**2. The contribution of astrocytes in the neuroinflammatory response**

### **2.1. Activation profile of astrocytes in human and animal models of ALS**

Under normal and healthy conditions, astrocytes, which are the most abundant cell type within the CNS, are typically found in a resting state. Activation of astrocytes follows an acute or chronic injury, where the cells adopt a different morphology, become proliferative, express the intermediate filament glial fibrillary acidic protein (GFAP) release pro-inflammatory

cytokines and growth factors as well as produce nitric oxide (NO)(reviewed in [3]). The phenomenon of astrocytosis has been well characterized in both ALS patients and animal models. Analysis of human ALS brains reveals the presence of reactive astrocytes within the subcortical white matter in a widespread fashion [4]. Importantly, the same brain regions from patients with non-ALS neurological disorders display a distinct histopathology, suggesting that the ALS astrocytosis is not simply an indirect result of the ongoing neurodegenerative process [4]. Similarly, the cortical gray matter tissue and the primary motor area from both sporadic and familial ALS patients are characterized by the omnipresence of reactive astrocytes [5, 6]. Studies performed on spinal cords from ALS patients show the occurrence of astrocytosis in both the ventral and dorsal horn region of the spinal cord [7, 8]. In addition to the abovementioned post-mortem observations, in vivo brain imaging of ALS patients using deuteriumsubstituted [11C](L)-deprenyl positron emission tomography has allowed the visualization of astrocytosis in live patients [9]. Hence, a thorough analysis of the CNS of ALS patients has uncovered and highlighted astrocytosis as a *bona fide* feature of ALS pathology, whether sporadic or inherited. While human ALS tissue represents most accurately the hallmarks that typify the disease, the caveat is that it limits our knowledge of the cellular events that occur prior to disease onset.

that mutant astrocytes contribute to progression, but not onset of the disease [17]. However, knocking down the mutant SOD1 in astrocytes of the *SOD1G85R* mouse model results in increased survival by delaying disease onset as well as the early stage of the disease [18]. Despite minor differences between the targeted disease stages in both models, the key finding is that mutant SOD1-expressing astrocytes regulate the disease progression of murine ALS.

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Another approach used to address the astrocytic-induced motoneuron loss in ALS is the *in vitro* co-culture of both cell types. Indeed, when cultured alone, primary *SOD1G93A* astrocytes express high levels of pro-inflammatory effectors such as tumor necrosis factor alpha (TNFα), interferon gamma (IFNγ), interleukins (IL)-1 beta (IL-1β) and -18 (IL-18), 5-lipoxygenase (5- LOX), leukotriene B4, cyclooxygenase (COX-2) and prostaglandin E2 (PGE2), thus displaying an inflammatory phenotype with potential neurotoxic effects [19]. Consequently, primary wildtype and mutant motoneurons or motoneurons derived from murine or human embry‐ onic stem cells show decreased survival when cultured in the presence of astrocytes expressing different mutated forms of SOD1 [20-24]. While the above-mentioned *in vivo* and *in vitro* studies suggest a contributory role for astrocytes in ALS pathogenesis, the targeted ablation of GFAPexpressing proliferating astrocytes in *SOD1G93A* mice has no effect on the onset or the progres‐ sion of the neurodegenerative process [25]. Recently, a subtype of astrocytes from spinal cord cultures of *SOD1G93A* rats that displayed an aberrant phenotype has been isolated (termed Aba cells). Aba cells, that highly express S100β and connexin-43, but weakly express GFAP, are distinguished by their increased proliferative abilities and the absence of replicative senes‐ cence. Specifically, they are localized in proximity of motoneurons *in vivo*, increase drastically upon disease onset and demonstrate a greater neurotoxicity compared to non-Aba astrocytes isolated from *SOD1G93A* rats [26]. Combined, these studies suggest that different subpopula‐ tions of astrocytes with different functional features and different cellular origin coexist during

An additional important feature of the astrocytic contribution in ALS relates to the observation that the expression of SOD1G85R solely in astrocytes does not give rise to motoneuron loss despite the fact that astrocytosis occurs prominently [27]. Likewise, the specific expression of SOD1G37R in spinal cord motoneurons or the accumulation of SOD1G93A in postnatal motoneur‐ ons does not impact motor function, neurodegeneration or disease onset and progression [28, 29]. Together, these observations therefore point to the critical communication that takes place between astrocytes and motoneurons, which might in turn lead to the initiation of neuronal

The glutamate hypothesis proposes that a glutamate imbalance, leading to a calcium (Ca2+) mediated excitotoxic insult, represents a major mechanism of motoneuron injury [30]. Astrocytes actively participate in modulating neuronal excitability and neurotransmission by controlling the extracellular levels of ions and neurotransmitters. The astroglial glutamate transporter excitatory amino-acid transporter 2 (EAAT2) in humans or glutamate transporter 1 (GLT-1) in rodents is the primary means of maintaining low extracellular glutamate levels. EAAT2/GLT-1 rapidly removes glutamate from the extracellular milieu and thereby prevents

**2.3. Misregulation of neuronal transmission by astrocytes**

the pathological processes.

death pathways.

