**4. Excitotoxicity**

#### **4.1. Glutamate**

A dominant hypothesis of ALS pathogenesis is glutamate excitotoxicity. Glutamate is the major excitatory neurotransmitter found in mammalian central nervous system (CNS) however, in high concentrations is toxic to motor neurons. Some of the evidence supporting glutamate excitotoxicity was based on the observation that exposure of neuronal cell cultures to excess glutamate leads to cell death (Choi et al 1988). A similar observation was made in anterior horn cells in tissue cultures of rat spinal cord where incubation with high concentra‐ tions of glutamate is associated with cell loss (Silani et al 2000). In addition, defects in glutamate transport leading to elevated glutamate levels have been reported in mSOD1 mice and significant number of patients with sALS (Dunlop et al., Lin et al., Rothstein et al.,). Elevated glutamate levels were found in serum and spinal fluid of patients with sALS (Al-Chalabi, et al 2000; Rothstein et al., 1990; Shaw, et al, 1995). Another study showed that 40% of about 400 patients with sALS have an elevation in glutamate levels that correlates with the severity of the disease (Spreux-Varoquax et al., 2002)

The mechanism of glutamate neurotoxicity remains elusive. Excessive glutamate levels lead to activation of glutamate ionotropic AMPA receptors in neurons and glial cells. AMPA receptor activation triggers mitochondrial changes such as reduction in ATP synthesis, decreased cellular oxygen consumption, oxydative phosphorylation uncoupling, and increase in mitochondrial reactive oxygen species (ROS) production, causing a loss in the mitochondrial calcium buffer properties and apotosis (Heath and Shaw 2002) (Fig. 1). Rapid clearance of glutamate at the synapsis cleft is an essential step in the prevention of motor neuron excito‐ toxicity. This step accomplished by the astrocyte glutamate transporter excitatory amino acid-2 (EAAT2) (Rothstein et,al 1996). In transgenic mice, depletion of EAAT2 has been implicated with neuronal death (Rothstein et,al 1996). Abnormalities in EAAT2 expression were identified in two rodent models of fALS. In the SOD1G85R transgenic mice a ~ 50% decrease in EAAT2 expression was observed in the spinal cord at the end of the disease (Bruijin et al., 1997), while in the spinal cord ventral horn of transgenic SOD1G93A rats EAAT2 expression was decreased before the symptomatic stage of the disease and was almost undetectable at the end of the disease (Howland et al., 2002). Reduction in the expression of EAAT2 was found in motor neuron disease (Fray et al 1998) and decreased glutamate transport was identified in motor cortex and spinal cord in ALS (Rothstein et al., 1992) (Fig. 1).

#### **4.2. Glutamate receptor**

in ALS and it has been proposed that in fALS mutant SOD1 produces motor neuron death by allowing peroxynitrite formation and catalyzing tyrosine nitration, which in turn inhibits trophic signals (Estevez et al., 1999; Beckman et al., 1993; Crow et al., 1997; Ischiropoulos et al., 1992; Franco and Estevez, 2011). Motor neurons are highly dependent on a continuous supply of trophic factors to survive both *in vivo* and *in vitro*. Deprivation of trophic support *in vivo* by ventral root avulsion in adult animals and axotomy in newborns, but not in adults, triggers apoptosis (Li et al, 1994; Oppenheim, 1997; Gould and Oppenheim, 2011). Induction of apoptosis in these conditions is preceded by induction of neuronal nitric oxide synthase (nNOS) and nitric oxide production. Motor neuron death induced by trophic factor deprivation requires protein synthesis and caspase activation both *in vivo* and *in vitro* (Milligan et al., 1994; Li et al, 1998; Yaginuma et al, 2001)*.* Cultured motor neurons deprived of trophic factors induce nNOS expression, production of nitric oxide and peroxynitrite formation that is followed by tyrosine nitration, which precedes motor neuron death (Estevez et al., 1998). Inhibition of nitric oxide production and peroxynitrite formation prevents rather than delays motor neuron death, suggesting that peroxynitrite is acting at decision-making points in the apoptotic cascade. Deprivation of trophic factors activates the Fas pathway in motor neurons, and inhibition of the Fas pathway prevents motor neuron death. Fas activation in motor neurons triggers two parallel pathways: the classical extrinsic pathway recruiting FADD and Caspase 8; and a seemingly motor neuron specific pathway, that activates DAXX/ASK1/p38 and the induction of neuronal NOS, increasing production of nitric oxide, peroxynitrite

A dominant hypothesis of ALS pathogenesis is glutamate excitotoxicity. Glutamate is the major excitatory neurotransmitter found in mammalian central nervous system (CNS) however, in high concentrations is toxic to motor neurons. Some of the evidence supporting glutamate excitotoxicity was based on the observation that exposure of neuronal cell cultures to excess glutamate leads to cell death (Choi et al 1988). A similar observation was made in anterior horn cells in tissue cultures of rat spinal cord where incubation with high concentra‐ tions of glutamate is associated with cell loss (Silani et al 2000). In addition, defects in glutamate transport leading to elevated glutamate levels have been reported in mSOD1 mice and significant number of patients with sALS (Dunlop et al., Lin et al., Rothstein et al.,). Elevated glutamate levels were found in serum and spinal fluid of patients with sALS (Al-Chalabi, et al 2000; Rothstein et al., 1990; Shaw, et al, 1995). Another study showed that 40% of about 400 patients with sALS have an elevation in glutamate levels that correlates with the severity of

The mechanism of glutamate neurotoxicity remains elusive. Excessive glutamate levels lead to activation of glutamate ionotropic AMPA receptors in neurons and glial cells. AMPA receptor activation triggers mitochondrial changes such as reduction in ATP synthesis,

formation and tyrosine nitration (Raoul et al, 2002).

