# Neuro-glial interactions at the nodes of Ranvier: implication in health and diseases.

Catherine Faivre-Sarrailh, Jérôme J Devaux

► To cite this version:

Catherine Faivre-Sarrailh, Jérôme J Devaux. Neuro-glial interactions at the nodes of Ranvier: implication in health and diseases.. Frontiers in Cellular Neuroscience, 2013, 7, pp.196. 10.3389/fncel.2013.00196. hal-00966194

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# Neuro-glia interactions at the nodes of Ranvier: implication in health and diseases

Catherine Faivre-Sarrailh and Jérôme J. Devaux*

Aix-Marseille Université, CNRS, CRN2M-UMR7286, Marseille, France

**Edited by:**

Martin Stangel, Hannover Medical School, Germany

**Reviewed by:**

Laurence Goutebroze, INSERM
UMRS 839, France
Edgar Meinl, Ludwig Maximilian
University Munich, Germany

**\*Correspondence:**

Jérôme J. Devaux, Centre de
Recherche en Neurobiologie et
Neurophysiologie de Marseille,
Aix-Marseille Université, CNRS,
CRN2M-UMR7286, Boulevard Pierre
Dramard, 13344 Marseille Cedex 15,
France
e-mail: jerome.devaux@univ-amu.fr

Specific cell adhesion molecules (CAMs) are dedicated to the formation of axo-glia contacts at the nodes of Ranvier of myelinated axons. They play a central role in the organization and maintenance of the axonal domains: the node, paranode, and juxtaparanode. In particular, CAMs are essential for the accumulation of voltage-gated sodium channels at the nodal gap that ensures the rapid and saltatory propagation of the action potentials (APs). The mechanisms regulating node formation are distinct in the central and peripheral nervous systems, and recent studies have highlighted the relative contribution of paranodal junctions and nodal extracellular matrix. In addition, CAMs at the juxtaparanodal domains mediate the clustering of voltage-gated potassium channels which regulate the axonal excitability. In several human pathologies, the axo-glia contacts are altered leading to disruption of the nodes of Ranvier or mis-localization of the ion channels along the axons. Node alterations and the failure of APs to propagate correctly from nodes to nodes along the axons both contribute to the disabilities in demyelinating diseases. This article reviews the mechanisms regulating the association of the axo-glia complexes and the role of CAMs in inherited and acquired neurological diseases.

**Keywords:** node of Ranvier, ion channel, axon-glia interactions, neurological disease, extracellular matrix

## INTRODUCTION

In vertebrate, most axons are insulated by myelin sheaths and the action potentials (APs) are regenerated at the nodes of Ranvier which enable the rapid saltatory propagation of the nerve impulses. The myelin is formed by glial cells: Schwann cells in peripheral nervous system (PNS) and oligodendrocytes in central nervous system (CNS). The mechanisms underlying myelin formation are complex and involve interactions between neurons and glial cells. Myelination begins with the contact and recognition of the axon by the glial processes. The glial processes then wrap around the axon, form multiple layers of myelin, and elongate along the axon. Simultaneously, the myelinating glial cells organize the axonal domains: nodes, paranodes, and juxtaparanodes. First, the voltage-gated Na⁺ (Nav) channels are aggregated at hemi-nodes which border the myelinated segments (Vabnick et al., 1996). These hemi-nodes then fuse into a node of Ranvier as the myelin segments grow and approach each other (See **Figure 2**). At both sides of the nodes, the myelin spirals around the axon forming paranodal loops. Electron microscopic observations revealed that the paranodal loops form septate-like junctions with the axon (Einheber et al., 1997). These junctions may preclude current leakage across the paranodes and favor rapid propagation. Recent evidences indicate that the association of Contactin-1/Caspr-1/Neurofascin-155 (NF155) is required for the formation of the septate-like junctions. In addition, these junctions favor the sequestration of the voltage-gated potassium channels (VGKCs; Kv), Kv1.1/Kv1.2/Kv1.6, in the juxtaparanodal regions (Vabnick et al., 1999). The localization of the Nav and Kv channels is strongly dependent on cell adhesion molecules (CAMs) at nodes, paranodes, and juxtaparanodes. Alterations of

the axo-glia interaction contribute to the etiology of numerous neurological diseases. This article reviews recent findings documenting the implication of CAMs in axon specialization and in neurological diseases.

## MOLECULAR ORGANIZATION OF THE AXONAL DOMAINS OF MYELINATED FIBERS

### NEUROFASCIN-186, NrCAM, AND GLIOMEDIN: STRUCTURE AND FUNCTION AT PNS NODES

During development, the clustering of Nav is strongly dependent on the axo-glia contact at PNS nodes of Ranvier (Melendez-Vasquez et al., 2001), but also on two scaffolding proteins, ankyrin-G and βIV-spectrin, which links the nodal proteins to the actin cytoskeleton (Jenkins and Bennett, 2002; Komada and Soriano, 2002; Yang et al., 2004; Devaux, 2010). In the PNS, the myelinating Schwann cells form the nodal microvilli which face the nodes of Ranvier. Several CAMs expressed at nodal axolemma or secreted by Schwann cells at the nodal lumen mediate the axo-glia contact and the clustering of Nav channels (Nav1.2 and Nav1.6) at nodes of Ranvier (Caldwell et al., 2000; Boiko et al., 2001). Neurofascin-186 (NF186) and NrCAM belong to the L1-family of CAMs and are concentrated at the nodes of Ranvier (Davis et al., 1996). NF186 is expressed at the nodal axolemma only. By contrast, NrCAM exists as both an axonal form and a form secreted by the Schwann cell microvilli (Feinberg et al., 2010).

