**5. Pre-clinical therapies targeting neuroinflammation**

#### **5.1. Pharmacological targeting of the neuroinflammatory response**

In light of the salient evidence supporting the contribution of neuroinflammation in ALS, several drug- or cell-based therapeutic approaches have been evaluated in ALS mice for their ability to modulate the pathologic process. Those that have shown a positive effect on astrocytosis and microgliosis are described below and have been categorized based on their desired functional target.

reduces astrocytosis [185]. Importantly, celecoxib-treated *SOD1G93A* spinal cords display reduced levels of PGE2, a potent pro-inflammatory mediator as well as a signal for glutamate

**Figure 3.** Potential Mechanisms by which peripheral and central immunity might contribute to the neurodegenerative process in ALS. Both neuroprotective and neurotoxic functions can be proposed for the involvement of lymphocytes in

TNFα

IL-17

Tc17 Th17

IL-1β IL-6

BDNF GDNF

Treg

TGF-β IL-10 IL-4

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neurotoxic microglia (M1) Nox2+

LPS

extracellular mutant SOD1

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motoneuron

neuroprotection

CTL Th1

Fas

IFNγ perforin

IL-17 IL-22

NK cells

cytotoxicity

autoantibodies (Ca2+ channels,

complement activation

Fas…)

FasL

**+**

**+**

Lenalidomide, an immunomodulatory drug with pleiotropic properties derived from thali‐ domide, has been evaluated in mutant SOD1 mice due to its inhibitory effect on TNFα production by monocytes [186]. A lenalidomide-diet given to *SOD1G93A* mice retards disease onset, ameliorates motoneuron survival and extends survival by 18%. A significant decrease in IL-1α and TNFα as well as an increase in IL-1 receptor antagonist (IL-1RA) and TGF-β1 is observed in the spinal cord of Lenalidomide-treated mice [187]. In a similar study, the lifespan of ALS mice treated with lenalidomide at onset of symptoms is increased by 12%. Concomi‐ tantly, there is an improved survival of motoneurons, decreased levels of the pro-inflammatory cytokines TNFα and Fas associated Factor as well as an increased expression of the anti-

Epigallocathecin gallate (EGCG) is a green tea polyphenol that can prevent microglial neurotoxicity through the modulation of TNFα mRNA transcription and release as well as iNOS production [189]. The daily oral administration of EGCG to *SOD1G93A* mice daily from asymptomatic to endstage delays disease onset and lifespan by approximately 10 and 14%,

release from astrocytes [67].

ALS pathogenesis (as described in section 3).

inflammatory cytokines TGF-β3 and IL-1RA [188].

In order to mitigate the detrimental effects of the overactive p75NTR pathway in ALS, an antagonist that mimics the short NGF β loop region that binds the p75NTR has been utilized [183]. Unfortunately, the intraperitoneal (i.p.) delivery of the p75NTR antagonist from asymp‐ tomatic stage up until the endpoint of the disease does not improve the phenotype or survival of *SOD1G93A* mice [183]. However, antisense peptide nucleic acid-based silencing of p75NTR following early systemic i.p. administration delays by about 10% both onset and progression of the disease [184]. Although, alternative route of administration or development of more efficient molecules should be assessed, p75NTR represents a therapeutic target that needs to be further explored.

COX-2 appears as an appealing therapeutic target for ALS as it promotes both pro-inflamma‐ tory events and astrocytic glutamate release [60, 67]. Celecoxib, a COX-2 inhibitor, fed to *SOD1G93A* mice from asymptomatic to end-stage results in a delayed onset and an increased lifespan of approximately 25%. Celecoxib treatment prevents loss of spinal motoneurons and

directly trigger motoneuron death through the LIGHT/LT-βR pathway or potentiate a cytotoxic Th1/CTL response via the combined action of other NK-related cytokines such as IL-17 or IL-22 [173]. Of note, NK cells also produce IL-4 upon activation, which as described earlier, mediates a neuroprotective effect. Therefore, NK cells represent an appealing branch

In addition to the adaptive immune system, several studies suggest that humoral immunity and immunoglobulins could also contribute to the disease. Autoantibodies to voltage-gated Ca2+ or K+ channels have been described in ALS patients, which induce specific motoneuron alterations both *in vitro* and *in vivo* after passive transfer in mice [174-178]. Accordingly, C5a and other complement activation products released after activation of the classical comple‐ ment pathway by antibodies are elevated in the CSF and spinal cord of ALS mice and patients and specific inhibition of C5a receptor ameliorates disease in *SOD1G93A* mice [179, 180]. Additionally, abnormal levels of anti-Fas antibodies, able to induce neuronal apoptosis *in vitro*, have been detected in the serum of patients with ALS [181, 182]. Thus, both the innate and adaptive immune system appear to have deleterious consequences on the survival and

