosis in the spinal cord ventral horn and increases lifespan (Hetz et al. 2007).

(Martin et al. 2007).

The role of caspases in ALS is more controversial. While several authors have reported activation of caspases and a functional role for these activated proteases in animal models of the disease (Friedlander et al. 1997; Li et al. 2000; Pasinelli et al. 1998; Nagai et al. 2001; Pasinelli et al. 2000), others were unable to find a functional role for the enzymes and report that motor neuron death in the G93A mouse model is independent of caspase activation

To study the role of mutant SOD1 in different cell types involved in the pathology of ALS, chimeric mice were developed with mixtures of normal and SOD1 mutant-expressing cells. Normal motor neurons in SOD1 mutant chimeras develop features of ALS pathology. However, non-neuronal cells that do not express mutant SOD1 delay degeneration and significantly extend survival of mutant expressing motor neurons (Clement et al. 2003). When primary mouse spinal motor neurons express mutant human SOD1, the cells are not triggered to degenerate, however when rodent astrocytes express mutated SOD1, they kill

Further evidence for the role of apoptosis in the pathogenesis of ALS comes from the delay in onset and progression of the disease due to over-expression of the anti-apoptotic protein Bcl-2 in a transgenic mouse model of ALS (Vukosavic et al. 2000). More recently it was reported that Bcl-2 binds both wild-type and mutant SOD1 *in vitro* and *in vivo*. Because Bcl-2 associated with mutant SOD1 is present in protein aggregates located in mitochondria from the spinal cord of ALS patient and animal models of the disease, it was suggested that entrapment of Bcl-2 by SOD1 leads to the depletion of the anti-apoptotic protein in motor neurons, increasing the vulnerability of these cells (Pasinelli et al. 2004). However, no evidence for the aggregates selectively located in motor neuron mitochondria has been reported. Conversely, BIM, a member of the pro-apoptotic family of Bcl-2 proteins, is upregulated in the SOD1G93A familial ALS mouse model during the symptomatic stage of the disease, and its expression is required to trigger cell death induced by SOD1 mutants *in vitro*. Genetic deletion of BIM in an animal model of ALS results in reduced cellular

superoxide that makes nitric oxide toxic to motor neurons (Noh and Koh 2000; Tammariello, Quinn, and Estus 2000). In motor neurons, activation of the p75 neurotrophic receptor results in apoptosis in different conditions (Ricart et al. 2006; Pehar et al. 2004; Pehar et al. 2007; Wiese et al. 1999). At least in part this toxicity is mediated by induction of superoxide production by mitochondria (Pehar et al. 2007). In summary, inhibition of nitric oxide production blocks motor neuron death induced by ventral root avulsion, and deletion of SOD increases the sensitivity of motor neurons to the same noxious stimulus. Moreover, motor neuron degeneration after ventral root avulsion is preceded by increased tyrosine nitration and the activation of the death receptor pathways. In aggregate these results reveal that motor neuron death *in vitro* and *in vivo* occurs largely by the same mechanisms and through the activation of the same signaling pathways.

### **7. ALS and reactive nitrogen species**

A pathological condition associated with an increased expression of neuronal NOS and nitrotyrosine in motor neurons is amyotrophic lateral sclerosis (ALS)(Abe et al. 1995; Beal et al. 1997; Chou, Wang, and Komai 1996; Chou, Wang, and Taniguchi 1996, , Barber, 2010 #1694). As for today more than 25 reports have found increased levels of nitrotyrosine immunoreactivity or free nitrotyrosine in tissue from patients and animal models of ALS. The definitive evidence for the presence of nitrotyrosine, at least in a transgenic mouse model of ALS, was provided by mass spectrometry studies identifying some of the nitrated proteins and the nitrated residues. Other studies confirmed the identity of the nitrated proteins showing that motor neurons in pre-symptomatic mutant SOD1 mice generate superoxide, NO and ONOO- at higher levels than control motor neurons. In addition, nitration of Cox-I, SOD2 and -synuclein occurs in pre-symptomatic mutant SOD1 mice suggesting a role for peroxynitrite in the pathogenesis of the disease (Martin et al. 2007). ALS is a neurodegenerative disease characterized by the death of pyramidal neurons in the motor cortex and motor neurons in the brain stem and ventral spinal cord. About 2% of all ALS cases are due to the presence of one of more than 100 mutations in the gene encoding Cu,Zn SOD (Cleveland and Rothstein 2001; Traub, Mitsumoto, and Rowland 2011). When expressed in mice and rats, some of the human ALS-linked SOD mutations produce a motor neuron disease reminiscent of ALS (Gurney et al. 1994; Dal Canto and Gurney 1995; Wong et al. 1995; Bruijn et al. 1997); these are currently the most widely accepted models for the disease. It is generally accepted that the toxic effect of the mutations is due to a gain-offunction (Cleveland and Rothstein 2001). Growing evidence implicates apoptosis as the mechanism of motor neuron death in the ALS. The fact that the morphological and biochemical characteristics of apoptosis only last upwards of 24 hours in conjunction with the slow progression of the disease, which implicate that only a few motor neurons are dying at a time, make the definitive detection of apoptosis in post mortem tissue from ALS patients challenging (Sathasivam, Ince, and Shaw 2001). However, a comprehensive analysis of degenerating motor neurons in ALS patients revealed their apoptotic morphology in the ventral horn of the spinal cord and motor cortex, combined with an increase in DNA fragmentation and caspase 3 activation (Martin 1999). Further analysis of the post mortem human tissue showed increased formation of Bax-Bax homodimers and a

decrease in Bcl-2-Bax heterodimers in motor neurons, suggestive of an increased proapoptotic tone in the disease (Martin 1999).

superoxide that makes nitric oxide toxic to motor neurons (Noh and Koh 2000; Tammariello, Quinn, and Estus 2000). In motor neurons, activation of the p75 neurotrophic receptor results in apoptosis in different conditions (Ricart et al. 2006; Pehar et al. 2004; Pehar et al. 2007; Wiese et al. 1999). At least in part this toxicity is mediated by induction of superoxide production by mitochondria (Pehar et al. 2007). In summary, inhibition of nitric oxide production blocks motor neuron death induced by ventral root avulsion, and deletion of SOD increases the sensitivity of motor neurons to the same noxious stimulus. Moreover, motor neuron degeneration after ventral root avulsion is preceded by increased tyrosine nitration and the activation of the death receptor pathways. In aggregate these results reveal that motor neuron death *in vitro* and *in vivo* occurs largely by the same mechanisms

A pathological condition associated with an increased expression of neuronal NOS and nitrotyrosine in motor neurons is amyotrophic lateral sclerosis (ALS)(Abe et al. 1995; Beal et al. 1997; Chou, Wang, and Komai 1996; Chou, Wang, and Taniguchi 1996, , Barber, 2010 #1694). As for today more than 25 reports have found increased levels of nitrotyrosine immunoreactivity or free nitrotyrosine in tissue from patients and animal models of ALS. The definitive evidence for the presence of nitrotyrosine, at least in a transgenic mouse model of ALS, was provided by mass spectrometry studies identifying some of the nitrated proteins and the nitrated residues. Other studies confirmed the identity of the nitrated proteins showing that motor neurons in pre-symptomatic mutant SOD1 mice generate superoxide, NO and ONOO- at higher levels than control motor neurons. In addition, nitration of Cox-I, SOD2 and -synuclein occurs in pre-symptomatic mutant SOD1 mice suggesting a role for peroxynitrite in the pathogenesis of the disease (Martin et al. 2007). ALS is a neurodegenerative disease characterized by the death of pyramidal neurons in the motor cortex and motor neurons in the brain stem and ventral spinal cord. About 2% of all ALS cases are due to the presence of one of more than 100 mutations in the gene encoding Cu,Zn SOD (Cleveland and Rothstein 2001; Traub, Mitsumoto, and Rowland 2011). When expressed in mice and rats, some of the human ALS-linked SOD mutations produce a motor neuron disease reminiscent of ALS (Gurney et al. 1994; Dal Canto and Gurney 1995; Wong et al. 1995; Bruijn et al. 1997); these are currently the most widely accepted models for the disease. It is generally accepted that the toxic effect of the mutations is due to a gain-offunction (Cleveland and Rothstein 2001). Growing evidence implicates apoptosis as the mechanism of motor neuron death in the ALS. The fact that the morphological and biochemical characteristics of apoptosis only last upwards of 24 hours in conjunction with the slow progression of the disease, which implicate that only a few motor neurons are dying at a time, make the definitive detection of apoptosis in post mortem tissue from ALS patients challenging (Sathasivam, Ince, and Shaw 2001). However, a comprehensive analysis of degenerating motor neurons in ALS patients revealed their apoptotic morphology in the ventral horn of the spinal cord and motor cortex, combined with an increase in DNA fragmentation and caspase 3 activation (Martin 1999). Further analysis of the post mortem human tissue showed increased formation of Bax-Bax homodimers and a decrease in Bcl-2-Bax heterodimers in motor neurons, suggestive of an increased pro-

and through the activation of the same signaling pathways.

