**3. Oxidative stress**

by impairing mitochondria or activating proteases, cytosolic phospholipase A2, kinases,

Glutamate transporter 1 (GLT-1), also known as excitatory amino acid transporter 2 (EAAT2), and glutamate-aspartate transporter (GLAST), the primary transporters of glutamate into astrocytes, plays a central role in regulating the extracellular levels of glutamate [4-5]. The expression of GLT-1 was markedly reduced in the motor cortex and the spinal cord of sporadic and familial ALS patients [6]. In mutant SOD1 mice, the levels and the activity of EAAT2 were reduced in the spinal cord [7-8]. The levels of extracellular glutanmate increased in the plasma and the cerebrospinal fluid of ALS patients [9-10] and of mutant SOD1-expressing rodent models [7,11-12]. Reducing the expression of EAAT2 with antisense oligonucleotide reduced transporter activity induces neuronal death in vitro and in vivo [13]. Crossing transgenic mice that overexpress EAAT2 with SOD1G93A mice caused delayed motor deficit [14]. In addition, increasing the expression of GLT-1 significantly extended the survival of mutant SOD1 mice [15]. More recently, a sumoylated fragment of EAAT2 cleaved to by activating caspase-3 was shown to cause motor neuron death [16]. This implies that reduced glutamate uptake into

endonucleases, and nuclear factor kappa B [3].

astrocytes mediates degeneration of spinal motor neurons in ALS.

degeneration and shortened the survival time.

*2.1.3. Therapies related to glutamate-mediated excitotoxicity*

*2.1.2. Mediation of motor neuron degeneration by the Ca2+ permeability of AMPA receptors*

Ca2+-permeable AMPA glutamate receptors appear to mediate chronic motor neuron degen‐ eration in ALS. AMPA receptors consist of heteromeric combinations of four sub-units, GluR1-4 [17]. The glutamate (Q)/arginine (R)-editing of the GluR2 mRNA provides a positively charged form of GluR2 protein with arginine, which is responsible for Ca2+ impermeability [18]. When AMPA receptors contain reduced levels of Q/R-edited GluR2, the AMPA receptor complex becomes more permeable to Ca2+ [18]. The motor neuron of ALS patients showed evidence of defective editing of the pre-mRNA of GluR2 [19]. While lack of GluR2 accelerated motor neuron degeneration and shortened the life span of the SOD1 mice, overexpression of GluR2 delayed the disease onset and reduced the mortality of mutant SOD1 mice [20-21]. Moreover, the GluR2-N transgenic mice that expressed GluR2 gene encoding a asparagine at the Q/R site showed late-onset degeneration of the spinal motor neurons and motor function deficit [22]. Crossbreeding GluR2-N mice with mutant SOD1 mice aggravated motor neuron

Although riluzole, the only approved disease-modifying therapy available to ALS patients since 1995, has been shown to inhibit glutamate release, subsequent studies demonstrated that riluzole inhibited AMPA receptors and presynaptic NMDA receptors [23-24]. Administration of riluzole significantly improved the motor neuron survival, motor function, and life expect‐ ancy of mutant SOD1 mice [25]. Similar beneficial effects of AMPA receptor antagonists such as memantine, 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide (NBQX), and talampanel have been verified in mutant SOD1 mice [26-28]. The B-lactam

