Axonal Conduction and Injury in Multiple Sclerosis: the Role of Sodium Channels

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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 Multiple sclerosis (MS) is the most common neurological contribute to axonal degeneration, and the possibility of + Small gaps in the myelin cause of disability in young adults in industrialized societies. a role for a third Na channel isoform in the mistuning of sheath along myelinated fibres. It is usually diagnosed between the ages of 20 and 40, and cerebellar Purkinje neurons, which perturbs the pattern Nodes of Ranvier extend ~1 is called ‘multiple’ sclerosis because most patients have of activity of these cells. μm along the fibre, and are separated by segments of multiple attacks separated both in time and in space, in + myelin that extend for tens or, which different parts of the CNS can be involved (for Na channels and axonal conduction more commonly, hundreds of example, involvement of the optic nerve can cause uni- The disease process in MS attacks myelinated axons, micrometres. lateral visual loss, whereas involvement of spinal sensory denuding them of myelin or causing them to degener- tracts can cause numbness). Early in its course, MS often ate. Normal myelinated axons exhibit a clustering of Internodal domains + μ –2 Regions of the axon between displays a relapsing–remitting pattern, with patients losing Na channels (~1,000 m ) in the axon membrane the nodes of Ranvier. functions such as vision or motor function, then recover- at the nodes of Ranvier, with a much lower density ing these capabilities during remissions. Later in some (< 25 channels μm–2) in internodal domains where the Saltatory conduction patients (secondary progressive MS), or at the beginning axon is covered by myelin1,2. This arrangement (FIG. 1a) A process of rapid impulse saltatory conduction conduction that is conferred on of disease onset in others (primary progressive MS), there supports in the normal myelinated axons by myelin sheaths, in is cumulative acquisition of neurological deficits which axon. However, it is less well-suited to the functional which the action potential do not remit. Although the cause of MS is unknown, and needs of the demyelinated axon, in which impedance leaps discontinously and multiple etiologies including autoimmunity, infectious mismatch and loss of the myelin capacitative shield permit rapidly from one node of agents, environmental triggers and hereditary factors have current to be dissipated through formerly myelinated Ranvier to the next. been proposed, there is substantial evidence to indicate portions of the axon membrane where Na+ channel that dysregulated immune responses, including immune density is low, impairing the conduction of action mechanisms directed against myelin proteins, have a role potentials (FIG. 1b). Department of Neurology and in triggering disease onset. Following the loss of myelin in MS, remyelination does Center for Neuroscience and Recent studies have identified changes in the expres- not always occur and in many lesions the myelin is not Regeneration Research, Yale sion pattern of specific Na+ channel isoforms as an impor- replaced. Surprisingly, although demyelination causes School of Medicine, New tant contributor to remission and progression in MS, and symptoms such as visual loss (when the optic nerve is Haven, Connecticut 06510, + and the Rehabilitation there is evidence suggesting that aberrantly expressed Na involved) or weakness (when the corticospinal tract Research Center, Veterans channels might also contribute to cerebellar dysfunction is involved), remissions can occur in the absence of remy- Affairs Medical Center, in MS. In this article, I discuss the multiple roles of Na+ elination. For example, vision can recover in some patients West Haven, Connecticut channels in the pathophysiology of MS, including the in which demyelination affects a substantial length 06516, USA. + e-mail: adaptive role of some Na channel isoforms in restor- (a centimetre or more, thereby encompassing the [email protected] ing conduction in chronically demyelinated axons, the territory of many myelin segments) of all the axons doi:10.1038/nrn2023 maladaptive role of other Na+ channel isoforms that within the optic nerve3. Recovery of clinical function in

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a Nav1.6 Myelinated Caspr Node internode

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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 Impedance mismatch (red, upper right panel) and Nav1.2 channels ( red, lower right panel) along optic nerve axons (arrows) in experimental A phenomenon in which, owing autoimmune encephalomyelitis (EAE) is shown. d | Degeneration of axons also occurs in multiple sclerosis, and produces to non-uniform properties, non-remitting, permanent loss of function. Images in part c reproduced, with permission, from REF. 34 © (2003) Oxford there is a sudden drop in Univ. Press. electrical resistance or rise in capacitance along a cable or nerve fibre. Impedance + mismatch occurs at the border cases such as this requires the restoration of secure action Na channels and axonal degeneration between normally myelinated potential conduction along at least some of the demyeli- Although largely eclipsed by an emphasis on