**7. ALS and reactive nitrogen species** 

apoptotic tone in the disease (Martin 1999).

Further evidence for the participation of apoptosis in ALS was provided by the use of transgenic models of the disease (Shibata 2001). Histological analysis of motor neurons from transgenic mice carrying G93A SOD mutant shows decreased expression of the antiapoptotic proteins Bcl-2 and Bcl-xl and increased expression of the pro-apoptotic Bcl-2 family members Bax and Bad, which expression was attenuated following over-expression of Bcl-2 (Vukosavic et al. 1999; Vukosavic et al. 2000). Genetic deletion of Bax in transgenic mice for the G93A ALS-linked mutant SOD delays onset and extends the lifespan of the animals, but it does not prevent the disease in spite of preventing motor neuron death (Gould et al. 2006). These results are a clear testimony to the complexity of the disease process. Although motor neurons are protected against apoptosis, other abnormalities such as neuromuscular denervation and mitochondrial vacuolization are still occurring (Gould et al. 2006). Other studies suggest that alterations of the neuromuscular junction are between the first symptoms of the disease both in humans and animals models of the disease (Fischer et al. 2004). However, it is important to remember that in the human disease motor neurons do die. The artificial deletion of a gene that regulates apoptosis may just inhibit the final step in the death process, without affecting upstream pathways responsible for the death of the cells. In addition, these results suggest that reactive oxygen and nitrogen species formed during the disease process are not final effectors of cell death, but rather upstream triggers of the activation of signaling pathways resulting in motor neuron death.

Further evidence for the role of apoptosis in the pathogenesis of ALS comes from the delay in onset and progression of the disease due to over-expression of the anti-apoptotic protein Bcl-2 in a transgenic mouse model of ALS (Vukosavic et al. 2000). More recently it was reported that Bcl-2 binds both wild-type and mutant SOD1 *in vitro* and *in vivo*. Because Bcl-2 associated with mutant SOD1 is present in protein aggregates located in mitochondria from the spinal cord of ALS patient and animal models of the disease, it was suggested that entrapment of Bcl-2 by SOD1 leads to the depletion of the anti-apoptotic protein in motor neurons, increasing the vulnerability of these cells (Pasinelli et al. 2004). However, no evidence for the aggregates selectively located in motor neuron mitochondria has been reported. Conversely, BIM, a member of the pro-apoptotic family of Bcl-2 proteins, is upregulated in the SOD1G93A familial ALS mouse model during the symptomatic stage of the disease, and its expression is required to trigger cell death induced by SOD1 mutants *in vitro*. Genetic deletion of BIM in an animal model of ALS results in reduced cellular apoptosis in the spinal cord ventral horn and increases lifespan (Hetz et al. 2007).

The role of caspases in ALS is more controversial. While several authors have reported activation of caspases and a functional role for these activated proteases in animal models of the disease (Friedlander et al. 1997; Li et al. 2000; Pasinelli et al. 1998; Nagai et al. 2001; Pasinelli et al. 2000), others were unable to find a functional role for the enzymes and report that motor neuron death in the G93A mouse model is independent of caspase activation (Martin et al. 2007).

To study the role of mutant SOD1 in different cell types involved in the pathology of ALS, chimeric mice were developed with mixtures of normal and SOD1 mutant-expressing cells. Normal motor neurons in SOD1 mutant chimeras develop features of ALS pathology. However, non-neuronal cells that do not express mutant SOD1 delay degeneration and significantly extend survival of mutant expressing motor neurons (Clement et al. 2003). When primary mouse spinal motor neurons express mutant human SOD1, the cells are not triggered to degenerate, however when rodent astrocytes express mutated SOD1, they kill

Reactive Nitrogen Species in Motor Neuron Apoptosis 323

toxicity in transgenic motor neurons can be reversed by copper chelators and scavenging of superoxide and peroxynitrite (Sahawneh et al. 2010), suggesting production of superoxide

Fig. 7. Zn-deficient SOD as a catalyst for peroxynitrite formation and tyrosine nitration.

(Fukada et al. 2001; Witan et al. 2008; Witan et al. 2009; Sahawneh et al. 2010).

Intracellular delivery of wild type Cu,Zn-containing human SOD (Cu,Zn SOD) to motor neurons isolated from transgenic rats overexpressing G93A mutant SOD, has no effect on survival whether the cells are cultured in the presence or absence of trophic factors. However, these motor neurons carrying both, mutant and Cu,Zn-containing SOD are more sensitive to nitric oxide toxicity than transgenic motor neurons without intracellular delivered Cu,Zn SOD (Sahawneh et al. 2010). These *in vitro* results closely mimic the *in vivo* observations of acceleration of death when transgenic mice for mutant SOD are crossbreed with mice transgenic for wild type SOD (Jaarsma et al. 2000; Fukada et al. 2001; Deng et al. 2006). There is some controversy over the effects of wild type SOD on the survival of mice carrying the SODG85R were one group reported acceleration of disease (Wang et al. 2009) and other claims no effect in a different SODG85R line (Bruijn et al. 1998). The mechanism through which coexpression of Cu,Zn SOD wild type increases the toxicity of mutant SOD is controversial, with some groups arguing that aggregation plays a role (Deng et al. 2006; Furukawa et al. 2006), while others conclude that increased solubility of the enzyme contributes to the enhanced toxicity (Fig 8)

The mechanisms of mutant SOD-induced motor neuron death in the presence of nitric oxide and Zn-deficient SOD stimulation of motor neuron apoptosis in culture seem identical (Estévez et al. 1999; Raoul et al. 2002; Sahawneh et al. 2010). The dependence on copper and the production of superoxide and peroxynitrite in both conditions indicate that nitrative stress plays a key role in the induction of cell death. In addition, Zn-deficient SOD toxicity is prevented by peptides that inhibit nitration (Ye et al. 2007; Sahawneh et al. 2010), as is the increased toxicity after the addition of Cu,Zn SOD (Sahawneh et al. 2010). Biochemical and biophysical studies indicate that the formation of a dimer between Zn-deficient SOD and Cu,Zn SOD increases the stability of the Zn-deficient SOD monomer (Beckman 1996; Roberts et al. 2007), in agreement with the *in vivo* and *in vitro* studies showing that wild type

in transgenic motor neurons.

spinal primary and embryonic mouse stem cell-derived motor neurons (Nagai et al. 2007). In addition, when G93A mutant embryonic stem cells are cultured as motor neurons through *in vitro* differentiation, co-cultures with G93A mutant glial cells lead to a decrease in survival of the motor neurons (Di Giorgio et al. 2007). Using conditional knockout to delete the mutant SOD in specific cell types it was found that the expression of mutant SOD in motor neurons plays a key role on the onset of the disease and the early phases of disease progression. When the levels of mutant SOD were reduced in microglia, the onset and early phases of the disease were not affected, but the later stage of the disease was slowed down. Therefore, onset and progression of ALS are dependent on distinct cell types, indicating the occurrence of a non-cell-autonomous death of motor neurons (Boillee et al. 2006; Boillee, Vande Velde, and Cleveland 2006).

### **8. SOD1 toxicity**

In spite of all the studies done to date on mutant SOD1 in ALS, the mechanism of SOD toxicity remains elusive and highly controversial. The devolpment of an antibody that recognizes the monomer misfolded forms of SOD1 showed the presence of the misfolded monomer in three ALS mouse models with G37R, G85R and G93A SOD1 mutations as well as in a human individual with an A4V SOD1 mutation (Rakhit et al. 2007). One of the hypotheses for mutant SOD toxicity proposes that an aberrant SOD chemistry, which allows small molecules such us peroxynitrite or hydrogen peroxide to produce damaging free radicals, is responsible for the toxic gain-of-function (Beckman and Crow 1993; Lyons et al. 1996). This aberrant chemistry could be the result of a reversal in SOD function caused by the loss of the structural zinc atom (Estévez et al. 1999; Beckman et al. 2001), since the mutant enzymes show a lower affinity for zinc than the wild type SOD, thus increasing the probability for the formation of Zn-deficient SOD (Lyons et al. 1996). There is great controversy about whether this proposed mechanism can take place and its relevance in the pathogenesis of ALS (Subramaniam et al. 2002), but the hypothesis also has its supporters (Liochev and Fridovich 2003).

The Zn-deficient hypothesis was tested using cultured motor neurons and liposomes for the intracellular release of the enzyme (Estévez et al. 1999). These experiments revealed that Zn-deficient human SOD, either mutant or wild type are equally toxic to motor neurons in culture by a mechanism requiring copper and the production of peroxynitrite (Estévez et al. 1999). On the other hand, Cu,Zn mutant and wild type SOD are equally protective for trophic factor deprived motor neurons (Estévez et al. 1999). Based on these results and the characteristics of the altered chemistry of the Zn-deficient SOD, the conclusion was that Zndeficient SOD produces superoxide using intracellular reducing activities leading to the formation of peroxynitrite (Fig 7).