Figure 3 illustrates the potential mechanisms implicating different populations of immune cells

In light of the salient evidence supporting the contribution of neuroinflammation in ALS, several drug- or cell-based therapeutic approaches have been evaluated in ALS mice for their ability to modulate the pathologic process. Those that have shown a positive effect on astrocytosis and microgliosis are described below and have been categorized based on their

In order to mitigate the detrimental effects of the overactive p75NTR pathway in ALS, an antagonist that mimics the short NGF β loop region that binds the p75NTR has been utilized [183]. Unfortunately, the intraperitoneal (i.p.) delivery of the p75NTR antagonist from asymp‐ tomatic stage up until the endpoint of the disease does not improve the phenotype or survival of *SOD1G93A* mice [183]. However, antisense peptide nucleic acid-based silencing of p75NTR following early systemic i.p. administration delays by about 10% both onset and progression of the disease [184]. Although, alternative route of administration or development of more efficient molecules should be assessed, p75NTR represents a therapeutic target that needs to be

COX-2 appears as an appealing therapeutic target for ALS as it promotes both pro-inflamma‐ tory events and astrocytic glutamate release [60, 67]. Celecoxib, a COX-2 inhibitor, fed to *SOD1G93A* mice from asymptomatic to end-stage results in a delayed onset and an increased lifespan of approximately 25%. Celecoxib treatment prevents loss of spinal motoneurons and

**5. Pre-clinical therapies targeting neuroinflammation**

**5.1. Pharmacological targeting of the neuroinflammatory response**

of the immunopathology that could be considered as a therapeutic target for ALS.

maintenance of motoneurons in ALS.

112 Current Advances in Amyotrophic Lateral Sclerosis

in ALS pathogenesis.

desired functional target.

further explored.

**Figure 3.** Potential Mechanisms by which peripheral and central immunity might contribute to the neurodegenerative process in ALS. Both neuroprotective and neurotoxic functions can be proposed for the involvement of lymphocytes in ALS pathogenesis (as described in section 3).

reduces astrocytosis [185]. Importantly, celecoxib-treated *SOD1G93A* spinal cords display reduced levels of PGE2, a potent pro-inflammatory mediator as well as a signal for glutamate release from astrocytes [67].

Lenalidomide, an immunomodulatory drug with pleiotropic properties derived from thali‐ domide, has been evaluated in mutant SOD1 mice due to its inhibitory effect on TNFα production by monocytes [186]. A lenalidomide-diet given to *SOD1G93A* mice retards disease onset, ameliorates motoneuron survival and extends survival by 18%. A significant decrease in IL-1α and TNFα as well as an increase in IL-1 receptor antagonist (IL-1RA) and TGF-β1 is observed in the spinal cord of Lenalidomide-treated mice [187]. In a similar study, the lifespan of ALS mice treated with lenalidomide at onset of symptoms is increased by 12%. Concomi‐ tantly, there is an improved survival of motoneurons, decreased levels of the pro-inflammatory cytokines TNFα and Fas associated Factor as well as an increased expression of the antiinflammatory cytokines TGF-β3 and IL-1RA [188].

Epigallocathecin gallate (EGCG) is a green tea polyphenol that can prevent microglial neurotoxicity through the modulation of TNFα mRNA transcription and release as well as iNOS production [189]. The daily oral administration of EGCG to *SOD1G93A* mice daily from asymptomatic to endstage delays disease onset and lifespan by approximately 10 and 14%, respectively. EGCG also moderately mitigates motoneuron loss and reduces microglia activation [190].

Another potential protein therapy is the administration of the activated protein C (APC), a plasma protease with anti-coagulant, neuroprotective and anti-inflammatory functions (reviewed in [206]). A daily i.p. injection of APC to symptomatic *SOD1G93A* mice until death slows disease progression, leading to a 25% increase in lifespan [207]. Further, APC appears to exert its beneficial effects via a downregulation of mutant SOD1 expression in both moto‐

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Anakinra (Kineret), a recombinant form of human IL-1RA, that inhibits the pro-inflammatory activity of both IL-1α and IL-1β, is approved by the U.S food and drug administration for rheumatoid arthritis [208]. When administrated by i.p. daily to asymptomatic stage to *SOD1G93A* mice, it ameliorates motor function and prolongs lifespan by approximately 4% [121].