demyelina- and demyelinated parts of nated axons. Setting the stage for an understanding of the tion, it has been appreciated since the time of Charcot axons in disorders such as role of Na+ channels in this process, an early longitudinal that, in addition to becoming demyelinated, some axons multiple sclerosis, and contributes to conduction current analysis showed that some chronically demyelinated degenerate in MS. Recent studies have focused new failure. axons can recover the ability to conduct action potentials attention on axonal degeneration in MS (FIG. 1d), and in a continuous manner4, and early cytochemical studies have underscored its frequency and occurrence early Capacitative shield showed that, after demyelination, the denuded axon in the course of the disease9,10. Importantly, studies in The electrical shield provided by the myelin that surrounds membrane can develop higher-than-normal densities of animal models and in human MS patients have shown + 5 the axon, which prevents loss Na channels . Immunocytochemical studies using pan- that axonal loss produces non-remitting, persistent of current through the specific Na+ channel antibodies6,7 subsequently confirmed neurological deficits11,12. This has led to considerable membrane capacitance of the the appearance of increased numbers of Na+ channels interest in the development of protective strategies aimed axon. in experimentally demyelinated axons. Saxitoxin bind- at preventing degeneration of axons, and thereby slowing Longitudinal current ing studies also demonstrated a fourfold increase in the or halting the progression of disability in MS. analysis number of Na+ channels in demyelinated lesions from MS The available evidence suggests that Na+ channels are A method in which extracellular patients8. Taken together, these results indicated that Na+ important participants in axonal degeneration in MS electrodes are used to measure channel expression is increased in at least some demy- (FIG. 2). As described below, there is evidence for energy electrical currents as they flow (FIG. 1c) along nerve fibres and, thereby, elinated axons . However, these early studies did failure within the CNS in MS. Early studies on white + to infer the presence of nodes not reveal the molecular identity of the new axonal Na matter tracts in vitro used anoxia, which produces energy or foci of node-like membrane. channels along demyelinated axons. failure, as a highly reproducible, quantifiable insult, and

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to have a protective effect in an experimental model of Microglia MS, experimental autoimmune encephalomyelitis (EAE), where they prevent degeneration of CNS axons, maintain axonal conduction and improve clinical outcome. Hypoxia/ Altered Inflammation Nav ischaemia gene A link to energy failure expression Because Na+/K+ ATPase — which serves to pump Na+ NO and K+ in opposite directions across the cell mem- Na+/K+ ATPase Nav1.6 brane — is required for extrusion of Na+ that enters the NCX 2+ Ca axon, an inadequate ATP supply might be expected to exacerbate the effects of persistent Na+ influx. Nitric oxide Na+ Ca2+ influx Run-down (NO) is present at increased concentrations in acute MS + of ATPase Peristant Na lesions20 and is known to have a deleterious effect on influx CICR mitochondria21. Kapoor et al.22 proposed that NO injures Energy failure Mitochondrial Ca2+ axons by damaging the mitochondria within them, Depolarization Activation of NOS Activation of proteases thereby reducing ATP levels and producing a gradual Activation of lipases decrease in Na+/K+ ATPase activity, which limits the + Axon 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 Nav1.2 is indeed the case. Further supporting the idea that Na+ Less persistent Na+ current supports conduction influx after exposure to NO exceeds the ability of ATP- Figure 2 | Model of functional effects of expression of Nav1.2 and Nav1.6 dependent mechanisms for extrusion, TTX has been channels along demyelinated axons. Following demyelination, voltage-gated sodium shown to preserve ATP levels, concurrent with protecting (Nav) 1.2 channels, expressed diffusely along some axons, support recovery of action white matter axons from NO-induced injury23. potential conduction. Nav1.6 channels, however, produce a persistent Na+ current that Another link to energy failure has been provided by can drive the Na+–Ca2+ exchanger (NCX) to operate in a reverse mode, importing Ca2+ global transcript profiling of brain tissue from MS and triggering injurious secondary cascades and axonal injury as indicated in the figure. patients24, which demonstrated decreased mRNA levels In addition, NO-induced mitochondrial damage, changes in mitochondrial gene for nuclear-encoded mitochondrial genes in MS lesions; expression, and hypoxia/ischaemia due to perivascular inflammation seem to contribute notably, the activities of respiratory gene complexes + + to axonal energy failure, which in turn leads to loss of function of Na /K ATPase and I and III (membrane-bound redox carriers within mito- impaired ability of the axon to maintain resting potential and to export Na+. Ca2+ influx chondria that have essential roles in electron transport 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 thereby in ATP production) were decreased in this and the activation of