**5. Inflammation**

#### **5.1. Microglia and astrocyte**

Microglia activation is an early event in all forms of pathology. Thus, activated microglia was initially considered a sensitive marker to identify sites that were predestined for tissue. The classical bone-marrow-derived microglial cells dwell in the gray matter and have ramified (highly branched) structures with a small portion of perinuclear cytoplasm and a small, dense, and heterochromatic nucleus. In many CNS pathologies, the cells increase, and this may arise from either local proliferation or recruitment from the blood, or both. The morphology of microglia becomes reactive under pathological conditions that were determined as infiltration of blood-derived cells, local BBB [120], or presence of damaged neurons. Microglia near areas of neuronal injury tend to have more amoeboid features with intense cell bodies and reduced numbers of shortened and thick processes [121] that lead to a structural morphology similar to that of macrophages. A shift in the active style of microglia affects neural, vascular, and blood-borne cells due to several secretions that include pro-inflammatory cytokines and chemokines, nitric oxide, and reactive oxygen intermediates. Astrocytes have many essential physiological functions in the CNS such as provision of trophic support for neurons, conduct of synaptic formation and plasticity, and regulation of the cerebral blood flow. Due to their strategic structure, they are in close contact with CNS resident cells and blood vessels [122-123]. An inflammatory insult causes proliferation of astrocytes and morphological changes. Astroglial activation is recognized via increased expression of the intermediate 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 features of neurodegenerative diseases that include AD, PD, and ALS.

#### **5.2. Evidence of inflammation in ALS**

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

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

Microglia activation is an early event in all forms of pathology. Thus, activated microglia was initially considered a sensitive marker to identify sites that were predestined for tissue. The classical bone-marrow-derived microglial cells dwell in the gray matter and have ramified (highly branched) structures with a small portion of perinuclear cytoplasm and a small, dense, and heterochromatic nucleus. In many CNS pathologies, the cells increase, and this may arise from either local proliferation or recruitment from the blood, or both. The morphology of microglia becomes reactive under pathological conditions that were determined as infiltration of blood-derived cells, local BBB [120], or presence of damaged neurons. Microglia near areas of neuronal injury tend to have more amoeboid features with intense cell bodies and reduced numbers of shortened and thick processes [121] that lead to a structural morphology similar to that of macrophages. A shift in the active style of microglia affects neural, vascular, and blood-borne cells due to several secretions that include pro-inflammatory cytokines and chemokines, nitric oxide, and reactive oxygen intermediates. Astrocytes have many essential physiological functions in the CNS such as provision of trophic support for neurons, conduct of synaptic formation and plasticity, and regulation of the cerebral blood flow. Due to their strategic structure, they are in close contact with CNS resident cells and blood vessels [122-123]. An inflammatory insult causes proliferation of astrocytes and morphological changes. Astroglial activation is recognized via increased expression of the intermediate

of caspase cascade in motor neuron death.

42 Current Advances in Amyotrophic Lateral Sclerosis

did not show beneficial effects [119].

**5. Inflammation**

**5.1. Microglia and astrocyte**

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

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 [138].

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

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 expression of SOD1 mutation in mice led to the ALS phenotype, with marginal effects [141-142]. Specific expression of mutant SOD1 within neurons using *Nefl* (neurofilament light chain) promoters did not cause motor neuron degeneration in transgenic mice [142]. Consis‐ tently, selective expression of mutant SOD1 in microglia or astrocytes did not kill motor neurons [141,143].These non-cell-autonomous deaths of motor neurons were supported by an analysis of chimeric mice that had mixed populations of normal cells and cells that expressed mutant SOD1 [144]. Conditional knockout of mutant SOD1 in motor neurons using an *Isl1* promoter-driven *Cre* transgene that is expressed in the spinal cord delayed the disease onset in and prolonged the survival of mutant SOD1 transgenic mice. On the other hand, however, selective removal from cells of the myeloid lineage that included microglia using a *Cd11b* promoter-driven *Cre* transgene did not delay the disease onset but extended its progress [139]. In the same lineages, selective viral vector-mediated delivery of small interfering RNAs against human SOD1 in motor neurons delayed the disease onset but did not modify the disease progression once it started [145], whereas silencing of mutant SOD1 within myeloid cells or astrocytes slowed the disease progression rather than the disease onset [139-140]. After all the bone marrow of mutant-SOD1-expressing PU-/- mice, which lacked myeloid and lymphoid cells, were replaced with wild-type-SOD1 bone marrow, their disease progression and survival improved [143], which suggests that microglia and astrocytes were not sufficient for the initiation of motor neuron death, but hastened the disease progression.