Both NF186 and NrCAM bind Gliomedin, an extracellular matrix component secreted by the Schwann cell microvilli (**Figure 1A**). Gliomedin contains a coiled-coil, two collagen-like, and one olfactomedin domain (Eshed et al., 2005). Gliomedin exists as both transmembrane and secreted forms (Eshed et al.,
A

PNS node

<figure><img src="image_3.png" /></figure>

B

CNS node

<figure><img src="image_4.png" /></figure>

C

Paranode (CNS/PNS)

<figure><img src="image_5.png" /></figure>

D

Juxtaparanode (CNS/PNS)

<figure><img src="image_6.png" /></figure>

**FIGURE 1** | Organization of CNS and PNS nodes of Ranvier. (A) At PNS nodes, NF186 binds Gliomedin (Gldn) and NrCAM which are secreted by Schwann cells in the nodal gap lumen. The cytoplasmic region of axonal NF186 and NrCAM bind ankyrin-G, which anchors the nodal complex to βIV-spectrin and to the actin cytoskeleton. Ankyrin-G enables the clustering of Nav and Kv7.2/7.3 channels at nodes. (B) In the CNS, Tenascin-R (TN-R), Brevican (Bcan), Versican (Vcan), and Phosphacan (Phcan) are enriched in the extracellular matrix surrounding the nodes, and stabilize the nodal complex.

These molecules bind NF186, NrCAM, and Contactin-1 which are expressed at CNS nodes. (C) The complex Contactin-1/Caspr-1/NF155 forms the septate-like junctions at both PNS and CNS paranodes. This complex is stabilized by the cytosolic protein 4.1B which co-localizes with ankyrin-B, αII- and βII-spectrin at both paranodes and juxtaparanodes. (D) The complex Contactin-2/Caspr-2 enables the sequestration of Kv1.1/Kv1.2/Kv1.6 channels at juxtaparanodes, but also of PSD-93 and PSD-95. ADAM22 and Connexin-29 (Cx29) are also enriched at juxtaparanodes.

2007; Maertens et al., 2007). However, solely the secreted form, generated by proteolytic cleavage with furin and BMP-1 enzymes, is detected at the nodes of Ranvier. The release of the C-terminal olfactomedin domain favors its oligomerization, its incorporation in the extracellular matrix, and its interaction with NF186. The interactions between Gliomedin, NF186, and NrCAM are critical for the initial clustering of the Nav channels at hemi-nodes. In the developing sciatic nerve or in myelinating co-cultures of dorsal root ganglion (DRG) with Schwann cells, the clustering of nodal components (Nav channels, ankyrin-G, NF186, NrCAM, and Gliomedin) is first detected at hemi-nodes at the edge of each myelinated segment (See **Figure 2**). Deficiency in Gliomedin, NF186, or NrCAM prevents the initial clustering of the Nav channels at hemi-nodes both *in vivo* and *in vitro* (Feinberg et al., 2010). Nonetheless, Nav channel aggregation is not prevented at mature nodes in Gliomedin- or NrCAM-deficient animals. As detailed below, mature nodes are flanked by paranodal septate junctions that likely mediate a barrier to the lateral diffusion of the nodal components. Thus, the organization of the PNS nodes depends on axo-glial contacts at nodes and paranodes. The role of NF186 in

the organization of mature PNS nodes is, however, controversial. Some studies have shown that NF186 is crucial for the formation of PNS nodes (Dzhashiashvili et al., 2007; Thaxton et al., 2011), but others have shown that deleting NF186 does not alter nodal organization which is maintained by paranodal junctions (Sherman et al., 2005; Zonta et al., 2008; Feinberg et al., 2010).

Recent evidences have underpinned the mechanisms regulating the targeting of nodal components at PNS nodes (Zhang et al., 2012). It appears that nodal CAMs (NF186, NrCAM, and Gliomedin) accumulate to nascent nodes from local sources via diffusion trapping. Nav channels and ankyrin-G, by contrast, are transported to the nodes, and show a slow turnover in mature nodes. The exact mechanisms regulating the selective incorporation of the transported proteins at nodes remained, however, to be elucidated.

The nodal CAMs present several interacting modules which participate in the axo-glial contact. NF186 contains a mucin-related domain, three Fibronectin type III (FnIII) and six Ig domains (Figure 1). NrCAM is composed of four FnIII and six Ig domains (Figure 1). The Ig domains of NrCAM and NF186
<figure><img src="image_7.png" /><figcaption>**FIGURE 2 |** Soluble FnIII domains of NF186 inhibit the clustering of Gliomedin and Nav channels at hemi-nodes. These are PNS myelinating co-cultures of DRG neurons with Schwann cells that have been triple-stained for MBP (blue), Caspr or Gliomedin (red), and Nav channels (green). Myelination was induced with ascorbic acid after 7 days *in vitro*. Co-cultures were treated with control Fc or with the FnIII domains of NF186 fused with Fc (NF186Fn-Fc) from day 7 to day 24.</figcaption></figure>

Gliomedin (Gldn) and Nav channels are clustered at hemi-nodes and
flanked the paranodes and myelin borders in myelinating co-cultures.
Incubation with NF186Fn-Fc abrogated the clustering of Gliomedin and
Nav channels at hemi-nodes, but not at mature nodes of Ranvier. This
indicated that the interaction between NF186 and Gliomedin is crucial for
the formation of hemi-node clusters. Scale bar: 10 µm. Adapted from
Labasque et al. (2011).

are crucial for their heterophilic interaction (Volkmer et al., 1996). Particularly, NF186 interacts with NrCAM in *trans* via its Ig1–4 domains (Labasque et al., 2011). Deletion of the Ig domains of NF186 abolishes its accumulation at nodes (Dzhashiashvili et al., 2007), indicating that the Ig domains are crucial for the targeting at nodes. In addition, the FnIII domains of both NF186 and NrCAM are implicated in Gliomedin binding (Labasque et al., 2011). Soluble FnIII domains of NF186 has been shown to inhibit the clustering of Nav channels at hemi-nodes in myelinating co-cultures (Figure 2). This indicates that the nodal complex assemble via multiple locking modules.