NO synthase (NOS), proteases and lipases. Nav channels are also tissue, suggesting that the changes could have functional involved in the activation of microglia and macrophages, which contribute to the significance in MS. This study also revealed fragmented production of NO, and in phagocytosis by these cells. neurofilaments, depolymerized microtubules and reduced organelle content within residual demyelinated axons, suggestive of Ca2+-mediated axonal injury. On the indicated that Ca2+-mediated injury to myelinated CNS basis of these results, the authors of the study proposed axons can be triggered by a sustained Na+ influx, which that an inadequate axonal ATP supply contributes to the drives reverse operation of the Na+–Ca2+ exchanger, an degeneration of demyelinated axons in MS because it antiporter molecule that can import damaging levels of impairs Na+/K+ ATPase activity, and thereby limits or Ca2+ into axons13. The timing of Na+ influx, which persists prevents extrusion of axoplasmic Na+ (REF. 24). throughout one hour of anoxia, and the prolonged effect of the Na+ channel blocker tetrodotoxin (TTX), which Molecular specificity can block this influx throughout the anoxic period, The results described above suggest that Na+ channels suggest the involvement of a persistent (non-inactivating) have an important role in axonal conduction and axonal Na+ conductance, and in fact a TTX-sensitive persistent degeneration in MS, but which isoforms are involved? conductance can be measured along the trunks of optic At least nine different genes encode distinct voltage- nerve axons14. Furthermore, electron microprobe analysis gated sodium (Nav) channels (the neuronal channels has demonstrated a continuous rise in intra-axonal Na+, Nav1.1 to Nav1.3 and Nav1.6 to Nav1.9, the muscle which is paralleled by a rise in Ca2+ levels within anoxic Na+ channel Nav1.4, and the cardiac channel Nav1.5), myelinated axons15. all of which share a common overall motif but possess As might be expected in view of the participation of different amino acid sequences, voltage dependencies Na+ channels in axonal injury, pharmacological block- and kinetics25. Nav1.1, Nav1.2 and Nav1.6 channels ing of Na+ channels with agents that include TTX, are expressed widely in neurons in the PNS and CNS. quarternary local anaesthetics such as lidocaine and Nav1.2 and Nav1.6 are the predominant Na+ channel Electron microprobe procaine, or the clinically used anticonvulsants phenytoin isoforms found in axonal membranes in the CNS. By A non-invasive tool that permits the measurement of and carbamazepine, prevents axonal degeneration in contrast, Nav1.7, Nav1.8 and Nav1.9 are expressed pref- 13,16,17 the elemental composition anoxic CNS white matter . As discussed below, erentially in dorsal root ganglion (DRG) and trigemi- of tissues. phenytoin18 and flecainide19 have recently been shown nal ganglion neurons (and in sympathetic ganglion

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neurons in the case of Nav1.7) and, although present in mice with EAE. This study showed that 92% of β-APP- peripheral axons, are not present in CNS axons25. positive axons in EAE express Nav1.6 (either alone (56%) or Studies using isoform-specific antibodies have co-expressed with Nav1.2 (36%)); by contrast, less shown that both Nav1.2 and Nav1.6 channels are than 2% of β-APP-positive axons express Nav1.2 in present in normal myelinated axons and their premy- the absence of Nav1.6. Co-expression of Nav1.6 and the elinated precursors in the CNS, but that each isoform Na+–Ca2+ exchanger was seen in 74% of β-APP-positive is present at different stages of development. At early axons, in contrast to only 4% of β-APP-negative axons. developmental stages before glial ensheathment, a Therefore, Nav1.6 and the Na+–Ca2+ exchanger are low density of Na+ channels26, which are now known co-expressed in injured axons in EAE. to be Nav1.2 channels27,28, are present along the entire length of pre-myelinated central axons, and support Nav1.2 and Nav1.6 in MS action potential conduction prior to myelination29,30. Although EAE is commonly studied as an animal Nav1.2 channels are also distributed diffusely along model of MS, there is no perfect animal model of the non-myelinated mature CNS axons27,31,32. As myelina- human disorder. There is a need for direct analysis tion proceeds, there is a loss of Nav1.2 channels and of human tissue — this is challenging because brain tissue an aggregation of Nav1.6 channels at mature nodes of is now rarely biopsied in patients with MS or suspected Ranvier27,28, so that in mature myelinated axons there MS (in large part because modern imaging methods are few if any Nav1.2 channels, and Nav1.6 is the pre- such as MRI have facilitated diagnosis), and because the dominant Na+ channel isoform at fully-formed nodes time lag between death and removal of tissue for post- of Ranvier33 (FIG. 1). mortem study, which is usually at least a few hours, limits the utility of post-mortem tissue for molecular analysis. Nav1.2 and Nav1.6 in EAE Lesion-to-lesion variability, both between patients and EAE, in which animals such as mice or rats are inocu- within patients, further complicates the analysis of human lated with components of white matter and subsequently MS tissue38. Nevertheless, some clues can be gleaned from develop an