Review

Neuroinflammation in amyotrophic lateral sclerosis: role of

glial activation in motor neuron disease
Thomas Philips, Wim Robberecht

Neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis (ALS) Lancet Neurol 2011; 10: 253–63
are characterised by the appearance of reactive microglial and astroglial cells, a process referred to as neuroinflammation. VIB Vesalius Research Center,
In transgenic mouse models of mutant SOD1-associated familial ALS, reactive microglial cells and astrocytes actively Leuven, Belgium (T Philips MSc,
Prof W Robberecht MD); and
contribute to the death of motor neurons. The biological processes that drive this glial reaction are complex and have
Laboratory of Neurobiology,
both beneficial and deleterious effects on motor neurons. Therapeutic interventions targeting these cells are being Department of Neurology,
explored. An improved understanding of the biological processes that cause neuroinflammation will help to define its K U Leuven, Leuven, Belgium
medical importance and to identify the therapeutic potential of interfering with this reaction. (T Philips, W Robberecht)
Correspondence to:
Prof Wim Robberecht, Neurology
Introduction humans and in animal models) are likely to be relevant to
Department, University Hospital
Neurodegenerative diseases such as Alzheimer’s disease, studies of sporadic ALS. Leuven, Herestraat 49,
Parkinson’s disease, and amyotrophic lateral sclerosis A common characteristic of ALS and other 3000 Leuven, Belgium
(ALS) represent a biomedical challenge; their frequent neurodegenerative disorders is the occurrence of a wim.robberecht@uz.
kuleuven.be
occurrence in the growing population of elderly people neuroinflammatory reaction consisting of activated glial
has become a concern for western health-care systems. cells, mainly microglia and astrocytes, and T cells. This
Identification of strategies for treatment of these inflammatory reaction has recently received attention as
disorders is an important task for translational an unexpected potential target for the treatment of these
neuroscience. ALS is a degenerative disorder, mainly but diseases. For ALS, knowledge of the contribution of
not exclusively affecting the motor neurons in the spinal microglia, astrocytes, and inflammatory T cells to the
cord, brainstem, and cortex. This degeneration results in degeneration of motor neurons has expanded greatly, and
fasciculations, muscle weakness and muscle atrophy, has resulted in clinical trials of drugs targeting
and hyper-reflexia with spasticity. Although the course of neuroinflammatory processes in patients with ALS. In
ALS is variable, median survival of patients is less than this Review, we attempt to address the clinical importance
3 years. A minority of patients with ALS have concomitant of this neuroinflammatory response in patients with ALS.
frontotemporal dementia, but more subtle frontal
executive difficulties occur in many.1 Key players in neuroinflammation
The disease is familial in 10% of patients and is usually Microglia are of mesenchymal origin and are the resident
characterised by a dominant pattern of inheritance. macrophages in the nervous system; they constantly
Several ALS-causing genes (in familial ALS) or ALS- monitor the extracellular environment, closely interact
associated genes (in sporadic and familial ALS) have been with astrocytes and neurons, and can be identified as
identified.2 Mutations in SOD1 are by far the most ramified CD11b (also known as ITGAM) expressing cells.
common cause of familial ALS (20%), whereas mutations Microglia are activated by a range of signals,11 and are the
in TARDBP (5%), FUS (5%), and ANG (<1%) are less first line of defence against infection or injury to the
common. VCP3 and OPTN4 mutations have been recently nervous system. Although the microglial response is
added to the steadily growing list of genes associated with diverse, generally, upon activation, microglia acquire an
ALS. Mice and rats that overexpress the human mutant amoeboid appearance and secrete proinflammatory
superoxide dismutase 1 (SOD1) protein are used molecules such as tumour necrosis factor (TNF) α,
extensively to study ALS;5,6 they develop adult-onset, interferon γ, and interleukin 1β; they upregulate oxidant
progressive, and ultimately fatal muscle weakness and molecules such as NO and O2, which can protect against
atrophy, caused by prominent motor-neuron degeneration. invading organisms. This proinflammatory reaction clears
Mice and rats with mutant forms of TAR DNA binding hazards and repairs any damage, and resolves when anti-
protein 43 (TDP-43) have become available only recently.7,8 inflammatory molecular networks outweigh proinflam-
The cause of sporadic ALS remains unknown and no matory molecular networks, a hitherto poorly understood
reliable animal model for this form of the disease is shift. Trophic and anti-inflammatory factors such as
available. Conformational changes similar to those in insulin-like growth factor 1 (IGF1), interleukin 4, and
mutant SOD1 have been seen in wild-type SOD1 from interleukin 10, also released by microglia, contribute to
samples of spinal cord from patients with sporadic ALS, repair and limitation of inflammation. Microglial
suggesting that modified wild-type SOD1 could contribute proinflammatory or anti-inflammatory processes are
to its pathogenic mechanism.9 Of note, most patients influenced by surrounding astrocytes and inflammatory
with sporadic ALS have TDP-43-containing inclusions in T-cell subsets, which can affect their phagocytic and
their motor neurons, despite not harbouring mutations antigen-presenting cell properties. Microglia can thus exert
in this protein.10 Findings from studies of familial ALS (in a deleterious (so-called M1 microglia) and a benign

