**3. A role for microglia in neuroinflammation**

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

Microglia are often termed the immune cells of the CNS as they constantly monitor the neuronal environment in a resting state and become activated upon acute or chronic neuronal damage, eliciting a strong pro-inflammatory response (reviewed in [86]). In ALS patients, reactive microglia are observed in the motor cortex, the motor nuclei of the brainstem, the ventral horn of the spinal cord, along the entire corticospinal tract and within the CSF [87-89]. Given the relationship between astrocytes and microglia [17, 84] and the importance of astrocytosis in ALS, it has been hypothesized that microgliosis may also participate in ALS pathogenesis.

To better understand at which developmental point of the disease reactive microglia appear, microgliosis has been characterized in rodent ALS models at various stages of the disease. Microgliosis occurs in pre-symptomatic and symptomatic *SOD1G93A* spinal cords as well as within various CNS compartments [90-93]. Similarly, *SOD1G37R* mice display microgliosis at both onset and early-stage of the disease [94]. An in-depth characterization of microgliosis in *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 exerting differential effects on the degenerating motoneurons.

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

**reactive astrocytes**

nucleus

increased Ca2+ influx

Wnt β−catenin

cyclin D1

FGFR1

GluR2 decrease

MAO-B

ROS

EAAT2/GLT-1 loss-of-function

glutamate accumulation

> RNA editing

NO

PGE2 LTB4

**3. A role for microglia in neuroinflammation**

**3.1. Activation profile in human and animal models of ALS**

CD40

well as by releasing soluble factors that are toxic to motoneurons (as described in section 2).

CD40L

COX-2 5-LOX

**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

Microglia are often termed the immune cells of the CNS as they constantly monitor the neuronal environment in a resting state and become activated upon acute or chronic neuronal damage, eliciting a strong pro-inflammatory response (reviewed in [86]). In ALS patients, reactive microglia are observed in the motor cortex, the motor nuclei of the brainstem, the ventral horn of the spinal cord, along the entire corticospinal tract and within the CSF [87-89]. Given the relationship between astrocytes and microglia [17, 84] and the importance of astrocytosis in ALS, it has been hypothesized that microgliosis may also participate in ALS

To better understand at which developmental point of the disease reactive microglia appear, microgliosis has been characterized in rodent ALS models at various stages of the disease. Microgliosis occurs in pre-symptomatic and symptomatic *SOD1G93A* spinal cords as well as within various CNS compartments [90-93]. Similarly, *SOD1G37R* mice display microgliosis at both onset and early-stage of the disease [94]. An in-depth characterization of microgliosis in

iNOS

motoneuron

death

NGF

p75NTR

IL-1β

TNFα

IFNγ

LIGHT

LT-βR

activation

104 Current Advances in Amyotrophic Lateral Sclerosis

microglia

recruitment activation

pathogenesis.

immune cells

O2 . -

oxidative damage

mitochondrial dysfunction

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

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

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 dying motoneurons. Indeed, various misregulated pathways within ALS microglia have been identified that may influence motoneuron survival.

mice display an upregulation of P2X4, P2X7 and P2Y6 receptors [125]. Notably, the immunor‐ eactivity of P2X is increased within spinal cord microglia of ALS patients [126]. Activation of P2X7 in *SOD1G93A* microglial cells produces significantly higher levels of TNFα, which has a neurotoxic effect on motoneuron cultures [127], and of COX-2, compared to non-mutant microglia [125]. In addition, a reduced ATP hydrolysis activity, possibly implicating the ecto-NTPDase CD39, is observed in mutant SOD1 microglia, suggesting that a potentiation of a purinergic-mediated inflammation can participate to the neuroinflammatory state of micro‐ glial cells. Since ATP induces an astrocytic neurotoxic phenotype through P2X7 [128], it is thus feasible to hypothesize that increased extracellular ATP in ALS, whether exacerbated by