inflammatory disorder that includes demy- a recent analysis39 of spinal cord and optic nerve tissue elination and axonal degeneration, is commonly used obtained by rapid autopsy from patients who died with as a model of MS. The Na+ channel isoforms expressed disabling secondary progressive MS. along demyelinated axons in EAE have recently been Acute MS lesions in this study displayed a pattern of identified in studies that used subtype-specific immuno- Na+ channel expression similar to that seen in EAE. In cytochemical methods and in situ hybridization34,35. control white matter, there was abundant myelin basic These studies did not show Nav1.1 or Nav1.3 channels protein (MBP; a marker for myelin) and there were foci of along axons in control animals or in animals with EAE; Nav1.6 at the nodes of Ranvier. By contrast, in acute MS the latter result might not have been predicted because lesions (which could be identified on the basis of attenu- expression of Nav1.3 channels is upregulated in DRG ated MBP immunostaining, evidence of inflammation cells and their axons after axonal injury36. and recent phagocytosis of myelin), Nav1.6 and Nav1.2 Importantly, however, these studies revealed upregu- were expressed along extensive regions of demyelinated lated expression of Nav1.2 and Nav1.6 over long regions axons, often running tens of micrometres (FIG. 3a–d). of demyelinated CNS axons in EAE, with domains of Zones of Nav1.6 or Nav1.2 channel immunostaining were Nav1.2 or Nav1.6 immunoreactivity extending for tens in some cases bounded by damaged myelin (FIG. 3a,b) or of micrometres along the fibre axis in tracts such as the contactin associated protein (Caspr) (FIG. 3c,d), a constitu- optic nerve and dorsal columns (FIG. 1c). An increase in ent of paranodal domains40, confirming the identity of these Nav1.2 channel mRNA levels within retinal ganglion profiles as axons in which the myelin had been damaged. cells, the cells of origin of optic nerve axons34, showed So, in both EAE and MS, Nav1.6 and Nav1.2 were identi- that neuronal transcription of the gene encoding Nav1.2 fied as the Na+ channel isoforms that are expressed along channels was upregulated. demyelinated axons. Several lines of evidence suggest that expression of Craner et al.39 also examined Na+ channel expression Nav1.6 along extensive axonal domains is associated in β-APP-positive axons in these lesions. Almost all with axonal injury in EAE. As described above, a β-APP immunopositive axons in these MS lesions showed sustained Na+ influx that flows through Na+ channels extensive regions of expression of Nav1.6; by contrast, can trigger Ca2+-mediated injury of white matter few β-APP immunopositive axons expressed Nav1.2 axons by driving the Na+–Ca2+ exchanger to operate immunostaining. Moreover, Nav1.6 and the Na+–Ca2+ in a reverse (Ca2+-importing) mode13. Nav1.6 channels exchanger tended to be colocalized in β-APP-positive produce a larger persistent current than Nav1.2 chan- axons within the MS lesions that were studied (FIG. 3e–g). nels37. So, co-expression of Nav1.6 channels and the So, as in EAE, in these acute MS lesions there was an Na+–Ca2+ exchanger would be expected to predispose association between axonal injury and co-expression demyelinated axons to import injurious levels of Ca2+. To of Nav1.6 channels and the Na+–Ca2+ exchanger. Paranodal domain determine whether Nav1.6 channels and the Na+–Ca2+ The part of the axon, at the exchanger are, in fact, co-expressed in degenerating Nav1.2 channels and restoration of conduction ends of the internodes, where 35 the axon and the myelin form a axons in EAE, Craner et al. immunolocalized these The extensive distribution of Nav1.2 channels, for tens of relatively tight seal (the molecules and β-amyloid precursor protein (β-APP), micrometres along demyelinated but apparently uninjured paranodal junction). a marker of axonal injury, in spinal cord axons from axons in EAE and MS, is similar to the diffuse pattern

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+ a e that can carry Na inward through these channels at a Nav1.6 range of membrane potentials between –65 and –40 mV, μ Nav1.6 MBP 10 m suggesting that, in axons that are depolarized after injury, Nav1.6 channels can drive reverse Na+/Ca2+ exchange b even in the absence of action potential activity37. μ Recent evidence raises the possibility that persist- Nav1.2 MBP 10 m f NCX ent current through Nav1.6 channels might be further c 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 Nav1.6 Caspr Ca2+ levels early in the course of injury43. If this occurs in g β-APP d MS, it could introduce a feedforward process that would further increase the current flowing through Nav1.6 channels. Calmodulin, a Ca2+-binding protein that inter- μ Nav1.2 Caspr 5 m acts with and regulates many proteins within cells, also interacts with Nav1.6 and increases the Nav1.6 chan- Figure 3 | Nav1.6 and Nav1.2 channel expression along demyelinated axons in nel current amplitude in a Ca2+-dependent manner44, active MS lesions. Panels (a) and (b) show active multiple sclerosis (MS) lesions in spinal and might additionally amplify the persistent current cord tissue, in which axons display residual damaged myelin (green), establishing these produced by these channels after initial injury to axons. profiles as axons, next to extensive