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

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

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

(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

Several lines of evidence support the role of oxidative stress in mutant SOD1 toxicity, but some evidence suggest that interactions other than the redox properties of the enzyme stimulate oxidative stress by different mechanisms. Mutant SOD1 can induce oxidative stress by disruption of the redox-sensitive regulation of NADPH oxidase (Nox) in microglial cells. Noxs are transmembrane proteins that catalyze the reduction of oxygen to superoxide using NADPH as an electron donor (Brown and Griendling, 2009). Superoxide is then converted to hydrogen peroxide by SOD1. Under reducing conditions, SOD1 regulates Nox2 activation by binding and stabilizing Rac1. The oxidation of Rac1 by hydrogen peroxide disrupts the complex with SOD1 and inactivates Nox2. Upon expression of certain ALS SOD1 mutants, the dissociation of Rac1 from SOD1 is impaired and Nox2 remains active (Figure 1C). In addition, the expression of Nox2 is upregulated in the SOD1G93A mouse model and in ALS patients. In fact, gene deletion of Nox1 or Nox2 provides the larger protection to date in animal models of

Mitochondria are one of the major sources of cellular ROS formed as by-products of oxidative phosphorylation. Abnormalities in the mitochondrial structure, localization and number as well as altered activity of the electron transport chain have been described in both, sporadic and familial ALS (Manfredi and Xu, 2005). The mitochondrial electron transport chain and ATP synthesis are severely impaired at disease onset in spinal cord and brain of SOD1G93A transgenic mice (Lin and Beal, 2006). Both, wild type and mutant SOD localize in mitochondria in the central nervous system (Higgins et al., 2002). Mutant human SOD1 was found in the mitochondrial outer membrane, intermembrane space and matrix in transgenic mice, while inactive mutant SOD1 accumulates and forms aggregates in the mitochondrial matrix in the brain (Vijayvergiya et al., 2005). Aggregates of the mutant enzyme are also selectively found in the mitochondrial outer membrane in spinal cord from mouse models of ALS (Liu et al., 2004). Interestingly, the anti-apoptotic protein Bcl-2 binds to mutant SOD1 and aggregates in spinal cord mitochondria from patients and a mouse model of ALS, suggesting that mutant SOD1 may be toxic by depleting motor neurons of this anti-apoptotic protein (Pasinelli et al., 2004). Mutant SOD1 targeted to the mitochondrial intermembrane space in NSC34 cells induces cell death upon exposure of the cells to hydrogen peroxide (Magrane et al., 2009). In addition, the increase in carbonylated proteins and lipid hydroperoxides in mitochondria, as well as the abnormally high rates of production of hydrogen peroxide in SOD1G93A transgenic

**3. Regulation of NADPH oxidase activity by mutant SOD1**

source of debate and remains to be determined.

146 Current Advances in Amyotrophic Lateral Sclerosis

ALS (Harraz et al., 2008; Marden et al., 2007).

**4. Mutant SOD1 translocation to mitochondria**

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

last few years, a role for microglia and astrocytes in the induction of motor neuron death has

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Activated microglia is found in the spinal cord of SOD1G93A transgenic mice, suggesting that it may play a role in the neurodegeneration of neighboring motor neurons (Beers et al., 2006). Reducing the expression of mutant SOD1 in microglia and peripheral macrophages in chimeric mice leads to a delay in the late progression of ALS but has little effect on the onset and early disease progression (Boillee et al., 2006). Likewise, in the PU.1(-/-)/SOD1G93A mice unable to synthesize myeloid cells, the replacement of microglia, monocyte, and macrophage lineages with genotypically identical wild type cells slows disease progression and extends overall survival (Beers et al., 2006), suggesting that non cell-autonomous effects contribute to ALS progression independently of disease onset. Comparable findings were observed in co-culture studies where glial cells expressing mutant-SOD1 had a direct adverse effect on motor neuron survival (Di Giorgio et al., 2007). Microglia expressing G93A-SOD1 is toxic to primary motor neurons *in vitro.* In addition, SODG93A microglia show an increase in superoxide and nitric oxide production and release respect to wild type microglia. Treatment with lipopolysaccharide further increases SODG93A microglia activation and induction of motor neuron death (Beers et al., 2006). Hence, mutant SOD1-expressing microglia is activated, is more susceptible to activation, and it is capable of inducing motor neuron death *in vitro* (Beers et al., 2006). Interestingly, PU.1(-/-) mice transplanted with bone marrow from a SOD1G93A donor do not develop clinical or pathological evidence of ALS, suggesting that expression of mutant SOD1 in microglia is not enough to induce motor neuron disease *in vivo* (Beers et al., 2006). The fact that expression of mutant SOD1 in microglia alone does not induce motor neuron degeneration suggests that motor neurons and other glial cells play a role in the pathological process. Indeed, motor neurons expressing mutant SOD1 are more susceptible to cell death induced by