Other extracellular matrix components and their receptors
may be necessary for the proper formation or stability of the
Schwann cell microvilli, such as laminins and dystroglycan. Spe-
cific laminin isoforms (α2, α5, γ5) are expressed in the basal
lamina above the nodes of Ranvier (Feltri and Wrabetz, 2005).
In addition, members of the dystrophin-dystroglycan complex
are present at nodes. Mice deficient in laminin-α2 or dystrogly-
can show severe alteration of microvilli and Nav channel clusters
(Saito et al., 2003; Occhi et al., 2005). Similar alterations are also
observed in patients with merosin-deficient congenital muscular
dystrophy type 1A which is associated with a mutation in the gene
encoding laminin-α2 (Occhi et al., 2005). Because Gliomedin and
NrCAM are secreted in the extracellular lumen, it is plausible that
the extracellular matrix may stabilize the organization of the nodal
components. The proteoglycans syndecan-3 and -4 and Perlecan
are also enriched in the perinodal processes of Schwann cells early
during development (Goutebroze et al., 2003; Melendez-Vasquez
et al., 2005; Bangratz et al., 2012). However, the function of these
latter components remains to be determined.

NF186, NrCAM, AND BREVICAN/VERSCAN COMPLEX: STRUCTURE AND FUNCTION AT CNS NODES

At CNS nodes, the molecular mechanisms implicated in the nodal
clustering of Nav channels are different from those involved in the
PNS. In the CNS, myelin sheaths are produced by oligodendro-
cytes, and the nodal gap is contacted by perinodal astrocyte pro-
cesses. In addition, the extracellular matrix in the nodal gap differs
from that in the PNS. The CNS nodes express NF186 and NrCAM,
but lack Gliomedin (Figure 1). The CNS nodal axolemma also
expresses a high molecular weight form of Contactin-1 (Rios et al.,

2000), an Ig CAM implicated in the assembly of the septate-like
junctions at paranodes (see below). In addition, several secreted
proteins are found in the perinodal extracellular matrix surround-
ing the CNS nodes: Tenascin-R, Brevican, Versican, phosphacan,
Bral1, and Neurocan (Weber et al., 1999; Bekku et al., 2009; Dours-
Zimmermann et al., 2009; Susuki et al., 2013; **Figure 1**). Brevican
and Versican are chondroitin-sulfate proteoglycans that bind
hyaluronic acid to form a negatively charged complex with Bral1,
the brain-specific hyaluronan-binding link protein. Phosphacan is
a chondroitin-sulfate proteoglycan which is the secreted form of
the receptor-like protein tyrosine-phosphatase-β, and which binds
Tenascin-R and Contactin-1 with high-affinity (Barnea et al., 1994;
Grumet et al., 1994; Peles et al., 1995; Revest et al., 1999). Finally,
Tenascin-R is a trimeric glycoprotein consisting of EGF-like and
FnIII repeats that may act as a cross-linker between proteogly-
can complexes, and which is also able to bind Neurofascin and
Contactin-1 (Zisch et al., 1992; Volkmer et al., 1998). These nega-
tively charged matrix components may provide a diffusion barrier
around the nodes underlying the accumulation of cations during
saltatory conduction (Bekku et al., 2010), but also the stabilization
of the nodal complex (Susuki et al., 2013).

In contrast to the PNS, the aggregation of the Nav channels at CNS nodes appears subsequently to the formation of the paranodal junctions (Rasband et al., 1999; Jenkins and Bennett, 2002). Disruption of the paranodal junctions in Caspr-1-deficient mice is associated with important abnormalities at CNS nodes, including Nav channels dispersion and persistent expression of the immature Nav1.2 rather than the mature Nav1.6 subunits (Rios et al., 2003). By contrast, PNS node organization is unaffected in these animals. The axo-glial contact at nodes also participates in CNS node formation. Neurofascin-deficient mice, which lack NF186 at nodes and NF155 at paranodes, show disrupted nodal and paranodal complexes at PNS and CNS. Transgenic expression of NF186 in neurons or NF155 in glial cells can rescue the accumulation of Nav channels at CNS nodes in Neurofascin-deficient mice (Zonta et al., 2008). In contrast to the PNS, the partners of NF186 at CNS node are yet unknown. NF186 can bind directly to Bral1, Brevican, Versican, and Tenascin-R (Volkmer et al., 1998; Hedstrom et al., 2007). However, during development, these perinodal matrix components assemble at nodes after the clustering of NF186 and Nav channels in the optic nerve. Thus, these matrix components may
rather be implicated in the maintenance of the nodal structure. In keeping, Nav channels are properly clustered at CNS nodes in Tenascin-R-, Versican-, and Bral-1-deficient mice, despite the loss or dispersion of Tenascin-R and Phosphacan at nodes (Weber et al., 1999; Dours-Zimmermann et al., 2009; Bekku et al., 2010). By contrast, the disruption of the paranodal complex and of the perinodal matrix in Caspr-1/Brevican/Versican triple knock-out mice induces a significant decrease in the number of Nav channel clusters (Susuki et al., 2013). These results lead to the suggestion that the formation of the paranodal diffusion barrier is the primary mechanism enabling the clustering of Nav channels at CNS nodes, whereas nodal axo-glia! contact may be a secondary mechanism which allows the maintenance of Nav clusters at nodes or their formation in absence of paranodes.

CASPR-1, CONTACTIN-1, AND NF155: STRUCTURE AND FUNCTION AT
PARANODES

A peculiar type of cell-cell junctions named the septate-like junc-
tions are encountered at paranodes in both the CNS and PNS
(Einheber et al., 1997). The septate-like junctions seal the termi-
nal loops of myelinated segments to the axolemma on both sides
of the nodal gap. These paranodal junctions are characterized by
intermembrane transverse bands and derive from an ancestral type
of junctions observed in invertebrates, the septate junctions, that
provides paracellular barrier between epithelial cells or between
glial cells insulating axon fascicles (Hortsch and Margolis, 2003;
Faivre-Sarrailh et al., 2004). In vertebrates, the paranodes act as a
fence separating the nodal and juxtaparanodal domains enriched
in Nav and Kv channels, respectively and as an electrical barrier
that promotes AP propagation. The molecular composition of the
paranodal junctions consists of a ternary complex of glycopro-
teins highly conserved during evolution: Caspr-1, Contactin-1,
and NF155. Deficiency in either Contactin-1, or Caspr-1, or Neu-
rofascin in mice induces severe neurological defects, disruption of
the septate-like junctions, and a reduction of nerve conduction
velocity (Bhat et al., 2001; Boyle et al., 2001; Sherman et al., 2005;
Zonta et al., 2008; Pillai et al., 2009).