which belonged to alterations in their microglia/macrophage activation profiles [153]; elevated levels of complementary proteins in their sera [154]; increased IL-13-producing T cells and circulating neutrophils [155-156]; and higher production of CD8+ T cells in the lymphocytes [157]. Monocyte chemoattractant protein (MCP)-1 and RANTES were abundant in the cerebrospinal fluid and sera of ALS patients [158-161]. Increased MCP-1 was shown in the microglia of mutant SOD1 mice [162-163]. Moreover, the higher LPS level in the plasma of ALS patients was proportional to the total abnormally activated monocyte/macrophage contents of the peripheral blood [164]. Long-term exposure to LPS also furthered the disease progres‐ sion in animal ALS models, which implies that systemic inflammation connected to peripheral factors and innate immunity in the CNS concurrently influences the disease course [165]. With aging, the blood-brain barrier (BBB) is less tight and thus, more vulnerable to systemic inflammation. The collapse of BBB or of the blood-spinal cord barrier (BSCB) was shown in animal ALS models or human ALS patients using evans blue leakage and immunohistochem‐ istry against the anti-CD44 antibody, respectively [166-167]. Under these conditions, periph‐ eral-inflammation-inducing factors were very apparent in the CNS and thereby affected the

Multiple Routes of Motor Neuron Degeneration in ALS

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

45

Minocycline, which is believed to attenuate microglia activation, or celecoxib, a cox-2 inhibitor, showed beneficial effects in mutant SOD1 mice [115-117,168-169]. Clinical studies on the two drugs did not disprove, however, their therapeutic property in ALS patients. Thalidomide, glatiramer acetate, and ONO-2506 also supported the causative role of the inflammation in the pathology in ALS mice that showed improved motor function and survival [170-171], but their

Mitochondria constitute approximately 25% of the cytoplasmic volume in most eukaryotic cells and produce cellular energy in the form of ATP via electron transport and oxidative phosphorylation. 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 have been recognized as target organelles for the regulation and execution of cell death under pathological conditions [172-173]. There are many mitochondria in the motor neurons because of the high rate of metabolic demand therein, which implies that motor neurons are susceptible to functional or morphological alteration in mitochondria. Mtochondrial abnormality may play a crucial role in the pathologic mechanism of motor neuron diseases and of ALS. Studies with ALS patients and animal ALS models have been performed to examine both the mor‐ phologic and functional abnormalities of the mitochondria [174]. Morphological abnormality in the organelle that includes a fragmented network, swelling, and increased cristae has been observed in the soma and proximal axons of ventral motor neurons of sporadic ALS (sALS) patients [39]. In ALS patients, a reduction in complex IV of the electron transport chain activity

neurodegeneration.

*5.2.3. Therapies for inflammation in ALS*

beneficial effects were not linked to the ALS patients.

**6. Mitochondrial pathology in ALS**

#### *5.2.2. Systemic inflammation*

Damaged or aged brains continuously suffer from systemic inflammation connected with peripheral factors, regardless of the presence of innate inflammation in the CNS [146-147]. Three critical components are directly correlated with the synthesis of cytokines and inflam‐ matory mediators in the brain parenchyma to communicate an inflammatory signal to the brain and to trigger tissue injury. First, inflammatory responses in the thoracic-abdominal cavity are transduced into the brain via vagal-nerve sensory afferents, and then the outflow of a vagal efferent seems to manipulate these events through acetylcholine secretion, which acts on alpha 7 nicotinic receptors of macrophages [148]. Second, cytokines and inflammatory mediators from the specific area of the inflammation are put into the blood and communicate with macrophages and other cells in the circumventricular organs, which lack a patent bloodbrain barrier [149]. Third, the cytokines or inflammatory mediators themselves might directly communicate with the brain endothelium via receptors expressed on the endothelium [150]. Several pieces of evidence showed that a systemic immune response is related to a clinically symptomatic feature of a neurodegenerative disease such as AD. In accordance with frequently circulating cytokines in the blood or CSF of AD patients, the abundance of pro-inflammatory factors preceded the clinical onset of dementia in the subjects [151]. Aged people with systemic infections have a double risk of developing AD. Similarly, the correlation of clinical events with systemic immunity was experimentally evaluated in an animal that was challenged with systemic stimulation. Infection of aged rats with LPS revealed neuronal loss in the brain and the memory deficits [152]. Thus, it can be said that systemic inflammation contributes to the onset and progression of neurodegenerative diseases. In recent clinical and pathological studies, ALS patients revealed dysregulation of their systemic inflammatory components, which belonged to alterations in their microglia/macrophage activation profiles [153]; elevated levels of complementary proteins in their sera [154]; increased IL-13-producing T cells and circulating neutrophils [155-156]; and higher production of CD8+ T cells in the lymphocytes [157]. Monocyte chemoattractant protein (MCP)-1 and RANTES were abundant in the cerebrospinal fluid and sera of ALS patients [158-161]. Increased MCP-1 was shown in the microglia of mutant SOD1 mice [162-163]. Moreover, the higher LPS level in the plasma of ALS patients was proportional to the total abnormally activated monocyte/macrophage contents of the peripheral blood [164]. Long-term exposure to LPS also furthered the disease progres‐ sion in animal ALS models, which implies that systemic inflammation connected to peripheral factors and innate immunity in the CNS concurrently influences the disease course [165]. With aging, the blood-brain barrier (BBB) is less tight and thus, more vulnerable to systemic inflammation. The collapse of BBB or of the blood-spinal cord barrier (BSCB) was shown in animal ALS models or human ALS patients using evans blue leakage and immunohistochem‐ istry against the anti-CD44 antibody, respectively [166-167]. Under these conditions, periph‐ eral-inflammation-inducing factors were very apparent in the CNS and thereby affected the neurodegeneration.