The Neuroinflammation in the Physiopathology of Amyotrophic Lateral Sclerosis

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107

To our knowledge, there is presently no direct assessment of the influence of microglia on motoneuron electrophysiology. However, studies on peripheral nerve injury or spinal cord injury show that microglia activation has prominent effects on neuronal inhibitory control. Importantly, loss of inhibitory control is a contributing mechanism to the motoneuron

Loss of neuronal inhibitory control occurs by several means including decrease in gammaaminobutyric acid (GABA)ergic interneurons [130] combined with changes in the expression of the GABAA receptor mRNA subunit [131]. GABAA and glycine receptors are chloride (Cl-

channels and the expression of cation-chloride co-transporter contributes to inhibitory effects

spinal microglia following peripheral nerve injury induces a decrease in KCC2 expression among dorsal horn nociceptive neurons [134]. KCC2 decrease is induced by the brain-derived neurotrophic factor (BDNF) and this is consistent with the previous observation that BDNF can be produced by non-neuronal cells involved in immune responses, including T and B lymphocytes, monocytes and microglia [135, 136]. BDNF produces a depolarizing shift in the

prompts an inversion of inhibitory GABA currents that contributes to neuropathic pain following nerve injury [135]. Decrease in KCC2 expression is thus responsible for the excitatory effects of GABA on neurons. Microglia activation and BDNF secretion are mediated through ATP activation of microglial P2X receptors. As described earlier, P2X receptors might be involved in ALS pathology since a higher density of P2X7-immunoreactive microglial cells/ macrophages are found in affected regions of spinal cords from ALS patients [126]. Levels of BDNF have been found to be increased in microglial cells isolated from ALS mice at the onset of disease and KCC2 is decreased in vulnerable motoneurons in *SOD1G93A* mice [96, 137]. Additionally, BDNF might play a role in the microglia's influence on motoneuron electric activity as suggested by work on spasticity. Spasticity is characterized by a velocity-dependent increase in muscle tone resulting from hyperexcitable stretch reflexes, spasms and hypersen‐

anion reversal potential of dorsal horn lamina I neurons due to an increase in [Cl-

channels inhibits neuron excitability by hyperpolarizing membrane

]i

)

)-

following the opening of GABAA and glycine

from mature neurons [133]. Stimulation of

is maintained by the potassium (K+

]i

. This shift

motoneurons and/or microglia contributes to the pathogenic microgliosis.

**3.4. The potential influence of microglia on neuronal excitability**

hyperexcitability that typifies ALS pathogenesis in humans [129].

currents [132]. Indeed, the entry of Cl-

potential. Under physiological condition, low [Cl-

chloride co-transporter KCC2 that extrudes Cl-

of these Cl-

receptor-gated Cl-

#### *3.3.1. Endoplasmic reticulum stress*

When a cell starts to excessively accumulate misfolded or unfolded proteins, the over-activated endoplasmic reticulum (ER) stress induces apoptosis (reviewed in [110]). Importantly, ER stress is an established characteristic of ALS pathogenesis (reviewed in [111]). In spinal cord microglia of both sporadic ALS patients and symptomatic *SOD1G93A* mice, there is an increased expression of C/EBP homologous protein (CHOP) [112], a member of the apoptotic ER stress pathway (reviewed in [113]). It remains unclear however if the aberrant levels of CHOP reflect an upstream defect in protein folding or if they directly participate in microglial neurotoxicity. It is noteworthy that the exposure of microglial cells to IFNγ induces iNOS expression, and the subsequent increased NO production can cause an ER stress response involving CHOP [114]. Interestingly, the analysis of selectively vulnerable motoneurons from low-expression *SOD1G93A*, high-expression *SOD1G93A* and *SOD1G85R* mice shows the initiation of a specific ER stress response accompanied by microglial activation [115]. Thus, the interaction between ALS motoneurons and microglia may be important in the modulation of the neurodegenerative process.

