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.     

Internodal specializations of myelinated axons in the central nervous system

We have examined the localization of contactin-associated protein (Caspr), the Shaker-type potassium channels, Kv1.1 and Kv1.2, their associated beta subunit, Kvbeta2, and Caspr2 in the myelinated fibers of the CNS. Caspr is localized to the paranodal axonal membrane, and Kv1.1, Kv1.2, Kvbeta2 and Caspr2 to the juxtaparanodal membrane. In addition to the paranodal staining, an internodal strand of Caspr staining apposes the inner mesaxon of the myelin sheath. Unlike myelinated axons in the peripheral nervous system, there was no internodal strand of Kv1.1, Kv1.2, Kvbeta2, or Caspr2. Thus, the organization of the nodal, paranodal, and juxtaparanodal axonal membrane is similar in the central and peripheral nervous systems, but the lack of Kv1.1/Kv1.2/Kvbeta2/Caspr2 internodal strands indicates that the oligodendrocyte myelin sheaths lack a trans molecular interaction with axons, an interaction that is present in Schwann cell myelin sheaths.     

Protein 4.1B contributes to the organization of peripheral myelinated axons

Neurons are characterized by extremely long axons. This exceptional cell shape is likely to depend on multiple factors including interactions between the cytoskeleton and membrane proteins. In many cell types, members of the protein 4.1 family play an important role in tethering the cortical actin-spectrin cytoskeleton to the plasma membrane. Protein 4.1B is localized in myelinated axons, enriched in paranodal and juxtaparanodal regions, and also all along the internodes, but not at nodes of Ranvier where are localized the voltage-dependent sodium channels responsible for action potential propagation. To shed light on the role of protein 4.1B in the general organization of myelinated peripheral axons, we studied 4.1B knockout mice. These mice displayed a mildly impaired gait and motility. Whereas nodes were unaffected, the distribution of Caspr/paranodin, which anchors 4.1B to the membrane, was disorganized in paranodal regions and its levels were decreased. In juxtaparanodes, the enrichment of Caspr2, which also interacts with 4.1B, and of the associated TAG-1 and Kv1.1, was absent in mutant mice, whereas their levels were unaltered. Ultrastructural abnormalities were observed both at paranodes and juxtaparanodes. Axon calibers were slightly diminished in phrenic nerves and preterminal motor axons were dysmorphic in skeletal muscle. βII spectrin enrichment was decreased along the axolemma. Electrophysiological recordings at 3 post-natal weeks showed the occurrence of spontaneous and evoked repetitive activity indicating neuronal hyperexcitability, without change in conduction velocity. Thus, our results show that in myelinated axons 4.1B contributes to the stabilization of membrane proteins at paranodes, to the clustering of juxtaparanodal proteins, and to the regulation of the internodal axon caliber.     

Clustering and activity tuning of Kv1 channels in myelinated hippocampal axons

Precise localization of axonal ion channels is crucial for proper electrical and chemical functions of axons. In myelinated axons, Kv1 (Shaker) voltage-gated potassium (Kv) channels are clustered in the juxtaparanodal regions flanking the node of Ranvier. The clustering can be disrupted by deletion of various proteins in mice, including contactin-associated protein-like 2 (Caspr2) and transient axonal glycoprotein-1 (TAG-1), a glycosylphosphatidylinositol-anchored cell adhesion molecule. However, the mechanism and function of Kv1 juxtaparanodal clustering remain unclear. Here, using a new myelin coculture of hippocampal neurons and oligodendrocytes, we report that tyrosine phosphorylation plays a critical role in TAG-1-mediated clustering of axonal Kv1.2 channels. In the coculture, myelin specifically ensheathed axons but not dendrites of hippocampal neurons and clustered endogenous axonal Kv1.2 into internodes. The trans-homophilic interaction of TAG-1 was sufficient to position Kv1.2 clusters on axonal membranes in a neuron/HEK293 coculture. Mutating a tyrosine residue (Tyr⁴⁵⁸) in the Kv1.2 C terminus or blocking tyrosine phosphorylation disrupted myelin- and TAG-1-mediated clustering of axonal Kv1.2. Furthermore, Kv1.2 voltage dependence and activation threshold were reduced by TAG-1 coexpression. This effect was eliminated by the Tyr⁴⁵⁸ mutation or by cholesterol depletion. Taken together, our studies suggest that myelin regulates both trafficking and activity of Kv1 channels along hippocampal axons through TAG-1.     