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 Review
(so-called M2 microglia) function, depending on their
intrinsic properties, interaction with the cellular
microenvironment, and presence of pathogenic factors.11–14
Astrocytes are ectodermal cells and are probably about
ten times as numerous as neurons. Astroglial cells have
many complex functions in the nervous system, such as
regulating extracellular neurotransmitter concentrations,
exerting metabolic or ionic homoeostatic functions, and
providing trophic support for surrounding neurons.15
Astroglial activation is characterised by a prominent
increase in expression of the intermediate filament glial
fibrillary acidic protein (GFAP) and the marker aldehyde
dehydrogenase 1 family, member L1 (ALDH1L1).
Astrocytes are not immune cells per se, but can, in specific
conditions, contribute to the immune response.16
Several other cell types are associated with
neuroinflammation. First, T cells are key contributors to
this reaction. T-cell subpopulations that infiltrate the
CNS modulate the neuroinflammatory reaction
differently, depending on stage of disease progression.17,18
Second, NG2 (also known as CSPG4) expressing glial
cells, oligodendrocyte precursor cells that are widely
distributed in the adult nervous system, have been
reported to differentiate into astrocytes and even neurons
in specific conditions;19,20 their role in neuroinflammation
is still poorly understood, as is the role of oligodendrocytes.
Third, the roles of ependymal and subependymal
(precursor) cells in the glial reaction and in neuro-
degeneration are poorly understood.21
In neurodegenerative diseases, the neuroinflammatory
reaction is not transient, but sustained, probably because
of the persistent presence of its precipitating factor. In
the familial dominant forms of these disorders, this
factor is believed to be the disease-causing mutant protein
(eg, amyloid β1–42 for Alzheimer’s disease, mutant
α-synuclein for Parkinson’s disease, and mutant SOD1
for ALS). This inflammatory reaction, which is supposed
to combat the precipitating factor, is believed to turn into
a hazardous process and contribute to neuronal damage.
Glial activation in ALS
Many studies have characterised the activation of
microglia and astrocytes, and the appearance of
lymphocytes in post-mortem tissue of patients with ALS
and in the spinal cord of transgenic mice that express a
mutant form of human SOD1.22–29 Clear upregulation of
expression of CD11b, IBA1 (ionised calcium-binding
adapter molecule 1), and CD68 markers for microglia,
and of GFAP and ALDH1L1 markers for astrocytes, is
reported consistently. Additionally, these cells change
their morphological appearance from a so-called
surveying state, characterised by a small cell body and
thin processes, to an activated state, characterised by
enlargement of the cell body and thickening of processes.
Microglial activation is associated with infiltration of
helper T cells and cytotoxic T cells.23 Concomitant with
increased infiltration of inflammatory T cells,
254
parenchymous microglia acquire properties of antigen-
presenting cells, shown by upregulation of the expression
of markers such as CD11c (also known as ITGAX), CD86,
and intracellular adherin molecule 1 (ICAM1);17,30–32 this
suggests that these microglia interact closely with the
T-cell infiltrates.17,30,31
In spinal cords of patients with ALS, astrocytosis is not
restricted to the ventral horn but is also evident in the
dorsal horn and at sites where fibres of the corticospinal
tract enter the grey matter.28 In the brain, astrocytosis
occurs in both cortical grey matter26 and subcortical white
matter,27 and is not restricted to the motor cortex.
Microgliosis occurs in the motor cortex, the motor nuclei
of the brainstem, along the corticospinal tract, and in the
ventral horn of the spinal cord29 where microglia might
interact with T-cell infiltrates.23 These findings from
studies of human beings are based on post-mortem
tissue and thus represent the final stages of the disease.
Study of microglial activation is difficult during early
disease states and during disease progression. By use of
¹¹C-PK11195 PET imaging, Turner and colleagues33
identified in-vivo microglial activation in the motor
cortices, dorsolateral prefrontal cortices, and thalami of
patients with ALS; there was a significant association
between microgliosis and damage to upper motor
neurons, but not to lower motor neurons.33 This technique
could be applicable to the study of microgliosis in patients
with ALS over time, to assess the relation between
microgliosis and disease progression. An association
between systemic immune activation and rate of disease
progression was detected.34
Changes during disease progression have been studied
in rodent models (figure 1A–F). In G93A mutant SOD1
transgenic mice, microglial cells become activated before
motor neurons disappear from the lumbar spinal cord at
about 90 days of age, and thus well before clinical disease
onset.24,25 Astrocytic activation occurs concomitantly with
a decrease in neuronal cell bodies.35 Of note, astrocytosis
is initially most prominent in the ventral horn
(figure 1D–F). Numbers of activated microglia and
astrocytes increase further until the end stage of
disease.25,30 Increase in the number and activation of
myeloid cells is not restricted to the CNS: activated
macrophages are readily detected in the sciatic nerve of
SOD1-mutant mice, from the presymptomatic stage of
disease onwards.36 In SOD1 G93A mice, an increase in
T-cell numbers occurs concomitantly with microglial
activation, similar to that seen in human beings.17,18 Some
discrepancy regarding the nature of this increase in
T cells exists: one study reported an increase in both
CD4+ and CD8+ T cells early in disease,17 whereas another
study detected only CD8+ cytotoxic T cells at end-stage
disease.18 During this process of activation, microglia and
astrocytes upregulate expression of a whole subset of
cell-surface markers—chemokines and cytokines.
Several studies have shown that an increased number
of microglial cells results from local proliferation of
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A B C