#### *3.3.2. CD14-toll-like receptor signaling*

Once the ligand-dependent CD14 lipopolysaccharide (LPS) receptor located at the microglial surface [116] is activated, it initiates a pro-inflammatory signaling cascade dependent on Tolllike receptors (TLRs), specifically TLR2 and TLR4 [117, 118]. Interestingly, the neurotoxic activation of microglia by extracellular *SOD1G93A* is mediated by the CD14-TLR pathway [105, 119]. Indeed, immortalized microglia cells expressing mutant SOD1 display an increased TLR2 stimulation and subsequent release of pro-inflammatory cytokines, including TNFα and IL-1β. Importantly, an analysis of spinal cord microglia from sporadic ALS patients shows an enhanced TLR2 immunoreactivity [120]. Recently, it has been shown that the endocytosis of extracellular mutant SOD1 by microglia is required for the activation of caspase-1, which is required for the maturation of IL-1β [121]. This can be paralleled with the finding that the microgliosis caused by fibrillar amyloid beta (Aβ), the main component of the aggregates that are a pathological signature of Alzheimer's disease, also requires CD14, TLR2 and TLR4 [122]. All together, these studies suggest that microglia may participate in motoneuron loss following the specific activation of the CD14-TLR pathway by secreted SOD1 mutant, therefore propa‐ gating pro-inflammatory stimuli.

#### *3.3.3. Purinergic signaling*

The release of extracellular nucleoside di- and tri-phosphates by degenerating neurons can elicit the activation of microglia through the ionotropic P2X and metabotropic P2Y purinergic receptors. A general alarm signal for microglia is ATP, which can subsequently elicit a proinflammatory response, chemotaxis and phagocytosis (reviewed in [123, 124]). Embryonic immortalized microglia and neonatal primary microglial cultures isolated from mutant *SOD1* mice display an upregulation of P2X4, P2X7 and P2Y6 receptors [125]. Notably, the immunor‐ eactivity of P2X is increased within spinal cord microglia of ALS patients [126]. Activation of P2X7 in *SOD1G93A* microglial cells produces significantly higher levels of TNFα, which has a neurotoxic effect on motoneuron cultures [127], and of COX-2, compared to non-mutant microglia [125]. In addition, a reduced ATP hydrolysis activity, possibly implicating the ecto-NTPDase CD39, is observed in mutant SOD1 microglia, suggesting that a potentiation of a purinergic-mediated inflammation can participate to the neuroinflammatory state of micro‐ glial cells. Since ATP induces an astrocytic neurotoxic phenotype through P2X7 [128], it is thus feasible to hypothesize that increased extracellular ATP in ALS, whether exacerbated by motoneurons and/or microglia contributes to the pathogenic microgliosis.

#### **3.4. The potential influence of microglia on neuronal excitability**

dying motoneurons. Indeed, various misregulated pathways within ALS microglia have been

When a cell starts to excessively accumulate misfolded or unfolded proteins, the over-activated endoplasmic reticulum (ER) stress induces apoptosis (reviewed in [110]). Importantly, ER stress is an established characteristic of ALS pathogenesis (reviewed in [111]). In spinal cord microglia of both sporadic ALS patients and symptomatic *SOD1G93A* mice, there is an increased expression of C/EBP homologous protein (CHOP) [112], a member of the apoptotic ER stress pathway (reviewed in [113]). It remains unclear however if the aberrant levels of CHOP reflect an upstream defect in protein folding or if they directly participate in microglial neurotoxicity. It is noteworthy that the exposure of microglial cells to IFNγ induces iNOS expression, and the subsequent increased NO production can cause an ER stress response involving CHOP [114]. Interestingly, the analysis of selectively vulnerable motoneurons from low-expression *SOD1G93A*, high-expression *SOD1G93A* and *SOD1G85R* mice shows the initiation of a specific ER stress response accompanied by microglial activation [115]. Thus, the interaction between ALS motoneurons and microglia may be important in the modulation of the neurodegenerative

