Microglia centered pathogenesis in ALS: insights in cell interconnectivity

1Amyotrophic lateral sclerosis (ALS) is a non-cell-autonomous disease targeting motor neurons (MNs) and neighboring glia, with microgliosis directly contributing to neurodegeneration. Indeed, the neurodegenerative process in ALS was shown to be accompanied by a sustained inflammation in the brain and spinal cord (SC). Recently, microglia were suggested to be implicated in ALS initiation, as well as to lose their surveillance capacity by switching from an activated to a neurodegenerative phenotype as the disease progresses. Therefore, a better therapeutic strategy should envisage the recovery of healthy microglia from those transformed cells near the most affected MNs in the SC. In such way we may preserve the passage of toxic mediators to environmental cells, and maintain the homeostatic conditions.

2For the vast majority of patients with ALS, the etiology of the disorder is unknown. Actually, only some ALS cases (less than 10%) have been linked to mutations in a number of genes, including in the enzyme Cu, Zn superoxide dismutase 1 (SOD1), TAR DNA binding protein (TDP-43), fused in sarcoma (FUS), optineurin (OPTN), valosin-containing protein (VCP), ubiquilin 2 (UBQLN2), profilin 1 (PFN1), and chromosome 9 open reading frame 72 (C9ORF72) repeat expansions. Interestingly, both the non-genetic and the genetic forms of ALS are suggested to have common pathogenic mechanisms, as well as similar clinical courses and dysfunctional features, such as the abnormal accumulation of neurofilaments in degenerating MNs. Actually, cytoplasmic aggregation of nuclear TDP-43 and FUS in the degenerating neurons and glia of ALS patients, and release of the accumulated cytoplasmic mutant SOD1 (mSOD1) to the extracellular space that can be taken up by other cells, are common features. The identification of C9ORF72 repeat expansions in patients with ALS but without a family history of ALS challenged the division between genetic (familial) and non-genetic (sporadic) cases. As indicated by, the true substrate of ALS may reside in a pathogenic signature of nuclear protein mishandling.

3There are several in vitro and in vivo models of MN degeneration. In vitro experimental models include SC cultures, NSC-34 cell line expressing the mSOD1 and organotypic cultures, while the axotomy-induced MN death, the naturally occurring ALS models, and the transgenic models are the most commonly used in vivo models. Among the various transgenic models used in the study of ALS pathogenesis, the transgenic rodent overexpressing mSOD1, in particular the SOD1G93A strain, is the most utilized and characterized. Transgenic mice containing other mSOD1 genes (G85R, G37R, D90A, or G93A missense mutations or truncated SOD1) and the related mutant (G86R) mouse have also shown progressive neurodegeneration of the motor system and resemblance to ALS (for review, see ). Distinctive injurious effects between SOD1G93A and SOD1H46R on two different genetic backgrounds were recently recognized. Additionally developed models are based on TDP-43 and FUS mutations, but none of these models is currently used to study the pathogenesis of ALS and to test new drugs. Thus, the human mSOD1 murine model is the most widely used in the evaluation of the involved molecular targets, biomarkers and novel drugs/treatments for ALS. Apart from developing loss of MNs and symptoms that resemble human ALS by mSOD1, the model evidences molecular links between genetic and non-genetic cases of ALS. To note, that non-genetic perturbations of the wild-type (wt) SOD1 protein may lead to SOD1 misfolding with a conformation much similar to genetic SOD1 variants. Therefore, in this review we will summarize the most recent developments obtained in the SOD1G93A transgenic model to give consistency and cohesion between the data disclosed, and because we admit that common factors and pathways are shared in both genetic and non-genetic derived ALS cases, in particular changes in microglia performance and in neuron–glia communication.

4It was initially considered that the selective death of MNs expressing the mutant protein was the player in the disease onset. However, non-cell-autonomous processes associated with mSOD1 in glial cells are believed to be implicated not only in disease progression and extent, but also to be related with the onset and early stage of the disease, thus underlying MN dysfunction and loss. Indeed, healthy glia evidenced to delay the progression of the disease and the replacement of mSOD1 microglia by wt microglia slowed disease progression and prolonged mice survival. This finding is in line with previous studies showing that mSOD1 in microglia leads to the disease and that the reduction of the mutant levels in the cells slows ALS progression. Indeed, damage to MNs by neighboring cells expressing mSOD1 seems to be required for MN degeneration. Accumulating knowledge on the active participation of different microglia phenotypes in ALS was recently obtained when microglia were isolated from SOD1G93A rats at presymptomatic, symptom onset and end-stage periods. Microglia were shown to be regionally different and to evidence a heterogeneity of phenotypes with the disease progression. Thus, it will be interesting to investigate the influence of changes caused by aging in the performance of the mutated microglia, namely on the interconnectivity with neurons and other glial cells. Moreover, our preliminary data indicate a decreased phagocytic and migration ability of healthy microglia by the soluble factors (SFs) released by NSC-34 cells expressing mSOD1, thus causing the loss of important microglia properties. In addition, when this microglia was co-cultured with the mMNs we observed a reduction of the activation of matrix metalloproteinases (MMP)-2 and -9 in the extracellular media after cultivation for 4 days in vitro (DIV), evidencing the beneficial effect of the healthy microglia in decreasing mMN stress.

5Finally, we provide an outlook on the extent to which a diverse cellular environment may determine different pathological windows along disease progression opening new opportunities to explore distinct therapeutic approaches to either trigger a less reactive microglia phenotype at the early-onset, or a recovery of microglia dynamics at late-stage ALS. It will be also summarized recent advances in regenerative medicine technologies with potential to reverse or halt ALS progression by slowing MN death.

6Neuronal homeostasis and survival were shown to be compromised in ALS due to multiple aberrant biological processes and to deregulated communication between neurons and glial cells in the brain and in SC by the disease. ALS, once considered a MN disease, is now known to have multiple influences and regarded as a multi-cellular/multi-systemic disease. In fact, MN death seems to be driven by a convergence of damaging mechanisms, including glial cell pathology and inflammatory conditions, such as microglial activation or the invasion of lymphocytes, and calcium dysregulation. It can be also determined by excitotoxicity due to the selective loss of the astrocytic glutamate transporter GLT-1, and consequent accumulation of extracellular glutamate. Our late results with astrocytes isolated from SOD1G93A mice and cultivated for 13 DIV suggest that both GLT-1 and glutamate aspartate transporter (GLAST) are compromised (personal communication) and corroborate other findings indicating that expression of such transporters are less potently activated by lipopolysaccharide (LPS) in astrocytes from the mSOD1 model than in those from the wt mice. Other contributing processes include from neurovascular changes and compromised barriers of the central nervous system (CNS), to dysfunctional communication between neurons, abnormal neuron–glia interactions, and microglia and astroglia loss-of-function.

7In fact, for the optimal functioning of the CNS, i.e., brain and SC, accounts the constant immune surveillance promoted by cells such as microglia, and the blood–brain barrier (BBB), the blood–SC barrier (BSCB), and the blood–cerebrospinal fluid (BCSF) barrier that uniquely shield CNS from potential mediators of infection and damage. Recently, brain pericytes, as important components of the neurovascular unit in the CNS, have shown to have pleiotropic and regulatory activities in brain vessel function and homeostasis, blood flow and barrier function. Astrocytes also participate in maintaining homeostasis and supporting neuronal function. In addition to microglia, neurons have lately been indicated to intervene in immune responses through control of T cells infiltration into the CNS and glial cell immunoreactivity. This integrative network sustains ionic, energetic, and redox homeostasis for proper function.

8Moreover, ALS progression was suggested to involve cell-to-cell transmission of mSOD1 aggregates involving MNs, microglia and astrocytes, similarly to prion disease. Interestingly, the transcellular spread of SOD1 aggregates evidenced to not require cell-to-cell contacts but to depend from their fragmentation and extracellular release. However, additional studies should demonstrate that similar findings also occur in vivo. Intriguingly, while extracellular mSOD1G93A has shown to not have direct toxic effects on MNs, it morphologically and functionally activates microglia, supporting the non-cell-autonomous nature of MN toxicity in ALS. Indeed, it was shown that this extracellular mSOD1 can be endocytosed into microglia, determining the activation of caspase-1 and the up-regulation of interleukin (IL)-1β.