4 Current Advances in Amyotrophic Lateral Sclerosis

the disease (Spreux-Varoquax et al., 2002)

**4. Excitotoxicity**

**4.1. Glutamate**

An alteration in the expression of the glutamate receptor was found in motor neurons expressing mutant SOD1, suggesting that excitotoxicity is not only induced by increased glutamate levels but also by alterations in the glutamate signaling pathway (Spalloni et al., 2004). In oocytes co-expressing A4V or I113T-SOD1 with EAAT2, the mutants but not the wild type SOD1 selectively inactivated the glial glutamate transporter in the presence of hydrogen peroxide. This suggests that EAAT2 may be a target for mutant SOD1 toxicity (Trotti et al., 1999). On the other hand, overexpression of EAAT2 in mutant SOD1 expressing mice delayed the onset of motor neuron disease and decreased caspase 3 activation, the final step of the apoptotic pathway (Guo et al 2003). In motor cortex and spinal cord extracts from ALS patients, 25% of the patients showed almost complete loss of EAAT2 protein, and 80% of the patients showed some sort of protein abnormality (Rothstein et al., 1995) (Fig.1).

Glutamate receptor dysfunction is other possible route of excitotoxicity. Glutamate toxicity in motor neurons is primarily mediated via alpha-amino-3-hydroxy-5-methyl-4 isoxazole propionic acid (AMPA) receptors (Van Den Bosch et al., 2000). In patients with ALS, a deficiency in the AMPA receptor mRNA expression was found in spinal motor neurons (Kawahara, et al., 2004). This defect results in an increase in calcium influx through the receptor leading to cell damaged. The increased entry in calcium in addition to the reduction in the calcium buffer capacity due to abnormal mitochondria result in an increase in free intracellular calcium levels, leading to motor neuron death (Bogaert et al., 2010) (Fig.1). Additionally, the expression of the glutamate receptor subunits is reduced in ALS motor neurons (Williams et al 1997). Another pathway leading to excitotoxicity is via deficiency in glutamate dehydro‐ genase activity (Pioro et al., 1999).

The modest protection conferred by the antigluaminergic drug riluzole in ALS patients as well as in mutant SOD1 mice seems to support the effect of glutamate toxicity in the pathogenesis of ALS (Lacomblez et al., 1996; Gurney et al., 1996). However, whether riluzole protects by a mechanism related to its antiglutaminergic properties needs to be established.

resulting in mitochondrial protein retention and mitochondrial dysfunction with damaged of the mitochondrial membrane and loss of mitochondrial membrane potential and swelling (Wong et al., 1995; Kong et al., 1998). Severe mitochondrial morphological changes were found in NSC34 cells overexpressing mutant SOD1 (Raimondi et al., 2006; Menzies et al., 2002). In addition, mitochondrial swelling and vacuolization in motor neuron axons and dendrites were reported in mSOD1 mice even before disease onset (Wong et al., 1995; Kong et al., 1998; Borthwick et al., 1999). The presence of abnormal mitochondrial clusters was also described in mutant SOD1 rat motor axons (Sotelo-Silveira et al., 2009) as well as in lumbar spinal cord motor neurons and proximal axons of sALS patients (Sasaki et al., 1996; Hirano et al., 1984).

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Abnormal respiratory complex activities, disrupted redox homeostasis and decreased ATP production were described in ALS (Borthwick et al., 1999; Jung et al., 2002; Bowling et al., 1993; Ferri et al., 2006). Biochemical studies showed several abnormalities in mitochondrial electron transport chain. The enzymatic activity of the electron transport chain complexes I, II, IV was reduced in mSOD1 mice and cell cultures from patients with fALS (Jung et al., 2002; Mattiazzi et al., 2002). The interaction between cytochrome c and the inner mitochondrial membrane in addition to the activity of complex IV were reduced in the SOD1G93A transgenic mice (Kirkinezos et al., 2005). Decreased oxygen consumption, lack of ADP-dependent respiratory control, and decreased membrane potential were also reported in mutant SOD1

Mitochondria play an important role in the intracellular calcium homeostasis as a calcium buffer, accumulating or releasing calcium depending on the cytosolic levels. Abnormalities in mitochondrial calcium homeostasis were reported in ALS patients and in mutant SOD1 animals (Kruman et al., 1999; Carri et al., 1997; Reiner et al, 1995; Jaiswal et al., 2009). The release of calcium from the mitochondria leads to excessive intracellular calcium levels. This abnormal calcium homeostasis induces motor neuron death through several mechanisms including: 1) toxic generation of reactive oxygen species (ROS), as reported in SOD1G93A transgenic mice (Kruman et al., 1999); 2) release of cytochrome c from the mitochondria (Martin et al., 2009); 3) glutamate excitotoxicity (Nicholls et al., 2003), and others. All these mechanisms may have a special role in motor neurons because these cells contain less mitochondrial density per volume compared to non-neuronal cells, thus making neurons more deficient in mitochondrial calcium buffering properties (Grosskreutz et al., 2007) (Fig. 2). In addition, ALS patients show a deficiency in calcium binding proteins calbidin and paralbumin in cortical motor and spinal motor neurons. These two proteins regulate intracellular calcium levels and their deficiency may result in neuronal loss. On the contrary, oculomotor neurons or neuron from the Onuff's nuclei contains normal levels of calbidin and paralbumin levels and they are preserved despite ALS progression (Alexianu, et al. 1994; Celio, 1990; Ince et al., 1993, Palecek et al., 1999).

**5.2. Electron transport chain**

**5.3. Calcium homeostasis**

rat spinal astrocytes (Cassina et al., 2008) (Fig. 2).