Further confirmation of an altered mutant SOD chemistry was found using cultured motor neurons isolated from a transgenic mice model of human ALS in conjunction with wild type SOD (Raoul et al. 2002). Nitric oxide is not toxic to non-transgenic mouse and rat motor neurons (Estévez, Spear, Manuel, Radi, et al. 1998; Estévez et al. 2000; Raoul et al. 2002). In contrast, motor neurons isolated from transgenic rats and mice carrying human ALS-linked SOD G85R and G93A die after exposure to nanomolar steady state concentrations of nitric oxide (Raoul et al. 2002; Sahawneh et al. 2010). Transgenic motor neurons carrying mutant SOD are also ten times more sensitive to Fas-induced apoptosis, while transgenic motor neurons carrying human wild type SOD are 100 times more resistant than non-transgenic motor neurons to Fas-mediated apoptosis (Raoul et al. 2002; Raoul et al. 2006). Nitric oxide

spinal primary and embryonic mouse stem cell-derived motor neurons (Nagai et al. 2007). In addition, when G93A mutant embryonic stem cells are cultured as motor neurons through *in vitro* differentiation, co-cultures with G93A mutant glial cells lead to a decrease in survival of the motor neurons (Di Giorgio et al. 2007). Using conditional knockout to delete the mutant SOD in specific cell types it was found that the expression of mutant SOD in motor neurons plays a key role on the onset of the disease and the early phases of disease progression. When the levels of mutant SOD were reduced in microglia, the onset and early phases of the disease were not affected, but the later stage of the disease was slowed down. Therefore, onset and progression of ALS are dependent on distinct cell types, indicating the occurrence of a non-cell-autonomous death of motor neurons (Boillee et al. 2006; Boillee,

In spite of all the studies done to date on mutant SOD1 in ALS, the mechanism of SOD toxicity remains elusive and highly controversial. The devolpment of an antibody that recognizes the monomer misfolded forms of SOD1 showed the presence of the misfolded monomer in three ALS mouse models with G37R, G85R and G93A SOD1 mutations as well as in a human individual with an A4V SOD1 mutation (Rakhit et al. 2007). One of the hypotheses for mutant SOD toxicity proposes that an aberrant SOD chemistry, which allows small molecules such us peroxynitrite or hydrogen peroxide to produce damaging free radicals, is responsible for the toxic gain-of-function (Beckman and Crow 1993; Lyons et al. 1996). This aberrant chemistry could be the result of a reversal in SOD function caused by the loss of the structural zinc atom (Estévez et al. 1999; Beckman et al. 2001), since the mutant enzymes show a lower affinity for zinc than the wild type SOD, thus increasing the probability for the formation of Zn-deficient SOD (Lyons et al. 1996). There is great controversy about whether this proposed mechanism can take place and its relevance in the pathogenesis of ALS (Subramaniam et al. 2002), but the

The Zn-deficient hypothesis was tested using cultured motor neurons and liposomes for the intracellular release of the enzyme (Estévez et al. 1999). These experiments revealed that Zn-deficient human SOD, either mutant or wild type are equally toxic to motor neurons in culture by a mechanism requiring copper and the production of peroxynitrite (Estévez et al. 1999). On the other hand, Cu,Zn mutant and wild type SOD are equally protective for trophic factor deprived motor neurons (Estévez et al. 1999). Based on these results and the characteristics of the altered chemistry of the Zn-deficient SOD, the conclusion was that Zndeficient SOD produces superoxide using intracellular reducing activities leading to the

Further confirmation of an altered mutant SOD chemistry was found using cultured motor neurons isolated from a transgenic mice model of human ALS in conjunction with wild type SOD (Raoul et al. 2002). Nitric oxide is not toxic to non-transgenic mouse and rat motor neurons (Estévez, Spear, Manuel, Radi, et al. 1998; Estévez et al. 2000; Raoul et al. 2002). In contrast, motor neurons isolated from transgenic rats and mice carrying human ALS-linked SOD G85R and G93A die after exposure to nanomolar steady state concentrations of nitric oxide (Raoul et al. 2002; Sahawneh et al. 2010). Transgenic motor neurons carrying mutant SOD are also ten times more sensitive to Fas-induced apoptosis, while transgenic motor neurons carrying human wild type SOD are 100 times more resistant than non-transgenic motor neurons to Fas-mediated apoptosis (Raoul et al. 2002; Raoul et al. 2006). Nitric oxide

hypothesis also has its supporters (Liochev and Fridovich 2003).

Vande Velde, and Cleveland 2006).

formation of peroxynitrite (Fig 7).

**8. SOD1 toxicity** 

toxicity in transgenic motor neurons can be reversed by copper chelators and scavenging of superoxide and peroxynitrite (Sahawneh et al. 2010), suggesting production of superoxide in transgenic motor neurons.

Fig. 7. Zn-deficient SOD as a catalyst for peroxynitrite formation and tyrosine nitration.

Intracellular delivery of wild type Cu,Zn-containing human SOD (Cu,Zn SOD) to motor neurons isolated from transgenic rats overexpressing G93A mutant SOD, has no effect on survival whether the cells are cultured in the presence or absence of trophic factors. However, these motor neurons carrying both, mutant and Cu,Zn-containing SOD are more sensitive to nitric oxide toxicity than transgenic motor neurons without intracellular delivered Cu,Zn SOD (Sahawneh et al. 2010). These *in vitro* results closely mimic the *in vivo* observations of acceleration of death when transgenic mice for mutant SOD are crossbreed with mice transgenic for wild type SOD (Jaarsma et al. 2000; Fukada et al. 2001; Deng et al. 2006). There is some controversy over the effects of wild type SOD on the survival of mice carrying the SODG85R were one group reported acceleration of disease (Wang et al. 2009) and other claims no effect in a different SODG85R line (Bruijn et al. 1998). The mechanism through which coexpression of Cu,Zn SOD wild type increases the toxicity of mutant SOD is controversial, with some groups arguing that aggregation plays a role (Deng et al. 2006; Furukawa et al. 2006), while others conclude that increased solubility of the enzyme contributes to the enhanced toxicity (Fig 8) (Fukada et al. 2001; Witan et al. 2008; Witan et al. 2009; Sahawneh et al. 2010).

The mechanisms of mutant SOD-induced motor neuron death in the presence of nitric oxide and Zn-deficient SOD stimulation of motor neuron apoptosis in culture seem identical (Estévez et al. 1999; Raoul et al. 2002; Sahawneh et al. 2010). The dependence on copper and the production of superoxide and peroxynitrite in both conditions indicate that nitrative stress plays a key role in the induction of cell death. In addition, Zn-deficient SOD toxicity is prevented by peptides that inhibit nitration (Ye et al. 2007; Sahawneh et al. 2010), as is the increased toxicity after the addition of Cu,Zn SOD (Sahawneh et al. 2010). Biochemical and biophysical studies indicate that the formation of a dimer between Zn-deficient SOD and Cu,Zn SOD increases the stability of the Zn-deficient SOD monomer (Beckman 1996; Roberts et al. 2007), in agreement with the *in vivo* and *in vitro* studies showing that wild type

Reactive Nitrogen Species in Motor Neuron Apoptosis 325

formation of reactive nitrogen species and tyrosine nitration. Most evidence indicates that the source of the toxic nitric oxide is the neuronal isoform of NOS. However, the knockdown of the exon 1 in neuronal NOS, which greatly decreases the enzyme levels, has no effect on the survival of transgenic mouse models of ALS (Facchinetti et al. 1999). On the other hand, inhibitors of neuronal NOS are equally protective to motor neurons isolated from wild type and mice deficient for neuronal NOS (Ricart and Estevez, unpublished observations). These results suggest that the beta isoform of the neuronal NOS, which lacks exon 1, is the responsible for the production of the toxic nitric oxide in motor neurons. If this is the case, then these results may explain why the partial neuronal NOS deficiency is not protective in ALS. The multiple controversies in the role of reactive nitrogen species in ALS are not likely to end soon and will stimulate much needed

This work was supported by the NIH/NIDNS (NS36761). We thanks the constructive criticism of Thong Ma and Mary Ann Sahawneh during the preparation of this manuscript.

Abe, K, L H Pan, M Watanabe, T Kato, and Y Itoyama. 1995. Induction of nitrotyrosine-like

Alvarez, B, G Ferrer-Sueta, B A Freeman, and R Radi. 1999. Kinetics of peroxynitrite reaction with amino acids and human serum albumin. *J. Biol. Chem.* 274 (2):842-848. Ara, J, S Przedborski, A B Naini, V Jackson-Lewis, R R Trifiletti, J Horwitz, and H

Aslan, M., T. M. Ryan, T. M. Townes, L. Coward, M. C. Kirk, S. Barnes, C. B. Alexander, S. S.

Bar, P. R. 2000. Motor neuron disease in vitro: the use of cultured motor neurons to study

Barthelemy, C. Catherine, Christopher E. Henderson, and B. Brigitte Pettmann. 2004. Foxo3a

Beal, M F, L J Ferrante, S E Browne, R T Matthews, N W Kowall, and R H Brown. 1997.

