Sebastian Poliak

The local differentiation of myelinated axons at nodes of Ranvier

Efficient and rapid propagation of action potentials in myelinated axons depends on the molecular specialization of the nodes of Ranvier. The nodal region is organized into several distinct domains, each of which contains a unique set of ion channels, cell-adhesion molecules and cytoplasmic adaptor proteins. Voltage-gated Na+ channels — which are concentrated at the nodes — are separated from K+ channels — which are clustered at the juxtaparanodal region — by a specialized axoglial contact that is formed between the axon and the myelinating cell at the paranodes. This local differentiation of myelinated axons is tightly regulated by oligodendrocytes and myelinating Schwann cells, and is achieved through complex mechanisms that are used by another specialized cell–cell contact — the synapse.

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.

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.

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.

Distinct claudins and associated PDZ proteins form different autotypic tight junctions in myelinating Schwann cells.

The apposed membranes of myelinating Schwann cells are joined by several types of junctional specializations known as autotypic or reflexive junctions. These include tight, gap, and adherens junctions, all of which are found in regions of noncompact myelin: the paranodal loops, incisures of Schmidt-Lanterman, and mesaxons. The molecular components of autotypic tight junctions have not been established. Here we report that two homologues of Discs Lost–multi PDZ domain protein (MUPP)1, and Pals-associated tight junction protein (PATJ), are differentially localized in myelinating Schwann cells and associated with different claudins. PATJ is mainly found at the paranodal loops, where it colocalized with claudin-1. MUPP1 and claudin-5 colocalized in the incisures, and the COOH-terminal region of claudin-5 interacts with MUPP1 in a PSD-95/Disc Large/zona occludens (ZO)-1 (PDZ)-dependent manner. In developing nerves, claudin-5 and MUPP1 appear together in incisures during the first postnatal week, suggesting that they coassemble during myelination. Finally, we show that the incisures also contain four other PDZ proteins that are found in epithelial tight junctions, including three membrane-associated guanylate-kinase proteins (membrane-associated guanylate-kinase inverted-2, ZO-1, and ZO-2) and the adaptor protein Par-3. The presence of these different tight junction proteins in regions of noncompact myelin may be required to maintain the intricate cytoarchitecture of myelinating Schwann cells.

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.

Multiple Molecular Interactions Determine the Clustering of Caspr2 and Kv1 Channels in Myelinated Axons

Clustering of Kv1 channels at the juxtaparanodal region (JXP) in myelinated axons depends on their association with the Caspr2/TAG-1 adhesion complex. The interaction between these channels and Caspr2 was suggested to depend on PDZ (PSD-95/Discs large/zona occludens-1) scaffolding proteins. Here, we show that at a subset of the JXP, PSD-93 colocalizes with Caspr2, K+ channels and its related protein postsynaptic density protein-95 (PSD-95). The localization of PSD-93 and PSD-95 depends on the presence of Caspr2, as both scaffolding proteins failed to accumulate at the JXP in mice lacking either Caspr2 or TAG-1. In contrast, Caspr2 and K+ channels still colocalized and associated in PSD-93, PSD-95 or double PSD-93/PSD-95 null mice. To directly evaluate the role of PDZ domain proteins in the function of Caspr2, we examined the ability of transgenic Caspr2 molecules lacking either their cytoplasmic domain (Caspr2dCT), or their PDZ-binding sequence (Caspr2dPDZ), to restore Kv1 channel clustering in Caspr2 null mice. We found that while Kv1 channels were distributed throughout internodes in nerves expressing Caspr2dCT, they were clustered at the JXP in axons expressing a full-length Caspr2 (Caspr2FL) or the Caspr2dPDZ transgene. Further proteomic analysis revealed that Caspr2 interacts with a distinct set of scaffolding proteins through its PDZ- and protein 4.1-binding sequences. These results demonstrate that while the molecular assembly of the JXP requires the cytoplasmic domain of Caspr2, its carboxy-terminal PDZ-binding motif is dispensable for Kv1 channel clustering. This mechanism is clearly distinct from the one operating at the axon initial segment, which requires PSD-93 for Kv1 channel clustering.

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.

Internodal specializations of myelinated axons in the central nervous system

Abstract. We have examined the localization of contactin-associated protein (Caspr), the Shaker-type potassium channels, Kv1.1 and Kv1.2, their associated β subunit, Kvβ2, and Caspr2 in the myelinated fibers of the CNS. Caspr is localized to the paranodal axonal membrane, and Kv1.1, Kv1.2, Kvβ2 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, Kvβ2, 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/Kvβ2/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.