*2.1.1. Abnormal glutamate re-uptake in ALS*

36 Current Advances in Amyotrophic Lateral Sclerosis

#### **3.1. Homeostasis and generation of free radicals in cells**

Free radicals, including reactive oxygen species (ROS) and reactive nitrogen species (RNS), are characterized by unpaired electrons in their outer orbit. The most common cellular free radicals are hydroxyl (OH ) radicals, superoxide (O2 - ) anions, and nitric monoxide (NO ). Although hydrogen peroxide (H2O2) and peroxynitrite (ONOO-) are literally not free radicals, they are deemed to generate free radicals through various chemical reactions in many cases. Free radicals are cleared through several defense mechanisms, as follows: (1) catalytic removal of reactive species by enzymes such as superoxide dismutase, catalase, and peroxidase; (2) scavenging of reactive species by low-molecular-weight agents that were either synthesized in vivo (including glutathione, α-keto acids, lipoic acid, and coenzyme Q) or obtained from the diet [including ascorbate (vitamin C) and α-tocopherol (vitamin E)]; and (3) minimization of the availability of pro-oxidants such as transition metals [30]. CNS, which is mainly composed of polyunsaturated fatty acids (PUFAs), is readily susceptible to oxidative damage because the system demands a high metabolic oxidative rate with limited anti-oxidants and has a high transition metal content that acts as a potent pro-oxidant through the Haber-Weiss reaction or the Fenton reaction [51]. Upon shifting to pro-oxidants, CNS is promptly attacked by ROS that includes H2O2, NO, O2 - , and highly reactive OH and NO and undergoes serious functional abnormality that is directly related to the demise of the course of neurons.

#### **3.2. Evidence of oxidative stress in ALS**

There is extensive evidence of the causative role of oxidative stress in motor neuron degener‐ ation in ALS. The 3-nitrotyrosine(3-NT) level was elevated in subjects with both sporadic and familial cases of ALS, and the immunoreactivity of 3-NT became more evident within large motor neurons in the ventral horn of the lumbar spinal cord [31-32]. Higher carbonylation of proteins with the use of 2,4-dinitrophenylhydrazine (DNPH) was detected in the spinal cord in sporadic ALS [33]. Elevation of 8-hydroxy-2-deoxyguanosine (8-OHdG) was found in the CSF, serum, and urine of ALS patients [34]. The 4-hydroxynonenal level increased in the serum of ALS patients [35]. Transgenic ALS mice overexpression of the human mutant SOD1 revealed oxidative damage to proteins, lipids, and DNA [36-37].

#### *3.2.1. Role of mitochondria in oxidative stress*

Mitochondria produce ATP using about 90% of the O2 that is taken up by neurons. During electron transfer in the inner membrane of the organelle, electrons spontaneously leak from the electron transport chain and react with available O2 to produce superoxide, which makes mitochondria the major cellular sources of ROS. Mitochondria exist in the motor neurons due to the high rate of metabolic demand, which makes motor neurons more vulnerable to cumulative oxidative stress. Free radicals that accumulate over time decrease mitochondrial efficacy and increase the production of mutated mitochondrial DNA related to the aging process, although mitochondria have their own specific anti-oxidants that consist of SOD1, SOD2, glutathioneperoxidase, and peroxiredoxin 3 and can usually combat the high rate of ROS production [38]. Morphological abnormality in the organelle, which includes a fragment‐ ed network and swelling, and increased cristae have been observed in the soma and proximal axons of ventral motor neurons of sporadic ALS (sALS) patients [39]. In the axon and soma of motor neurons of mice that expressed SOD1G93A and SOD1G37R [40-41], membrane vacuoles derived from degenerating mitochondria were reported. Morphological alteration in mito‐ chondria was also illustrated in NSC34 motor-neuron-like cells that expressed SOD1G93A [42-43]. Mutant SOD1 that was localized in mitochondria was associated with increased oxidative damage, decreased respiratory activity of the mitochondria, and architectural change. The interaction of mutant SOD1 and mitochondria was enough to result in motor neuron death in neuroblastoma cells [44]. Mitochondrial SOD1 and its chaperone protein named copper chaperone for SOD1 (CCS) are co-localized in the mitochondrial inter-mem‐ brane space [45]. The aggregates of mutant SOD1 were shown within the mitochondria in the spinal cord of SOD1G93A mice before the onset of the symptoms [46-47] and were implicated in increased oxidative damage, decreased respiratory activity of mitochondria [48], and mito‐ chondrial swelling and vacuolization [47].