The CD40 costimulatory pathway, which plays an important role in B and T cell activation [209], has been proposed to contribute to ALS pathogenesis. The weekly delivery of a blocking anti-CD40L antibody by i.p. injection starting at an asymptomatic stage delays onset and prolongs survival by approximately 7%. Consistently, anti-CD40L delivery reduces signifi‐ cantly the percentage of peripheral CD8+ T cells as well as GFAP+ astrocytes and Mac2+ microglia in the spinal cord [66], suggesting that the CD40 pathway, an integral component of

While drugs and protein therapy target the misregulated pathways within astrocytes and microglia, the aim of cell therapy is to replace these aberrantly functional cells by healthy ones or use implanted cells as a therapeutic platform to deliver neurotrophic support, thus hope‐

Glial cell therapy has indeed been evaluated by isolating human neural progenitor cells (hNPCs) and genetically modifying them to express GDNF [210, 211]. Prior to direct injection in the spinal cord of *SOD1G93A* rats, hNPCs were pre-differentiated into astrocytes [210]. Despite the fact that the hNPC injection did not increase the lifespan of *SOD1G93A* rats, they do localize within both the grey and white matter of the spinal cord and survive until the death of the animal. Further investigation of this method reveals that hNPCs preserve dying motoneuron cell bodies in *SOD1G93A* rats without improving their innervations at the neuromuscular junction [212]. In *SOD1G93A* mice, GDNF-expressing hNPCs also migrate to the spinal cord where a subset of them differentiates into astrocytes, again without improving survival or neurodegeneration [213]. However, the lack of beneficial outcome is most likely due to the regional specificity of GDNF's biological activity, as intramuscular but not intraspinal delivery of GDNF exerts its neuroprotective effect [214, 215]. Another astrocyte precursor with potential benefits is the glial-restricted progenitors (GRPs), isolated from the spinal cord of embryonic rats [216]. The transplantation of GRPs in the ventral horn of *SOD1G93A* rats shows that these cells differentiate into astrocytes and can survive and migrate along the spinal cord [217]. Importantly, GRP-recipient ALS rats survive longer as well as show a slower cervical neuro‐

neurons and microglia, thus resulting in delayed neuroinflammatory events [207].

neuroimmunity, is a potential therapeutic target in ALS.

**5.3. Cell therapy perspectives**

*5.3.1. Glial precursor cells*

fully alleviating neuroinflammation in ALS.

Pioglitazone is a drug that was initially developed to treat type II diabetes patients that also exerts ant-inflammatory and neuroprotective activities (reviewed in [191]). For these reasons, it has been hypothesized that it may improve ALS pathology. Indeed, pioglitazone-fed *SOD1G93A* mice have a delayed onset of 10% and a prolonged lifespan of about 8% [192]. Pioglitazone significantly reduces microgliosis and astrocytosis in *SOD1G93A* mice as well as alters the expression profile of spinal cord lysates from pro-inflammatory to anti-inflammatory [192, 193]. Further analysis of spinal cords reveals that pioglitazone may act through the inhibition of the p38 kinase, NF-κB and STAT3 pathways [167, 193, 194].

Olesoxime has previously been selected as a neuroprotective agent via a motoneuron survivalbased screen [195]. Interestingly, *SOD1G93A* mice fed an olesoxime diet from asymptomatic stage to end-stage survive 10% longer than non-treated mice and also demonstrate a reduction in both astrocytosis and microgliosis [195, 196].

Dicatechol nordihydroguaiaretic acid (NDGA) is a selective inhibitor of 5-LOX that presents TNFα antagonizing activity in microglial cells. *SOD1G93A* mice on an NDGA-diet from pre‐ symptomatic stage to end-point have a 32% increase in median lifespan as well as a reduced motoneuron loss and astrocytosis [197].

Minocycline is a member of the tetracycline molecules that can enter the CNS and mediates inflammation and microgliosis (reviewed in [198]). Asymptomatic *SOD1G93A* mice that received daily minocycline by i.p. injection have a delayed disease onset, a 16% increase in lifespan as well as a preservation of spinal motoneurons [199]. Similarly, minocycline-fed late presymptomatic *SOD1G37R* mice display a 6% longer survival, an increased number of spinal cord motoneurons and a reduced microgliosis [200]. However, a minocycline diet in symptomatic *SOD1G93A* has no effect on survival while amplifying both astrocytosis and microgliosis [201]. These results strikingly illustrate the time-dependent dynamics of the neuroinflammation response, highlighting not only the requirement to target the most pertinent therapeutic molecular and cellular effectors but to also do so at the proper stage of the disease.