#### *5.2.3. Therapies for inflammation in ALS*

expression of SOD1 mutation in mice led to the ALS phenotype, with marginal effects [141-142]. Specific expression of mutant SOD1 within neurons using *Nefl* (neurofilament light chain) promoters did not cause motor neuron degeneration in transgenic mice [142]. Consis‐ tently, selective expression of mutant SOD1 in microglia or astrocytes did not kill motor neurons [141,143].These non-cell-autonomous deaths of motor neurons were supported by an analysis of chimeric mice that had mixed populations of normal cells and cells that expressed mutant SOD1 [144]. Conditional knockout of mutant SOD1 in motor neurons using an *Isl1* promoter-driven *Cre* transgene that is expressed in the spinal cord delayed the disease onset in and prolonged the survival of mutant SOD1 transgenic mice. On the other hand, however, selective removal from cells of the myeloid lineage that included microglia using a *Cd11b* promoter-driven *Cre* transgene did not delay the disease onset but extended its progress [139]. In the same lineages, selective viral vector-mediated delivery of small interfering RNAs against human SOD1 in motor neurons delayed the disease onset but did not modify the disease progression once it started [145], whereas silencing of mutant SOD1 within myeloid cells or astrocytes slowed the disease progression rather than the disease onset [139-140]. After all the bone marrow of mutant-SOD1-expressing PU-/- mice, which lacked myeloid and lymphoid cells, were replaced with wild-type-SOD1 bone marrow, their disease progression and survival improved [143], which suggests that microglia and astrocytes were not sufficient for the

initiation of motor neuron death, but hastened the disease progression.

Damaged or aged brains continuously suffer from systemic inflammation connected with peripheral factors, regardless of the presence of innate inflammation in the CNS [146-147]. Three critical components are directly correlated with the synthesis of cytokines and inflam‐ matory mediators in the brain parenchyma to communicate an inflammatory signal to the brain and to trigger tissue injury. First, inflammatory responses in the thoracic-abdominal cavity are transduced into the brain via vagal-nerve sensory afferents, and then the outflow of a vagal efferent seems to manipulate these events through acetylcholine secretion, which acts on alpha 7 nicotinic receptors of macrophages [148]. Second, cytokines and inflammatory mediators from the specific area of the inflammation are put into the blood and communicate with macrophages and other cells in the circumventricular organs, which lack a patent bloodbrain barrier [149]. Third, the cytokines or inflammatory mediators themselves might directly communicate with the brain endothelium via receptors expressed on the endothelium [150]. Several pieces of evidence showed that a systemic immune response is related to a clinically symptomatic feature of a neurodegenerative disease such as AD. In accordance with frequently circulating cytokines in the blood or CSF of AD patients, the abundance of pro-inflammatory factors preceded the clinical onset of dementia in the subjects [151]. Aged people with systemic infections have a double risk of developing AD. Similarly, the correlation of clinical events with systemic immunity was experimentally evaluated in an animal that was challenged with systemic stimulation. Infection of aged rats with LPS revealed neuronal loss in the brain and the memory deficits [152]. Thus, it can be said that systemic inflammation contributes to the onset and progression of neurodegenerative diseases. In recent clinical and pathological studies, ALS patients revealed dysregulation of their systemic inflammatory components,

*5.2.2. Systemic inflammation*

44 Current Advances in Amyotrophic Lateral Sclerosis

Minocycline, which is believed to attenuate microglia activation, or celecoxib, a cox-2 inhibitor, showed beneficial effects in mutant SOD1 mice [115-117,168-169]. Clinical studies on the two drugs did not disprove, however, their therapeutic property in ALS patients. Thalidomide, glatiramer acetate, and ONO-2506 also supported the causative role of the inflammation in the pathology in ALS mice that showed improved motor function and survival [170-171], but their beneficial effects were not linked to the ALS patients.