Figure 1. **Induction of motor neuron death by glutamate excitotoxicity**. Red arrows/lines indicate the pathways that are affected or induced in ALS. **Figure 1. Induction of motor neuron death by glutamate excitotoxicity.** Red arrows/lines indicate the pathways that are affected or induced in ALS.

#### The mitochondrion is a vital organelle with multiple functions within cells. Mitochondria are the main source of ATP, maintain calcium homeostasis and participate in calcium signaling, and play a key role in the intrinsic apoptotic pathway. Mitochondrial malfunction turns motor neuron more vulnerable to damage, especially in aging and stress neurons. Mitochondrial malfunction is **5. Mitochondrial abnormalities**

**5. Mitochondrial abnormalities** 

**5.1. Mitochondrial morphology**  Indeed, mitochondria morphological and ultrastructural changes as well as bioenergetic malfunction have been reported in ALS. SOD1 is localized mainly in the cytoplasm, but has been found also in the mitochondria and other organelles (Okado et al., 2001; Sturtz et al., 2001). Mutant SOD1 protein is present in the mitochondrial intermembrane space, matrix and outer membrane of mitochondria (Higgins, et al, 2002; Vijayvergiya et al., 2005; Vande Velde et al., 2008; Kawamata et al., 2008). This abnormal SOD1 The mitochondrion is a vital organelle with multiple functions within cells. Mitochondria are the main source of ATP, maintain calcium homeostasis and participate in calcium signaling, and play a key role in the intrinsic apoptotic pathway. Mitochondrial malfunction turns motor neuron more vulnerable to damage, especially in aging and stress neurons. Mitochondrial malfunction is an important hypothesis in ALS pathogenesis (Bruijn et al., 2004; Manfredi et al., 2005).

protein may fail to fold properly resulting in mitochondrial protein retention and mitochondrial dysfunction with damaged of the

(Borthwick et al., 1999; Jung et al., 2002; Bowling et al., 1993; Ferri et al., 2006). Biochemical studies showed several abnormalities in mitochondrial electron transport chain. The enzymatic activity of the electron transport chain complexes I, II, IV was reduced in mSOD1 mice and cell cultures from patients with fALS (Jung et al., 2002; Mattiazzi et al., 2002). The interaction between cytochrome c and the inner mitochondrial membrane in addition to the activity of complex IV were reduced in the SOD1G93A transgenic mice (Kirkinezos et al., 2005). Decreased oxygen consumption, lack of ADP-dependent respiratory control, and

an important hypothesis in ALS pathogenesis (Bruijn et al., 2004; Manfredi et al., 2005).

#### mitochondrial membrane and loss of mitochondrial membrane potential and swelling (Wong et al., 1995; Kong et al., 1998). Severe mitochondrial morphological changes were found in NSC34 cells overexpressing mutant SOD1 (Raimondi et al., 2006; Menzies et **5.1. Mitochondrial morphology**

al., 2002). In addition, mitochondrial swelling and vacuolization in motor neuron axons and dendrites were reported in mSOD1 mice even before disease onset (Wong et al., 1995; Kong et al., 1998; Borthwick et al., 1999). The presence of abnormal mitochondrial clusters was also described in mutant SOD1 rat motor axons (Sotelo-Silveira et al., 2009) as well as in lumbar spinal cord motor neurons and proximal axons of sALS patients (Sasaki et al., 1996; Hirano et al., 1984). **5.2. Electron transport chain**  Abnormal respiratory complex activities, disrupted redox homeostasis and decreased ATP production were described in ALS Indeed, mitochondria morphological and ultrastructural changes as well as bioenergetic malfunction have been reported in ALS. SOD1 is localized mainly in the cytoplasm, but has been found also in the mitochondria and other organelles (Okado et al., 2001; Sturtz et al., 2001). Mutant SOD1 protein is present in the mitochondrial intermembrane space, matrix and outer membrane of mitochondria (Higgins, et al, 2002; Vijayvergiya et al., 2005; Vande Velde et al., 2008; Kawamata et al., 2008). This abnormal SOD1 protein may fail to fold properly

decreased membrane potential were also reported in mutant SOD1 rat spinal astrocytes (Cassina et al., 2008) (Fig. 2).

resulting in mitochondrial protein retention and mitochondrial dysfunction with damaged of the mitochondrial membrane and loss of mitochondrial membrane potential and swelling (Wong et al., 1995; Kong et al., 1998). Severe mitochondrial morphological changes were found in NSC34 cells overexpressing mutant SOD1 (Raimondi et al., 2006; Menzies et al., 2002). In addition, mitochondrial swelling and vacuolization in motor neuron axons and dendrites were reported in mSOD1 mice even before disease onset (Wong et al., 1995; Kong et al., 1998; Borthwick et al., 1999). The presence of abnormal mitochondrial clusters was also described in mutant SOD1 rat motor axons (Sotelo-Silveira et al., 2009) as well as in lumbar spinal cord motor neurons and proximal axons of sALS patients (Sasaki et al., 1996; Hirano et al., 1984).

#### **5.2. Electron transport chain**

Abnormal respiratory complex activities, disrupted redox homeostasis and decreased ATP production were described in ALS (Borthwick et al., 1999; Jung et al., 2002; Bowling et al., 1993; Ferri et al., 2006). Biochemical studies showed several abnormalities in mitochondrial electron transport chain. The enzymatic activity of the electron transport chain complexes I, II, IV was reduced in mSOD1 mice and cell cultures from patients with fALS (Jung et al., 2002; Mattiazzi et al., 2002). The interaction between cytochrome c and the inner mitochondrial membrane in addition to the activity of complex IV were reduced in the SOD1G93A transgenic mice (Kirkinezos et al., 2005). Decreased oxygen consumption, lack of ADP-dependent respiratory control, and decreased membrane potential were also reported in mutant SOD1 rat spinal astrocytes (Cassina et al., 2008) (Fig. 2).