regions of diffuse expression of voltage-gated sodium Irrespective of whether these amplification mecha- (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 contactin- nisms are recruited in MS, the physiological data associated protein (Caspr; green), without overlap, consistent with the expression of together with the immunolocalization results support Nav1.6 and Nav1.2 channels in the demyelinated axon membrane. Panels e–g show the proposal that Nav1.6 channels, when co-expressed co-expression of the Na+–Ca2+ exchanger and Nav1.6 channels in β-amyloid precursor with the Na+–Ca2+ exchanger along demyelinated axons, protein (APP)-positive axons in MS. The images show representative axons in MS spinal can contribute to axonal injury (FIG. 2). Consistent with cord white matter immunostained for Nav1.6 channels (red; e) NCX (green; f) and β-APP the idea that the presence of Nav1.6 channels, but not (blue; g). MBP, myelin basic protein; NCX, Na+–Ca2+ exchanger. Modified, with permission, Nav1.2 channels, predisposes axons to injury, it has from REF. 39 © (2004) National Academy of Sciences. been shown that dysmyelinated CNS axons express Nav1.2 channels rather than Nav1.6 channels27,31, and are substantially less sensitive than myelinated axons of distribution of Nav1.2 channels along pre-myelinated to anoxic injury45. Also consistent with this proposal is axons27 and non-myelinated axons in the CNS31,41,42. Action the observation that demyelinated CNS axons are less potential conduction is known to occur in these fibres29,30, sensitive than myelinated axons to anoxic injury46, an suggesting that Nav1.2 might support this function. observation that might be explained by the expression Nav1.2 channels display activation and availability of Nav1.2 channels along demyelinated axons that have (steady-state inactivation) properties that are more survived rather than degenerated. depolarized than for Nav1.6 channels so that Nav1.2 chan- It is also possible that some axons degenerate in MS nels show less inactivation with modest depolarization. in the absence of demyelination. De Luca et al.47 found Nav1.2 channels, however, show greater accumulation only a weak correlation between axonal loss and plaque of inactivation, and so are less available to open and load in post-mortem MS tissue, raising the possibility contribute to action potentials at high frequencies (20–100 that demyelination might not be a prime determinant Hz)37. These observations suggest that Nav1.2 channels of axonal degeneration in MS. The Na+–Ca2+ exchanger might support action potential conduction along demy- is in fact present at intact nodes where Nav1.6 channels elinated axons even in the context of any depolarization are aggregated in normal white matter48. Therefore, that occurs in the fibres as a result of an injury, but that if the inadequacy of ATP supply described by Dutta conduction in these axons would be limited to lower et al.24 in MS occurs in neurons in which axons are not frequencies. Importantly, Nav1.2 channels produce demyelinated, the axons might be poised to undergo a smaller persistent current than Nav1.6 channels37. Ca2+-mediated injury. Therefore, demyelinated axons expressing Nav1.2 would be expected to be subjected to a smaller sustained Na+ Chaos in the cerebellum influx, a factor that could encourage their survival, as has Dysregulated Na+ channel expression might also con- been observed in immuno histochemical studies on EAE tribute to cerebellar dysfunction in MS. There are and MS35,39. a number of indications in the clinical literature of a different pathophysiology for cerebellar deficits in MS, Nav1.6 and axonal injury compared with other types of clinical deficits. First, As described above, Nav1.6 produces a large, persistent clinical abnormalities due to cerebellar dysfunction in Plaque load Na+ conductance that could trigger reverse Na+–Ca2+ MS tend to be persistent, even early in the course of the An aggregate measure of the 2+ number and volume of lesions exchange and consequent Ca entry that results in the disease, in contrast to other types of clinical deficits which 13,14 49 in a brain with multiple injury of myelinated axons . The biophysical properties tend to be remitting . Second, cerebellar dysfunction sclerosis. of Nav1.6 channels predict a persistent ‘window’ current is occasionally observed in patients with MS in whom

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Ataxia cerebellar lesions cannot be visualized through channel Nav1.8 was found in Purkinje cells of the Taiep 53 Loss of coordination of muscle neuro imaging. Third, there are reports of occasional rat , a dysmyelinating model in which myelin initially movements, produced most patients with MS who have paroxysmal bouts of ataxia ensheaths CNS axons but subsequently degenerates commonly by dysfunction of similar to those seen in the episodic ataxias, a group due to an inherited abnormality of oligodendrocytes. the cerebellum (cerebellar of disorders caused by hereditary channelopathies, Black et al.54 subsequently used in situ hybridization ataxia) or defective sensory input (sensory ataxia). and the paroxysmal attacks have been reported to and immunocytochemical methods to examine the respond favourably to treatment with Na+ channel cerebellums of mice with EAE and humans with progres- blockers