Astrocytes are the most abundant non-neuronal cells in the nervous system. The co-culture of normal primary embryonic or stem cell-derived motor neurons with astrocytes expressing mutant SOD1 result in motor neuron death. The death pathway is triggered by a toxic factor released by the astrocytes (Aebischer et al., 2011; Nagai et al., 2007). A population of pheno‐ typically aberrant astrocytes was recently described in the SOD1G93A mouse model of ALS (Diaz-Amarilla et al., 2011). These astrocytes, referred to as "AbA cells", have an increased proliferative capacity and secrete soluble factors that are 10 times more potent than neonatal SOD1G93A astrocytes for the induction of motor neuron death. AbA cells are present in degenerating spinal cord of SODG93A rats surrounding affected motor neurons, and their number increases dramatically after disease onset, highlighting the importance of this finding. Interestingly, the levels of interferon-γ (IFNγ) are significantly increased in mutant SOD1 expressing astrocytes, and IFNγ induces motor neuron death (Aebischer et al., 2011), suggest‐ ing that this cytokine may be one of the toxic factors mediating induction of cell death (Figure

*5.2.1. Role of microglia in the induction of motor neuron death*

exposure to nitric oxide or Fas activation (Raoul et al., 2002).

*5.2.2. Role of astrocytes in the induction of motor neuron death*

become evident.

**Figure 1. Role of oxidative stress in mutant SOD1 toxic gain-of-function**. A. The toxic gain-of-function depends on the redox properties of the enzyme and relies on the copper atom. The mutant SOD1 translocates to mitochondria, while the metal-deficient enzyme may translocate and bind copper in the organelle. B. Zn-deficient SOD1 as catalyst of ROS production and tyrosine nitration. C. Mutant SOD1 regulation of ROS production by Noxs. NO2-Tyr: nitrotyro‐ sine, HO. : hydroxyl radical, O2: molecular oxygen, ONOO- : peroxynitrite, NO. : nitric oxide, O2.-: superoxide.

neurons as well as embryonic stem cell-derived motor neurons expressing mutant SOD1 showed changes characteristic of neurodegeneration (Di Giorgio et al., 2007; Raoul et al., 2002). Primary embryonic motor neurons from SOD1G93A and SOD1G85R transgenic animals exposed to endogenously produced or exogenously added nitric oxide show an increased susceptibility to cell death in culture (Raoul et al., 2002). Thus, motor neurons expressing mutant SOD1 are susceptible to cell death stimulated by oxidative stress.

#### **5.2. Expression of mutant SOD1 in glial cells**

Neighboring cells also seem to play a role in mutant SOD1 toxicity. Normal motor neurons in the context of a mutant SOD1-expressing chimera show signs of neurodegeneration, while non-neuronal cells negative for mutant SOD1 expression delay neuronal degeneration and significantly extend survival of mutant-expressing motor neurons (Clement et al., 2003). In the last few years, a role for microglia and astrocytes in the induction of motor neuron death has become evident.

