**2. Pathology**

ALS is characterized by upper motor neuron (corticospinal motor neurons) and lower motor neuron (bulbospinal motor neurons) degeneration and death as well as reactive gliosis

© 2013 Rossi et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

replacing death neurons (Leigh and Garafolo, 1995). As corticospinal motor neuron degenerate the cells suffer from a retrograde axonal loss with secondary myelin pallor and gliosis. These changes are most severed at the brainstem and upper spinal cord, but are extended throughout the spinal cord (Brownell et al., 1970). ALS motor cortex shows astrocytic gliosis, especially in the deeper layers at the gray matter and underlying the subcortical white matter. Irregular immunoreactivity to GFAP is identified in the motor strip (Kamo, et al. 1987; Ince, 2000). The lysosomal marker CD68 also revealed that most of the glial response at the cortical and spinal tracts corresponds to microglia activation and active macrophages (Cagnin et al., 2001; Sitte et al., 2001). ALS affects spinal motor neurons of the ventral horn and brainstem motor neurons. The autopsy of ALS patients shows loss of motor neurons and atrophic motor neurons with basophilic appearance suggesting a programmed cell mechanism (Martin, 1999). The ventral roots become thin with loss of large myelinated fibers in motor nerves leading to denervation atrophy with evidence of reinnervation in affected muscles. Frontal temporal dementia ALS (FTD-ALS) is a neurodegenerative disorder associated with ALS that presents typical patho‐ logical findings of the disease in addition to neuronal loss of the frontal or temporal cortex, hippocampus and amygdale, and spongiform changes of the neocortex with (Leigh PN and Garofolo, 1995). Non-motor findings encountered in ALS pathology are posterior columns demyelination and reduced density of myelinated sensory fibers (Ince, 2007)

*2.1.1.2. Fused in Sarcoma protein (FUS)*

*2.1.2. Bunina bodies*

C (Okamoto et al., 1993).

**3. Oxidative stress**

ing in the cells.

by the oxidant peroxynitrite (ONOO-

TDP-43 and ubiquitin (Chaudhuri et al., 1995).

*2.1.3. Hyaline Conglomerate Inclusions (HCI)*

contrary of UBI. (Ince PG and Wharton S, 2007).

Recently, mutations in the gene codifying for the fused in sarcoma protein (FUS) have been linked to fALS. Indeed, spinal cord LMNs in fALS and sALS but not in mSOD1-fALS are immunoreactive for FUS inclusions. These inclusions also present immunoreactivity for

Bunina bodies are eosinophilic paracrystalline bodies present in the LMNs of many cases of ALS (Piao et al., 2003). They are immunoreactive for a cysteine protease inhibitor called cystatin

HCI consist of intracellular accumulation of intermediate filament proteins, especially hyperphosphorylated neurofilament subunits and peripherin (Corbo and Hays, 2002), and are found in the motor cortex neurons (Troost et al., 1992). HCI are much less frequently encoun‐ tered in spinal motor neurons than UBI and they are mainly associated with some types of mSOD1 fALS. They form a larger conglomeration than UBI and are positive for silver staining,

Mutations in the gene of copper/zinc superoxide dismutase type 1 (SOD1) are the most common cause of fALS (Rothstein, 2009; Boillee and Cleveland, 2008; Robberecht and Phillips, 2013). Recent reports indicate that SOD1 mutations may also be the cause of between 0.7 - 4% cases of sporadic ALS (sALS) (Robberecht and Phillips, 2013). SOD1 is primarily an antioxidant

hydrogen peroxide (H2O2). However, SOD1-linked fALS is most likely not caused by loss of the normal SOD1 activity, but rather by a gain of a toxic function. One of the hypotheses for mutant SOD-linked fALS toxicity proposes that an aberrant SOD1 chemistry is responsible for the toxic gain-of-function, which allows small molecules such us peroxynitrite or hydrogen peroxide to produce damaging free radicals. Other hypotheses for mutant SOD1 neurotoxicity include inhibition of the proteasome activity, mitochondrial damage, and formation of intracellular aggregates. SOD1 aggregation is an early event in ALS and could mediate motor neuron degeneration via sequestration of cellular components, decreasing chaperone activity and the ubiquitin proteasome pathway. Also, SOD1 mutations seem to disrupt RNA process‐

