motor neurons - astrocyte - microglia - amyotrophic lateral sclerosis - motor neuron
disease
neurçnios motores - astrócitos - microglia - esclerose lateral amiotrófica - doença
do neurçnio motor
Motor neuron disease represents an important group of adult-onset progressive neurodegenerative
motor conditions, typified primarily by upper and lower motor neuron compromise in
amyotrophic lateral sclerosis (ALS)[1],[2]. Amyotrophic lateral sclerosis results from progressive neurodegenerative processes
of the central nervous system (CNS) involving the motor cortex, brainstem motor nuclei
and anterior horn of spinal cord[3]. Specific clinical, electrophysiological and neuroimaging findings guarantee a definitive
diagnosis by using the revised El Escorial diagnostic criteria for ALS[1]. Although alpha motor neuron and cortical upper motor neuron diseases establish
the motor clinical commitment, its pathophysiological mechanisms are wider and involve
a complex network of cell interactions, non-neuronal cell roles and molecular mechanisms
involving dysfunction of glial cells. Despite great improvement in its proper clinical,
neurophysiological and radiological diagnosis, pharmacologic management is still based
on symptom exclusion therapy measures[4].
Different mechanisms have been postulated as dysfunctions in ALS including motor neuron
oxidative stress (including endoplasmatic reticulum stress), glutamate direct excitotoxicity,
dysfunction in electrolytes and vesicular homeostasis, disruption of axonal transport
(of proteins and mitochondria along microtubules), exosome and vesicular trafficking
(including dysregulated endosomal trafficking), motor neuron apoptosis (including
mitochondria-mediated mechanisms), dysfunction in the ubiquitin-proteasome system
and in autophagy, mitochondrial dysfunction, aberrant RNA metabolism and processing,
and glial cell pathology[5],[6],[7],[8]. There is also growing evidence that other underlying hypothetical mechanisms affecting,
primarily, the glial cells should include emergent latent virus infection and misfolded
infectious protein (prion-like effect)[7]. This review aims to summarize the most important data established on the role of
glial cell types in ALS.
PRIMARY GLIAL CELL TYPES AND FUNCTIONS RELATED TO MOTOR NEURODEGENERATION
PRIMARY GLIAL CELL TYPES AND FUNCTIONS RELATED TO MOTOR NEURODEGENERATION
Most information about physiopathological mechanisms involved in ALS has been through
post-mortem histopathological evaluation and from mouse models related to different
induced mutations, mainly the human transgenic mouse model with G93A mutation in the
gene SOD1 (Cu-Zn superoxide dismutase-1), involved in an autosomal inherited form of ALS, with
overexpression of SOD1 protein. In motor neuron disease linked to TAR DNA-binding
protein 43 (TDP43), fused in sarcoma protein (FUS), ALS2 gene, VAPB gene, OPTN gene and C9ORF72 gene (chromosome 9 open reading frame 72) mechanisms[7],[9], few data regarding the involvement of glial cells are available, compared with
cases related to SOD1 gene mutations.
There is no doubt about the importance of cellular and non-neuronal extracellular
microenvironments in the regulation of mechanisms involved in neuron survival[10] and in early stage activation of astroglial and microglial cells in ALS pathogenesis[11]. Changes in the adequate formation and balance of the neuron-glia network and abnormal
assembly of its components represent one of the main mechanisms related to different,
slowly progressive, focal and global neuropsychiatric dysfunctions, such as ALS[10].
Although nearly all glial cells are involved in ALS pathogenesis, astrocytes and microglia
have major roles[12] ([Figure 1]). Protoplasmic and fibrous astrocytes, microglial cells and oligodendrocytes represent
the major types of glial cells in the CNS and most non-neuronal pathological processes
related to neurodegeneration involve these cell types. The role of ependymal cells
and choroid plexus epithelial cells are not yet understood, specifically in ALS pathogenesis,
and have not been adequately studied[10]. Mechanisms involving other glial cell types including Schwann cells, oligodendrocytes
and NG2 (nerve-glia factor 2 proteglycan antibody) positive cells are also not well
established[12].