9Another interesting concept is that astrocyte and microglia activation, which is regulated by a variety of signaling pathways, should not be considered merely as pernicious for CNS homeostasis, once it promotes metabolic support, wound healing and repair. In fact, the production of cytokines and chemokines initiate and coordinate diverse cellular and intercellular actions. Although neurons and glial cells of the CNS express receptors for cytokines and chemokines, the biological consequence of receptor activation is not fully understood. In contrast, it must be considered as part of the pathological processes the excessive or deregulated signaling pathways leading to microglia and astrocyte abnormalities that culminate in abnormal CNS function.

10Thus, progressive neurodegeneration of MNs in ALS may result from a combination of intrinsic MN vulnerability to mSOD1 aggregates and of non-cell-autonomous toxicity derived from neighboring cells (Tables 1 and 2). Therefore, pathological changes in ALS indicate a broken homeostasis in the CNS. We will focus on how CNS homeostasis is lost in ALS and in what way BBB and neural networks dysregulation contribute to neurodegeneration in ALS.

11A certain number of cases are linked to mutations in SOD1 and candidate mechanisms are the formation of protein aggregates and pro-oxidant effects. Therefore, most of the experiments are generally conducted in the MN-like hybridoma cell line NSC-34 expressing human mSOD1 (hSOD1G93A) or in the transgenic mice generated by that also over-express hSOD1G93A.

12SOD1 aggregates are observed in both genetic and non-genetic ALS cases, but their contribution to MN toxicity remains to be established, although deregulation of Golgi, endoplasmic reticulum (ER), and mitochondria, together with axonal transport defects have been indicated (for review, see; ). Interestingly, when working with the NSC-34/hSOD1G93A cells we found that one of the most sensitive indicators of SOD1 accumulation and neuronal dysfunction was the elevation of MMP-9, but not of MMP-2 that remained unchanged. Elevation of the MMP-9 levels was previously observed in the SC of ALS mice from pre-symptomatic phase, predominantly in MNs but also in glia. These authors suggest that circulating MMP-9 is associated with the disease onset and is mainly derived from degenerating SC MNs and circulating cells. Its inhibition was shown to enhance animal survival in more than 30% (for review, see ). It was also found in the CNS, muscles, plasma, and skin of patients. Major inducers are reactive oxygen species (ROS) and cytokines released by microglia. Once MMP-9 is indicated to promote the regeneration of the injured neuron, it may be hypothesized that its increase results from a MN response to the pathology. However, it was recently claimed that MMP-9 is indeed a determinant of the selective neurodegeneration. The Authors demonstrated that MMP-9 is only expressed by the selectively vulnerable fast MNs and can originate ER stress and axonal dye-back. This finding provides a basis for considering MMP-9 as a candidate target for novel therapeutic approaches to ALS.

13Oxidative damage at the level of proteins, lipids, and DNA was also observed in the transgenic hSOD1 mice models where mSOD1 revealed to be the most severely oxidized protein mouse. Also accounting to MN disturbance is the failure of the Keap1/Nrf2/ARE system in regulating stress proteins, as evidenced along disease progression in MNs from the SC of hSOD1G93A mice. In addition, failure in MN autophagy was revealed as critical in the pathogenesis and progression of ALS (for review, see;; ).

14Currently, there is no cure for the ALS disease, although Riluzole, the only drug approved by the U.S. Food and Drug Administration (FDA), was shown to prolong median patient survival by 2–3 months and to be more effective when administered at an early stage of the disease. More potent therapeutic strategies may derive from better target identification and clarification of signaling mechanisms able to be modulated and from the use of cell-based therapies, such as the administration of mononuclear cells from human umbilical cord blood and of mesenchymal stromal (stem) cells, as referred to in Section “Challenges to Nerve Regeneration in ALS.” Bellow we introduce what is known about the most prominent MN membrane proteins and SFs suggested to be implicated in ALS pathology, but still deserving to be more explored.

15Glutamate excitotoxicity is one primarily cause of neuronal death by necrosis and apoptosis and a critical player in ALS onset and progression. Indeed the blockade of the glutamate transporter in a SC organotypic slice model from SOD1G93A rats has shown to result in an increased survival of MNs. Glutamate, by activating the glutamatergic ionotropic receptor N-methyl-D-aspartate (NMDAR), triggers the influx of Ca2+ into neurons, increasing its intracellular levels (for review, see ). In these conditions, changes in mitochondria dynamic properties are produced leading to excessive oxidative phosphorylation and increased generation of ROS and reactive nitrogen species (RNS) that culminate in apoptosis. In parallel, the NMDAR excitotoxicity also results in the release of Ca2+ from the ER. In addition, it was shown that ER stress resulting from the accumulation of aggregated mSOD1 and dysfunction of the unfolded protein response (UPR) activation contributes to the apoptotic signaling cascade in ALS (for review, see ).

16Besides the synaptic glutamate pool directly implicated in the excitatory neurotransmission it has been lately additionally considered the extra-synaptic glutamate pool that influences cell communication. This pool, mainly derived from astrocyte and microglia release, is greatly increased by pathological stimuli (for review, see ). Besides being implicated in many physiological conditions glutamate contribution to neuron–glia alterations of homeostasis and to the pathophysiology of neurodegenerative diseases, such as ALS, needs to be explored in the near future, due to controversial results. In fact, although linkage between glutamate and neuroinflammation was suggested to have a role in potentiating MN death in a model mimicking ALS disease, other Authors have demonstrated that glutamate by itself was not able to induce MN damage in ALS. Indeed, it has been questioned the involvement of glutamate in ALS-induced MN death and the cytotoxicity of the cerebrospinal fluid (CSF) from patients with ALS evidenced to not be related to glutamate.

17Fractalkine (FKN) mRNA is dominantly expressed in neuronal cells, particularly in those at the cortex, hippocampus, caudate putamen, thalamus, and olfactory bulb. FKN mRNA was also detected in unstimulated astrocytes (more) and microglia (less). Interestingly, FKN immunoreactivity and mRNA was observed also in the rat SC and dorsal root ganglia neurons, but not in glia, and levels were not enhanced by neuropathic conditions. FKN, or CX3CL1, exists as a membrane-bound and soluble protein (sFKN) allowing both adhesive and chemoattractive properties. In fact, the sFKN has potent chemoattractant activity, recruiting CXCR1-expressing T cells, monocytes, and microglia to the injured neurons, as well as in regulating the phagocytic capacity of microglia. sFKN was shown to increase upon stimulation of glutamate and to induce proliferation of human microglia. The constitutive expression of FKN and its receptor CX3CR1 in microglia indicates its involvement in fundamental processes of communication between neurons and microglia.

18Under stimulation, such as an excitotoxic stimulus, the membrane-bound form of FKN is rapidly cleaved from cultured neurons and significantly reduces neuronal NMDA-induced apoptosis. Indeed attenuated glutamate-induced neuronal cell death was observed after treatment of primary neuron–microglia co-cultures with sFKN. Proteolytic cleavage of CX3CL1 is mediated by action of metalloproteinase ADAM10, ADAM17, cathepsin S (CatS), and MMP-2. Thus, cleavage of FKN may be prevented by the inhibition of MMPs. The source of CatS is the activated microglial cells that upon stimulation with LPS secrete sFKN to the extracellular media. Similarly, stromal cell-derived factor-1 (SDF-1) was indicated to stimulate the expression of ADAM17 and to increase sFKN, while up-regulating FKN expression.

19In addition, FKN has a neuroprotective function by inhibiting the nitric oxide (NO) production and the expression of inducible NO synthase (iNOS) mRNA in activated microglia. Interestingly, it was observed that FKN expression was reduced in the brain of aged rats probably accounting for the increase in microglial activation in such condition. In fact, treatment with FKN has attenuated the age-related increase in microglial activation.

20Taken together, FKN seems to have both intrinsic and anti-inflammatory properties in the CNS and to act by interfering with toxic microglial–neuron interactions. Whether FKN may have a role in the development of ALS is still not known.