The axonal Caspr-1 and Contactin-1 form *cis*-heteromers that
are targeted to the paranodal junctions during myelination and
interact in *trans* with the glial expressed NF155 (Rios et al., 2000;
Charles et al., 2002). NF155 is a 155-kDa splice variant obtained
from the same gene as NF186, but which is expressed only by
the myelinating glial cells (Tait et al., 2000). Caspr-1 belongs to
the neurexin family and is composed of a discoidin domain,
and several laminin-G and EGF-like modules (Menegoz et al.,
1997; Peles et al., 1997; **Figure 1**). Caspr-1 contains a cyto-
plasmic motif for binding to the scaffolding 4.1B protein and
co-localizes with ankyrin-B, αII- and βII-spectrin at paranodes
(Ogawa et al., 2006). Contactin-1 and NF155 both contain six Ig
domains and four FnIII domains (**Figure 1**), however, Contactin-1
is a glycosyl-phosphatidyl-inositol anchored protein. The assem-
bly and targeting of the Caspr-1/Contactin-1/NF155 complex at
paranodes is a tightly controlled process. First, Contactin-1 is
required for the transport of the Contactin-1/Caspr-1 complex
to the axonal membrane (Faivre-Sarrailh et al., 2000). This com-
plex is addressed to the cell surface with ER-type mannose-rich
N-glycans that favor its interaction with NF155 (Bonnon et al.,

2007). In addition, selective modules are required for the asso-
ciation of NF155 with the Contactin-1/Caspr-1 complex. The Ig
domains of Contactin-1 mediate its interaction with NF155 and
Caspr-1. Also, the Ig domains 5 and 6 of Neurofascin are implicated
in its interaction with Contactin-1. Mutant mice with deletion of
these Ig domains show a disruption of the paranodal septate-like
junctions (Thaxton et al., 2010).

Worth noting, paranodal proteins are lipid raft-associated pro-
teins and this localization may favor the maintenance of paranodal
junctions (Ogawa and Rasband, 2009; Labasque and Faivre-
Sarrailh, 2010). Indeed, the deletion of MAL, a raft-associated
proteolipid, results in the disorganization of the paranodal septate-
like junctions (Schaeren-Wiemers et al., 2004). Also, the mainte-
nance of paranodal junctions appears to be dependent on myelin
galactolipids (Popko, 2000; Ishibashi et al., 2002). Mice lacking raft
gangliosides, notably GM1 and GD1a, show alterations in Caspr-
1/NF155 aggregation at paranodes (Susuki et al., 2007a). In mice
lacking Caspr-1 or gangliosides, the partition of NF155 into lipid
rafts is strongly attenuated.

CONTACTIN-2 AND CASPR-2 AT JUXTAPARANODES

The juxtaparanodal regions are adjacent to the paranodes and are
recovered by compact myelin. The juxtaparanodes are enriched
in Shaker-type Kv1 channels, mainly Kv1.1, Kv1.2, and Kv1.6 sub-
units, but also Kv1.4 in a subtype of sensory fibers (Rasband et al.,
1998; Rasband and Trimmer, 2001). These channels may stabilize
conduction by dampening repetitive firing and maintaining the
internodal resting potential, particularly during development and
in small diameter axons (Rasband et al., 1998; Devaux et al., 2002;
Devaux and Gow, 2008). A heteromeric complex of Contactin-2
(also known as TAG-1) and Caspr-2 is implicated in the forma-
tion of juxtaparanodes in both CNS and PNS (Poliak et al., 2003;
Traka et al., 2003). These molecules are homologs of Contactin-1
and Caspr-1, respectively. Contactin-2 is expressed at the axonal
and glial membranes at juxtaparanodes and displays homophilic
binding activity which mediates adhesive contact. Contactin-2
exists as a glycosyl-phosphatidyl-inositol anchored form, as well
as a released form (Furley et al., 1990). Within the axonal mem-
brane, Contactin-2 forms a *cis*-complex with Caspr-2 via its Ig
domains which allows the formation of a ternary complex with
the glial-secreted Contactin-2 (Savvaki et al., 2010). Disruption of
Caspr-2 or Contactin-2 in knock-out mice prevents the accumula-
tion of Kv1 channels at juxtaparanodes and induces their diffusion
along the internodes. Albeit, the mis-localization of Kv1 chan-
nels does not affect nerve conduction (Poliak et al., 2003; Traka
et al., 2003), it was reported that Contactin-2-deficient animals
show behavioral deficits and defects in sensori-motor gating and
motor coordination (Savvaki et al., 2008). Strikingly, the trans-
genic expression of Contactin-2 exclusively in oligodendrocytes is
sufficient to rescue juxtaparanode formation and the behavioral
deficits in Contactin-2-deficient mice (Savvaki et al., 2010). These
data highlight the importance of glial-secreted Contactin-2.

A number of scaffolding proteins (4.1B, ankyrin-B, αII- and βII-
spectrin) are expressed at juxtaparanodes with Caspr-2, but also at
paranodes (Denisenko-Nehrbass et al., 2003; Ogawa et al., 2006).
In 4.1B-null mice, the accumulation of Caspr-2, Contactin-2, and
Kv1.1/Kv1.2 at juxtaparanodes is abolished, indicating that 4.1B
protein is crucial for the formation of juxtaparanodal domains (Horresh et al., 2010; Buttermore et al., 2011; Cifuentes-Diaz et al., 2011a; Einheber et al., 2013). In addition, the membrane-associated guanylate kinases PSD-93 and PSD-95 are concentrated at juxtaparanodes (Ogawa et al., 2010). However, these proteins are not required for Kv1 and Caspr-2 clustering at juxtaparanodes (Horresh et al., 2010; Ogawa et al., 2010).

The juxtaparanodal complex also comprises disintegrin and metalloproteinase 22 (ADAM22). The deletion of ADAM22 results in the loss of PSD-93 and -95 at juxtaparanodes, but does not affect the localization of Kv1 channels and Caspr-2. The exact function of disintegrin and ADAM22 at juxtaparanodes, thus, remains to be determined.

