**2. Excitotoxicity**

#### **2.1. Glutamate neurotoxicity**

Glutamate mediates excitatory synaptic transmission by activating the ionotropic glutamate receptors that are sensitive to N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-meth‐ yl-4-isoxazolepropionic acid (AMPA), or kainate. While the ionotropic glutamate receptors constitute fast excitatory synapses in the brain and the spinal cord, the glutamate receptors are excessively activated under pathological conditions such as hypoxic ischemia, trauma, and epilepsy, which triggers degeneration of neurons and oligodendrocytes. Extensive evidence supports the causative role of Ca2+-permeable ionotropic glutamate receptors in motor neuron degeneration in ALS patients. Intracellular Ca2+ overload causes catastrophic neuronal death

© 2013 Shin and Lee; 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.

by impairing mitochondria or activating proteases, cytosolic phospholipase A2, kinases, endonucleases, and nuclear factor kappa B [3].

antibiotic cefriaxone increased GLT-1 expression in spinal cord culture and in normal rats. The cefriaxone treatment delayed motor deficits with marginal survival in SOD1G93A mice [15]. An adaptive design Phase II/III study revealed good tolerability over 20 weeks [29]. The

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

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

) anions, and nitric monoxide (NO ). Although

Multiple Routes of Motor Neuron Degeneration in ALS

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

37

, and highly reactive OH and NO and undergoes serious


extened phase III of this study is ongoing.

are hydroxyl (OH ) radicals, superoxide (O2

by ROS that includes H2O2, NO, O2

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

*3.2.1. Role of mitochondria in oxidative stress*

oxidative damage to proteins, lipids, and DNA [36-37].

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


functional abnormality that is directly related to the demise of the course of neurons.

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

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

**3. Oxidative stress**

#### *2.1.1. Abnormal glutamate re-uptake in ALS*

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 astrocytes mediates degeneration of spinal motor neurons in ALS.

#### *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 degeneration and shortened the survival time.

#### *2.1.3. Therapies related to glutamate-mediated excitotoxicity*

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 antibiotic cefriaxone increased GLT-1 expression in spinal cord culture and in normal rats. The cefriaxone treatment delayed motor deficits with marginal survival in SOD1G93A mice [15]. An adaptive design Phase II/III study revealed good tolerability over 20 weeks [29]. The extened phase III of this study is ongoing.
