**2. Mechanisms of motor neuron death in ALS**

After one hundred fifty years since the first ALS description of the disease, the cause of motor neuron degeneration remains unknown, but progress in neuroscience and clinical research has identified several mechanisms that seem to be involved in the cell death process, such as glutamate-mediated excitotoxicity, inflammatory events, axonal transport deficits, oxidative stress, mitochondrial dysfunction and energy failure.

### **2.1 Excitotoxicity**

Based on the reduction of glutamate transporter-1 (GLT1 in rodents and excitatory amino acid transporter 2 or EAAT2 in human) content detected post-mortem in motor cortex and spinal cord of ALS patients (Rothstein et al., 1992; Rothstein et al., 1995) and on the increase of glutamate concentration in the cerebrospinal fluid (CSF) of about 40% of ALS patients (Shaw et al., 1995b; Spreux-Varoquaux et al., 2002), one proposed mechanism to explain

Role of Mitochondrial Dysfunction in Motor Neuron Degeneration in ALS 199

Fig. 1. Scheme of the main proposed mechanisms involved in motor neuron death.

Description in the text.

motor neuron death in ALS is glutamate-mediated excitotoxicity. This hypothesis has been generally accepted, although some data from our laboratory do not support it because a chronic increase in extracellular glutamate due to glutamate transport inhibition in the spinal cord in vivo was innocuous for motor neurons (Tovar-y-Romo et al., 2009b). However, overactivation of glutamate ionotropic receptors by agonists leads to neuronal death by augmenting the influx of Ca2+ into motor neurons. Experimental models in vivo have shown that of three major glutamate ionotropic receptor types, NMDA (N-methyl-Daspartate), kainate and AMPA (α-amino-3-hydroxy-5-isoxazolepropionate), the Ca2+ permeable AMPA receptor seems to be particularly involved in motor neuron death, because the selective blockade of Ca2+-permeable AMPA receptors or the chelation of intracellular Ca2+ prevents the motor neuron loss and the consequent paralysis induced by the infusion of AMPA into the rat lumbar spinal cord (Corona & Tapia, 2004, 2007; Tovar-y-Romo et al., 2009a). The Ca2+ permeability of this receptor is governed by the presence of the GluR2 subunit and its edition in the Q/R (glutamine/arginine) site of the second transmembrane domain (Burnashev et al., 1992; Corona & Tapia, 2007; Hollmann et al., 1991; Hume et al., 1991).

Increases in cytoplasmic Ca2+ concentration can be buffered by mitochondria, but when maintained for prolonged periods can cause mitochondrial swelling and dysfunction. These alterations are associated with deficits in mitochondrial ATP synthesis and energetic failure (this topic will be discussed later). The energetic deficits have been mainly associated with cell death process similar to necrosis (Kroemer et al., 2009; Martin, 2010). On the other hand, mitochondrial damage has also been linked to the release of proapoptotic factors such as cytochrome c and apoptosis-inducing factor (Martin et al., 2009). Cytochrome c involvement has been stressed because of its role in triggering the caspases pathway, which leads to apoptotic cellular death. In the cytoplasm cytochrome c promotes the formation of the apoptosome complex and activates caspase-3. The necrosis and apoptosis pathways are illustrated in Fig. 1.

### **2.2 Axonal transport deficits**

Because of the structural and functional characteristics of motor neuron axons, the role of axonal transport is essential for the communication between the neuronal soma and the periphery, as well as for the anterograde and retrograde dispersive distribution of cargo intracellular structures such as vesicles or organelles. Changes in the speed of anterograde and retrograde transport (Breuer & Atkinson, 1988; Breuer et al., 1987; Sasaki & Iwata, 1996), as well as neurofilament disorganization and accumulation of mitochondria, vesicles and smooth endoplasmic reticulum have been described in peripheral nerves of ALS patients (Hirano et al., 1984a, b; Sasaki & Iwata, 1996). These alterations in axonal transport have been observed also in transgenic models of FALS, which have allowed the study of their progression and the molecular machinery involved (Bilsland et al., 2010; Brunet et al., 2009; Collard et al., 1995; De Vos et al., 2007; Ligon et al., 2005; Perlson et al., 2009; Pun et al., 2006; Tateno et al., 2009; Warita et al., 1999; Williamson & Cleveland, 1999). In mutant SOD1 (mSOD1) rodents, some motor proteins such as: dynein, dynactin, kinesin, myosin, actin, and microtubules and neurofilaments are affected by mSOD1 aggregates (Breuer & Atkinson, 1988; Breuer et al., 1987; Collard et al., 1995; Ligon et al., 2005; Sasaki & Iwata, 1996; Williamson & Cleveland, 1999; Zhang et al., 2007).