D E F

Figure 1: Microgliosis and astrogliosis in the pathogenesis of ALS

At the asymptomatic stage of disease (postnatal day 40) of G93A mutant SOD1 transgenic mice, ramified microglia are in a surveying state, expressing low amounts
of microglial activation markers, such as ionised calcium-binding adapter molecule 1 (Iba1; A). At onset, the number of microglia increases and they acquire an
amoeboid morphology. They increase levels of microglial activation markers and become more reactive (B). At the end stage of disease, many amoeboid microglia are
present throughout the ventral horn of the spinal cord (C). Resting astrocytes express low amounts of glial fibrillary acidic protein (GFAP), if any, at the asymptomatic
disease stage (D). Astrocytes increase the expression of GFAP when activated and become more reactive, acquiring a thick cell body from which thick processes
extend (E). Astrocytosis is even more prominent at the end stage of disease, with highly reactive GFAP-expressing astrocytes throughout the entire ventral horn (F).
Scale bar represents 100 μm in large panels and 20 μm in insets. ALS=amyotrophic lateral sclerosis.

resident microglia.30,37 Although microglial turnover is
very low in normal conditions, proliferation increases
greatly in a mouse model of ALS.30 Studies of
transplantation of bone-marrow cells in lethally irradiated
SOD1-mutant mice suggest that myeloid precursor cells
are recruited from the blood, infiltrate the spinal cord,
and differentiate, and thus contribute to microgliosis.36–39
However, this finding is likely to be an artifact of the
breakdown of the blood–brain barrier by irradiation or
engraftment of progenitors in the bloodstream, which
are absent under normal conditions.40,41 Even after
irradiation and transplantation, contribution of blood-
derived cells to parenchymatous-myeloid-cell populations
is small and is likely to be limited to perivascular cells,
both in healthy and diseased cells.37,42
Unlike microglia, astrocytes do not proliferate as the
disease progresses in SOD1-mutant mice.30,43 These cells
differentiate from GFAP-negative precursor cells, or
alternatively, resident astrocytes upregulate expression of
this filament above the detection limit when activated in
disease conditions. Candidate precursors are
(sub)ependymal cells, radial glia, and oligodendrocyte
precursor cells expressing NG2. NG2 cells generate
astrocytes in vitro44 and during embryonic development,20
but they do not seem to be able to generate them in the
adult CNS.19,45 Ependymal cells, which line the central
canal of the spinal cord, are known to have stem-cell-like
characteristics and might contribute to an increase in the
number of astrocytes in patients with ALS. This increase
is similar to what happens in experimental stroke or after
spinal-cord injury, whereby ependymal cells lining the
lateral ventricle or central canal, respectively, can give
rise to astrocytes.46,47 One study did not find evidence for
such increase in SOD1-mutant mice, but more studies
are needed to clarify the contribution of these cells to the
glial reaction.
Effects of glial cells on motor-neuron
degeneration
SOD1-mutant mice with motor-neuron degeneration
express the transgene ubiquitously. Selective expression
of mutant SOD1 in motor neurons did not result in loss
of motor neurons48,49 or gave rise to mild abnormalities
only.50,51 Selective expression in astrocytes or microglia
did not result in motor-neuron degeneration either.52,53
These studies provided evidence that expression in motor
neurons is necessary, but that surrounding cells play an
important part in motor-neuron degeneration. The
notion that motor-neuron death is non-cell autonomous
was supported further by a study that used chimeric mice
in which only some cells expressed mutant SOD1.54
Direct evidence for a role of non-neuronal cells in
rodent models of ALS came from experiments on mice
that expressed mutant SOD1 that could be deleted in a
cell-specific way. Selective deletion of mutant SOD1
from motor neurons, or neurons generally, delayed
disease onset or onset and early disease, respectively.55,56
This result agreed with the finding that selective

