Juxtaparanodal clustering of Shaker-like K+ channels in myelinated axons depends on Caspr2 and TAG-1

In myelinated axons, K+ channels are concealed under the myelin sheath in the juxtaparanodal region, where they are associated with Caspr2, a member of the neurexin superfamily. Deletion of Caspr2 in mice by gene targeting revealed that it is required to maintain K+ channels at this location. Furthermore, we show that the localization of Caspr2 and clustering of K+ channels at the juxtaparanodal region depends on the presence of TAG-1, an immunoglobulin-like cell adhesion molecule that binds Caspr2. These results demonstrate that Caspr2 and TAG-1 form a scaffold that is necessary to maintain K+ channels at the juxtaparanodal region, suggesting that axon-glia interactions mediated by these proteins allow myelinating glial cells to organize ion channels in the underlying axonal membrane.     

Neurofascins are required to establish axonal domains for saltatory conduction

Voltage-gated sodium channels are concentrated in myelinated nerves at the nodes of Ranvier flanked by paranodal axoglial junctions. Establishment of these essential nodal and paranodal domains is determined by myelin-forming glia, but the mechanisms are not clear. Here, we show that two isoforms of Neurofascin, Nfasc155 in glia and Nfasc186 in neurons, are required for the assembly of these specialized domains. In Neurofascin-null mice, neither paranodal adhesion junctions nor nodal complexes are formed. Transgenic expression of Nfasc155 in the myelinating glia of Nfasc-/- nerves rescues the axoglial adhesion complex by recruiting the axonal proteins Caspr and Contactin to the paranodes. However, in the absence of Nfasc186, sodium channels remain diffusely distributed along the axon. Our study shows that the two major Neurofascins play essential roles in assembling the nodal and paranodal domains of myelinated axons; therefore, they are essential for the transition to saltatory conduction in developing vertebrate nerves.     

Cellular contacts in myelinated fibers of the peripheral nervous system

Myelination allows the fast propagation of action potentials at a low energetic cost. It provides an insulating myelin sheath regularly interrupted at nodes of Ranvier where voltage-gated Na+ channels are concentrated. In the peripheral nervous system, the normal function of myelinated fibers requires the formation of highly differentiated and organized contacts between the myelinating Schwann cells, the axons and the extracellular matrix. Some of the major molecular complexes that underlie these contacts have been identified. Here we review current knowledge in this field.     

Caspr2, a new member of the neurexin superfamily, is localized at the juxtaparanodes of myelinated axons and associates with K+ channels

Rapid conduction in myelinated axons depends on the generation of specialized subcellular domains to which different sets of ion channels are localized. Here, we describe the identification of Caspr2, a mammalian homolog of Drosophila Neurexin IV (Nrx-IV), and show that this neurexin-like protein and the closely related molecule Caspr/Paranodin demarcate distinct subdomains in myelinated axons. While contactin-associated protein (Caspr) is present at the paranodal junctions, Caspr2 is precisely colocalized with Shaker-like K+ channels in the juxtaparanodal region. We further show that Caspr2 specifically associates with Kv1.1, Kv1.2, and their Kvbeta2 subunit. This association involves the C-terminal sequence of Caspr2, which contains a putative PDZ binding site. These results suggest a role for Caspr family members in the local differentiation of the axon into distinct functional subdomains.     

The Nodes of Ranvier: Molecular Assembly and Maintenance

Action potential (AP) propagation in myelinated nerves requires clustered voltage gated sodium and potassium channels. These channels must be specifically localized to nodes of Ranvier where the AP is regenerated. Several mechanisms have evolved to facilitate and ensure the correct assembly and stabilization of these essential axonal domains. This review highlights the current understanding of the axon intrinsic and glial extrinsic mechanisms that control the formation and maintenance of the nodes of Ranvier in both the peripheral nervous system (PNS) and central nervous system (CNS).Axons conduct electrical signals, called action potentials (APs), among neurons in a circuit in response to sensory input, and between motor neurons and muscles. In mammals and other vertebrates, many axons are myelinated. Myelin, made by Schwann cells and oligodendrocytes in the peripheral nervous system (PNS) and central nervous system (CNS), respectively, is a multilamellar sheet of glial membrane that wraps around axons to increase transmembrane resistance and decrease membrane capacitance. Although myelin is traditionally viewed as a passive contributor to nervous system function, it is now recognized that myelinating glia also play many active roles including regulation of axon diameter, axonal energy metabolism, and the clustering of ion channels at gaps in the myelin sheath called nodes of Ranvier. Together, the active and passive properties conferred on axons by myelin, result in axons with high AP conduction velocities, low metabolic demands, and reduced space requirements as compared with unmyelinated axons. Thus, myelin and the clustering of ion channels in axons permitted the evolution of the complex nervous systems found in vertebrates. This review highlights the current understanding of the axonal intrinsic and glial extrinsic mechanisms that control the formation and maintenance of the nodes of Ranvier in both the PNS and CNS.     