#### **5.2. Advances and possible applications of protein therapy**

In addition to chemical compounds, the therapeutic delivery of proteins has also been assessed as a potential modulator of neuroinflammation in ALS. Indeed, the granulocyte-colony stimulating factor (G-CSF), a hematopoietic growth factor, has been delivered to *SOD1G93A* mice by osmotic pump starting at asymptomatic stage for a continuance of 8 weeks [202]. G-CSFrecipient ALS mice display a delay in disease onset as well as an increased motoneuron survival. The time to clinical endstage is increased by 10% in *SOD1G93A* mice receiving G-CSF. Importantly, while G-CSF was initially used for its neuroprotective effects and its ability to readily cross the blood-brain barrier [203, 204], further characterization of treated *SOD1G93A* mice shows a reduced spinal cord astrocytosis and microgliosis as well as an increased availability of migratory healing monocytes, suggesting that G-CSF may be beneficial in ALS via its modulation of neuroinflammation [205].

Another potential protein therapy is the administration of the activated protein C (APC), a plasma protease with anti-coagulant, neuroprotective and anti-inflammatory functions (reviewed in [206]). A daily i.p. injection of APC to symptomatic *SOD1G93A* mice until death slows disease progression, leading to a 25% increase in lifespan [207]. Further, APC appears to exert its beneficial effects via a downregulation of mutant SOD1 expression in both moto‐ neurons and microglia, thus resulting in delayed neuroinflammatory events [207].

Anakinra (Kineret), a recombinant form of human IL-1RA, that inhibits the pro-inflammatory activity of both IL-1α and IL-1β, is approved by the U.S food and drug administration for rheumatoid arthritis [208]. When administrated by i.p. daily to asymptomatic stage to *SOD1G93A* mice, it ameliorates motor function and prolongs lifespan by approximately 4% [121].

The CD40 costimulatory pathway, which plays an important role in B and T cell activation [209], has been proposed to contribute to ALS pathogenesis. The weekly delivery of a blocking anti-CD40L antibody by i.p. injection starting at an asymptomatic stage delays onset and prolongs survival by approximately 7%. Consistently, anti-CD40L delivery reduces signifi‐ cantly the percentage of peripheral CD8+ T cells as well as GFAP+ astrocytes and Mac2+ microglia in the spinal cord [66], suggesting that the CD40 pathway, an integral component of neuroimmunity, is a potential therapeutic target in ALS.

### **5.3. Cell therapy perspectives**

respectively. EGCG also moderately mitigates motoneuron loss and reduces microglia

Pioglitazone is a drug that was initially developed to treat type II diabetes patients that also exerts ant-inflammatory and neuroprotective activities (reviewed in [191]). For these reasons, it has been hypothesized that it may improve ALS pathology. Indeed, pioglitazone-fed *SOD1G93A* mice have a delayed onset of 10% and a prolonged lifespan of about 8% [192]. Pioglitazone significantly reduces microgliosis and astrocytosis in *SOD1G93A* mice as well as alters the expression profile of spinal cord lysates from pro-inflammatory to anti-inflammatory [192, 193]. Further analysis of spinal cords reveals that pioglitazone may act through the

Olesoxime has previously been selected as a neuroprotective agent via a motoneuron survivalbased screen [195]. Interestingly, *SOD1G93A* mice fed an olesoxime diet from asymptomatic stage to end-stage survive 10% longer than non-treated mice and also demonstrate a reduction

Dicatechol nordihydroguaiaretic acid (NDGA) is a selective inhibitor of 5-LOX that presents TNFα antagonizing activity in microglial cells. *SOD1G93A* mice on an NDGA-diet from pre‐ symptomatic stage to end-point have a 32% increase in median lifespan as well as a reduced

Minocycline is a member of the tetracycline molecules that can enter the CNS and mediates inflammation and microgliosis (reviewed in [198]). Asymptomatic *SOD1G93A* mice that received daily minocycline by i.p. injection have a delayed disease onset, a 16% increase in lifespan as well as a preservation of spinal motoneurons [199]. Similarly, minocycline-fed late presymptomatic *SOD1G37R* mice display a 6% longer survival, an increased number of spinal cord motoneurons and a reduced microgliosis [200]. However, a minocycline diet in symptomatic *SOD1G93A* has no effect on survival while amplifying both astrocytosis and microgliosis [201]. These results strikingly illustrate the time-dependent dynamics of the neuroinflammation response, highlighting not only the requirement to target the most pertinent therapeutic

molecular and cellular effectors but to also do so at the proper stage of the disease.