The generation of both mouse and rat models of ALS has helped elucidate more precisely the contributory role of astrocytosis during the neurodegenerative process. Analysis of different *superoxide dismutase 1* (*SOD1*) mutant mouse models identifies astrocytic alterations such as reactive morphological changes, proliferation as well as the presence of SOD1- and ubiquitinpositive inclusions, as occurring prior or close-to axonal degeneration and neuronal loss [10-13]. Furthermore, the process of astrocytosis significantly intensifies as the disease progresses [10, 11]. Three-dimensional reconstruction of *SOD1G93A* spinal cord sections shows that astrocytic processes actually target and envelop pathological vacuoles within the degen‐ erating neurons [11]. Similarly to the murine models, the transgenic *SOD1G93A* rats also display signs of astrocytosis prior to significant motoneuron loss. As the disease progresses, there is an increase of astrocytic hypertrophy and proliferation as well as an accumulation of ubiquitin and tau-positive aggregates [14, 15]. Thus, while the human data provided the first insights into astrocytosis as a pathological hallmark of ALS, the observation in pre-clinical models of astrocytic inflammation prior to neurodegeneration strengthened the proposed contributory role of astrocytes in ALS pathogenesis.

#### **2.2. A role for astrocytes in ALS pathogenesis**

Once the astrocytic histopathology was thoroughly characterized in both human and animal ALS models, a comprehensive assessment of its functional influence on motoneuron loss thus ensued. One of the first indications of astrocyte-dependent neurodegeneration in ALS comes from the generation of chimeric mice, composed of both normal cells and SOD1 mutantexpressing cells [16]. This study demonstrates that mutant SOD1-positive motoneurons surrounded by wildtype non-neuronal cells have a better survival rate than those enclosed by mutant SOD1-positive non-neuronal cells [16]. A complementary approach consisting in deleting the human mutant SOD1 specifically within astrocytes of the *SOD1G37R* mice suggests that mutant astrocytes contribute to progression, but not onset of the disease [17]. However, knocking down the mutant SOD1 in astrocytes of the *SOD1G85R* mouse model results in increased survival by delaying disease onset as well as the early stage of the disease [18]. Despite minor differences between the targeted disease stages in both models, the key finding is that mutant SOD1-expressing astrocytes regulate the disease progression of murine ALS.

cytokines and growth factors as well as produce nitric oxide (NO)(reviewed in [3]). The phenomenon of astrocytosis has been well characterized in both ALS patients and animal models. Analysis of human ALS brains reveals the presence of reactive astrocytes within the subcortical white matter in a widespread fashion [4]. Importantly, the same brain regions from patients with non-ALS neurological disorders display a distinct histopathology, suggesting that the ALS astrocytosis is not simply an indirect result of the ongoing neurodegenerative process [4]. Similarly, the cortical gray matter tissue and the primary motor area from both sporadic and familial ALS patients are characterized by the omnipresence of reactive astrocytes [5, 6]. Studies performed on spinal cords from ALS patients show the occurrence of astrocytosis in both the ventral and dorsal horn region of the spinal cord [7, 8]. In addition to the abovementioned post-mortem observations, in vivo brain imaging of ALS patients using deuteriumsubstituted [11C](L)-deprenyl positron emission tomography has allowed the visualization of astrocytosis in live patients [9]. Hence, a thorough analysis of the CNS of ALS patients has uncovered and highlighted astrocytosis as a *bona fide* feature of ALS pathology, whether sporadic or inherited. While human ALS tissue represents most accurately the hallmarks that typify the disease, the caveat is that it limits our knowledge of the cellular events that occur

The generation of both mouse and rat models of ALS has helped elucidate more precisely the contributory role of astrocytosis during the neurodegenerative process. Analysis of different *superoxide dismutase 1* (*SOD1*) mutant mouse models identifies astrocytic alterations such as reactive morphological changes, proliferation as well as the presence of SOD1- and ubiquitinpositive inclusions, as occurring prior or close-to axonal degeneration and neuronal loss [10-13]. Furthermore, the process of astrocytosis significantly intensifies as the disease progresses [10, 11]. Three-dimensional reconstruction of *SOD1G93A* spinal cord sections shows that astrocytic processes actually target and envelop pathological vacuoles within the degen‐ erating neurons [11]. Similarly to the murine models, the transgenic *SOD1G93A* rats also display signs of astrocytosis prior to significant motoneuron loss. As the disease progresses, there is an increase of astrocytic hypertrophy and proliferation as well as an accumulation of ubiquitin and tau-positive aggregates [14, 15]. Thus, while the human data provided the first insights into astrocytosis as a pathological hallmark of ALS, the observation in pre-clinical models of astrocytic inflammation prior to neurodegeneration strengthened the proposed contributory

Once the astrocytic histopathology was thoroughly characterized in both human and animal ALS models, a comprehensive assessment of its functional influence on motoneuron loss thus ensued. One of the first indications of astrocyte-dependent neurodegeneration in ALS comes from the generation of chimeric mice, composed of both normal cells and SOD1 mutantexpressing cells [16]. This study demonstrates that mutant SOD1-positive motoneurons surrounded by wildtype non-neuronal cells have a better survival rate than those enclosed by mutant SOD1-positive non-neuronal cells [16]. A complementary approach consisting in deleting the human mutant SOD1 specifically within astrocytes of the *SOD1G37R* mice suggests

prior to disease onset.

98 Current Advances in Amyotrophic Lateral Sclerosis

role of astrocytes in ALS pathogenesis.