Beckman, J S. 1996. Nitric oxide: Principles and actions. In *The Physiological and Pathological Chemistry of Nitric Oxide*, edited by J. R. Lanacaster. New York: Academic Press. Repeated Author. 1996. The Physiological and Pathological Chemistry of Nitric Oxide. In *Nitric Oxide: Principles and Actions*, edited by J. L. Jr: Academic Press Inc.

tetrahydropyridine(MPTP). *Proc Natl Acad Sci USA* 95 (13):7659-63.

amyotrophic lateral sclerosis. *Eur J Pharmacol* 405 (1-3):285-95.

polymerization. *J Biol Chem* 278 (6):4194-204.

immunoreactivity in the lower motor neuron of amyotrophic lateral sclerosis.

Ischiropoulos. 1998. Inactivation of tyrosine hydroxylase by nitration following exposure to peroxynitrite and 1-methyl-4-phenyl-1,2,3,6-

Rosenfeld, and B. A. Freeman. 2003. Nitric oxide-dependent generation of reactive species in sickle cell disease. Actin tyrosine induces defective cytoskeletal

induces motoneuron death through the Fas pathway in cooperation with JNK.

Increased 3-nitrotyrosine in both sporadic and familial amyotrophic lateral

research to found a cure for this devastating disease.

*Neurosci. Lett.* 199:152-154.

*BMC neuroscience* 5 (1):48.

sclerosis. *Ann Neurol* 42:644-654.

**9. Acknowledgements** 

**10. References** 

SOD increases the stability and solubility of the mutant protein (Fukada et al. 2001; Witan et al. 2008; Witan et al. 2009).

Fig. 8. Proposed mechanisms for the toxicity induced by mutant SOD.

Moreover, in spite of Cu,Zn SOD being a very stable enzyme, the half-life for exchange between this enzyme and Zn-deficient SOD at 37°C is surprisingly fast at 13-17 min when determined using differential mobility gel electrophoresis and 14 min by FRET (Roberts et al. 2007; Sahawneh et al. 2010). An important observation is that the reassociation of Cu,Zn SOD monomers is approximately 10,000 times slower than the reassociation of apoSOD monomers (Lindberg et al. 2004; Svensson et al. 2006), suggesting that the alterations of the dimer interface in the apoSOD are responsibly for the faster association of the monomers even though the stability of the dimer is diminished when compared with Cu,Zn SOD. It is possible that a similar mechanism occurs in the formation of the dimers between Cu,Zn and Zn-deficient SOD, which will explain the increased toxicity by a nitrative mechanism.

In addition, supporting the hypothesis that the increase in stability of Zn-deficient SOD is key in enhancing its toxicity, the substitution of the cysteine residues in positions 6 and 111 by alanine and serine in the human SOD increases its thermostability, and also increases the toxicity of the wild type SOD and SOD1A4V when they are Zn-deficient, but has no effect when the enzymes have the full content of metals (Sahawneh et al. 2010). Other studies reveal that mutation of these two cysteines decreases the stability of the SOD protein but prevents its aggregation (Lindberg et al. 2004; Svensson et al. 2006; Lepock, Frey, and Hallewell 1990). Zn-deficient enzyme with the double cysteine mutation shows more toxicity than the equivalent Zn-deficient SOD with the cysteine residues. In this model, the increased stability of the Zn-deficient form of the SOD is also responsible for the toxicity by a mechanism requiring the production of nitric oxide, superoxide and peroxynitrite followed by tyrosine nitration (Sahawneh et al. 2010).

In summary, reactive nitrogen species and some of their products, such as nitrotyrosine play a causal role in the activation of motor neuron death pathways rather than just having an effector activity. In addition, the abundant evidence indicating the formation of reactive nitrogen species and nitrotyrosine in pathological conditions also suggests that nitrotyrosine is more than a footprint in these conditions. In ALS is well documented the formation of reactive nitrogen species and tyrosine nitration. Most evidence indicates that the source of the toxic nitric oxide is the neuronal isoform of NOS. However, the knockdown of the exon 1 in neuronal NOS, which greatly decreases the enzyme levels, has no effect on the survival of transgenic mouse models of ALS (Facchinetti et al. 1999). On the other hand, inhibitors of neuronal NOS are equally protective to motor neurons isolated from wild type and mice deficient for neuronal NOS (Ricart and Estevez, unpublished observations). These results suggest that the beta isoform of the neuronal NOS, which lacks exon 1, is the responsible for the production of the toxic nitric oxide in motor neurons. If this is the case, then these results may explain why the partial neuronal NOS deficiency is not protective in ALS. The multiple controversies in the role of reactive nitrogen species in ALS are not likely to end soon and will stimulate much needed research to found a cure for this devastating disease.

### **9. Acknowledgements**

This work was supported by the NIH/NIDNS (NS36761). We thanks the constructive criticism of Thong Ma and Mary Ann Sahawneh during the preparation of this manuscript.

### **10. References**

324 Amyotrophic Lateral Sclerosis

SOD increases the stability and solubility of the mutant protein (Fukada et al. 2001; Witan et

Moreover, in spite of Cu,Zn SOD being a very stable enzyme, the half-life for exchange between this enzyme and Zn-deficient SOD at 37°C is surprisingly fast at 13-17 min when determined using differential mobility gel electrophoresis and 14 min by FRET (Roberts et al. 2007; Sahawneh et al. 2010). An important observation is that the reassociation of Cu,Zn SOD monomers is approximately 10,000 times slower than the reassociation of apoSOD monomers (Lindberg et al. 2004; Svensson et al. 2006), suggesting that the alterations of the dimer interface in the apoSOD are responsibly for the faster association of the monomers even though the stability of the dimer is diminished when compared with Cu,Zn SOD. It is possible that a similar mechanism occurs in the formation of the dimers between Cu,Zn and Zn-deficient SOD, which will explain the increased toxicity by a nitrative mechanism. In addition, supporting the hypothesis that the increase in stability of Zn-deficient SOD is key in enhancing its toxicity, the substitution of the cysteine residues in positions 6 and 111 by alanine and serine in the human SOD increases its thermostability, and also increases the toxicity of the wild type SOD and SOD1A4V when they are Zn-deficient, but has no effect when the enzymes have the full content of metals (Sahawneh et al. 2010). Other studies reveal that mutation of these two cysteines decreases the stability of the SOD protein but prevents its aggregation (Lindberg et al. 2004; Svensson et al. 2006; Lepock, Frey, and Hallewell 1990). Zn-deficient enzyme with the double cysteine mutation shows more toxicity than the equivalent Zn-deficient SOD with the cysteine residues. In this model, the increased stability of the Zn-deficient form of the SOD is also responsible for the toxicity by a mechanism requiring the production of nitric oxide, superoxide and peroxynitrite

In summary, reactive nitrogen species and some of their products, such as nitrotyrosine play a causal role in the activation of motor neuron death pathways rather than just having an effector activity. In addition, the abundant evidence indicating the formation of reactive nitrogen species and nitrotyrosine in pathological conditions also suggests that nitrotyrosine is more than a footprint in these conditions. In ALS is well documented the

Fig. 8. Proposed mechanisms for the toxicity induced by mutant SOD.

followed by tyrosine nitration (Sahawneh et al. 2010).

al. 2008; Witan et al. 2009).


Reactive Nitrogen Species in Motor Neuron Apoptosis 327

Chou, S. M., H. S. Wang, and A. Taniguchi. 1996. Role of SOD-1 and nitric oxide/cyclic

Clement, A. M., M. D. Nguyen, E. A. Roberts, M. L. Garcia, S. Boillee, M. Rule, A. P.

Crow, J P, M J Strong, Y Zhuang, Y Ye, and J S Beckman 1997. Superoxide dimutase

Crow, J. P., J. S. Beckman, and J. M. McCord. 1995. Sensitivity of the essential zinc-thiolate

Dal Canto, M.C. , and M.E. Gurney. 1995. Neuropathological changes in two lines of mice

Dawson, V L, and T M Dawson. 1996. Nitric Oxide Toxicity in Central Nervous System

Dawson, V. L., and T. M. Dawson. 1996. Nitric oxide in neuronal degeneration. *Proc Soc Exp* 

Dawson, V.L., and T.M. Dawson. 1996. Nitric oxide neurotoxicity. *J Chem. Neuroanat* 10:179-

Deng, Han-Xiang, Yong Shi, Yoshiaki Furukawa, Hong Zhai, Ronggen Fu, Erdong Liu,

Di Giorgio, Francesco Paolo, Monica A. Carrasco, Michelle C. Siao, Tom Maniatis, and Kevin

Eiserich, J P, A G Estévez, T V Bamberg, Y Z Ye, P H Chumley, J S Beckman, and B A

Eiserich, J. P., M. Hristova, C. E. Cross, A. D. Jones, B. A. Freeman, B. Halliwell, and A. van

Estévez, A G, J P Crow, J B Sampson, C Reiter, Y-X Zhuang, G J Richardson, M M Tarpey, L

stem cell-based ALS model. *Nat Neurosci* 10 (5):608-614.

myeloperoxidase in neutrophils. *Nature* 391 (6665):393-7.