Defining the role of oxidative stress, and particularly nitrative stress in neurodegeneration has been extremely difficult because of the multiplicity of potential targets that can be damaged by oxidation and nitration. Certain proteins are particularly susceptible to tyrosine nitration

). Tyrosine nitration is a well-established, early biomarker

.-) to oxygen (O2) and

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metalloenzyme that catalyzes the conversion of superoxide radical (O2

#### **2.1. Inclusion bodies**

The hallmark finding of lower motor neuron (LMN) pathology in ALS is the presence of intracellular inclusion bodies in neuronal soma and proximal dendrites as well as glia (Barbeito et al., 2004).

#### *2.1.1. Ubiquitylated Inclusions (UBI)*

UBI are the most common and specific inclusion in ALS, found at LMN of the spinal cord and brainstem (Matsumoto et al., 1993) and also at the corticospinal tract upper motor neurons (UMN) (Sasaki and Maruyama, 1994). UBI morphological spectrum goes from thread-like ubiquitylated profiles, through skeins of different compactness to more spherical bodies (Ince et al, 1998). The compacted lesions may be eosinophilic, basophilic and "Lewy-like" in appearance. The composition of UBI remains unknown but several proteins were identified in UBI such as ubiquitin (Leigh et al., 1991), peripherin (He and Hays, 2004), Cu/Zn SOD1 (Shibata 1996) and dorfin (Niwa et al., 2002). UBI are present in near 100% of sALS (Ince et al., 2003). However, UBI are found in FTD with ubiquitin positive/tau negative inclusions. In both fALS and sporadic types of ALS-FTD, UBI are found in cortical frontal and temporal lobe neurons.

#### *2.1.1.1. TAR DNA binding Protein 43 (TDP-43)*

TDP-43 is a major component of ubiquinated inclusions in sALS, FTD with ubiquitin-positive but tau-negative inclusions (non-tau FTD), FTD-ALS, and non-SOD1 fALS. TDP-43 inclusions are practically not present in mSOD1-related fALS.

#### *2.1.1.2. Fused in Sarcoma protein (FUS)*

Recently, mutations in the gene codifying for the fused in sarcoma protein (FUS) have been linked to fALS. Indeed, spinal cord LMNs in fALS and sALS but not in mSOD1-fALS are immunoreactive for FUS inclusions. These inclusions also present immunoreactivity for TDP-43 and ubiquitin (Chaudhuri et al., 1995).

#### *2.1.2. Bunina bodies*

replacing death neurons (Leigh and Garafolo, 1995). As corticospinal motor neuron degenerate the cells suffer from a retrograde axonal loss with secondary myelin pallor and gliosis. These changes are most severed at the brainstem and upper spinal cord, but are extended throughout the spinal cord (Brownell et al., 1970). ALS motor cortex shows astrocytic gliosis, especially in the deeper layers at the gray matter and underlying the subcortical white matter. Irregular immunoreactivity to GFAP is identified in the motor strip (Kamo, et al. 1987; Ince, 2000). The lysosomal marker CD68 also revealed that most of the glial response at the cortical and spinal tracts corresponds to microglia activation and active macrophages (Cagnin et al., 2001; Sitte et al., 2001). ALS affects spinal motor neurons of the ventral horn and brainstem motor neurons. The autopsy of ALS patients shows loss of motor neurons and atrophic motor neurons with basophilic appearance suggesting a programmed cell mechanism (Martin, 1999). The ventral roots become thin with loss of large myelinated fibers in motor nerves leading to denervation atrophy with evidence of reinnervation in affected muscles. Frontal temporal dementia ALS (FTD-ALS) is a neurodegenerative disorder associated with ALS that presents typical patho‐ logical findings of the disease in addition to neuronal loss of the frontal or temporal cortex, hippocampus and amygdale, and spongiform changes of the neocortex with (Leigh PN and Garofolo, 1995). Non-motor findings encountered in ALS pathology are posterior columns

demyelination and reduced density of myelinated sensory fibers (Ince, 2007)

The hallmark finding of lower motor neuron (LMN) pathology in ALS is the presence of intracellular inclusion bodies in neuronal soma and proximal dendrites as well as glia (Barbeito