was observed in the serum of sporadic ALS patients, which suggests a possible risk factor and the disturbance of iron homeostasis [61-62]. Ferritin was upregulated just prior to the endstage disease in SOD1-G93A mice, which supports increased Fe levels [63]. In the same animal model, increased iron was evident in the spinal cord at the ages of 90 and 120 days, with the onset of the symptoms and in the late stage, due to the disease progress. The increased iron levels were attenuated by iron chelators, which improved the motor function and the survival [64]. mRNAs associated with iron homeostasis (e.g., DMT1, TfR1, the iron exporter Fpn, and CP) also increased with a caudal-to-rostral gradient, with the highest levels rostrally in the cervical region in SOD1G37R [65]. HFE protein is a membrane protein that can influence cellular iron uptake, and mutated HFE is well recognized in haemochromatosis, a genetic disorder due to the irregular accumulation of free forms of Fe in parenchymal tissue. In studies of sporadic ALS patients, both the prevalence of HFE mutation and its polymorphisms (e.g., H63D) were evident [66-67]. Therefore, HFE polymorphisms in ALS may be associated with the altered Fe homeostasis and oxidative stress in this disease. Although abnormal iron homeostasis was evident, the iron regulation mechanisms for motor neuron death must be

Multiple Routes of Motor Neuron Degeneration in ALS

http://dx.doi.org/10.5772/56625

39

Human SOD1 mutation has a toxic gain-of-function that may be due to loss of the active site of copper binding that converts the SOD1 itself to pro-oxidant proteins and participates in ROS generation [68]. Several pieces of evidence have been suggested to show that higher interaction of mutant SOD1 with mitochondria may induce mitochondrial dysfunction and selectively lead to excessive oxidative stress in motor neurons [46]. Reduced transcription factor nuclear erythroid 2-related factor 2 (Nrf2) mRNA and protein expression has been reported in the spinal cord of ALS patients [69]. Crossbreeding SOD1G93A mice with overexpressed Nrf2 extended their survival [70], which suggests that increasing the Nef2 activity may be a novel therapeutic target. Nrf2 activation increases the expression of anti-oxidant proteins due to its interaction with the anti-oxidant-response element (ARE) after its translocation to the nucleus. In another reported mechanism of oxidative stress, the activity of NADPH oxidase (Nox) increased in both sALS patients and mutant SOD1 mice. Expressed Nox in activated microglia may influence motor neuron death. Deletion of either Nox1 or Nox2 prolonged the survival of mutant SOD1G93A mice [71-72]. Protein aggregation is a common pathological feature in ALS patients and animal ALS models. TAR DNA-binding protein-43 (TDP-43) or mutant SOD1 is a constituent of inclusions in ALS patients and mutant SOD1 mice [73-74]. Mutant SOD1

Several anti-oxidants have been tested using animal ALS models (Table 1). Completed, ongoing, or planned trials explored, are exploring, or will explore the value of anti-oxidants. Vitamin E, the most potent natural scavenger of ROS and RNS, delayed their clinical onset and slowed the disease progression in mutant SOD1 mice [25]. Long-term vitamin E supplements reduced the risk of death from ALS in ALS-free subjects [75-76]. Unfortunately, two vitamin

explained.

*3.2.3. Possible mechanisms related to oxidative stress in ALS*

itself caused oxidative damage of proteins in mutant SOD1 mice [37].