Figure 1 The ALS molecular profile. Glial cells interact with neurons in many different ways
to maintain neural tissue health, for which a peculiar cytoarchitecture is required,
as shown. Molecular mechanisms and molecules recently implicated in the pathophysiology
of motor neuron disease are listed, according to cell types.
Neuropathological studies have shown that reactive astrocytes, microglia activation,
and macrophage and T-lymphocyte infiltration of neural tissues overcoming classical
motor neuron dysfunction in ALS[3] ([Figure 2]). There is a fine immune cross-talk in ALS pathogenesis in a suggestive immunopattern
shown by reactive microglia (with a mixture of minor expression of CX3CL1 and its
receptor and CD200 and its receptor and major expression of IL-6, IL-12, TNF-α and
reactive oxygen species); activated macrophages (with higher expression levels of
IL-1β, COX2 and glycoprotein CD68), reactive astrocytes (raised SDF-1α); and reactive
T-lymphocytes (starting with a preponderance of regulatory T lymphocytes, and, during
progression, with a predominance of homolog effector T lymphocytes with reduction
of their neuroprotector effect with raised reactive oxygen species, inducible nitric
oxide synthase and NOX2/phagocyte oxidase gp91phox)[3],[13].
Figure 2 Glial cells functioning in healthy subjects and in ALS patients. a1. Astrocyte: nourishes
motor neurons through the synthesis of amino acids, neurotransmitters and neurotrophic
molecules; clears extracellular glutamate via specific transporters (EAAT2); offers
antioxidant defenses. a2. Reactive astrocyte: reduced production of neurotrophic factors;
inefficient glutamate clearance; induces metabolic pathways that favor oxidative stress.
b. Motor neuron. c1. Microglia: expresses ligands and receptors with neuroprotective
roles (CX3CL1/ CX3CL1R, CD200/CD200R); minimal production of oxygen and nitrogen reactive
species. c2. Reactive microglia: increased synthesis and release of oxidant agents
(H2O2, NO2) and cytokines (IL-6, IL-12, IL-23, TNF-α). d1. Monocyte: no chemotactic
stimulus. d2. Macrophage: invades neural tissue, secretes molecules that perpetuate
inflammation and neuron loss (IL-1 beta), increased expression of COX-2. e1. T-lymphocyte:
neuroprotective production of IGF-1, brain-derived neurotrophic factor and glial-derived
neutrotrophic factor. e2. Reactive T-lymphocyte: secretes IL-4, IL-10, TGF-β; increased
expression of INOS and NOX-2. f. Skeletal muscle fiber.
Environmental factors and extraneuronal disturbances are also essential in its immunopathogenesis.
For example, it has also been proven that the direct modulatory effect of vitamin
D in glial cell function in patients with ALS affects different cell and extracellular
matrix mechanisms: cell-signaling mechanisms (glutamate, matrix metalloproteinases,
mitogen-activated protein kinase pathways, Wnt/β-catenin signaling pathway, prostaglandins
and reactive oxygen species release, and nitric oxide synthase), major histocompatibility
complex class II molecules, toll-like receptors, poly (ADP-ribose) polymerase-1, heme
oxygenase-1, calcium-binding proteins, and a reduced form of nicotinamide adenine
dinucleotide phosphate[14].
At different stages of evolution, there are remarkable changes in neuroinflammation
patterns and cell activation in ALS. Briefly, there is no doubt that the balance of
astrocyte neuroprotective or microglia pro-inflammatory functions generate a progression,
or decrease, in the rate of primary intrinsic motor neuron degeneration[15],[16]. During the late stages of ALS, there is a wide increase in cytotoxic T cell infiltration
of the spinal cord leading to a pro-neurotoxic profile of cytokines and chemokines
and decreases in local levels of neuroprotective neurotrophic factors, such as GLT1
(SLC1A2) and GLAST (SLC1A3)[16]. Next, we will sumarize the most relevant physiopathological functions described
in each glial cell type.