**2.2. A role for astrocytes in ALS pathogenesis**

Another approach used to address the astrocytic-induced motoneuron loss in ALS is the *in vitro* co-culture of both cell types. Indeed, when cultured alone, primary *SOD1G93A* astrocytes express high levels of pro-inflammatory effectors such as tumor necrosis factor alpha (TNFα), interferon gamma (IFNγ), interleukins (IL)-1 beta (IL-1β) and -18 (IL-18), 5-lipoxygenase (5- LOX), leukotriene B4, cyclooxygenase (COX-2) and prostaglandin E2 (PGE2), thus displaying an inflammatory phenotype with potential neurotoxic effects [19]. Consequently, primary wildtype and mutant motoneurons or motoneurons derived from murine or human embry‐ onic stem cells show decreased survival when cultured in the presence of astrocytes expressing different mutated forms of SOD1 [20-24]. While the above-mentioned *in vivo* and *in vitro* studies suggest a contributory role for astrocytes in ALS pathogenesis, the targeted ablation of GFAPexpressing proliferating astrocytes in *SOD1G93A* mice has no effect on the onset or the progres‐ sion of the neurodegenerative process [25]. Recently, a subtype of astrocytes from spinal cord cultures of *SOD1G93A* rats that displayed an aberrant phenotype has been isolated (termed Aba cells). Aba cells, that highly express S100β and connexin-43, but weakly express GFAP, are distinguished by their increased proliferative abilities and the absence of replicative senes‐ cence. Specifically, they are localized in proximity of motoneurons *in vivo*, increase drastically upon disease onset and demonstrate a greater neurotoxicity compared to non-Aba astrocytes isolated from *SOD1G93A* rats [26]. Combined, these studies suggest that different subpopula‐ tions of astrocytes with different functional features and different cellular origin coexist during the pathological processes.

An additional important feature of the astrocytic contribution in ALS relates to the observation that the expression of SOD1G85R solely in astrocytes does not give rise to motoneuron loss despite the fact that astrocytosis occurs prominently [27]. Likewise, the specific expression of SOD1G37R in spinal cord motoneurons or the accumulation of SOD1G93A in postnatal motoneur‐ ons does not impact motor function, neurodegeneration or disease onset and progression [28, 29]. Together, these observations therefore point to the critical communication that takes place between astrocytes and motoneurons, which might in turn lead to the initiation of neuronal death pathways.

#### **2.3. Misregulation of neuronal transmission by astrocytes**

The glutamate hypothesis proposes that a glutamate imbalance, leading to a calcium (Ca2+) mediated excitotoxic insult, represents a major mechanism of motoneuron injury [30]. Astrocytes actively participate in modulating neuronal excitability and neurotransmission by controlling the extracellular levels of ions and neurotransmitters. The astroglial glutamate transporter excitatory amino-acid transporter 2 (EAAT2) in humans or glutamate transporter 1 (GLT-1) in rodents is the primary means of maintaining low extracellular glutamate levels. EAAT2/GLT-1 rapidly removes glutamate from the extracellular milieu and thereby prevents excitotoxic injury to neurons that occurs by overstimulation of the post-synaptic N-methyl-Daspartic acid (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/ kainate ionotropic glutamate receptors [31, 32]. Decreased expression of EAAT2/GLT-1, which leads to elevated levels of extracellular glutamate, has been found in a vast majority of sporadic and familial ALS patients as well as ALS mice and rats [10, 33-35], suggesting the participation of astrocytes in glutamate-induced excitotoxicity.

tivity within the grey and white matter [43], suggesting that the astrocytosis in ALS might in fact be a responsive phenomenon. Conversely, many research groups have identified specific factors that are abnormally regulated in ALS astrocytes that could potentially trigger the

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Type I, II and III IFNs are an important family of immunomodulatory cytokines (reviewed in [44]). Elevated levels of IFNγ, a potent pro-inflammatory mediator, are found in the CSF of ALS patients, in the serum as the disease progresses and in spinal cord of sporadic ALS patients [45-47]. Further, the analysis of spinal cord sections from ALS patients shows that IFNγ is detected in ventral horn neurons, glial cells and plausibly immune cells [47]. In addition, the IFNγ-inducible protein, IP-30 and the interferon-stimulated gene 15 (ISG15) are significantly upregulated in human ALS spinal cord [48, 49]. In spinal cord extracts and serum of ALS mice, elevated levels of IFNγ mRNA and protein are also documented [24, 50, 51]. The expression of IFNγ is found within motoneurons and astrocytes of *SOD1G93A* and *SOD1G85R* spinal cords at both disease onset and symptomatic stages [24]. Similarly, a gene expression array analysis of pre-symptomatic *SOD1G93A* spinal cord reveals an induction of several genes regulated by type I IFNα, IFNβ and type II IFNγ, with specifically an increased expression of ISG15 in spinal cord astrocytes. Further, the phosphorylation of signal transducer and activator of transcrip‐ tion (STAT) 1 and 2, downstream effectors of IFNs [52], and STAT4, an inducer of IFNγ, is also elevated in *SOD1G93A* spinal cords [51]. Functionally, the genetic deletion of *Ifnα/β receptor 1* in *SOD1G93A* mice significantly prolongs life expectancy [49]. Importantly, astrocytic IFNγ triggers a motoneuron-selective death pathway via the activation of lymphotoxin beta receptor (LTβR) by LIGHT. LIGHT is also upregulated in sporadic ALS spinal cords and the genetic ablation of *Light* in *SOD1G93A* mice delays disease progression [24]. Combined, these observa‐ tions in rodent and human models of the disease suggest that the neuroinflammatory role of

