Faculty Bibliography

The node of Ranvier is a distinct domain of myelinated axons that is highly enriched in sodium channels and is critical for impulse propagation. During development, the channel subtypes expressed at the node undergo a transition from Nav1.2 to Nav1.6. Specialized junctions that form between the paranodal glial membranes and axon flank the nodes and are candidates to regulate their maturation and delineate their boundaries. To investigate these roles, we characterized node development in mice deficient in contactin-associated protein (Caspr), an integral junctional component. Paranodes in these mice lack transverse bands, a hallmark of the mature junction, and exhibit progressive disruption of axon-paranodal loop interactions in the CNS. Caspr mutant mice display significant abnormalities at central nodes; components of the nodes progressively disperse along axons, and many nodes fail to mature properly, persistently expressing Nav1.2 rather than Nav1.6. In contrast, PNS nodes are only modestly longer and, although maturation is delayed, eventually all express Nav1.6. Potassium channels are aberrantly clustered in the paranodes; these clusters are lost over time in the CNS, whereas they persist in the PNS. These findings indicate that interactions of the paranodal loops with the axon promote the transition in sodium channel subtypes at CNS nodes and provide a lateral diffusion barrier that, even in the absence of transverse bands, maintains a high concentration of components at the node and the integrity of voltage-gated channel domains

Three cell adhesion molecules are present at the axoglial junctions that form between the axon and myelinating glia on either side of nodes of Ranvier. These include an axonal complex of contacin-associated protein (Caspr) and contactin, which was proposed to bind NF155, an isoform of neurofascin located on the glial paranodal loops. Here, we show that NF155 binds directly to contactin and that surprisingly, coexpression of Caspr inhibits this interaction. This inhibition reflects the association of Caspr with contactin during biosynthesis and the resulting expression of a low molecular weight (LMw), endoglycosidase H-sensitive isoform of contactin at the cell membrane, which remains associated with Caspr but is unable to bind NF155. Accordingly, deletion of Caspr in mice by gene targeting results in a shift from the LMw- to a HMw-contactin glycoform. These results demonstrate that Caspr regulates the intracellular processing and transport of contactin to the cell surface, thereby affecting its ability to interact with other cell adhesion molecules

Neurotrimin (Ntm) and the limbic system-associated membrane protein (LAMP) are members of the IgLON (LAMP, OBCAM, Ntm) family of glycorylphosphatidylinositol anchored neural cell adhesion molecules. We previously reported that LAMP and Ntm promote adhesion and neurite outgrowth via a homophilic mechanism, suggesting that these proteins promote the formation of specific neuronal circuits by homophilic interactions. In this report, we have further characterized the expression and binding specificity of Ntm. Using a newly generated monoclonal antibody to Ntm, we demonstrated that this protein is largely expressed in a complementary pattern to that of LAMP in the nervous system, with co-expression at a few sites. Ntm is expressed at high levels in sensory-motor cortex and, of particular note, is transiently expressed in neurons of cortical barrel fields and corresponding thalamic 'barreloids.' Binding of a recombinant, soluble form of Ntm to CHO cells expressing either Ntm or LAMP demonstrates that Ntm and LAMP interact both homophilically and heterophilically. In contrast to conventional growth-promoting activity of Ig superfamily members, LAMP strongly inhibits the outgrowth of Ntm-expressing dorsal root ganglion (DRG) neurons in a heterophilic manner. These anatomical and functional data support the concept that homophilic and heterophilic interactions between IgLON family members are likely to play a role in the specification of neuronal projections via growth promoting and inhibiting effects, respectively.

