REVIEWS
Axonal conduction and injury
in multiple sclerosis: the role of
sodium channels
Stephen G. Waxman
Abstract | Multiple sclerosis (MS) is the most common cause of neurological disability in
young adults. Recent studies have implicated specific sodium channel isoforms as having an
important role in several aspects of the pathophysiology of MS, including the restoration of
impulse conduction after demyelination, axonal degeneration and the mistuning of Purkinje
neurons that leads to cerebellar dysfunction. By manipulating the activity of these channels
or their expression, it might be possible to develop new therapeutic approaches that will
prevent or limit disability in MS.
Nodes of Ranvier
Small gaps in the myelin
sheath along myelinated fibres.
Nodes of Ranvier extend ~1
μm along the fibre, and are
separated by segments of
myelin that extend for tens or,
more commonly, hundreds of
micrometres.
Internodal domains
Regions of the axon between
the nodes of Ranvier.
Saltatory conduction
A process of rapid impulse
conduction that is conferred on
axons by myelin sheaths, in
which the action potential
leaps discontinously and
rapidly from one node of
Ranvier to the next.
Department of Neurology and
Center for Neuroscience and
Regeneration Research, Yale
School of Medicine, New
Haven, Connecticut 06510,
and the Rehabilitation
Research Center, Veterans
Affairs Medical Center,
West Haven, Connecticut
06516, USA.
e-mail:
[email protected]
doi:10.1038/nrn2023
Multiple sclerosis (MS) is the most common neurological
cause of disability in young adults in industrialized societies.
It is usually diagnosed between the ages of 20 and 40, and
is called ‘multiple’ sclerosis because most patients have
multiple attacks separated both in time and in space, in
which different parts of the CNS can be involved (for
example, involvement of the optic nerve can cause unilateral visual loss, whereas involvement of spinal sensory
tracts can cause numbness). Early in its course, MS often
displays a relapsing–remitting pattern, with patients losing
functions such as vision or motor function, then recovering these capabilities during remissions. Later in some
patients (secondary progressive MS), or at the beginning
of disease onset in others (primary progressive MS), there
is cumulative acquisition of neurological deficits which
do not remit. Although the cause of MS is unknown, and
multiple etiologies including autoimmunity, infectious
agents, environmental triggers and hereditary factors have
been proposed, there is substantial evidence to indicate
that dysregulated immune responses, including immune
mechanisms directed against myelin proteins, have a role
in triggering disease onset.
Recent studies have identified changes in the expression pattern of specific Na+ channel isoforms as an important contributor to remission and progression in MS, and
there is evidence suggesting that aberrantly expressed Na+
channels might also contribute to cerebellar dysfunction
in MS. In this article, I discuss the multiple roles of Na+
channels in the pathophysiology of MS, including the
adaptive role of some Na+ channel isoforms in restoring conduction in chronically demyelinated axons, the
maladaptive role of other Na+ channel isoforms that
932 | DECEMBER 2006 | VOLUME 7
contribute to axonal degeneration, and the possibility of
a role for a third Na+ channel isoform in the mistuning of
cerebellar Purkinje neurons, which perturbs the pattern
of activity of these cells.
Na+ channels and axonal conduction
The disease process in MS attacks myelinated axons,
denuding them of myelin or causing them to degenerate. Normal myelinated axons exhibit a clustering of
Na+ channels (~1,000 μm–2) in the axon membrane
at the nodes of Ranvier, with a much lower density
(< 25 channels μm–2) in internodal domains where the
axon is covered by myelin1,2. This arrangement (FIG. 1a)
supports saltatory conduction in the normal myelinated
axon. However, it is less well-suited to the functional
needs of the demyelinated axon, in which impedance
mismatch and loss of the myelin capacitative shield permit
current to be dissipated through formerly myelinated
portions of the axon membrane where Na+ channel
density is low, impairing the conduction of action
potentials (FIG. 1b).
Following the loss of myelin in MS, remyelination does
not always occur and in many lesions the myelin is not
replaced. Surprisingly, although demyelination causes
symptoms such as visual loss (when the optic nerve is
involved) or weakness (when the corticospinal tract
is involved), remissions can occur in the absence of remyelination. For example, vision can recover in some patients
in which demyelination affects a substantial length
(a centimetre or more, thereby encompassing the
territory of many myelin segments) of all the axons
within the optic nerve3. Recovery of clinical function in
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a
Nav1.6
Caspr
Myelinated
Node internode
?
5 μm
b
?
c
?
d
10 μm
Impedance mismatch
A phenomenon in which, owing
to non-uniform properties,
there is a sudden drop in
electrical resistance or rise in
capacitance along a cable or
nerve fibre. Impedance
mismatch occurs at the border
between normally myelinated
and demyelinated parts of
axons in disorders such as
multiple sclerosis, and
contributes to conduction
failure.
Capacitative shield
The electrical shield provided
by the myelin that surrounds
the axon, which prevents loss
of current through the
membrane capacitance of the
axon.
Longitudinal current
analysis
A method in which extracellular
electrodes are used to measure
electrical currents as they flow
along nerve fibres and, thereby,
to infer the presence of nodes
or foci of node-like membrane.