21High-mobility group box 1 (HMGB1) protein, also known as amphoterin, is an inflammatory factor that can be released by astrocytes, microglia, and neurons, mainly when cells are dying (for review, see ). HMGB1 has dual activities depending on whether is alone (probably promoting inflammation resolution and tissue regeneration) or forming complexes with several proinflammatory mediators (potentiating inflammation and promoting innate immune cell activation). Nuclear HMGB1 regulates transcription of different sets of genes, including proinflammatory genes. Beneficial effects were observed in early CNS development but increased levels of HMGB1 were shown to be correlated with apoptosis and degeneration of neurons. When neurons are injured, secretion of HMBG1 activates microglia through receptor for advanced glycation end products (RAGE), Toll-like receptors (TLRs) 2, 4, and 9, as well as Mac1 receptors, as depicted in Figure 1. Release of inflammatory mediators by activated microglia further induces neuronal necrosis. HMGB1 was indicated to decrease in neurons and to increase in astrocytes with aging. When expression and localization of HMBG1 was evaluated in the lumbar SC of SOD1G93A transgenic mice, although intense reactivity was found, no differences were obtained between controls and the SOD1 mice. However, since HMBG1 was identified in the cytoplasm of astrocytes and microglia in SC samples from ALS patients it may trigger TLR signaling pathways. The finding was observed at the late ALS phase and, thus, it will be important to follow the TLR/RAGE cascade in animal models at different stages of the disease. In contrast, a progressive reduction of HMGB1 immunopositive MNs was found at advanced stages and may reflect the loss of MNs, reduced synthesis or enhanced released of the cytokine. Additional studies are needed to investigate the causative hypothesis indicated for the decreased HMGB1 immunoreactivity in MNs, inasmuch since it can also have beneficial effects on neuroregeneration.

22CCL21 was shown to be implicated in signaling neuronal injury to microglia through the receptor CXCR3. CCL21 expression was demonstrated to increase in cortical neurons, in vitro, 2 h after excitotoxic stimulus. Intriguingly, these authors demonstrated its location within vesicles that are transported along the neuronal process till presynaptic structures. This chemokine is considered a chemotactic agent important to drive microglia to the site of lesion. Astrocytes were also indicated to have the receptor CXCR3, but unless high levels of CCL21 are produced, no relevant changes can be observed, indicating separate functions from microglia. CCL21 expression in CNS revealed to induce a massive brain inflammation, but not lymphocytic infiltration in transgenic mice expressing the chemokine. Interestingly, CCL21/CXCR3 signaling axis was never explored in ALS.

23The membrane glycoprotein CD200 is expressed in neurons and in endothelial cells and its receptor CD200R is restricted to cells of myeloid origin including macrophages and microglia. CD200 was also evidenced to be induced by kainic acid in microglia and the authors have suggested that microglia are maintained in an activated state with autocrine signaling by interactions between microglial CD200 and CD200R, as well as in a surveillant/quiescent state by interactions between neuronal CD200 and microglial CD200R. In addition, they also demonstrated that IL-4 leads to an increased expression of CD200 what may be the mechanism down-regulating microglia activation whenever IL-4 is produced. In conclusion, deficits in CD200–CD200R system exacerbate microglia activation and the release of proinflammatory cytokines (for review, see ). Curiously, multivariate analyses of gene expression have previously identified alterations on the CD200R expression in presymptomatic SOD1 mice model. Thus, more studies on the role of CD200/CD200R in ALS are needed to evaluate whether its disruption is implicated in ALS.

24Neuregulins have been showing to be implicated in a wide range of neurological and psychiatric disorders including multiple sclerosis, schizophrenia, and AD. Neuregulin precursors are expressed predominantly in cortical neurons, but also accumulate at the surface of white matter astrocytes. CSF neuregulin was found reduced in ALS and increased in AD. Neuregulin-1 (NRG1) is a growth and differentiation factor that binds to erbB receptors in microglia. NRG1–erbB signaling is activated after peripheral nerve injury and contributes to microgliosis and neuropathic pain. Recently, it was suggested that NRG1 released from damaged neurons and other cells in the SC triggers microglial activation leading to progressive MN degeneration in ALS. Indeed, it was observed that in the early stage of ALS there was activation of NRG1 receptors on microglia. Later on, a reduced membrane-bound NRG1 and an increased mRNA expression were noticed in the SC. More studies are, however, needed to sustain the NRG1 contribution to ALS pathogenesis.

25Major histocompatibility complex (MHC) class I and class II molecules present fragments of peptide antigens to CD8+ or CD4+ T cells, respectively, and cells lacking such molecules are unable to interact with immunocompetent T cells. Presentation of antigenic peptides to CD8+ cells is mediated by β2-microglobulin, which is non-covalently bound to MHC class I. Despite being recently observed during hippocampal neurodevelopment, adult neurons do not constitutively express MHC class I. In contrast, spinal MNs were shown to display a high constitutive expression of both MHC class I and β2-microglobulin mRNAs. A latest paper describes a strong up-regulation of β2-microglobulin in MNs during disease progression in the SOD1G93A mice model, which revealed to be important for the ALS mouse survival. Therefore, β2-microglobulin target driven therapies may be of help in strategies counteracting ALS.

26It is believed that non-neuronal cells expressing mSOD1 may secrete toxic mediators or fail to secrete trophic factors, or both, resulting in either reduced function or survival of MNs. Indeed, several SFs were shown to drive axonal degeneration and regional-specific microglia activation expressing SODG93A was suggested to be implicated. This calls our attention for the existence of multiple factors, and MNs and glial cells interplay in contributing to ALS onset and progression. Microglia activation and dysfunction have lately been related with the onset, but mostly with the progression, of several neurodegenerative diseases. Particularly, in ALS, it has been suggested that besides MNs, also glia and muscles are implicated in the disease.

27Prominent neuroinflammation is a pathological hallmark in human ALS and mouse models of the disease. Gliosis and accumulation of large numbers of activated microglia and astrocytes can be observed in CNS and in SC areas. Active contributions of glial cells in ALS pathology have recently been reviewed. Conflicting results were published on whether the expression of mSOD1 in astrocytes and microglia really contributes to the progression of ALS disease. Some argues against and others in favor (for review, see ). Overall, it will be important to identify the molecules released by glia, as well as the ones secreted by dysfunctional neurons targeting microglia or even acting retrogradely.

28Glial cells (astrocytes, microglia, oligodendrocytes, and Schwann cells), as well as neurons are known to express different members of the TLR family. Increasing evidence indicates that, in the absence of pathogens, TLR signaling can be activated by molecules released by the injured tissue, namely the HMGB1 protein, one of the damage-associated molecular patterns (DAMPs) molecules. Recent findings have underlined the activation of TLRs and RAGE signaling pathways in ALS. TLR2, TLR4, and RAGE expression was increased in reactive glial cells in both gray (ventral horn) and white matter in SC. TLR2 was predominantly detected in cells of the microglia/macrophage lineage, whereas TLR4 and RAGE were strongly expressed in astrocytes. Activated macrophages and microglia (personal communication) release HMBG1 that promotes the secretion of IL-1β and IL-18 further inducing the necrosis of neighboring neurons and the amount of extracellular HMBG1, which binds to microglial Mac1 leading to the activation of nuclear factor-κB (NF-κB) pathway and inflammasome, forming a vicious circle that sustains progressive neurodegeneration (Figure 1). Recent data have demonstrated that NF-κB activation in wt microglia causes MN death providing a therapeutic target for ALS.

29When evaluating whether extracellular mSOD1 caused a direct or indirect injury to MNs, it was observed that neurodegeneration was mediated by microglia and concerted activation of CD14/TLR pathway involving both TLR2 and TLR4. In addition, granulocyte macrophage-colony stimulating factor (GM-CSF), a pleiotropic cytokine predominantly released by astrocytes that up-regulates TLR4 and CD14 expression in microglia, may function to exacerbate TLR signaling in ALS disease. Indeed, when GM-CSF was blocked it was observed a delayed onset and increased life span in the ALS mice. The activation of these pathways may contribute to the progression of inflammation and to a further injury to MNs.

