REVIEWS
Mechanisms of neurodegeneration and
axonal dysfunction in multiple sclerosis
Manuel A. Friese, Benjamin Schattling and Lars Fugger
Abstract | Multiple sclerosis (MS) is the most frequent chronic inflammatory disease of the CNS, and
imposes major burdens on young lives. Great progress has been made in understanding and moderating
the acute inflammatory components of MS, but the pathophysiological mechanisms of the concomitant
neurodegeneration—which causes irreversible disability—are still not understood. Chronic inflammatory
processes that continuously disturb neuroaxonal homeostasis drive neurodegeneration, so the clinical
outcome probably depends on the balance of stressor load (inflammation) and any remaining capacity for
neuronal self-protection. Hence, suitable drugs that promote the latter state are sorely needed. With the aim
of identifying potential novel therapeutic targets in MS, we review research on the pathological mechanisms of
neuroaxonal dysfunction and injury, such as altered ion channel activity, and the endogenous neuroprotective
pathways that counteract oxidative stress and mitochondrial dysfunction. We focus on mechanisms inherent
to neurons and their axons, which are separable from those acting on inflammatory responses and might,
therefore, represent bona fide neuroprotective drug targets with the capability to halt MS progression.
Friese, M. A. et al. Nat. Rev. Neurol. 10, 225–238 (2014); published online 18 March 2014; doi:10.1038/nrneurol.2014.37
Introduction
Zentrum für Molekulare
Neurobiologie
Hamburg,
Universitätsklinikum
Hamburg-Eppendorf,
Falkenried 94,
D‑20251 Hamburg,
Germany (M.A.F., B.S.).
Nuffield Department of
Clinical Neurosciences
and Medical Research
Council Human
Immunology Unit,
Weatherall Institute of
Molecular Medicine,
John Radcliffe Hospital,
University of Oxford,
Oxford OX3 9DS, UK
(L.F.).
Correspondence to:
M.A.F.
manuel.friese@
zmnh.uni-hamburg.de
Multiple sclerosis (MS) is a disseminated, chronic, inflam­
matory demyelinating disease of the CNS with progres­
sive neuroaxonal degeneration. The disease nearly always
starts before the age of 40 years, afflicts approximately
three females per one male, and is a major cause of dis­
ability.1 MS is considered to be an autoimmune disease,
initiated by T cells targeting self antigens in the CNS in
genetically susceptible individuals. The first lesions are
typically focal areas of demyelination in the white matter,
known as plaques. Depending on the locations of these
plaques, the resulting clinical neurological manifest­
ations are notoriously variable, and usually result from
immune cell invasion across the blood–brain barrier.
This process eventually leads to continuous activation
of CNS-homing and CNS-resident innate immune cells
(macrophages and microglia) in the brain parenchyma,
with ensuing demyelination and neurodegeneration.1,2
However, we still do not fully understand the relation­
ships between the inflammatory lesions, demyelination,
and the neuro­degenerative changes that correlate best
with clinical disability. Although demyelination with loss
of trophic support from oligodendrocytes clearly contrib­
utes to axonal degeneration,3,4 axonal and neuronal injury
also occurs without demyelination.5–10
Since modulation of peripheral inflammatory
responses or demyelination seems to be insufficient to
inhibit neuronal loss, we aim to identify potential novel
therapeutic targets in MS by reviewing work on patho­
logical mechanisms of neuroaxonal dysfunction and
Competing interests
The authors declare no competing interests.
injury, which have the potential to be translated into
druggable approaches. In particular, we point to evi­
dence that axonal and neuronal impairment are early and
independent contributors to the progression of MS,
and assess pathophysiological mechanisms of intrinsic
neurodegeneration, which could occur under chronic
inflammatory insults.
Early and continuous neurodegeneration
Clinical evidence
In most patients, MS starts with a relapsing–remitting
course (RRMS), with subacute episodes of neurologi­
cal symptoms that subside spontaneously to apparently
normal baseline function. After 15–25 years, however,
the relapses typically shift into inexorably progressive
neurodegeneration, which is termed secondary progres­
sive MS (SPMS; Figure 1a).11 10–15% of patients enter
this neurodegenerative phase directly from the onset
of clinical disease—a condition known as primary pro­
gressive MS (PPMS). While the length of the relapsing–­
remitting phase varies greatly, both the inevitability and
the rate of neurological decline are highly consistent,
regardless of the preceding disease course and severity.1,12
These clinical observations have led to the sugges­
tion that the underlying neurodegeneration depends
on the patient’s age and not on the number of relapses
or inflammation. Notably, it transpires that neither the
risk of entering the secondary progressive phase nor
the latency of entering progression is related to total
relapse number during the relapsing–remitting phase
(Figure 1b).13,14 This dissociation between relapses and
progression implies that relapses might not be a valid
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Key points
■■ Neuronal and axonal degeneration in multiple sclerosis (MS) is a slow
process initiated by acute lymphocytic inflammation, and subsequently driven
by chronically smouldering, diffuse parenchymal myeloid and meningeal
lymphocytic inflammation
■■ Oxidative stress, mitochondrial injury and subsequent ion channel dysfunction
secondary to chronic inflammation seem to have a constant impact on neurons
and axons, leading to their demise during progressive MS
■■ Several ion channels show compensatory changes in response to
the inflammatory stimulus by altering their relative distribution in the
neuron—a process that eventually becomes maladaptive and perpetuates
neuroaxonal injury
■■ Several neuroprotective pathways have been identified in MS, but these
pathways become overridden, resulting in neuronal degeneration that is
probably mediated by the initiation of apoptosis and Wallerian degeneration
■■ The balance between continuous inflammatory stressors and intrinsic buffering
mechanisms depends partly on age, sex and genetic factors, which eventually
determine the clinical course of MS
■■ In an animal model of MS, few molecular targets with proven neuroprotective
properties that are separable from their impact on inflammatory responses
have been identified; these molecules include CyPD, ASIC1 and TRPM4
surrogate outcome measure for the late disability of MS.
In support of this notion are the minimal—or at least
unknown—long-term effects on disability or rate of
clini­cal progression of current disease-modifying treat­
ments initiated early during clinically isolated syndrome
(CIS) or RRMS. 15–17 Nevertheless, once progression
starts, no current drug has been found to affect its rate.
These findings have led to the hypothesis that MS is a
two-stage disease, starting with an inflammatory phase
and later entering a supposedly distinct neurodegenera­
tive phase. However, advances in imaging and neuro­
pathology in early MS are making it increasingly clear
that neurodegeneration starts directly at onset of disease,
so is already apparent by the time of clinical diagnosis,
and that the transition from RRMS to SPMS is likely to
Paraclinical evidence from MRI and biomarkers
Several classic MRI modalities detecting hypointense
T1 ‘black holes’, cortical lesions and atrophy of the brain
and spinal cord, as well as novel analytical methods,
provide data that correlate with clinical disability, and
have been used to show that neurodegeneration is often
already evident at the time of diagnosis (Figure 2). 18
Cerebral and spinal cord atrophy has proved to be the
most important marker of neurodegeneration, and can
be detected in patients with CIS, as well as at the earliest
stages of RRMS.19,20 Various studies have demonstrated
that this atrophy also affects the grey matter, again from
the ­earliest stages of disease.19
Unmyelinated CNS axons are uniquely accessible in
the retinal nerve fibre layer (RNFL), and optical coher­
ence tomography (OCT) now allows their measure­
ment at micrometre resolution, offering the potential to
monitor neurodegeneration in a novel and simple way.