ASTROCYTE ROLE IN ALS PATHOGENESIS
ASTROCYTE ROLE IN ALS PATHOGENESIS
Although classically related to neuronal regulation functions, astrocytes are involved
in regulation of the extracellular microenvironment in neurotransmitters at synapses
and during their developmental stages; control of signaling of CNS vascular development
(including modulation of blood-brain-barrier development); neurotrophic support and
stimulation for diverse neurons, maintenance of intercellular signaling (including
modulation of excitatory synaptic transmission via release and propagation of glutamatergic
stimuli); and neurometabolites and ionic regulation and homeostasis (acid and fluid
equilibrium)[3],[8],[13],[17],[18],[19].
The main mechanism involving astrocytes in ALS pathophysiology is dysfunction of glutamate
transporters[20] with loss of the astroglial glutamate transporter EAAT2 (by aberrant RNA splicing,
exon skipping and intron retention) in the motor cortex and in the anterior horn of
the spinal cord, disclosing its important function in excitotoxic damage in sporadic
ALS. As previously mentioned, astrocytes participate in glutamate clearance from the
synaptic clefts providing more balanced levels of extracellular excitatory neurotransmission,
the defective reuptake of which being the key mechanism in mouse models linked to
EAAT2 transporter dysfunctions. It has also been proved in mutant SOD1 mice that astrocytes directly regulate the expression of glutamate receptor subunit
GluR2 in AMPA receptors of motor neurons[12]. Other proven mechanisms included impaired release of multiple neurotrophic factors,
including glial-derived neutrotrophic factor, ciliary neurotrophic factor, vascular
endothelial growth factor and brain-derived neurotrophic factor[12]. Astrocytes with SOD1 gene mutation produce reactive oxygen species and soluble molecules with a selective
toxicity pattern to spinal cord motor neurons. Another proven mechanism in the SOD1 mouse model includes activation of the pro-nerve growth factor (NGF)-p75 receptor-signaling
pathway involved in direct astrocyte toxicity to motor neurons[8].
It has also been established that high levels of cyclooxygenase 2 is involved in prostaglandin
E2 synthesis and hyperstimulation of NMDA glutamate receptor activation of COX2 and
subsequent production of reactive oxygen species and prostaglandin E2, and this enhances
glutamate release from astrocytes[20].
THE ROLE OF MICROGLIA IN ALS PATHOGENESIS
THE ROLE OF MICROGLIA IN ALS PATHOGENESIS
Microglia represent the major primary resident phagocytic immune cells of the CNS
associated with some astrocyte immune functions, secreting pro-inflammatory immune
response molecules including cytokines and chemokines and anti-inflammatory molecules
during resolution of neural damage, and stimulating the release of neurotrophic growth
factors. Microglia result from differentiation of precursors of the monocyte/mesodermal
lineage of hematopoietic stem cells that normally protect against microbial infection,
abnormal aggregated protein, immunoglobulin-antigen complexes and microhemorrhagic
content[3],[21].
Microglial activation is a common hallmark of many neurodegenerative diseases, including
ALS, despite the fact that it results mainly from proliferation of myeloid precursor
cells[22]. Activated microglia releases proinflammatory cytokines (tumor necrosis factor-α,
interleukin-1β, interleukin-12, interferon-γ), mitogenic factors (monocyte chemoattractant
protein 1, macrophage colony stimulating factor), neurotrophic factors (insulin-like
growth factor-1), and anti-inflammatory cytokines (tumor growth factor-β)[12]. There is a direct neuropathological correlation of microglial activation with severity
of upper motor neuron damage. Diminishing the toxicity of mutant SOD1 transgene within
microglia of mice has been shown to significantly slow disease progression of ALS[23]. It has also been shown that the CCAAT/enhancer binding protein-β is enhanced in
activated microglial cells of the spinal cord of ALS mouse models with SOD1 gene mutation, promoting higher expression of nitric oxide synthase-2, cyclooxygenase-2
and upregulation of other proinflammatory gene expression[24].