The low affinity p75 neurotrophin receptor (p75NTR) has a well-described role in mediating neuronal death signaling (reviewed in [53]). In symptomatic *SOD1G93A* mice and in ALS patients, p75NTR is overexpressed within spinal motoneurons [54]. Correspondingly, the immunoreactivity of nerve growth factor (NGF), a p75NTR ligand [55], is increased in spinal cord astrocytes of symptomatic *SOD1G93A* mice and in primary *SOD1G93A* astrocyte cultures [56, 57]. Further, the excessive expression of fibroblast growth factor 1 (FGF-1) by *SOD1G93A* motoneurons stimulates the nuclear accumulation of FGF receptor 1 (FGFR1) in astrocytes, consequently triggering astrocytic NGF production [58]. Importantly, primary *SOD1G93A* motoneuron cultures are hypersensitive to the NGF-p75NTR apoptotic signaling [59]. Thus, the astrocyte-dependent activation of the neurotoxic NGF-p75NTR pathway might participate to

motoneuron loss that typifies the disease.

IFNs may contribute to the neurodegenerative process in ALS.

*2.4.2. The contribution of nerve growth factor*

the neurodegeneration that typifies ALS.

*2.4.1. The interferon response*

In addition to the relationship between glutamate excitotoxicity and glutamate transporter loss, other glutamatergic pathways have been implicated in motoneuron degeneration. Functional AMPA receptors consist of various combinations of four subunits (designated glutamate receptor (GluR)1-4) and are involved in fast excitatory synaptic transmission in the CNS [36]. The GluR2 subunit is functionally dominant and renders AMPA receptors imper‐ meable to Ca2+, preventing Ca2+ influx-induced toxicity. Thus, high levels of GluR2 in neuronal tissues might confer neuroprotection against glutamate-induced excitotoxicity. Within normal human spinal motoneurons, there is a low relative abundance of the GluR2 subunit mRNA compared to other GluR subunits and to other neuronal tissues, which may make them unduly susceptible to Ca2+-mediated toxic events following glutamate receptor activation [37]. However, work from another group does not observe any significant quantitative changes in GluR2 mRNA within spinal cord motoneurons, suggesting that a selective decrease of the GluR2 subunit might not be the only mechanism mediating the AMPA receptor-dependent neurotoxicity in ALS [38]. Indeed, it has been demonstrated that RNA editing of GluR2 mRNA at the glutamine/arginine (Q/R) site is decreased in autopsy-obtained spinal motoneurons from patients with sporadic ALS [39], a molecular event that confers Ca2+ permeability to the GluR2 receptor [40]. Therefore, reductions in both GluR2 expression and GluR2 Q/R site editing may contribute to increased Ca2+ influx and neurotoxicity through AMPA receptors in ALS.

The molecular basis for lower GluR2 abundance in motoneurons compared to other CNS neurons has been investigated using two different rat strains that show differential vulnera‐ bility to AMPA-mediated excitotoxicity [41]. It has thus been demonstrated that astrocytes derived from the ventral spinal cord, but not those derived from the dorsal spinal cord, cerebellum, or the cortex, have the ability to regulate GluR2 expression in motoneurons. Interestingly, expression of mutant SOD1 abolishes their GluR2-regulating capacity. Al‐ though, the astrocytic factor responsible for GluR2 regulation in motoneurons remains to be identified, the regulation of motoneuron electrical activity through neuronal GluR2 expression and the uptake of glutamate by the glial transporter EAAT2/GLT-1 are major mechanisms by which astrocytes may mediate excitotoxic neurodegeneration in ALS.

#### **2.4. Additional mechanisms of astrocytic neurotoxicity**

While the astrocytic influence on neuronal excitability is seldom disputed, various reports suggest that they may also participate in the neurodegenerative process via the release of neurotoxic factors. Typically, the activation and/or reaction of astrocytes that characterize neuroinflammation occurs following a CNS injury, including chronic neurodegenerative diseases (reviewed in [42]). In experiments where the spinal cords of neonatal rats were injected with cerebrospinal fluid (CSF) from ALS patients, there is an increased GFAP immunoreac‐ tivity within the grey and white matter [43], suggesting that the astrocytosis in ALS might in fact be a responsive phenomenon. Conversely, many research groups have identified specific factors that are abnormally regulated in ALS astrocytes that could potentially trigger the motoneuron loss that typifies the disease.

#### *2.4.1. The interferon response*

excitotoxic injury to neurons that occurs by overstimulation of the post-synaptic N-methyl-Daspartic acid (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/ kainate ionotropic glutamate receptors [31, 32]. Decreased expression of EAAT2/GLT-1, which leads to elevated levels of extracellular glutamate, has been found in a vast majority of sporadic and familial ALS patients as well as ALS mice and rats [10, 33-35], suggesting the participation