Cellular contacts in myelinated fibers of the peripheral nervous system

Myelination allows the fast propagation of action potentials at a low energetic cost. It provides an insulating myelin sheath regularly interrupted at nodes of Ranvier where voltage-gated Na+ channels are concentrated. In the peripheral nervous system, the normal function of myelinated fibers requires the formation of highly differentiated and organized contacts between the myelinating Schwann cells, the axons and the extracellular matrix. Some of the major molecular complexes that underlie these contacts have been identified. Compact myelin which forms the bulk of the myelin sheath results from the fusion of the Schwann cell membranes through the proteins P0, PMP22 and MBP. The basal lamina of myelinating Schwann cells contains laminin-2 which associates with the glial complex dystroglycan/DPR2/L-periaxin. Non compact myelin, found in paranodal loops, periaxonal and abaxonal regions, and Schmidt-Lanterman incisures, presents reflexive adherens junctions, tight junctions and gap junctions, which contain cadherins, claudins and connexins, respectively. Axo-glial contacts determine the formation of distinct domains on the axon, the node, the paranode, and the juxtaparanode. At the paranodes, the glial membrane is tightly attached to the axolemma by septate-like junctions. Paranodal and juxtaparanodal axoglial complexes comprise an axonal transmembrane protein of the NCP family associated in cis and in trans with cell adhesion molecules of the immunoglobulin superfamily (IgSF-CAM). At nodes, axonal complexes are composed of Na+ channels and IgSF-CAMs. Schwann cell microvilli, which loosely cover the node, contain ERM proteins and the proteoglycans syndecan-3 and -4. The fundamental role of the cellular contacts in the normal function of myelinated fibers has been supported by rodent models and the detection of genetic alterations in patients with peripheral demyelinating neuropathies such as Charcot-Marie-Tooth diseases. Understanding more precisely their molecular basis now appears essential as a requisite step to further examine their involvement in the pathogenesis of peripheral neuropathies in general.     

Developmental clustering of ion channels at and near the node of Ranvier.

Voltage-gated Na(+) and K(+) channels are localized to distinct subcellular domains in mammalian myelinated nerve fibers. Specifically, Na(+) channels are clustered in high densities at nodes of Ranvier, while K(+) channels are found in juxtaparanodal zones just beyond regions of axoglial contact where sequential layers of the myelin sheath terminate. Specific targeting, clustering, and maintenance of these channels in their respective domains are essential to achieve high conduction velocities of action potential propagation. The cellular, molecular, and developmental mechanisms that exist to achieve this neuronal specialization are discussed and reviewed. Current evidence points to a prominent role in channel clustering played by myelinating glial cells, and sites of axoglial contact in particular.     

Nr-CAM and neurofascin interactions regulate ankyrin G and sodium channel clustering at the node of Ranvier.

Voltage-dependent sodium (Na(+)) channels are highly concentrated at nodes of Ranvier in myelinated axons and play a key role in promoting rapid and efficient conduction of action potentials by saltatory conduction. The molecular mechanisms that direct their localization to the node are not well understood but are believed to involve contact-dependent signals from myelinating Schwann cells and interactions of Na(+) channels with the cytoskeletal protein, ankyrin G. Two cell adhesion molecules (CAMs) expressed at the axon surface, Nr-CAM and neurofascin, are also linked to ankyrin G and accumulate at early stages of node formation, suggesting that they mediate contact-dependent Schwann cell signals to initiate node development. To examine the potential role of Nr-CAM in this process, we treated myelinating cocultures of DRG (dorsal root ganglion) neurons and Schwann cells with an Nr-CAM-Fc (Nr-Fc) fusion protein. Nr-Fc had no effect on initial axon-Schwann cell interactions, including Schwann cell proliferation, or on the extent of myelination, but it strikingly and specifically inhibited Na(+) channel and ankyrin G accumulation at the node. Nr-Fc bound directly to neurons and clustered and coprecipitated neurofascin expressed on axons. These results provide the first evidence that neurofascin plays a major role in the formation of nodes, possibly via interactions with Nr-CAM.     