Indeed, in patients with MS who have no prior clinical
history of optic neuritis, the estimated annual thinning of
the RNFL is approximately 2 μm, compared with 0.2 μm
per year in disease-free controls (Figure 3a,b).18,21
Independent evidence for ongoing neurodegeneration
has come from biomarker studies measuring different
neuron-specific and axon-specific proteins in cerebro­
spinal fluid (CSF). The most robust marker to date seems
to be the concentration of neurofilament heavy chain in
the CSF, which correlates with the extent of disability.
Indeed, levels of the protein are already significantly
increased in patients with CIS.22 Neurofilament light
chains have shown similarly increased CSF levels at all
stages of MS; in patients with RRMS, the levels decreased
after treatment with natalizumab.23
b 100
1–2
2–4
≥5
80
80
RRMS
SPMS
60
50% RR
40
Median time to SP = 15 years
20
0
10
60
40
20
50% SP
0
Patient survival (%)
Patients converting to SPMS (%)
a 100
be the point at which the compensatory bypassing of
neuronal injury exceeds its capacity.
0
20
30
40
50
0
Disease duration (years)
10
20
30
40
50
Time (years)
Figure 1 | Clinical correlates of neurodegeneration in MS. a | Rate of conversion to SPMS. In a population-based series
of 806 patients with RRMS with 28-year follow up, median time to conversion to SPMS (defined as at least 1 year of
deterioration) was 15 years, shown here as a cumulative percentage in a Kaplan–Meier analysis. b | In the same cohort
as in part a, patients with RRMS reached onset of progression within a similar time frame, regardless of categorization as
having low (1–2), intermediate (3–4) or high (≥5) total number of relapses. Data are depicted as Kaplan–Meier survival
curves. Estimated mean times from disease onset to Disability Status Scale 6 (requiring aid for walking) were similar
between the groups: 1–2 relapses, 15.6 mean years; 3–4 relapses, 15.7 mean years; ≥ 5 relapses, 15.9 mean years.
Abbreviations: MS, multiple sclerosis; RR, relapsing–remitting; SP, secondary progressive. Part a adapted by permission
from BMJ Publishing Group Limited. J. Neurol. Neurosurg. Psychiatry, Scalfari, A. et al. 85, 67–75 © 2013. Part b by
permission of Oxford University Press: Scalfari, A. et al. The natural history of multiple sclerosis: a geographically based
study 10: relapses and long-term disability. Brain 133 (Pt 7) (2010), 1914–1929.
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Figure 2 | Radiological correlates of neurodegeneration in multiple sclerosis. Black
holes on T1-weighted spin-echo MRI in multiple sclerosis represent severe tissue
destruction, including axonal and neuronal loss (boxes). Sequential T1-weighted
spin-echo MRI scans, obtained at 2‑month intervals, show evolution of black holes
from a new lesion, starting with a new gadolinium-enhancing (bright) lesion that
represents an active inflammatory process with a breakdown of the blood–brain
barrier, evolving into hypointense non-contrast-enhanced images at all subsequent
time points. Permission obtained from Nature Publishing Group © Barkhof, F. et al.
Nat. Rev. Neurol. 5, 256–266 (2009).
Neuropathological evidence
Historically, the white matter plaques, with primary
demyelin­ation and astrocytic scarring, dominated think­
ing on MS pathology, and degeneration of neurons and
their axons was overlooked.24 However, early involvement
of axons is now accepted; indeed, their loss is the best cor­
relate of the irreversible neurological disability in MS.25
Acute axon injury is commonly found in white matter
inflammatory plaques in early RRMS, but is less evident
in chronic inactive lesions and normal-appearing white
matter (NAWM; Figure 3c,d). Although NAWM is defined
as macroscopically normal and microscopically normally
myelinated, most samples actually show significantly
decreased densities of axons.5–9 This finding might partly
be explained by Wallerian degeneration, which occurs
after transections have separated distal axon segments
from their cell bodies.26 However, diffuse axon injury in
NAWM is not associated with white matter plaques, but
correlates instead with diffusely scattered inflammation
throughout the CNS. Therefore, this process seems to be
partially independent of demyelination.5–10
Imaging and neuropathological studies have rediscov­
ered extensive grey matter pathology in MS, often with
demyelinated cortical plaques and transected axons,
apoptotic neurons,27 and reduced neuronal density with
atrophy.24,28 The grey matter lesions are usually wide­
spread and random, include the deep grey matter, and
show intense microglial and immune cell infiltration.29
Chronic grey matter lesions, however, tend to have less
immune cell infiltration than do chronic white matter
lesions. 24 Pathological evidence indicates very early
involvement of the cortex, sometimes even before any
white matter lesions are found in brain biopsies29 and,
since cortical lesions are seen particularly in SPMS
and PPMS, they could be important pathological cor­
relates of the irreversible disability.7,30 Supporting this
idea, cortical grey matter lesions with widespread neuro­
nal loss are associated with diffuse meningeal inflam­
mation that is, in turn, closely correlated with the rate
of clinical progression in SPMS and PPMS, suggest­
ing that soluble factors produced by the inflammatory
cells in the subarachnoid compartment have a crucial
role in neurodegeneration.31,32
Taken together, these findings imply that neuro­
degeneration in MS is at least partially independent of
demyelination, and is a slow process initiated by acute
lymphocytic inflammation and subsequently driven by
chronically smouldering, diffuse parenchymal myeloid
and meningeal lymphocytic inflammation. According to
this model, inflammation can drive neurodegeneration
at any stage of the disease.
Genetic evidence
The suggestion that inflammation is causally linked to
neurodegeneration has recently been supported by a cor­
relation between meningeal inflammation and the extent
of small-fibre axonal loss in the lumbar spinal cord. This
phenomenon can, however, only be detected in indi­
viduals who are positive for HLA-DRB1*15,33 the most
important genetic risk factor for MS.34 To date, almost
all genome-wide association studies (GWAS) have com­
pared MS patients with healthy controls, thereby focusing
on initiating events rather than on severity and, thus, the
neurodegenerative pathways involved in MS progres­
sion.34 Only three GWAS have focused on MS sever­
ity, 35–37 and showed no consistent associ­ations with
any of the genes known to predispose to MS onset,37
although GRIN2A, which encodes the NR2A subunit of
N‑methyl‑d-aspartate (NMDA)-type glutamate recep­
tors, showed an association in two of the three studies.35,37
Moreover, MS patients with allelic variants in a gene
network that led to higher glutamate levels in the brain
showed greater brain atrophy over 1 year of follow-up
in comparison to patients who did not carry these risk
alleles.38 Clearly, these data need to be replicated in larger
studies, which might identify other interesting candidate
genes that are associated with MS severity.
Although the above-delineated studies provide mostly
phenomenological and correlative evidence, they have
prompted hypothesis-driven molecular studies, which
have provided partial insights into the inflammationinduced neurodegenerative and compensatory neuro­
protective pathways, which we will describe in the
next section.
Molecular pathways of neurodegeneration
Inflammation in the CNS leads to innumerable molecu­
lar changes (Figure 4). Immune cells secrete neurotoxic
products—including reactive oxygen species (ROS),
glutamate, cytokines and chemokines—that direct the
evolution of immune responses and also alter cellular
metabolism in neurons and their axons. The short-term
effects of these immune cell products are important for
tissue defence and resolution; however, in the long-term,
they evoke intrinsic stress responses and activate other
homeostatic processes. Dissecting these pathways in MS
is very difficult because of the complexity of the disease,
the limited access to the target tissue, and the clinical and
genetic heterogeneity of the condition.