In addition to the relationship between glutamate excitotoxicity and glutamate transporter loss, other glutamatergic pathways have been implicated in motoneuron degeneration. Functional AMPA receptors consist of various combinations of four subunits (designated glutamate receptor (GluR)1-4) and are involved in fast excitatory synaptic transmission in the CNS [36]. The GluR2 subunit is functionally dominant and renders AMPA receptors imper‐ meable to Ca2+, preventing Ca2+ influx-induced toxicity. Thus, high levels of GluR2 in neuronal tissues might confer neuroprotection against glutamate-induced excitotoxicity. Within normal human spinal motoneurons, there is a low relative abundance of the GluR2 subunit mRNA compared to other GluR subunits and to other neuronal tissues, which may make them unduly susceptible to Ca2+-mediated toxic events following glutamate receptor activation [37]. However, work from another group does not observe any significant quantitative changes in GluR2 mRNA within spinal cord motoneurons, suggesting that a selective decrease of the GluR2 subunit might not be the only mechanism mediating the AMPA receptor-dependent neurotoxicity in ALS [38]. Indeed, it has been demonstrated that RNA editing of GluR2 mRNA at the glutamine/arginine (Q/R) site is decreased in autopsy-obtained spinal motoneurons from patients with sporadic ALS [39], a molecular event that confers Ca2+ permeability to the GluR2 receptor [40]. Therefore, reductions in both GluR2 expression and GluR2 Q/R site editing may contribute to increased Ca2+ influx and neurotoxicity through AMPA receptors in ALS.

The molecular basis for lower GluR2 abundance in motoneurons compared to other CNS neurons has been investigated using two different rat strains that show differential vulnera‐ bility to AMPA-mediated excitotoxicity [41]. It has thus been demonstrated that astrocytes derived from the ventral spinal cord, but not those derived from the dorsal spinal cord, cerebellum, or the cortex, have the ability to regulate GluR2 expression in motoneurons. Interestingly, expression of mutant SOD1 abolishes their GluR2-regulating capacity. Al‐ though, the astrocytic factor responsible for GluR2 regulation in motoneurons remains to be identified, the regulation of motoneuron electrical activity through neuronal GluR2 expression and the uptake of glutamate by the glial transporter EAAT2/GLT-1 are major mechanisms by

While the astrocytic influence on neuronal excitability is seldom disputed, various reports suggest that they may also participate in the neurodegenerative process via the release of neurotoxic factors. Typically, the activation and/or reaction of astrocytes that characterize neuroinflammation occurs following a CNS injury, including chronic neurodegenerative diseases (reviewed in [42]). In experiments where the spinal cords of neonatal rats were injected with cerebrospinal fluid (CSF) from ALS patients, there is an increased GFAP immunoreac‐

which astrocytes may mediate excitotoxic neurodegeneration in ALS.

**2.4. Additional mechanisms of astrocytic neurotoxicity**

of astrocytes in glutamate-induced excitotoxicity.

100 Current Advances in Amyotrophic Lateral Sclerosis

Type I, II and III IFNs are an important family of immunomodulatory cytokines (reviewed in [44]). Elevated levels of IFNγ, a potent pro-inflammatory mediator, are found in the CSF of ALS patients, in the serum as the disease progresses and in spinal cord of sporadic ALS patients [45-47]. Further, the analysis of spinal cord sections from ALS patients shows that IFNγ is detected in ventral horn neurons, glial cells and plausibly immune cells [47]. In addition, the IFNγ-inducible protein, IP-30 and the interferon-stimulated gene 15 (ISG15) are significantly upregulated in human ALS spinal cord [48, 49]. In spinal cord extracts and serum of ALS mice, elevated levels of IFNγ mRNA and protein are also documented [24, 50, 51]. The expression of IFNγ is found within motoneurons and astrocytes of *SOD1G93A* and *SOD1G85R* spinal cords at both disease onset and symptomatic stages [24]. Similarly, a gene expression array analysis of pre-symptomatic *SOD1G93A* spinal cord reveals an induction of several genes regulated by type I IFNα, IFNβ and type II IFNγ, with specifically an increased expression of ISG15 in spinal cord astrocytes. Further, the phosphorylation of signal transducer and activator of transcrip‐ tion (STAT) 1 and 2, downstream effectors of IFNs [52], and STAT4, an inducer of IFNγ, is also elevated in *SOD1G93A* spinal cords [51]. Functionally, the genetic deletion of *Ifnα/β receptor 1* in *SOD1G93A* mice significantly prolongs life expectancy [49]. Importantly, astrocytic IFNγ triggers a motoneuron-selective death pathway via the activation of lymphotoxin beta receptor (LTβR) by LIGHT. LIGHT is also upregulated in sporadic ALS spinal cords and the genetic ablation of *Light* in *SOD1G93A* mice delays disease progression [24]. Combined, these observa‐ tions in rodent and human models of the disease suggest that the neuroinflammatory role of IFNs may contribute to the neurodegenerative process in ALS.

#### *2.4.2. The contribution of nerve growth factor*

The low affinity p75 neurotrophin receptor (p75NTR) has a well-described role in mediating neuronal death signaling (reviewed in [53]). In symptomatic *SOD1G93A* mice and in ALS patients, p75NTR is overexpressed within spinal motoneurons [54]. Correspondingly, the immunoreactivity of nerve growth factor (NGF), a p75NTR ligand [55], is increased in spinal cord astrocytes of symptomatic *SOD1G93A* mice and in primary *SOD1G93A* astrocyte cultures [56, 57]. Further, the excessive expression of fibroblast growth factor 1 (FGF-1) by *SOD1G93A* motoneurons stimulates the nuclear accumulation of FGF receptor 1 (FGFR1) in astrocytes, consequently triggering astrocytic NGF production [58]. Importantly, primary *SOD1G93A* motoneuron cultures are hypersensitive to the NGF-p75NTR apoptotic signaling [59]. Thus, the astrocyte-dependent activation of the neurotoxic NGF-p75NTR pathway might participate to the neurodegeneration that typifies ALS.