Figure 1 | Na+ channel organization of myelinated and demylinated axons. a | Voltage-gated sodium (Nav) channels,
now identified as the Nav1.6 isoform, are aggregated at a high density in the normal axon membrane at the nodes of
Ranvier, but are sparse in the paranodal and internodal axon membranes under the myelin. Right panel shows clustering of
Nav1.6 channels (red) at a node of Ranvier, bounded by Caspr (a constituent of the paranodal apparatus; green) in
paranodal regions, in a normal myelinated axon. Fluorescence and differential contrast images are merged to show the
myelin sheath. b | The acutely demyelinated axon has a low Na+ channel density, a factor that contributes to conduction
failure. c | Some demyelinated axons acquire higher-than-normal densities of Na+ channels in regions where myelin has
been lost, supporting the restoration of conduction that contributes to clinical remissions. Extensive expression of Nav1.6
(red, upper right panel) and Nav1.2 channels ( red, lower right panel) along optic nerve axons (arrows) in experimental
autoimmune encephalomyelitis (EAE) is shown. d | Degeneration of axons also occurs in multiple sclerosis, and produces
non-remitting, permanent loss of function. Images in part c reproduced, with permission, from REF. 34 © (2003) Oxford
Univ. Press.
cases such as this requires the restoration of secure action
potential conduction along at least some of the demyelinated axons. Setting the stage for an understanding of the
role of Na+ channels in this process, an early longitudinal
current analysis showed that some chronically demyelinated
axons can recover the ability to conduct action potentials
in a continuous manner4, and early cytochemical studies
showed that, after demyelination, the denuded axon
membrane can develop higher-than-normal densities of
Na+ channels5. Immunocytochemical studies using panspecific Na+ channel antibodies6,7 subsequently confirmed
the appearance of increased numbers of Na+ channels
in experimentally demyelinated axons. Saxitoxin binding studies also demonstrated a fourfold increase in the
number of Na+ channels in demyelinated lesions from MS
patients8. Taken together, these results indicated that Na+
channel expression is increased in at least some demyelinated axons (FIG. 1c). However, these early studies did
not reveal the molecular identity of the new axonal Na+
channels along demyelinated axons.
NATURE REVIEWS | NEUROSCIENCE
Na+ channels and axonal degeneration
Although largely eclipsed by an emphasis on demyelination, it has been appreciated since the time of Charcot
that, in addition to becoming demyelinated, some axons
degenerate in MS. Recent studies have focused new
attention on axonal degeneration in MS (FIG. 1d), and
have underscored its frequency and occurrence early
in the course of the disease9,10. Importantly, studies in
animal models and in human MS patients have shown
that axonal loss produces non-remitting, persistent
neurological deficits11,12. This has led to considerable
interest in the development of protective strategies aimed
at preventing degeneration of axons, and thereby slowing
or halting the progression of disability in MS.
The available evidence suggests that Na+ channels are
important participants in axonal degeneration in MS
(FIG. 2). As described below, there is evidence for energy
failure within the CNS in MS. Early studies on white
matter tracts in vitro used anoxia, which produces energy
failure, as a highly reproducible, quantifiable insult, and
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to have a protective effect in an experimental model of
MS, experimental autoimmune encephalomyelitis (EAE),
where they prevent degeneration of CNS axons, maintain
axonal conduction and improve clinical outcome.
Microglia
Hypoxia/
Inflammation
ischaemia
Altered
gene
expression
Nav
NO
Nav1.6
Na+/K+ ATPase
Run-down
of ATPase
NCX
Na+
Ca2+
Ca2+ influx
+
Peristant Na
influx
Energy failure
Depolarization
CICR
Mitochondrial Ca2+
Activation of NOS
Activation of proteases
Activation of lipases
Axon
Nav1.2
Less persistent Na+ current
supports conduction
Figure 2 | Model of functional effects of expression of Nav1.2 and Nav1.6
channels along demyelinated axons. Following demyelination, voltage-gated sodium
(Nav) 1.2 channels, expressed diffusely along some axons, support recovery of action
potential conduction. Nav1.6 channels, however, produce a persistent Na+ current that
can drive the Na+–Ca2+ exchanger (NCX) to operate in a reverse mode, importing Ca2+
and triggering injurious secondary cascades and axonal injury as indicated in the figure.
In addition, NO-induced mitochondrial damage, changes in mitochondrial gene
expression, and hypoxia/ischaemia due to perivascular inflammation seem to contribute
to axonal energy failure, which in turn leads to loss of function of Na+/K+ ATPase and
impaired ability of the axon to maintain resting potential and to export Na+. Ca2+ influx
into the axon in the context of these impaired homeostatic mechanisms has a number of
effects, including triggering calcium-induced calcium release (CICR) from internal stores,
and the activation of NO synthase (NOS), proteases and lipases. Nav channels are also
involved in the activation of microglia and macrophages, which contribute to the
production of NO, and in phagocytosis by these cells.