Not surprisingly, most of our current knowledge
about neurodegeneration in MS originates from experi­
mental autoimmune encephalomyelitis (EAE), the
animal model of MS, which mimics many clinical and
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Box 1, and have thereby identified proven neuroaxonal
mechanisms of injury or protection during chronic CNS
inflammation in vivo (Figure 5, Table 1, Supplementary
Table 1 online). Moreover, we have included selected
studies that have a compelling rationale but do not yet
comply with these criteria and, therefore, need further
investigations to demonstrate neuroprotective properties.
a
Thickness (μm)
b 200
100
0
0
T
120 150 180 210 240
N
I
T
Position on retinal map
Pathologically thinned RNFL
Normal RNFL thickness
RNFL thickness in optic neuritis
Borderline RNFL thickness
30
60
S
90
0
1
2
Axons (%)
d 100
c
0
0
*
50
0
1
*
1 2
NAWM
2
*
Lesion
Figure 3 | Pathological correlates of neurodegeneration
in MS. a | RNFL thickness (red and yellow area indicated by
arrow), assessed by optical coherence tomography, provides
an estimate of the extent of neurodegeneration. Retinogram
shows imaged layers of a normal retina. b | Blue line
indicates RNFL thickness in an eye affected by optic neuritis,
superimposed over normative data for an age-matched
control. c | Representative axonal changes in acute MS
lesions. Normal appearance of axons (stage 0; top), focal
swellings (stage 1; middle) or fragmentation (stage 2;
bottom) in Bielschowsky silver impregnation. d | Axonal injury
is also prevalent in NAWM, but stages 1 and 2 are much
more frequently detected in acute MS lesions. Asterisks
indicate significant differences between NAWM and lesions.
Abbreviations: I, inferior sector; MS, multiple sclerosis;
N, nasal sector; NAWM, normal-appearing white matter;
RNFL, retinal nerve fibre layer; S, superior sector; T, temporal
sector. Permission for parts a and b obtained from Nature
Publishing Group © Barkhof, F. et al. Nat. Rev. Neurol. 5,
256–266 (2009). Permission for parts c and d obtained from
Nature Publishing Group © Nikic, I. et al. Nat. Med. 17,
495–499 (2011).
neuropathological features of the disease.39 To identify
truly neurodegenerative pathways in MS or EAE, one
must be aware of indirect effects of inflammatory pro­
cesses, which could account for several reports of ‘neuro­
degeneration’ or ‘neuroprotection’. Since it is difficult to
predict whether in vitro experiments mimicking only
part of the inflammatory environment will translate
into the same pathophysiological correlate in EAE or
MS, we will primarily discuss in vivo studies that have
provided novel conceptual insights into oxidative stress,
mito­chondrial dysfunction and altered ion channel activ­
ity. These studies fulfil the strict quality criteria listed in
228 | APRIL 2014 | VOLUME 10
Oxidative stress
Neuropathological studies have implicated activated
macro­phages and microglia in driving ongoing neuro­
degeneration. Given that active neurodegenerative
processes in RRMS and SPMS correlate closely with
inflammation,5 great interest has been expressed in the
neurotoxic products released by such innate immune
cells. ROS and reactive nitrogen species (RNS) such as
nitric oxide (NO) are produced by macrophages and
micro­glia in MS and EAE lesions, and their oxidation
products correlate with inflammation.40–42 Moreover,
neurons with intense cytoplasmic accumulation of oxi­
dized phospho­lipids and DNA strand breaks are abun­
dant in active cortical MS lesions, implying that oxidative
damage is highly prevalent.43
Besides being a by-product of cellular respiration, ROS
can be synthesized specifically in activated macrophages
and microglia by enzymes including myeloperoxidase,
xanthine and NADPH oxidases (NOX). 44,45 Activated
microglia in both active and slowly expanding lesions
show upregulation of NOX2 subunits, and also of NOX1
and NOX organizer 1.45 The importance of NOX2 in
oxidative tissue damage is supported by the milder EAE
course in mice that were deficient in gp91phox, which
encodes a NOX2 subunit, although neither neuroaxonal
degeneration nor the extent of inflammatory infiltrates
was assessed. 46 NO donors have also been shown to
cause reversible conduction block in axons and to drive
neuronal degeneration in rat spinal cords.47 An in vivo
imaging study revealed that application of oxygen and
nitrogen donors to the spinal cords of healthy mice
was sufficient to induce EAE-like axonal injury in the
absence of demyelin­ation.42 In turn, scavengers that
reduced levels of ROS and RNS in EAE mice were able
to attenuate focal axonal degeneration without altering
the numbers of immune cells in acute EAE lesions.
Iron accumulation in the physiologically ageing human
brain and in patients with MS can further amplify ROSand-RNS-mediated injury 48 by generating toxic react­
ants, as experimentally shown in spinal cord injury.49 In
the CNS, iron is stored in oligodendrocytes, and injury
of these cells during MS releases Fe2+ into the extra­
cellular space, where it is taken up by activated macro­
phages and microglia.50,51 In turn, the macrophages and
microglia degenerate,52 thereby further contributing to
Fe2+ accumu­lation in the extracellular space and also in
axons.48 Eventually, this process might contribute to oxi­
dative neuronal damage.53 Importantly, this iron accumu­
lation correlates with early axonal injury, and is particular
prominent in active lesions of patients with acute MS and
short disease duration.48 In chronic MS, however, a sig­
nificant decrease in iron levels is observed in the NAWM,
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Reactive
oxygen
species
Chronic
CNS
inflammation
Reactive
nitrogen
species
Oxidative
stress
Hypoxia
Mitochondrial
damage and
dysfunction
Cytokines
Demyelination
Glutamate
Ca2+ influx
Ion channel
redistribution
Energy
deficiency
Ion
imbalance
2+
Ca /Na+
overload
Activation
of degrading
enzymes
Oncotic
cell
swelling
Neuroaxonal
damage by
apoptosis
and
necrosis
Figure 4 | Cascades leading to inflammation-induced neuroaxonal injury. The scheme illustrates the prevailing hypothetical
sequence of events eventually leading to neuroaxonal degeneration in multiple sclerosis. Chronic CNS inflammation lies at the
root of deregulation of neuronal and axonal metabolism. The cascade culminates in the hallmarks of inflammation-induced
neurodegeneration described in this Review.
corresponding with disease duration,48 questioning a
direct role for iron accumulation in MS progression with
advancing age. Moreover, iron is important for several
cellular processes and homeo­stasis in the CNS, sound­
ing a note of caution regarding the use of iron-chelating
therapies in patients with MS. Fe2+ accumulation in
the CNS of EAE animals is scarce,54 so its pathogenic
role during inflammation-induced neurodegeneration
remains e­ nigmatic and difficult to test.
Intrinsic and extrinsic oxidants are major stressors
in cell physiology, and dynamic cellular programmes
have evolved to moderate their long-term deleterious
effects. One such programme is orchestrated by nuclear
factor erythroid 2‑related factor 2 (NRF2), a transcrip­
tion factor that induces such antioxidant enzymes as
heme oxygenase 1 (HMOX1), which scavenge free radi­
cals and remove damaged proteins.55 HMOX1 levels are
increased in MS lesions56 and in the CNS of EAE mice,57
and are also found to be elevated in the CSF of patients
with MS.58 Exacerbation of EAE in both Nrf2 and Hmox1
knockout mice underscores the therapeutic potential of
Box 1 | Criteria verifying direct neurodegeneration or neuroprotection in EAE
Very few studies on defined molecules allow direct inference regarding
neurodegeneration or neuroprotection; that is, direct injury or preservation of
axons and neurons without altering the accompanying CNS inflammation in vivo.