Molecular domains of myelinated axons

Myelinated axons are organized into specific domains as the result of interactions with glial cells. Recently, distinct protein complexes of cell adhesion molecules, Na(+) channels and ankyrin G at the nodes, Caspr and contactin in the paranodes, and K(+) channels and Caspr2 in the juxtaparanodal region have been identified, and new insights into the role of the paranodal junctions in the organization of these domains have emerged.     

Myelinating Schwann cells determine the internodal localization of Kv1.1, Kv1.2, Kvβ2, and Caspr

We examined the localization of Caspr and the K⁺ channels Kv1.1 and Kv1.2, all of which are intrinsic membrane proteins of myelinated axons in the PNS. Caspr is localized to the paranode; Kv1.1, Kv1.2 and their β2 subunit are localized to the juxtaparanode. Throughout the internodal region, a strand of Caspr staining is flanked by a double strand of Kv1.1/Kv1.2/Kvβ2 staining. This tripartite strand apposes the inner mesaxon of the myelin sheath, and forms a circumferential ring that apposes the innermost aspect of Schmidt-Lanterman incisures. The localization of Caspr and Kv1.2 are not disrupted in mice with null mutations of the myelin associated glycoprotein, connexin32, or Kv1.1 genes. At all of these locations, Caspr and Kv1.1/Kv1.2/Kvβ2 define distinct but interrelated domains of the axonal membrane that appear to be organized by the myelin sheath.     

On the molecular architecture of myelinated fibers

Schwann cells and oligodendrocytes make the myelin sheaths of the PNS and CNS, respectively. Their myelin sheaths are structurally similar, consisting of multiple layers of specialized cell membrane that spiral around axons, but there are several differences. (1) CNS myelin has a ”radial component” composed of a tight junction protein, claudin-11/oligodendrocyte-specific protein. (2) Schwann cells have a basal lamina and microvilli. (3) Although both CNS and PNS myelin sheaths have incisures, those in the CNS lack the structural as well as the molecular components of ”reflexive” adherens junctions and gap junctions. In spite of their structural differences, the axonal membranes of the PNS and CNS are similarly organized. The nodal axolemma contains high concentrations of voltage-dependent sodium channels that are linked to the axonal cytoskeleton by ankyrin_G. The paranodal membrane contains Caspr/paranodin, which may participate in the formation of axoglial junctions. The juxtaparanodal axonal membrane contains the potassium channels Kv1.1 and Kv1.2, their associated β2 subunit, as well as Caspr2, which is closely related to Caspr. The myelin sheath probably organizes these axonal membrane-related proteins via trans interactions. Accepted: 25 November 1999     

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.     

Contactin orchestrates assembly of the septate-like junctions at the paranode in myelinated peripheral nerve

Rapid nerve impulse conduction depends on specialized membrane domains in myelinated nerve, the node of Ranvier, the paranode, and the myelinated internodal region. We report that GPI-linked contactin enables the formation of the paranodal septate-like axo-glial junctions in myelinated peripheral nerve. Contactin clusters at the paranodal axolemma during Schwann cell myelination. Ablation of contactin in mutant mice disrupts junctional attachment at the paranode and reduces nerve conduction velocity 3-fold. The mutation impedes intracellular transport and surface expression of Caspr and leaves NF155 on apposing paranodal myelin disengaged. The contactin mutation does not affect sodium channel clustering at the nodes of Ranvier but alters the location of the Shaker-type Kv1.1 and Kv1.2 potassium channels. Thus, contactin is a crucial part in the machinery that controls junctional attachment at the paranode and ultimately the physiology of myelinated nerve.     