George H. Gorrie, Mohammad S. Khan, Wu-Yen Hung, Eileen H. Bigio, Thomas Lukas, Mauro C. Dal Canto, Thomas V. O'Halloran, and Teepu Siddique. 2006. Conversion to the amyotrophic lateral sclerosis phenotype is associated with intermolecular linked insoluble aggregates of SOD1 in mitochondria. *PNAS* 103

Eggan. 2007. Non-cell autonomous effect of glia on motor neurons in an embryonic

Freeman. 1999. Microtubule dysfunction by posttranslational nitrotyrosination of atubulin: a nitric oxide-dependent mechanism of cellular injury. *Proc. Natl. Acad. Sci.* 

der Vliet. 1998. Formation of nitric oxide-derived inflammatory oxidants by

Barbeito, and J S Beckman. 1999. Induction of nitric oxide-dependent apoptosis in motor neurons by zinc-deficient superoxide dismutase. *Science* 286:2498-2500.

of SOD1 mutant motor neurons in ALS mice. *Science* 302 (5642):113-7. Cleveland, D W, and J D Rothstein. 2001. From Charcot to Lou Gehrig: Deciphering Selective Motor Neuron Death in ALS. *Nature Neuroscience Review* 2 (11):806-819. Colasanti, M., and H. Suzuki. 2000. The dual personality of NO. *Trends Pharmacol Sci* 21

neurofilament L. *J Neurochem* 69 (5):1945-1953.

Cultures. *Methods in Neurosciences* 30:26-43.

*Biochemistry* 34 (11):3544-52.

*Brain Res.* 676:25-40.

*Biol Med* 211 (1):33-40.

(18):7142-7147.

*USA* 96:6365-6370.

190.

Suppl:16-26.

(7):249-52.

GMP cascade on neurofilament aggregation in ALS/MND. *J Neurol Sci* 139

McMahon, W. Doucette, D. Siwek, R. J. Ferrante, R. H. Brown, Jr., J. P. Julien, L. S. Goldstein, and D. W. Cleveland. 2003. Wild-type nonneuronal cells extend survival

catalyzes nitration of tyrosines by peroxinitrite in the rod and head domains of

moiety of yeast alcohol dehydrogenase to hypochlorite and peroxynitrite.

carrying a transgene for mutant human Cu, Zn SOD, and in mice overexpressing wild type human SOD: a model of familial amyotrophic lateral sclerosis (FALS).


Beckman, J S, T W Beckman, J Chen, P M Marshall, and B A Freeman. 1990. Apparent

by nitric oxide and superoxide. *Proc. Natl. Acad. Sci. (USA)* 87 (4):1620-1624. Beckman, J S, and J P Crow. 1993. Pathological implications of nitric oxide, superoxide and

Beckman, J S, A G Estévez, J P Crow, and L Barbeito. 2001. Superoxide dismutase and the

Beckman, J S, and W H Koppenol. 1996. Nitric oxide, superoxide, and peroxynitrite -- the good, the bad, and the ugly. *Am. J. Physiol.* 271 (Cell Physiol. 40):C1424-C1437. Beckman, J. S., H. Ischiropoulos, L. Zhu, M. van der Woerd, C. Smith, J. Chen, J. Harrison, J.

nitration of phenolics by peroxynitrite. *Arch Biochem Biophys* 298 (2):438-445. Beckman, J. S., and W. H. Koppenol. 1996. Nitric oxide, superoxide, and peroxynitrite: the

Boillee, Severine, Christine Vande Velde, and DonW Cleveland. 2006. ALS: A Disease of Motor Neurons and Their Nonneuronal Neighbors. *Neuron* 52 (1):39-59. Boillee, Severine, Koji Yamanaka, Christian S. Lobsiger, Neal G. Copeland, Nancy A.

Bonfoco, E, D Krainc, M Ankarcrona, P Nicotera, and S A Lipton. 1995. Apoptosis and

Bruijn, L I, M W Becher, M K Lee, K L Anderson, N A Jenkins, N G Copeland, S S Sisodia, J

Bruijn, L I, M K Houseweart, S Kato, K L Anderson, S D Anderson, E Ohama, A G Reaume,

Cappelletti, G., M. G. Maggioni, G. Tedeschi, and R. Maci. 2003. Protein tyrosine nitration is

Carreras, Maria Cecilia, and Juan Jose Poderoso. 2007. Mitochondrial nitric oxide in the signaling of cell integrated responses. *Am J Physiol Cell Physiol* 292 (5):C1569-1580. Casanovas, A., J. Ribera, M. Hukkanen, V. Riveros-Moreno, and J. E. Esquerda. 1996.

motoneuron cell death after neonatal axotomy. *Neuroscience* 71 (2):313-25. Chang, W., D. R. Webster, A. A. Salam, D. Gruber, A. Prasad, J. P. Eiserich, and J. C.

Chou, S. M., H. S. Wang, and K. Komai. 1996. Colocalization of NOS and SOD1 in

an immunohistochemical study. *J Chem Neuroanat* 10 (3-4):249-58.

Disease with SOD1-Containing Inclusions. *Neuron* 18 (2):327-338.

peroxynitrite formation. *Biochem. Soc. Trans.* 21 (2):330-334.

good, the bad, and ugly. *Am J Physiol* 271 (5 Pt 1):C1424-37.

312 (5778):1389-1392.

281:1851-1854.

30698.

*Cell Res* 288 (1):9-20.

*Natl. Acad. Sci. U.S.A.* 92 (16):7162-7166.

death of motoneurons in ALS. *TINS* 24 (11):S15-S20.

hydroxyl radical production by peroxynitrite: Implications for endothelial injury

C. Martin, and M. Tsai. 1992. Kinetics of superoxide dismutase and iron- catalyzed

Jenkins, George Kassiotis, George Kollias, and Don W. Cleveland. 2006. Onset and Progression in Inherited ALS Determined by Motor Neurons and Microglia. *Science*

necrosis: Two distinct events induced, respectively, by mild and intense insults with *N*-methyl-D-aspartate or nitric oxide/superoxide in cortical cell cultures. *Proc.* 

D Rothstein, D R Borchelt, D L Price, and D W Cleveland. 1997. ALS-Linked SOD1 Mutant G85R Mediates Damage to Astrocytes and Promotes Rapidly Progressive

R W Scott, and D W Cleveland. 1998. Aggregation and Motor Neuron Toxicity of an ALS-Linked SOD1 Mutant Independent from Wild-Type SOD1. *Science*

triggered by nerve growth factor during neuronal differentiation of PC12 cells. *Exp* 

Prevention by lamotrigine, MK-801 and N omega-nitro-L-arginine methyl ester of

Bulinski. 2002. Alteration of the C-terminal amino acid of tubulin specifically inhibits myogenic differentiation. *Journal of Biological Chemistry* 277 (34):30690-

neurofilament accumulation within motor neurons of amyotrophic lateral sclerosis:


Reactive Nitrogen Species in Motor Neuron Apoptosis 329

Hetz, C., P. Thielen, J. Fisher, P. Pasinelli, R. H. Brown, S. Korsmeyer, and L. Glimcher. 2007.

Hughes, R A, M Sendtner, and H Thoenen. 1993. Members of several gene families influence survival of rat motoneurons *in vitro* and *in vivo*. *J. Neurosci. Res.* 36:663-671. Ignarro, L. J. 1990. Biosynthesis and metabolism of endothelium-derived nitric oxide. *Annu* 

Ischiropoulos, H. 1998. Biological tyrosine nitration: A pathophysiological function of nitric

Ischiropoulos, H., and J. S. Beckman. 2003. Oxidative stress and nitration in

Jaarsma, Dick, Elize D Haasdijk, J A C Grashorn, Richard Hawkins, Wim van Duijn, Hein W

Kaal, E. C., A. S. Vlug, M. W. Versleijen, M. Kuilman, E. A. Joosten, and P. R. Bar. 2000.

new model for amyotrophic lateral sclerosis. *J Neurochem* 74 (3):1158-65. Le-Niculescu, H., E. Bonfoco, Y. Kasuya, F. X. Claret, D. R. Green, and M. Karin. 1999.

neurodegeneration: Cause, effect, or association? *Journal of Clinical Investigation* 111

Verspaget, Jacqueline London, and Jan C Holstege. 2000. Human Cu/Zn superoxide dismutase (SOD1) overexpression in mice causes mitochondial vacuolization, axonal degeneration, and premature motor neuron death and accelerates motor neuron disease in mice expressing a familial amyotrophic lateral

Chronic mitochondrial inhibition induces selective motoneuron death in vitro: a

Withdrawal of survival factors results in activation of the JNK pathway in neuronal cells leading to Fas ligand induction and cell death. *Mol Cell Biol* 19 (1):751-63. Lepock, J. R., H. E. Frey, and R. A. Hallewell. 1990. Contribution of conformational stability

and reversibility of unfolding to the increased thermostability of human and bovine superoxide dismutase mutated at free cysteines. *J Biol Chem* 265 (35):21612-8. Li, L., R. W. Oppenheim, M. Lei, and L. J. Houenou. 1994. Neurotrophic agents prevent

motoneuron death following sciatic nerve section in the neonatal mouse. *J Neurobiol*

pathway of developing motoneurons deprived of trophic support. *J Neurobiol* 46

E. Stieg, J. P. Lee, S. Przedborski, and R. M. Friedlander. 2000. Functional role of caspase-1 and caspase-3 in an ALS transgenic mouse model. *Science* 288 (5464):335-

superoxide dismutase: disulfide reduction prevents dimerization and produces

amyotrophic lateral sclerosis: evaluation of oxidative hypotheses. *Free Radic Biol* 

Bredesen, E B Gralla, and J S Valentine. 1996. Mutations in copper-zinc superoxide

Li, L., R. W. Oppenheim, and C. E. Milligan. 2001. Characterization of the execution

Li, M., V. O. Ona, C. Guegan, M. Chen, V. Jackson-Lewis, L. J. Andrews, A. J. Olszewski, P.