#### *2.4.3. Cyclooxygenase-2*

COX-2 is a pro-inflammatory enzyme that converts arachidonic acid into prostanoids such as PGE2, a potent inflammatory mediator (reviewed in [60]). In the anterior horn region of the spinal cord of *SOD1G93A* mice, at both the early and end stage of the disease, COX-2 immunor‐ eactivity is elevated in astrocytes [61]. Similarly, spinal cord astrocytes from sporadic ALS patients also display increased COX-2 expression [61, 62]. The expression of COX-2 can be modulated by the binding of CD40, a member of the TNF family (reviewed in [63]), with its ligand CD40L [64]. Interestingly, spinal cord astrocytes of symptomatic *SOD1G39A* mice show an upregulation of CD40, concomitant with COX-2 astrocytic expression. Moreover, the activation of COX-2 in astrocytes upon CD40 stimulation leads to motoneuron death *in vitro* [65], suggesting that an astrocytic CD40-COX-2 pathway could also participate in ALS pathogenesis. The contribution of the CD40/CD40L pathway has recently been proposed in ALS mice, though its role in astrocytic neurotoxicity role has not been established [66]. Finally, another facet of the COX-2 pathway relates to the ability of PGE2 to promote glutamate release from astrocytes, emphasizing further the complex multimodality of neuroinflammatory signals [67].

displayed a higher content of MAO-B compared to microglial cells [8, 78]. Finally, an epide‐ miological analysis has uncovered that the MAO-B allelic phenotype influences the age of ALS onset [79]. Excessive astrocytic MAO-B expression, which results in elevations of extracellular ROS levels, may have damaging effects on neighboring motoneurons. Additional mechanisms could also involve mitochondrial dysfunction by the selective inhibition of respiratory complex I, which further leads to increased production of superoxide as well as microglial

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While there is a vast amount of research on the mitochondrial dysfunction in ALS motoneurons (reviewed in [81]), not much is known about the impact of toxic genetic mutations on the mitochondria of astrocytes. There is evidence however, that ALS astrocytes do in fact display pathological mitochondrial dysfunction that subsequently leads to oxidative damage, sus‐ taining their reactive status. Indeed, primary astrocytes isolated from the cerebral cortex of neonatal rats and overexpressing *SOD1G93A* display a decreased mitochondrial respiration rate, an increased superoxide formation and a decreased membrane potential [82]. In co-culture experiments, modulating the mitochondrial defects of SOD1G93A-expressing astrocytes via small chemical compounds improves astrocytic-dependent motoneuron survival. Conversely, induction of mitochondrial damage to wildtype astrocytes increases motoneuron death [82]. Thus, organelle dysfunction within ALS astrocytes may be an important contributor to the neurodegenerative process. In addition, a positive amplification system could take place during the degenerative process, since the inflammatory mediator NO, mainly produced by the inducible form of nitric oxide synthase (iNOS) in reactive astrocytes, can in turn induce

A fundamental role for astrocytes in the neuroinflammation process is the recruitment of microglia [84], the resident macrophages of the CNS (reviewed in [85]). In *SOD1G37R* mice where the mutant *SOD1* gene is specifically deleted in astrocytes, the delay in the progression of the later stages of disease is accompanied with an inhibition of microglial activation and microgliadependent detrimental NO production [17]. Thus, astrocytosis in ALS may promote neuroin‐ flammation events through microglial recruitment, which in turn may participate directly or indirectly to the motoneuron loss in ALS. Several pro-inflammatory contributors including TNFα, IFNγ, IL-1β and NO, which are aberrantly produced by mutant astrocytes can indeed enhance the activation of microglia. The specific role of microglia in the neuroinflammatory

Figure 1 illustrates the potential non-cell-autonomous mechanisms implicating reactive

activation [80].

*2.4.6. Mitochondrial dysfunctions*

mitochondrial dysfunction in astrocytes [83].

aspects of ALS will therefore be discussed below.

astrocytes in the selective death of motoneurons in ALS.

*2.4.7. Activation of microglial cells*

#### *2.4.4. The Wnt/β-catenin signaling pathway*

The canonical Wnt/β-catenin transduction pathway, which comprises multiple Wnt genes, regulates many biological functions (reviewed in [68]), including neuronal survival, as demonstrated by its involvement in other neurodegenerative disease such as Alzheimer's disease and Parkinson's disease [69, 70]. In the ventral region of symptomatic *SOD1G93A* spinal cords, there is an increase in the number of Wnt3a- and β-catenin-positive astrocytes [71]. An upregulation of Wnt2 and Wnt7 within astrocytes of symptomatic *SOD1G93A* spinal cords is also reported [72]. Among its biological functions, the Wnt/β-catenin pathway mediates the activity of cyclin D1 [73], a nuclear transcription factor important for cell cycle regulation (reviewed in [74]). The upregulation of cyclin D1 in *SOD1G93A* astrocytes suggests that the increased activation of the Wnt/β-catenin/cyclin D1 may plausibly direct astrocytosis [71]. Interestingly, a study performed in colorectal cancer cell lines uncovers the possible regulation of COX-2 by the Wnt/β-catenin pathway [75]. Thus, an astrocytic increased activation of Wnt and β-catenin may not only impact cyclin D1 expression but potentially that of COX-2, for which a possible role in ALS neurodegeneration has been described above.