Electron microprobe
A non-invasive tool that
permits the measurement of
the elemental composition
of tissues.
indicated that Ca2+-mediated injury to myelinated CNS
axons can be triggered by a sustained Na+ influx, which
drives reverse operation of the Na+–Ca2+ exchanger, an
antiporter molecule that can import damaging levels of
Ca2+ into axons13. The timing of Na+ influx, which persists
throughout one hour of anoxia, and the prolonged effect
of the Na+ channel blocker tetrodotoxin (TTX), which
can block this influx throughout the anoxic period,
suggest the involvement of a persistent (non-inactivating)
Na+ conductance, and in fact a TTX-sensitive persistent
conductance can be measured along the trunks of optic
nerve axons14. Furthermore, electron microprobe analysis
has demonstrated a continuous rise in intra-axonal Na+,
which is paralleled by a rise in Ca2+ levels within anoxic
myelinated axons15.
As might be expected in view of the participation of
Na+ channels in axonal injury, pharmacological blocking of Na+ channels with agents that include TTX,
quarternary local anaesthetics such as lidocaine and
procaine, or the clinically used anticonvulsants phenytoin
and carbamazepine, prevents axonal degeneration in
anoxic CNS white matter13,16,17. As discussed below,
phenytoin18 and flecainide19 have recently been shown
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A link to energy failure
Because Na+/K+ ATPase — which serves to pump Na+
and K+ in opposite directions across the cell membrane — is required for extrusion of Na+ that enters the
axon, an inadequate ATP supply might be expected to
exacerbate the effects of persistent Na+ influx. Nitric oxide
(NO) is present at increased concentrations in acute MS
lesions20 and is known to have a deleterious effect on
mitochondria21. Kapoor et al.22 proposed that NO injures
axons by damaging the mitochondria within them,
thereby reducing ATP levels and producing a gradual
decrease in Na+/K+ ATPase activity, which limits the
ability of axons to extrude Na+. This hypothesis predicts
that Na+ channel blockers should protect axons from
NO-induced injury, and Kapoor et al.22 showed that this
is indeed the case. Further supporting the idea that Na+
influx after exposure to NO exceeds the ability of ATPdependent mechanisms for extrusion, TTX has been
shown to preserve ATP levels, concurrent with protecting
white matter axons from NO-induced injury23.
Another link to energy failure has been provided by
global transcript profiling of brain tissue from MS
patients24, which demonstrated decreased mRNA levels
for nuclear-encoded mitochondrial genes in MS lesions;
notably, the activities of respiratory gene complexes
I and III (membrane-bound redox carriers within mitochondria that have essential roles in electron transport
and thereby in ATP production) were decreased in this
tissue, suggesting that the changes could have functional
significance in MS. This study also revealed fragmented
neurofilaments, depolymerized microtubules and
reduced organelle content within residual demyelinated
axons, suggestive of Ca2+-mediated axonal injury. On the
basis of these results, the authors of the study proposed
that an inadequate axonal ATP supply contributes to the
degeneration of demyelinated axons in MS because it
impairs Na+/K+ ATPase activity, and thereby limits or
prevents extrusion of axoplasmic Na+ (REF. 24).
Molecular specificity
The results described above suggest that Na+ channels
have an important role in axonal conduction and axonal
degeneration in MS, but which isoforms are involved?
At least nine different genes encode distinct voltagegated sodium (Nav) channels (the neuronal channels
Nav1.1 to Nav1.3 and Nav1.6 to Nav1.9, the muscle
Na+ channel Nav1.4, and the cardiac channel Nav1.5),
all of which share a common overall motif but possess
different amino acid sequences, voltage dependencies
and kinetics25. Nav1.1, Nav1.2 and Nav1.6 channels
are expressed widely in neurons in the PNS and CNS.
Nav1.2 and Nav1.6 are the predominant Na+ channel
isoforms found in axonal membranes in the CNS. By
contrast, Nav1.7, Nav1.8 and Nav1.9 are expressed preferentially in dorsal root ganglion (DRG) and trigeminal ganglion neurons (and in sympathetic ganglion
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neurons in the case of Nav1.7) and, although present in
peripheral axons, are not present in CNS axons25.
Studies using isoform-specific antibodies have
shown that both Nav1.2 and Nav1.6 channels are
present in normal myelinated axons and their premyelinated precursors in the CNS, but that each isoform
is present at different stages of development. At early
developmental stages before glial ensheathment, a
low density of Na+ channels26, which are now known
to be Nav1.2 channels27,28, are present along the entire
length of pre-myelinated central axons, and support
action potential conduction prior to myelination29,30.
Nav1.2 channels are also distributed diffusely along
non-myelinated mature CNS axons27,31,32. As myelination proceeds, there is a loss of Nav1.2 channels and
an aggregation of Nav1.6 channels at mature nodes of
Ranvier27,28, so that in mature myelinated axons there
are few if any Nav1.2 channels, and Nav1.6 is the predominant Na+ channel isoform at fully-formed nodes
of Ranvier33 (FIG. 1).
Paranodal domain
The part of the axon, at the
ends of the internodes, where
the axon and the myelin form a
relatively tight seal (the
paranodal junction).