motor neuron death in ALS is glutamate-mediated excitotoxicity. This hypothesis has been generally accepted, although some data from our laboratory do not support it because a chronic increase in extracellular glutamate due to glutamate transport inhibition in the spinal cord in vivo was innocuous for motor neurons (Tovar-y-Romo et al., 2009b). However, overactivation of glutamate ionotropic receptors by agonists leads to neuronal death by augmenting the influx of Ca2+ into motor neurons. Experimental models in vivo have shown that of three major glutamate ionotropic receptor types, NMDA (N-methyl-Daspartate), kainate and AMPA (α-amino-3-hydroxy-5-isoxazolepropionate), the Ca2+ permeable AMPA receptor seems to be particularly involved in motor neuron death, because the selective blockade of Ca2+-permeable AMPA receptors or the chelation of intracellular Ca2+ prevents the motor neuron loss and the consequent paralysis induced by the infusion of AMPA into the rat lumbar spinal cord (Corona & Tapia, 2004, 2007; Tovar-y-Romo et al., 2009a). The Ca2+ permeability of this receptor is governed by the presence of the GluR2 subunit and its edition in the Q/R (glutamine/arginine) site of the second transmembrane domain (Burnashev et al., 1992; Corona & Tapia, 2007; Hollmann et al.,

Increases in cytoplasmic Ca2+ concentration can be buffered by mitochondria, but when maintained for prolonged periods can cause mitochondrial swelling and dysfunction. These alterations are associated with deficits in mitochondrial ATP synthesis and energetic failure (this topic will be discussed later). The energetic deficits have been mainly associated with cell death process similar to necrosis (Kroemer et al., 2009; Martin, 2010). On the other hand, mitochondrial damage has also been linked to the release of proapoptotic factors such as cytochrome c and apoptosis-inducing factor (Martin et al., 2009). Cytochrome c involvement has been stressed because of its role in triggering the caspases pathway, which leads to apoptotic cellular death. In the cytoplasm cytochrome c promotes the formation of the apoptosome complex and activates caspase-3. The necrosis and apoptosis pathways are

Because of the structural and functional characteristics of motor neuron axons, the role of axonal transport is essential for the communication between the neuronal soma and the periphery, as well as for the anterograde and retrograde dispersive distribution of cargo intracellular structures such as vesicles or organelles. Changes in the speed of anterograde and retrograde transport (Breuer & Atkinson, 1988; Breuer et al., 1987; Sasaki & Iwata, 1996), as well as neurofilament disorganization and accumulation of mitochondria, vesicles and smooth endoplasmic reticulum have been described in peripheral nerves of ALS patients (Hirano et al., 1984a, b; Sasaki & Iwata, 1996). These alterations in axonal transport have been observed also in transgenic models of FALS, which have allowed the study of their progression and the molecular machinery involved (Bilsland et al., 2010; Brunet et al., 2009; Collard et al., 1995; De Vos et al., 2007; Ligon et al., 2005; Perlson et al., 2009; Pun et al., 2006; Tateno et al., 2009; Warita et al., 1999; Williamson & Cleveland, 1999). In mutant SOD1 (mSOD1) rodents, some motor proteins such as: dynein, dynactin, kinesin, myosin, actin, and microtubules and neurofilaments are affected by mSOD1 aggregates (Breuer & Atkinson, 1988; Breuer et al., 1987; Collard et al., 1995; Ligon et al., 2005; Sasaki & Iwata, 1996; Williamson &

1991; Hume et al., 1991).

illustrated in Fig. 1.

**2.2 Axonal transport deficits** 

Cleveland, 1999; Zhang et al., 2007).

Fig. 1. Scheme of the main proposed mechanisms involved in motor neuron death. Description in the text.