Lindberg, M. J., J. Normark, A. Holmgren, and M. Oliveberg. 2004. Folding of human

Lyons, T J, H Liu, J J Goto, A Nersissian, J A Roe, J A Graden, C Café, L M Ellerby, D E

marginally stable monomers. *Proc Natl Acad Sci U S A* 101 (45):15893-8. Liochev, S. I., and I. Fridovich. 2003. Mutant Cu,Zn superoxide dismutases and familial

of amyotrophic lateral sclerosis. *Cell Death Differ* 14 (7):1386-1389.

oxide and reactive oxygen species. *Arch Biochem Biophys* 356:1-11.

dismutase mutation. *Science* 264 (5166):1772-1775.

sclerosis mutant SOD1. *Neurobiol Disease* 7:623-643.

*Rev Pharmacol Toxicol* 30:535-60.

(2):163-169.

25 (7):759-66.

(4):249-64.

*Med* 34 (11):1383-9.

9.

Motor neuron degeneration in mice that express a human Cu,Zn superoxide

The proapoptotic BCL-2 family member BIM mediates motoneuron loss in a model


Estévez, A G, R Radi, L Barbeito, J T Shin, J A Thompson, and J S Beckman. 1995.

Estévez, A G, J B Sampson, Y-X Zhuang, N Spear, G J Richardson, J P Crow, M M Tarpey, L

Estévez, A G, N Spear, S Machelle Manuel, R Radi, C E Henderson, L Barbeito, and J S

Estévez, A. G. , N. Spear, S. Machelle Manuel, L. Barbeito, R. Radi, and J. S. Beckman. 1998.

Fischer, Lindsey R., Deborah G. Culver, Philip Tennant, Albert A. Davis, Minsheng Wang,

Friedlander, R M, J Wang, V Gagliardini, R H Jr Brown, and J Yuan. 1997. Inhibition of ICE

Fukada, K., S. Nagano, M. Satoh, C. Tohyama, T. Nakanishi, A. Shimizu, T. Yanagihara, and

familial amyotrophic lateral sclerosis mice. *Eur J Neurosci* 14 (12):2032-6. Furukawa, Yoshiaki, Ronggen Fu, Han-Xiang Deng, Teepu Siddique, and Thomas V.

Go, Y. M., R. P. Patel, M. C. Maland, H. Park, J. S. Beckman, V. M. Darley-Usmar, and H. Jo.

Greenacre, S A, and H Ischiropoulos. 2001. Tyrosine nitration: localisation, quantification,

Groves, J T, and S S Marla. 1995. Peroxynitrite-induced DNA strand scission mediated by a

Gurney, M E, H Pu, A Y Chiu, M C Dal Canto, C Y Polchow, D D Alexander, J Caliendo, A

death of motor neurons in culture. *Progress in Brain Research* 118:269-280. Facchinetti, F., M. Sasaki, F. B. Cutting, P. Zhai, J. E. MacDonald, D. Reif, M. F. Beal, P. L.

of familial amyotrophic lateral sclerosis. *Neuroscience* 90 (4):1483-92.

induced by trophic factor deprivation. *J. Neurosci.* 18 (3):923-931.

withdrawal. *Free Rad. Biol. Med.* 28 (3):437-446.

man. *Experimental Neurology* 185 (2):232-240.

spinal cords of model mice. *PNAS* 103 (18):7148-7153.

Mouse Model of ALS. *J. Neurosci.* 26 (34):8774-8786.

manganese porphyrin. *J. Am. Chem. Soc.* 117:9578-9579.

slows ALS in mice. *Nature* 388:31.

(6):541-581.

(4):1543-1550.

Peroxynitrite-induced cytotoxicity in PC12 cells: evidence for an apoptotic mechanism differentially modulated by neurotrophic factors. *J. Neurochem.* 65

Barbeito, and J S Beckman. 2000. Liposome-delivered superoxide dismutase prevents nitric oxide-dependent motor neuron death induced by trophic factor

Beckman. 1998. Nitric oxide and superoxide contribute to motor neuron apoptosis

Role of endogenous nitric oxide and peroxinitrite formation in the survival and

Huang, T. M. Dawson, M. E. Gurney, and V. L. Dawson. 1999. Lack of involvement of neuronal nitric oxide synthase in the pathogenesis of a transgenic mouse model

Amilcar Castellano-Sanchez, Jaffar Khan, Meraida A. Polak, and Jonathan D. Glass. 2004. Amyotrophic lateral sclerosis is a distal axonopathy: evidence in mice and

S. Sakoda. 2001. Stabilization of mutant Cu/Zn superoxide dismutase (SOD1) protein by coexpressed wild SOD1 protein accelerates the disease progression in

O'Halloran. 2006. From the Cover: Disulfide cross-linked protein represents a significant fraction of ALS-associated Cu, Zn-superoxide dismutase aggregates in

1999. Evidence for peroxynitrite as a signaling molecule in flow-dependent activation of c-Jun NH(2)-terminal kinase. *Am J Physiol* 277 (4 Pt 2):H1647-53. Gould, Thomas W., Robert R. Buss, Sharon Vinsant, David Prevette, Woong Sun, C. Michael

Knudson, Carol E. Milligan, and Ronald W. Oppenheim. 2006. Complete Dissociation of Motor Neuron Death from Motor Dysfunction by Bax Deletion in a

consequences for protein function and signal transduction. *Free Radical Research* 34

Hentati, Y W Kwon, H-X Deng, W Chen, P Zhai, R L Sufit, and T Siddique. 1994.

Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation. *Science* 264 (5166):1772-1775.


Reactive Nitrogen Species in Motor Neuron Apoptosis 331

Neff, N. T., D. Prevette, L. J. Houenou, M. E. Lewis, M. A. Glicksman, Q. W. Yin, and R. W.

Noh, K. M., and J. Y. Koh. 2000. Induction and activation by zinc of NADPH oxidase in

Novikov, L, L Novikova, and J-O Kellerth. 1995. Brain-derived neurotrophic factor promotes

Oppenheim, R. W. 1991. Cell death during development of the nervous system. *Annu Rev* 

Pacher, Pal, Joseph S. Beckman, and Lucas Liaudet. 2007. Nitric Oxide and Peroxynitrite in

Padmaja, S, and R E Huie. 1993. The reaction of nitric oxide with organic peroxyl radicals.

Pasinelli, P , D R Borchelt, M K Houseweart, D W Cleveland, and R H Brown Jr. 1998.

Pasinelli, P., M. E. Belford, N. Lennon, B. J. Bacskai, B. T. Hyman, D. Trotti, and R. H. Brown,

Paxinou, E., Q. Chen, M. Weisse, B. I. Giasson, E. H. Norris, S. M. Rueter, J. Q. Trojanowski,

Pehar, M., P. Cassina, M. R. Vargas, R. Castellanos, L. Viera, J. S. Beckman, A. G. Estevez,

Pehar, Mariana, Marcelo R. Vargas, Kristine M. Robinson, Patricia Cassina, Pablo J. Diaz-

Pennica, D, V Arce, T A Swanson, R Vejsada, R A Pollock, M Armanini, K Dudley, H S

aggregate with Bcl-2 in spinal cord mitochondria. *Neuron* 43 (1):19-30. Pasinelli, P., M. K. Houseweart, R. H. Brown, and D. W. Cleveland. 2000. Caspase-1 and-3

*of Sciences of the United States of America* 97 (25):13901-13906.

intracellular nitrative insult. *J Neurosci* 21 (20):8053-61.

Apoptosis. *J. Neurosci.* 27 (29):7777-7785.

motoneurons. *Neuron* 17 (1):63-74.