Glial and neuronal isoforms of Neurofascin have distinct roles in the assembly of nodes of Ranvier in the central nervous system

Rapid nerve impulse conduction in myelinated axons requires the concentration of voltage-gated sodium channels at nodes of Ranvier. Myelin-forming oligodendrocytes in the central nervous system (CNS) induce the clustering of sodium channels into nodal complexes flanked by paranodal axoglial junctions. However, the molecular mechanisms for nodal complex assembly in the CNS are unknown. Two isoforms of Neurofascin, neuronal Nfasc186 and glial Nfasc155, are components of the nodal and paranodal complexes, respectively. Neurofascin-null mice have disrupted nodal and paranodal complexes. We show that transgenic Nfasc186 can rescue the nodal complex when expressed in Nfasc(-/-) mice in the absence of the Nfasc155-Caspr-Contactin adhesion complex. Reconstitution of the axoglial adhesion complex by expressing transgenic Nfasc155 in oligodendrocytes also rescues the nodal complex independently of Nfasc186. Furthermore, the Nfasc155 adhesion complex has an additional function in promoting the migration of myelinating processes along CNS axons. We propose that glial and neuronal Neurofascins have distinct functions in the assembly of the CNS node of Ranvier.     

Gliomedin mediates Schwann cell-axon interaction and the molecular assembly of the nodes of Ranvier

Accumulation of Na(+) channels at the nodes of Ranvier is a prerequisite for saltatory conduction. In peripheral nerves, clustering of these channels along the axolemma is regulated by myelinating Schwann cells through a yet unknown mechanism. We report the identification of gliomedin, a glial ligand for neurofascin and NrCAM, two axonal immunoglobulin cell adhesion molecules that are associated with Na+ channels at the nodes of Ranvier. Gliomedin is expressed by myelinating Schwann cells and accumulates at the edges of each myelin segment during development, where it aligns with the forming nodes. Eliminating the expression of gliomedin by RNAi, or the addition of a soluble extracellular domain of neurofascin to myelinating cultures, which caused the redistribution of gliomedin along the internodes, abolished node formation. Furthermore, a soluble gliomedin induced nodal-like clusters of Na+ channels in the absence of Schwann cells. We propose that gliomedin provides a glial cue for the formation of peripheral nodes of Ranvier.     

Clustering of neuronal sodium channels requires contact with myelinating Schwann cells

Efficient and rapid conduction of action potentials by saltatory conduction requires the clustering of voltage-gated sodium channels at nodes of Ranvier. This clustering results from interactions between neurons and myelinating glia, although it has not been established whether this glial signal is contact-dependent or soluble. To investigate the nature of this signal, we examined sodium channel clustering in co-cultures of embryonic rat dorsal root ganglion neurons and Schwann cells. Cultures maintained under conditions promoting or preventing myelination were immunostained with antibodies against the α subunit of the sodium channel and against ankyrin_G, a cytoskeletal protein associated with these channels. Consistent with previous in vivo studies (Vabnick et al., 1996), sodium channels and ankyrin G cluster at the onset of myelination. These clusters form adjacent to the ends of the myelinating Schwann cells and appear to fuse to form mature nodes. In contrast, sodium channels and ankyrin G do not cluster in neurons grown alone or in co-cultures where myelination is precluded by growing cells in defined media. Conditioned media from myelinating co-cultures also failed to induce sodium channel or ankyrin G clusters in cultures of neurons alone. Finally, no clusters develop in the amyelinated portions of suspended fascicles of dorsal root ganglia explants despite being in close proximity to myelinated segments in other areas of the dish. These results indicate that clustering of sodium channels requires contact with myelinating Schwann cells.     

Polarized domains of myelinated axons

The entire length of myelinated axons is organized into a series of polarized domains that center around nodes of Ranvier. These domains, which are crucial for normal saltatory conduction, consist of distinct multiprotein complexes of cell adhesion molecules, ion channels, and scaffolding molecules; they also differ in their diameter, organelle content, and rates of axonal transport. Juxtacrine signals from myelinating glia direct their sequential assembly. The composition, mechanisms of assembly, and function of these molecular domains will be reviewed. I also discuss similarities of this domain organization to that of polarized epithelia and present emerging evidence that disorders of domain organization and function contribute to the axonopathies of myelin and other neurologic disorders.     