*3.2.4. Therapeutic drugs for oxidative stress in ALS*

#### *3.2.2. Role of transition metals in oxidative stress*

Redox-active transition metals are useful but harmful trace elements. Copper and iron are abundant (~0.1-0.5 mM) in the brain and have been implicated in the generation of ROS in various neurodegenerative diseases that include Alzheimer's disease and Parkinson's disease [49-50]. These transition metals mediate the formation of a hydroxyl radical through the ironcatalyzed or copper-catalyzed Haber-Weiss reactions [51]. Once copper ions are transported into the cell, they must be delivered to specific targets (e.g., SOD1 and cytochrome c oxidase) or stored in copper scavenging systems (e.g., GSH and metallothioneins) [52-53]. When these events are out of control, the cells have an uncomfortable abundance of toxic and radicalgenerating metal ions. FALS-linked SOD1 mutation has weaker binding affinity to copper ions, which are readily libertated to increase oxidative stress in cells expressed with fALS-SOD1 [54]. The detrimental role of copper in fALS pathogenesis was supported by several experiments that used copper chelators, which delayed the disease onset and prolonged the survival of fALS-G93A mice [55], prevented peroxidase activity by expressing fALS-SOD1 A4V and G93A in vitro [56], and reduced elevated ROS production in the lymphoblasts of fALS patients [57]. Iron is vital for all living organisms because it has an essential role in oxygen transport and electron transfer, and is a cofactor in many enzyme systems that include DNA synthesis. Iron homeostasis and its regulatory system [58] was readily disrupted in the development and progress of neurodegenerative diseases such as AD or PD [59-60]. Recently, several pieces of evidence supported the concept that iron is dysregulated in ALS. An increased ferritin level was observed in the serum of sporadic ALS patients, which suggests a possible risk factor and the disturbance of iron homeostasis [61-62]. Ferritin was upregulated just prior to the endstage disease in SOD1-G93A mice, which supports increased Fe levels [63]. In the same animal model, increased iron was evident in the spinal cord at the ages of 90 and 120 days, with the onset of the symptoms and in the late stage, due to the disease progress. The increased iron levels were attenuated by iron chelators, which improved the motor function and the survival [64]. mRNAs associated with iron homeostasis (e.g., DMT1, TfR1, the iron exporter Fpn, and CP) also increased with a caudal-to-rostral gradient, with the highest levels rostrally in the cervical region in SOD1G37R [65]. HFE protein is a membrane protein that can influence cellular iron uptake, and mutated HFE is well recognized in haemochromatosis, a genetic disorder due to the irregular accumulation of free forms of Fe in parenchymal tissue. In studies of sporadic ALS patients, both the prevalence of HFE mutation and its polymorphisms (e.g., H63D) were evident [66-67]. Therefore, HFE polymorphisms in ALS may be associated with the altered Fe homeostasis and oxidative stress in this disease. Although abnormal iron homeostasis was evident, the iron regulation mechanisms for motor neuron death must be explained.

#### *3.2.3. Possible mechanisms related to oxidative stress in ALS*

the electron transport chain and react with available O2 to produce superoxide, which makes mitochondria the major cellular sources of ROS. Mitochondria exist in the motor neurons due to the high rate of metabolic demand, which makes motor neurons more vulnerable to cumulative oxidative stress. Free radicals that accumulate over time decrease mitochondrial efficacy and increase the production of mutated mitochondrial DNA related to the aging process, although mitochondria have their own specific anti-oxidants that consist of SOD1, SOD2, glutathioneperoxidase, and peroxiredoxin 3 and can usually combat the high rate of ROS production [38]. Morphological abnormality in the organelle, which includes a fragment‐ ed network and swelling, and increased cristae have been observed in the soma and proximal axons of ventral motor neurons of sporadic ALS (sALS) patients [39]. In the axon and soma of motor neurons of mice that expressed SOD1G93A and SOD1G37R [40-41], membrane vacuoles derived from degenerating mitochondria were reported. Morphological alteration in mito‐ chondria was also illustrated in NSC34 motor-neuron-like cells that expressed SOD1G93A [42-43]. Mutant SOD1 that was localized in mitochondria was associated with increased oxidative damage, decreased respiratory activity of the mitochondria, and architectural change. The interaction of mutant SOD1 and mitochondria was enough to result in motor neuron death in neuroblastoma cells [44]. Mitochondrial SOD1 and its chaperone protein named copper chaperone for SOD1 (CCS) are co-localized in the mitochondrial inter-mem‐ brane space [45]. The aggregates of mutant SOD1 were shown within the mitochondria in the spinal cord of SOD1G93A mice before the onset of the symptoms [46-47] and were implicated in increased oxidative damage, decreased respiratory activity of mitochondria [48], and mito‐