Nav1.2 and Nav1.6 in EAE
EAE, in which animals such as mice or rats are inoculated with components of white matter and subsequently
develop an inflammatory disorder that includes demyelination and axonal degeneration, is commonly used
as a model of MS. The Na+ channel isoforms expressed
along demyelinated axons in EAE have recently been
identified in studies that used subtype-specific immunocytochemical methods and in situ hybridization34,35.
These studies did not show Nav1.1 or Nav1.3 channels
along axons in control animals or in animals with EAE;
the latter result might not have been predicted because
expression of Nav1.3 channels is upregulated in DRG
cells and their axons after axonal injury36.
Importantly, however, these studies revealed upregulated expression of Nav1.2 and Nav1.6 over long regions
of demyelinated CNS axons in EAE, with domains of
Nav1.2 or Nav1.6 immunoreactivity extending for tens
of micrometres along the fibre axis in tracts such as the
optic nerve and dorsal columns (FIG. 1c). An increase in
Nav1.2 channel mRNA levels within retinal ganglion
cells, the cells of origin of optic nerve axons34, showed
that neuronal transcription of the gene encoding Nav1.2
channels was upregulated.
Several lines of evidence suggest that expression of
Nav1.6 along extensive axonal domains is associated
with axonal injury in EAE. As described above, a
sustained Na+ influx that flows through Na+ channels
can trigger Ca 2+ -mediated injury of white matter
axons by driving the Na+–Ca2+ exchanger to operate
in a reverse (Ca2+-importing) mode13. Nav1.6 channels
produce a larger persistent current than Nav1.2 channels37. So, co-expression of Nav1.6 channels and the
Na+–Ca2+ exchanger would be expected to predispose
demyelinated axons to import injurious levels of Ca2+. To
determine whether Nav1.6 channels and the Na+–Ca2+
exchanger are, in fact, co-expressed in degenerating
axons in EAE, Craner et al.35 immunolocalized these
molecules and β-amyloid precursor protein (β-APP),
a marker of axonal injury, in spinal cord axons from
NATURE REVIEWS | NEUROSCIENCE
mice with EAE. This study showed that 92% of β-APPpositive axons in EAE express Nav1.6 (either alone (56%) or
co-expressed with Nav1.2 (36%)); by contrast, less
than 2% of β-APP-positive axons express Nav1.2 in
the absence of Nav1.6. Co-expression of Nav1.6 and the
Na+–Ca2+ exchanger was seen in 74% of β-APP-positive
axons, in contrast to only 4% of β-APP-negative axons.
Therefore, Nav1.6 and the Na+–Ca2+ exchanger are
co-expressed in injured axons in EAE.
Nav1.2 and Nav1.6 in MS
Although EAE is commonly studied as an animal
model of MS, there is no perfect animal model of the
human disorder. There is a need for direct analysis
of human tissue — this is challenging because brain tissue
is now rarely biopsied in patients with MS or suspected
MS (in large part because modern imaging methods
such as MRI have facilitated diagnosis), and because the
time lag between death and removal of tissue for postmortem study, which is usually at least a few hours, limits the
utility of post-mortem tissue for molecular analysis.
Lesion-to-lesion variability, both between patients and
within patients, further complicates the analysis of human
MS tissue38. Nevertheless, some clues can be gleaned from
a recent analysis39 of spinal cord and optic nerve tissue
obtained by rapid autopsy from patients who died with
disabling secondary progressive MS.
Acute MS lesions in this study displayed a pattern of
Na+ channel expression similar to that seen in EAE. In
control white matter, there was abundant myelin basic
protein (MBP; a marker for myelin) and there were foci of
Nav1.6 at the nodes of Ranvier. By contrast, in acute MS
lesions (which could be identified on the basis of attenuated MBP immunostaining, evidence of inflammation
and recent phagocytosis of myelin), Nav1.6 and Nav1.2
were expressed along extensive regions of demyelinated
axons, often running tens of micrometres (FIG. 3a–d).
Zones of Nav1.6 or Nav1.2 channel immunostaining were
in some cases bounded by damaged myelin (FIG. 3a,b) or
contactin associated protein (Caspr) (FIG. 3c,d), a constituent of paranodal domains40, confirming the identity of these
profiles as axons in which the myelin had been damaged.
So, in both EAE and MS, Nav1.6 and Nav1.2 were identified as the Na+ channel isoforms that are expressed along
demyelinated axons.
Craner et al.39 also examined Na+ channel expression
in β-APP-positive axons in these lesions. Almost all
β-APP immunopositive axons in these MS lesions showed
extensive regions of expression of Nav1.6; by contrast,
few β-APP immunopositive axons expressed Nav1.2
immunostaining. Moreover, Nav1.6 and the Na+–Ca2+
exchanger tended to be colocalized in β-APP-positive
axons within the MS lesions that were studied (FIG. 3e–g).
So, as in EAE, in these acute MS lesions there was an
association between axonal injury and co-expression
of Nav1.6 channels and the Na+–Ca2+ exchanger.