Caspase-1 is activated in neural cells and tissue with amyotrophic lateral sclerosisassociated mutations in copper-zinc superoxide dismutase. *Proc Natl Acad Sci USA*

Jr. 2004. Amyotrophic lateral sclerosis-associated SOD1 mutant proteins bind and

are sequentially activated in motor neuron death in Cu,Zn superoxide dismutasemediated familial amyotrophic lateral sclerosis. *Proceedings of the National Academy* 

V. M. Lee, and H. Ischiropoulos. 2001. Induction of alpha-synuclein aggregation by

and L. Barbeito. 2004. Astrocytic production of nerve growth factor in motor neuron apoptosis: implications for amyotrophic lateral sclerosis. *J Neurochem* 89

Amarilla, Tory M. Hagen, Rafael Radi, Luis Barbeito, and Joseph S. Beckman. 2007. Mitochondrial Superoxide Production and Nuclear Factor Erythroid 2-Related Factor 2 Activation in p75 Neurotrophin Receptor-Induced Motor Neuron

Phillips, A Rosenthal, A C Kato, and C E Henderson. 1996. Cardiotrophin-1, a cytokine present in embryonic muscle, supports long-term survival of spinal

agents that promote motoneuron survival. *J Neurobiol* 24 (12):1578-88. Newbern, J., A. Taylor, M. Robinson, M. O. Lively, and C. E. Milligan. 2007. c-Jun N-

degeneration in motoneurons. *Neuroscience* 147 (3):680-692.

embarrassment of riches. *Neuron* 17 (2):195-197.

Health and Disease. *Physiol. Rev.* 87 (1):315-424.

*Biochem. Biophys. Res. Commun.* 195:539-544.

*Neurosci* 14:453-501.

95 (26):15763-8.

(2):464-73.

cultured cortical neurons and astrocytes. *J Neurosci* 20 (23):RC111.

motoneurons after ventral root avulsion. *Neurosci. Lett.* 200 (1):45-48. Oppenheim, R W. 1996. Neurotrophic survival molecules for motoneurons: an

Oppenheim. 1993. Insulin-like growth factors: putative muscle-derived trophic

terminal kinase signaling regulates events associated with both health and

survival and blocks nitric oxide synthase expression in adult rat spinal

dismutase that cause amyotrophic lateral sclerosis alter the zinc binding site and the redox behavior of the protein. *Proc. Natl. Acad. Sci. U.S.A.* 93:12240-12244.


Martin, L. J. 1999. Neuronal death in amyotrophic lateral sclerosis is apoptosis: possible

Martin, L. J., K. Chen, and Z. Liu. 2005. Adult motor neuron apoptosis is mediated by nitric

Martin, L. J., A. Kaiser, and A. C. Price. 1999. Motor neuron degeneration after sciatic nerve

Martin, Lee J. , Zhiping Liu, Kevin Chen, Ann C. Price, Pan. Yan, Jason A. Swaby, and W.

Medema, J. P., C. Scaffidi, F. C. Kischkel, A. Shevchenko, M. Mann, P. H. Krammer, and M.

Milligan, C. E., R. W. Oppenheim, and L. M. Schwartz. 1994. Motoneurons deprived of

Milligan, C. E., D. Prevette, H. Yaginuma, S. Homma, C. Cardwell, L. C. Fritz, K. J.

Moncada, S , R M J Palmer, and E A Higgins. 1991. Nitric oxide: physiology,

Morishima, Y., Y. Gotoh, J. Zieg, T. Barrett, H. Takano, R. Flavell, R. J. Davis, Y. Shirasaki,

Nagai, M, M Aoki, I Miyoshi, M Kato, P Pasinelli, N Kasai, R H Jr. Brown, and Y Itoyama.

Nagai, Makiko, Diane B. Re, Tetsuya Nagata, Alcmene Chalazonitis, Thomas M. Jessell,

Nauser, T., and W. H. Koppenol. 2002. The rate constant of the reaction of superoxide with

pathophysiology, phamacology. *Pharmacol Rev* 43 (2):109-142.

signaling complex (DISC). *Embo J* 16 (10):2794-804.

death. *J Neurobiol* 25 (8):1005-16.

Fas ligand. *J Neurosci* 21 (19):7551-60.

neuron disease. *J Neurosci* 21 (21):9246-9254.

vitro. *Neuron* 15 (2):385-93.

(5):615-622.

106 (16):4084-4086.

*Arch. Biochem. Biophys.* 377 (2):350-356.

(5):459-71.

25 (27):6449-59.

(2):185-201.

46.

dismutase that cause amyotrophic lateral sclerosis alter the zinc binding site and the redox behavior of the protein. *Proc. Natl. Acad. Sci. U.S.A.* 93:12240-12244. MacMillan-Crow, L A, J S Greendorfer, S M Vickers, and J A Thompson. 2000. Tyrosine

nitration of c-SRC tyrosine kinase in human pancreatic ductal adenocarcinoma.

contribution of a programmed cell death mechanism. *J Neuropathol Exp Neurol* 58

oxide and Fas death receptor linked by DNA damage and p53 activation. *J Neurosci*

avulsion in adult rat evolves with oxidative stress and is apoptosis. *J Neurobiol* 40

Christopher Golden. 2007. Motor neuron degeneration in amyotrophic lateral sclerosis mutant superoxide dismutase-1 transgenic mice: Mechanisms of mitochondriopathy and cell death. *The Journal of Comparative Neurology* 500 (1):20-

E. Peter. 1997. FLICE is activated by association with the CD95 death-inducing

trophic support in vitro require new gene expression to undergo programmed cell

Tomaselli, R. W. Oppenheim, and L. M. Schwartz. 1995. Peptide inhibitors of the ICE protease family arrest programmed cell death of motoneurons in vivo and in

and M. E. Greenberg. 2001. Beta-amyloid induces neuronal apoptosis via a mechanism that involves the c-Jun N-terminal kinase pathway and the induction of

2001. Rats expressing human cytosolic copper-zinc superoxide dismutase transgenes with amyotrophic lateral sclerosis: associated mutations develop motor

Hynek Wichterle, and Serge Przedborski. 2007. Astrocytes expressing ALS-linked mutated SOD1 release factors selectively toxic to motor neurons. *Nat Neurosci* 10

nitrogen monoxide: Approaching the diffusion limit. *Journal of Physical Chemistry A*


Reactive Nitrogen Species in Motor Neuron Apoptosis 333

Sahawneh, M. A., K. C. Ricart, B. R. Roberts, V. C. Bomben, M. Basso, Y. Ye, J. Sahawneh, M.

Sathasivam, S., P. G. Ince, and P. J. Shaw. 2001. Apoptosis in amyotrophic lateral sclerosis: a review of the evidence. *Neuropathology and Applied Neurobiology* 27 (4):257-274. Schopfer, F. J., P. R. Baker, and B. A. Freeman. 2003. NO-dependent protein nitration: a cell

Sendtner, M., G. Pei, M. Beck, U. Schweizer, and S. Wiese. 2000. Developmental motoneuron

Shacka, J. J., M. A. Sahawneh, J. D. Gonzalez, Y. Z. Ye, T. L. D'Alessandro, and A. G. Estévez.

Shibata, Noriyuki. 2001. Transgenic mouse model for familial amyotrophic lateral sclerosis

Shin, J T, L Barbeito, L A MacMillan-Crow, J S Beckman, and J A Thompson. 1996. Acidic

Spear, N, A G Estévez, L Barbeito, J S Beckman, and G V W Johnson. 1997. Nerve growth

Subramaniam, J R, W E Lyons, J Liu, T B Bartnikas, J Rothstein, D L Price, D W Cleveland, J

Sunayama, J., F. Tsuruta, N. Masuyama, and Y. Gotoh. 2005. JNK antagonizes Akt-mediated

Svensson, A. K., O. Bilsel, E. Kondrashkina, J. A. Zitzewitz, and C. R. Matthews. 2006.

Takakura, K., J. S. Beckman, L. A. MacMillan-Crow, and J. P. Crow. 1999. Rapid and

Tammariello, S. P., M. T. Quinn, and S. Estus. 2000. NADPH oxidase contributes directly to

Traub, R., H. Mitsumoto, and L. P. Rowland. 2011. Research advances in amyotrophic lateral sclerosis, 2009 to 2010. *Current neurology and neuroscience reports* 11 (1):67-77. Ugolini, G., C. Raoul, A. Ferri, C. Haenggeli, Y. Yamamoto, D. Salaun, C. E. Henderson, A.

dependent on phosphatidylinositol-3 kinase. *J. Neurochem.* 69 (1):53-59. Spear, N, A G Estévez, R Radi, and J S Beckman. 1997. Peroxynitrite and Cell signalling. In

with superoxide dismutase-1 mutation. *Neuropathology* 21 (1):82-92.

murine fibroblasts. *Arch. Biochem. Biophys.* 335 (1):32-41. Snyder, S. H. 1993. Janus faces of nitric oxide. *Nature* 364 (6438):577.

cell death and neurotrophic factors. *Cell Tissue Res* 301 (1):71-84.