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.     

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.     

ELECTRON MICROSCOPE STUDIES OF THE FORMATION OF NODES OF RANVIER IN MOUSE SCIATIC NERVES

Observations with the electron microscope of longitudinal sections of the sciatic nerves of infant mice during the period of early myelin formation are described. These observations are interpreted in relation to previous studies of transverse sections, and a general picture of the formation of an internodal length of the myelin sheath in three dimensions is formulated. In general, an internodal length of myelin sheath is attained by the spiral wrapping of the infolded Schwann cell surface; the increase in length of the internode during maturation is at least partially explained by the increased length of axon covered by the overlapping of successive layers during the wrapping of the infolded Schwann cell surface; and the nodes of Ranvier refer to the structure complex at the junctions of adjacent non-syncytial Schwann cells. The fact that the mode of formation of myelin brings each of its layers into intimate contact with the axon surface at the nodes is emphasized because of the possible functional significance of this arrangement. The manner of origin of Schmidt-Lantermann clefts remains obscure. Certain isolated observations provide evidence for the possibility that occasional internodes of myelin may form from several small segments of myelin within a single Schwann cell.     

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.     

Autophagy aids membrane expansion by neuropathic Schwann cells

Demyelinating peripheral neuropathies associated with abnormal expression of peripheral myelin protein 22 (PMP22) involve the formation of cytosolic protein aggregates within Schwann cells. Towards developing a therapy for these progressive neurodegenerative diseases, we assessed whether pharmacological activation of autophagy by rapamycin (RM) could prevent protein aggregation and enhance Schwann cell myelination. Indeed, we found that glial cells from neuropathic mice activate autophagy in response to RM and produce abundant myelin internodes. Lentivirus-mediated shRNA shutdown of Atg12 abrogates the improvements in myelin production, demonstrating that autophagy is critical for the observed benefits.     

The Axonal Membrane Protein Caspr, a Homologue of Neurexin IV, Is a Component of the Septate-like Paranodal Junctions That Assemble during Myelination

We have investigated the potential role of contactin and contactin-associated protein (Caspr) in the axonal–glial interactions of myelination. In the nervous system, contactin is expressed by neurons, oligodendrocytes, and their progenitors, but not by Schwann cells. Expression of Caspr, a homologue of Neurexin IV, is restricted to neurons. Both contactin and Caspr are uniformly expressed at high levels on the surface of unensheathed neurites and are downregulated during myelination in vitro and in vivo. Contactin is downregulated along the entire myelinated nerve fiber. In contrast, Caspr expression initially remains elevated along segments of neurites associated with nascent myelin sheaths. With further maturation, Caspr is downregulated in the internode and becomes strikingly concentrated in the paranodal regions of the axon, suggesting that it redistributes from the internode to these sites. Caspr expression is similarly restricted to the paranodes of mature myelinated axons in the peripheral and central nervous systems; it is more diffusely and persistently expressed in gray matter and on unmyelinated axons. Immunoelectron microscopy demonstrated that Caspr is localized to the septate-like junctions that form between axons and the paranodal loops of myelinating cells. Caspr is poorly extracted by nonionic detergents, suggesting that it is associated with the axon cytoskeleton at these junctions. These results indicate that contactin and Caspr function independently during myelination and that their expression is regulated by glial ensheathment. They strongly implicate Caspr as a major transmembrane component of the paranodal junctions, whose molecular composition has previously been unknown, and suggest its role in the reciprocal signaling between axons and glia.     

K+ channel distribution and clustering in developing and hypomyelinated axons of the optic nerve

The localization of Shaker-type K⁺ channels in specialized domains of myelinated central nervous system axons was studied during development of the optic nerve. In adult rats Kv1.1, Kv1.2, Kv1.6, and the cytoplasmic β-subunit Kvβ2 were colocalized in juxtaparanodal zones. During development, clustering of K⁺ channels lagged behind that for nodal Na⁺ channels by about 5 days. In contrast to the PNS, K⁺ channels were initially expressed fully segregated from nodes and paranodes, the latter identified by immunofluorescence of Caspr, a component of axoglial junctions. Clusters of K⁺ channels were first detected at postnatal day 14 (P14) at a limited number of sites. Expression increased until all juxtaparanodes had immunoreactivity by P40. Developmental studies in hypomyelinating Shiverer mice revealed dramatically disrupted axoglial junctions, aberrant Na⁺ channel clusters, and little or no detectable clustering of K⁺ channels at all ages. These results suggest that in the optic nerve, compact myelin and normal axoglial junctions are essential for proper K⁺ channel clustering and localization.     