Nav1.2 channels and restoration of conduction
The extensive distribution of Nav1.2 channels, for tens of
micrometres along demyelinated but apparently uninjured
axons in EAE and MS, is similar to the diffuse pattern
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e Nav1.6
a
Nav1.6
MBP
10 μm
MBP
10 μm
b
Nav1.2
f NCX
c
Nav1.6
Caspr
g β-APP
d
Nav1.2
5 μm
Caspr
Figure 3 | Nav1.6 and Nav1.2 channel expression along demyelinated axons in
active MS lesions. Panels (a) and (b) show active multiple sclerosis (MS) lesions in spinal
cord tissue, in which axons display residual damaged myelin (green), establishing these
profiles as axons, next to extensive regions of diffuse expression of voltage-gated sodium
(Nav) 1.6 channels (red; a) and Nav1.2 channels (red; b). Extensive regions of Nav1.6
channels (red; c) or Nav1.2 channels (red; d) in some axons are bounded by contactinassociated protein (Caspr; green), without overlap, consistent with the expression of
Nav1.6 and Nav1.2 channels in the demyelinated axon membrane. Panels e–g show
co-expression of the Na+–Ca2+ exchanger and Nav1.6 channels in β-amyloid precursor
protein (APP)-positive axons in MS. The images show representative axons in MS spinal
cord white matter immunostained for Nav1.6 channels (red; e) NCX (green; f) and β-APP
(blue; g). MBP, myelin basic protein; NCX, Na+–Ca2+ exchanger. Modified, with permission,
from REF. 39 © (2004) National Academy of Sciences.
of distribution of Nav1.2 channels along pre-myelinated
axons27 and non-myelinated axons in the CNS31,41,42. Action
potential conduction is known to occur in these fibres29,30,
suggesting that Nav1.2 might support this function.
Nav1.2 channels display activation and availability
(steady-state inactivation) properties that are more
depolarized than for Nav1.6 channels so that Nav1.2 channels show less inactivation with modest depolarization.
Nav1.2 channels, however, show greater accumulation
of inactivation, and so are less available to open and
contribute to action potentials at high frequencies (20–100
Hz)37. These observations suggest that Nav1.2 channels
might support action potential conduction along demyelinated axons even in the context of any depolarization
that occurs in the fibres as a result of an injury, but that
conduction in these axons would be limited to lower
frequencies. Importantly, Nav1.2 channels produce
a smaller persistent current than Nav1.6 channels37.
Therefore, demyelinated axons expressing Nav1.2 would
be expected to be subjected to a smaller sustained Na+
influx, a factor that could encourage their survival, as has
been observed in immunohistochemical studies on EAE
and MS35,39.
Plaque load
An aggregate measure of the
number and volume of lesions
in a brain with multiple
sclerosis.
Nav1.6 and axonal injury
As described above, Nav1.6 produces a large, persistent
Na+ conductance that could trigger reverse Na+–Ca2+
exchange and consequent Ca2+ entry that results in the
injury of myelinated axons13,14. The biophysical properties
of Nav1.6 channels predict a persistent ‘window’ current
936 | DECEMBER 2006 | VOLUME 7
that can carry Na+ inward through these channels at a
range of membrane potentials between –65 and –40 mV,
suggesting that, in axons that are depolarized after injury,
Nav1.6 channels can drive reverse Na+/Ca2+ exchange
even in the absence of action potential activity37.
Recent evidence raises the possibility that persistent current through Nav1.6 channels might be further
increased by secondary injury to the channel itself, due
to Ca2+-induced proteolytic cleavage of the domain
III–IV linker that damages the channels’ inactivation
mechanism, and is triggered by the rise in intra-axonal
Ca2+ levels early in the course of injury43. If this occurs in
MS, it could introduce a feedforward process that would
further increase the current flowing through Nav1.6
channels. Calmodulin, a Ca2+-binding protein that interacts with and regulates many proteins within cells, also
interacts with Nav1.6 and increases the Nav1.6 channel current amplitude in a Ca2+-dependent manner44,
and might additionally amplify the persistent current
produced by these channels after initial injury to axons.
Irrespective of whether these amplification mechanisms are recruited in MS, the physiological data
together with the immunolocalization results support
the proposal that Nav1.6 channels, when co-expressed
with the Na+–Ca2+ exchanger along demyelinated axons,
can contribute to axonal injury (FIG. 2). Consistent with
the idea that the presence of Nav1.6 channels, but not
Nav1.2 channels, predisposes axons to injury, it has
been shown that dysmyelinated CNS axons express
Nav1.2 channels rather than Nav1.6 channels27,31, and
are substantially less sensitive than myelinated axons
to anoxic injury45. Also consistent with this proposal is
the observation that demyelinated CNS axons are less
sensitive than myelinated axons to anoxic injury46, an
observation that might be explained by the expression
of Nav1.2 channels along demyelinated axons that have
survived rather than degenerated.