Of interest, the loss of the paranodal septate-like junctions in Caspr-1 and Contactin-1 deficient mice induces the re-location of the juxtaparanodal proteins near the nodes (Bhat et al., 2001; Boyle et al., 2001). The role of 4.1B in paranode formation or maintenance is uncertain. Nonetheless, the transgenic expression of Caspr-1 lacking the 4.1-binding module in Caspr-null mice restores paranode formation, but does not restore the accumulation of Kv1 channels at juxtaparanodes (Horresh et al., 2010). Altogether, these studies indicate that the organization and maintenance of juxtaparanodes depend on the combination of three distinct processes: assembly of an axo-gial complex at juxtaparanodes, the linkage of this complex to the cytoskeleton, and the sequestration of this complex by the paranodal diffusion barrier.

## IMPLICATIONS OF CAMS IN INHERITED AND ACQUIRED NEUROLOGICAL DISORDERS

### NODE ALTERATIONS IN INHERITED DEMYELINATING DISORDERS

Although nodal paranodal CAMs are not the priming factors in human inherited demyelinating pathologies, it has come to light during the last decade that demyelination not solely affects the biological properties of the myelinated axons but also results in the redistribution or disorganization of the nodal and paranodal components. These latter changes likely participate to the conduction deficits and give important clues about the mechanisms dictating node formation or re-formation during remyelination. Here, we will focus on two human pathologies: the demyelinating forms of Charcot-Marie-Tooth (CMT) disease and Pelizaeus–Merzbacher disease.

Charcot–Marie-Tooth type 1 are inherited demyelinating diseases affecting peripheral nerves which are caused in most patients by mutations in *Pmp22* (CMT1A), *MPZ* (CMT1B), and *GJB1* genes (CMT1X; see for review Suter and Scherer, 2003). *Trembler-J* mice are an animal model of CMT1A and show a point mutation in *Pmp22* that is also found in a family with CMT1A (Suter et al., 1992; Valentijn et al., 1992). In these animals, peripheral axons show important segmental demyelination, a reduction in the internodal length, but also a shortening of the paranodal regions (Devaux and Scherer, 2005). These latter alterations are associated with abnormally distributed Kv1.1 and Kv1.2 channels which often flank the nodes or diffuse in demyelinated segments. In demyelinated segments, Nav channels do not diffuse along the axons, but remain clustered at hemi-nodes bordering the Schwann cells (Devaux and Scherer, 2005) and co-localize with Gliomedin (our unpublished observations). These results

indicate that despite the paranodal alterations and demyelination, the preservation of the axo-gial contact at nodes is sufficient to enable the clustering of Nav channels in these animals. Interestingly, hemi-nodes and nodes contain two unusual subunits, Nav1.8 and Kv3.1b (Devaux and Scherer, 2005), which are normally absent from PNS nodes. Similar alterations were also found in P0-deficient mice, an animal model of CMT1B. In these animals, most axons exhibit disrupted paranodes and abnormally distributed Kv1.1/Kv1.2 channels (Ulzheimer et al., 2004). In addition, Nav1.8 subunits were found co-expressed with Nav1.6 at nodes and hemi-nodes bordering the Schwann cells in P0-deficient mice. Immunohistological studies of skin biopsies from CMT1A and CMT1B patients have further confirmed that such alterations also take place in human patients. Indeed, segmental demyelination, reduction in the internodal length, and paranodal alterations have been documented in these patients (Li et al., 2005; Bai et al., 2006; Saporta et al., 2009). In particular, reorganization of Kv1.1/Kv1.2 channels was observed in CMT1A patients (Li et al., 2005), whereas, aberrant expression of Nav1.8 subunits at nodes was found in CMT1B (Saporta et al., 2009). Altogether, these findings indicate that demyelination and/or remyelination affects the distribution and composition of ion channels in peripheral axons.

Animal models of Pelizaeus–Merzbacher disease have further revealed some of the mechanisms responsible for the maintenance of Nav channel clusters in the CNS. Pelizaeus–Merzbacher disease is a leukodystrophy associated with mutations in the *PLP* gene. *Myelin-deficient* (*md*) rats and *jimpy* mice are animal models of Pelizaeus–Merzbacher disease, and show severe phenotypes caused by mutations in the *PLP* gene. In both strains, severe dysmyelination occurs during the first post-natal weeks due to spontaneous oligodendrocyte cell death (Knapp, 1986; Grinspan et al., 1998). At P21, few myelinated axons are found in the spinal cord of these animals, and are ensheathed by only a few myelin wraps. Nevertheless, Nav channels and ankyrin-G remain clustered at node-like structures, even in regions devoid of oligodendrocytes (Mathis et al., 2001; Arroyo et al., 2002). By contrast, paranodal regions are severely impacted in the spinal cord of these animals. Caspr-1/Contactin-1/NF155 clusters are not detected, and no septate-like junctions are observed by electron microscopy. Hence, the localization of the Kv1.1/Kv1.2 subunits is strongly altered in *md* rats and *jimpy* mice, and Kv1.1/Kv1.2 subunits abutted the node-like clusters of Nav, Kv7.2/Kv7.3, and Kv3.1b channels (Mathis et al., 2001; Arroyo et al., 2002; Devaux et al., 2003, 2004). These results show that node-like clusters of Nav channels can maintain, at least temporarily, in the absence of myelin sheaths and paranodal junctions in *jimpy* and *md* animals. The mechanisms responsible for the maintenance of these node-like structures are, however, unclear. It is plausible that the presence of astrocyte processes contacting the node or the preservation of the extracellular matrix components (Brevican, Phosphacan, and Versican) maintain these node-like clusters.