30Oligodendrocytes in CNS, and Schwann cells in the peripheral nervous system (PNS), are responsible for the myelin sheaths surrounding neurons which provide electrical insulation essential for rapid signal conduction. Schwann cells also participate in the clearance of debris and in guiding the axon after neuron damage. In the SC injury, reactive gliosis emerges in the lesion accompanied by the up-regulation of chondroitin sulfate proteoglycans (CSPGs) in oligodendrocytes and Schwann cells.

31Considering the regenerative capacity of NG2 cells, one of the first cells responding to any alteration in CNS environment, it will be important to clarify their role in ALS since they can be mobilized to originate cells or to release factors. Nevertheless, NG2 cells were shown to remain committed to an oligodendrocyte lineage in both controls and mutant ALS mice, indicating their scarce participation. However, increased proliferation rate of NG2 revealed to mediate an elevated number of early-born oligodendrocytes that degenerate, resulting in gray matter demyelination in ALS mice and human CNS.

32Although only a few studies have examined whether oligodendrocytes or myelin sheaths have a role in ALS, the myelin abnormalities consisting in loss of compact myelin and lamellae detachment in the SC of pre-symptomatic SOD1 transgenic rats and aggravated at symptomatic stages suggest that it may be an interesting target for further study. Biochemical examinations of the myelin structural components revealed a decrease in the phospholipid content along disease progression, as well as in cholesterol (already in the early presymptomatic stage) and cerebrosides (in the paralyzed animals). Degeneration of oligodendrocytes was observed in human patients and mice models of ALS prior to the disease. Cells that were lost were replaced by newly differentiated oligodendrocyte precursor cells, which, however, evidenced reduced myelin basic protein. This finding is suggestive that they may contribute to MN degeneration in ALS. Moreover, these cells evidenced a loss of the monocarboxylate transporter 1 (MCT1), thus compromising the supply of lactate to MNs (Table 2).

33Little is known about Schwann cell involvement in ALS pathology. Schwann cells are located near the MN axons and are known to bridge between denervated and reinnervated endplates, and to guide axonal sprouts. Expression of mSOD1 in perisynaptic Schwann cells was suggested to interfere with the trophic maintenance of normal or regenerating motor axons. In vivo evidence suggests that glial fibrillary acidic protein (GFAP) up-regulation in the stressed/proliferating Schwann cells may be the underlying pathological events. Conversely, different results were obtained in SOD1G73R mice where elimination of mutant SOD1G37R from Schwann cells failed to slow disease progression. Nevertheless, found that knockdown of mSOD1 in Schwann cells of SODG85R transgenic mice delayed disease onset and extended survival indicating that SOD1G85R expression is neurotoxic. These results imply that diverse mutations confer different outcomes to cell toxicity and, in the case of Schwann cells, oxidative damage seems to be an important feature in the context of ALS.

34Astrocytes are a potential source of both pro- and anti-inflammatory cytokines and are ideally placed in close proximity to BBB and BSCB, thus translating signals from the periphery to the CNS. Although not being immune cells, they can also contribute to the immune response. Astroglial activation, or astrogliosis, is characterized by hyperplasia, hypertrophy of cell bodies and cytoplasmic processes, up-regulation of intermediate filament proteins, namely GFAP and vimentin, mediating a histologically apparent glial scar at the lesion site in the damaged SC. Reactive astrogliosis in ALS was first revealed in 1990s by increased GFAP staining in the subcortical white matter, and later on similarly observed in the ventral and dorsal horns of the SC, as well as in the transition between gray matter and anterior and lateral funiculi, where the dystrophy of neuritis exists. SC astrocytes were shown to assume a neurotoxic phenotype in response to extracellular ATP. In SOD1G93A astrocytes this activation is mediated through P2X7 receptor signaling. Reactive astrocytes surround degenerating MNs in patients and transgenic animal models of ALS, and in particular those localized in the ventral SC of SOD1G93A mice, are a source of nerve growth factor (NGF) and NO, which are both required for MN apoptosis (Table 2). Interestingly, the NO-induced MN death was shown to be mediated by astrocytes expressing SOD1 and TDP-43 mutations. have shown that the increase in total NGF is due to the secreted pro-NGF fraction before cleavage to the mature form, achieving a twofold increased ratio in the SOD1G93 mice as compared to control conditions. Such finding may derive from the increased ALS-associated MMP-9, one of the enzymes that degrade mature NGF in the extracellular space. In this scenario, neuroprotection of MNs could be achieved by promoting pro-NGF cleavage.

35Recently, it was demonstrated that loss of GFAP did not affect disease onset and marginally shorten the SOD1H46R mice survival, indicating that GFAP only plays modulatory effects, at least in this mice. On the contrary, astrogliosis was observed in the SOD1G93A mice in either symptomatic or presymptomatic phase, preceding microglial activation. Indeed, depending on the ALS animal model used, astrocyte activation was observed to occur earlier or later and to be rather complex, increasing and decreasing in waves at different times throughout the disease (for review, see ). Despite no differences in the astrocyte number, increased GFAP labeling in the ventral horn of lumbar SC from SOD1G93A mice was observed before MN loss. Astrocytes derived from such mice evidenced to uptake glutamate less efficiently and showed a reduced trophic response to activation, deficiently protecting MNs. Attesting this low astrocyte efficiency, the mainly expressed astroglial S100B protein was found to be decreased in CSF and serum from ALS patients. Interestingly, exposure of primary cultures of astrocytes from ALS patients to CSF evidenced to enhance GFAP and S100B expression. Similarly to neurons, astrocytes have demonstrated constitutive and regulated expression of FKN, which may control the migration and function of the microglia. Additional contribution of astrocytes to MN death involves the release of ROS mediated by mitochondrial dysfunction and of D-serine that is a co-activator of the NMDARs, thus exacerbating glutamate excitotoxicity, among other factors as indicated in Table 2 and reviewed in.

36A recent publication has characterized a specific astrocytic phenotype (aberrant astrocytes) obtained from primary SC cultures of SOD1G93A symptomatic rats. These aberrant astrocytes isolated on the basis of marked proliferative capacity and lack of replicative senescence, lack GLT-1 and the NG2 marker and were shown to release toxic factors accounting for a MN hostile environment. This specific astrocyte phenotype expressing increased S100B and connexin-43 (Cx-43) is abundant in the symptomatic phase of the disease and seems to be located close to MNs, representing a new potential target for delaying ALS progression.

37Data in postmortem tissues of ALS patients revealed changes in the morphology of astrocytes together with elevated GFAP and aldehyde dehydrogenase family 1, member L1 (ALDH1L1), corroborating findings in animal models of ALS. Indeed, mSOD1 gene excision from microglia and selective reduction in astrocytes significantly slowed disease progression. However, there is still some controversy on whether astrogliosis is detrimental or beneficial, what surely will depend from spectrum intensity and toxicity potential of the aberrant phenotype.

38Microglia are the immune resident cells of the CNS with both a supporting role to neurons and astrocytes, and immunological properties with either neuroprotective or neurotoxic potential. Though microglia activation was indicated to precede astrocyte reactivity, reduced neuroprotective behavior of mSOD1 microglia in a resting-state, and at the disease end-stage was documented. If confirmed, boosting of microglia with stimulatory factors may reveal to be clinically useful.

39In a recent paper, verified that incubation of microglia with aggregated SOD1 first drives its location at the membrane level, and later within the cell. Moreover, supernatants of these SOD1 activated microglia caused a significant decrease in MN viability, which was not related with tumor necrosis factor-α (TNF-α) secretion, NO, or superoxide anion radical. The toxic factors involved are currently unknown, although it was shown that expression of mSOD1 increases TNF-α secretion, which may induce neurotoxicity by increasing the glutamate release by microglia in an autocrine manner. Therefore, a very early stage of the disease may rely in this link between protein aggregation and microglial activation. Indeed, neurotoxic potential of mutant microglia in MN degeneration in ALS is a very well documented issue: (1) mSOD1 acting on microglia is required to cause the disease; (2) limiting mutant damage to microglia slows progression; replacement of mSOD1 microglia for wt microglia delays disease and prolongs mice survival.