In addition to chemical compounds, the therapeutic delivery of proteins has also been assessed as a potential modulator of neuroinflammation in ALS. Indeed, the granulocyte-colony stimulating factor (G-CSF), a hematopoietic growth factor, has been delivered to *SOD1G93A* mice by osmotic pump starting at asymptomatic stage for a continuance of 8 weeks [202]. G-CSFrecipient ALS mice display a delay in disease onset as well as an increased motoneuron survival. The time to clinical endstage is increased by 10% in *SOD1G93A* mice receiving G-CSF. Importantly, while G-CSF was initially used for its neuroprotective effects and its ability to readily cross the blood-brain barrier [203, 204], further characterization of treated *SOD1G93A* mice shows a reduced spinal cord astrocytosis and microgliosis as well as an increased availability of migratory healing monocytes, suggesting that G-CSF may be beneficial in ALS

**5.2. Advances and possible applications of protein therapy**

via its modulation of neuroinflammation [205].

inhibition of the p38 kinase, NF-κB and STAT3 pathways [167, 193, 194].

in both astrocytosis and microgliosis [195, 196].

motoneuron loss and astrocytosis [197].

activation [190].

114 Current Advances in Amyotrophic Lateral Sclerosis

While drugs and protein therapy target the misregulated pathways within astrocytes and microglia, the aim of cell therapy is to replace these aberrantly functional cells by healthy ones or use implanted cells as a therapeutic platform to deliver neurotrophic support, thus hope‐ fully alleviating neuroinflammation in ALS.

#### *5.3.1. Glial precursor cells*

Glial cell therapy has indeed been evaluated by isolating human neural progenitor cells (hNPCs) and genetically modifying them to express GDNF [210, 211]. Prior to direct injection in the spinal cord of *SOD1G93A* rats, hNPCs were pre-differentiated into astrocytes [210]. Despite the fact that the hNPC injection did not increase the lifespan of *SOD1G93A* rats, they do localize within both the grey and white matter of the spinal cord and survive until the death of the animal. Further investigation of this method reveals that hNPCs preserve dying motoneuron cell bodies in *SOD1G93A* rats without improving their innervations at the neuromuscular junction [212]. In *SOD1G93A* mice, GDNF-expressing hNPCs also migrate to the spinal cord where a subset of them differentiates into astrocytes, again without improving survival or neurodegeneration [213]. However, the lack of beneficial outcome is most likely due to the regional specificity of GDNF's biological activity, as intramuscular but not intraspinal delivery of GDNF exerts its neuroprotective effect [214, 215]. Another astrocyte precursor with potential benefits is the glial-restricted progenitors (GRPs), isolated from the spinal cord of embryonic rats [216]. The transplantation of GRPs in the ventral horn of *SOD1G93A* rats shows that these cells differentiate into astrocytes and can survive and migrate along the spinal cord [217]. Importantly, GRP-recipient ALS rats survive longer as well as show a slower cervical neuro‐ degeneration and a reduced spinal cord microgliosis. In *SOD1G93A* mice however, while the GRPs efficiently differentiate into astrocytes, survive, and locate to both grey and white matter of the spinal cord, they do not influence lifespan and do not prevent motoneuron loss [218].

opment of neuronal-specific therapies toward those targeting more general phenomena

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The pattern of neurodegeneration in ALS has been described as overall linear, albeit with some variations [228, 229]. One of the biggest discrepancies between individuals is the evolution of the disease, which ranges from death in less than 6 months to a limited handicap after more than 10 years following the initial diagnosis. Either rapid or slow, the topography of neuro‐ degenerative events is rather reproducible, usually spreading from one limb to the opposite one and then to another level. Thus, from the moment a patient presents himself at the clinic with symptoms, there is a progressive extension and diffusion of the pathological process. This spreading of neurodegeneration over time may result from the infiltration and migration of non-neuronal cells or by the exchange of molecules from one cell to another. This hypothesis, based on pre-clinical and clinical observations, highlights the importance of developing