PC12 cells. *Cell Death Differ* 13:1506-1514.

New york: Chapman&Hall.

dismutase. *J Mol Biol* 364 (5):1084-102.

neurons. *J Neurosci* 20 (1):RC53.

*Neurosci* 23 (24):8526-31.

by peroxynitrite. *Arch Biochem Biophys* 369 (2):197-207.

(4):301-307.

enhancing protein stability. *J Biol Chem* 285 (44):33885-97.

(12):646-54.

C. Franco, J. S. Beckman, and A. G. Estevez. 2010. Cu,Zn superoxide dismutase (SOD) increases toxicity of mutant and Zn-deficient superoxide dismutase by

signaling event or an oxidative inflammatory response? *Trends Biochem Sci* 28

2006. Two distinct signaling pathways regulate peroxynitrite-induced apoptosis in

fibroblast growth factor enhances peroxynitrite-induced apoptosis in primary

factor protects PC12 cells against peroxynitrite-induced apoptosis via a mechanism

*Oxidative Stress and Signal Transduction*, edited by H. J. Forman and E. Cadenas.

D Gitlin, and P C Wong. 2002. Mutant SOD1 causes motor neuron disease independent of copper chaperone-mediated copper loading. *Nature Neurosci* 5

survival signals by phosphorylating 14-3-3. *The Journal of cell biology* 170 (2):295-304.

Mapping the folding free energy surface for metal-free human Cu,Zn superoxide

irreversible inactivation of protein tyrosine phosphatases PTP1B, CD45, and LAR

oxidative stress and apoptosis in nerve growth factor-deprived sympathetic

C. Kato, B. Pettmann, and A. O. Hueber. 2003. Fas/tumor necrosis factor receptor death signaling is required for axotomy-induced death of motoneurons in vivo. *J* 


Radi, R, J S Beckman, K M Bush, and B A Freeman. 1991. Peroxynitrite oxidation of

Repeated Author. 1991. Peroxynitrite-induced membrane lipid peroxidation: The cytotoxic potential of superoxide and nitric oxide. *Arch. Biochem. Biophys.* 288 (2):481-487. Radi, R. 2004. Nitric oxide, oxidants, and protein tyrosine nitration. *Proc Natl Acad Sci U S A*

Radi, R., A. Cassina, R. Hodara, C. Quijano, and L. Castro. 2002. Peroxynitrite reactions and

Rae, T. D., P. J. Schmidt, R. A. Pufahl, V. C. Culotta, and T. V. O'Halloran. 1999.

Rakhit, R., J. Robertson, C. Vande Velde, P. Horne, D. M. Ruth, J. Griffin, D. W. Cleveland,

pathological monomer-misfolded SOD1 in ALS. *Nature medicine* 13 (6):754-9. Ramachandran, Anup, Anna-Liisa Levonen, Paul S. Brookes, Erin Ceaser, Sruti Shiva, Maria

Undetectable intracellular free copper: the requirement of a copper chaperone for

N. R. Cashman, and A. Chakrabartty. 2007. An immunological epitope selective for

Cecilia Barone, and Victor Darley-Usmar. 2002. Mitochondria, nitric oxide, and cardiovascular dysfunction. *Free Radical Biology and Medicine* 33 (11):1465-1474. Raoul, C, C E Henderson, and B Pettmann. 1999. Programmed cell death of embryonic motoneurons triggered though the Fas death receptor. *J Cell Biol* 147 (5):1049-1062. Raoul, C, B Pettmann, and C E Henderson. 2000. Active killing of neurons during

development and following stress: a role for p75NTR and Fas? *Curr Op Neurobiol* 10

and G. Haase. 2006. Chronic activation in presymptomatic amyotrophic lateral sclerosis (ALS) mice of a feedback loop involving Fas, Daxx, and FasL. *PNAS* 103

and Anne-Odile Hueber. 2005. Expression of a dominant negative form of Daxx <I>in vivo</I> rescues motoneurons from Fas (CD95)-induced cell death. *Journal of* 

deLapeyrière, Chistopher E Henderson, Georg Haase, and Brigitte Pettmann. 2002. Motoneuron death triggered by a specific pathway downstream of Fas: potentiation

G Flood, M F Beal, R H Jr Brown, R W Scott, and W D Snider. 1996. Motor neurons in Cu/Zn superoxide dimutase-deficient mice develop normally but exhibit

L Carroll, and Alvaro G Estévez. 2006. Interactions between b-neuregulin and neurotrophins in motor neuron apoptosis. *Journal of Neurochemistry* 97 (1):222-233. Roberts, Blaine R., John A. Tainer, Elizabeth D. Getzoff, Dean A. Malencik, Sonia R.

Anderson, Valerie C. Bomben, Kathrin R. Meyers, P. Andrew Karplus, and Joseph S. Beckman. 2007. Structural Characterization of Zinc-deficient Human Superoxide Dismutase and Implications for ALS. *Journal of Molecular Biology* 373 (4):877-890.

Raoul, C., E. Buhler, C. Sadeghi, A. Jacquier, P. Aebischer, B. Pettmann, C. E. Henderson,

Raoul, Cedric, Catherine Barthelemy, Arnaud Couzinet, David Hancock, Brigitte Pettmann,

Raoul, Cédric, Alvaro G Estévez, Hiroshi Nishimune, Don W Cleveland, Odile

Reaume, A G, J L Elliott, E K Hoffman, N W Kowall, R J Ferrante, D F Siwek, H M Wilcox, D

Ricart, Karina C., Richard J. Pearson, Liliana Viera, Patricia Cassina, Andrés Kamaid, Steven

enhanced cell death after axonal injury. *Nature Genetics* 13:43-47.

by ALS-linked SOD1 mutations. *Neuron* 35:1067-1083.

formation in mitochondria. *Free Radic Biol Med* 33 (11):1451-64.

superoxide dismutase. *Science* 284 (5415):805-8.

266 (7):4244-4250.

101 (12):4003-4008.

(1):111-117.

(15):6007-6012.

*Neurobiology* 62 (2):178-188.

sulfhydryls. The cytotoxic potential of superoxide and nitric oxide. *J. Biol. Chem.*


**14** 

*Japan* 

Yoshiaki Furukawa

*Department of Chemistry, Keio University* 

**Protein Aggregates in Pathological Inclusions** 

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder that is characterized by a progressive loss of upper and/or lower motor neurons (Bruijn et al., 2004). Dysfunction and death of these neurons lead to muscle weakness, atrophy and spasticity. A fatal event for the majority of patients is a failure of the respiratory muscles, which generally occurs within one to five years of disease onset. The typical age of onset is between 50 and 60 years, and the prevalence rate is 5 – 10 cases per 100,000 populations (de Belleroche et al., 1996). No effective cures for this disease are currently available, and the pathomechanism still remains controversial. The majority of ALS cases have no genetic component (sporadic ALS, sALS),

Historically, ALS has been described by Charcot and Joffroy in 1869 (Charcot & Joffroy, 1869), and linkage analysis of fALS families was performed in 1991, by which the genetic locus was identified to be linked to chromosome 21q (Siddique et al., 1991). In 1993, Rosen et al. (Rosen et al., 1993) and Deng et al. (Deng et al., 1993) have found that mutations in the Cu,Zn-superoxide dismutase (SOD1) gene, which lies on chromosome 21q, are associated with fALS. Because SOD1-related fALS exhibited several clinicopathological similarities to sALS, various animal models including rodents, worms and flies have been constructed, in which mutant forms of SOD1 are expressed. Using these models, furthermore, various drugs have been continuingly tested to cure or alleviate ALS. In 2001, ALS2 (or called alsin) has been also identified as a new gene associated with a rare, recessively inherited and slowly progressed juvenile onset form of ALS, which is, however, significantly different from the disease phenotypes of sALS (Hadano et al., 2001; Yang et al., 2001). Accordingly, studies on mutant SOD1 have served as a "gold standard" for a long time and provided

Recent progress on genetic analysis has fuelled the identification of other genes responsible for fALS: for example, **T**AR **D**NA-binding **p**rotein-43 (TDP-43) gene reported in 2008 (Gitcho et al., 2008; Kabashi et al., 2008; Sreedharan et al., 2008; Van Deerlin et al., 2008), **Fu**sed in **S**arcoma (FUS) gene in 2009 (Kwiatkowski et al., 2009; Vance et al., 2009). Each of TDP-43 and FUS mutations describes approximately 4 % of total fALS cases, which is a smaller number than that of SOD1 mutations (~20 % of total fALS cases). Unlike SOD1, however, TDP-43 and/or FUS pathologies are observed in many of sALS patients (Deng et al., 2011b; Mackenzie et al., 2007) and also in other neurodegenerative diseases (Lagier-

while about 10 % are inherited in a dominant manner (familial ALS, fALS).

valuable insight into molecular pathomechanisms of ALS.

**1. Introduction** 

**of Amyotrophic Lateral Sclerosis** 

