Molecular changes in neurons in multiple sclerosis: Altered axonal expression of Nav1.2 and Nav1.6 sodium channels and Na؉͞Ca2؉ exchanger Matthew J. Craner*†, Jia Newcombe‡, Joel A. Black*†, Caroline Hartle‡, M. Louise Cuzner‡, and Stephen G. Waxman*†§ *Department of Neurology and Paralyzed Veterans of America͞Eastern Paralyzed Veterans Association Neuroscience Research Center, Yale School of Medicine, New Haven, CT 06510; †Rehabilitation Research Center, Veterans Affairs Medical Center, West Haven, CT 06516; and ‡Department of Neuroinflammation, Institute of Neurology, University College London, London WC1N 3BG, United Kingdom Communicated by Dominick P. Purpura, Albert Einstein College of Medicine, Bronx, NY, April 19, 2004 (received for review February 12, 2004) Although voltage-gated sodium channels are known to be de- density of sodium channels in chronically demyelinated axons ployed along experimentally demyelinated axons, the molecular but do not reveal the isoforms of the channels. A 4-fold increase identities of the sodium channels expressed along axons in human in saxitoxin-binding sites in demyelinated white matter from MS demyelinating diseases such as multiple sclerosis (MS) have not patients also suggests the deployment of new Na channels (30) been determined. Here we demonstrate changes in the expression but gives no clues about the channel isoform(s) that are ex- of sodium channels in demyelinated axons in MS, with Nav1.6 pressed. confined to nodes of Ranvier in controls but with diffuse distribu- Recent studies have demonstrated up-regulated expression of tion of Na 1.2 and Na 1.6 along extensive regions of demyelinated v v Nav1.2 and Nav1.6 along extensive regions of demyelinated axons axons within acute MS plaques. Using triple-labeled fluorescent in EAE (4, 31). Nav1.2 channels produce rapidly activating and immunocytochemistry, we also show that Nav1.6, which is known inactivating currents (32–34) and appear to support action- ؉͞ 2؉ to produce a persistent sodium current, and the Na Ca ex- potential conduction, which occurs before myelination (35, 36), changer, which can be driven by persistent sodium current to suggesting that newly produced Nav1.2 channels can support import damaging levels of calcium into axons, are colocalized with conduction in demyelinated axons. Na 1.6 channels, on the other ␤ v -amyloid precursor protein, a marker of axonal injury, in acute MS hand, produce a persistent current in addition to rapidly acti- lesions. Our results demonstrate the molecular identities of the vating and inactivating currents (33, 37, 38), and Nav1.6 is sodium channels expressed along demyelinated and degenerating coexpressed together with the Naϩ͞Ca2ϩ exchanger (NCX) ؉͞ 2؉ axons in MS and suggest that coexpression of Nav1.6 and Na Ca along degenerating axons in EAE (31). exchanger is associated with axonal degeneration in MS. In this study, we present an analysis of Nav1.6 and Nav1.2 sodium channels, and of the NCX, in white matter from the demyelinating diseases ͉ action potential conduction ͉ axonal human CNS, obtained postmortem from patients with secondary degeneration progressive MS, and from control subjects with no neurological disease. We demonstrate that there are changes in the pattern of ine genes encode distinct voltage-gated sodium channels expression of sodium channels along demyelinated CNS axons in N(Nav1.1–Nav1.9) with a common motif but with different MS, with expression of Nav1.6 confined to nodes of Ranvier in amino acid sequences and physiological characteristics (1). control white matter and with diffuse expression of both Nav1.2 Nav1.6 is the major sodium channel, which is clustered at the and Nav1.6 along extensive regions of demyelinated axons in MS. nodes of Ranvier (2), although Nav1.2 is also present at some We also show that Nav1.6 (but not Nav1.2), coexpressed with the nodes (3, 4). However, the identity of sodium-channel isoforms NCX, is associated with axonal injury in MS. that are present along demyelinated axons in disorders such as multiple sclerosis (MS) has not been established. In MS, the loss Materials and Methods of myelin produces failure of axonal action-potential conduction MS Tissue. Postmortem cervical spinal cord and optic nerve that is associated with clinical exacerbations, but axonal con- tissue, acquired by means of a rapid protocol from patients with duction can recover as a result of expression of new sodium disabling secondary progressive MS (n ϭ 7; 46.1 Ϯ 6.5 yr, mean channels along demyelinated axons, providing a substrate for disease duration 14.0 Ϯ 3.7 yr) and from controls (n ϭ 6; 66.4 Ϯ remission of clinical deficits (5). Axonal degeneration also 6.0 yr) with no neurological disease, was obtained from the occurs in MS, contributes to persistent neurological deficits NeuroResource tissue bank (Institute of Neurology, London) (6–9), and may involve persistently activated sodium channels 3 ϩ ϩ (39); 1-cm tissue blocks were placed in OCT mounting medium that drive injurious reverse Na ͞Ca2 exchange (10, 11). Block- (Lamb, London) on cork discs, then gently stirred