#### *5.2.1. Role of microglia in the induction of motor neuron death*

Activated microglia is found in the spinal cord of SOD1G93A transgenic mice, suggesting that it may play a role in the neurodegeneration of neighboring motor neurons (Beers et al., 2006). Reducing the expression of mutant SOD1 in microglia and peripheral macrophages in chimeric mice leads to a delay in the late progression of ALS but has little effect on the onset and early disease progression (Boillee et al., 2006). Likewise, in the PU.1(-/-)/SOD1G93A mice unable to synthesize myeloid cells, the replacement of microglia, monocyte, and macrophage lineages with genotypically identical wild type cells slows disease progression and extends overall survival (Beers et al., 2006), suggesting that non cell-autonomous effects contribute to ALS progression independently of disease onset. Comparable findings were observed in co-culture studies where glial cells expressing mutant-SOD1 had a direct adverse effect on motor neuron survival (Di Giorgio et al., 2007). Microglia expressing G93A-SOD1 is toxic to primary motor neurons *in vitro.* In addition, SODG93A microglia show an increase in superoxide and nitric oxide production and release respect to wild type microglia. Treatment with lipopolysaccharide further increases SODG93A microglia activation and induction of motor neuron death (Beers et al., 2006). Hence, mutant SOD1-expressing microglia is activated, is more susceptible to activation, and it is capable of inducing motor neuron death *in vitro* (Beers et al., 2006). Interestingly, PU.1(-/-) mice transplanted with bone marrow from a SOD1G93A donor do not develop clinical or pathological evidence of ALS, suggesting that expression of mutant SOD1 in microglia is not enough to induce motor neuron disease *in vivo* (Beers et al., 2006). The fact that expression of mutant SOD1 in microglia alone does not induce motor neuron degeneration suggests that motor neurons and other glial cells play a role in the pathological process. Indeed, motor neurons expressing mutant SOD1 are more susceptible to cell death induced by exposure to nitric oxide or Fas activation (Raoul et al., 2002).

#### *5.2.2. Role of astrocytes in the induction of motor neuron death*

neurons as well as embryonic stem cell-derived motor neurons expressing mutant SOD1 showed changes characteristic of neurodegeneration (Di Giorgio et al., 2007; Raoul et al., 2002). Primary embryonic motor neurons from SOD1G93A and SOD1G85R transgenic animals exposed to endogenously produced or exogenously added nitric oxide show an increased susceptibility to cell death in culture (Raoul et al., 2002). Thus, motor neurons expressing

: peroxynitrite, NO.

: nitric oxide, O2.-: superoxide.

**Figure 1. Role of oxidative stress in mutant SOD1 toxic gain-of-function**. A. The toxic gain-of-function depends on the redox properties of the enzyme and relies on the copper atom. The mutant SOD1 translocates to mitochondria, while the metal-deficient enzyme may translocate and bind copper in the organelle. B. Zn-deficient SOD1 as catalyst of ROS production and tyrosine nitration. C. Mutant SOD1 regulation of ROS production by Noxs. NO2-Tyr: nitrotyro‐

Neighboring cells also seem to play a role in mutant SOD1 toxicity. Normal motor neurons in the context of a mutant SOD1-expressing chimera show signs of neurodegeneration, while non-neuronal cells negative for mutant SOD1 expression delay neuronal degeneration and significantly extend survival of mutant-expressing motor neurons (Clement et al., 2003). In the

mutant SOD1 are susceptible to cell death stimulated by oxidative stress.

**5.2. Expression of mutant SOD1 in glial cells**

: hydroxyl radical, O2: molecular oxygen, ONOO-

148 Current Advances in Amyotrophic Lateral Sclerosis

sine, HO.

Astrocytes are the most abundant non-neuronal cells in the nervous system. The co-culture of normal primary embryonic or stem cell-derived motor neurons with astrocytes expressing mutant SOD1 result in motor neuron death. The death pathway is triggered by a toxic factor released by the astrocytes (Aebischer et al., 2011; Nagai et al., 2007). A population of pheno‐ typically aberrant astrocytes was recently described in the SOD1G93A mouse model of ALS (Diaz-Amarilla et al., 2011). These astrocytes, referred to as "AbA cells", have an increased proliferative capacity and secrete soluble factors that are 10 times more potent than neonatal SOD1G93A astrocytes for the induction of motor neuron death. AbA cells are present in degenerating spinal cord of SODG93A rats surrounding affected motor neurons, and their number increases dramatically after disease onset, highlighting the importance of this finding. Interestingly, the levels of interferon-γ (IFNγ) are significantly increased in mutant SOD1 expressing astrocytes, and IFNγ induces motor neuron death (Aebischer et al., 2011), suggest‐ ing that this cytokine may be one of the toxic factors mediating induction of cell death (Figure 2). The role of astrocytes in the induction of motor neuron death was recently confirmed in astrocytes generated from post-mortem tissue of familial and sporadic ALS patients, addi‐ tionally providing an *in vitro* model system for the study of these mechanisms (Haidet-Phillips et al., 2011).