such as carbamazepine50,51. These clinical sive MS, and observed the presence of Nav1.8 mRNA observations raise the possibility that, in addition and protein (FIG. 4A), which are not present in normal to demyelination and axonal degeneration, molecu- Purkinje neurons. Annexin II/p11, a protein that binds to lar changes in cerebellar neurons might contribute the amino (N)-terminus of Nav1.8 and facilitates inser- to the pathophysiology of MS52. tion of functional Nav1.8 channels into the neuronal Consistent with these clinical observations, upregu- cell membrane55, was also shown to be upregulated lated expression of the sensory neuron specific (SNS) within Purkinje cells in EAE and MS, and — as shown in FIG. 4A — is colocalized with Nav1.8 (REF. 56). The coor- dinated upregulation of Nav1.8 and its binding partner A annexin II/p11 suggest that functional Nav1.8 channels Control EAE d 10 could be inserted into Purkinje cell membranes in MS GFP Aa Ab 0 and its animal models. –10 The Nav1.8 channel was initially termed SNS because it is selectively expressed in the healthy nervous system –20 in DRG neurons. It is distinguished from other Nav –30

Vm (mV) channels by resistance to TTX and steady-state inactiva- Ac Ad –40 tion with voltage-dependence that is more depolarized –50 than for other Na+ channels, a property that enables –60 these channels to open even when the cell membrane is 0 250 500 750 1,000 depolarized to the extent that other Na+ channel Time (ms) isoforms are inactivated57,58. Nav1.8 is also unique in displaying rapid recovery from inactivation59,60. Nav1.8 10 Ae Af Nav1.8/GFP produces most of the current underlying the depolar- 0 izing upstroke of the action potential in cells in which –10 it is present61,62, and supports repetitive firing in these –20 cells, even when depolarization inactivates the other Na+ 50 μm –30 channels present61. Vm (mV) –40 Electrogenesis in Purkinje cells depends, in part, bcControl Purkinje cell +Nav1.8 + 63–65 –50 on the activity of Na channels , and mutations of Na+ channels that are normally expressed in Purkinje –60 0 mV 0 mV 0mV 0 250 500 750 1,000 cells produce substantial changes in patterns of firing Time (ms) in these cells, which can result in clinical cerebellar 65,66 10 mV dysfunction, including ataxia . Recordings from 50 ms 0.08 Purkinje cells transfected with Nav1.8 in vitro67 and 0.04 68 nA from Purkinje cells in vivo in animals with EAE 0.00 0 250 500 750 1,000 suggest that expression of Nav1.8 can substantially Time (ms) perturb their pattern of firing. The firing patterns

+ of Purkinje neurons transfected in vitro with Nav1.8 Figure 4 | Sensory neuron specific Na channel Nav1.8 is aberrantly expressed 67 within cerebellar Purkinje neurons in MS and its experimental models. Voltage- are altered in several ways : first, by increased action gated sodium (Nav) 1.8 (Ab) and annexinII/p11, a binding partner which facilitates the potential duration and amplitude (FIG. 4b,c); second, insertion of functional Nav1.8 channels into the cell membrane (Ad), are co-expressed in by fewer action potentials that consist of multiple Purkinje cells in experimental autoimmune encephalomyelitis (EAE). The merged images spikes and a reduction in the number of spikes in are shown in yellow (Af). Neither of these molecules can be detected within control these conglomerate action potentials; and third, by Purkinje neurons (Aa,Ac,Ae). Expression of Nav1.8 alters action potential electrogenesis the generation of sustained, pacemaker-like impulse in Purkinje neurons (b,c). Current clamp recordings show spontaneous action potentials in trains in response to depolarization, which are not control Purkinje neurons lacking Nav1.8 (b), and two days after transfection with Nav1.8 seen in the absence of Nav1.8 (FIG. 4d). Purkinje cells (c). Action potentials in control Purkinje neurons lacking Nav1.8 (b, left) show less in mice with EAE show similar changes in their overshoot (dotted lines indicate 0 mV) and tend to be conglomerate, consisting of two or firing patterns68. 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 The trigger for upregulated expression of Nav1.8 neurons transfected with Nav1.8 show sustained repetitive firing (d, middle), not present within Purkinje cells in MS and its animal models is in the absence of Nav1.8 (d, top), in response to the injection of a depolarizing current not known. Nerve growth factor (NGF) is known to (d, bottom). GFP, green fluorescent protein; Vm, membrane potential. Panels Aa–f upregulate transcription of the Nav1.8 gene69,70. Levels reproduced, with permission, from REF. 56 © (2003) Lippincott, Williams & Wilkins. of NGF are increased in humans with MS71 and in Panels b–d reproduced, with permission, from REF. 67 © (2003) Elsevier Science. animal models of MS, where expression of the p75