40Microgliosis at sites of MN injury is a neuropathological hallmark of ALS and recent therapeutic interventions are looking into factors capable of skewing microglia neurotoxic potential into a neuroprotective phenotype. However, a trophic role for activated microglia has been suggested at early stages of the disease (for review, see ). In the SOD1G93A mice, it was observed a decrease in microglia number in the entire SC at the pre-symptomatic age. The authors identified two diverse microglia subpopulations (low and high Iba1 expression cells), suggesting a distinct microglia involvement at pre- and early-symptomatic ALS stages. Interestingly, microglia heterogeneity observed between cervical and lumbar SC regions in ALS mice may derive from different environmental specificities determined by local MN/astrocyte/lymphocyte disposition and activation. While cortical microglia appear unaffected by the disease, additional studies evidenced an increased microglial number in the lumbar SC at symptom onset, and neither the typical inflammatory nor the anti-inflammatory phenotypes were identified at end-stage. In addition, elevated TNF-α gene expression and immunoreactivity were observed in lumbar SC of mSOD1 mice and related with invading microglia. Early microglia activation in ALS may be further explored in vivo by positron emission tomography (PET). The possibilities of PET suggest its valuable contribution to monitor the progression of the disease and the efficacy of the therapy in use.

41Context-dependent neuroprotective and neurotoxic properties. An important neuroprotective role of microglia is phagocytosis. Following signaling by neuronal FKN, milk-fat globule EGF factor-8 protein (MFG-E8, SED1) is up-regulated and serve as a bridge via specific integrins between apoptotic neurons and microglia (Figure 1). The authors hypothesize that MFG-E8 is assembled on the surface of exosomes and apoptotic neurons so microglia can recognize their target cells. MFG-E8 seems to be essential for microglia engulfment and removal of the dying neurons. An interesting concept is that inflammatory microglia can also phagocyte viable neurons through MFG-E8 mediation accounting for a reduced number of neurons. Phagocytic ability may, however, be lost if the cell is continuously stressed by a neurotoxic stimulus.

42Our recent data with the neurotoxic unconjugated bilirubin have evidenced that following the phagocytic ability at eliminating cell debris microglia changes to a more inflammatory phenotype and even to a senescent-like cell morphology and death if the duration of exposure is prolonged. However, microglia behavior in the presence of unconjugated bilirubin is modulated by the presence of both astrocytes and neurons. Also to consider is that during the inflammatory phenotype, microglia may intervene in the glutamate homeostasis, but may also contribute to neurite degeneration through the release of NO. Interestingly, microglia phagocytic features were shown to occur early in ALS disease. Microglia were revealed to aggregate, proliferate, and phagocyte in the lumbar SC of pre-symptomatic mutant SOD1H46R transgenic mice. However, in other studies microglia have shown to contribute to MN death and to decrease in number within disease progression, thus contributing to the disease propagation. By using in vivo imaging by two-photon laser-scanning microscopy and axonal transection, it was observed different phases of microglia-mediated inflammation in the ALS mice model. Indeed, a first phase (preclinical) with highly reactive microglia was followed by another (clinical stage) with morphologically transformed microglia presenting reduced surveillance activity and reactivity. Regional, temporal, and immune environmental differences may contribute to changes in microglia phenotypes and response heterogeneity, thus requiring differentiated immunomodulatory or even combinatory therapeutic approaches along ALS disease progression.

43Microglia phenotypes. Activation of microglia may be observed through the up-regulation of CD11b, Iba1, and CD68 markers. Primary microglia differ from other blood macrophages in the expression of CD11b/CD45low/high and CD68low/high (for review, see ), although not specific for microglia in a pathologic brain. These cells show a round morphology with an enlarged cell body and smaller and thicker processes in the resting/quiescent state (M0; Figure 2). When activated in response to an insult or injury, microglia are capable of acquiring diverse and complex phenotypes, allowing them to participate in the cytotoxic response, immune regulation, and injury resolution. Microglia may then favor the entrance of inflammatory T cells with which the cells seem to interact. Recent work have classified and characterized M1 and M2 phenotypes (Table 2) (for review, see ). The first is cytotoxic, characterized by the release of proinflammatory cytokines and influenced by T helper cell type 1 (Th1) that release GM-CSF and interferon-γ (IFN-γ), triggering M1 proliferation. Cytotoxic M1 markers include IL-1β, IL-6, TNF-α, iNOS, COX2, and CX3CL1, and known inducers are the TLR4 agonist LPS and IFN-γ.

44The M2, promoted by the cytokines IL-4 and IL-13 released by Th2, contribute to neuroprotection once they also secrete anti-inflammatory cytokines, such as IL-4 and IL-10, and growth factors such as insulin-like growth factor 1 (IGF-1). Indeed, IL-4 was shown to protect MNs from the injury produced by LPS-activated microglia. Thus, M2 polarization may be desirable, although excessive or prolonged M2 polarization may become prejudicial in allowing unwanted fibrotic responses and scarring, not facilitating axonal growth. However, there are three M2 phenotypes: the M2a or alternate activation repair/regeneration/remodeling phenotype, the M2b immunoregulatory and the M2c acquired-deactivating (Figure 2). Reliable markers for M2a stimulated by IL-4 and IL-13 are high IL-1 receptor antagonist (IL-1Ra) and high arginase (Arg1). M2b is stimulated by immune complexes, TLR agonists and IL-1R ligands. Useful markers are IL-1Ra and SOCS3. Characterization of M2c stimulated by IL-10, transforming growth factor-β (TGF-β) and glucocorticoids is obtained through the increased levels of anti-inflammatory cytokines (IL-10, TGF-β), low levels of pro-inflammatory cytokines and enhanced IL-4Rα, Arg1, SOCS3, and CD206. While LPS, IL-1β, TNF-α, and IFN-γ lead to cytotoxic M1 and immunomodulatory M2b activation states, IL-4 mainly triggers M2a phenotype.

45Therefore, balance between M1 and M2 phenotypes may be a desirable therapeutic goal. Interestingly, mSOD1 microglia isolated from ALS mice at disease onset showed higher levels of Ym1, CD163, and brain-derived neurotrophic factor (BDNF) (M2 markers) and lower levels of Nox2 mRNA (M1 marker) as compared to the end-stage disease. Interestingly, when co-cultured with wt MNs the first microglia phenotype exerted neuroprotection while the M1 phenotype was neurotoxic, supporting the pathoprogression-related changes in microglia. We may probably consider that microglia display the M2 phenotype at an early stage of the disease switching to the M1 phenotype during the late rapid phase (reviewed in ). However, we should also consider that microglia may become functionally impaired at the end-stage, as recently observed in the SOD1G93A SC microglia. Different roles of microglia depending on the neurological disease context was recently suggested by based on the observation by FACS-transcriptome comparisons that SOD1G93A microglia show a unique phenotype that differs from M1 or M2 macrophages, and from activation with LPS. Robust up-regulation of MMP-12, IGF-1 and osteopontin were pointed as hallmarks. Transcriptomic technology has been carried out to examine the gene expression changes of ALS tissue as compared to controls (reviewed in ) and in the future may provide insights into the microglia phenotype profiling prior to disease onset and along ALS progression to assist in the development of new treatments for ALS at different stages.

46Inflammatory microRNA profiling. MicroRNAs (miRNAs), small, non-coding RNAs, have been recently pointed to mediate cell-to-cell communication. Inflammatory phenotypes are miR-155, miR-21, miR-146a/b, and miR-124. Recent studies demonstrated the miR-124 involvement in promoting microglia quiescence by skewing their polarization from an M1 to an M2 phenotype and that miR-155 together with miR-124 are likely to be directly related to M1 and M2 phenotypes, respectively. Up-regulation of miR-155 is induced by TNF-α and Il-1β, as well as by HMGB1 through the activation of the TLR2/MyD88/miR-155 pathway, while it is down-regulated by TGF-β. Increase of miR-146a expression was shown to occur in the aged mice, as well as after LPS stimulation, and is related with the microglia phagocytic potential. Moreover, up-regulation of miR-21 in murine models of SC injury was pointed as a modulator of the pro-reactive effects of inflammatory signaling cascades. Dysregulation of miRNAs expression was lately found in the SC of ALS patients and in microglia isolated from the hSOD1G93A mice. Promising candidates that were found to be altered in patients were miR-146a*, miR-524-5p, and miR-582-3p, while those up-regulated in the mice model were miR-155, miR-146b, miR-22, miR-365, and miR-125b, which need to be further investigated for their relevance as ALS biomarkers and therapeutic targets. Actually, it was recently discovered that inhibition of miR-155 prolongs survival in the mSOD1 mice.