#### **5.3. Calcium homeostasis**

Figure 1. **Induction of motor neuron death by glutamate excitotoxicity**. Red arrows/lines indicate the pathways that are affected or induced in

**Figure 1. Induction of motor neuron death by glutamate excitotoxicity.** Red arrows/lines indicate the pathways

The mitochondrion is a vital organelle with multiple functions within cells. Mitochondria are the main source of ATP, maintain calcium homeostasis and participate in calcium signaling, and play a key role in the intrinsic apoptotic pathway. Mitochondrial malfunction turns motor neuron more vulnerable to damage, especially in aging and stress neurons. Mitochondrial malfunction is an important hypothesis in ALS pathogenesis (Bruijn et al., 2004; Manfredi et

Indeed, mitochondria morphological and ultrastructural changes as well as bioenergetic malfunction have been reported in ALS. SOD1 is localized mainly in the cytoplasm, but has been found also in the mitochondria and other organelles (Okado et al., 2001; Sturtz et al., 2001). Mutant SOD1 protein is present in the mitochondrial intermembrane space, matrix and outer membrane of mitochondria (Higgins, et al, 2002; Vijayvergiya et al., 2005; Vande Velde et al., 2008; Kawamata et al., 2008). This abnormal SOD1 protein may fail to fold properly

Astrocyte

**ROS Ca2+** EAAT2

AMPA receptor Glutamate Mitochondrion

**CELL DEATH**

The mitochondrion is a vital organelle with multiple functions within cells. Mitochondria are the main source of ATP, maintain calcium homeostasis and participate in calcium signaling, and play a key role in the intrinsic apoptotic pathway. Mitochondrial malfunction turns motor neuron more vulnerable to damage, especially in aging and stress neurons. Mitochondrial malfunction is

Indeed, mitochondria morphological and ultrastructural changes as well as bioenergetic malfunction have been reported in ALS. SOD1 is localized mainly in the cytoplasm, but has been found also in the mitochondria and other organelles (Okado et al., 2001; Sturtz et al., 2001). Mutant SOD1 protein is present in the mitochondrial intermembrane space, matrix and outer membrane of mitochondria (Higgins, et al, 2002; Vijayvergiya et al., 2005; Vande Velde et al., 2008; Kawamata et al., 2008). This abnormal SOD1 protein may fail to fold properly resulting in mitochondrial protein retention and mitochondrial dysfunction with damaged of the mitochondrial membrane and loss of mitochondrial membrane potential and swelling (Wong et al., 1995; Kong et al., 1998). Severe mitochondrial morphological changes were found in NSC34 cells overexpressing mutant SOD1 (Raimondi et al., 2006; Menzies et al., 2002). In addition, mitochondrial swelling and vacuolization in motor neuron axons and dendrites were reported in mSOD1 mice even before disease onset (Wong et al., 1995; Kong et al., 1998; Borthwick et al., 1999). The presence of abnormal mitochondrial clusters was also described in mutant SOD1 rat motor axons (Sotelo-Silveira et al., 2009) as well as in lumbar spinal

Abnormal respiratory complex activities, disrupted redox homeostasis and decreased ATP production were described in ALS (Borthwick et al., 1999; Jung et al., 2002; Bowling et al., 1993; Ferri et al., 2006). Biochemical studies showed several abnormalities in mitochondrial electron transport chain. The enzymatic activity of the electron transport chain complexes I, II, IV was reduced in mSOD1 mice and cell cultures from patients with fALS (Jung et al., 2002; Mattiazzi et al., 2002). The interaction between cytochrome c and the inner mitochondrial membrane in addition to the activity of complex IV were reduced in the SOD1G93A transgenic mice (Kirkinezos et al., 2005). Decreased oxygen consumption, lack of ADP-dependent respiratory control, and

decreased membrane potential were also reported in mutant SOD1 rat spinal astrocytes (Cassina et al., 2008) (Fig. 2).

an important hypothesis in ALS pathogenesis (Bruijn et al., 2004; Manfredi et al., 2005).

Synap c cle

**Ca2+ Ca2+**

cord motor neurons and proximal axons of sALS patients (Sasaki et al., 1996; Hirano et al., 1984).

ALS.

al., 2005).

Impulse

6 Current Advances in Amyotrophic Lateral Sclerosis

that are affected or induced in ALS.

**5. Mitochondrial abnormalities** 

**5. Mitochondrial abnormalities**

**5.1. Mitochondrial morphology** 

**5.2. Electron transport chain** 

**5.1. Mitochondrial morphology**

Mitochondria play an important role in the intracellular calcium homeostasis as a calcium buffer, accumulating or releasing calcium depending on the cytosolic levels. Abnormalities in mitochondrial calcium homeostasis were reported in ALS patients and in mutant SOD1 animals (Kruman et al., 1999; Carri et al., 1997; Reiner et al, 1995; Jaiswal et al., 2009). The release of calcium from the mitochondria leads to excessive intracellular calcium levels. This abnormal calcium homeostasis induces motor neuron death through several mechanisms including: 1) toxic generation of reactive oxygen species (ROS), as reported in SOD1G93A transgenic mice (Kruman et al., 1999); 2) release of cytochrome c from the mitochondria (Martin et al., 2009); 3) glutamate excitotoxicity (Nicholls et al., 2003), and others. All these mechanisms may have a special role in motor neurons because these cells contain less mitochondrial density per volume compared to non-neuronal cells, thus making neurons more deficient in mitochondrial calcium buffering properties (Grosskreutz et al., 2007) (Fig. 2). In addition, ALS patients show a deficiency in calcium binding proteins calbidin and paralbumin in cortical motor and spinal motor neurons. These two proteins regulate intracellular calcium levels and their deficiency may result in neuronal loss. On the contrary, oculomotor neurons or neuron from the Onuff's nuclei contains normal levels of calbidin and paralbumin levels and they are preserved despite ALS progression (Alexianu, et al. 1994; Celio, 1990; Ince et al., 1993, Palecek et al., 1999).