Demyelination results in severe disability in many neurodegenerative diseases and nervous system infections, and it is typically mediated by inflammatory responses. Mycobacterium leprae, the causative organism of leprosy, induced rapid demyelination by a contact-dependent mechanism in the absence of immune cells in an in vitro nerve tissue culture model and in Rag1-knockout (Rag1(-/-)) mice, which lack mature B and T lymphocytes. Myelinated Schwann cells were resistant to M. leprae invasion but undergo demyelination upon bacterial attachment, whereas nonmyelinated Schwann cells harbor intracellular M. leprae in large numbers. During M. leprae-induced demyelination, Schwann cells proliferate significantly both in vitro and in vivo and generate a more nonmyelinated phenotype, thereby securing the intracellular niche for M. leprae

Sodium channel subtypes are frequently coexpressed within neurons. Recent studies demonstrate that targeting of specific channel subtypes to distinct membrane domains of axons is regulated by local signals from myelinating glia; they also show that channel subtypes are sequentially expressed at nodes of Ranvier, indicating an unexpected regulation in the composition of these domains

During development, neuregulin-1 promotes Schwann cell proliferation and survival; its role in later events of Schwann cell differentiation, including myelination, is poorly understood. Accordingly, we have examined the effects of neuregulin-1 on myelination in neuron-Schwann cell cocultures. Glial growth factor (GGF), a neuregulin-1 isoform, significantly inhibited myelination by preventing axonal segregation and ensheathment. Basal lamina formation was not affected. Treatment of established myelinated cultures with GGF resulted in striking demyelination that frequently began at the paranodes and progressed to the internode. Demyelination was dose dependent and accompanied by dedifferentiation of Schwann cells to a promyelinating stage, as evidenced by reexpression of the transcription factor suppressed cAMP-inducible POU; a significant proportion of cells with extensive demyelination also proliferated. Two other Schwann cell mitogens, fibroblast growth factor-2 and transforming growth factor-beta, inhibited myelination but did not cause demyelination, suggesting this effect is specific to the neuregulins. The neuregulin receptor proteins, erbB2 and erbB3, are expressed on ensheathing and myelinating Schwann cells and rapidly phosphorylated with GGF treatment. GGF treatment of myelinating cultures also induced phosphorylation of phosphatidylinositol 3-kinase, mitogen-activated protein kinase, and a 120-kD protein. These results suggest that neuronal mitogens, including the neuregulins, may inhibit myelination during development and that activation of mitogen signaling pathways may contribute to the initial demyelination and subsequent Schwann cell proliferation observed in various pathologic conditions

In the adult peripheral nerve, microvillous processes of myelinating Schwann cells project to the nodes of Ranvier; their composition and physiologic function have not been established. As the ezrin-radixin-moesin (ERM) proteins are expressed in the microvilli of many epithelial cells, we have examined the expression and distribution of these proteins in Schwann cells and neurons in vitro and in vivo. Cultured Schwann cells express high levels of all three proteins and the ezrin-binding protein 50, whereas neurons express much lower, although detectable, levels of radixin and moesin. Ezrin is specific for Schwann cells. All three ERM proteins are expressed predominantly at the membrane of cultured Schwann cells, notably in their microvilli. In vivo, the ERM proteins are concentrated strikingly in the nodal processes of myelinating Schwann cells. Because these processes are devoid of myelin proteins, they represent a unique compartment of the myelinating Schwann cell. During development, the ERM proteins become concentrated at the ends of Schwann cells before myelin basic protein expression, demonstrating that Schwann cells are polarized longitudinally at the onset of myelination. ERM-positive Schwann cell processes overlie and are associated closely with nascent nodes of Ranvier, identified by clusters of ankyrin G. Ankyrin accumulation at the node precedes that of Caspr at the paranodes and therefore does not depend on the presence of mature paranodal junctions. These results demonstrate that nodes of Ranvier in the peripheral nervous system form in contact with specialized processes of myelinating Schwann cells that are highly enriched in ERM proteins

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

Myelinated fibers are organized into distinct domains that are necessary for saltatory conduction. These domains include the nodes of Ranvier and the flanking paranodal regions where glial cells closely appose and form specialized septate-like junctions with axons. These junctions contain a Drosophila Neurexin IV-related protein, Caspr/Paranodin (NCP1). Mice that lack NCP1 exhibit tremor, ataxia, and significant motor paresis. In the absence of NCP1, normal paranodal junctions fail to form, and the organization of the paranodal loops is disrupted. Contactin is undetectable in the paranodes, and K(+) channels are displaced from the juxtaparanodal into the paranodal domains. Loss of NCP1 also results in a severe decrease in peripheral nerve conduction velocity. These results show a critical role for NCP1 in the delineation of specific axonal domains and the axon-glia interactions required for normal saltatory conduction