**8. RNA metabolism disorders**

Ubiquinated intracytoplamsmatic inclusions containing trans-activation response DNAbinding protein of 43 KDa (TDP-43), encoded by the TARDBP gene in chromosome-1, had been identified in motor neurons of patients with sALS and frontal lobar degeneration (FTLD) linked to TDP-43 pathology (FTLD-TDP) (Neumann et al., 2006). TDP-43 positive inclusions were also identified in patients with non-SOD1 fALS. Gene mutations of the TDP-43 gene probably accounts for 5% of patients with fALS. All the cases of sALS and SOD1 negative fALS have neural and glial inclusions immunoreactive to both ubiquitin and TDP-43 whereas positive SOD1 mutations in fALS were absent of TDP-43 immunoreactivity. (MacKenzie et al., 2007; Tran et al., 2007). TDP-43 inclusions were also identified in patients with Guamanian 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 death, but the mechanism remains elusive.

#### **9. Non-cell autonomous mechanisms**

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

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

Other beneficial growth factors are innsuline growth factor-1, glial cell line –derived neuro‐

Ubiquinated intracytoplamsmatic inclusions containing trans-activation response DNAbinding protein of 43 KDa (TDP-43), encoded by the TARDBP gene in chromosome-1, had been identified in motor neurons of patients with sALS and frontal lobar degeneration (FTLD) linked to TDP-43 pathology (FTLD-TDP) (Neumann et al., 2006). TDP-43 positive inclusions were also identified in patients with non-SOD1 fALS. Gene mutations of the TDP-43 gene probably accounts for 5% of patients with fALS. All the cases of sALS and SOD1 negative fALS have neural and glial inclusions immunoreactive to both ubiquitin and TDP-43 whereas positive SOD1 mutations in fALS were absent of TDP-43 immunoreactivity. (MacKenzie et al., 2007; Tran et al., 2007). TDP-43 inclusions were also identified in patients with Guamanian

perfusion.

of angiogenesis (Poesen et al., 2008).

10 Current Advances in Amyotrophic Lateral Sclerosis

**8. RNA metabolism disorders**

trophic factor, and brain derived neurotrophic factor.

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 transgenic mutant SOD1 mice. The expression of mutant SOD1 restricted to motor neurons *in vivo* was not enough or caused a mild neurodegeneration (Jaarsma et. al.; 2008). Indeed, when mutant SOD1 expression was reduced in microglia and macrophages there was a reduction in motor neuron degeneration (Boillee 2006, Wang 2009). In addition, mutant SOD1 expression in astrocytes is required to cause neurodegeneration by release of toxic factors (Gong et. al., 2000; Nagai et al. 2007). Co-cultures of healthy motor neurons with astrocytes expressing mutant SOD1 resulted in more than 50% motor neuron death (Marchetto et al, 2008), while astrocytes obtained from postmortem tissue from patients with fALS and sALS were both toxic to motor neurons (Haidet-Phillips et al., 2011). In agreement, mutant SOD1 knockdown in astrocytes attenuated toxicity towards motor neurons, suggesting that the mutant enzyme plays a role in both fALS and sALS (Phillips et al., 2011). SOD1G93A glial-restricted precursor cells transpanted into the cervical spinal cord of wild type rats survived and differentiated efficiently into astrocytes. These graft-derived SOD1G93A astrocytes induced host ubiquitina‐ tion and death of motor neurons, reactive astrocytosis, and reduction of the glial glutamate transporter GLT-1 expression that was associated with animal limb weakness and respiratory dysfunction (Papadeas et al., 2011). The SOD1G93A astrocyte-induced motor neuron death may be madiated by host microglial activation (Papadeas et al., 2011).

neurodegeneration, but is activated in the first steps of neurodegeneration. An increase in the immunostaining for GFAP and CD11 suggests the presence of reactive astrocytes and micro‐ glia in SOD1 transgenic mice (Fischer et. al, 2004). Increase in NGF, a sign of reactive atrocytes, leads to aptoptosis in ALS through a pathway involving activation of p75 (Pehar et al., 2004). Additionally, in ALS animal models mutant SOD1-expressing astrocytes are neurotoxic to motor neurons, and reducing mutant SOD1 expression decreases motor neuron degeneration and increases animal life span (Lepore at al., 2008, Barbeito et al., 2010). The release of proinflammatory cytokines, oxidative stressors such as prostanglandins, leukotrienes, and reactive nitrogen species (RNS) is toxic to motor neurons (Henkel, et al., 2009). In *in vitro* studies, normal motor neurons die through a pro-apoptotic Bax pathway when co-cultured with astrocytes expressing mutant SOD1 (Nagai at al., 2007). In *in vivo* studies, microglia releases pro-inflammatory cytokines such us TNF-α and IL-1β as well as ROS (Henkel 2009) whereas in ALS patients, there is an increase of pro-inflammatory cytokines and prostran‐ glandin E2 (Papadimitou, 2010). Media obtained from activated microglia causes motor neuron death by activation of TNF-α and NMDA receptors (Moisee and Strong 2006). In mouse model of ALS, a reduction in the expression of mutant SOD1 by microglia does not change age of symptoms onset, but slowed down disease progression (Boillee et al. 2006b). Motor neurons expressing mutant SOD1 are more susceptible to Fas ligand and NO-triggered cell death (Raoul et al., 2002), suggesting that in the context of ALS progression, motor neurons express‐ ing mutant SOD1 are more vulnerable to external stimuli such as ROS, RNS and toxic factors