It is also possible that some axons degenerate in MS
in the absence of demyelination. De Luca et al.47 found
only a weak correlation between axonal loss and plaque
load in post-mortem MS tissue, raising the possibility
that demyelination might not be a prime determinant
of axonal degeneration in MS. The Na+–Ca2+ exchanger
is in fact present at intact nodes where Nav1.6 channels
are aggregated in normal white matter48. Therefore,
if the inadequacy of ATP supply described by Dutta
et al.24 in MS occurs in neurons in which axons are not
demyelinated, the axons might be poised to undergo
Ca2+-mediated injury.
Chaos in the cerebellum
Dysregulated Na+ channel expression might also contribute to cerebellar dysfunction in MS. There are
a number of indications in the clinical literature of a
different pathophysiology for cerebellar deficits in MS,
compared with other types of clinical deficits. First,
clinical abnormalities due to cerebellar dysfunction in
MS tend to be persistent, even early in the course of the
disease, in contrast to other types of clinical deficits which
tend to be remitting49. Second, cerebellar dysfunction
is occasionally observed in patients with MS in whom
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Ataxia
Loss of coordination of muscle
movements, produced most
commonly by dysfunction of
the cerebellum (cerebellar
ataxia) or defective sensory
input (sensory ataxia).
cerebellar lesions cannot be visualized through
neuroimaging. Third, there are reports of occasional
patients with MS who have paroxysmal bouts of ataxia
similar to those seen in the episodic ataxias, a group
of disorders caused by hereditary channelopathies ,
and the paroxysmal attacks have been reported to
respond favourably to treatment with Na + channel
blockers such as carbamazepine50,51. These clinical
observations raise the possibility that, in addition
to demyelination and axonal degeneration, molecular changes in cerebellar neurons might contribute
to the pathophysiology of MS52.
Consistent with these clinical observations, upregulated expression of the sensory neuron specific (SNS)
A
Control
d
EAE
Aa
Ab
10
GFP
0
Vm (mV)
–10
Ac
–20
–30
–40
Ad
–50
–60
0
250
500
750
1,000
Time (ms)
Ae
10
Af
Nav1.8/GFP
0
Vm (mV)
–10
50 μm
b Control Purkinje cell
–20
–30
–40
c +Nav1.8
–50
–60
0 mV
0 mV
0
0mV
250
500
750
1,000
750
1,000
Time (ms)
nA
10 mV
50 ms
0.08
0.04
0.00
0
250
500
Time (ms)
Figure 4 | Sensory neuron specific Na+ channel Nav1.8 is aberrantly expressed
within cerebellar Purkinje neurons in MS and its experimental models. Voltagegated sodium (Nav) 1.8 (Ab) and annexinII/p11, a binding partner which facilitates the
insertion of functional Nav1.8 channels into the cell membrane (Ad), are co-expressed in
Purkinje cells in experimental autoimmune encephalomyelitis (EAE). The merged images
are shown in yellow (Af). Neither of these molecules can be detected within control
Purkinje neurons (Aa,Ac,Ae). Expression of Nav1.8 alters action potential electrogenesis
in Purkinje neurons (b,c). Current clamp recordings show spontaneous action potentials in
control Purkinje neurons lacking Nav1.8 (b), and two days after transfection with Nav1.8
(c). Action potentials in control Purkinje neurons lacking Nav1.8 (b, left) show less
overshoot (dotted lines indicate 0 mV) and tend to be conglomerate, consisting of two or
more spikes in 62% of cells (b, right). By contrast, conglomerate action potentials contain
fewer spikes and are less common (15%) in Purkinje cells that express Nav1.8. Purkinje
neurons transfected with Nav1.8 show sustained repetitive firing (d, middle), not present
in the absence of Nav1.8 (d, top), in response to the injection of a depolarizing current
(d, bottom). GFP, green fluorescent protein; Vm, membrane potential. Panels Aa–f
reproduced, with permission, from REF. 56 © (2003) Lippincott, Williams & Wilkins.
Panels b–d reproduced, with permission, from REF. 67 © (2003) Elsevier Science.
NATURE REVIEWS | NEUROSCIENCE
channel Nav1.8 was found in Purkinje cells of the Taiep
rat53, a dysmyelinating model in which myelin initially
ensheaths CNS axons but subsequently degenerates
due to an inherited abnormality of oligodendrocytes.
Black et al.54 subsequently used in situ hybridization
and immunocytochemical methods to examine the
cerebellums of mice with EAE and humans with progressive MS, and observed the presence of Nav1.8 mRNA
and protein (FIG. 4A), which are not present in normal
Purkinje neurons. Annexin II/p11, a protein that binds to
the amino (N)-terminus of Nav1.8 and facilitates insertion of functional Nav1.8 channels into the neuronal
cell membrane55, was also shown to be upregulated
within Purkinje cells in EAE and MS, and — as shown in
FIG. 4A — is colocalized with Nav1.8 (REF. 56). The coordinated upregulation of Nav1.8 and its binding partner
annexin II/p11 suggest that functional Nav1.8 channels
could be inserted into Purkinje cell membranes in MS
and its animal models.