Once the ligand-dependent CD14 lipopolysaccharide (LPS) receptor located at the microglial surface [116] is activated, it initiates a pro-inflammatory signaling cascade dependent on Tolllike receptors (TLRs), specifically TLR2 and TLR4 [117, 118]. Interestingly, the neurotoxic activation of microglia by extracellular *SOD1G93A* is mediated by the CD14-TLR pathway [105, 119]. Indeed, immortalized microglia cells expressing mutant SOD1 display an increased TLR2 stimulation and subsequent release of pro-inflammatory cytokines, including TNFα and IL-1β. Importantly, an analysis of spinal cord microglia from sporadic ALS patients shows an enhanced TLR2 immunoreactivity [120]. Recently, it has been shown that the endocytosis of extracellular mutant SOD1 by microglia is required for the activation of caspase-1, which is required for the maturation of IL-1β [121]. This can be paralleled with the finding that the microgliosis caused by fibrillar amyloid beta (Aβ), the main component of the aggregates that are a pathological signature of Alzheimer's disease, also requires CD14, TLR2 and TLR4 [122]. All together, these studies suggest that microglia may participate in motoneuron loss following the specific activation of the CD14-TLR pathway by secreted SOD1 mutant, therefore propa‐

The release of extracellular nucleoside di- and tri-phosphates by degenerating neurons can elicit the activation of microglia through the ionotropic P2X and metabotropic P2Y purinergic receptors. A general alarm signal for microglia is ATP, which can subsequently elicit a proinflammatory response, chemotaxis and phagocytosis (reviewed in [123, 124]). Embryonic immortalized microglia and neonatal primary microglial cultures isolated from mutant *SOD1*

identified that may influence motoneuron survival.

*3.3.1. Endoplasmic reticulum stress*

106 Current Advances in Amyotrophic Lateral Sclerosis

*3.3.2. CD14-toll-like receptor signaling*

gating pro-inflammatory stimuli.

*3.3.3. Purinergic signaling*

process.

To our knowledge, there is presently no direct assessment of the influence of microglia on motoneuron electrophysiology. However, studies on peripheral nerve injury or spinal cord injury show that microglia activation has prominent effects on neuronal inhibitory control. Importantly, loss of inhibitory control is a contributing mechanism to the motoneuron hyperexcitability that typifies ALS pathogenesis in humans [129].

Loss of neuronal inhibitory control occurs by several means including decrease in gammaaminobutyric acid (GABA)ergic interneurons [130] combined with changes in the expression of the GABAA receptor mRNA subunit [131]. GABAA and glycine receptors are chloride (Cl- ) channels and the expression of cation-chloride co-transporter contributes to inhibitory effects of these Cl currents [132]. Indeed, the entry of Cl following the opening of GABAA and glycine receptor-gated Cl channels inhibits neuron excitability by hyperpolarizing membrane potential. Under physiological condition, low [Cl- ]i is maintained by the potassium (K+ ) chloride co-transporter KCC2 that extrudes Cl from mature neurons [133]. Stimulation of spinal microglia following peripheral nerve injury induces a decrease in KCC2 expression among dorsal horn nociceptive neurons [134]. KCC2 decrease is induced by the brain-derived neurotrophic factor (BDNF) and this is consistent with the previous observation that BDNF can be produced by non-neuronal cells involved in immune responses, including T and B lymphocytes, monocytes and microglia [135, 136]. BDNF produces a depolarizing shift in the anion reversal potential of dorsal horn lamina I neurons due to an increase in [Cl- ]i . This shift prompts an inversion of inhibitory GABA currents that contributes to neuropathic pain following nerve injury [135]. Decrease in KCC2 expression is thus responsible for the excitatory effects of GABA on neurons. Microglia activation and BDNF secretion are mediated through ATP activation of microglial P2X receptors. As described earlier, P2X receptors might be involved in ALS pathology since a higher density of P2X7-immunoreactive microglial cells/ macrophages are found in affected regions of spinal cords from ALS patients [126]. Levels of BDNF have been found to be increased in microglial cells isolated from ALS mice at the onset of disease and KCC2 is decreased in vulnerable motoneurons in *SOD1G93A* mice [96, 137]. Additionally, BDNF might play a role in the microglia's influence on motoneuron electric activity as suggested by work on spasticity. Spasticity is characterized by a velocity-dependent increase in muscle tone resulting from hyperexcitable stretch reflexes, spasms and hypersen‐ sitivity to normally innocuous sensory stimulations. Spasticity develops following spinal cord injury and is also regarded as an ALS clinical symptom [138]. The main mechanisms hypothe‐ sized to be responsible for spasticity are increased motoneuron excitability and increased synaptic inputs in response to muscle stretch due to reduced inhibitory mechanisms. Recently, it has been demonstrated that, following spinal cord injury, increased levels of BDNF mediated spasticity, due to post-transcriptional down regulation of KCC2 [139]. Together, these studies suggest that reactive microglia in ALS may exert an aberrant effect on the electrical activity of motoneurons and highlight the importance of furthering our understanding of this functional interaction.