Since many tested molecules are expressed in both CNS-resident cells and
the immune system, studies on these molecules and their pathways reporting
neurodegenerative or neuroprotective processes in the context of EAE must fulfil
at least one of the following criteria to verify their direct neuronal involvement:
■■ Reports of altered neuroaxonal damage with unchanged CNS inflammation
during the disease. However, due to the difficulties of analysing the entire EAErelevant immune cell functions, and the possibility that reduced (neuronal)
tissue damage results in an attenuated reactive inflammatory response, further
measures must be taken to prove a direct neuronal phenotype by interfering with
defined molecular pathways
■■ Generation of tissue-specific (conditional) transgenic animals with exclusive
expression or deletion of the respective molecule in neurons
■■ Bone marrow chimaeric animals, which harbour an immune system that is
replaced by haematopoietic stem cells of a different genotype, resulting in mice
with a wild-type immune system and genetically altered CNS-resident cells.
However, the caveat remains that CNS-resident immune cells, such as microglial
cells, are not replaced by this procedure
■■ A further valid approach to decipher immune and neurobiological effects is
adoptive transfer EAE experiments, whereby the animals are passively immunized
by ex vivo-activated, CNS-specific T cells from appropriate donor animals
Abbreviation: EAE, experimental autoimmune encephalomyelitis.
enhancing these antioxidant pathways, 57,59 but both of
these knockout mice show elevated immune responses
in the EAE model, thereby preventing imputation of their
enhanced neuronal injury to a compromised neuronal
antioxidative response.57,59
Notably, in mice, Nrf2 is induced in neurons by the
drug dimethyl fumarate (DMF).60 This effect has been
proposed to contribute to an attenuated EAE disease
course in DMF-treated mice, and might, therefore, partly
underlie the beneficial effects of this drug in patients
with MS.60 No differences were reported in immune cell
infiltration for DMF-treated versus control mice, but the
assessments were performed 74 days after EAE induc­
tion, by which time most of the inflammatory responses
have already subsided from the CNS. Other studies have
reported that DMF induces Hmox1 in dendritic cells,
resulting in an anti-inflammatory phenotype of den­
dritic cells and, subsequently, T cells, but this agent also
inhibits macrophage infiltration.61,62 This finding might
explain, at least in part, the ameliorated EAE course in
DMF-treated animals.
The hypothesis that antioxidative pathways protect
neurons from inflammatory-mediated oxidative stress
is attractive, but whether these pathways are enhanced
in neurons is not yet known, and experimental data are
lacking to support the idea that neuronal NRF2 induction
by DMF results in neuroprotection in CNS inflammation
in vivo. However, given that mitochondrial dysfunction
is a major component in MS pathophysiology (Box 2),
and mitochondria have long been known to be particu­
larly vulnerable to damage from prolonged exposure
to oxidants,63 the concept of ROS-and-RNS-mediated
neurodegeneration in chronic CNS ­inflammation is
particularly appealing.
Mitochondrial dysfunction and energy deficit
According to one hypothesis, damage to mitochon­
dria is the main cause of tissue injury in MS (Box 2).64
Mitochondria are derived from aerobic bacteria that
infiltrated proto-eukaryotes around 109 years ago, and
they still have a separate genome and synthesize ATP by
oxidative phosphorylation (OXPHOS). Normal cellular
respiration yields ROS as by-products that require detoxi­
fication. Lacking protective histones, mitochondrial DNA
(mtDNA) can rapidly accumulate mutations when ROS
levels are increased, which can eventually compromise
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b
Glu
Microglia
Glu
Monocyte
Myelin
GluR
NOS
Lymphocyte
H+
Glu
H+
H+
VGCC
ASIC1
Ca2+
Na+/K+ATPase
NCX
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Axon
Neuron
Nav1.2
Nav1.6
TRPM4 Nav1.8
Na+
Reverse
Na+
Na+
Cell
swelling
ATP
Calpain
Oligodendrocyte
Blood
vessel
Axon
Myelin
Neuron
a
Axon
c
Demyelination
ROS
OXPHOS
CyPD
Mutated
mtDNA
Dying
back
mtPTP
Presynaptic
neuron
Necrosis
ATP
Apoptosis
DR4/5
CD200R
CD200
WldS
Resveratrol
Bcl-2
Neuroaxonal
injury
H+
Glu
NO
CB1R
ROS
Cannabinoids
Cytokines
Figure 5 | Neuronal injury and counteracting pathways in chronic CNS inflammation. Chronic inflammation in multiple sclerosis
is primarily driven by activated parenchymal macrophages and microglia, and meningeal lymphocytes. a | Inflammatory ROS and
NO production leads to mtPTP opening, accumulation of mtDNA mutations and oxidized and misfolded proteins, and necrosis.
Compromised mitochondria disturb OXPHOS, leading to further ROS elevation and decreased ATP production. Apoptosis is
induced partly by DR4/5 engagement. b | Excess extracellular Glu activates Glu receptors, leading to neuroaxonal Ca2+ influx.
Membrane depolarization activates VGCC and increases Nav activity. ASIC1 contributes to further Ca2+ influx, and ASIC1 and
TRPM4 potentiate Na+ influx. Reduced ATP levels lower Na+/K+-ATPase activity, resulting in reverse operation of NCX and fatally
increasing intracellular Ca2+ and Na+ levels. Continuously elevated Ca2+ activates degradative enzymes and NOS. These
processes lead to neuronal apoptosis and necrosis. c | Excitotoxicity associated with Ca2+ and Na+ overload is counterbalanced
by prosurvival gene expression, cannabinoid system activity, and inhibition of Wallerian degeneration. Continuous inflammation
overrides these buffering systems, resulting in substantial neuroaxonal damage. Abbreviations: ASIC1, acid-sensing ion
channel 1; CyPD, cyclophilin D; DR4/5, death receptor 4 and 5; Glu, glutamate; mtDNA, mitochondrial DNA; mtPTP,
mitochondrial permeability transition pore; Nav, voltage-gated sodium channel; NCX, Na+/Ca2+-exchanger; NO, nitric oxide;
NOS, NO synthase; OXPHOS, oxidative phosphorylation; ROS, reactive oxygen species; TRPM4, transient receptor potential
cation channel subfamily M member 4; VGCC, voltage-gated calcium channels.
OXPHOS efficiency.65 Both excessive ROS production
and mitochondrial deficiency normally seem to increase
with age.63 ROS-inflicted mtDNA mutations compromise
OXPHOS, initiating a vicious circle that results in mito­
chondrial collapse with consequent reductions in cellular
fitness (Figure 5a).65 This effect is further mimicked in
mice66 and humans67 with mutations in mtDNA polymer­
ase, which accumulate excessive mtDNA mutations and
exhibit signs of premature ageing of a type that can also
be detected in the brains of patients with MS.