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delivery of small interfering RNA against human SOD1
to motor neurons of SOD1-mutant mice delayed disease
onset, but did not modify the disease course once
started.57 Conversely, diminished expression of mutant
SOD1 in myeloid cells or astrocytes significantly slowed
disease progression rather than onset.55,56 Of note,
deletion of mutant SOD1 from myeloid cells results in
loss of mutant SOD1 not only in microglial cells in the
CNS, but also in peripheral macrophages, such as those
in the sciatic nerve, which might contribute to the
increase in survival.36
A series of sophisticated transplantation experiments
further substantiated the role of microglia. Mutant-SOD1-
expressing PU–/– neonatal mice, which lack myeloid cells,
T cells, and B cells, were transplanted with wild-type-
SOD1 bone marrow. All myeloid cells in these mice were
wild-type donor-derived, whereas all host cells expressed
mutant SOD1; this significantly slowed disease
progression and improved survival.52
Several studies report inhibition of myeloid-cell
proliferation by treating mice expressing thymidine
kinase with ganciclovir, under control of the CD11b
promoter. In models for Alzheimer’s disease, stroke, and
infection, ablation of proliferating microglia significantly
worsened the phenotype.58–60 By contrast, in SOD1-mutant
mice this significantly reduced the number of activated
microglia (and astrocytes), but did not rescue motor
neurons or their axons.30
Contribution of microglia to motor-neuron degeneration
seems to be T-cell dependent.61 T cells in the CNS are
rarely detected at early disease stages, but readily infiltrate
the spinal cord as disease progresses (figure 2A–C).17,18
When T cells are depleted in SOD1-mutant mice (by
crossbreeding them with RAG2–/– knockout mice [also
deleting B cells] or with CD4–/– knockout mice [only
deleting CD4+ T cells]), disease course (but not onset) is
significantly worsened.18 CD11b-positive immunoreactivity
of microglia in these animals is greatly reduced, but the
cells that are present can become highly active, because
they express more proinflammatory factors such as Nox2
(also known as CYBB) and TNFα.18 Transplantation of
bone marrow from either mutant or wildtype SOD1
donors antagonised this worsening. A similar worsening
of disease was noted when deleting the T-cell receptor b
subunit from mutant SOD1 mice.17 By contrast, adoptive
transfer of activated T regulatory cells or T effector cells
from wild-type donor mice delayed loss of motor function
and extended survival.62 Thus (CD4+) T cells, regardless
of whether they express a mutant protein or not, are
protective in this model of ALS, by enhancing the M2
character of microglia over their M1 character (figure 2B).
This dual action of microglia most probably explains why
simple ablation of microglia in SOD1-mutant mice does
not affect disease course; the contribution of T cells might
change over time. Cytotoxic T cells at the end stage of
disease can predominate, explaining why such
neuroprotective action ultimately fails (figure 2C).18
256
The contribution of astrocytes has been investigated in
transplantation studies, albeit to a lesser degree than for
microglia. Wild-type glial-restricted precursor cells have
been transplanted in the cervical spinal cord of SOD1-
mutant rats.63 These cells differentiated into GFAP-
expressing astrocytes and rescued motor neurons, leading
to a significant increase in survival.63 Increased glutamate
scavenging activity of glial-restricted precursor cells might
underlie this benefit. Whether and how microglia and
astrocytes functionally interact is unknown; both cell
types release factors that can influence the other.55,64
Results from most of these studies suggest that mutant
proteins in motor neurons determine disease onset and
early stages of disease, whereas mutant SOD1 in
microglia and astrocytes mainly determines disease
progression and duration. However, in chimeric mice
that express mutant SOD1 in all oligodendrocytes and
motor neurons, and in various other neuronal and non-
neuronal cells, onset of disease was significantly delayed
in comparison with mice that ubiquitously express
mutant SOD1, and thus was not determined by expression
of mutant SOD1 in motor neurons only.65 Similarly, in
some models, expression of mutant SOD1 in microglia
and astrocytes determined disease onset or early disease
stage, which could have been mediated by absence of
potential neuroprotective residual dismutase activity in
dismutase-inactive SOD1-mutant mice.64,66
Factors that influence the neuroinflammatory
response
A wide range of factors has been suggested to induce a
neuroinflammatory response in patients with ALS and to
mediate the hazardous and beneficial effects of glial cells
on motor neurons.67 Chemokines such as chemokine
(C-C) motif ligand 2 (CCL2) are greatly upregulated in
spinal cords of SOD1-mutant mice and of patients with
ALS.31,32 Similarly, colony stimulating factor 1 (CSF1) is
increased greatly in spinal cords of SOD1-mutant mice.68
These factors could contribute to the increased
proliferation and activation of microglia. Treatment of
SOD1-mutant mice with CSF1, which increases microglial
proliferation and expression of phagocytotic and
proinflammatory cytokines, exacerbated disease.69 By
contrast, in an APP(Swe)/PS1 mouse model of
Alzheimer’s disease, CSF1 treatment decreased amyloid-β
burden and cognitive loss, thus increasing neuro-
protection.70 This finding shows a dual role for microglia
in neurodegeneration, mediating neuroprotective action
versus neurotoxic action depending on interaction with
their surroundings and timing of activation.
A logical assumption is that damaged motor neurons
initiate the inflammatory response. A good candidate for
a possible messenger associated with induction of the
neuroinflammatory state of microglia and astrocytes is
mutant SOD1. Mutant SOD1, but not wild-type SOD1, is
released by motor neurons through a chromogranin
chaperone-like process.71 In-vitro data suggest that this
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A B C
Blood vessel Inflammatory T cell Th

Th Tc
+ + Tc ?