## ANTIBODIES AGAINST CASPR-2 AND CONTACTIN-2 IN PERIPHERAL NERVE HYPEREXCITABILITY AND AUTOIMMUNE ENCEPHALITIS

Numerous studies have implicated the molecular complex found at juxtaparanodes, named the VGKC complex, as an autoimmune
target in generalized neuromyotonia (Isaac's syndrome), per-
sistent facial myokymia, Morvan's syndrome, and in limbic
encephalitis. Neuromyotonia and myokymia are peripheral nerve
hyperexcitabilities characterized by repetitive muscle contrac-
tions (Gutmann and Gutmann, 2004). Neuromyotonia and
myokymia are often linked to impaired function of the Kv1
channels. Neuromyotonia is also observed in Morvan's syn-
drome in which it is associated to confusion, autonomic distur-
bance, and delirium or insomnia (Newsom-Davis et al., 2003).
By contrast, limbic encephalitis are characterized by amne-
sia, confusion, seizures, and psychosis (Buckley et al., 2001;
Vincent et al., 2004). Originally, it was suspected that anti-
bodies targeting Kv1.1/Kv1.2/Kv1.6 subunits may be the caus-
ing agents in these disorders (Shillito et al., 1995; Hart et al.,
1997; Buckley et al., 2001; Vincent et al., 2004; Kleopa et al.,
2006). However, recent investigations revealed that most patients
with anti-VGKC-complex antibodies present antibodies against
Leucine-rich glioma inactivated 1 (LGI-1), a secreted protein
associated with presynaptic Kv1 channels (Irani et al., 2010;
Lai et al., 2010). In addition, many patients present antibod-
ies against the juxtaparanodal CAMs: Caspr-2 and Contactin-2
(Irani et al., 2010; Lancaster et al., 2011). These findings further
emphasized that axonal CAMs are implicated in excitability dis-
orders. Worth noting, sera from patients with neuromyotonia,
Morvan's syndrome, or limbic encephalitis recognize cell sur-
face antigens and stain the juxtaparanodes in the PNS (Kleopa
et al., 2006; Lancaster et al., 2011). In addition, most of these
patients responded to immunotherapy (Irani et al., 2010; Lai et al.,
2010; Lancaster et al., 2011), suggesting that the autoantibod-
ies are pathogenics and may induce the down-regulation of the
Caspr-2/Contactin-2/Kv1 channel complex. In keeping with this
view, sera from patients with neuromyotonia and anti-VGKC-
complex antibodies significantly decreased the density of the
potassium currents in PC-12, NB-1, or CHO-K1 cells express-
ing Kv1.1/Kv1.6 cells when the cells were incubated for 3 days
with the sera (Sonoda et al., 1996; Nagado et al., 1999). However,
these sera did not directly block the potassium currents in these
cells.

The fact that antibodies to Caspr-2 or Contactin-2 are associ-
ated with peripheral nerve hyperexcitabilities originating in motor
axons suggest that these antibodies are susceptible to diffuse across
the paranodal barrier and act on the juxtaparanodal Kv1 channels.
Recent studies indicate that the paranodal regions is not as tightly
sealed as originally thought (Devaux and Gow, 2008; Mierzwa
et al., 2010), thus it is plausible that serum IgG in patients with
Morvan's syndrome may slowly diffuse toward the juxtaparan-
odes. However, the exact pathogenic mechanisms remain to be
clarified as well as the epitopes recognized by the antibodies. In
some patients, antibodies to Caspr-2 are associated with thymo-
mas (Vincent and Irani, 2010), suggesting a reaction against tumor
antigens.

## NODAL ALTERATIONS AND AUTOIMMUNITY AGAINST CAMS IN MULTIPLE SCLEROSIS

Multiple sclerosis (MS) is an immune-mediated disease character-
ized by CNS demyelination, inflammation, axonal degeneration,
and cortical lesions which may lead to numbness, paralysis,

blindness, and other deficits. Alterations of the nodes of Ranvier
have been documented in MS, and Nav channels appear to dif-
fuse along the demyelinated axons in white matter lesions (Moll
et al., 1991; Craner et al., 2004; Coman et al., 2006). In addition,
the paranodal length is increased within demyelinating lesions,
and NF155 immunoreactivity spreads along the internodes, par-
ticularly in damaged or stressed axons (Howell et al., 2006). Worth
noting, paranodal alterations precede the dismantling of the node,
and result in the incursion of the juxtaparanodal Kv1 channels at
nodes and paranodes both in MS and in animal models of MS, the
experimental autoimmune encephalomyelitis (EAE; Howell et al.,
2006; Zoupi et al., 2013). It is very likely that the disruption of the
nodal aggregates of Nav channels participates to the conduction
and locomotor deficits in MS patients. Similarly, the alterations
of the paranodal axo-glia junctions and the redistribution of the
Kv1 channels might contribute to the conduction defects. Sev-
eral mechanisms may be responsible for these alterations. First,
microglia infiltration has been found to correlate with nodal and
paranodal alterations in MS patients and in EAE (Howell et al.,
2010). Particularly, the inhibition of microglia activation mini-
mized the nodal/paranodal alterations in animal model of MS.
This indicates that inflammation can participate in MS etiology
by affecting node organization.

Secondly, autoimmune attack against the nodal/paranodal
compartments may favor node disruption. Autoantibodies against
Neurofascin (NF186 and NF155) have been detected in a few
patients with MS (Mathey et al., 2007; Elliott et al., 2012). The
immunoabsorption of MS sera over immobilized NF155 abol-
ished the demyelinating and axopathic activities of the serum
in one patient (Elliott et al., 2012). Hence, antibodies to NF155
may participate to the nodal/paranodal alterations. However, the
prevalence of such antibodies appears to be low in MS patients,
as three recent studies indicate that Neurofascin is not the domi-
nant target of antibodies in MS (Devaux et al., 2012; Elliott et al.,
2012; Kawamura et al., 2013). Interestingly, the prevalence of anti-
bodies against NF155 is very high (86%) in patients presenting
combined central and peripheral demyelination (Kawamura et al.,
2013). These patients show a good response to intravenous Ig
injection (IVIg) and plasma exchange, suggesting that these anti-
bodies may participate in the demyelination process. The passive
transfer of anti-NF155 antibodies in rats does not exert pathogenic
effects (Lindner et al., 2013). However, the passive transfer of anti-
NF186 antibodies in rats exacerbates the clinical signs of EAE
and induces axonal loss (Mathey et al., 2007; Lindner et al., 2013).
It is thus likely that antibodies to Neurofascin are pathogenics
and participate to the etiology of MS and other demyelinating
disorders.