As transport in axons is highly energy-demanding,
these structures are extremely sensitive to fluctuations in
energy supply; indeed, compromised mitochondrial trans­
port is an early change in inflammatory EAE lesions,42
230 | APRIL 2014 | VOLUME 10
possibly mediated by translocation of histone deacetylase 1
from the nucleus to the axoplasm, where it hinders kinesin
motor protein functions.68 Resveratrol attenuates neuronal
damage in optic neuritis during EAE without affecting the
inflammatory infiltrate in the optic nerve;69 it protects by
indirectly activating sirtuin 1 (SIRT1), a NAD+-dependent
deacetylase that promotes mitochondrial function.70
In addition to excessive ROS, mitochondrial damage or
death can be triggered by the mitochondrial per­meability
transition (MPT), which depends on the matrix protein
cyclophilin D (CyPD). MPT leads to instantaneous col­
lapse of the mitochondrial trans­membrane potential,
equilibration of ionic gradients, and cessation of OXPHOS
with subsequent necrosis. EAE is milder in Cypd-deficient
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Table 1 | Genetic evidence: molecules mediating neurodegeneration or neuroprotection in EAE
Gene modulation*
Outcome
Evidence for neurospecific effect
Reference
BCL2 overexpression
under NSE promoter
Reduction in clinical impairment;
reduced axonal damage
Overexpression of human BCL2 only in neurons
(NSE promotor); no differences in T‑cell
proliferation and ‘delayed-type hypersensitivity’
Offen et al.
(2000)129
Asic1–/–
Reduction in clinical impairment;
reduced neuroaxonal damage
No significant difference in immune cell
infiltrate; adoptive transfer EAE
Friese et al.
(2007)72
Cnr1–/– (CB1)
Reduction in clinical impairment on
cannabinoid treatment (is dependent
on CB1 in neurons)
Conditional knockout in neurons (nestin
promotor) and T cells (Lck promotor)
Maresz et al.
(2007)139
Trpm4–/–
Reduction in clinical impairment;
reduced neuroaxonal damage
No significant differences in immune cell
infiltrate, T‑cell activation or cytokine
production; bone marrow transplantation
Schattling
et al. (2012)94
Ppid–/– (CyPD)
Reduction in clinical impairment;
reduced neuroaxonal damage
No significant differences in immune cell
infiltrate and antigen-specific T‑cell proliferation
Forte et al.
(2007)71
Expression of the
fusion protein Wlds
Reduction in clinical impairment;
reduced neuroaxonal damage
No significant differences in the area of
inflammation and T‑cell infiltration
Kaneko et al.
(2006)140
Reduction in clinical impairment;
reduced demyelination and neuroaxonal
damage; reduced microglia activation by
neuronal inhibition
No significant differences in T‑cell proliferation,
cytokine production and ‘delayed-type
hypersensitivity’
Chitnis et al.
(2007)131
Scn10a–/– (Nav1.8)
Reduction in clinical impairment
Immune cell effects were not tested, but Nav1.8
is probably not expressed in immune cells
Shields et al.
(2012)91
Scn2b–/–
Reduction in lethality and clinical
impairment; reduced neuroaxonal damage
No significant differences in immune cell
infiltrate, T‑cell proliferation and cytokine release
O’Malley et al.
(2009)83
*Affected proteins are given in brackets, where relevant. Abbreviation: EAE, experimental autoimmune encephalomyelitis.
mice, and axon damage is decreased despite unaffected
immune cell infiltration.71 Furthermore, neurons lacking
CyPD are more resistant to damage mediated by ROS
and RNS.71
Eventually, because of impaired mitochondrial
OXPHOS and MPT, the high demands for neuroaxonal
ATP can no longer be met, and chronic hypoxia ensues.
This shortfall has been one of the prevailing explana­
tions for neurodegeneration in MS,64 and is supported
by hypoxic gene expression signatures, including induc­
tion of hypoxia-inducible factor‑1α (HIF‑1α) in EAE.72
In addition, some studies in the brains of patients with
MS have revealed nuclear translocation of HIF‑1α that
indicates its activation,73,74 which would promote tissue
oxygenation and glucose delivery.
Box 2 | Mitochondrial damage in multiple sclerosis
Chronic inflammation leads to increased local levels of reactive oxygen species and
reactive nitrogen species, increasing the risk of mitochondrial damage. Remarkably,
mitochondria from the motor cortex of patients with MS show abnormal reductions
in nuclear DNA-encoded complex I and III activities.141 Moreover, mitochondrial
DNA (mtDNA) gradually accumulates deletions in neurons in the grey matter of
patients with secondary progressive MS (SPMS) compared with age-matched
controls.142 These deletions are not limited to cortical lesions, but are found
throughout the grey matter—and primarily in layer VI—in SPMS. Layer VI consists
mainly of motor neurons, and borders on the subcortical white matter, suggesting
involvement of white matter processes such as production of toxic agents by
macrophages and microglia within active white matter lesions.142 Additional loss of
the mtDNA-encoded catalytic subunits of complex IV might be incurred, especially
in demyelinated axons in active and chronic active lesions with high densities
of macrophages and microglia.107,143 On the other hand, total mitochondrial
content and complex IV activity can be increased in apparently undamaged but
demyelinated axons in chronic inactive lesions, probably in response to increased
energy needs,107 and might even normalize in remyelinating axons.144
Given that mitochondria are key regulators of cell
survival and death, a detailed understanding of specific
damaging pathways seems to be central in designing
strategies to halt inflammation-induced neuroaxonal
failure. Moreover, as the newest calculations of energy
consumption by cortical neurons show that 75% of total
neural energy is used for ionic equilibrium during infor­
mation processing,75 ion channel malfunction seems to be
central during the energy shortfall, as we will now discuss.
Ion channel dysfunction
Both energy imbalance and demyelination during chronic
CNS inflammation lead to activation, dysfunction and
maldistribution of several ion channels (Supplementary
Table 1 online), evoking downstream mechanisms that
mostly converge on Ca2+ overload (Figure 5b). This over­
load seems to be the key instigator of neurotoxicity, initi­
ating a vicious circle by activating degradative enzymes,
compromising mitochondrial function and impairing
axonal transport, all of which results in a further increase
in Ca2+ levels. Sodium MRI has revealed increased Na+
concentrations within MS lesions, NAWM and grey
matter, which were particularly pronounced in patients
with SPMS,76 providing evidence for ion dysregulation
in progressive MS.
Sodium channels
Concentration of Na+ channels at nodes of Ranvier allows
accelerated ‘saltatory’ conduction from node to node by
myelinated fibres. When it enters the axon, Na+ is rapidly
exchanged for extracellular K + by Na+/K+-ATPase at
the internodal axolemma.77 This ongoing ion exchange
is important for axonal polarization, which in turn is
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needed for transmission of action potentials, but con­
sumes large amounts of neuronal energy, leading to the
assumption that excessive Na+ influx results in aggravated
energy run down.75 These observations have also resulted
in the concept that demyelination not only exposes axon
segments to extrinsic stressors, but also vastly increases
the energy demand for signal conduction, as Na+ channels
spread along the denuded axolemma.78 However, it has
now been shown that action potentials consume much
less energy 79,80 than was previously thought.81 Therefore,
Na+ influx-related energy run down remains speculative,
as there is a low correlation between the membrane area
and the predicted energy consumption of a neuron.75
Another postulate is that the Na +/Ca 2+ exchanger
(NCX) operates in reverse when Na+ levels rise in axons,
thereby increasing intracellular Ca2+ levels. This sug­
gestion is supported by colocalization of voltage-gated
Na+ channel (Nav)1.2, Nav1.6 and NCX subunits along
demyelin­ated axons in MS plaques78 and EAE lesions,82
as well as by the beneficial effects of gene knockouts
and Nav/NCX blockers in EAE. Deficiency of the Navmodulating β2 subunit (Scn2b) prevents Nav1.6 upregula­
tion in EAE lesions and reduces both axon degeneration
and clinical scores.83 Moreover, the class I antiarrhyth­
mic flec­ainide,84,85 Na+-channel antagonistic anticonvul­
sants86–88 and the α‑aminoamide derivative safinamide85
all reduce axon injury and neurological disability in mice.