Ince et al., 1993, Palecek et al., 1999).

**5.3. Calcium homeostasis** 

Mitochondria play an important role in the intracellular calcium homeostasis as a calcium buffer, accumulating or releasing calcium depending on the cytosolic levels. Abnormalities in mitochondrial calcium homeostasis were reported in ALS patients and in mutant SOD1 animals (Kruman et al., 1999; Carri et al., 1997; Reiner et al, 1995; Jaiswal et al., 2009). The release of calcium from the mitochondria leads to excessive intracellular calcium levels. This abnormal calcium homeostasis induces motor neuron death through several mechanisms including: 1) toxic generation of reactive oxygen species (ROS), as reported in SOD1G93A transgenic mice (Kruman et al., 1999); 2) release of cytochrome c from the mitochondria (Martin et al., 2009); 3) glutamate excitotoxicity (Nicholls et al., 2003), and others. All these mechanisms may have a special role in motor neurons because these cells contain less mitochondrial density per volume compared to non-neuronal cells, thus making neurons more deficient in mitochondrial calcium buffering properties (Grosskreutz et al., 2007) (Fig. 2). In addition, ALS patients show a deficiency in calcium binding proteins

their deficiency may result in neuronal loss. On the contrary, oculomotor neurons or neuron from the Onuff's nuclei contains normal levels of calbidin and paralbumin levels and they are preserved despite ALS progression (Alexianu, et al. 1994; Celio, 1990;

> mice overexpressing or with reduced expression of NF subunits (Collard et al., 1995; Cote et al., 1993). In neurons, mitochondria are frequently found in axon terminals due to the high demand of ATP and calcium handling at the synapses (Shepherd et al., 1998; Rowland et al., 2000). Mitochondria are transported in both anterograde and retrograde directions via kinesin and dynein motor complexes (Nangaku et al., 1994; Zhang, et al 2004; Varadi et al., 2004). The disruption of the mitochondrial axonal transport has been implicated in neurodegenerative diseases including ALS (Hollenbeck et al., 2005; De Vos et al., 2007; Magrane et al. 2009). Mitochondria display saltatory movement along microtubules. In SOD1G93A transgenic mice and cortical neurons transfected with G93A-SOD1, mitochondrial transport was selectively reduced in the anterograde direction (De Vos et al., 2007). In addition, in NSC34 cells overex‐ pressing mutant SOD1 mitochondrial transport was altered in both anterograde and retro‐ grade directions (Magrane et al., 2009). Abnormalities in axonal transport cause abnormal renewal of mitochondria and autophagosomes at distal motor axons resulting in mitochondrial accumulation, deficit in energy production, accumulation of ROS, and released of proapoptotic agents leading to neuronal death. Slow axonal transport impairment has been described as one of the earliest pathological events in mSOD1 mice (Williamson et al., 1999; Zhang et al. 1997). Fast axonal transport is mediated by kinesin while dynein motor complexes mediate the transport of membrane-bound organelles necessary for axonal and synaptic functions. In patients and transgenic rodent models of ALS there are impairments in the kinesin-mediated anterograde transport and dynein-mediated retrograde axonal transport (Williamson et al., 1999; Breuer et al 1987; Breuer et al., 1988; Collard et al., 1995; Sasaki et al., 1996; Ligon et al., 2005; Parkhouse et al., 2008). Disruption of kinesin heavy chain KIF5B causes perinuclear clustering of mitochondria in mice neurons, indicating that KIF5B is essential for mitochondrial dispersion (Tanaka et al., 1998). Abnormalities in the transport of other proteins such as dynactin, myosin and actine were also identified in transgenic mutant SOD1 models (LaMonte et al., 2002). Dynactin mutations have been associated with autosomal familiar motor neuron disease (Puls et al 2003; Puls et al 2005). Peripherin, another intermediate transport filament was found in neuronal inclusions of sALS (Corbo et al., 1992). Overexpression of peripherin in transgenic mice is associated with axonal degeneration. Inflammatory cytokines increased peripherin levels, suggesting an association between inflammation and axonal

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Several growth factors (GFs) has been investigated and potentially implicated in the patho‐ gensis of ALS. One of the most studied GFs is the vascular endothelial growth factor (VEGF), a protein involved in vasculogenesis and angiogenesis, and in restoration of oxygen supply upon limited blood circulation. Animal data suggest that VEGF may be neuroprotector. Overexpression of VEGF delayed onset and progression motor neuron disease, as shown in a double transgenic mice generated by crossing mice expressing human mutant SOD1 with mice overexpressing neuronal VEGF (mSOD1/VEGF). The mSOD1/VEGF transgenic mice showed a delayed in motor neuron loss, motor impairment, and a prolonged survival compared with

transport disorders (Sterneck, et al 1996).