Molecular organization of axo-glial junctions

Axo-glial interactions are required for the organization of highly specialized molecular domains in myelinated axons. The molecular composition of these domains includes cell adhesion molecules, ion channels and cytoskeletal proteins. Recent genetic and molecular studies provide new insights into how these macromolecular complexes are assembled and organized into functional domains, and how the loss of individual components affects domain organization and function. More importantly, the key molecular components identified at the vertebrate axo-glial septate junctions are also present at the Drosophila septate junctions. In addition, new roles for axo-glial paranodal septate junctions have emerged, which suggest that the paranodal region may act as an ionic barrier and a molecular fence in myelinated axons.     

Compact myelin dictates the differential targeting of two sodium channel isoforms in the same axon

Voltage-dependent sodium channels are uniformly distributed along unmyelinated axons, but are highly concentrated at nodes of Ranvier in myelinated axons. Here, we show that this pattern is associated with differential localization of distinct sodium channel alpha subunits to the unmyelinated and myelinated zones of the same retinal ganglion cell axons. In adult axons, Na(v)1.2 is localized to the unmyelinated zone, whereas Na(v)1.6 is specifically targeted to nodes. During development, Na(v)1.2 is expressed first and becomes clustered at immature nodes of Ranvier, but as myelination proceeds, Na(v)1.6 replaces Na(v)1.2 at nodes. In Shiverer mice, which lack compact myelin, Na(v)1.2 is found throughout adult axons, whereas little Na(v)1.6 is detected. Together, these data show that sodium channel isoforms are differentially targeted to distinct domains of the same axon in a process associated with formation of compact myelin.     

Development of nodes of Ranvier

The architecture and function of the nodes of Ranvier depend on several specialized cell contacts between the axon and myelinating glial cells. These sites contain highly organized multimolecular complexes of ion channels and cell adhesion molecules, closely connected with the cytoskeleton. Recent findings are beginning to reveal how this organization is achieved during the development of myelinated nerves. The role of membrane proteins involved in axoglial interactions and of associated cytoplasmic molecules is being elucidated, while studies of mutant mice have underlined the importance of glial cells and the specific role of axonal proteins in the organization of axonal domains.     

Localization of Phospholipid Synthesis to Schwann Cells and Axons

Quantitative electron microscopic autoradiography was used to detect and characterize endoneurial sites of lipid synthesis in mouse sciatic nerve. Six tritiated phospholipid precursors (choline, serine, methionine, inositol, glycerol, and ethanolamine) and a protein precursor (proline) were individually injected into exposed nerves and after 2 h the mice were perfused with buffered aldehyde. The labeled segments of nerve were prepared for autoradiography with procedures that selectively remove nonincorporated precursors and other aqueous metabolites, while preserving nerve lipids (and proteins). At both the light and electron microscope levels, the major site of phospholipid and protein synthesis was the crescent-shaped perinuclear cytoplasm of myelinating Schwann cells. Other internodal Schwann cell cytoplasm, including that in surface channels, Schmidt-Lanterman incisures, and paranodal regions, was less well labeled than the perinuclear region. Newly formed proteins were selectively located in the Schwann cell nucleus. Lipid and protein formation was also detected in unmyelinated fiber bundles and in endoneurial and perineurial cells. Tritiated inositol was selectively incorporated into phospholipids in both myelinated axons and unmyelinated fibers. Like inositol, glycerol incorporation appeared particularly active in unmyelinated fibers. Quantitative autoradiographic analyses substantiated the following points: myelinating Schwann cells dominate phospholipid and protein synthesis, myelinated axons selectively incorporate tritiated inositol, phospholipid precursors label myelin sheaths and myelinated axons better than proline.     