– –
–
Microglia Astrocyte

– – – –

+ + + + + +

Motor neuron

Figure 2: Interaction of inflammatory T cells with microglia and astrocytes during ALS pathogenesis
At asymptomatic disease stage, almost no T cells are present in the spinal cord; surveying glial cells are not activated and they scan the surrounding
microenvironment and resolve microdamage in the spinal cord (A). At early disease stages, some inflammatory T cells infiltrate the spinal cord from the blood. These
T cells are mainly CD4-expressing helper T cells that interact with surrounding astrocytes and microglia; their neuroprotective effects (thick arrow, +) outweigh their
neurotoxic action (dashed arrow, –), because deletion of inflammatory T cells increases expression of proinflammatory markers and decreases levels of
neuroprotective GLT1 and GLAST (B). At the end stage of disease, more T cells infiltrate the spinal cord, of which a substantial proportion are cytotoxic T cells. These
cells could mediate production of a net neurotoxic non-neuronal environment (thick arrows, –) instead of a net neuroprotective non-neuronal environment (dashed
arrows, +), surrounding motor neurons and resulting in increased motor-neuron death (C). Th=helper T cell. Tc=cytotoxic T cell.

extracellular mutant SOD1 exerts a toxic action on motor
neurons, but only in the presence of microglial cells via a
CD14–TLR (toll-like receptor) mediated mechanism,
increasing levels of reactive oxygen species.72 In-vivo
overexpression of chromogranin A accelerates disease
progression by increasing the amount of misfolded SOD1
species.73 To investigate whether chromogranins also play
a part in human ALS, a genetic association study of single
nucleotide polymorphisms in chromogranin genes was
done. A chromogranin B variant (P413L) was shown to be
a risk factor and a modifier of disease onset for patients
with ALS.74 Disappointingly, a recent study did not
confirm these results.75
Microglia in the spinal cords of patients with ALS
upregulate the expression of antigen-presenting and
dendritic cell markers, such as CD86 and CD11c, possibly
linking microglial activation to T-cell activation. Reduction
in the number of T cells results in a substantial increase
in the expression of neurotoxic molecules such as Nox2
and TNFα, and a concomitant reduction in neurotrophic
factors such as IGF1, glial-cell-derived neurotrophic
factor (GDNF), brain-derived neurotrophic factor
(BDNF), and GLT1 (also known as SLC1A2) and GLAST
(also known as SLC1A3);17,18 this might explain the
beneficial effect of T cells. CD4+ T cells express high
levels of the anti-inflammatory factor interleukin 4, which
might induce the neuroprotective phenotype in the
surrounding microglia.17 Of note, T cells also influence
astroglial cell behaviour, reducing their GFAP expression
at early stages of disease, but not at the end stage of
disease;18 they modulate the contribution of astrocytes to
the inflammatory response by modulating their levels of
GLT1 expression and release of neurotrophic factors.18
Overexpression of mutant SOD1 seems to make
microglia more neurotoxic than wild-type or non-
transgenic microglia.55,76 Many studies have reported
aberrant expression of a range of proinflammatory
cytokines such as interleukin 1β and TNFα by cultured
microglia that express mutant SOD1.77,78 Similarly, many
types of proinflammatory mediators are increased in
patients with ALS and in rodent models.68,79–82 Additionally,
levels of superoxide and nitrite or nitrate might increase
because of elevated levels of NADPH oxidase and nitric
oxide synthase 2 (NOS2) activity, respectively.52,83 Reactive
oxygen species and nitrate compounds induce lipid
peroxidation and protein carbonylation, impeding
integrity and function of neurons and glial cells.
Increased expression of cyclo-oxygenase 2 (COX2), an
important enzyme for proinflammatory-prostanoid
synthesis, has been reported both in patients with ALS
and in SOD1-mutant mice.84 The prostaglandin inhibitors
celecoxib and nimesulide were efficient in delaying
disease onset and improving survival of SOD1-mutant
mice, by blocking COX2 activity.85,86 However, the drugs
did not affect disease progression, which is a more
relevant parameter when extrapolating results from
mouse trials to human studies.
Another interesting system is the Fas pathway; it links
microglial function to oxidative molecules such as NO,
and to an apoptotic pathway to which motor neurons seem