**7. Growth factors**

Figure 2. **Mitochondrial abnormalities associated with mutant SOD1 (mSOD1)**. Mutant SOD1 translocates to mitochondrial intermembrane space and matrix, and is associated with the mitochondrial outer membrane. The expression of mutant SOD1 is linked to decrease of mitochondrial membrane potential and electron transport chain activity. The release of calcium and cytochrome c to the cytosol, and the production of ROS lead to cell death in ALS. **Figure 2. Mitochondrial abnormalities associated with mutant SOD1 (mSOD1).** Mutant SOD1 translocates to mi‐ tochondrial intermembrane space and matrix, and is associated with the mitochondrial outer membrane. The expres‐ sion of mutant SOD1 is linked to decrease of mitochondrial membrane potential and electron transport chain activity. The release of calcium and cytochrome c to the cytosol, and the production of ROS lead to cell death in ALS.

involved in axonal anterograde and retrograde transport between soma and motor axons. NFs are intermediate filaments made

#### Transport of proteins, vesicles, and organelles between cell body and terminal axons is a vital process in neuronal development, function and survival. Cytoskeletal proteins such as neurofilament (NFs) confer structure and shape to motor neurons, and are **6. Axonal transport abnormalities**

**6. Axonal transport abnormalities** 

from the assembly of light, medium, and heavy subunits (Maragakis and Galvez-Jimenez, 2012). Disorganization of NFs affects axonal transport resulting in axonal strangulation and accumulation of axonal cargo (Collard et al., 1995). A hallmark of ALS pathology is the abnormal accumulation of NFs in the neuronal cell bodies and proximal axons. Animal models and patients with ALS show that axonal transport is a critical component in ALS pathogenesis (Morrison et al., 1998; Lin et al., 2006). Transgenic models of fALS and sALS are associated with mutations of the heavy NF subunits (Figlewicz et al., 1994; Al-Chalabi et al., 1999). In addition, reduction in light subunit mRNA levels was found in motor neurons from the spinal cord of patients with ALS (Wong et al., 2000). Abnormal axonal transport, vacuolization and degeneration of axons and motor neurons have been reported in transgenic mice overexpressing or with reduced expression of NF subunits (Collard et al., 1995; Cote et al., 1993). In neurons, mitochondria are frequently found in axon terminals due to the high demand of ATP and calcium handling at the synapses Transport of proteins, vesicles, and organelles between cell body and terminal axons is a vital process in neuronal development, function and survival. Cytoskeletal proteins such as neurofilament (NFs) confer structure and shape to motor neurons, and are involved in axonal anterograde and retrograde transport between soma and motor axons. NFs are intermediate filaments made from the assembly of light, medium, and heavy subunits (Maragakis and Galvez-Jimenez, 2012). Disorganization of NFs affects axonal transport resulting in axonal strangulation and accumulation of axonal cargo (Collard et al., 1995). A hallmark of ALS pathology is the abnormal accumulation of NFs in the neuronal cell bodies and proximal axons. Animal models and patients with ALS show that axonal transport is a critical component in ALS pathogenesis (Morrison et al., 1998; Lin et al., 2006). Transgenic models of fALS and sALS are associated with mutations of the heavy NF subunits (Figlewicz et al., 1994; Al-Chalabi et al., 1999). In addition, reduction in light subunit mRNA levels was found in motor neurons from the spinal cord of patients with ALS (Wong et al., 2000). Abnormal axonal transport, vacuolization and degeneration of axons and motor neurons have been reported in transgenic

mice overexpressing or with reduced expression of NF subunits (Collard et al., 1995; Cote et al., 1993). In neurons, mitochondria are frequently found in axon terminals due to the high demand of ATP and calcium handling at the synapses (Shepherd et al., 1998; Rowland et al., 2000). Mitochondria are transported in both anterograde and retrograde directions via kinesin and dynein motor complexes (Nangaku et al., 1994; Zhang, et al 2004; Varadi et al., 2004). The disruption of the mitochondrial axonal transport has been implicated in neurodegenerative diseases including ALS (Hollenbeck et al., 2005; De Vos et al., 2007; Magrane et al. 2009). Mitochondria display saltatory movement along microtubules. In SOD1G93A transgenic mice and cortical neurons transfected with G93A-SOD1, mitochondrial transport was selectively reduced in the anterograde direction (De Vos et al., 2007). In addition, in NSC34 cells overex‐ pressing mutant SOD1 mitochondrial transport was altered in both anterograde and retro‐ grade directions (Magrane et al., 2009). Abnormalities in axonal transport cause abnormal renewal of mitochondria and autophagosomes at distal motor axons resulting in mitochondrial accumulation, deficit in energy production, accumulation of ROS, and released of proapoptotic agents leading to neuronal death. Slow axonal transport impairment has been described as one of the earliest pathological events in mSOD1 mice (Williamson et al., 1999; Zhang et al. 1997). Fast axonal transport is mediated by kinesin while dynein motor complexes mediate the transport of membrane-bound organelles necessary for axonal and synaptic functions. In patients and transgenic rodent models of ALS there are impairments in the kinesin-mediated anterograde transport and dynein-mediated retrograde axonal transport (Williamson et al., 1999; Breuer et al 1987; Breuer et al., 1988; Collard et al., 1995; Sasaki et al., 1996; Ligon et al., 2005; Parkhouse et al., 2008). Disruption of kinesin heavy chain KIF5B causes perinuclear clustering of mitochondria in mice neurons, indicating that KIF5B is essential for mitochondrial dispersion (Tanaka et al., 1998). Abnormalities in the transport of other proteins such as dynactin, myosin and actine were also identified in transgenic mutant SOD1 models (LaMonte et al., 2002). Dynactin mutations have been associated with autosomal familiar motor neuron disease (Puls et al 2003; Puls et al 2005). Peripherin, another intermediate transport filament was found in neuronal inclusions of sALS (Corbo et al., 1992). Overexpression of peripherin in transgenic mice is associated with axonal degeneration. Inflammatory cytokines increased peripherin levels, suggesting an association between inflammation and axonal transport disorders (Sterneck, et al 1996).

### **7. Growth factors**

**5.3. Calcium homeostasis** 

Ince et al., 1993, Palecek et al., 1999).

8 Current Advances in Amyotrophic Lateral Sclerosis

cell death in ALS.