As described in section 2.3, the excitotoxic theory suggests that glutamate accumulates within the intercellular space and induce a pathological synaptic excitotoxic transmission, leading to motoneuron death [230]. This hypothesis motivated a series of clinical trials with riluzole, a potent glutamate antagonist, culminating in the demonstration that riluzole is efficient in slowing down disease progression [231]. To date, only riluzole is marketed as a *bona fide* treatment for ALS. While riluzole is thought to reduce glutamate release in neurons via the inhibition of voltage-gated sodium channels [232], riluzole may also have some important antiinflammatory functions. Indeed, riluzole significantly decreases IL-1β, TNFα and iNOS levels as well as increases IL-10 levels in LPS-activated microglial cells [233]. Another neuroprotective mechanism mediated by riluzole may include the production of BDNF and GDNF by astro‐ cytes, as demonstrated in cultures [234]. In experimental autoimmune encephalomyelitis (EAE), a commonly used murine model of multiple sclerosis, riluzole administration signifi‐ cantly ameliorates motor functions. Importantly, the decrease in the clinical severity of riluzole-treated EAE mice is associated with a diminished inflammatory response and a marked reduction in lymphocytes infiltrating the spinal cord [235]. Together, these results suggest a more complex mode of action for riluzole where a modulation of inflammation

Other immunomodulatory agents have also been tested in ALS clinical trials, but their therapeutic benefits have not been as promising as those demonstrated by riluzole [236]. Indeed, immunosuppressants such as cyclosporine or cyclophosphamide as well as the more aggressive total lymphoid irradiation were not successful. The intravenous immunoglobulin G (IVIg) treatment has been proposed to suppress inflammatory responses by inducing an IFNγ-refractory state in macrophages [237]. Interestingly, an open-label pilot study of IVIg administration in ALS patients led to a transient clinical improvement in subjects with bulbar-ALS but not in patients with lower signs, suggesting that immunomodulation may have therapeutic potential [238]. Nevertheless, the combined administration of cyclophosphamide and IVIg in another cohort of 7 patients with upper and lower signs did not lead to clinical improvement [239]. These studies highlight the importance of a better identification of targets

characterizing ALS, including neuroinflammation.

therapies that modulate immunity and/or neuroinflammation.

should be acknowledged as one of its therapeutic activity.

Human umbilical cord blood cells (hUCBCs) also have the potential to differentiate into glial cells (reviewed in [219]). Pre-symptomatic *SOD1G93A* mice received either native hUCBCs or cells engineered to overexpress vascular endothelial growth factor (VEGF) and/or FGF [220]. Two weeks following the orbital injection, analysis of the spinal cords reveals the presence of the transplanted hUCBCs with the non-modified cells preferentially differentiating into microglia while the cells expressing the growth factors became astrocytes [220]. Importantly, the administration of hUCBCs to pre-symptomatic and symptomatic *SOD1G93A* mice via intravenous injections delays disease progression, increases lifespan by approximately 8%, prevents motoneuron loss and reduces both astrocytosis and microgliosis [221].

#### *5.3.2. Mesenchymal stem cells*

Mesenchymal stem cells (MSCs) are multipotent stem cells that can differentiate in a broad variety of cells and instigate a reparative environment. MSCs have been therapeutically assessed in light of their immunosuppressive capacities, limiting inflammatory responses in their surroundings [222]. MSCs isolated from rat muscle injected into the CSF of *SOD1G93A* rats subsequently localize to the spinal cord and adopt astrocytic characteristics [223]. The injected ALS rats display motor deficits at the same age than vehicle-injected animals. However, the MSC-injected *SOD1G93A* rats show an increased survival of approximately 11%, an increased number of motoneurons as well as a reduced neuroinflammation [223]. Similarly, the intra‐ venous injection of MSCs after the onset of disease in *SOD1G93A* mice increases lifespan by 13% and reduces both astrocytosis and microgliosis [224]. Alternatively, a combination of intra‐ spinal and intravenous transplantation of MSCs has been evaluated in *SOD1G93A* rats at disease onset and leads to a 6% increased in survival [225]. Similarly, the intracisternal delivery at asymptomatic stage of human MSCs derived from ALS patients also prolongs survival ALS mice by about 6% [226]. Further, intramuscular administration of MSCs genetically engineered to produce GDNF in asymptomatic *SOD1G93A* rats does not influence the time of disease onset but prolongs survival of implanted ALS rats. However, the beneficial effect of MSCs trans‐ plantation was not correlated with decreased astrocytosis or microgliosis [227]. Although this pre-clinical evidence proposes MSC-based therapy as a potential means to intervene in the course of disease, the neuroprotective mechanisms involved remain elusive, especially regarding the immunosuppressive abilities of MSCs.
