**4. Apoptosis**

#### **4.1. Evidence of apoptosis in ALS**

Kerr et al. (1972)[82] reported electron microscopic features of shrinkage necrosis or apoptosis that are expected to play a role in the regulation of the number of cells under physiological and pathological conditions. The apoptotic cells were accompanied by condensation of the nucleus and the cytoplasm, nuclear fragmentation, and aggregated condensation of nuclear chromatin. Interestingly, apoptosis is prevented by inhibitors of protein and mRNA synthesis, and thus, appears to require the expression and activation of death-regulating proteins in neurons and non-neuronal cells [83-84]. The morphological and molecular features of apop‐ tosis have been reported in the nervous system during the development of various neurolog‐ ical diseases. Apoptosis is probably correlated with the demise of motor neurons in ALS. Degenerating motor neurons in the spinal cord and the motor cortex are illustrated by the dark and shrunken cytoplasm and nuclei, chromatin condensation, and apoptotic bodies in the cells. Various pro-apoptosis proteins are activated in the ALS-injured area, and protein synthesis inhibitors attenuate ALS-related neuronal death.

#### *4.1.1. Death receptor Fas*

The death receptor Fas (CD95 or APO-1) belongs to the tumor necrosis factor (TNF) receptor superfamily and functions as a key determinant of cell fate under physiological and patho‐ logical conditions [86-87]. The Fas ligand (Fas-L) activates Fas in an autocrine or paracrine manner, which leads to the trimerization of Fas with Fas-associating protein within the death domain (FADD) and procaspase-8. Fas activation has been shown as an obligatory step in apoptosis in neurons deprived of trophic factors [88-90]. Fas antibodies were more frequently found in the serum of sporadic or familial ALS patients than in that of the normal controls [91], which also induced apoptosis in the human neuroblastoma cell line and in neuron-glia cocultured cells of the spinal cord of rat embryos [92]. Primary motor neurons of mouse embryos that expressed mutant SOD1 were susceptible to Fas-induced death [93]. Continuous silencing of the Fas receptor on the motor-neuron-ameliorated motor function and survival of SOD1G93A mice using small interfering RNA-mediated interference supported the role of Faslinked motor neuron degeneration in ALS [94]. In SOD1G93A mice, a Fas pathway is required to allow Fas interaction with FADD, which in turn recruits caspase-8 as one of the downstream effectors. In addition, TIMP-3 controls Fas-mediated apoptosis by inhibiting the MMP-3 mediated shedding activity in the Fas ligand on the cell surface [95]. The FASS/FADDmediated motor neuron degeneration was attenuated by Lithium treatment in SOD1G93A mice [96]. A Fas/NO feedback loop with downstream Daxx and P38 was proposed as another Fas pathway of motor neuron death in mutant SOD1 mice [97].

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

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 N-

Kerr et al. (1972)[82] reported electron microscopic features of shrinkage necrosis or apoptosis that are expected to play a role in the regulation of the number of cells under physiological and pathological conditions. The apoptotic cells were accompanied by condensation of the nucleus and the cytoplasm, nuclear fragmentation, and aggregated condensation of nuclear chromatin. Interestingly, apoptosis is prevented by inhibitors of protein and mRNA synthesis, and thus, appears to require the expression and activation of death-regulating proteins in neurons and non-neuronal cells [83-84]. The morphological and molecular features of apop‐ tosis have been reported in the nervous system during the development of various neurolog‐ ical diseases. Apoptosis is probably correlated with the demise of motor neurons in ALS. Degenerating motor neurons in the spinal cord and the motor cortex are illustrated by the dark and shrunken cytoplasm and nuclei, chromatin condensation, and apoptotic bodies in the cells. Various pro-apoptosis proteins are activated in the ALS-injured area, and protein synthesis

The death receptor Fas (CD95 or APO-1) belongs to the tumor necrosis factor (TNF) receptor superfamily and functions as a key determinant of cell fate under physiological and patho‐ logical conditions [86-87]. The Fas ligand (Fas-L) activates Fas in an autocrine or paracrine manner, which leads to the trimerization of Fas with Fas-associating protein within the death domain (FADD) and procaspase-8. Fas activation has been shown as an obligatory step in apoptosis in neurons deprived of trophic factors [88-90]. Fas antibodies were more frequently found in the serum of sporadic or familial ALS patients than in that of the normal controls [91], which also induced apoptosis in the human neuroblastoma cell line and in neuron-glia cocultured cells of the spinal cord of rat embryos [92]. Primary motor neurons of mouse embryos that expressed mutant SOD1 were susceptible to Fas-induced death [93]. Continuous silencing of the Fas receptor on the motor-neuron-ameliorated motor function and survival of SOD1G93A mice using small interfering RNA-mediated interference supported the role of Faslinked motor neuron degeneration in ALS [94]. In SOD1G93A mice, a Fas pathway is required to allow Fas interaction with FADD, which in turn recruits caspase-8 as one of the downstream effectors. In addition, TIMP-3 controls Fas-mediated apoptosis by inhibiting the MMP-3 mediated shedding activity in the Fas ligand on the cell surface [95]. The FASS/FADDmediated motor neuron degeneration was attenuated by Lithium treatment in SOD1G93A

acetylcystein were not effective in the clinical trial phase II.