**6. Axonal transport abnormalities** 

**6. Axonal transport abnormalities**

Mitochondria play an important role in the intracellular calcium homeostasis as a calcium buffer, accumulating or releasing calcium depending on the cytosolic levels. Abnormalities in mitochondrial calcium homeostasis were reported in ALS patients and in mutant SOD1 animals (Kruman et al., 1999; Carri et al., 1997; Reiner et al, 1995; Jaiswal et al., 2009). The release of calcium from the mitochondria leads to excessive intracellular calcium levels. This abnormal calcium homeostasis induces motor neuron death through several mechanisms including: 1) toxic generation of reactive oxygen species (ROS), as reported in SOD1G93A transgenic mice (Kruman et al., 1999); 2) release of cytochrome c from the mitochondria (Martin et al., 2009); 3) glutamate excitotoxicity (Nicholls et al., 2003), and others. All these mechanisms may have a special role in motor neurons because these cells contain less mitochondrial density per volume compared to non-neuronal cells, thus making neurons more deficient in mitochondrial calcium buffering properties (Grosskreutz et al., 2007) (Fig. 2). In addition, ALS patients show a deficiency in calcium binding proteins calbidin and paralbumin in cortical motor and spinal motor neurons. These two proteins regulate intracellular calcium levels and their deficiency may result in neuronal loss. On the contrary, oculomotor neurons or neuron from the Onuff's nuclei contains normal levels of calbidin and paralbumin levels and they are preserved despite ALS progression (Alexianu, et al. 1994; Celio, 1990;

Figure 2. **Mitochondrial abnormalities associated with mutant SOD1 (mSOD1)**. Mutant SOD1 translocates to mitochondrial intermembrane space and matrix, and is associated with the mitochondrial outer membrane. The expression of mutant SOD1 is linked to decrease of mitochondrial membrane potential and electron transport chain activity. The release of calcium and cytochrome c to the cytosol, and the production of ROS lead to

**Figure 2. Mitochondrial abnormalities associated with mutant SOD1 (mSOD1).** Mutant SOD1 translocates to mi‐ tochondrial intermembrane space and matrix, and is associated with the mitochondrial outer membrane. The expres‐ sion of mutant SOD1 is linked to decrease of mitochondrial membrane potential and electron transport chain activity.

Transport of proteins, vesicles, and organelles between cell body and terminal axons is a vital process in neuronal development, function and survival. Cytoskeletal proteins such as neurofilament (NFs) confer structure and shape to motor neurons, and are involved in axonal anterograde and retrograde transport between soma and motor axons. NFs are intermediate filaments made from the assembly of light, medium, and heavy subunits (Maragakis and Galvez-Jimenez, 2012). Disorganization of NFs affects axonal transport resulting in axonal strangulation and accumulation of axonal cargo (Collard et al., 1995). A hallmark of ALS pathology is the abnormal accumulation of NFs in the neuronal cell bodies and proximal axons. Animal models and patients with ALS show that axonal transport is a critical component in ALS pathogenesis (Morrison et al., 1998; Lin et al., 2006). Transgenic models of fALS and sALS are associated with mutations of the heavy NF subunits (Figlewicz et al., 1994; Al-Chalabi et al., 1999). In addition, reduction in light subunit mRNA levels was found in motor neurons from the spinal cord of patients with ALS (Wong et al., 2000). Abnormal axonal transport, vacuolization and degeneration of axons and motor neurons have been reported in transgenic

The release of calcium and cytochrome c to the cytosol, and the production of ROS lead to cell death in ALS.

Transport of proteins, vesicles, and organelles between cell body and terminal axons is a vital process in neuronal development, function and survival. Cytoskeletal proteins such as neurofilament (NFs) confer structure and shape to motor neurons, and are involved in axonal anterograde and retrograde transport between soma and motor axons. NFs are intermediate filaments made from the assembly of light, medium, and heavy subunits (Maragakis and Galvez-Jimenez, 2012). Disorganization of NFs affects axonal transport resulting in axonal strangulation and accumulation of axonal cargo (Collard et al., 1995). A hallmark of ALS pathology is the abnormal accumulation of NFs in the neuronal cell bodies and proximal axons. Animal models and patients with ALS show that axonal transport is a critical component in ALS pathogenesis (Morrison et al., 1998; Lin et al., 2006). Transgenic models of fALS and sALS are associated with mutations of the heavy NF subunits (Figlewicz et al., 1994; Al-Chalabi et al., 1999). In addition, reduction in light subunit mRNA levels was found in motor neurons from the spinal cord of patients with ALS (Wong et al., 2000). Abnormal axonal transport, vacuolization and degeneration of axons and motor neurons have been reported in transgenic mice overexpressing or with reduced expression of NF subunits (Collard et al., 1995; Cote et al., 1993). In neurons, mitochondria are frequently found in axon terminals due to the high demand of ATP and calcium handling at the synapses