47Autotaxin. Autotaxin (ATX) is a secreted lysophospholipase D that converts lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA), a phospholipid growth factor that activates several transduction pathways, and is involved in migration, proliferation, and survival of various cells. LPA acts as an autocrine and/or paracrine signaling molecule mediating a broad range of intracellular signaling cascades, especially the RHOA pathway (reviewed in ). LPA receptors are expressed by astrocytes, oligodendrocytes, and microglia.

48Data suggest that ATX levels increase within reactive astrocytes following neurotrauma, but other studies have obtained a down-regulation instead. Most fascinating, it was observed that overexpression of ATX inhibits microglial activation and protects the cell against oxidative stress. Thus, reduced expression of ATX in microglia may contribute to a sustained neuroinflammation condition. Since ATX is also a motility driver that stimulates cell invasion it can be involved in microglia migration, a property not exploited till now. Although ATX is considered an inflammatory mediator we were not able to find studies demonstrating its relevance or not in ALS.

49BBB and BSCB are dynamic and complex interfaces between the blood and the CNS. Endothelial cells and tight junctional complexes physically limit solute exchanges between the blood and the brain. These cells together with pericytes, astrocytes, neurons, microglia, and oligodendrocytes and the basement membrane form the neurovascular unit (for review, see; ). The interaction between all these components provides a sustainable environment for neural function while restricting permeability and transport. Alterations in barrier properties and dynamics were observed in transgenic SOD1 rats. In particular, it was noticed a reduction of endothelial tight junctions, together with the disruption of the neurovascular unit and up-regulation of MMP-9 prior to MN degeneration. MMP-9 activation was observed in blood vessel-like structures and microglia. Increased microvascular microglia, expressing CX3CR1 and weakly labeling Iba1, were detected in the SCs from the ALS mice model and suggested to have a bone marrow origin. In addition, it was recently demonstrated that SC microglia in the mSOD1 mice promote the recruitment of inflammatory monocytes into the CNS well before the onset of the disease, triggering microglia apoptosis (Table 2). Actually, the BSCB has been shown to be disrupted in different mSOD1 mice before MN degeneration, thus favoring blood monocytes infiltration. To that it may account the 54% reduction in pericytes at the BSCB level in patients with ALS. In fact, some authors consider ALS as a neurovascular disease with evidence indicating impairment of all neurovascular unit components, including the BBB and the BSCB in both ALS patients and animal models. Disruption of BBB and BSCB was observed in the G93A mice by electron microscopy in places of MN degeneration at both early and late disease stages. The damage of barriers besides allowing the entrance of T lymphocytes, such as CD4+ (helper/inducer) and CD8+ (cytotoxic) into the brain parenchyma, may also permit the entrance of harmful substances that will contribute to disrupt neuronal homeostasis and accelerate MN degeneration. The impact of microvascular damage in ALS pathology is a novel and promising research topic in that interactions with glia may determine neuroprotective or cytotoxic cell phenotypes with consequences on MN survival.

50Communication between neurons and microglia is essential for maintaining homeostasis in the CNS and altered cross-talk is implicated in the pathogenesis of ALS and other MN diseases. Dysregulated neuron–neuron signaling and neuron–microglia cross-talk in ALS, and other neurodegenerative diseases, may derive from: (i) secretion of toxic mediators or fail to secrete trophic factors, or both, resulting in reduced function and survival of MNs; (ii) SFs released by neurons and their action on microglia receptors; (iii) changes in direct cellular interactions.

51Reduced expression of trophic factors such as BDNF, fibroblast growth factor 2 (FGF2), and IGF-1 was found in the SC of the newborn rat after intrathecal administration of CSF from ALS patients. To note, that the release of mSOD1 was shown to trigger microgliosis and neuronal death and increased levels of ROS/RNS, mainly of NO, were shown to play a critical role in the earliest stages of neuronal dysfunction in ALS. NO mainly produced by glia through the activation of iNOS can react with superoxide producing the potent oxidant peroxynitrite, which mediates apoptosis or necrosis, depending on the concentration. Peroxynitrite also induces toxic glial phenotypes further propagating oxidative damage and cellular dysfunction. Interestingly, the same concentrations of NO may either promote the survival of MNs healthy conditions or induce apoptosis and glia reactivity when in the presence of stressors. Up-regulation of neuronal NO synthase (nNOS) was shown to occur in MNs before iNOS in either pre-symptomatic ALS animal models or in patients and to be associated with MN loss.

52Activated microglia and astrocytes amplify the initial damage to the MNs by activating AP-1 and NF-κB through production of proinflammatory cytokines and apoptosis-triggering molecules such as TNF-α and Fas ligand (FASL). TNF-α and IL-1β exert neurotoxic effects in vitro, but deletion of the individual genes seems to not affect the course of the disease. In addition, dying MNs release ATP that can further activate glia through the purinergic receptor P2X7 expressed by both microglia and astrocytes (Figure 2). Classical microglia activation results in upregulation of MHC class II proteins that are involved in presentation of antigens to T lymphocytes. Microglia activation is mediated by the release of SFs and/or expression of surface receptors by neurons and astrocytes, such as CX3CL1, CD200, and CCL21. Microglia also express a diverse set of pattern recognition receptors (PRRs) for pathogen-associated molecular patterns (PAMPs) and endogenous ligands derived from injury that include TLRs and inflammasome. In addition, emerging evidence suggests that members of the nuclear receptor (NR) family of transcription factors, many of which are ligand-dependent, control the activation of microglia under physiological and pathological conditions (for review, see ).

53Neuroinflammation involves the activation and proliferation of microglia and the infiltration of T cells into the brain and SC. In these conditions, astrocytes and microglia release IL-1β, TNF-α, and IL-6 (Figure 2). Cytokines can then up-regulate oxidative stress by NO and superoxide (O2-) generation (for review, see ). Repair and limitation of the damage is promoted by the release of trophic and anti-inflammatory factors. Deleterious microglia M1 and benign M2 phenotypes are influenced by astrocytes and T cell subsets. In ALS, it is still currently unknown the precise function of microglia and astrocytes and how they mediate neuroinflammation and contribute to the pathology. Identification of the factors driving microglia phenotype and consequent functional changes, once known, may advance our knowledge on their role and on ways to modulate these cells. In addition, it will be important to clarify whether inflammation contributes to ALS pathogenesis or in opposite is a protective response, and if different individual immune responses to the disease are also implicated, so that immunomodulatory therapies can be pursued.

54Evidence for autoimmunity in ALS was proposed in 1990s (for review, see ). Inflammation is not a resultant from MN degeneration since it regulates the balance between neuroprotection and neurotoxicity. Initially, the diverse populations and phenotypes of CD4+ T cells that cross-talk with microglia can slow disease progression, but later they may contribute to the acceleration of the disease. The inflammatory process involves infiltration of T cell subpopulations at sites of neuronal injury in the brain parenchyma. T cells may damage MNs by cell–cell contact or cytokine secretion through the activation of microglia. CD8+ cytotoxic T cells and natural killer T (NKT) cells were found significantly increased in patients with ALS, while regulatory T (Treg) cells were decreased. It was suggested that CD4+ T cells may trigger oxidative phosphorylation in microglia and CD8+ may stimulate phagocytosis accordingly to data obtained by FACS-transcriptome comparisons in cells isolated from the SC of SOD1G93A mice.

55The naive T cells, or Th0 cells, expand and differentiate into at least four functionally distinct subsets upon stimulation: Th1, Th2, Treg, and Th17 cells. Th1 cells secret IFN-γ and turn resting microglia into M1 phenotype; in contrast Th2 and Treg cells release IL-4 which induces activation of resting microglia into M2 phenotype (for review, see ). Th1 and Th17 are CD4+ T cells that produce proinflammatory cytokines, causing damage while CD4+CD25high T lymphocytes (Treg) suppress Th1 cell effector function.