Role of Mitochondrial Dysfunction in Motor Neuron Degeneration in ALS 201

Mitochondria, ROS and glutamate-induced excitotoxicity are closely related and this is relevant in ALS, because the mitochondrion is the main oxygen consumer and it is also the main producer of ROS. These species can be produced in neurons and in non-neuronal cells and can cause failure in the glutamate uptake system of both motor neurons and astroglia (Rao et al., 2003; Trotti et al., 1996, 1998; Volterra et al., 1994; Zagami et al., 2009). This may contribute to an excitotoxic condition due to increased concentration of extracellular glutamate. ROS production and its effects on motor neurons and non-neuronal cells are

A mechanism of non-cell-autonomous death associated with motor neuron degeneration in both FALS and SALS is the participation of non-neuronal cells in inflammatory events (Boillee et al., 2006a; Boillee et al., 2006b; Hall et al., 1998; Yamanaka et al., 2008; Yang et al., 2011). The main histopathological feature of inflammation is the proliferation of reactive astrogliosis and of activated microglial cells, associated with alterations in their cellular functions, such as glutamate reuptake failure and release of proapoptotic and proinflammatory factors (Sanagi et al., 2010; Sargsyan et al., 2005; Sofroniew, 2005). Molecules associated with inflammatory process, such as interleukins 6, 12, 15, 17A, 23, C4d and C3d complement proteins, as well as tumor necrosis factor-alpha, have been found in blood and CSF from ALS patients (Almer et al., 2002; Fiala et al., 2010; Henkel et al., 2004; Kawamata et al., 1992; McGeer et al., 1991; Moreau et al., 2005; Rentzos et al., 2010a, b). The finding of increased levels of granzymes A, B in serum (Ilzecka, 2011) and decrease in cytochrome c levels in the CSF (Ilzecka, 2007), suggests an apoptotic process in human disease. The proliferation of activated non-neuronal cells has been associated with the disease severity (Clement et al., 2003). Nevertheless, alteration in their functions may be more important than their proliferation (Lepore et al., 2008). In experimental models of FALS (mSOD1) it has been attempted to prevent the motor neuron loss through the use of drugs with anti-inflammatory properties, such as minocycline (Keller et al., 2010; Kriz et al., 2002; Neymotin et al., 2009; Van Den Bosch et al., 2002; Zhu et al., 2002). This drug was effective in delaying the motor neurons loss when given prior to the symptoms onset, but when given at late stages it exaggerated the neuroinflammatory response and accelerated the progression of the symptoms (Keller et al., 2010). In this transgenic ALS model, apoptosis processes can be triggered by non-neuronal cells through the extrinsic apoptotic pathway, via the release from activated glial cells of several death ligands (for example FasL) that bind to their respective death receptor (Fas) and trigger the cleavage of caspase-8

(Locatelli et al., 2007; Petri et al., 2006; Raoul et al., 2006) (Fig. 1).

and apoptotic death, thus playing a central role in neuronal survival.

**3. Mitochondrial dysfunction in ALS and in experimental motor neuron** 

A convergent point of the deleterious mechanisms discussed above is the mitochondrion. This organelle is the main energy producer in eukaryotic cells and plays a fundamental role in normal cell physiology. Among the functions mitochondria carry out, besides ATP synthesis, intracellular Ca2+buffering has been recognized as another relevant factor for the protection against deleterious processes such as oxidative stress, excitotoxicity and necrotic

illustrated in Fig. 1.

**2.4 Inflammation** 

**degeneration** 

These deficits may affect the renewal of organelles in the axon terminals of motor neurons, leading to accumulation of damaged mitochondria or autophagosomes, increased ROS production, disruption of microtubule formation and stability (Julien & Mushynski, 1998), as well as damage of presynaptic structure such as swelling of axon terminals (Komatsu et al., 2007). Accumulation of damaged mitochondria may result in energetic failure (Liu et al., 2004; Martin et al., 2009; Menzies et al., 2002a, b; Pasinelli et al., 2004; Wong et al., 1995; Zhu et al., 2002) and in the release of proapoptotic factors (Pasinelli et al., 2004) (Fig. 1, bottom left). These alterations may be involved in the distal neurophaty and impairment of muscular reinnervation observed in ALS.

### **2.3 Oxidative stress**

Another mechanism implicated in motor neuron degeneration in ALS that involves both motor neurons and non-neuronal cells is oxidative stress. Reactive oxygen species (ROS) arise in cells as aerobic metabolism by-products, mostly due to the leakage of electrons from the mitochondrial respiratory chain, resulting in an incomplete reduction of molecular oxygen during the oxidative phosphorylation, generating the superoxide radical anion (O2•-). The O2•- anion reacts quickly with the nitric oxide radical (NO•, produced by nitric oxide synthase, NOS) to form peroxynitrite (ONOO-). Meanwhile, the product of O2• dismutation, H2O2, slowly decomposes to form the highly reactive hydroxyl radical (•OH). Both ONOO- and •OH are highly reactive and can damage proteins, membranes and DNA by oxidation. Cellular mechanisms to combat the constant production of free radicals are: 1) enzymes such as SOD, catalase and peroxidase, which catalytically remove reactive species; 2) reducing agents synthesized in vivo, such as glutathione, -keto acids, lipoic acid and coenzyme Q, and compounds obtained from the diet, such as ascorbate (vitamin C) and tocopherol (vitamin E); and 3) chaperone heat shock proteins which remove or facilitate repair of damaged proteins. Oxidative stress arises from an imbalance between ROS production and its control mechanisms.