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72 Channelopathies neurotrophin receptor is also upregulated . Reasoning An immune connection + Disorders due to mutations of that NGF might trigger upregulation of Nav1.8 in Although neuroscientists tend to think of Na chan- ion channels (inherited Purkinje cells in EAE and MS, Damarjian et al.73 exam- nels as ‘neuronal’ molecules, Na+ channels are present channelopathies), or due to ined the relationship between upregulation of Nav1.8 in, and seem to regulate the activity of, microglia and altered channel function and expression of the NGF receptors p75 and TrkA macrophages77, two immune cell types that contribute attributable to exposure to toxins or antibodies, or to in the cerebellum in EAE. Consistent with previous to axonal degeneration in EAE and MS. Microglia and dyregulated channel studies74,75 they observed low levels of p75 and TrkA macrophages are closely associated with degenerating expression after tissue injury in control Purkinje neurons. However, there was a axons in MS9,78,79, and have been suggested to produce (acquired channelopathies). significant upregulation of p75 within Purkinje cells in axonal injury by multiple mechanisms, including the + 80 Electrogenesis EAE and a high level of expression of p75 in Purkinje induction of CD4 T-cell proliferation , production of 81 82,83 The production of electrical cells that expressed Nav1.8. These observations raise pro-inflammatory cytokines and NO , antigen presen- signals — for example, action the possibility that NGF could trigger upregulated tation84 and phagocytosis85. Patch-clamp studies86,87 have potentials — by cells such as expression of Nav1.8 within Purkinje cells in EAE shown that Nav channels are present in microglia. In a neurons. and MS. Some support for this hypothesis is provided recent immunocytochemical study, Craner et al.77 showed by the observation that antisense knockdown of p75 that Nav1.6 channels are indeed present in microglia, at ameliorates disease severity in EAE76. 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 a 8 b Control MS patients, compared with control patients without MS neurological disease (FIG. 5a). Proposing that Na+ chan- 50 6 p < 0.001 nels are important for the function of these cells, they asked whether Na+ channel blockade might attenuate the

–1 40 inflammatory activity of these cells, and observed a 40% 4 30 reduction in phagocytic activity of cultured microglia following exposure to TTX (FIG. 5b) and a 75% decrease in 2 20 the number of inflammatory cells in EAE after treatment with phenytoin (FIG. 5c). The researchers also observed Normalized optical intensity 10 that activation of microglia from med mice (which lack 0 Number of beads cell functional Nav1.6) is reduced compared with wild-type 0 mice (in which Nav1.6 is present), and showed that the Resting TTX suppressing effect of TTX on microglial activation is Activated

Intermediate not present in med mice, confirming a role for Nav1.6 Macrophages ACM/LPS ACM/LPS Microglia in microglial activation. Further studies are underway to examine the mechanism by which Nav1.6 (and c Control EAE EAE-Phenytoin possibly other Na+ channel isoforms) modulates the activ- OX42 CD45 ity 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 25 μm In the face of the results described in this article, it should + Figure 5 | Na+ channels are present in EAE and MS, and contribute to microglia/ not come as a surprise that Na channels are being inves- macrophage activation and function. a | Progressive upregulation of voltage-gated tigated as potential therapeutic targets in MS. Subtype- sodium (Nav) 1.6 protein (detected by immunocytochemistry and shown in terms of specific blockers of Nav1.6 channels (and possibly normalized optical density) with activation of microglia and macrophages in acute blockers of the persistent component of the current multiple sclerosis (MS) lesions, compared with resting microglia in controls with no produced by Nav1.6 channels) might be predicted to neurological disease. b | Activation and phagocytic function of microglia/macrophages have a protective effect, either by preventing axonal are attenuated by Na+ channel blockade. Administration of tetrodotoxin (TTX) (upper degeneration through direct action on axons or by blunt- panel) to lipopolysaccharide (LPS)-stimulated microglia in primary culture attenuates ing the activity of microglia and macrophages in MS. phagocytic function, as seen by a decreased number of latex beads phagocytosed per cell Although subtype-specific blockers are not yet avail- compared with cells not treated with TTX (lower panel). The histogram shows significant + 18 c able, the nonspecific Na channel blockers phenytoin reduction in the degree of phagocytosis after treatment with TTX. | Phenytoin reduces 19 inflammatory infiltrate in experimental autoimmune encephalomyelitis (EAE). The panels and flecainide have been shown to be protective in show images of control, EAE, and phenytoin-treated EAE spinal cord immunostained for mouse and rat models of EAE. Administration of these anti-CD45 (green) and anti-OX42 (blue). Administration of phenytoin results in a marked agents in the rodent models of MS results in a reduced reduction in inflammatory infiltrate. ACM, astrocyte conditional medium. Modified, with degree of axonal degeneration in CNS white matter permission, from REF. 77 © (2005) Wiley-Liss. as assessed after 28–30 days of disease. For example,