The Nav1.8 channel was initially termed SNS because
it is selectively expressed in the healthy nervous system
in DRG neurons. It is distinguished from other Nav
channels by resistance to TTX and steady-state inactivation with voltage-dependence that is more depolarized
than for other Na+ channels, a property that enables
these channels to open even when the cell membrane is
depolarized to the extent that other Na + channel
isoforms are inactivated57,58. Nav1.8 is also unique in
displaying rapid recovery from inactivation59,60. Nav1.8
produces most of the current underlying the depolarizing upstroke of the action potential in cells in which
it is present61,62, and supports repetitive firing in these
cells, even when depolarization inactivates the other Na+
channels present61.
Electrogenesis in Purkinje cells depends, in part,
on the activity of Na+ channels63–65, and mutations of
Na+ channels that are normally expressed in Purkinje
cells produce substantial changes in patterns of firing
in these cells, which can result in clinical cerebellar
dysfunction, including ataxia 65,66. Recordings from
Purkinje cells transfected with Nav1.8 in vitro67 and
from Purkinje cells in vivo in animals with EAE 68
suggest that expression of Nav1.8 can substantially
perturb their pattern of firing. The firing patterns
of Purkinje neurons transfected in vitro with Nav1.8
are altered in several ways67: first, by increased action
potential duration and amplitude (FIG. 4b,c); second,
by fewer action potentials that consist of multiple
spikes and a reduction in the number of spikes in
these conglomerate action potentials; and third, by
the generation of sustained, pacemaker-like impulse
trains in response to depolarization, which are not
seen in the absence of Nav1.8 (FIG. 4d). Purkinje cells
in mice with EAE show similar changes in their
firing patterns68.
The trigger for upregulated expression of Nav1.8
within Purkinje cells in MS and its animal models is
not known. Nerve growth factor (NGF) is known to
upregulate transcription of the Nav1.8 gene69,70. Levels
of NGF are increased in humans with MS 71 and in
animal models of MS, where expression of the p75
VOLUME 7 | DECEMBER 2006 | 937
© 2006 Nature Publishing Group
REVIEWS
neurotrophin receptor is also upregulated72. Reasoning
that NGF might trigger upregulation of Nav1.8 in
Purkinje cells in EAE and MS, Damarjian et al.73 examined the relationship between upregulation of Nav1.8
and expression of the NGF receptors p75 and TrkA
in the cerebellum in EAE. Consistent with previous
studies74,75 they observed low levels of p75 and TrkA
in control Purkinje neurons. However, there was a
significant upregulation of p75 within Purkinje cells in
EAE and a high level of expression of p75 in Purkinje
cells that expressed Nav1.8. These observations raise
the possibility that NGF could trigger upregulated
expression of Nav1.8 within Purkinje cells in EAE
and MS. Some support for this hypothesis is provided
by the observation that antisense knockdown of p75
ameliorates disease severity in EAE76.
Channelopathies
Disorders due to mutations of
ion channels (inherited
channelopathies), or due to
altered channel function
attributable to exposure to
toxins or antibodies, or to
dyregulated channel
expression after tissue injury
(acquired channelopathies).
Electrogenesis
The production of electrical
signals — for example, action
potentials — by cells such as
neurons.
b
8
Control
MS
50
2
Macrophages
Activated
Intermediate
Resting
0
30
20
10
0
ACM/LPS
TTX
4
p < 0.001
40
ACM/LPS
6
Number of beads cell–1
Normalized optical intensity
a
Microglia
c
OX42
Control
EAE
EAE-Phenytoin
CD45
25 μm
Figure 5 | Na+ channels are present in EAE and MS, and contribute to microglia/
macrophage activation and function. a | Progressive upregulation of voltage-gated
sodium (Nav) 1.6 protein (detected by immunocytochemistry and shown in terms of
normalized optical density) with activation of microglia and macrophages in acute
multiple sclerosis (MS) lesions, compared with resting microglia in controls with no
neurological disease. b | Activation and phagocytic function of microglia/macrophages
are attenuated by Na+ channel blockade. Administration of tetrodotoxin (TTX) (upper
panel) to lipopolysaccharide (LPS)-stimulated microglia in primary culture attenuates
phagocytic function, as seen by a decreased number of latex beads phagocytosed per cell
compared with cells not treated with TTX (lower panel). The histogram shows significant
reduction in the degree of phagocytosis after treatment with TTX. c | Phenytoin reduces
inflammatory infiltrate in experimental autoimmune encephalomyelitis (EAE). The panels
show images of control, EAE, and phenytoin-treated EAE spinal cord immunostained for
anti-CD45 (green) and anti-OX42 (blue). Administration of phenytoin results in a marked
reduction in inflammatory infiltrate. ACM, astrocyte conditional medium. Modified, with
permission, from REF. 77 © (2005) Wiley-Liss.