The involvement of oxidative stress in ALS pathogenesis is supported by abundant evidence that has been reported in both SALS and FALS patients, where several indicators of increased oxidative damage have been found: 1) In postmortem central nervous system (CNS) tissue samples (mainly spinal cord) these markers include oxidized DNA (Ferrante et al., 1997b; Fitzmaurice et al., 1996), lipid peroxidation (Siciliano et al., 2002), protein glycoxidation (Shibata et al., 2001), elevated protein carbonylation (Ferrante et al., 1997b; Shaw et al., 1995a), and increased protein tyrosine nitration; remarkably, nitrotyrosine immunoreactivity was more densely detected in motor neurons (Abe et al., 1995; Abe et al., 1997; Beal et al., 1997; Ferrante et al., 1997a). 2) Oxidation markers in CSF, plasma and blood from living ALS patients during the course of the disease have also been described. The most relevant are oxidized DNA (Bogdanov et al., 2000; Ihara et al., 2005), hydroxyl and ascorbate free radicals (Ihara et al., 2005), lipid peroxidation (Baillet et al., 2010; Bogdanov et al., 2000; Bonnefont-Rousselot et al., 2000; Ihara et al., 2005; Oteiza et al., 1997; Simpson et al., 2004; Smith et al., 1998), and a remarkable elevation of 3-nitrotyrosine levels in CSF (Tohgi et al., 1999). However, in other study, 3-nitroyrosine was not different between the CSF of ALS patients and control subjects (Ryberg et al., 2004). Increased oxidative damage to proteins, lipids and DNA has also been demonstrated in CNS tissue of transgenic mouse model of FALS expressing mSOD1 (Andrus et al., 1998; Casoni et al., 2005; Liu et al., 1999; Liu et al., 1998; Poon et al., 2005).

Mitochondria, ROS and glutamate-induced excitotoxicity are closely related and this is relevant in ALS, because the mitochondrion is the main oxygen consumer and it is also the main producer of ROS. These species can be produced in neurons and in non-neuronal cells and can cause failure in the glutamate uptake system of both motor neurons and astroglia (Rao et al., 2003; Trotti et al., 1996, 1998; Volterra et al., 1994; Zagami et al., 2009). This may contribute to an excitotoxic condition due to increased concentration of extracellular glutamate. ROS production and its effects on motor neurons and non-neuronal cells are illustrated in Fig. 1.

### **2.4 Inflammation**

200 Amyotrophic Lateral Sclerosis

These deficits may affect the renewal of organelles in the axon terminals of motor neurons, leading to accumulation of damaged mitochondria or autophagosomes, increased ROS production, disruption of microtubule formation and stability (Julien & Mushynski, 1998), as well as damage of presynaptic structure such as swelling of axon terminals (Komatsu et al., 2007). Accumulation of damaged mitochondria may result in energetic failure (Liu et al., 2004; Martin et al., 2009; Menzies et al., 2002a, b; Pasinelli et al., 2004; Wong et al., 1995; Zhu et al., 2002) and in the release of proapoptotic factors (Pasinelli et al., 2004) (Fig. 1, bottom left). These alterations may be involved in the distal neurophaty and impairment of

Another mechanism implicated in motor neuron degeneration in ALS that involves both motor neurons and non-neuronal cells is oxidative stress. Reactive oxygen species (ROS) arise in cells as aerobic metabolism by-products, mostly due to the leakage of electrons from the mitochondrial respiratory chain, resulting in an incomplete reduction of molecular oxygen during the oxidative phosphorylation, generating the superoxide radical anion (O2•-). The O2•- anion reacts quickly with the nitric oxide radical (NO•, produced by nitric oxide synthase, NOS) to form peroxynitrite (ONOO-). Meanwhile, the product of O2• dismutation, H2O2, slowly decomposes to form the highly reactive hydroxyl radical (•OH). Both ONOO- and •OH are highly reactive and can damage proteins, membranes and DNA by oxidation. Cellular mechanisms to combat the constant production of free radicals are: 1) enzymes such as SOD, catalase and peroxidase, which catalytically remove reactive species; 2) reducing agents synthesized in vivo, such as glutathione, -keto acids, lipoic acid and coenzyme Q, and compounds obtained from the diet, such as ascorbate (vitamin C) and tocopherol (vitamin E); and 3) chaperone heat shock proteins which remove or facilitate repair of damaged proteins. Oxidative stress arises from an imbalance between ROS