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to be particularly sensitive.87 The role of the NADPH-
oxidase subunits of the NADPH-oxidase complex is
notable; expression of mutant SOD1 increases the
activation of the Nox2 (also known as GP91-PHOX)
subunit of this complex, which leads to elevated levels of
reactive oxygen species.88 Inhibition or deletion of NADPH
oxidase leads to a substantial delay in disease progression
and an increase in survival of SOD1-mutant mice, as
reported by some researchers,88,89 but not all,83 most likely
because of the genetic background differences between
the mice used for this latter study. In mouse models of
Alzheimer’s disease, Nox2 activation is upregulated in
response to accumulation of amyloid β. Treatment of
these mice with ibuprofen attenuated activation of
NADPH oxidase and microgliosis, and prevented oxidative
damage and increased plaque clearance.90
Many other studies have focused on the role of a (usually
single) potentially relevant proinflammatory factor
associated with microglial neurotoxicity. Generally, deletion
of one factor (eg, TNFα,91 interleukin 1β,78,82 or NOS292,93)
resulted in either no or a small effect on survival, and
furthermore, the results were sometimes dependent on
the SOD1 mutation studied;78,82,92,93 it is likely that targeting
a single factor is insufficient because of functional
abundance and compensation. For example, TNFα signals
through different receptors and mediates different
functional effects depending on location and timing of
expression. These differences might explain why deletion
of TNFα expression in a SOD1-mutant mouse (G37R) did
not affect the disease,91 but inhibition of its action at a
specific disease stage is more successful. Moreover, other
factors could compensate for the absence of TNFα, as
shown with interleukin 1β and toll-like receptor 2 (TLR2),
which are upregulated after TNFα is knocked out.91
Conversely, treating SOD1-mutant mice with anti-
inflammatory drugs that might have effects on several
aspects of the inflammatory response has proven to be
efficacious. Treatment of SOD1-mutant mice with
nordihydroguaiaretic acid, thalidomide, or lenalidomide,
which all inhibit TNFα production but also modulate
other cytokine expression levels, delayed disease onset
and improved survival.94,95 Lenalidomide was even
successful in extending survival when given after disease
onset, which is of relevance for many patients with ALS
for which treatment can be started only after symptom
onset.96 Minocycline, an anti-inflammatory drug that
non-specifically affects microglial activation, significantly
improved lifespan of SOD1-mutant mice.97–99 Thus,
inhibition of the inflammatory reaction generally, rather
than targeting a specific inflammatory mediator, seems
to be more effective. Disappointingly, a study in mice
using optimised biological parameters failed to reproduce
the previously published results of these drugs.100
Additionally, almost all of these compounds affected
disease onset in mice, but not disease progression. Not
surprisingly, therefore, the results from clinical trials of
minocycline and celecoxib were negative.101.102
258
Surprisingly, microglia expressing mutant SOD1 can
also increase expression of factors that confer
neuroprotection. These microglia can increase expression
of IGF1 in vivo,17 although its release reduced levels in
vitro.76 Adeno-associated-virus-mediated delivery of IGF1
increases survival of SOD1 G93A mice.103,104 Similarly,
microglia upregulate expression of the neuroprotective
molecule progranulin as disease progresses.105,106
Therefore, unselective targeting of microglia, and
inhibition of their hazardous effects and their protective
properties, might not be an effective strategy against
motor-neuron degeneration.
Pathways through which activated astrocytes might
contribute to motor-neuron death are beginning to
emerge. Mutant SOD1 seems to result in a loss of the
normal beneficial effects of astrocytes on motor neurons,
and in the release of toxic factors by activated astrocytes.
Normally, astrocytes protect neurons from excitotoxicity, a
mechanism believed to play a pathogenic part in both
human ALS and rodent models (figure 3A–B).107 This
protective action seems to be compromised in patients
with ALS and in SOD1-mutant mice; there is a significant
decrease in expression of the glutamate transporter EAAT2
(GLT1 in mice).108–111 Normally functioning presynaptic
neurons can modulate astrocytic GLT1 or EAAT2 levels by
inducing recruitment of astroglial kappa B-motif binding
phosphoprotein (KBBP) factor to the GLT1 promotor,
increasing GLT1 expression.112 Reduction of neuronal
signalling after a neurodegenerative insult will result in
reduced transcription of the glutamate transporter. Raised
glutamate concentrations in the synaptic cleft, which
result from this loss of scavenging function, might
overstimulate AMPA and NMDA receptors on motor
neurons, resulting in increased entry of calcium into
motor neurons and cell death.113 Overexpression of GLT1
in astrocytes114 or increasing its expression by treatment
with ceftriaxone115 positively affected motor-neuron
degeneration in SOD1-mutant mice. An increase in
EAAT2 function might also explain the beneficial effect of
transplantation of glial-restricted precursor cells into the
spinal cords of SOD1-mutant rats.63 Furthermore,
astrocytes normally reduce the Ca²+ permeability of AMPA-
type glutamate receptors by inducing the expression of the
Glur2 (also known as Gria2) subunit in motor neurons.116
This action is stopped in the presence of mutant SOD1,
making motor neurons vulnerable to excitotoxicity.116
These studies suggest that astrocytes protect motor
neurons from excitotoxic cell death, a function that is lost
in the presence of mutant SOD1.
Astrocytes modulate neuronal integrity by release of
neurotrophic factors, of which GDNF, BDNF, IGF1, and
vascular endothelial growth factor (VEGF) are well
studied examples. Exogenous administration of these
factors increases survival of motor neurons in the mutant
SOD1 model.103,104,117–119
Apart from losing their normal beneficial effects on
neurons, astrocytes affected by ALS also seem to release
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factors that are hazardous to motor neurons. In vitro,