Lastly, a hypothetical scenario relates to the defect in astrocytic glutamate transporter and the neurotoxic accumulation of the excitatory amino acid that we have mentioned above. It has been demonstrated that TNFα promotes the release of glutamate by activated microglia through the cystine/glutamate exchanger (Xc)[140]. Though the implication of the Xc system in ALS has not yet been investigated, it is intriguing that the Aβ peptide induces a neurotoxic phenotype in microglia through the Xc-mediated release of glutamate Therefore, system Xc represents a potential mechanism of microglia-mediated excitotoxicity that warrants further study [141].

The potential non-cell-autonomous mechanisms involving microglial cells in the selective degeneration of motoneurons in ALS are illustrated in Figure 2.

> attenuate toxic microglial responses. Contribution of the innate immune system is also suggested by the presence of immunoglobulins and complement deposition as well as a significant increase of NK cells in the blood of ALS patients [87, 144, 152]. While these investigations of ALS samples and tissues do not assess the contributory role of the immune

> **Figure 2.** Proposed mechanisms by which microglial activation and inflammation contribute to the neurodegenera‐ tive process in ALS. Microglia can influence astrocytes and immune cells as well as directly impact motoneuron viability

proIL-1β

extracellular mutated SOD1

TLR2/4 CD14

COX-2

system Xc

activation

glutamate

excitotoxicity

increased Ca2+ influx

The Neuroinflammation in the Physiopathology of Amyotrophic Lateral Sclerosis

cystine

**activated microglia**

caspase-1

ER stress

oxidative damage

TNFR

TNFα

IL-1β prostanoids

immune cells

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109

motoneuron

BDNF

iNOS NO

IFNR

IFNγ

KCC2 downregulation

Cl-

P2X7 P2X4 P2Y6

GABAAR GlyR

reduced ATP hydrolysis

astrocytes motoneurons lymphocytes

P2X7 ATP

neurotoxicity astrocyte

via several mechanisms.

In support of what is observed in humans, ALS rodent models also display a particular immunological phenotype. Indeed, *SOD1G93A* mice demonstrate that the inflammatory cell subtypes are phenotypicaly and functionally different depending upon the disease stage [96]. During the initial stages, infiltrating CD4+ T cells are almost mainly Th2 (IL-4+) while as the disease progresses there is a skew toward Th1 (IFNγ+) cells and CD8+ T cells (both IL-17A positive and negative)[106, 153]. Alteration in inflammatory cell subtypes is associated with, and maybe driven by, differences in Tregs. Interestingly, early symptomat‐ ic *SOD1G93A* mice have increased numbers of Tregs and a decreased proliferation of effectors T lymphocytes (Teffs), whereas decreased numbers of Tregs and increased proliferation of Teffs is found in end-stage animals [151, 154]. The innate immune system is also affected in ALS rodents, displayed by the substantial increase of NK and NKT in the spinal cord

system to the disease pathogenesis, they do highlight its active presence.

of *SOD1G93A* mice [155, 156].