#### *2.4.5. Monoamine oxidase-B*

Monoamine oxidase-B (MAO-B) is an outer mitochondrial membrane-bound enzyme that catalyzes the oxidative deamination of biogenic amines, thus producing reactive oxygen species (ROS). MAO-B is primarily found in the CNS where it localizes mainly in astrocytes and radial glial [76]. The spinal cord lumbar region from symptomatic ALS patients displays more MAO-B, due to the general astrocyte proliferation and to a cell-intrinsic increased expression [77]. Using 3 H-L deprenyl *in vitro* autoradiography, a more in-depth follow-up study in the *post-mortem* ALS CNS reveals an increased expression in the corticospinal tract, the ventral white matter and in the vicinity of motoneurons. Further, reactive astrocytes displayed a higher content of MAO-B compared to microglial cells [8, 78]. Finally, an epide‐ miological analysis has uncovered that the MAO-B allelic phenotype influences the age of ALS onset [79]. Excessive astrocytic MAO-B expression, which results in elevations of extracellular ROS levels, may have damaging effects on neighboring motoneurons. Additional mechanisms could also involve mitochondrial dysfunction by the selective inhibition of respiratory complex I, which further leads to increased production of superoxide as well as microglial activation [80].

#### *2.4.6. Mitochondrial dysfunctions*

*2.4.3. Cyclooxygenase-2*

102 Current Advances in Amyotrophic Lateral Sclerosis

signals [67].

*2.4.4. The Wnt/β-catenin signaling pathway*

*2.4.5. Monoamine oxidase-B*

expression [77]. Using 3

COX-2 is a pro-inflammatory enzyme that converts arachidonic acid into prostanoids such as PGE2, a potent inflammatory mediator (reviewed in [60]). In the anterior horn region of the spinal cord of *SOD1G93A* mice, at both the early and end stage of the disease, COX-2 immunor‐ eactivity is elevated in astrocytes [61]. Similarly, spinal cord astrocytes from sporadic ALS patients also display increased COX-2 expression [61, 62]. The expression of COX-2 can be modulated by the binding of CD40, a member of the TNF family (reviewed in [63]), with its ligand CD40L [64]. Interestingly, spinal cord astrocytes of symptomatic *SOD1G39A* mice show an upregulation of CD40, concomitant with COX-2 astrocytic expression. Moreover, the activation of COX-2 in astrocytes upon CD40 stimulation leads to motoneuron death *in vitro* [65], suggesting that an astrocytic CD40-COX-2 pathway could also participate in ALS pathogenesis. The contribution of the CD40/CD40L pathway has recently been proposed in ALS mice, though its role in astrocytic neurotoxicity role has not been established [66]. Finally, another facet of the COX-2 pathway relates to the ability of PGE2 to promote glutamate release from astrocytes, emphasizing further the complex multimodality of neuroinflammatory

The canonical Wnt/β-catenin transduction pathway, which comprises multiple Wnt genes, regulates many biological functions (reviewed in [68]), including neuronal survival, as demonstrated by its involvement in other neurodegenerative disease such as Alzheimer's disease and Parkinson's disease [69, 70]. In the ventral region of symptomatic *SOD1G93A* spinal cords, there is an increase in the number of Wnt3a- and β-catenin-positive astrocytes [71]. An upregulation of Wnt2 and Wnt7 within astrocytes of symptomatic *SOD1G93A* spinal cords is also reported [72]. Among its biological functions, the Wnt/β-catenin pathway mediates the activity of cyclin D1 [73], a nuclear transcription factor important for cell cycle regulation (reviewed in [74]). The upregulation of cyclin D1 in *SOD1G93A* astrocytes suggests that the increased activation of the Wnt/β-catenin/cyclin D1 may plausibly direct astrocytosis [71]. Interestingly, a study performed in colorectal cancer cell lines uncovers the possible regulation of COX-2 by the Wnt/β-catenin pathway [75]. Thus, an astrocytic increased activation of Wnt and β-catenin may not only impact cyclin D1 expression but potentially that of COX-2, for

Monoamine oxidase-B (MAO-B) is an outer mitochondrial membrane-bound enzyme that catalyzes the oxidative deamination of biogenic amines, thus producing reactive oxygen species (ROS). MAO-B is primarily found in the CNS where it localizes mainly in astrocytes and radial glial [76]. The spinal cord lumbar region from symptomatic ALS patients displays more MAO-B, due to the general astrocyte proliferation and to a cell-intrinsic increased

study in the *post-mortem* ALS CNS reveals an increased expression in the corticospinal tract, the ventral white matter and in the vicinity of motoneurons. Further, reactive astrocytes

H-L deprenyl *in vitro* autoradiography, a more in-depth follow-up

which a possible role in ALS neurodegeneration has been described above.