In addition to the humoral response, T-cell response against
Contactin-2 has also been reported in MS (Derfuss et al., 2009).
The adoptive transfer of Contactin-2-reactive T-cells induces
EAE in rats characterized by inflammation of the gray matter.
Moreover, Contactin-2-reactive T-cells enhance the demyelinat-
ing activity of anti-MOG antibodies by damaging the blood-brain
barrier. Taken together, these findings suggest that reactive T-cells
may contribute to the pathology of MS. It now appears essential
to determine whether other axonal or glial CAMs are the targets
of autoimmunity in MS.
AUTOIMMUNITY TO CAMS IN IMMUNE-MEDIATED DEMYELINATING NEUROPATHIES

A large catalog of neurological disorders affecting peripheral
nerves is suspected to be immune-mediated. Among these,
autoimmune reaction against the nodes of Ranvier is implicated
in Guillain–Barré syndrome (GBS) and chronic inflammatory
demyelinating polyradiculoneuropathies (CIDP; Santoro et al.,
1990; Griffin et al., 1996; Hafer-Macko et al., 1996a,b; Cifuentes-
Diaz et al., 2011b). The causes and pathogenesis of GBS and CIDP
remain largely unknown. The presence of inflammatory infiltrates,
the deposition of IgG and IgM in nerve biopsies, and the response
to IVIg and steroids suggest an autoimmune origin (Dalakas and
Engel, 1980; Schmidt et al., 1996; Bouchard et al., 1999; also see
for review Hughes and Cornblath, 2005; Mehndiratta and Singh,
2007). In particular, the deposition of complement on the abax-
onal surface of the Schwann cells in GBS patients (Hafer-Macko
et al., 1996b; Lu et al., 2000; Wanschitz et al., 2003) has suggested
that the pathology is humorally mediated.

Several recent studies have revealed that autoantibodies in GBS
and CIDP patients target CAMs located at the nodes of Ranvier
and paranodes (Pruss et al., 2011; Devaux et al., 2012; Ng et al.,
2012; Querol et al., 2012; **Figure 3**). In particular, serum IgG in
nearly 40 % of GBS and 30% of CIDP patients from a Japanese
cohort bind the nodal or paranodal regions of peripheral nerve
fibers (Devaux et al., 2012). Also, the serum IgG in nearly 40 % of

CIDP patients from a French cohort label the nodal or paranodal
regions (our unpublished observations). These results indicate
that the node of Ranvier is the target of the immune attack in
many GBS and CIDP patients. Gliomedin, Neurofascin, Caspr-
1, and Contactin-1 have been identified as the target antigens in
some GBS and CIDP patients (Pruss et al., 2011; Devaux et al.,
2012; Ng et al., 2012; Querol et al., 2012; **Figure 3**). The propor-
tion of patients with antibodies against these CAMs is relative low
and ranges from 1 to 8%. Nevertheless, antibodies to Gliomedin
and Contactin-1 are mostly associated with the demyelinating
form of GBS, acute inflammatory demyelinating polyneuropa-
thy (AIDP), and with CIDP (Devaux et al., 2012; Querol et al.,
2012). Particularly, Querol et al. (2012) have shown that anti-
bodies to Contactin-1 are associated with a specific sub-form of
CIDP characterized by an aggressive onset and a poor response to
IVIg. In their study, Ng et al. (2012) have examined the prevalence
of antibodies against Neurofascin and found that the reactivity
against NF155 is more frequent in patients with CIDP. Worth not-
ing, the CIDP patients had IgG4 against NF155. These antibodies
may have an antigen-blocking function, as IgG4 does not bind Fc
receptors and does not activate the complement pathway (Nirula
et al., 2011). Altogether, this suggests that immune attack against
nodal or paranodal CAMs could be a common mechanism mediat-
ing paranodal demyelination in some sub-forms of demyelinating
neuropathies.

<figure><img src="image_8.png" /></figure>

**FIGURE 3** | Antibodies target nodal CAMs in GBS patients and animal models. (A) Mouse sciatic nerve fibers were incubated with sera (green) from AIDP (left panels) or AMAN (right panels) patients which are reactive against Contactin-1 and Neurofascin, respectively. Fibers were stained for Caspr (red) to label the paranodes. Pre-incubation of the sera with soluble Contactin-1-Fc or NF186-Fc abolished the binding of the IgG at nodes (arrowheads) and paranodes (double arrowheads). (B) Animal models of GBS were used to evaluate the pathogenic action of anti-Gliomedin antibodies. In animals immunized against P2 peptide (EAN-P2), Nav channels (green) are clustered at nodes (arrowheads) and

at hemi-nodes bordering the Schwann cells in demyelinated axons (bar
with arrows). The injection of anti-Gliomedin IgG (here 6 days after IgG
injection) induces the dispersion of Nav channels in demyelinated
segments (between arrows). (C) Node disruption is associated with an
important conduction slowing and loss in ventral roots of EAN-P2 animals
injected with anti-Gliomedin IgG. The amplitude of the nerve potentials
progressively decreased 1, 3, and 6 days post-injection (dpi) of
anti-Gliomedin IgG. Gray arrows indicate the latency of control nerves.
Scale bars: 10 µm. Adapted from Lonigro and Devaux (2009); Devaux
(2012), and Devaux et al. (2012).
Animal models of GBS have further confirmed that autoantibodies to nodal/paranodal CAMs have pathogenic functions. Experimental allergic neuritis (EAN) is induced by immunization of Lewis rats against the P2 peptide (EAN-P2) or purified myelin fraction (EAN-PM) that causes a demyelinating pathology reminiscent of AIDP (Uyemura et al., 1982; Hahn et al., 1988, 1991). Of interest, node disruptions are observed in EAN-PM animals and are associated with antibodies against NF186 and Gliomedin (Lonigro and Devaux, 2009). In these animals, the disappearance of NF186 and Gliomedin at nodes precedes demyelination, and results in the loss of Nav channels in demyelinated segments and in severe conduction defects (Novakovic et al., 1998; Lonigro and Devaux, 2009). By contrast, EAN-P2 animals do not exhibit nodal alterations and antibodies to nodal components, despite the presence of segmental demyelination. This work emphasizes that antibodies to nodal CAMs may participate to conduction defects by dismantling axo-gial attachment at nodes and paranodes. Further, it was found that immunization against Gliomedin, but not NF186, induces a chronic neuropathy with conduction block and nodal dysfunctions (Devaux, 2012). Most importantly, the passive transfer of anti-Gliomedin IgG in EAN-P2 animals induced demyelination, nodal disruption, and an important conduction loss (Figure 3; Devaux, 2012). These results showed that primary immune reaction against a nodal CAM can be responsible for the initiation or progression of a demyelinating form of peripheral neuropathy. The passive transfer of antibodies to Neurofascin has also been found to exacerbate the pathology of EAN-P2 (Ng et al., 2012), indicating that these antibodies are pathogenics. In animals injected with anti-Gliomedin IgG, an important deposition of IgG was found at nodes preceding demyelination, but no important deposition of complement (Devaux, 2012). These results suggest that anti-CAMs IgG may induce demyelination by directly blocking the antigen or through the recruitment of macrophages.