deficient SOD1 reacts with hydrogen peroxide, produces superoxide and peroxynitrite, and is able to catalyze tyrosine nitration, altering the cellular redox balance. In addition, although not related to the redox properties of the enzyme, the interaction of mutant SOD1 with mitochondria and Nox, the two major sources of cellular ROS, further support the involvement of oxidative stress in the toxic gain-of-function. The cell type affected by mutant SOD1 is also controversial. A picture in which several cell types are affected and play a role at different stages of the disease seems to be emerging. In this context, during onset and early stages of the disease SOD1-expressing motor neurons undergo neurodegeneration and cell death by cell-autonomous processes. The activation of microglia and astrocytes may work as an amplification mechanism in the induction of motor neuron death in the late progression of the

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María Clara Franco1,2, Cassandra N. Dennys1,2, Fabian H. Rossi1,2 and Alvaro G. Estévez1,2

1 Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida,

[1] Aebischer, J, Cassina, P, Otsmane, B, Moumen, A, Seilhean, D, Meininger, V, Barbei‐ to, L, Pettmann, B, & Raoul, C. (2011). IFNgamma triggers a LIGHT-dependent selec‐ tive death of motoneurons contributing to the non-cell-autonomous effects of mutant

[2] Barber, S. C, & Shaw, P. J. (2010). Oxidative stress in ALS: key role in motor neuron

[3] Beal, M. F, Ferrante, L. J, Browne, S. E, Matthews, R. T, Kowall, N. W, & Brown, R. H. (1997). Increased 3-nitrotyrosine in both sporadic and familial amyotrophic lateral

[4] Beckman, J. S, Carson, M, Smith, C. D, & Koppenol, W. H. (1993). ALS, SOD and per‐

[5] Beckman, J. S, Estévez, A. G, Barbeito, L, & Crow, J. P. (2002). CCS knockout mice establish an alternative source of copper for SOD in ALS. *Free Rad. Biol. Med.* , 33,

injury and therapeutic target. *Free radical biology & medicine* , 48, 629-641.

disease (Figure 2).

**Author details**

Orlando, FL, USA

**References**

2 Orlando VA Healthcare System, Orlando, USA

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

oxynitrite. *Nature* 364:584.

1433-1435.

SOD1. *Cell death and differentiation* , 18, 754-768.

**Figure 2. Model for the role of motor neurons and glia in the ROS-mediated ALS progression.** Different cell types are affected and play a role at different stages of the disease. During the onset and early disease progression, motor neurons undergo degeneration and cell death by cell-autonomous mechanisms. Later in the disease progression, acti‐ vated microglia and astrocytes release ROS, RNS, and toxic factors that magnify the injury (cell death amplification mechanisms).

## **6. Conclusion**

In summary, the mechanism of mutant SOD1 toxicity is unknown and highly controversial but there is strong evidence suggesting that the mutant SOD1 toxic gain-of-function is related to an alteration of its redox properties and the induction of oxidative stress. In this scenario, the aberrant chemistry of mutant SOD1 turns the enzyme from antioxidant to pro-oxidant. Zndeficient SOD1 reacts with hydrogen peroxide, produces superoxide and peroxynitrite, and is able to catalyze tyrosine nitration, altering the cellular redox balance. In addition, although not related to the redox properties of the enzyme, the interaction of mutant SOD1 with mitochondria and Nox, the two major sources of cellular ROS, further support the involvement of oxidative stress in the toxic gain-of-function. The cell type affected by mutant SOD1 is also controversial. A picture in which several cell types are affected and play a role at different stages of the disease seems to be emerging. In this context, during onset and early stages of the disease SOD1-expressing motor neurons undergo neurodegeneration and cell death by cell-autonomous processes. The activation of microglia and astrocytes may work as an amplification mechanism in the induction of motor neuron death in the late progression of the disease (Figure 2).
