Edited by:
Reviewed by:
*Correspondence:
This article was submitted to the journal Frontiers in Cellular Neuroscience.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Amyotrophic lateral sclerosis (ALS) is the most common and most aggressive form of adult motor neuron (MN) degeneration. The cause of the disease is still unknown, but some protein mutations have been linked to the pathological process. Loss of upper and lower MNs results in progressive muscle paralysis and ultimately death due to respiratory failure. Although initially thought to derive from the selective loss of MNs, the pathogenic concept of non-cell-autonomous disease has come to the forefront for the contribution of glial cells in ALS, in particular microglia. Recent studies suggest that microglia may have a protective effect on MN in an early stage. Conversely, activated microglia contribute and enhance MN death by secreting neurotoxic factors, and impaired microglial function at the end-stage may instead accelerate disease progression. However, the nature of microglial–neuronal interactions that lead to MN degeneration remains elusive. We review the contribution of the neurodegenerative network in ALS pathology, with a special focus on each glial cell type from data obtained in the transgenic SOD1G93A rodents, the most widely used model. We further discuss the diverse roles of neuroinflammation and microglia phenotypes in the modulation of ALS pathology. We provide information on the processes associated with dysfunctional cell–cell communication and summarize findings on pathological cross-talk between neurons and astroglia, and neurons and microglia, as well as on the spread of pathogenic factors. We also highlight the relevance of neurovascular disruption and exosome trafficking to ALS pathology. The harmful and beneficial influences of NG2 cells, oligodendrocytes and Schwann cells will be discussed as well. Insights into the complex intercellular perturbations underlying ALS, including target identification, will enhance our efforts to develop effective therapeutic approaches for preventing or reversing symptomatic progression of this devastating disease.
Amyotrophic lateral sclerosis (ALS) is a non-cell-autonomous disease targeting motor neurons (MNs) and neighboring glia, with microgliosis directly contributing to neurodegeneration (
For 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 (
There are several
It 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 (
Finally, 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.
Neuronal 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, 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 (
Moreover, ALS progression was suggested to involve cell-to-cell transmission of mSOD1 aggregates involving MNs, microglia and astrocytes, similarly to prion disease (
Another 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.
Thus, 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 (
Functional alterations of motor neurons (MNs) in amyotrophic lateral sclerosis (ALS): candidate molecular targets.
Changes in MN signaling by ALS | ALS stages | Reference |
---|---|---|
Elevation of matrix metalloproteinase (MMP)-9 | Pre-symptomatic phase | |
Impaired antioxidative Keap1/Nrf2/ARE system | Along disease progression | |
Oxidative and nitrosative stress | Early phase | |
Glutamate excitotoxicity | Onset and progression | |
Release of ATP | Along disease progression | |
Protein misfolding, aggregation, and accumulation | Onset and progression | |
Changes in fractalkine (CX3CL1), CD200, and CCL21 | Not clarified | – |
Decreased high-mobility group box protein 1 (HMGB1) cellular expression | Advanced phase | |
Release of neuregulin-1 (NRG1) | Along disease progression | |
Up-regulation of major histocompatibility complex (MHC) class I and β2-microglobulin mRNAs | Along disease progression |
Glial impairment and deregulated glia–motor neuron (MN) interconnectivity in amyotrophic lateral sclerosis (ALS).