Redox-active transition metals are useful but harmful trace elements. Copper and iron are abundant (~0.1-0.5 mM) in the brain and have been implicated in the generation of ROS in various neurodegenerative diseases that include Alzheimer's disease and Parkinson's disease [49-50]. These transition metals mediate the formation of a hydroxyl radical through the ironcatalyzed or copper-catalyzed Haber-Weiss reactions [51]. Once copper ions are transported into the cell, they must be delivered to specific targets (e.g., SOD1 and cytochrome c oxidase) or stored in copper scavenging systems (e.g., GSH and metallothioneins) [52-53]. When these events are out of control, the cells have an uncomfortable abundance of toxic and radicalgenerating metal ions. FALS-linked SOD1 mutation has weaker binding affinity to copper ions, which are readily libertated to increase oxidative stress in cells expressed with fALS-SOD1 [54]. The detrimental role of copper in fALS pathogenesis was supported by several experiments that used copper chelators, which delayed the disease onset and prolonged the survival of fALS-G93A mice [55], prevented peroxidase activity by expressing fALS-SOD1 A4V and G93A in vitro [56], and reduced elevated ROS production in the lymphoblasts of fALS patients [57]. Iron is vital for all living organisms because it has an essential role in oxygen transport and electron transfer, and is a cofactor in many enzyme systems that include DNA synthesis. Iron homeostasis and its regulatory system [58] was readily disrupted in the development and progress of neurodegenerative diseases such as AD or PD [59-60]. Recently, several pieces of evidence supported the concept that iron is dysregulated in ALS. An increased ferritin level

chondrial swelling and vacuolization [47].

38 Current Advances in Amyotrophic Lateral Sclerosis

*3.2.2. Role of transition metals in oxidative stress*

Human SOD1 mutation has a toxic gain-of-function that may be due to loss of the active site of copper binding that converts the SOD1 itself to pro-oxidant proteins and participates in ROS generation [68]. Several pieces of evidence have been suggested to show that higher interaction of mutant SOD1 with mitochondria may induce mitochondrial dysfunction and selectively lead to excessive oxidative stress in motor neurons [46]. Reduced transcription factor nuclear erythroid 2-related factor 2 (Nrf2) mRNA and protein expression has been reported in the spinal cord of ALS patients [69]. Crossbreeding SOD1G93A mice with overexpressed Nrf2 extended their survival [70], which suggests that increasing the Nef2 activity may be a novel therapeutic target. Nrf2 activation increases the expression of anti-oxidant proteins due to its interaction with the anti-oxidant-response element (ARE) after its translocation to the nucleus. In another reported mechanism of oxidative stress, the activity of NADPH oxidase (Nox) increased in both sALS patients and mutant SOD1 mice. Expressed Nox in activated microglia may influence motor neuron death. Deletion of either Nox1 or Nox2 prolonged the survival of mutant SOD1G93A mice [71-72]. Protein aggregation is a common pathological feature in ALS patients and animal ALS models. TAR DNA-binding protein-43 (TDP-43) or mutant SOD1 is a constituent of inclusions in ALS patients and mutant SOD1 mice [73-74]. Mutant SOD1 itself caused oxidative damage of proteins in mutant SOD1 mice [37].