A
mutant SOD1-expressing astrocytes release more
neurotoxic factors than do wild-type expressing Astrocyte
astrocytes;120,121 researchers excluded many potential
neurotoxic mediators, but the toxic factors still need GLT1
identification. Similar to microglia, astrocytes upregulate
expression of NOS2, promoting generation of reactive KBBP
oxygen and nitrogen species.122 Increase in expression of
GLT1 3
molecules related to oxidative stress could be countered 1 2
by astrocytic upregulation of the antioxidant Nrf2 (also
known as NFE2L2), which delays disease onset in SOD1-
mutant mice.123 NMDA

From animal models to therapeutic strategies AMPA

Results from many studies investigating beneficial 4

effects of immune-active or anti-inflammatory
compounds, and of transplantation of myeloid (and
astroglial) cells in mice and rats, have encouraged
clinicians to explore the neuroinflammatory response in
patients with ALS as a potential therapeutic target; so far,
results of these studies have been disappointing. General
immunosuppression or immunomodulation (using
cyclophosphamide, ciclosporin, interferon beta, and
many others) was reported to be ineffective, although
some studies were probably underpowered. Compounds
with specific targets such as minocycline, believed to
reduce microglial activation,97–99 or celecoxib, a COX2
inhibitor,85,86 were studied in patients with ALS because
of beneficial effects on disease onset in mice, but were
reported to be ineffective.101,102 These findings might not
be surprising, because these drugs did not affect disease
progression in animal models, but only delayed onset.
Antioxidants such as vitamin E, coenzyme Q10, and
creatine, used to scavenge toxic mediators of
neuroinflammation, were ineffective.124–126 Results from
two studies using ONO-2506 (ONO, Osaka, Japan), a
molecule believed to enhance the protective effect of
astrocytes, were negative (Ludolph A, University Hospital
and Medical Faculty of Ulm, Germany, personal
communication). Treatment with glatiramer acetate, a
compound that modulates microglial function, was
negative.127 A trial investigating the effect of ceftriaxone
is ongoing.128
Correction of the motor-neuron microenvironment has
been attempted in a study in which patients underwent
whole-body irradiation and systematic bone-marrow
transplantation to replace endogenous microglial cells
with exogenous (non-ALS) microglial cells. No survival
benefit was noted.129 Studies investigating the feasibility
of non-ALS-astroglial-cell transplantation (eg, by
transplantion of glial-cell-restricted precursor cells) are
underway; non-genetic downregulation of mutant SOD1
expression in microglia might be possible. Treatment of
SOD1-mutant mice with activated protein C significantly
improves survival because of diminished mutant SOD1
concentrations in motor neurons and other cells,
including microglia.130 Whether a similar approach will
Presynaptic terminal Postsynaptic terminal
B
Astrocyte
GLT1
KBBP
5
6
NMDA
AMPA
[Ca2+]↑↑↑
Presynaptic terminal 7 Postsynaptic terminal
Figure 3: Glutamate-mediated excitotoxicity at the synapses between presynaptic and postsynaptic motor
neurons
Presynaptic neurons can induce KBBP translocation in astrocytes (A; 1), leading to increased transcription of GLT1
transporters, which are expressed on the membrane of these cells (2). GLT1 takes up extracellular glutamate,
protecting motor neurons from excitotoxicity. Astrocytes also regulate AMPA receptor GluR2 subunit levels (yellow)
on postsynaptic motor neurons, which makes motor neurons less vulnerable to overstimulation by glutamate
(3 and 4). In the presence of mutant superoxide dismutase 1 (SOD1), presynaptic regulation of GLT1 expression by
KBBP (B; 5) and GluR2 regulation (6) are decreased dramatically, increasing vulnerability of motor neurons to
glutamate-mediated excitotoxicity. Increased concentrations of glutamate in the synaptic cleft overstimulate
AMPA and NMDA receptors, leading to an increase in motor-neuron death (7). KBBP=kappa B-motif binding
phosphoprotein. AMPA=amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid. NMDA=N-Methyl-D-aspartic acid.
be of benefit for familial and perhaps sporadic cases of
ALS remains to be investigated.
Why do we have such problems translating findings
from robust animal studies into useful treatments for
patients? First, all the animal studies in which
encouraging results were obtained used the SOD1-
mutant mouse model for familial ALS; however, most
patients in clinical trials have sporadic ALS. Second,
very homogeneous cohorts of mice (eg, in terms of
genetics, food exposure, age) are used in treatment
trials, whereas even in well designed studies of patients
with ALS, the study population is surprisingly
heterogeneous. Third, treatment of mice is usually
initiated before disease onset, whereas patients often