While there is a vast amount of research on the mitochondrial dysfunction in ALS motoneurons (reviewed in [81]), not much is known about the impact of toxic genetic mutations on the mitochondria of astrocytes. There is evidence however, that ALS astrocytes do in fact display pathological mitochondrial dysfunction that subsequently leads to oxidative damage, sus‐ taining their reactive status. Indeed, primary astrocytes isolated from the cerebral cortex of neonatal rats and overexpressing *SOD1G93A* display a decreased mitochondrial respiration rate, an increased superoxide formation and a decreased membrane potential [82]. In co-culture experiments, modulating the mitochondrial defects of SOD1G93A-expressing astrocytes via small chemical compounds improves astrocytic-dependent motoneuron survival. Conversely, induction of mitochondrial damage to wildtype astrocytes increases motoneuron death [82]. Thus, organelle dysfunction within ALS astrocytes may be an important contributor to the neurodegenerative process. In addition, a positive amplification system could take place during the degenerative process, since the inflammatory mediator NO, mainly produced by the inducible form of nitric oxide synthase (iNOS) in reactive astrocytes, can in turn induce mitochondrial dysfunction in astrocytes [83].

#### *2.4.7. Activation of microglial cells*

A fundamental role for astrocytes in the neuroinflammation process is the recruitment of microglia [84], the resident macrophages of the CNS (reviewed in [85]). In *SOD1G37R* mice where the mutant *SOD1* gene is specifically deleted in astrocytes, the delay in the progression of the later stages of disease is accompanied with an inhibition of microglial activation and microgliadependent detrimental NO production [17]. Thus, astrocytosis in ALS may promote neuroin‐ flammation events through microglial recruitment, which in turn may participate directly or indirectly to the motoneuron loss in ALS. Several pro-inflammatory contributors including TNFα, IFNγ, IL-1β and NO, which are aberrantly produced by mutant astrocytes can indeed enhance the activation of microglia. The specific role of microglia in the neuroinflammatory aspects of ALS will therefore be discussed below.

Figure 1 illustrates the potential non-cell-autonomous mechanisms implicating reactive astrocytes in the selective death of motoneurons in ALS.

*SOD1G93A* mice via *in vivo* imaging by two-photon laser-scanning microscopy shows that microglia are highly reactive in pre-symptomatic stages while they lose their ability to respond to injury and to monitor the environment as the disease progresses [95]. Indeed, comparison of microglia populations during disease progression reveals that microglia isolated from either neonatal or early onset *SOD1G93A* mice display an alternatively activated M2 phenotype and enhance motoneuron survival while microglia isolated from either adult or endstage *SOD1G93A* mice have a classically activated M1 phenotype and induce motoneuron death [96, 97]. In the pre-symptomatic and symptomatic *SOD1G93A* rat model, microglia aggregates are detected in both the spinal cord and brainstem [98, 99]. Interestingly, the microglia in endstage *SOD1G93A* rats display a degenerative and apoptotic phenotype [98]. Further, in the lumbar spinal cord of pre-symptomatic *SOD1H46R* rats, the microglia express the proliferating marker Ki67 and the phagocytic markers ED1 and major histocompatibility complex (MHC) class II [100, 101]. The thorough investigation of microglial events in rodents therefore suggests that microgliosis not only typifies ALS but that the function of microglia changes during disease progression, thus

The Neuroinflammation in the Physiopathology of Amyotrophic Lateral Sclerosis

http://dx.doi.org/10.5772/56489

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Experimental endeavors have been undertaken to better understand the precise contribution of microglia in the neurodegenerative process. A key finding in support of the proposed direct contribution of microglia to ALS pathogenesis is in ALS mice where the mutant SOD1 (G37R or G85R) is specifically deleted from macrophages and microglial lineages [94, 102]. This results in a delay in the progression but not onset of the disease and a significant extension in lifespan. The importance of microgliosis in ALS pathology was also ascertained in *SOD1G93A* mice bred with *PU.1-/-* mice that lack CNS microglia at birth [103, 104]. While the bone marrow transplantation of *SOD1G93A* microglia into *PU.1-/-* mice did not induce neurodegeneration, the bone marrow transplantation of wildtype microglia into *SOD1G93A;PU.1-/-* mice improved survival compared to the bone marrow transplantation of *SOD1G93A* microglia [103]. Further, administration of extracellular murine SOD1G93A to primary cultures of microglia activates these cells and renders them neurotoxic [105]. However, phenotypical analysis of microglia in different regions of *SOD1G93A* spinal cord suggests that both neuroprotective and neurotoxic population of microglial cells may coexist during the disease [106]. In fact, the depletion of proliferative microglia does not prevent motoneuron degeneration [107]. Together, these studies suggest that microglia participate, through a complex balance between neuroprotec‐

While the injection of motoneuron-directed or ALS patient-derived immunoglobulin G into the spinal cord of mice initiates the recruitment of reactive microglia [108], a study looking at cerebral cortex of ALS patients shows that the phagocytosis of degenerating neurons is mediated by perivascular macrophages and not microglia [109]. This finding already suggest‐ ed that reactive microglia might play a more complex function in ALS than simply eliminating

exerting differential effects on the degenerating motoneurons.

tive and neurotoxic signals, in the course of the disease.

**3.3. Proposed mechanisms of microglial-derived neurotoxicity**

**3.2. A role for microglia in ALS pathogenesis**

**Figure 1.** Proposed mechanisms for astrocytic-mediated neuroinflammation and toxicity towards motoneurons. Reac‐ tive astrocytes contribute to the degenerative process by influencing the activity of microglial and immune cells as well as by releasing soluble factors that are toxic to motoneurons (as described in section 2).