938 | DECEMBER 2006 | VOLUME 7
An immune connection
Although neuroscientists tend to think of Na+ channels as ‘neuronal’ molecules, Na+ channels are present
in, and seem to regulate the activity of, microglia and
macrophages77, two immune cell types that contribute
to axonal degeneration in EAE and MS. Microglia and
macrophages are closely associated with degenerating
axons in MS9,78,79, and have been suggested to produce
axonal injury by multiple mechanisms, including the
induction of CD4+ T-cell proliferation80, production of
pro-inflammatory cytokines81 and NO82,83, antigen presentation84 and phagocytosis85. Patch-clamp studies86,87 have
shown that Nav channels are present in microglia. In a
recent immunocytochemical study, Craner et al.77 showed
that Nav1.6 channels are indeed present in microglia, at
levels that are increased during activation in EAE. This
study also showed an upregulation of Nav1.6 expression
within microglia and macrophages in acute lesions of
MS patients, compared with control patients without
neurological disease (FIG. 5a). Proposing that Na+ channels are important for the function of these cells, they
asked whether Na+ channel blockade might attenuate the
inflammatory activity of these cells, and observed a 40%
reduction in phagocytic activity of cultured microglia
following exposure to TTX (FIG. 5b) and a 75% decrease in
the number of inflammatory cells in EAE after treatment
with phenytoin (FIG. 5c). The researchers also observed
that activation of microglia from med mice (which lack
functional Nav1.6) is reduced compared with wild-type
mice (in which Nav1.6 is present), and showed that the
suppressing effect of TTX on microglial activation is
not present in med mice, confirming a role for Nav1.6
in microglial activation. Further studies are underway
to examine the mechanism by which Nav1.6 (and
possibly other Na+ channel isoforms) modulates the activity of microglia, macrophages and possibly other immune
cells in the CNS. Irrespective of the full repertoire of Na+
channel isoforms that are involved, these observations
suggest that, in addition to a direct neuroprotective action
on axons, Na+ channel blockers could attenuate axonal
injury by a second, parallel mechanism that reduces
inflammatory damage through an action on microglia or
macrophages.
Na+ channels as therapeutic targets
In the face of the results described in this article, it should
not come as a surprise that Na+ channels are being investigated as potential therapeutic targets in MS. Subtypespecific blockers of Nav1.6 channels (and possibly
blockers of the persistent component of the current
produced by Nav1.6 channels) might be predicted to
have a protective effect, either by preventing axonal
degeneration through direct action on axons or by blunting the activity of microglia and macrophages in MS.
Although subtype-specific blockers are not yet available, the nonspecific Na+ channel blockers phenytoin18
and flecainide19 have been shown to be protective in
mouse and rat models of EAE. Administration of these
agents in the rodent models of MS results in a reduced
degree of axonal degeneration in CNS white matter
as assessed after 28–30 days of disease. For example,
www.nature.com/reviews/neuro
© 2006 Nature Publishing Group
REVIEWS
phenytoin reduces the loss of dorsal corticospinal axons
from 63% to 25%, and of cuneate fasciculus axons
from 43% to 17%18. Electrophysiological experiments
show that axonal conduction is maintained in at least a
significant fraction of the surviving axons. Importantly,
treatment with these Na+ channel blockers improves
clinical outcome. A more recent study88 has shown that
the protective effect on axons and clinical improvement
persists for as long as 180 days in mice treated regularly
with phenytoin.
Questions and horizons
It is not yet clear whether Na+ channel blockers exert
their protective effect in EAE in vivo through a direct
action on axons, or by acting on immune cells such
as microglia or macrophages. Bechtold et al.19 noted
a protective effect of flecainide on neurological
symptoms early in the course of EAE (10–13 days
post disease induction) and suggested a possible
immunmodulatory effect. There is in fact evidence
suggesting a role for Na+ channels in the activation of
T cells89,90. Moreover, as discussed above, Craner et al.77
observed that treatment with phenytoin substantially
ameliorates the inflammatory cell infiltrate in EAE,
and noted that TTX significantly reduces the phagocytic function of activated microglia. Yet the protective
effect of Na+ channel blockers on axons in vitro13,16,17,
where inflammation is minimal or non-existent,
indicates that the mechanism of action of these agents
involves, at least in part, a direct effect on axons. It is
possible that Na+ channel blockers protect axons by
a dual mechanism, involving both a direct action on
axons and an immunomodulatory action on microglia
and/or macrophages.
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In addition to a protective action, the aberrant expression of Nav1.8 in Purkinje cells suggests that blockade
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Acknowledgements
Research in the author’s laboratory has been supported, in
part, by grants from the National Multiple Sclerosis Society
and the Medical Research Service and Rehabilitation
Research Service, the Department of Veteran Affairs, and by
gifts from Destination Cure and the Nancy Davis Foundation.
The Neuroscience and Regeneration Research Center is a
Collaboration of the Paralyzed Veterans of America and the
United Spinal Association with Yale University.
NATURE REVIEWS | NEUROSCIENCE
Competing interests statement
The author declares no competing financial interests.
DATABASES
The following terms in this article are linked online to:
Entrez Gene:
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene
med | Nav1.1 | Nav1.2 | Nav1.3 | Nav1.4 | Nav1.5 | Nav1.6 |
Nav1.7 | Nav1.8 | Nav1.9 | TrkA
OMIM:
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM
experimental autoimmune encephalomyelitis
FURTHER INFORMATION
Waxman’s laboratory: http://www.med.yale.edu/neurol/pvaepvacenter/index.html
Access to this links box is available online.
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