**6. Clinical aspects**

As discussed in the present book and chapter, the molecular pathways and cellular effectors responsible for ALS are numerous and their precise contributions to disease pathogenesis are still debated. While this has made translational therapy a challenge, it has shifted the devel‐ opment of neuronal-specific therapies toward those targeting more general phenomena characterizing ALS, including neuroinflammation.

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].

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%,

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

As discussed in the present book and chapter, the molecular pathways and cellular effectors responsible for ALS are numerous and their precise contributions to disease pathogenesis are still debated. While this has made translational therapy a challenge, it has shifted the devel‐

prevents motoneuron loss and reduces both astrocytosis and microgliosis [221].

*5.3.2. Mesenchymal stem cells*

116 Current Advances in Amyotrophic Lateral Sclerosis

regarding the immunosuppressive abilities of MSCs.

**6. Clinical aspects**

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 therapies that modulate immunity and/or neuroinflammation.

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 should be acknowledged as one of its therapeutic activity.

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 as well as a more efficient and specific design of therapies by specifically taking into account the clinical heterogeneity of the disease.

interesting results but there still remains a lot of work before a successful therapy targeting

The Neuroinflammation in the Physiopathology of Amyotrophic Lateral Sclerosis

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

119

In the present chapter, we have described the cellular and molecular events characterizing the neuroinflammation in ALS. We have also highlighted the beneficial potential of various therapeutic approaches specifically targeting these neuroinflammatory effectors. While the reports discussed herein support a role for astrocytes, microglia and immune cells in ALS, it remains unclear how they influence disease onset, progression or both. Hence, a thorough investigation of the neuroinflammatory pathways that impact neurodegeneration will ultimately enhance our understanding of how and when to therapeutically modulate this pathological process. Further, it is important to remember that the astrocytosis and microglio‐ sis that typify ALS stem from the chronicity of this neurodegenerative disorder and thus, there is an active communication with the neurotoxic environment that is composed of neurons, glial cells and immune cells. Therefore, it is with caution that we should proceed with defining a causal or consequential role for neuroinflammation in ALS, but instead, our focus should be

5-LOX, 5-lipoxygenase; Aβ, amyloid beta; ALS, amyotrophic lateral sclerosis; AMPA, αamino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; APC, activated protein C; BDNF, brain-derived neurotrophic factor; CHOP, C/EBP homologous protein; CNS, central nervous system; COX-2, cyclooxygenase; CSF, cerebrospinal fluid; CTL, cytotoxic T lymphocytes; EAE, experimental autoimmune encephalomyelitis; EGCG, Epigallocathecin Gallate; excitatory amino-acid transporter 2; FDA, food and drug administration; FGF, fibroblast growth factor; G-CSF, granulocyte-colony stimulating factor; GABA, gamma-aminobutyric acid; GDNF, glial-derived neurotrophic factor; GFAP, glial fibrillary acidic protein; GLT-1, glutamate transporter 1; GRP, glial-restricted progenitor; glutamate receptor, GluR; IL-1RA, IL-1 receptor antagonist, IFN, interferon; IL, interleukin; i.p., intraperitoneal; ISG15, interferon-stimulated

LPS, lipopolysaccharide; LT-βR, lymphotoxin beta receptor; MAO-B, monoamine oxidase-B; MHC, major histocompatibility complex; MSC, mesenchymal stem cell; NDGA, dicatechol nordihydroguaiaretic acid; NF-κB, nuclear factor kappa B; NK, natural killer; NMDA, Nmethyl-D-aspartic acid; NPC, neural progenitor cell; NO, nitric oxide; NTF, neurotrophic factor; p75NTR, p75 neurotrophin receptor; PGE2 prostaglandin E2; ROS, reactive oxygen species; SOD1, superoxide dismutase 1; STAT, signal transducer and activator of transcription; TDP-43, TAR DNA-binding protein 43; Teff, effectors T lymphocyte; TGF-β, transforming growth factor beta; TLR, Toll-like receptor; TNF, tumor necrosis factor; Treg, regulatory T lymphocyte; UCBC, umbilical cord blood cell; VEGF, vascular endothelial growth factor.

)-chloride co-transporter;

neuroinflammation is translated into humans.

on identifying its exact pathological contribution.

gene 15; IVIg, Intravenous immunoglobulin G; KCC, potassium (K+

**7. Future directions**

**List of abbreviations**

Among the drugs mentioned that have been evaluated in pre-clinical models, celecoxib and pioglitazone were both assessed in a randomized, double-blind, placebo-controlled trial, but gave disappointing results as there were no effects on motor function and survival rate [240, 241]. When minocycline was tested in a randomized placebo-controlled phase III trial, it not only did not show any benefits, it in fact displayed serious harmful effect in patients [242]. Thalidomide, an analogue of lenalidomide, which showed therapeutic potential in SOD1 mutant mice, was used in a single arm, open label phase II study. Unfortunately, similar to minocycline, thalidomide led to undesirable side effects, without any positive effects [243]. A pilot trial (double-blind, placebo-controlled, randomized) where G-CSF was administered to ALS patients for over 25 days does however show encouraging results on the prevention of degeneration of several white matter tracts [244]. This study supports a larger scale trial in which the immunomodulatory aspect of G-CSF should be further explored.

The translational therapy of neuroinflammatory and immunomodulatory effectors has thus shown both exciting and disappointing outcomes. There are many factors that could help explain the discrepancy between pre-clinical and clinical evaluations of potential therapies. Firstly, most drugs are typically assessed in the mutant SOD1 animal models. This poses an important caveat, as not only there exists an obvious difference between humans and animals, but SOD1 models also represent hereditary ALS, which account for only 4% of ALS cases. Thus, a drug may show a positive influence on an inherited disease model without having any effect on sporadic cases. There is therefore the risk of wrongly eliminating or pushing forward an ALS treatment due to the lack of diverse familial and sporadic pre-clinical models. Secondly, the exact timing of a specific treatment could also impact its efficiency. Indeed, as described in the present chapters, neuroinflammation consists of dynamic mechanisms, combining over time and space, different cell types with opposing neuroprotective and neurotoxic functions. Thus, depending on the desired target, the therapeutic window of various drugs may differ one from another. Thirdly, while establishing a dosage regimen (concentration of drug and treatment length) is amenable in pre-clinical models, determining the exact dose and duration of a therapy in human patients is somewhat more complex. Further, it remains unclear if the treatment of ALS patients should take place daily for several months or periodically in pulses. It thus becomes imperative to develop new analytical methods to adequately extract from preclinical studies the equivalent doses for humans as well as the optimal treatment protocol. Finally, in light of the high heterogeneity of ALS forms, it is possible that not all patients will respond equally to a particular therapy. Therefore, all of these parameters, including addi‐ tional ones not mentioned herein, have to be thoroughly considered and analyzed to ensure that we do not wrongly disregard or promote a drug.

When dealing with neuroinflammation in ALS, the therapeutic intervention is of a different kind than the previous major clinical trials. It is not compulsorily a matter of influencing the disease process, that is the motoneuron death itself, but a matter of stopping a potential amplification and/or diffusion phenomenon. In animal models, this strategy has given interesting results but there still remains a lot of work before a successful therapy targeting neuroinflammation is translated into humans.