The pathogenic mechanisms responsible for the production of anti-CAMs antibodies in GBS and CIDP patients are still elusive. Thus far, no clear correlation has been drawn between infectious agents and the presence of anti-CAMs antibodies. It is worth noting that an outbreak of polyradiculoneuropathy has been reported in a swine abattoir and was caused by aerosolized brain tissue (Meeusen et al., 2012). Nineteen of these patients presented antibodies to the VGKC-complex, and 2 out of 19 recognized Caspr-2. This emphasizes that the mechanisms leading to the production of anti-CAM IgG may be very broad as well as the number of target antigens, and the sub-forms of GBS and CIDP.

## NODAL ALTERATIONS IN IMMUNE-MEDIATED AXONAL NEUROPATHIES

Antibodies against NF186 have also been reported in patients with acute motor axonal neuropathy (AMAN; Devaux et al., 2012). AMAN is the most predominant form of GBS in China and Japan, and is characterized by extensive axonal degeneration. Most patients with AMAN show antibodies against the gangliosides GM1, GD1a, and GalNAc-GD1a (Yuki et al., 1997; Kuwabara et al., 1998; Ho et al., 1999). It is currently suspected that these antibodies bind the nodes of Ranvier and fix complement, then induce node elongation and axonal degeneration (Hafer-Macko et al., 1996a; Paparounas et al., 1999; O'Hanlon et al., 2003). In keeping, rabbits sensitized against GM1 develop an axonal neuropathy

similar to AMAN (Susuki et al., 2003). In these animals, the deposition of anti-GM1 antibodies and complement at nodes results in the disruption of the Nav channel clusters and in conduction block (Susuki et al., 2007b). In addition, anti-GD1a antibodies can induce node disruption *in vivo* and *in vitro* (McGonigal et al., 2010; Susuki et al., 2012). These findings indicate that autoimmune attack against the nodes of Ranvier can induce conduction deficits and cause human neuropathies. Thus far, it is unclear whether anti-NF186 antibodies also participate to the etiology of AMAN. The passive transfer of anti-NF186 IgG has been found to exacerbate the axonal loss in EAE (Mathey et al., 2007; Lindner et al., 2013). Because NF186 is located on the axolemma at PNS nodes, we can suspect that antibodies directed against this protein might also induce nodal disruption and axonal degeneration in peripheral nerves. It is thus plausible that in AMAN patients, a broad immune reaction against nodal glycolipids and glycoproteins is responsible for the pathology.

It is worth noting that many axonal neuropathies are associated with node dysfunctions, and are now classified as nodoparanodopathies (Uncini et al., 2013). For instance, antibodies to GD1b are associated with acute sensory ataxic neuropathy (Pan et al., 2001; Notturno et al., 2008) and result in nodal disruption and axonal degeneration of sensory axons in rabbits (Susuki et al., 2012). Also, alterations of the nodes of Ranvier have been documented in biopsies from patients with chronic idiopathic axonal polyneuropathies (Cifuentes-Diaz et al., 2011b). It would thus be interesting to determine the prevalence of antibodies against nodal/paranodal CAMs in these, but also in other idiopathic neuropathies.

## CONCLUDING REMARKS

Over the last decade, important works have unraveled the nature of the CAMs underlying the axo-gial contacts at nodes, paranodes, and juxtaparanodes. It appears that CAMs participate in the formation and in the stabilization of the axonal sub-domains in a very complex way, and require the cooperation of intracellular anchoring proteins, signaling molecules, and of the extracellular matrix. In the CNS and PNS, the mechanisms regulating the formation of the nodes are different, albeit the composition of the nodal membrane is very similar.

As reviewed here, the node of Ranvier is the epicenter of numerous neurological disorders. This is not surprising owing to the importance of the nodal and paranodal regions in the propagation of nerve impulse. Subtle changes in the biophysical properties or excitability of nerve fibers are likely to lead to broad neurological symptoms such as pain, numbness, confusion, ataxia, or epilepsy. In addition, immune attack against the nodes of Ranvier might be responsible for conduction loss and paralysis in demyelinating disorders and nodo-paranodopathies. Some of the target antigens have been identified, but many still remain to be unraveled. Future works should investigate the pathogenic mechanisms leading to autoimmunity toward nodal antigens.

## ACKNOWLEDGMENTS

This work was supported by the Association Française contre les Myopathies (MNM1 2012-14580) and the Association pour la Recherche sur la Sclérose en Plaques.
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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that

could be construed as a potential con-
flict of interest.

Received: 21 August 2013; accepted: 08
October 2013; published online: 29 Octo-
ber 2013.

Citation: Faivre-Sarrailh C and Devaux
JJ (2013) Neuro-glial interactions at
the nodes of Ranvier: implication in
health and diseases. Front. Cell. Neu-
rosci. 7:196. doi: 10.3389/fncel.2013.
00196

This article was submitted to the journal
Frontiers in Cellular Neuroscience.

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