Clustering of neuronal potassium channels is independent of their interaction with PSD-95

Voltage-dependent potassium channels regulate membrane excitability and cell-cell communication in the mammalian nervous system, and are found highly localized at distinct neuronal subcellular sites. Kv1 (mammalian Shaker family) potassium channels and the neurexin Caspr2, both of which contain COOH-terminal PDZ domain binding peptide motifs, are found colocalized at high density at juxtaparanodes flanking nodes of Ranvier of myelinated axons. The PDZ domain-containing protein PSD-95, which clusters Kv1 potassium channels in heterologous cells, has been proposed to play a major role in potassium channel clustering in mammalian neurons. Here, we show that PSD-95 colocalizes precisely with Kv1 potassium channels and Caspr2 at juxtaparanodes, and that a macromolecular complex of Kv1 channels and PSD-95 can be immunopurified from mammalian brain and spinal cord. Surprisingly, we find that the high density clustering of Kv1 channels and Caspr2 at juxtaparanodes is normal in a mutant mouse lacking juxtaparanodal PSD-95, and that the indirect interaction between Kv1 channels and Caspr2 is maintained in these mutant mice. These data suggest that the primary function of PSD-95 at juxtaparanodes lies outside of its accepted role in mediating the high density clustering of Kv1 potassium channels at these sites.     

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.     

Association of TAG-1 with Caspr2 is essential for the molecular organization of juxtaparanodal regions of myelinated fibers

Myelination results in a highly segregated distribution of axonal membrane proteins at nodes of Ranvier. Here, we show the role in this process of TAG-1, a glycosyl-phosphatidyl-inositol-anchored cell adhesion molecule. In the absence of TAG-1, axonal Caspr2 did not accumulate at juxtaparanodes, and the normal enrichment of shaker-type K+ channels in these regions was severely disrupted, in the central and peripheral nervous systems. In contrast, the localization of protein 4.1B, an axoplasmic partner of Caspr2, was only moderately altered. TAG-1, which is expressed in both neurons and glia, was able to associate in cis with Caspr2 and in trans with itself. Thus, a tripartite intercellular protein complex, comprised of these two proteins, appears critical for axo-glial contacts at juxtaparanodes. This complex is analogous to that described previously at paranodes, suggesting that similar molecules are crucial for different types of axo-glial interactions.     

Retention of a cell adhesion complex at the paranodal junction requires the cytoplasmic region of Caspr

An axonal complex of cell adhesion molecules consisting of Caspr and contactin has been found to be essential for the generation of the paranodal axo-glial junctions flanking the nodes of Ranvier. Here we report that although the extracellular region of Caspr was sufficient for directing it to the paranodes in transgenic mice, retention of the Caspr-contactin complex at the junction depended on the presence of an intact cytoplasmic domain of Caspr. Using immunoelectron microscopy, we found that a Caspr mutant lacking its intracellular domain was often found within the axon instead of the junctional axolemma. We further show that a short sequence in the cytoplasmic domain of Caspr mediated its binding to the cytoskeleton-associated protein 4.1B. Clustering of contactin on the cell surface induced coclustering of Caspr and immobilized protein 4.1B at the plasma membrane. Furthermore, deletion of the protein 4.1B binding site accelerated the internalization of a Caspr-contactin chimera from the cell surface. These results suggest that Caspr serves as a "transmembrane scaffold" that stabilizes the Caspr/contactin adhesion complex at the paranodal junction by connecting it to cytoskeletal components within the axon.