**4. Apoptosis**

**4.1. Evidence of apoptosis in ALS**

40 Current Advances in Amyotrophic Lateral Sclerosis

inhibitors attenuate ALS-related neuronal death.

*4.1.1. Death receptor Fas*

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 the life expectancy [108-109].

#### *4.1.3. Caspase cascade*

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 required for the mitochondria-dependent apoptosis pathway in a rodent ALS model [107]. Moreover, caspase-9 was simultaneously activated with a death receptor pathway that contained Fas, FADD, caspase-8, and caspase-3 in the ALS mice after their motor neuron death began [95-96]. Cleaved forms of caspase-12 were expressed presymptomatically in animal models, which shows evidence of ER stress [113]. A more advanced mechanism than that with caspases revealed that caspases such as caspase-3 or caspase-7 mediated TDP-43 cleavage [114], which was observed immunologically in an aggregated form in the cytoplasmic inclusions in ALS. Intraventricular administration of zVAD-fmk, a broad-spectrum caspase inhibitor, prolonged the survival of G93ASOD1 mice [111], which supports the causative role of caspase cascade in motor neuron death.

filament glial fibrillary acidic protein (GFAP) and the marker aldehyde dehydrogenase 1 family, member L1 (ALDH1L1). Although astrocytes are not immune cells, they can contribute to the immune response in pathological conditions. Microgliosis and astrocytosis are promient

Multiple Routes of Motor Neuron Degeneration in ALS

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

43

Several studies have shown the possibility that glial cells adjacent to degenerating motor neurons, mainly primed microglia and astrocytes, have causative roles in the course of disease propagation in ALS. Massive gliosis is apparent in pathologically vulnerable departments of CNS in both human ALS patients and ALS animal models [124-125]. Microglia antibodies have also been found in the CSF of an ALS patient [126]. Recently, the presence of activated microglia was visualized via positron emission tomography (PET), using [11C](R)-PK11195, in the motor cortex, dorsolateral prefrontal cortex, thalamus, and pos of living patients [127]. In the presymptomatic stage of the disease, TNF-α and M-CSF expression increased in a transgenic ALS model. Interestingly, the increase in the expressed TNF-α was found to be correlated to the severity of motor neuron loss [128]. The elevation of TNF-α and of its two receptors [TNFRI (p55TNF) and TNFRII ( p75TNF)] was observed in the serum of ALS patients, unlike in those of healthy controls [129]. To date, primed microglia-sensitive intracellular signaling that affectas ALS is authorized by the activation of p38 mitogen-activated protein kinase (p38MAPK), the translocation of the transcription factor NF-κB into the nucleus, and the upregulation of COX-2. The activation of NF-κB regulates the transcription of a wide range of inflammation-related genes that include inducible nitric oxide synthesis (iNOS), COX-2, MCP-1, MMP-9, IL-2, IL-6, IL-8, IL-12p40, IL-2 receptor, ICAM-1, TNF-α, and IFN-γ [130], which leads to the secretion of many inflammatory mediators. The aforementioned genes were shown to have changed in the tissues of ALS patients and hSOD1 transgenic mice [128,131-133]. COX-2 is inducible and is a rate-limiting enzyme of the synthesis pathways of the prostaglandins (PG) PGD2, PGE2, PGF2a, and PGI2 and thromboxane (TXA2). Prostaglan‐ dins play a role in various cellular effectors that include the instigation of inflammatory responses, the re-arrangement of cytoskeletons, and gene transcription changes [134]. COX-2 expression was significantly elevated in motor neuron and glial cells in the spinal cord of ALS patients [135-136], and the COX-2 activity increased in the spinal cord of ALS patients [137]. In addition, the PGE2 levels jumped up in the CSF of ALS patients by two to 10 times, compared with the controls [137]. The deletion of the prostaglandin E(2) EP2 receptor in SOD1G93A mice improved their motor function and prolonged their survival, which suggests that PGE2 signalling via the EP2 receptor acts as an inflammatory mediator in motor neuron degeneration

Aside from degenerating motor neurons, microglia and astrocytes concomitantly play a role in disease progression in ALS model mice. Recent reports emphasized the potential role of non-cell-autonomous mechanisms, which are harmonious with and critical in SOD1G93Ainduced cell-autonomous death signals [139-140]. Either neuron-specific or glia-specific

features of neurodegenerative diseases that include AD, PD, and ALS.

**5.2. Evidence of inflammation in ALS**

[138].

*5.2.1. Non-cell-autonomous neurotoxicity in ALS*

#### *4.1.4. Anti-apoptotic drugs served as therapy for ALS*

Even though minocycline has anti-inflammatory effects that prevent microglia proliferation, the drug prevented apoptotic motor neuron death by inhibiting cytochrome c release in mutant SOD1 mice [115]. The beneficial effects were proven in several studies to prolong survival and ameliorate the motor function [115-117]. Minocycline accelerated disease progression in a clinical trial, though [118]. TCH-346, a molecule that binds to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), was used in small samples in a Phase II/III randomized trial, but it did not show beneficial effects [119].
