Neuroinflammation is an inflammatory response that takes place within the central nervous system (CNS) during a neurodegenerative process or following a neuronal injury. The main effectors of neuroinflammation, which are astrocytes, microglia and immune cells can confer in a context- and time-dependent manner both neuroprotective and neurotoxic effects. It has now become evident that neuroinflammation is a prominent pathological hallmark of several neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease and Amyotrophic Lateral Sclerosis (ALS)(reviewed in [1, 2]). Indeed, reactive astrocytes and microglia as well as infiltrating T lymphocytes have been identified in ALS experimental models and patients. In the present chapter, we will describe the neuroinflammatory phenotype that characterizes ALS and discuss how the aberrant astrocytes, microglia and immune cells may actively participate in the neurodegenerative process. Further, we will examine the therapeutic potential of targeting neuroinflammation in both pre-clinical disease models and ALS patients.
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 ). 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 . 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 . 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 above-mentioned post-mortem observations, in vivo brain imaging of ALS patients using deuterium-substituted [11C](L)-deprenyl positron emission tomography has allowed the visualization of astrocytosis in live patients . Hence, a thorough analysis of the CNS of ALS patients has uncovered and highlighted astrocytosis as a
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
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 mutant-expressing cells . 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 . A complementary approach consisting in deleting the human mutant SOD1 specifically within astrocytes of the
Another approach used to address the astrocytic-induced motoneuron loss in ALS is the
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 . Likewise, the specific expression of SOD1G37R in spinal cord motoneurons or the accumulation of SOD1G93A in postnatal motoneurons 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 . 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-D-aspartic 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.
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 . The GluR2 subunit is functionally dominant and renders AMPA receptors impermeable 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 . 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 . 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 , a molecular event that confers Ca2+ permeability to the GluR2 receptor . 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 vulnerability to AMPA-mediated excitotoxicity . 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. Although, 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 ). In experiments where the spinal cords of neonatal rats were injected with cerebrospinal fluid (CSF) from ALS patients, there is an increased GFAP immunoreactivity within the grey and white matter , 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
Type I, II and III IFNs are an important family of immunomodulatory cytokines (reviewed in ). 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 . 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
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 ). In symptomatic
COX-2 is a pro-inflammatory enzyme that converts arachidonic acid into prostanoids such as PGE2, a potent inflammatory mediator (reviewed in ). In the anterior horn region of the spinal cord of
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 ), 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
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 . 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 . Using 3H-L deprenyl
2.4.6. Mitochondrial dysfunctions
While there is a vast amount of research on the mitochondrial dysfunction in ALS motoneurons (reviewed in ), 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, sustaining their reactive status. Indeed, primary astrocytes isolated from the cerebral cortex of neonatal rats and overexpressing
2.4.7. Activation of microglial cells
A fundamental role for astrocytes in the neuroinflammation process is the recruitment of microglia , the resident macrophages of the CNS (reviewed in ). In
Figure 1 illustrates the potential non-cell-autonomous mechanisms implicating reactive astrocytes in the selective death of motoneurons in ALS.
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 ). 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
3.2. A role for microglia in ALS pathogenesis
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
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 , a study looking at cerebral cortex of ALS patients shows that the phagocytosis of degenerating neurons is mediated by perivascular macrophages and not microglia . This finding already suggested 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.
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 ). Importantly, ER stress is an established characteristic of ALS pathogenesis (reviewed in ). In spinal cord microglia of both sporadic ALS patients and symptomatic
3.3.2. CD14-toll-like receptor signaling
Once the ligand-dependent CD14 lipopolysaccharide (LPS) receptor located at the microglial surface  is activated, it initiates a pro-inflammatory signaling cascade dependent on Toll-like receptors (TLRs), specifically TLR2 and TLR4 [117, 118]. Interestingly, the neurotoxic activation of microglia by extracellular
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 pro-inflammatory response, chemotaxis and phagocytosis (reviewed in [123, 124]). Embryonic immortalized microglia and neonatal primary microglial cultures isolated from mutant
3.4. The potential influence of microglia on neuronal excitability
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 .