#### *3.2.4. Therapeutic drugs for oxidative stress in ALS*

Several anti-oxidants have been tested using animal ALS models (Table 1). Completed, ongoing, or planned trials explored, are exploring, or will explore the value of anti-oxidants. Vitamin E, the most potent natural scavenger of ROS and RNS, delayed their clinical onset and slowed the disease progression in mutant SOD1 mice [25]. Long-term vitamin E supplements reduced the risk of death from ALS in ALS-free subjects [75-76]. Unfortunately, two vitamin E clinical trials failed to show the vitamin's efficacy in ALS patients due to impermeable BBB penetration [77]. Creatine, N-acetylcysteine, AEOL-10150, and edarabone have successfully improved the motor function and survival of mutant SOD1 mice [78-81]. Creatine and Nacetylcystein were not effective in the clinical trial phase II.

mice [96]. A Fas/NO feedback loop with downstream Daxx and P38 was proposed as another

Multiple Routes of Motor Neuron Degeneration in ALS

http://dx.doi.org/10.5772/56625

41

The physiological and pathological roles of the Bcl-2 family have been extensively reviewed [98-99]. The physical balance between anti-apoptotic and pro-apoptotic members of the Bcl-2 family generally appears to determine the fate of developing and mature cells. Anti- and proapoptotic proteins are separated by the presence or absence of Bcl-2 homology (BH) domains. There are four domains: BH1-BH4. Bcl-2 and Bcl-xL contain all four domains and are antiapoptotic. The pro-apoptotic Bcl-2 family includes Bax, Bcl-xs, Bak, Bad, and Bid and partici‐ pates in the neuronal death process. Unbalanced pro- or anti-apoptotic proteins activate caspase-realted apoptosis by releasing cytochrome c into cytosol. Bax is oligomerized, inserted into the outer membrane of mitochondria, and shown to induce cytochrome c release [100-101]. The ratio of the apoptotic cell death genes Bax to Bcl-2 increases at both the mRNA and protein levels in the spinal motor neurons of ALS patients and SOD1G93A mice [102-104]. Interest‐ ingly, mutant SOD1 was highly associated with Bcl-2 in the mitochondria, which resulted in conformational or phenotypic change of Bcl-2 that weakened the mitochondria in the spinal cord [105]. Blunt Bcl-2 may contribute to the activation of the mitochondrial apoptosis machinery such as caspase-9, caspase 3, and cytochrome c in the spinal motor neurons of ALS transgenic mice and humans with ALS [106-107]. To support this idea, Bcl-2 overexpression or Bax depletion crossbred with SOD1G93A mice delayed the onset of symptoms and extended

Caspases, a family of cysteine-dependent aspartate-directed proteases, mediate the propaga‐ tion and execution of apoptosis. They can be classified into initiator caspases and effector caspases [110]. Caspase-9 is an initiator caspase and is proteolytically activated by apaf-1, a cytoplasmic protein that is homologous to ced-4, and by cytochrome c. The latter is located in the intermembrane space of the mitochondria and released into the cytoplasm by the proapoptotic Bcl-2 (e.g., Bax) that is transported from the cytoplasm into the mitochondria in the early phase of apoptosis. Caspase-8, which is known as another initiator caspase, is activated through the interaction of procaspase-9 with the Fas receptor and the FADD adapter. Activated caspase-8 and caspase-9 can activate downstream caspases such as caspase-3, 6, and 7 that can cleave to a number of proteins that are essential to the structure, signal transduction, and cell cycle and terminate the overall apoptosis process. Under the ER (endoplasmic reticulum) stress, caspase-12 is activated with the cleavage (activation) of caspase-9 and caspase-3, regardless of the release of cytochrome c. Marginally, ER stress triggers caspase-8 activation, which results in a mitochondria-mediated pathway via Bid cleavage. The caspase-1, -3, and -9 activities were higher in the motor neurons of the spinal cord or the motor cortex of ALS patients than in those of the control [107,111]. Caspase-1 truncated Bid to be highly reactive [106]. The orderly activation of caspase-1 and -3 was evident, and their mRNAs were abundant in animal ALS models [111-112]. The sequential activation of caspase-9 to caspase-7 was

Fas pathway of motor neuron death in mutant SOD1 mice [97].

*4.1.2. Pro-apoptotic family of Bcl-2*

the life expectancy [108-109].

*4.1.3. Caspase cascade*