Several growth factors (GFs) has been investigated and potentially implicated in the patho‐ gensis of ALS. One of the most studied GFs is the vascular endothelial growth factor (VEGF), a protein involved in vasculogenesis and angiogenesis, and in restoration of oxygen supply upon limited blood circulation. Animal data suggest that VEGF may be neuroprotector. Overexpression of VEGF delayed onset and progression motor neuron disease, as shown in a double transgenic mice generated by crossing mice expressing human mutant SOD1 with mice overexpressing neuronal VEGF (mSOD1/VEGF). The mSOD1/VEGF transgenic mice showed a delayed in motor neuron loss, motor impairment, and a prolonged survival compared with mutant SOD1 transgenic mice (Wang et al., 2007). Intracerebroventricular administration of VEGF in a SOD1G93A rat model of ALS delayed motor neuron degeneration and onset of paralysis, improved motor performance, preserved neuromuscular junction, and extended survival (Storkebaum et al., 2005). This study also showed that in SOD1G93A mice, neurons expressing a transgenic VEGF receptor prolonged mice survival. Also supporting the VEGF neuroprotective role, a single injection of a VEGF-expressing lentiviral vector into several muscles of SOD1G93A mice delayed the onset as well as the progression of the disease even at onset of paralysis (Azzouz et al., 2004). Interestingly, mouse models in which the hypoxiaresponse element in the VEGF gene was deleted showed a decrease in VEGF expression in normoxia and under hypoxic conditions. This model resulted in a progressive motor neuron degeneration disease that resembles ALS (Oosthuyse, et al., 2001). A meta-analysis of over 900 individuals from Sweden and over 1,000 individuals from Belgium and England with a specific haplotype for VEGF associated with reduced circulating VEGF and VEGF gene transciption showed a two-fold increase in the risk of developing ALS for these individuals (Lambrechts et al., 2003). In another study, SOD1G93A mice crossed with VEGF haplotype mice showed a much more severe motor neuron degeneration. VEGF probably has neuronal direct and indirect neuroprotective effects preventing ischemic changes while regulating vascular perfusion.

parkinsonism-dementia complex, and familial British dementia (Sreedharan et al., 2008; Kabashi et al., 2008; Van Deerlin et al., 2008; Yokoseki et al., 2008; Rutherford et al., 2008; Del Bo et al., 2009; Hasegawa et al., 2007; Schwab et al., 2009). TDP-43 is a nuclear protein expressed in almost all tissues that binds to mRNA and DNA and regulates mRNA processing processes such as splicing, translation, and gene transcription. TDP-43 structure consists of two RNA recognition motifs (RRMs) that bind to nucleic acids, and a glycine rich domain containing the majority of ALS associated mutations (Cohen et al., 2012; Buratti et al., 2001). Genetic mutation of another RNA processing protein, fused in sarcoma /translated in liposarcoma (FUS/TLS), has been also associated with ALS (Kwiatkowski et al., 2009; Vance et al., 2009). The FUS/TLS has a similar structure to TDP-43 with RRM and glycin rich domains. FUS/TLS, also a nuclear protein, accumulates in intracytoplasmatic tau- and TDP-43 negative inclusions in patients with fALS, sALS, and frontal lobar degeneration FTLD-FUS (Mackenzie et al., 2010). TDP-43 and FUS/TLS stabilized mRNA encoding histone deacetylase 6 (HDAC6) involved in clearance of misfolded protein aggregates (Kim et.al, 2010, Fiesel, et al., 2010; Lee et al., 2010; Kawaguchi et al., 2003). TDP-43 binds to a wide range of RNA targets and promotes the synthesis of several proteins implicated in the neuronal development and integrity (Tollervey et al., 2011). TDP-43 expression is carefully controlled by a tightly autoregulated mechanism (Winton et al., 2008). Thus, TDP-43 abnormalities in RNA binding and autoregulation, and FUS/TLS may have a essential role in neuronal integrity. TDP-43 also has a protective effect on mitochondrial function; abnormal expression of mitochondrial fission/fusion proteins in transgenic mice expressing human wild-type TDP-43 transgene driven by mouse prion promoter had been reported (Xu et al., 2010). In cultured cells, exposure to stress caused TDP-43 to be relocated into stress granules (SGs). This abnormal localization of TDP-43 could start a pathological TDP-43 aggregation or TDP-43 interaction with other SGs-proteins. A similar process may occur with FUS/TLS protein (Bosco et al., 2010; Dormann et al., 2010). The formation of SGs may lead to pathological inclusion aggregations resulting in neuronal and glial cell damage. Hyperphosphorilated TDP-43 aggregates were identified in ALS spinal cord and FTLP-TDP brain tissue. TDP-43 glycin-rich domain, where most of the mutations had been identified, seems to be required for TDP-43 association with SGs. Expression of insoluble aggregates of TDP-43 terminal fragment was implicated in the generation of SGs (Liu-Yesucevitz et al., 2010). In addition, TDP-43 interacts with cytoplasmatic Ataxin-2 protein resulting in TDP-43 accumulation in misfolded aggregates. Mutant polyglutamine expansions within ataxin-2 enhanced the binding to TDP-43 facilitating the formation of aggregates in ALS patients (Elden,AC et al., 2010). The formation of these aggregates seems to be implicated in neuronal

Pathophysiology of Amyotrophic Lateral Sclerosis

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Evidence is accumulating indicating that motor neuron degeneration in ALS is not only restricted to neuronal autonomous cell death but it is rather a more complex process involving inflammatory neurotoxicity from non-neuronal glial cells such as astrocytes and microglia (Phani et al., 2012). Support for non-autonomous evidence comes from several studies in

death, but the mechanism remains elusive.

**9. Non-cell autonomous mechanisms**

Additionally, VEGF-B, a homolog of VEGF with minimal angiogenetic activity, was shown to be protective for cultured primary motor neurons. In addition, transgenic mice with deletion of the VEGF-B gene were implicated in an ALS-like pathogenesis, as shown crossing a VEGF-B knockout mouse with transgenic mice expressing human mutant SOD1 (mSOD1/VEGF-B-/-) (Poesen et al., 2008). mSOD1/VEGF-B-/- mice showed an earlier death and more severe motor neuron degeneration compared with mutant SOD1 transgenic mice. Intracerebroventricular administration of VEGF-B in a SOD1G93A rat model of ALS prolonged the survival of mutant SOD-expressing rats, suggesting that VEGF is neuroprotective by a mechanism independent of angiogenesis (Poesen et al., 2008).

Other beneficial growth factors are innsuline growth factor-1, glial cell line –derived neuro‐ trophic factor, and brain derived neurotrophic factor.