Loss of neuronal inhibitory control occurs by several means including decrease in gamma-aminobutyric acid (GABA)ergic interneurons  combined with changes in the expression of the GABAA receptor mRNA subunit . GABAA and glycine receptors are chloride (Cl-) channels and the expression of cation-chloride co-transporter contributes to inhibitory effects of these Cl- currents . 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 . Stimulation of spinal microglia following peripheral nerve injury induces a decrease in KCC2 expression among dorsal horn nociceptive neurons . 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 . 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 . 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
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). 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 .
The potential non-cell-autonomous mechanisms involving microglial cells in the selective degeneration of motoneurons in ALS are illustrated in Figure 2.
4. Involvement of neuroimmunity in motoneuron degeneration
4.1. Pathological phenotype of the immune system in ALS
In addition to astrocytes and microglia, immune cells may also play synergistic and critical roles in ALS neuroinflammation and disease progression. Presence of a systemic immune activation is suggested by abnormalities observed in the blood and CSF of ALS patients such as increased numbers of circulating lymphocytes (CD4+ helper T cells, CD8+ cytotoxic T lymphocytes (CTL) and natural killer (NK) cells), increased expression of MHC class II molecules on monocytes as well as higher levels of inflammatory chemokines and cytokines (regulated on activation normal T cell expressed and secreted (RANTES), monocyte chemotactic protein (MCP-1), IL-12, IL-15, IL-17 and IL-23)[142-146]. Further,
In support of what is observed in humans, ALS rodent models also display a particular immunological phenotype. Indeed,
Whether neuroinflammation is a cause or a consequence of motoneuron death is still debated. It is interesting to note that inflammation is not limited to the CNS but systemic with increased levels of plasma LPS associated with increased numbers of activated circulating monocytes and T lymphocytes that correlate with disease evolution [142, 157]. A thymic dysfunction is also observed in parallel to the neurodegenerative process in mutant SOD1 mice and ALS patient . In the CNS of ALS patients, TAR DNA-binding protein 43 (TDP-43) increased and interacts with nuclear factor kappa B (NF-κB) in glial and neuronal cells. LPS-activation of NF-κB in microglial cells expressing the TDP-43 mutant is associated with the production of pro-inflammatory cytokines, including TNFα, IL-1β, IL-6 and IFNγ . The central role of inflammation and NF-κB in ALS was recently confirmed by the description in familial ALS of mutations in the gene encoding optineurin, a negative regulator of TNF-induced NF-κB activation .
Altogether, the information from pre-clinical models and ALS patients suggest that systemic immune activation (innate and adaptive) might play a key role in ALS pathogenesis and may represent an interesting target for the development of novel treatments. However, a better understanding of the specific roles played by the different subtypes of immune cells is of utmost necessity. Indeed, accumulative evidence suggests that inflammatory cells mediate both protective and deleterious effects on motoneuron survival and that these functions vary during disease progression.
4.2. The protective function of the immune response in ALS
Protective immunity, a homeostatic phenomenon important in the repair of damaged tissues, results from both the clearance of debris and the effects of cytokines and growth factors delivered by inflammatory T-cells to the site of injury [161, 162]. The neuroprotective ability of immune cells is also evident in ALS. Indeed, when
4.3. The neurotoxic function of the immune response in ALS
T lymphocytes could mediate motoneuron damage either directly through cell-cell contact, secretion of cytokines or indirectly through activation of microglia and macrophages . As mentioned above, the effect of the immune system varies during disease progression from a protective role at early stages to a neurotoxic activity when disease accelerates . Neuroprotective activity has been associated with a Tregs/M2 response and expression of trophic and anti-inflammatory factors such as BDNF, GDNF and IL-4 whereas neurotoxic effects are associated with an M1/Th1/CTL pro-inflammatory immune response . Accordingly, mutated SOD1 Teffs proliferate to a greater extend and produce more IFNγ (Th1-driven) during the rapidly progressing phase than Teffs isolated during slowly progressing phase . Different death pathways can be induced by Th1/CTL lymphocytes and promote motoneuron loss in ALS. For instance, activation of Fas (CD95) has been demonstrated to trigger a motoneuron-restricted death pathway. Motoneurons expressing ALS-linked SOD1 mutants showed an increased susceptibility to Fas-mediated death through activation of an amplification loop [166-168]. Accordingly, mutant
T lymphocytes could also amplify the neuroinflammation in ALS via glial cells. Upon activation, microglia cells increase membrane expression of MHC class II molecules, becoming efficient antigen presenting cells able to actively drive T cell activation and differentiation. In turn, cytokines secreted by T cells modulate microglia phenotype and function. For instance, TNFα and IFNγ, two major pro-inflammatory cytokines produced by Th1 lymphocytes induce and activate M1 microglial cells and cause neurotoxicity toward motoneurons. Experimental studies in ALS mice demonstrated that inflammatory cell subtypes were phenotypicaly and functionally different depending upon the disease stage . At initial stages, microglia exhibits anti-inflammatory M2 phenotype (Ym1+, CD163+) and infiltrating T cells are almost exclusively CD4+ while end-stage disease is associated with a skew of microglia toward a pro-inflammatory M1 phenotype (Nox2+) and T lymphocytes are mainly Th1 cells .