56Interestingly, it was demonstrated that mSOD1 mice lacking CD4+ T cells evidence a faster disease progression and decreased microglia reactivity and as so, future therapeutic interventions should consider the benefits that T cells may have. Compromise of Treg lymphocytes with disease progression diminishes the secretion of IL-4 and fails in suppressing the toxic properties of microglia. Indeed, mSOD1 Treg suppress immune toxicity by inhibiting microglial activation, CD4+CD25- (T effector cells) proliferation, and the accompanying cytotoxicity, thus providing MN protection in ALS. As disease progresses, the supportive Treg/Th2/M2 changes to an injurious Th1/M1 response triggering increased TNF-α secretion that was shown to induce the dysfunction of Tregs. Therefore, elevation of Tregs in patients with ALS may trigger a longer life expectancy.

57It was before considered that the mutant protein was the responsible for the disease onset in MNs with microglia and astrocytes only determining disease progression and extent. However, expression of mSOD1 in microglia and astrocytes is now being related with the disease onset and early stage disease, while healthy SOD1 in those cells was shown to delay ALS progression.

58Several studies evidenced the demise of MNs in the presence of astrocytes harboring SOD1 mutations, attesting the non-cell-autonomous pathology in ALS, i.e., degeneration of MNs requires mSOD1 expression in other cells additionally to neurons. This finding was similarly obtained for astrocytes derived from ALS patients, which have shown to cause MN death. Deregulation between astrocytes and MNs communication seems to involve TGF-β signaling pathways and miR-124a mediated regulation. Evidence for the role of miRNAs in MN diseases is substantiated by the relevance that the proteins TDP-43 and FUS/TLS, responsible for the processing of miRNAs, RNA maturation and splicing, have recently acquired in ALS. A major challenge would be to establish circulating miRNAs as particularly accessible biomarkers to monitor ALS.

59Activated astrocytes (see Astrocyte Reactivity for more details) may also interfere with MNs function due to the reduced secretion of trophic factors such as BDNF, glial cell line-derived neurotrophic factor (GDNF) and vascular endothelial growth factor (VEGF), but this is not a clarified issue yet (for review, see ).

60Both in vitro and in vivo experiments have shown that astrocytes and microglia containing mSOD1 exert deleterious effects on MNs, by releasing proinflammatory factors. Microglia may respond earlier than astrocytes to injury and stress by first activating NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways, thus leading to a faster release of TNF-α and IL-1β. In that way one may believe that the activation of microglia precedes the reactivity of astrocytes and depends from the factors and cytokines that microglia release. Curiously, TNF-α and IL-1β released from activated microglia were reported to produce an inhibitory effect on Cx-43 expression, the main constitutive protein of gap junctions. Therefore, blockage of communication between astrocytes by the activated microglia can contribute to decrease the neuroprotective role of astrocytes. When considering the p25-mediated neuroinflammation, astrogliosis was shown to precede microglia activation, and apparently mediate the production of LPC, which as a chemoattractant for T cells may recruit peripheral cells into the brain. Recently, it was shown that astrocytes depend on functional microglia for response to LPS and to TLR2, 3, and 4 ligation. In their absence, astrocytes did not respond to TLR4 ligation and only weakly responded to TLR2 and 3 ligation. Thus, activation of astrocytes may be modulated by the proinflammatory cytokines released from microglia in an attempt to diminish the extent of excitotoxicity, but they can modulate microglia activation, as well. Which cells between microglia and astrocytes are first activated by the aggregated mSOD1 is a very controversial issue since different studies have obtained diverse profiles of glial activation in ALS models along disease progression. Some studies point out that microglia activation precede astrocyte reactivity. Others that astrogliosis is initiated in the symptomatic phase, while prominent microgliosis is only evident later at the moribund phase. However, since a close interaction/communication of both cells occurs in ALS, a better understanding of the benefits and risks of astrocyte and microglia activation in ALS will help to determine whether therapeutic strategies should envisage enhancing or impairing the actions of glial cells in ALS.

61Communication between MNs and microglia is essential for maintaining local homeostasis during both physiological and inflammatory conditions. Neuroprotective signaling from MN to microglia involve FKN and CD200, as previously mentioned. In addition, microglia “calming” effects during neuroinflammation are mediated through ATX. Insights into the complex intercellular perturbations and influence of alarming and calming factors underlying neurodegeneration will enhance our efforts toward target-driven directed therapeutic strategies in ALS.

62The neuroprotective/neurotoxic role of CX3CL1/CX3CR1 signaling is still a matter of debate once it seems to depend upon the CNS insult (for review, see ). Deletion of CX3CR1 in a transgenic model of ALS mice was shown to extend neuronal cell loss, suggesting that CX3CL1/CX3CR1 signaling limits microglial toxicity in ALS. Interestingly, it was also shown that treatment with LPS down-regulated the expression of CX3CR1, thus suppressing the functional response to FKN and potentiating LPS neurotoxic effects. Accordingly, in ALS, the disruption of CX3CL1/CX3CR1 signaling evidenced to promote neurodegeneration following LPS administration.

63Although it has been proposed that cathepsins compensate for each other because of their overlapping substrate specificities, there is increasing evidence that disturbance of the normal balance and extralysosomal localization of cathepsins contribute to age-related diseases (for review, see ). CatS that is a lysosomal/endosomal cysteine protease that degrades extracellular matrix proteins, even at neutral pH, was shown to be increasingly expressed by microglia upon LPS surcharge. However, some authors indicate that a further challenge with ATP is required to observe CatS release. This protease is preferentially expressed by antigen-presenting cells, including microglia.

64Microglial CatS seems to be responsible for the liberation of neuronal FKN (Figure 1), which via the specific receptor CX3CR1 in microglia activate p38 MAPK pathway leading to the release of mediators that interact with neurons. Several inhibitors have been tried to regulate the immune modulatory effects of CatS and a shift from a Th1/Th17 type response to a Th2 type of response was obtained.

65The up-regulation of cathepsins appears as a general transcriptional event in ALS. However, reference to CatS and microglia activation was not until now indicated in the context of ALS. Therefore, if CX3CL1/CX3CR1 demonstrates to have a role in any stage of ALS progression, inhibition of CatS may constitute a therapeutic approach for ALS.

66Exosomes are secretory vesicles deriving from late endosomes and multivesicular bodies that mediate neuron–glia communication, have 50–100 nm in size and carry specific protein and RNA cargo. Exosomes contain both miRNAs and mRNAs that may be delivered and be functional in another cell. Selectivity for miRNA incorporation into exosomes is proposed based on the fact that some are exclusive in exosomes derived from immature dendritic cells while others only exist in those from mature dendritic cells. Indeed, exosomes and miRNAs have been found to participate in cellular senescence and contribute to aging. In addition senescent cells produce high levels of exosomes thus interacting and inducing the senescence of neighboring cells. As so, exosomal miRNAs might become useful biomarkers of disease. Recently, it was suggested that exosomes may be important candidates to deliver siRNA and specific drugs, based on the perceived advantages of nanoparticle size and non-cytotoxicity, thus constituting a therapeutic platform.

67Microglia was shown to internalize oligodendroglial exosomes, thus participating in the degradation of oligodendroglial membrane. However, microglia also release exosomes, which generate IL-1β to the extracellular environment propagating inflammation. Increased exosome discharge by microglia was observed after stimulation with α-synuclein and such activated exosomes revealed an increased membrane content in TNF-α. Interestingly, it was shown that mouse MN-like NSC-34 cells overexpressing hSOD1G93A secrete SOD1 via exosomes probably accounting to cell–cell-mediated mutant toxicity in ALS pathogenesis. Therefore, exosomes from microglia may spread pathogenic factors, such as SOD1 and promote inflammation while influencing neuronal survival. In line with this, a latest study reports that cell-to-cell transmission of SOD1 misfolding is mediated by two non-exclusive mechanisms: through the release of protein aggregates that are taken up by macropinocytosis or via exosomes secreted from living cells.

68Reconstruction of neural network implies the restoration of tissue architecture and cell functionality. Cell replacement-based repair strategies have been tested both in vitro and in vivo and a major challenge resides in maintaining the cell function when transplanted to a broken parenchyma homeostasis. Confront with excessive neuroinflammation and cell senescence may compromise the success of the strategy. In particular, reactive astrocytes exacerbate inflammation and by forming glial scars impede regenerating axons from traversing the lesions, while myelin debris prevent axon growth and microglia lose the ability to migrate, phagocyte or sustain inflammation from spread. Therefore, the rewiring of the CNS to foster a permissive environment for neuroregeneration is the key to a successful functional integration and repair.