Importantly, peripheral immune cell compositions and
T‑cell responses in Scn2b-deficient mice were compar­able
to those in wild-type mice.83 Immune cell infiltration into
the CNS in EAE mice was not quantified, however, so the
possibility remains that channel modulation, as recorded
with the antagonists used to date, are actually acting as
immunosuppressants on Nav-expressing macro­phages
and microglia89—a notion that is supported by the acute
EAE exacerbations and increases in inflammation on
their withdrawal.85,86 Similarly, the anti­convulsant lamo­
trigine proved ineffective in SPMS, with no effect on cer­
ebral volume over 24 months. Indeed, this drug caused
transient early volume losses, suggesting water shifts or
‘pseudoatrophy’, again implying anti-inflammatory rather
than neuroprotective effects.90
Nav1.8 has a more definitive role in cerebellar dys­
function in MS. Normally restricted to the PNS, this
channel is ectopically expressed in cerebellar Purkinje
neurons in patients with MS and mice with EAE. In
mice, Nav1.8 overexpression clearly disrupts coordi­
nated motor behaviours; both treatment with a Nav1.8selective blocker and deficiency of the Scn10a gene,
which encodes Nav1.8, moderate EAE and alter Purkinje
neuron firing.91 These findings provide further evidence
for a distinct inflammation-induced ion channelopathy,
and suggest possible therapeutic options for treating
­cerebellar dysfunction in MS.
Nonselective cation channels
The transient receptor potential (TRP) superfamily of
ion channels enables individual cells to sense thermal
and chemical changes in their local environment.92
These channel properties allow neurons also to sense
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and respond to inflammation. Most of the TRP proteins
are nonselective cation channels, although member four
of the melastatin-like TRP subfamily (TRPM4) consti­
tutes an exception by just gating monovalent cations,
thereby being impermeable for Ca2+.93 As detailed above,
increased intracellular Ca2+ levels and ATP depletion
due to mitochondrial dysfunction are two major patho­
physiological alterations in neurons during chronic
CNS inflammation. Of note, the opening behaviour of
TRPM4 is altered by these two conditions: it can be acti­
vated by increasing intracellular Ca2+ levels but can also
be blocked by high cytosolic ATP levels.93
A study published in 2012 showed that genetic or
pharma­c ological inactivation of TRPM4 resulted
in reduced axonal and neuronal degeneration and
amelior­ated clinical EAE disease scores.94 Importantly,
the neuro­nal protection in Trpm4-deficient mice arose
without alterations in lesion or immune cell numbers,
antigen-specific T‑cell proliferation, cytokine profiles or
demyelination. The neuron-specific effect was further
validated by the generation of bone marrow chimaeric
mice and by in vitro studies. Importantly, TRPM4 is
expressed in neuronal somata in mouse and humans, but
is redistributed into axons during chronic inflammation
in MS and EAE, and colocalizes with the axonal injury
marker amyloid precursor protein (APP). Neuronal
damage probably occurs through TRPM4-dependent
neuronal ion influx with subsequent oncotic cell ­swelling
on intracellular Ca2+ stimulation.94
Potassium channels
Since K+ channels regulate synaptic transmission and
neuronal excitability, alterations in expression or acti­
vation, particularly of voltage-gated K+ (Kv) channels,
could possibly affect these functions. Congruously,
4‑aminopyridine, a nonspecific blocker of Kv channels,
is currently in use to improve the mobility of patients
with MS.95 Alterations in the neuronal expression pat­
terns of Kv1.2, Kv1.4 and Kv2.1 have been found during
chronic CNS inflammation, and could underlie the ben­
eficial effects of this therapy.96 Furthermore, selective
pharmacological blockade of Kv1.1, which is expressed
on neurons and astrocytes, attenuates the clinical EAE
phenotype and reduces brain damage, without affect­
ing peripheral T‑cell activation. 97 CNS immune cell
­infiltration was not quantified in this study.
The various members of the two-pore-domain K+
channel family are expressed in different body cells and, by
giving rise to leak K+ currents, control excitability. TWIKrelated acid-sensitive potassium channel 1 (TASK1) is
neuronally expressed and, due to its physicochemical
modulation (by pH and pO2, for example), it might alter
electrical excitability during chronic CNS inflamma­
tion.98 In support of this idea, gene deletion or pharma­
cological blockade of TASK1 ameliorates the EAE disease
course, although inactivation of the channel also leads to
repression of inflammatory responses, thereby hamper­
ing any conclusions regarding neuroprotective proper­
ties.99,100 Therefore, whether K+ channel blockers harbour
­neuroprotective properties in the strictest sense is unclear.
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Calcium channels
Several parallel sources contribute to neuroaxonal Ca2+
overloading; namely, entry through voltage-gated
Ca2+ channels (VGCCs), release from intracellular stores,
and other cation channels, among which glutamate-gated
receptors have a prominent role.
Depolarization of neurons and axons after substantial
cation influx will activate VGCCs, which might thereby
contribute to Ca2+ influx. Blocking of L‑type VGCCs with
bepridil or nitrendipine significantly moderated EAE,
and reduced inflammation and axon pathology.101 In nor­
mally myelinated axons, N‑type VGCCs are ubiquitously
expressed on nerve terminals and play an important part
in neurotransmitter release, but are absent from the
internodal axolemma.102 By contrast, prominent axonal
accumu­lation of the pore-forming subunit of N‑type
VGCC was reported in demyelinated axons and axonal
swellings in MS and EAE lesions, and integration into
the axonal membrane in EAE lesions has been shown.103
The maldistribution of VGCCs into demyelinated axons
might result in a pathological influx of Ca2+, contributing
to axonal demise. Supporting a role for these channels in
axonal degeneration, specific blockade of N‑type VGCCs
by use of ω‑conotoxin GVIA decreased axon and myelin
degeneration in an optic neur­itis model.104 However, as
with many other reports, it remains uncertain whether the
treatment was acting primarily on neurons and axons or
on immune cells, as substantially reduced macrophage–­
microglial inflammation was detected in the treatment
groups,101,104 precluding an interpretation with regard to
neuroprotective properties.
Locations of intracellular or intra-axonal stores of Ca2+
include the endoplasmic reticulum and mitochondria.
Both of these organelles seem to contribute to neuronal
Ca 2+ overload with subsequent neuroaxonal injury
in vitro105 but, other than indirect evidence for mitochon­
drial Ca2+ release,71 neither has been shown to operate
in vivo during CNS inflammation.
Glutamate is the main excitatory neurotransmitter in
the CNS. Its concentration is tightly regulated by several
mechanisms, including metabolic and transport pathways.