UBI are the most common and specific inclusion in ALS, found at LMN of the spinal cord and brainstem (Matsumoto et al., 1993) and also at the corticospinal tract upper motor neurons (UMN) (Sasaki and Maruyama, 1994). UBI morphological spectrum goes from thread-like ubiquitylated profiles, through skeins of different compactness to more spherical bodies (Ince et al, 1998). The compacted lesions may be eosinophilic, basophilic and "Lewy-like" in appearance. The composition of UBI remains unknown but several proteins were identified in UBI such as ubiquitin (Leigh et al., 1991), peripherin (He and Hays, 2004), Cu/Zn SOD1 (Shibata 1996) and dorfin (Niwa et al., 2002). UBI are present in near 100% of sALS (Ince et al., 2003). However, UBI are found in FTD with ubiquitin positive/tau negative inclusions. In both fALS and sporadic types of ALS-FTD, UBI are found in cortical frontal and temporal lobe

TDP-43 is a major component of ubiquinated inclusions in sALS, FTD with ubiquitin-positive but tau-negative inclusions (non-tau FTD), FTD-ALS, and non-SOD1 fALS. TDP-43 inclusions

**2.1. Inclusion bodies**

*2.1.1. Ubiquitylated Inclusions (UBI)*

2 Current Advances in Amyotrophic Lateral Sclerosis

*2.1.1.1. TAR DNA binding Protein 43 (TDP-43)*

are practically not present in mSOD1-related fALS.

et al., 2004).

neurons.

Bunina bodies are eosinophilic paracrystalline bodies present in the LMNs of many cases of ALS (Piao et al., 2003). They are immunoreactive for a cysteine protease inhibitor called cystatin C (Okamoto et al., 1993).

#### *2.1.3. Hyaline Conglomerate Inclusions (HCI)*

HCI consist of intracellular accumulation of intermediate filament proteins, especially hyperphosphorylated neurofilament subunits and peripherin (Corbo and Hays, 2002), and are found in the motor cortex neurons (Troost et al., 1992). HCI are much less frequently encoun‐ tered in spinal motor neurons than UBI and they are mainly associated with some types of mSOD1 fALS. They form a larger conglomeration than UBI and are positive for silver staining, contrary of UBI. (Ince PG and Wharton S, 2007).

### **3. Oxidative stress**

Mutations in the gene of copper/zinc superoxide dismutase type 1 (SOD1) are the most common cause of fALS (Rothstein, 2009; Boillee and Cleveland, 2008; Robberecht and Phillips, 2013). Recent reports indicate that SOD1 mutations may also be the cause of between 0.7 - 4% cases of sporadic ALS (sALS) (Robberecht and Phillips, 2013). SOD1 is primarily an antioxidant metalloenzyme that catalyzes the conversion of superoxide radical (O2 .-) to oxygen (O2) and hydrogen peroxide (H2O2). However, SOD1-linked fALS is most likely not caused by loss of the normal SOD1 activity, but rather by a gain of a toxic function. One of the hypotheses for mutant SOD-linked fALS toxicity proposes that an aberrant SOD1 chemistry is responsible for the toxic gain-of-function, which allows small molecules such us peroxynitrite or hydrogen peroxide to produce damaging free radicals. Other hypotheses for mutant SOD1 neurotoxicity include inhibition of the proteasome activity, mitochondrial damage, and formation of intracellular aggregates. SOD1 aggregation is an early event in ALS and could mediate motor neuron degeneration via sequestration of cellular components, decreasing chaperone activity and the ubiquitin proteasome pathway. Also, SOD1 mutations seem to disrupt RNA process‐ ing in the cells.

Defining the role of oxidative stress, and particularly nitrative stress in neurodegeneration has been extremely difficult because of the multiplicity of potential targets that can be damaged by oxidation and nitration. Certain proteins are particularly susceptible to tyrosine nitration by the oxidant peroxynitrite (ONOO- ). Tyrosine nitration is a well-established, early biomarker

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

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

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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

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‐

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.

cortex and spinal cord in ALS (Rothstein et al., 1992) (Fig. 1).

showed some sort of protein abnormality (Rothstein et al., 1995) (Fig.1).

**4.2. Glutamate receptor**

genase activity (Pioro et al., 1999).