69Redox changes within neurons, ER stress condition, and mitochondria dysfunction are accelerated by aging and are emerging as common features relevant to the pathogenesis of neurological disorders, including ALS. Actually, the variation in SOD1 activity in aging ALS patients, when compared to younger ones, point to an increased oxidative misbalance vulnerability. Nevertheless, it was recently indicated that the ALS incidence decline in the elderly, suggesting that the disease is not merely the result of aging. However, markers of senescence were found increased in satellite cells from ALS muscle biopsies suggesting a vulnerability to muscle atrophy. In addition, autophagic dysfunction and mitochondrial DNA damage in the CNS are prominently found in microglia with aging and may lead to a defective turnover of mitochondria and accumulation of hypergenerated ROS (reviewed in ). Interestingly, dysfunctional and senescent microglia may even release compounds that inhibit neuronal autophagy and neurogenesis (reviewed in ). Given the important and necessary functions of microglia in the CNS homeostasis it is of major relevance to understand the multiple stage-related microglia phenotypes in ALS, including the increased vulnerability to a senescent cell with the disease progression, as observed in other neurodegenerative and age-related CNS disorders.

70Recent data obtained in the mSOD1 mouse model suggest a dominant neuroinflammatory response in the CNS, with a reactive microglia in preclinical stages that turns into an irresponsive cell during disease progression, and a degenerative process in the PNS. Earlier studies evidenced that mutated SOD1 microglia have an age-dependent cytotoxic potential which reveals upon a stimulatory effect. Interestingly, extracellular SOD1G93A mediates the activation of CD14–TLR2 pathway with the consequent release of TNF-α and IL-1β, thus propagating the proinflammatory stimuli. However, since immunosuppressive strategies have not proven consistent efficacy, one may believe that microglia function may change along disease progression with consequent differential effects on MNs. Late data evidenced that SC microglia proliferate and that TNF-α mRNA expression decreases during disease progression in SOD1G93A rats. Indeed, microglia revealed to not be polarized to M1 or M2 phenotypes at any disease stage and CNS region evaluated. In addition, SC microglia evidenced to be irresponsive to experimental systemic inflammation at ALS end-stage. Diminished glial neuroprotection by senescent and/or dysfunctional microglia has been suggested to play a role in neurodegenerative diseases, mainly in the late stage. In such circumstances the aged microglia evidenced a “dystrophic” morphology with the loss of finely branched cytoplasmic processes, cytoplasmic beading/spheroid formation, and cytoplasmic fragmentation (cytorrhexis; Figure 2). Such severe abnormalities in microglia, including cell fusion (multinucleated giant cells) at the symptomatic stage and cytorrhexis at the end stage are indicative of microglial aberrant activation and degeneration, respectively, and were observed in SOD1G93A transgenic rats. Although prevalent in older human subjects this dystrophic cells may also in rare instances be observed in the young brain. Pathogenic miRNAs, such as miR-155, miR-146a, and miR-124 in microglia may be associated with the acquisition of a senescent phenotype. We recently observed that aged-cultured microglia exhibit lower phagocytic ability and higher expression levels of miR-146a than younger cells. Whether these findings are related with a dysfunctional microglia at ALS end-stages deserve to be further investigated.

71Although new oligodendrocytes were observed in the SC of SOD1G93A mice, they do not fully maturate, resulting in progressive demyelination and accelerated disease. The authors also found similar myelination defects in postmortem samples taken from SC and motor cortex from ALS patients. Actually, there are many potent inhibitors of axonal regeneration in the injured CNS including myelin-associated proteins, fibrinogen, and axonal guidance molecules, where epidermal growth factor receptor (EGFR) and eukaryotic ribosome biogenesis protein 1 (Erb1) may have a special role in reducing the effects of multiple inhibitors of axonal regeneration. However, the role of EGFR on the protection of MN synapses and survival extension in SOD1G93A mice is still a matter of debate; hence, it was reported that EGFR inhibitors failed to extend ALS mouse survival although influencing disease progression.

72Another important point to be considered is the decrease in the phagocytic clearance by the dysfunctional microglia that results in the accumulation of myelin debris, leading to oligodendrocyte differentiation arrest and decreased recruitment of oligodendrocyte precursor cells. Activated microglia were also shown to attenuate the proliferation of the oligodendrocyte precursor cells, thus concurring for demyelination progression, and reinforcing the recovery of healthy microglia as a potential therapeutic target in ALS. Indeed, studies on the global gene expression during demyelination and remyelination by microarray analysis reinforced that the primary function of microglia is the tolerance induction and support to regeneration.

73Cell replacement therapy has been suggested as a promising strategy for MN disease. The use of combined strategies to restore both the healthy state of MNs and glial cells, such as microglia, and their correct cross-talk to face persistent neurotoxic insults may even provide better benefits for this devastating disease.

74Mesenchymal stem cells (MSCs) isolated from the bone marrow of ALS patients did not show morphological or functional differences from those obtained from donors and seem to be useful for cell-based therapy for ALS patients. In a few well-monitored ALS patients the autologous transplantation of such MSCs into the SC evidenced to be well tolerated and to promote some clinical improvement.

75Another proposed strategy is the olfactory ensheathing cell (OEC) transplantation that although evidencing to slow the rate of ALS progression in a short period and to improve pulmonary function has been a matter of debate. Indeed, prominent glial and inflammatory reaction around the brain delivery track was observed in postmortem samples.

76Efficacy of transplantation-based astrocyte replacement was also evidenced as a promising therapy for slowing focal MN loss associated with ALS while also reducing microgliosis in the hSOD1G93A rodents. Effects may derive from the release of growth factors that are decreased in ALS and already evidenced to increase mSOD1 mice survival. However, limited efficacy was obtained in a later study. The use of induced pluripotent stem cell (iPSC) technologies may allow in the future the autologous cell transplantation in ALS patients, including MNs.

77More recently, depletion of microglia cells expressing mSOD1 with clodronate liposomes and subsequent transplantation with bone marrow cells (BMCs) expressing wtSOD1 was shown to trigger microglia replacement and to slow ALS disease progression in the SOD1G93A mice model. The method seems to afford better therapeutic effects than the one by using BMCs transplantation since microglia renewal is better achieved by tissue-resident microglia rather than by BMCs. Overall, the mechanisms and functional implications of microglia replacement require further elucidation, inasmuch because myeloid-derived infiltrating cells (monocyte-derived macrophages) revealed to be functionally distinct from the resident microglia assisting them. Clearly, additional research is required to address these issues and contribute to develop strategies able to stop, or at least delay, ALS progression.

78Amyotrophic lateral sclerosis is a fatal neurodegenerative disorder with limited identified targets, biomarkers, and therapeutic options. Therefore, a better comprehension of the underlying molecular mechanisms is necessary to develop novel etiological therapeutic strategies. Multiple studies suggest that a complex pathological interplay between MNs and glial cells, involving neuroinflammation and microglia physiopathological changes drive the performance of these glial cells before the ALS onset and during disease progression till late and end stages. In this context, we here summarized the growing body of evidence supporting the key role of microglia in the deregulated motor-neuron interconnectivity and in the dreadful chain of events leading to MN degeneration in ALS. As such, insights into the complex intercellular perturbations underlying ALS disease and centered on microglia phenotypic changes, and associated detrimental functions, will help on our efforts to develop effective therapeutic approaches for recovering adequate microglia function in initiation and progression phases of ALS. In conclusion, the role of microglia in keeping brain homeostasis leads to consider that healthy microglia may be used to replace the senescent or irresponsive cell. In addition, dysfunctional microglia may be differently modulated, either directly or indirectly, to be transformed in a less reactive cell to challenges in excessive and chronic neuroinflammation, or alternatively rejuvenated to enhance their capacity to fight the insult and improve disease outcomes. Considerably more research is necessary to realize the feasibility and usefulness of such strategies before their potential in clinic can be realized.

79The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.