Alterations in glutamate levels, which result in excessive
neuronal signalling, could lead to Ca2+-mediated excito­
toxicity,106 which is implicated by GWAS in patients with
MS, as specified above.40,42 On glutamate stimulation,
Ca2+ enters mainly through ionotropic glutamate recep­
tors, and several factors, such as cell type, developmen­
tal stage and the relative contributions of synaptic versus
extrasynaptic receptor activation, determine whether
prosurvival or death pathways are activated.106 Reduced
mitochondrial complex IV activity can further augment
glutamate-­mediated axon injury 107 and, in turn, glutamate
and abnormal NMDA receptor function contribute to
­dysfunctional mitochondrial activity in EAE.108
Glutamate levels are elevated in the CSF109 and brains38
of patients with MS, apparently deriving from dying
neurons and secretion by activated immune cells.110
Concomitantly, various cell types show upregulation of
the ionotropic NMDA, AMPA (α-amino‑3-hydroxy‑5methyl‑4-isoxazole propionic acid) and kainate receptors
in MS lesions.111 Moreover, disposal of glutamate prob­
ably declines, as expression of its transporters is decreased
in the CNS of patients with MS112 and EAE mice.113,114
Indeed, inhibition of NMDA and AMPA receptors
improves the outcome of EAE,115–121 and limits neuro­
degeneration also by preserving dendritic spines.120 Some
of these antagonists conferred clinical benefits without
affecting peripheral T‑lymphocyte activation,115,116,121
but monocyte–­microglial infiltration was substantially
reduced by glutamate receptor inhibition.121 Mice that were
genetically deficient for the enzyme d‑aspartate oxidase
released increased amounts of d‑aspartate and NMDA
in the CNS.108 These mice showed no difference in EAE
severity in comparison to wild-type mice, but did exhibit
accelerated disease progression. Therefore, the question
of whether inhibitors of ionotropic glutamate receptors
indeed act specifically as neuroprotectants remains enig­
matic, and further studies with conditional transgenic
animals, bone marrow chimaeras or adoptive transfer
EAE are needed to decipher the precise ­contribution of
glutamate receptors to direct neurodegeneration.
Chronic inflammation is also associated with a rise in
extracellular proton concentrations—another indepen­
dent stressor that activates a distinct class of cation chan­
nels, the acid-sensing ion channels (ASICs). These
channels belong to the degenerin/epithelial Na+ channel
(DEG/ENaC) family, are sensitive to amiloride, and are
present in peripheral and central neurons. 122 ASIC1a
seems to be essential for acid-activated currents in mouse
neurons, where it may heteromerize with ASIC2a. Besides
acid­osis, ASIC1a is activated by lactate, arachidonic acid
and decreased extracellular Ca2+, which are all features
of chronic inflammation.122 Interestingly, the pH drops
from around 7.4 to around 6.5 in inflammatory CNS
lesions.72 Although its main function is to gate Na+ cur­
rents, ASIC1a can also allow Ca2+ entry during acidosis.123
Notably, neuro­axonal degeneration is reduced—without
affecting the lymphocytic or myeloid inflammatory
­infiltrates—­in Asic1a-deficient mice and during treat­
ment with amiloride in several EAE models and neuro­
nal cultures.72,124 Importantly, like VGCC and TRPM4,
ASIC1 is found particularly in injured axons, and colocal­
izes with the axonal injury marker APP, in both EAE and
active MS lesions.124 However, mice deficient in ASIC2 are
not protected against EAE (M. A. Friese and L. Fugger,
unpublished data), so ASIC1 seems the more promis­
ing therapeutic target for strategies to protect neurons in
MS. Supporting this idea, in a pilot study, patients with
PPMS showed a reduction in whole-brain volume loss
during amiloride treatment, ­compared with their own
pre-treatment phase.125
Downstream of the Ca2+ overload in neuronal somata
and axons are Ca2+-dependent proteases such as cal­
pains, which degrade axon components. Inhibition of
these proteases improved clinical scores in a chronic EAE
model.126 Although calpain expression was also increased
in immune and glial cells during EAE,127 upregulation of
these proteases in neurons correlated most closely with the
extent of axonal degeneration in acute EAE.128 However,
calpain inhibitors also significantly reduced inflammatory
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Box 3 | Exploring neurotrophins as neuroprotectants
The few soluble neuroprotectants that have been identified include ciliary
neurotrophic factor (CNTF145) and brain-derived neurotrophic factor (BDNF146).
BDNF belongs to the family of neurotrophins and is secreted primarily by
neurons and glia. The wide-ranging actions of these factors are important for
the development and maintenance of the nervous system.
A single nucleotide polymorphism in the human BDNF gene converts valine to
methionine at codon 66 (Val66Met). The BDNFMet protein shows altered dendritic
trafficking and synaptic localization, and has a substantially reduced propensity
to activity-dependent release.147 Given that the Val66Met polymorphism has been
selected for during evolution,148 and in view of its association with changes in
brain structure, network and function in healthy humans and several neurological
and psychiatric disorders, it might have an important modifying influence on
health and disease.147 In patients with multiple sclerosis (MS), the BDNFMet
genotype is associated with preservation of grey matter volume, and correlates
inversely with autoimmune-induced lesions.149,150 BDNF has, therefore, been
tested as a potential neuroprotectant.151
BDNF is produced mainly by CNS-resident cells, but infiltrating immune
cells can also secrete BDNF in MS.152,153 Mice with Bdnf gene deletions in
either astrocytes or immune cells show enhanced experimental autoimmune
encephalomyelitis (EAE) and axon loss,146 and CNS-derived BDNF seems to
mediate axonal protection in EAE, as demonstrated by experiments with bone
marrow chimaeras.154 However, BDNF can be a double-edged sword, depending
on its levels and the availability of its receptors and target cells. Acting directly on
neurons, it mediates neuroprotection and neuroregeneration; however, it also
induces neurodegeneration after engaging with the TrkB receptor on astrocytes. 155
Therefore, the use of BDNF in MS demands caution. Two novel therapeutics,
alemtuzumab156 and laquinimod,157 increase BDNF secretion by peripheral
immune cells, and whether this action renders these drugs neuroprotective
should emerge from their forthcoming use in MS.
Box 4 | Cannabinoids and erythropoietin as neuroprotectants
Cannabinoids have been tested as neuroprotectants to counteract inflammatory
stressors (Figure 5c). CB1 receptors bind several different endocannabinoids
that are released locally on demand. These receptors are involved in controlling
excitotoxicity,158 and experimental autoimmune encephalomyelitis (EAE) is more
severe in Cb1-deficient mice than in controls, with greater neurodegeneration
and increased tumour necrosis factor-mediated excitotoxicity.159,160 In mice with
cell-type-specific knockouts, cannabinoid treatment-mediated neuroprotection
was shown to depend on CB1 expressed by neurons rather than by T cells.139
This neuroprotective effect is further supported by the fact that low, nonimmunosuppressive doses of cannabinoid receptor agonists can ameliorate
axonal loss and disability in EAE.161 Disappointingly, in a randomized,
double-blind, placebo-controlled study, the cannabinoid receptor agonist
Δ9-tetrahydrocannabinol showed no efficacy with regard to disability progression
over a 36-month period in patients with primary or secondary progressive
multiple sclerosis.162
Erythropoietin (EPO) shows more promising clinical signals. EPO and its receptor
are widely expressed in the nervous system and are upregulated after injury.
Since peripherally administered EPO crosses the blood–brain barrier and can
stimulate neurogenesis and neuronal differentiation, it is proposed to be a potent
neuroprotectant. Furthermore, EPO has antiapoptotic, antioxidant and antiinflammatory properties.163 Prompted by the beneficial effects of EPO derivatives
on inflammatory responses and neurodegeneration in the EAE model,164
a randomized phase II trial was initiated in which patients with optic neuritis
received recombinant human EPO or placebo daily for 3 days as an add-on therapy
to methylprednisolone.165 The EPO-treated patients showed a significant decrease
in retinal nerve fibre layer thinning after 16 weeks. Since long-term treatment of
EPO is associated with severe adverse effects, such as thromboembolic events
and oncogenic potential, the neuroprotective properties might be further explored
in long-term treatments with novel EPO derivatives and mimetics, which engage
the tissue-protective effects of EPO without activating haematopoietic and
coagulation pathways.163
234 | APRIL 2014 | VOLUME 10
infiltrates, thereby precluding any firm conclusions
regarding their potential neuroprotective activities.126
Adaptive changes in ion channels
On the basis of the evidence presented above, ion
channel dysregulation seems to be central to the process
of neuronal and axonal demise during MS. However,
it also transpires that several ion channels show adap­
tive changes to the inflammatory stimulus by altering
their relative distribution in the neuron. Nav,78 VGCC,103
TRPM494 and ASIC1a124 ion channels relocalize from the
somata and dendrites in healthy individuals to axons in
MS and EAE. Although these channels contribute to
axonal degeneration, it seems likely that their axonal
distribution represents an initially beneficial counter­
measure to preserve conductance and axonal integrity.