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phenytoin reduces the loss of dorsal corticospinal axons In addition to a protective action, the aberrant expres- from 63% to 25%, and of cuneate fasciculus axons sion of Nav1.8 in Purkinje cells suggests that blockade from 43% to 17%18. Electrophysiological experiments of this channel might be of symptomatic benefit. show that axonal conduction is maintained in at least a Whether Nav1.8-specific blockade, or nonspecific Na+ significant fraction of the surviving axons. Importantly, channel blockade, can reverse abnormal cerebellar treatment with these Na+ channel blockers improves function due to Nav1.8 misexpression in Purkinje cells clinical outcome. A more recent study88 has shown that is not yet known. Specific blockers of Nav1.8 are not the protective effect on axons and clinical improvement yet available, but this could change because the deploy- persists for as long as 180 days in mice treated regularly ment and functional role of Nav1.8 in nociceptive with phenytoin. neurons have made it an attractive molecular target. Improvement in cerebellar function in response to Questions and horizons blockade of Nav1.8 would strengthen the evidence for It is not yet clear whether Na+ channel blockers exert a role for this channel in symptom production in MS, their protective effect in EAE in vivo through a direct and might provide a new approach for the treatment of action on axons, or by acting on immune cells such ataxia in MS. as microglia or macrophages. Bechtold et al.19 noted Whether Nav1.6 or Nav1.8 channel-specific block- a protective effect of flecainide on neurological ade or nonspecific Na+ channel blockade will be useful symptoms early in the course of EAE (10–13 days clinically in MS remains to be determined. Several Na+ post disease induction) and suggested a possible channel blockers with relatively safe side effect profiles immunmodulatory effect. There is in fact evidence are already in clinical use, and clinical trials of these suggesting a role for Na+ channels in the activation of agents as potential axon-protective therapies for MS are T cells89,90. Moreover, as discussed above, Craner et al.77 being planned. The task of translation, from molecular observed that treatment with phenytoin substantially target to clinically effective therapy, is a significant chal- ameliorates the inflammatory cell infiltrate in EAE, lenge. Nonetheless, there is a promising precedent in and noted that TTX significantly reduces the phago- the efficacy of existing nonspecific Na+ blockers such cytic function of activated microglia. Yet the protective as phenytoin, carbamazepine and lamotrigine as anti- effect of Na+ channel blockers on axons in vitro13,16,17, epilepsy medications with a substantial therapeutic where inflammation is minimal or non-existent, window. Lessons learned from MS and its models might, indicates that the mechanism of action of these agents in fact, also be relevant to spinal cord injury and trans- involves, at least in part, a direct effect on axons. It is verse myelitis, where axonal degeneration, demyelination possible that Na+ channel blockers protect axons by and inflammation all occur91,92. Hopefully we will soon a dual mechanism, involving both a direct action on begin to learn whether Na+ channel blockers, either non- axons and an immunomodulatory action on microglia specific or subtype-specific, are of clinical value in the and/or macrophages. treatment of these disabling disorders.

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86. Korotzer, A. R. & Cotman, C. W. Voltage-gated 91. Waxman, S. G., Demyelination in spinal cord injury Competing interests statement currents expressed by rat microglia in culture. Glia 6, and multiple sclerosis: what can we do to enhance The author declares no competing financial interests. 81–88 (1992). functional recovery? J. Neurotrauma 9, S105–S117 87. Nörenberg W., Illes, P. & Gebicke-Haeter, P. J. Sodium (1992). DATABASES channels in isolated human brain macrophages 92. Popovich, P. G. & Jones, T. B. Manipulating The following terms in this article are linked online to: (microglia). Glia 10, 165–172 (1994). neuroinflammatory reactions in the injured spinal Entrez Gene: 88. Black, J. A., Liu, S., Hains, B. C., Saab, C. Y. & cord: back to basics. Trends Pharmacol. Sci. 24, http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene Waxman, S. G. Long-term protection of central axons 13–17 (2003). med | Nav1.1 | Nav1.2 | Nav1.3 | Nav1.4 | Nav1.5 | Nav1.6 | with phenytoin in monophasic and chronic-relapsing Nav1.7 | Nav1.8 | Nav1.9 | TrkA EAE. Brain 24 Aug 2006 (doi:10.1093/brain/ Acknowledgements OMIM: aw1216). Research in the author’s laboratory has been supported, in http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM 89. Lai, Z. F., Chen, Y. Z. & Nishimura, Y. An amiloride- part, by grants from the National Multiple Sclerosis Society experimental autoimmune encephalomyelitis sensitive and voltage-dependent Na+ channel in an and the Medical Research Service and Rehabilitation HLA-DR-restricted human T cell clone. J. Immunol. Research Service, the Department of Veteran Affairs, and by FURTHER INFORMATION 165, 83–90 (2000). gifts from Destination Cure and the Nancy Davis Foundation. Waxman’s laboratory: http://www.med.yale.edu/neurol/pva- 90. Khan, N. A. & Poisson, J. P. 5-HT3 receptor-channels The Neuroscience and Regeneration Research Center is a epvacenter/index.html coupled with Na+ influx in human T cells: role in T cell Collaboration of the Paralyzed Veterans of America and the Access to this links box is available online. activation. J. Neuroimmunol. 99, 53–60 (1999). United Spinal Association with Yale University.