Pathophysiology of Amyotrophic Lateral Sclerosis

http://dx.doi.org/10.5772/56562

13

Apoptosis is a programmed cell death cascade involved in several physiological processes during development and aging. Cell death by apoptosis sustains the homeostasis of cell population in tissues including cell turnover, hormone dependent- and chemical induced-cell death, and immune system development. The programmed cell death also functions as a defense when cells are damaged by disease or noxious stimuli (Elmore, S; 2007). Thereby, inappropriate apoptosis is a potential mechanism implicated in the pathogenesis of several neurodegenerative disorders, including ALS (Elmore, S; 2007). There are two main apoptotic pathways, the extrinsic or death receptor pathway and the intrinsic or mitochondrial pathway (Igney and Krammer, 2002). The extrinsic pathway involves transmembrane receptormediated interactions between ligands and death receptors resulting in transmission of death signals from cell surface to the intracellular signaling pathways (Locksley et al., 2001). The most studied ligand and death receptor association are Fas ligand and Fas receptor (FasL/FasR) and tumor necrosis factor (TNF) and its receptor (TNFL/TNFR) (Hsu et al 1995; Wajant, 2002). The intrinsic pathway consists of non-receptor-mediated stimuli that cause changes in the inner mitochondrial membrane. These changes include the opening of mitochondrial mem‐ brane pores leading to loss of transmembrane potential and released of pro-apoptotic proteins such as cytochrome c, Smac/DIABLO, HtrA2/Omi, and others ending with the activation of caspases (Sealens, et al., 2004; Du et al., 2000; Van Loo et al., 2002; Garrido at al., 2005). The

release by surrounding cells.

**10. Apoptosis**

Abnormalities in the immune system have also been observed in ALS patients. Blood samples of ALS patients have increased levels of CD4+ cells and reduced levels of CD8+ T lymphocytes. However, early in the disease when motor features are still mild there is a reduction in CD4+ / CD25+ T-regulatory cells (T-reg) and CD14+ monocytes. These observations suggest that the reduction in circulating T-reg cells could be due to the relocation of the cells into the central nervous system. Upon relocation, the T-reg cells would activate the innate immune cells like microglia, leading to the release of anti-inflammatory cytokines such as interleukin-10 and transforming growth factor-β to protect the affected area (Kipnis et al., 2004 and Mantonavi, et al., 2009). Indeed, immunostaining for the astrocytic marker glial fibrillary acid protein (GFAP) showed a significantly increased presence of astrocytes in the precentral gyrus of patients with both fALS and sALS. In addition, staining for activated microglia and macro‐ phages markers such as leukocyte common antigen (LCA), lymphocytes function associate molecule (LFA-1), complementary receptors CR3 (CD11b), and CR4 (CD11c) was also in‐ creased in motor cortex, brainstem, and corticospinal tract (Kawamata, et al., 1992; Papidimi‐ triou et al., 2010). Samples from brain and spinal cord from animal models and patient with ALS also showed a significant increase in activated or reactive astrocytes, an indication of neuroinflammation (Sta et al., 2011).

Astrocytes and microglia play an essential role in immune surveillance and response in the central nervous system. Reactive astrocytes recruited to the injured area reestablish the bloodbrain-barrier (BBB), release neurotrophins and growth factors (IGF-1), clear debri, and isolate the injured region through the formation of a glial scar (Papadimitriou et al 2010; Dong and Benviste 2001). Microglia are also activated in the presence of antigens exposed during neurodegeneration leading to the phagocytosis of cellular debri and the secretion of several neurotrophic factors, neurotrophins, and cytokines. However, a poor regulation of these factors could be harmful to motor neurons. Microglia seems to protect motor neurons from neurodegeneration, but is activated in the first steps of neurodegeneration. An increase in the immunostaining for GFAP and CD11 suggests the presence of reactive astrocytes and micro‐ glia in SOD1 transgenic mice (Fischer et. al, 2004). Increase in NGF, a sign of reactive atrocytes, leads to aptoptosis in ALS through a pathway involving activation of p75 (Pehar et al., 2004). Additionally, in ALS animal models mutant SOD1-expressing astrocytes are neurotoxic to motor neurons, and reducing mutant SOD1 expression decreases motor neuron degeneration and increases animal life span (Lepore at al., 2008, Barbeito et al., 2010). The release of proinflammatory cytokines, oxidative stressors such as prostanglandins, leukotrienes, and reactive nitrogen species (RNS) is toxic to motor neurons (Henkel, et al., 2009). In *in vitro* studies, normal motor neurons die through a pro-apoptotic Bax pathway when co-cultured with astrocytes expressing mutant SOD1 (Nagai at al., 2007). In *in vivo* studies, microglia releases pro-inflammatory cytokines such us TNF-α and IL-1β as well as ROS (Henkel 2009) whereas in ALS patients, there is an increase of pro-inflammatory cytokines and prostran‐ glandin E2 (Papadimitou, 2010). Media obtained from activated microglia causes motor neuron death by activation of TNF-α and NMDA receptors (Moisee and Strong 2006). In mouse model of ALS, a reduction in the expression of mutant SOD1 by microglia does not change age of symptoms onset, but slowed down disease progression (Boillee et al. 2006b). Motor neurons expressing mutant SOD1 are more susceptible to Fas ligand and NO-triggered cell death (Raoul et al., 2002), suggesting that in the context of ALS progression, motor neurons express‐ ing mutant SOD1 are more vulnerable to external stimuli such as ROS, RNS and toxic factors release by surrounding cells.