Reactive oxygen species and cytokines increase motor neuron susceptibility to glutamate
excitotoxicity and inhibit expression of astrocytic glutamate transporters diminishing
glutamate uptake and perpetuating this neurotransmitter’s neurotoxicity mechanism[25].
Nuclear factor-kappa B (NF-кB) is upregulated in the spinal cord of mouse models and
patients with ALS, although inhibitory effects of its pathway in astrocytes did not
prevent neurodegeneration or rescue motor neuron death induced by microglia. However,
the modulatory effect in NF-кB pathway impairs proinflammatory activation of microglia[26].
Neuropathological studies have also established the activation of microglia and T-cell
infiltration in different stages of ALS evolution[25]. It is widely known that the peripheral immune system represents a crucial stage
in the main pathophysiological mechanism: T-lymphocytes directly cross the bood-brain
barrier, and interact with resident primary microglia triggering two different immunophenotypes
depending on the clinical and pathological stage of the ALS: an M2 protective anti-inflammatory
profile in early processes involving regulatory T-cells and IGF-1, or, an M1 cytotoxic
profile in late processes induced by fractalkine (CX3CL1) and CD200 involving Th1
cells and interleukin, and other substances such as IL-1β, IL-6, IL-12, IL-23, reactive
oxygen species (mainly H2O2) and TNF-α[15],[27]. Microglia production of IL-10, induced by cytokines from leptomeningeal cells,
has also been shown.
OTHER GLIAL CELLS INVOLVED IN ALS PATHOGENESIS
OTHER GLIAL CELLS INVOLVED IN ALS PATHOGENESIS
NG2 cells, synantocytes or pericytes, participate in CNS immune mechanisms of defense,
producing new astrocytes, oligodendrocytes and neurons, in some situations and specific
areas of the CNS. NG2 cells become astrocytes by proinflammatory cytokine signaling
in most ALS stages[12]. Oligodendrocytes also regulate local neuronal microenvironments. They represent
the main myelinating cells of the CNS[28]. Oligodendrocytes are involved in mechanisms of central myelination and provide
metabolic sustenance to motor neurons. Amyotrophic lateral sclerosis does not represent
a primary oligodendrocyte disease.
The effective role of NG2+ cells in the astrogliosis process in ALS is still uncertain. NG2+ cells produce new oligodendrocytes, neurons and perhaps astrocytes after an injury,
changing the cellular microenvironment. In ALS, cell proliferation rates are enhanced
in regions of motor neuron degeneration, but myelination and remyelination defects
are also found[3]. Few studies have explored the role of myelinated oligodendrocytes in ALS pathogenesis,
which influence motor neurons through neurotrophic factors release and regeneration
after neuronal injury. It has also been shown how oligodendrocytes and their NG2+ progenitors mediate neuron loss in ALS, the abnormal rate of proliferation in mouse
models secondary to oligodendrocyte degeneration, and to the progressive neuroinflammatory
and neurodegenerative processes. The role of Schwann cells in ALS pathogenesis remains
undefined[12].
FINAL REMARKS
Motor neuron disease represents an important group of neurodegenerative disorders,
mainly represented by ALS. Primary alpha motor neuron involvement and degeneration
and secondary mechanisms including glial cell pathological processes represent the
most important features in the pathophysiology of ALS. Astrocyte and microglial dysfunctions
have been widely demonstrated in patients and animal models of ALS. More studies are
needed to establish specific molecular features linked to glial dysfunction at the
microcellular level and its extracellular involvement, to allow new pharmacological
perspectives and provide data about the natural history of the different genetic forms
and variants of ALS.