for9sin ing of sodium channels prevents axonal degeneration within isopentane precooled in liquid nitrogen before storage in air- white matter tracts in a variety of disease models (11–17), tight containers at Ϫ80°C. All tissue analyzed was characterized including a model of MS, experimental autoimmune encepha- ␮ lomyelitis (EAE) (18, 19). by oil red O and hematoxylin staining of 10 m sections taken in triplicate, i.e., immediately before, in the middle, and imme- During the development of myelinated CNS tracts, Nav1.2 channels [which are also present along unmyelinated axons (3, diately after serial sections cut from each tissue block. Acute MS 20, 21)] are initially expressed along premyelinated axons, with lesions with ongoing or recent demyelination were identified on the basis of the presence of substantial numbers (graded as Ն3 a transition to clusters of Nav1.6 at mature nodes of Ranvier (3, 22). In dysmyelinated axons from Shiverer mice, Nav1.2 channels are retained, and Nav1.6 is not expressed (3, 23), and in axons ͞Ϫ Abbreviations: MS, multiple sclerosis; MBP, myelin basic protein; ␤-APP, ␤-amyloid precur- from Plp mice, which myelinate normally and then lose their sor protein; NCX, Naϩ͞Caϩ exchanger; EAE, experimental autoimmune encephalomyelitis. myelin, there is a loss of Nav1.6 clustering and increased expres- §To whom correspondence should be addressed at: Department of Neurology LCI 707, Yale sion of Nav1.2 (24). Electrophysiological (25), cytochemical (26), School of Medicine, 15 York Street, New Haven, CT 06520-8018. E-mail: stephen.waxman and immunocytochemical (27–29) studies using pan-specific @yale.edu. sodium-channel antibodies demonstrate a higher-than-normal © 2004 by The National Academy of Sciences of the USA 8168–8173 ͉ PNAS ͉ May 25, 2004 ͉ vol. 101 ͉ no. 21 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0402765101 Downloaded by guest on October 1, 2021 ona0–5 scale) of oil red O-positive macrophages, containing neutral lipids resulting from myelin breakdown (40). Immunocytochemistry. Tissue sections were processed for immu- nocytochemistry as described (4, 31). Briefly, sections were incubated simultaneously or in combination with anti-myelin basic protein (MBP) mouse IgG (1:4,000; Sternberger Mono- clonals, Lutherville, MD), anti-␤-amyloid precursor protein (␤-APP) mouse IgG (1:100; Chemicon), anti-Caspr mouse IgG [1:500; provided by M. Rasband, University of Connecticut, Farmington (36)] or anti-NCX mouse IgM [NCX1 isoform, shown to be expressed in white matter axons (41)] (1:200; RDI, Flanders, NJ), anti-phosphorylated neurofilament (SMI-31, 1:20,000; Sternberger), anti-nonphosphorylated neurofilament (SMI-32, 1:20,000; Sternberger Monoclonals), and rabbit poly- clonal antibodies to Nav1.6 (residues 1042–1061; 1:100; Alomone, Jerusalem) or Nav1.2 (residues 467–485; 1:100; Alomone). Sections were then washed in PBS, incubated with appropriate secondary antibodies, comprising goat anti-rabbit IgG-Cy3 (1:2,000; Amersham Biosciences), goat anti-mouse IgG-Alexa Fluor 488 (1:1,000; Molecular Probes), goat anti- mouse IgM-Alexa Fluor 488 (1:1,000; Molecular Probes), and goat anti-mouse IgG-Cy5 (1:200, Rockland, Gilbertsville, PA), in blocking solution for 3 h, washed in PBS, and mounted. Biotin- ylated Ricinus communis agglutinin 1 (1:200; Vector Laborato- ries) was used as a microglial͞macrophage marker (42) and reacted with streptavidin-Cy5 (1:350; Amersham Biosciences) for detection. Control experiments, which included the omission of primary or secondary antibodies, showed no staining (data not shown). Tissue Analysis. For analysis of sections, multiple representative images were accrued by confocal microscopy with a Nikon Eclipse E600 microscope. Analysis was confined to images in which axons were sectioned longitudinally as evident from the presence of linear (presumably demyelinated) axonal profiles Fig. 1. Nav1.6 and Nav1.2 sodium channels are expressed along extensive with diffuse sodium-channel immunoreactivity, running for regions of demyelinated axons in MS. Shown are representative images Ͼ20–30 ␮m along the fiber tract within the plane of single demonstrating sections of white matter from spinal cord (A–D) and optic sections. nerve (E and F) immunostained for MBP (green) as a marker of myelination In contrast to control white matter in which there was focal and for sodium channels Nav1.6 (A, C, and E; red) and Nav1.2 (B, D, and F; red). Control spinal cord white matter (A and B) demonstrates robust MBP immu- expression of Nav1.6 and absence of Nav1.2 at nodes of Ranvier nostaining consistent with myelinated axons and does not display axonal (delineated by Caspr immunolabeling), we observed multiple profiles with diffuse (Ͼ10 ␮m) sodium-channel immunostaining. Small foci of ͞ extensive regions of diffuse Nav1.2 and or Nav1.6 sodium- Nav1.6 immunostaining (A; yellow arrows) are present, consistent with the NEUROSCIENCE channel immunoreactivity along demyelinated (demonstrated by focal distribution of Nav1.6 at nodes of Ranvier in control spinal cord white the lack of MBP immunostaining) axons (Figs.