Understanding this change in distribution, as well as
the sequence of pathological ion channel activation
and the relative contributions of the different channels
to MS‑related neurodegeneration, might open up new
possibilities for therapeutic intervention.
While ion channels represent promising targets mostly
for generating inhibitory drug compounds, hopes have
also been ascribed to endogenous soluble factors that
are physiologically produced by CNS or immune cells
to counteract neurodegeneration. These factors include
neuro­t rophins (Box 3), as well as cannabinoids and
erythropoietin (Box 4), which have already been tested
in ­clinical trials in MS.
Apoptosis and Wallerian degeneration
Eventually, most of the above-described neurodegenera­
tive pathways, combined with a lack of neuroprotective
support, result in a common final pathway of neuronal
and axonal demise, most probably mediated by the initi­
ation of apoptosis and Wallerian degeneration (Figure 5c).
DNA fragmentation reflecting activation of the apoptotic
signalling cascade in neurons is frequently detected in
MS lesions in areas of active demyelination and tissue
injury, but also to a lesser extent in the demyelinated
lesion centre, inactive cortical MS lesions and NAWM.27,43
Supporting evidence for the importance of apoptosis in
inflammation-induced neuronal cell death has come
from mice overexpressing the antiapoptotic protein B‑cell
lymphoma‑2 (Bcl‑2) speci­fically in neurons.129 In com­
parison with control mice, the transgenic mice displayed
attenuated EAE severity and reduced axonal loss without
exhibiting changes in immune response. Moreover,
the cytokine TRAIL, a member of the tumour necrosis
factor–nerve growth factor receptor superfamily that is
secreted by various immune cells, can induce caspasedependent apoptosis in neurons on binding to the death
receptors DR4 and DR5.130
Wallerian degeneration—an active process that is sim­
ilar to apoptosis—seems to drive axonal loss in patients
with MS26 and EAE mice.131 This dying-back process can
be attenuated in mice by the expression of the fusion
protein ‘Wallerian degeneration slow’ (Wlds). Wlds mice,
which carry a triplication of the fusion gene Ube4b/
Nmnat, show reduced neuroaxonal damage as well as
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ameliorated clinical impairment in EAE. Although this
neuronal protection occurs without changes in immune
cell infiltration, T‑cell proliferation or cytokine produc­
tion, Wlds mice display decreased levels of microglial and
macrophage activation in EAE lesions.131 Indeed, this
type of immune modulation seems to be directly con­
trolled by neuronal cells. Neurons of Wlds mice express
increased amounts of the non-signalling glycoprotein
CD200, which inactivates monocytes by binding to
the CD200 receptor,131 thereby providing a protection
mechanism by which neurons are able to control the
extent of CNS inflammation.
Conclusions and future prospects
Descriptive and correlative clinical, paraclinical and
neuro­pathological studies (Figures 1–3) indicate that
inflammation can cause white matter axon transection
as well as cortical and subcortical neuronal injury, even
from the onset of MS. Inflammation is composed of
several relentless stressors—such as electrons, protons or
­oxidants—that disturb complex interconnected neuro­
axonal metabolic pathways,132 to which mitochondrial
damage and glutamate metabolism seem to be central.
These events lead to energy deficits and Ca2+ overload.
Neurons and axons respond to these continuing inflam­
matory challenges via compensatory induction of protec­
tive pathways. However, several additional maladaptive
changes, most notably ion channel redistrib­ution along
inflamed axons, seem to accelerate degener­ation. Although
these alterations might have transient functional benefits,
­prolonged changes seem to be deleterious (Figure 4).
The balance between these continuous stressors and
intrinsic buffering mechanisms depends partly on age,
sex and genetic factors, which eventually determine
the clinical course. An obvious therapeutic goal is to
enhance any compensatory mechanisms and inhibit
maladaptive changes that perpetuate the stressor load.
However, inhibition of adaptive changes is fraught with
difficulties; for example, inhibition of Na+ channels has
resulted in initial worsening of paresis (as these channels
seem to secure conduc­tion along demyelinated axons),
but might provide long-term neuroprotection.90 Equally,
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maintain myelin and long-term axonal integrity.
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targeting of g­ lutamate c­ hannels is associated with severe
adverse effects.133
So far, few molecular targets have been identified
with proven neuroprotective properties that are sepa­
rable from their impact on inflammatory responses.
Attractive approaches would be to inhibit ROS produc­
tion or CyPD, ASIC1 or TRPM4 activity, or to explore
the tissue-protective effects of erythro­poietin (Figure 5,
Table 1, Supplementary Table 1 online). Several of these
targets are currently under investigation for drug develop­
ment. By contrast, no pharmacological agents are avail­
able that might be capable of inducing neuronal pathways
that increase tolerance towards inflammatory stressors.
Physical exertion has been shown to increase resist­
ance to degenerative disorders of ageing.134 Some crosssectional studies report positive associations between
aerobic fitness and grey matter density in trained versus
untrained patients with MS,135 and a randomized trial
has demonstrated a link between physical fitness and
improved cognition in patients with progressive MS.136
These findings are corroborated by the attenuation of
EAE with exercise and the consequent protection of axons
with enhancement of neuronal plasticity.137 Equally, exer­
cise potently rescues the premature ageing phenotype of
mice with mutations in mtDNA p
­ olymerase by inducing
mitochondrial biogenesis.138
Overall, the current dearth of treatment options for the
progressive phases of MS highlights the pressing need for
better understanding of neurodegenerative pathways and
protective mechanisms.
Review criteria
The cited articles in this Review were identified by a
search of the PubMed database, and were retrieved
as full-text articles. Search terms used were “multiple
sclerosis”, “neurodegeneration”, “axonal degeneration”,
“neuropathology”, “genetics”, “MRI”, “progression”, “ion
channels”, “oxidative injury”, “mitochondria”, “microglia”,
“inflammation”, “neuroprotection” and “neurotrophins”,
alone or in combination without limits on publication date
or language. We further analysed reference lists of key
papers to identify additional papers and cross-references.
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Acknowledgements
M.A.F. is supported by the Deutsche
Forschungsgemeinschaft Emmy Noether-Programme
(FR1720/3‑1), Gemeinnützige Hertie-Stiftung
(1.01.1/11/003 and P1130075), Werner Otto Stiftung
(1/81), Forschungs- und Wissenschaftsstiftung
Hamburg, Boehringer Ingelheim Stiftung Exploration
Grant and BMBF Biopharma (NEU2 programme). L.F. is
supported by the Wellcome Trust, the Medical
Research Council, the Lundbeck Foundation and the
Naomi Bransom Foundation.
Author contributions
M.A.F. researched most of the data and drafted the
article with substantial contributions from B.S. and
L.F. All authors contributed to discussion of the
content, reviewing, and editing of the manuscript
before submission.
Supplementary information is linked to the online
version of the paper at www.nature.com/nrneurol.
www.nature.com/nrneurol
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