Initiation of sodium channel clustering at the node of Ranvier in the mouse optic nerve

Ion fluxes in mammalian myelinated axons are restricted to the nodes of Ranvier, where, in particular, voltage-gated Na⁺ channels are clustered at a high density. The node of Ranvier is separated from the internode by two distinct domains of the axolemma, the paranode and the juxtaparanode. Each axonal domain is characterized by the presence of a specific protein complex. Although oligodendrocytes and/or myelin membranes are believed to play some instructive roles in the organization of axonal domains, the mechanisms leading to their localized distribution are not well understood. In this paper we focused on the involvement of myelin sheaths in this domain organization and examined the distribution of axonal components in the optic nerves of wild type, hypomyelinating jimpy mice and demyelinating PLP transgenic mice. The results showed that the clustering of Na⁺ channels does not require junction-like structures to be formed between the glial processes and axons, but requires mature oligodendrocytes to be present in close vicinity.     

Fibronectin type III-like domains of neurofascin-186 protein mediate gliomedin binding and its clustering at the developing nodes of Ranvier

The cell adhesion molecules (CAMs) of the immunoglobulin superfamily (Ig-CAMs) play a crucial role in the organization of the node of Ranvier in myelinated axons. In the peripheral nervous system, Gliomedin (Gldn) secreted by Schwann cell microvilli binds NgCAM-related CAM (NrCAM) and Neurofascin-186 (NF186) and direct the nodal clustering of voltage-gated sodium channels (Nav). NF186 is the single axonal Gldn partner to ensure Nav clustering at nodes, whereas NrCAM is only required in glial cells (Feinberg, K., Eshed-Eisenbach, Y., Frechter, S., Amor, V., Salomon, D., Sabanay, H., Dupree, J. L., Grumet, M., Brophy, P. J., Shrager, P., and Peles, E. (2010) Neuron 65, 490-502). The olfactomedin domain of Gldn is implicated in the interaction with nodal Ig-CAMs. However, the interacting modules of NrCAM or NF186 involved in Gldn association are unknown. Here, we report that fibronectin type III-like (FnIII) domains of both Ig-CAMs mediate their interaction with Gldn in pulldown and cell binding assays. Using surface plasmon resonance assays, we determined that NrCAM and NF186 display similar affinity constant for their association with Gldn (K(D) of 0.9 and 5.7 nm, respectively). We characterized the FnIII domains 1 and 2 of NF186 as interacting modules that ensure association with Gldn. We found that the soluble FnIII domains of NF186 (FnIII-Fc) bind on Schwann cells and inhibit Gldn and Nav clustering at heminodes, the precursors of mature nodes in myelinating cultures. Our study reveals the unexpected importance of FnIII domains of Ig-CAMs in the organization of nodes of Ranvier in peripheral axons. Thus, NF186 utilizes distinct modules to organize the multimeric nodal complex.     

Acetylcholinesterase clustering at the neuromuscular junction involves perlecan and dystroglycan.

Formation of the synaptic basal lamina at vertebrate neuromuscular junction involves the accumulation of numerous specialized extracellular matrix molecules including a specific form of acetylcholinesterase (AChE), the collagenic-tailed form. The mechanisms responsible for its localization at sites of nerve- muscle contact are not well understood. To understand synaptic AChE localization, we synthesized a fluorescent conjugate of fasciculin 2, a snake alpha-neurotoxin that tightly binds to the catalytic subunit. Prelabeling AChE on the surface of Xenopus muscle cells revealed that preexisting AChE molecules could be recruited to form clusters that colocalize with acetylcholine receptors at sites of nerve-muscle contact. Likewise, purified avian AChE with collagen-like tail, when transplanted to Xenopus muscle cells before the addition of nerves, also accumulated at sites of nerve-muscle contact. Using exogenous avian AChE as a marker, we show that the collagenic-tailed form of the enzyme binds to the heparan-sulfate proteoglycan perlecan, which in turn binds to the dystroglycan complex through alpha-dystroglycan. Therefore, the dystroglycan-perlecan complex serves as a cell surface acceptor for AChE, enabling it to be clustered at the synapse by lateral migration within the plane of the membrane. A similar mechanism may underlie the initial formation of all specialized basal lamina interposed between other cell types.     

Secreted gliomedin is a perinodal matrix component of peripheral nerves

The interaction between gliomedin and the axonodal cell adhesion molecules (CAMs) neurofascin and NrCAM induces the clustering of Na(+) channels at the nodes of Ranvier. We define new interactions of gliomedin that are essential for its clustering activity. We show that gliomedin exists as both transmembrane and secreted forms that are generated by proteolytic cleavage of the protein, and that only the latter is detected at the nodes of Ranvier. The secreted extracellular domain of gliomedin binds to Schwann cells and is incorporated into the extracellular matrix (ECM) in a heparin-dependent manner, suggesting the involvement of heparan sulfate proteoglycans (HSPGs). Furthermore, we show that the N-terminal region of gliomedin serves as an oligomerization domain that mediates self-association of the molecule, which is required for its binding to neurofascin and NrCAM. Our results indicate that the deposition of gliomedin multimers at the nodal gap by binding to HSPGs facilitates the clustering of the axonodal CAMs and Na(+) channels.     