www.thelancet.com/neurology Vol 10 March 2011 259

 Review
Search strategy and selection criteria for references
We searched Pubmed for published works using the following
search terms: “microglia,” “astrocytes”, “inflammation”,
“motor neuron degeneration”, and “amyotrophic lateral
sclerosis”. Only reports published in English, between
January, 1990, and December, 2010, that had clear in-vivo
relevance, and had potential for translation into human
beings were included.
have had the disease for years before experimental
treatment is started. Fourth, reported effects are often
related to disease onset and survival. Many studies
showed no benefit on disease progression, which is the
only relevant parameter for patients who already have
the disease. Additionally, many studies of mice were
probably not very robust in their outcomes, because the
number of mice included in these studies was too
small. Fifth, aside from offering a clinical benefit, very
few treatment strategies have been assessed to establish
whether they actually have the intended biological
effect. Sixth, pharmacokinetic and pharmacodynamic
differences between mice and humans are almost never
taken into account. Often, dosing and bioavailability
have not been studied appropriately. Interaction
between the drug being tested and riluzole, the only
registered drug for ALS, taken by many patients in
trials, is never addressed. Because of these factors,
therefore, failure to reproduce findings from mouse
models in human beings is not surprising.
Outstanding questions and future perpectives
Our understanding of neuroinflammation in mutant
SOD1 models has improved greatly. What was thought
of as a non-specific reaction has now emerged into an
area of intense research, the results of which are
relevant not only to motor-neuron degeneration, but to
neurodegeneration and neurobiological processes
generally. Additionally, the neuroinflammatory reaction
creates opportunities for intervention. However, our
understanding of neuroinflammation in ALS is far
from complete. We mention only a few of the issues
that need further study.
Further investigation into the two opposing effects—
deleterious and protective—of neuroinflammation is
crucial. Are these effects cell specific or exerted by
different cell types? Which factors determine whether
the M1 component or M2 characteristics predominate?
What is the origin of the neuroinflammatory reaction?
Where do the lymphocytes come from and when? Which
subtypes of T cells are involved? Which signals activate
microglia and astrocytes? What is the role of the NG2
progenitor cells? What is the nature of the interaction
between astrocytes and microglia, and which signalling
molecules mediate such interaction? What is the role of
oligodendrocytes?
260
Furthermore, we need to elucidate whether findings
from the mutant SOD1 model apply to other forms of
ALS. Is the same contribution of the neuroinflammatory
reaction seen in motor-neuron degeneration associated
with mutant TDP-43 and mutant FUS? Mouse models
used for studies of neuroinflammation overexpress
mutant SOD1; do findings hold in models in which one
copy of the mutant protein is present, such as is the case
for human familial ALS, and in conditions in which there
is no mutant protein, such as in sporadic ALS? Do non-
neuronal cells contribute in the same way and to the
same extent in sporadic disease? Absence of an
experimental model for sporadic ALS makes this very
difficult to study.
Crucial to the question of whether the neuro-
inflammatory reaction is a relevant therapeutic target
in humans is the development of a biomarker that
allows investigation of this response in patients from
disease onset, and during progression of motor-neuron
degeneration. Imaging studies using microglial or
astrocytic ligands might provide such important
information. Inflammatory markers in the CSF are
alternatives.
Finally, although almost all neurodegenerative diseases
are characterised by a neuroinflammatory reaction, there
are serious discrepancies between results obtained from
studies that manipulate this aspect of disease; this needs
to be addressed. Are these discrepancies genuine
differences? Do glial cells behave differently from one
form of neurodegeneration to another? What is the
mechanism of this difference?
Identification of the biological processes of the
inflammatory reaction in patients with ALS will greatly
increase our understanding of motor-neuron
degeneration. Additionally, it could generate new
approaches for treatment of patients with ALS and other
neurodegenerative disorders. Researchers and clinicians
should join forces to reach this goal.
Contributors
All authors contributed equally to the preparation of this Review.
Conflicts of interest
WR receives funding from KU Leuven, Flemish Institute of Biotechnology
(VIB), and FWO Flanders. TP declares no conflicts of interest.
Acknowledgments
This work was supported by funding from the Fund for Scientific
Research, Flanders (FWO-V 1.5.081.08), University of Leuven
(GOA/11/014), Interuniversity Attraction Poles (programme P6/43 of the
Belgian Federal Science Policy Office), and Health Seventh Framework
Programme (FP7/2007-2013; under grant agreement number 259867).
TP holds a PhD grant from the Institute for the Promotion of Innovation
through Science and Technology in Flanders (IWT-Vlaanderen).
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