The involvement of oxidative stress in ALS pathogenesis is supported by abundant evidence that has been reported in both SALS and FALS patients, where several indicators of increased oxidative damage have been found: 1) In postmortem central nervous system (CNS) tissue samples (mainly spinal cord) these markers include oxidized DNA (Ferrante et al., 1997b; Fitzmaurice et al., 1996), lipid peroxidation (Siciliano et al., 2002), protein glycoxidation (Shibata et al., 2001), elevated protein carbonylation (Ferrante et al., 1997b; Shaw et al., 1995a), and increased protein tyrosine nitration; remarkably, nitrotyrosine immunoreactivity was more densely detected in motor neurons (Abe et al., 1995; Abe et al., 1997; Beal et al., 1997; Ferrante et al., 1997a). 2) Oxidation markers in CSF, plasma and blood from living ALS patients during the course of the disease have also been described. The most relevant are oxidized DNA (Bogdanov et al., 2000; Ihara et al., 2005), hydroxyl and ascorbate free radicals (Ihara et al., 2005), lipid peroxidation (Baillet et al., 2010; Bogdanov et al., 2000; Bonnefont-Rousselot et al., 2000; Ihara et al., 2005; Oteiza et al., 1997; Simpson et al., 2004; Smith et al., 1998), and a remarkable elevation of 3-nitrotyrosine levels in CSF (Tohgi et al., 1999). However, in other study, 3-nitroyrosine was not different between the CSF of ALS patients and control subjects (Ryberg et al., 2004). Increased oxidative damage to proteins, lipids and DNA has also been demonstrated in CNS tissue of transgenic mouse model of FALS expressing mSOD1 (Andrus et al., 1998; Casoni et al., 2005; Liu et al., 1999;

muscular reinnervation observed in ALS.

production and its control mechanisms.

Liu et al., 1998; Poon et al., 2005).

**2.3 Oxidative stress** 

A mechanism of non-cell-autonomous death associated with motor neuron degeneration in both FALS and SALS is the participation of non-neuronal cells in inflammatory events (Boillee et al., 2006a; Boillee et al., 2006b; Hall et al., 1998; Yamanaka et al., 2008; Yang et al., 2011). The main histopathological feature of inflammation is the proliferation of reactive astrogliosis and of activated microglial cells, associated with alterations in their cellular functions, such as glutamate reuptake failure and release of proapoptotic and proinflammatory factors (Sanagi et al., 2010; Sargsyan et al., 2005; Sofroniew, 2005). Molecules associated with inflammatory process, such as interleukins 6, 12, 15, 17A, 23, C4d and C3d complement proteins, as well as tumor necrosis factor-alpha, have been found in blood and CSF from ALS patients (Almer et al., 2002; Fiala et al., 2010; Henkel et al., 2004; Kawamata et al., 1992; McGeer et al., 1991; Moreau et al., 2005; Rentzos et al., 2010a, b). The finding of increased levels of granzymes A, B in serum (Ilzecka, 2011) and decrease in cytochrome c levels in the CSF (Ilzecka, 2007), suggests an apoptotic process in human disease. The proliferation of activated non-neuronal cells has been associated with the disease severity (Clement et al., 2003). Nevertheless, alteration in their functions may be more important than their proliferation (Lepore et al., 2008). In experimental models of FALS (mSOD1) it has been attempted to prevent the motor neuron loss through the use of drugs with anti-inflammatory properties, such as minocycline (Keller et al., 2010; Kriz et al., 2002; Neymotin et al., 2009; Van Den Bosch et al., 2002; Zhu et al., 2002). This drug was effective in delaying the motor neurons loss when given prior to the symptoms onset, but when given at late stages it exaggerated the neuroinflammatory response and accelerated the progression of the symptoms (Keller et al., 2010). In this transgenic ALS model, apoptosis processes can be triggered by non-neuronal cells through the extrinsic apoptotic pathway, via the release from activated glial cells of several death ligands (for example FasL) that bind to their respective death receptor (Fas) and trigger the cleavage of caspase-8 (Locatelli et al., 2007; Petri et al., 2006; Raoul et al., 2006) (Fig. 1).