Mutant SOD1 cells | Loss of supportive functions | Contribution to ALS disease and MN death | Reference |
---|---|---|---|
Astrocytes | Deficient astrocyte-specific glutamate transporter EAAT2 (GLT-1) | Increase in the excitatory amino acid glutamate | |
Increased release of |
co-activator of the |
||
Mitochondrial dysfunction | Increased production of reactive oxygen species (ROS) | ||
Release of interferon-γ and transforming growth factor-β (TGF-β) | Increased neuroinflammation | ||
Ubiquitin- and active caspase-3-immunopositive | Degenerating astrocytes at the pre-symptomatic stage when MNs show axonal damage but are still alive | ||
Increased nerve growth factor (NGF) and NO production | MN apoptosis | ||
Astrocytes (aberrant) | Increased S100B and connexin-43 (Cx-43) | Decreased MN survival | |
Microglia (spinal cord – early stage) | Recruitment of peripheral monocytes to the CNS | Neuronal viability impairment | |
Microglia (spinal cord – end stage) | Decreased expression of M1 and M2 markers | Decreased reactivity to stimuli | |
Microglia (M2) – early stage | High levels of anti-inflammatory cytokines and neurotrophins | Enhancement of MN survival (neuroprotection) at ALS early stage | |
Microglia (M1) – progressive stage | Increased release of reactive oxygen species (ROS), tumor necrosis factor-α (TNF-α) and interleukin (IL)-1β | Toxicity to MN (death) in the late rapid phase of ALS | |
Dystrophic microglia – end stage | Decreased migration and phagocytosis by aging (not yet confirmed in ALS) | Neuronal degeneration by failure of the senescent microglia response to stimuli | |
Oligodendrocytes | Loss of the monocarboxylate transporter 1 (MCT1) | Decreased delivery of the metabolic substrate lactate to MNs and axonal sufferance | |
NG2+ cells | Increased proliferation rate and degeneration of early-born oligodendrocytes | Gray matter demyelination | |
Schwann cells | Signs of distress at the asymptomatic stage | Not known |
A 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) (
SOD1 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
Oxidative damage at the level of proteins, lipids, and DNA was also observed in the transgenic hSOD1 mice models (
Currently, 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 (
Glutamate 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 (
Besides 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
Fractalkine (FKN) mRNA is dominantly expressed in neuronal cells, particularly in those at the cortex, hippocampus, caudate putamen, thalamus, and olfactory bulb (
Under stimulation, such as an excitotoxic stimulus (
In 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 (
Taken together, FKN seems to have both intrinsic and anti-inflammatory properties in the CNS and to act by interfering with toxic microglial–neuron interactions (
High-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
CCL21 was shown to be implicated in signaling neuronal injury to microglia through the receptor CXCR3. CCL21 expression was demonstrated to increase in cortical neurons,
The 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 (
Neuregulins 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 (
Major 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 (
It 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 (
Prominent 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 (
Glial cells (astrocytes, microglia, oligodendrocytes, and Schwann cells), as well as neurons are known to express different members of the TLR family (
When 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 (
Oligodendrocytes 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 (
Considering 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 (
Although 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 (
Little 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 (
Astrocytes 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 (
Recently, 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 (
A recent publication has characterized a specific astrocytic phenotype (aberrant astrocytes) obtained from primary SC cultures of SOD1G93A symptomatic rats (
Data in
Microglia 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 (
In a recent paper,
Microgliosis 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
Our 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 (
The 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 (
Therefore, 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 (
Data suggest that ATX levels increase within reactive astrocytes following neurotrauma (
BBB 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
Communication 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 (
Reduced 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 (
Activated 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
Neuroinflammation 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 (
Evidence for autoimmunity in ALS was proposed in 1990s (for review, see
The 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
Interestingly, 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 (
It 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.
Several studies evidenced the demise of MNs in the presence of astrocytes harboring SOD1 mutations (
Activated 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
Both
Communication 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 (
The 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
Although 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
Microglial CatS seems to be responsible for the liberation of neuronal FKN (
The up-regulation of cathepsins appears as a general transcriptional event in ALS (
Exosomes 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 (
Microglia was shown to internalize oligodendroglial exosomes, thus participating in the degradation of oligodendroglial membrane (
Reconstruction of neural network implies the restoration of tissue architecture and cell functionality. Cell replacement-based repair strategies have been tested both
Redox changes within neurons (
Recent 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 (
Although new oligodendrocytes were observed in the SC of SOD1G93A mice, they do not fully maturate, resulting in progressive demyelination and accelerated disease (
Another 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 (
Cell 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.
Mesenchymal 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 (
Another proposed strategy is the olfactory ensheathing cell (OEC) transplantation that although evidencing to slow the rate of ALS progression in a short period (
Efficacy 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 (
More 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 (
Amyotrophic 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.
The 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.
This work was primarily supported by FEDER (COMPETE Programme) and by National grants from Fundação para a Ciência e a Tecnologia (FCT) through the Projects PTDC/SAU-FAR/118787/2010 and PEst-OE/SAU/UI4013/2011-2013 to Dora Brites. Ana R. Vaz was recipient of a post-doctoral fellowship from FCT (SFRH/BPD/76590/2011).