Optical measurement of conduction in single demyelinated axons

Demyelination was initiated in Xenopus sciatic nerves by an intraneural injection of lysolecithin over a 2-3-mm region. During the next week macrophages and Schwann cells removed all remaining damaged myelin by phagocytosis. Proliferating Schwann cells then began to remyelinate the axons, with the first few lamellae appearing 13 d after surgery. Action potentials were recorded optically through the use of a potential-sensitive dye. Signals could be detected both at normal nodes of Ranvier and within demyelinated segments. Before remyelination, conduction through the lesion occurred in only a small fraction of the fibers. However, in these particular cases we could demonstrate continuous (nonsaltatory) conduction at very low velocities over long (greater than one internode) lengths of demyelinated axons. We have previously found through loose patch clamp experiments that the internodal axolemma contains voltage-dependent Na+ channels at a density approximately 4% of that at the nodes. These channels alone, however, are insufficient for successful conduction past the transition point between myelinated and demyelinated regions. Small improvements in the passive cable properties of the axon, adequate for propagation at this site, can be realized through the close apposition of macrophages and Schwann cells. As the initial lamellae of myelin appear, the probability of success at the transition zone increases rapidly, though the conduction velocity through the demyelinated segment is not appreciably changed. A detailed computational model is used to test the relative roles of the internodal Na+ channels and the new extracellular layer. The results suggest a possible mechanism that may contribute to the spontaneous recovery of function often seen in demyelinating disease.     

Structure-function analysis of protein complexes involved in the molecular architecture of juxtaparanodal regions of myelinated fibers

Demyelinating disorders, including multiple sclerosis (MS), are common causes of neurological disability. One critical step towards the management and therapy of demyelinating diseases is to understand the basic functions of myelinating glia and their relationship with axons. Axons and myelinating glia, oligodendrocytes in the central (CNS) and Schwann cells in the peripheral (PNS) nervous systems, reciprocally influence each other's development and trophism. These interactions are critical for the formation of distinct axonal domains in myelinated fibers that ensure the rapid propagation of action potentials. Macromolecular complexes mediating axo-glial interactions in these domains have been identified, consisting of members of the immunoglobulin superfamily (IgSF) of adhesion molecules and the neurexin/NCP superfamily as well as other proteins. We have investigated the molecular details of axo-glial interactions in the juxtaparanodal region of myelinated fibers by utilizing domain-specific GFP constructs and immunoprecipitation assays on transfected cells. We have shown that the immunoglobulin domains of the IgSF member TAG-1/Cnt-2 are necessary and sufficient for the direct, cis interaction of this protein with Caspr2 and potassium channels.     

Transmembrane scaffolding proteins in the formation and stability of nodes of Ranvier

The function of myelinated fibers depends on the clustering of sodium channels at nodes of Ranvier, the integrity of the myelin sheath, and the existence of tight axoglial junctions at paranodes, on either sides of the nodes. While the ultrastructure of these regions has been known for several decades, recent progress has been accomplished in the identification of proteins essential for their organization, which depends on the interplay between axons and myelinating glial cells. Evolutionary conserved intercellular multimolecular complexes comprising proteins of the Neurexin IV/Caspr/paranodin (NCP) family and of the immunoglobulin-like cell adhesion molecules superfamily, are essential components for the axoglial contacts at the level of paranodes and juxtaparanodes. These complexes are able to interact with cytoplasmic proteins of the band 4.1 family, providing possible links to the axonal cytoskeleton. While the identification of these proteins represents a significant progress for understanding axoglial contacts, they also raise exciting questions concerning the molecular organization of these contacts and the mechanisms of their local enrichment.     

The “Lillie Transition”: models of the onset of saltatory conduction in myelinating axons

Almost 90 years ago, Lillie reported that rapid saltatory conduction arose in an iron wire model of nerve impulse propagation when he covered the wire with insulating sections of glass tubing equivalent to myelinated internodes. This led to his suggestion of a similar mechanism explaining rapid conduction in myelinated nerve. In both their evolution and their development, myelinating axons must make a similar transition between continuous and saltatory conduction. Achieving a smooth transition is a potential challenge that we examined in computer models simulating a segmented insulating sheath surrounding an axon having Hodgkin-Huxley squid parameters. With a wide gap under the sheath, conduction was continuous. As the gap was reduced, conduction initially slowed, owing to the increased extra-axonal resistance, then increased (the “rise”) up to several times that of the unmyelinated fiber, as saltatory conduction set in. The conduction velocity slowdown was little affected by the number of myelin layers or modest changes in the size of the “node,” but strongly affected by the size of the “internode” and axon diameter. The steepness of the rise of rapid conduction was greatly affected by the number of myelin layers and axon diameter, variably affected by internode length and little affected by node length. The transition to saltatory conduction occurred at surprisingly wide gaps and the improvement in conduction speed persisted to surprisingly small gaps. The study demonstrates that the specialized paranodal seals between myelin and axon, and indeed even the clustering of sodium channels at the nodes, are not necessary for saltatory conduction.