The neurotoxic effect of NK cells is suggested by the neuroprotective effect of the immunomodulation of NK cells, which increases lifespan of ALS mice and is accompanied by a reduced astrocytosis. While the pathological modalities of NK cells in ALS remain elusive, several hypothetical mechanisms can be raised. Indeed, activated NK (and to a lesser extent CD8+ T cells) inhibit neurite outgrowth of cerebellar neurons in a cell contact-dependent manner
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
Figure 3 illustrates the potential mechanisms implicating different populations of immune cells in ALS pathogenesis.
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.
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 . Unfortunately, the intraperitoneal (i.p.) delivery of the p75NTR antagonist from asymptomatic stage up until the endpoint of the disease does not improve the phenotype or survival of
COX-2 appears as an appealing therapeutic target for ALS as it promotes both pro-inflammatory events and astrocytic glutamate release [60, 67]. Celecoxib, a COX-2 inhibitor, fed to
Lenalidomide, an immunomodulatory drug with pleiotropic properties derived from thalidomide, has been evaluated in mutant SOD1 mice due to its inhibitory effect on TNFα production by monocytes . A lenalidomide-diet given to
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 . The daily oral administration of EGCG to
Pioglitazone is a drug that was initially developed to treat type II diabetes patients that also exerts ant-inflammatory and neuroprotective activities (reviewed in ). For these reasons, it has been hypothesized that it may improve ALS pathology. Indeed, pioglitazone-fed
Olesoxime has previously been selected as a neuroprotective agent via a motoneuron survival-based screen . Interestingly,
Dicatechol nordihydroguaiaretic acid (NDGA) is a selective inhibitor of 5-LOX that presents TNFα antagonizing activity in microglial cells.
Minocycline is a member of the tetracycline molecules that can enter the CNS and mediates inflammation and microgliosis (reviewed in ). Asymptomatic
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
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 ). A daily i.p. injection of APC to symptomatic
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 . When administrated by i.p. daily to asymptomatic stage to
The CD40 costimulatory pathway, which plays an important role in B and T cell activation , 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 significantly the percentage of peripheral CD8+ T cells as well as GFAP+ astrocytes and Mac2+ microglia in the spinal cord , suggesting that the CD40 pathway, an integral component of neuroimmunity, is a potential therapeutic target in ALS.
5.3. Cell therapy perspectives
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 hopefully 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
Human umbilical cord blood cells (hUCBCs) also have the potential to differentiate into glial cells (reviewed in ). Pre-symptomatic
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 . MSCs isolated from rat muscle injected into the CSF of
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 development of neuronal-specific therapies toward those targeting more general phenomena characterizing ALS, including neuroinflammation.
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 neurodegenerative 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 . 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 . To date, only riluzole is marketed as a
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 . 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 . 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 . 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 . 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.
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 . 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 . 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 . 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 pre-clinical 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 additional 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.
7. Future directions
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 microgliosis 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 on identifying its exact pathological contribution.
List of abbreviations
5-LOX, 5-lipoxygenase; Aβ, amyloid beta; ALS, amyotrophic lateral sclerosis; AMPA, α-amino-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 gene 15; IVIg, Intravenous immunoglobulin G; KCC, potassium (K+)-chloride co-transporter; 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, N-methyl-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.
Our work is supported by grants from the Institut National de la Santé et de la Recherche Médicale (Inserm), Association Française contre les Myopathies (AFM), Association Française pour la Recherche sur la SLA (ARS), Direction de l'Hospitalisation et de l'Organisation des soins (DHOS) and the Thierry Latran foundation. M.B is a recipient of a long-term EMBO Marie Curie Fellowship. We apologize to authors whose work could not have been cited due to space limitations.