Early events in node of Ranvier formation during myelination and remyelination in the PNS

Action potential conduction velocity increases dramatically during early development as axons become myelinated. Integral to this process is the clustering of voltage-gated Na(+) (Nav) channels at regularly spaced gaps in the myelin sheath called nodes of Ranvier. We show here that some aspects of peripheral node of Ranvier formation are distinct from node formation in the CNS. For example, at CNS nodes, Nav1.2 channels are detected first, but are then replaced by Nav1.6. Similarly, during remyelination in the CNS, Nav1.2 channels are detected at newly forming nodes. By contrast, the earliest Nav-channel clusters detected during developmental myelination in the PNS have Nav1.6. Further, during PNS remyelination, Nav1.6 is detected at new nodes. Finally, we show that accumulation of the cell adhesion molecule neurofascin always precedes Nav channel clustering in the PNS. In most cases axonal neurofascin (NF-186) accumulates first, but occasionally paranodal neurofascin is detected first. We suggest there is heterogeneity in the events leading to Nav channel clustering, indicating that multiple mechanisms might contribute to node of Ranvier formation in the PNS.     

Unique role of dystroglycan in peripheral nerve myelination,nodal structure,and sodium channel stabilization

Dystroglycan is a central component of the dystrophin-glycoprotein complex implicated in the pathogenesis of several neuromuscular diseases. Although dystroglycan is expressed by Schwann cells, its normal peripheral nerve functions are unknown. Here we show that selective deletion of Schwann cell dystroglycan results in slowed nerve conduction and nodal changes including reduced sodium channel density and disorganized microvilli. Additional features of mutant mice include deficits in rotorod performance, aberrant pain responses, and abnormal myelin sheath folding. These data indicate that dystroglycan is crucial for both myelination and nodal architecture. Dystroglycan may be required for the normal maintenance of voltage-gated sodium channels at nodes of Ranvier, possibly by mediating trans interactions between Schwann cell microvilli and the nodal axolemma.     

alphaII-spectrin is essential for assembly of the nodes of Ranvier in myelinated axons

Saltatory conduction in myelinated axons requires organization of the nodes of Ranvier, where voltage-gated sodium channels are prominently localized [1]. Previous results indicate that alphaII-spectrin, a component of the cortical cytoskeleton [2], is enriched at the paranodes [3, 4], which flank the node of Ranvier, but alphaII-spectrin's function has not been investigated. Starting with a genetic screen in zebrafish, we discovered in alphaII-spectrin (alphaII-spn) a mutation that disrupts nodal sodium-channel clusters in myelinated axons of the PNS and CNS. In alphaII-spn mutants, the nodal sodium-channel clusters are reduced in number and disrupted at early stages. Analysis of chimeric animals indicated that alphaII-spn functions autonomously in neurons. Ultrastructural studies show that myelin forms in the posterior lateral line nerve and in the ventral spinal cord in alphaII-spn mutants and that the node is abnormally long; these findings indicate that alphaII-spn is required for the assembly of a mature node of the correct length. We find that alphaII-spectrin is enriched in nodes and paranodes at early stages and that the nodal expression diminishes as nodes mature. Our results provide functional evidence that alphaII-spectrin in the axonal cytoskeleton is essential for stabilizing nascent sodium-channel clusters and assembling the mature node of Ranvier.     

MuSK is required for anchoring acetylcholinesterase at the neuromuscular junction

At the neuromuscular junction, acetylcholinesterase (AChE) is mainly present as asymmetric forms in which tetramers of catalytic subunits are associated to a specific collagen, collagen Q (ColQ). The accumulation of the enzyme in the synaptic basal lamina strictly relies on ColQ. This has been shown to be mediated by interaction between ColQ and perlecan, which itself binds dystroglycan. Here, using transfected mutants of ColQ in a ColQ-deficient muscle cell line or COS-7 cells, we report that ColQ clusterizes through a more complex mechanism. This process requires two heparin-binding sites contained in the collagen domain as well as the COOH terminus of ColQ. Cross-linking and immunoprecipitation experiments in Torpedo postsynaptic membranes together with transfection experiments with muscle-specific kinase (MuSK) constructs in MuSK-deficient myotubes or COS-7 cells provide the first evidence that ColQ binds MuSK. Together, our data suggest that a ternary complex containing ColQ, perlecan, and MuSK is required for AChE clustering and support the notion that MuSK dictates AChE synaptic localization at the neuromuscular junction.     

Molecular mechanisms of node of Ranvier formation

Action potential propagation along myelinated nerve fibers requires high-density protein complexes that include voltage-gated Na(+) channels at the nodes of Ranvier. Several complementary mechanisms may be involved in node assembly including: (1) interaction of nodal cell adhesion molecules with the extracellular matrix; (2) restriction of membrane protein mobility by paranodal junctions; and (3) stabilization of ion channel clusters by axonal cytoskeletal scaffolds. In the peripheral nervous system, a secreted glial protein at the nodal extracellular matrix interacts with axonal cell adhesion molecules to initiate node formation. In the central nervous system, both glial soluble factors and paranodal axoglial junctions may function in a complementary manner to contribute to node formation.     

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.     

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.     

Molecular constituents of the node of Ranvier

The interaction between neurons and glial cells that results in myelin formation represents one of the most remarkable intercellular events in development. This is especially evident at the primary functional site within this structure, the node of Ranvier. Recent experiments have revealed a surprising level of complexity within this zone, with several components, including ion channels, sequestered with a very high degree of precision and sharply demarcated borders. We discuss the current state of knowledge of the cellular and molecular mechanisms responsible for the formation and maintenance of the node. In normal axons, Na⁺ channels are present at high density within the nodal gap, and voltage-dependent K⁺ channels are sequestered on the internodal side of the paranode—a region known as the juxtaparanode. Modifying the expression of certain surface adhesion molecules that have been recently identified, markedly alters this pattern. There is a special emphasis on contactin, a protein with multiple roles in the nervous system. In central nervous system (CNS) myelinated fibers, contactin is localized within both the nodal gap and paranodes, and appears to have unique functions in each zone. New experiments on contactin-null mutant mice help to define these mechanisms.     

Disruption of perlecan binding and matrix assembly by post-translational or genetic disruption of dystroglycan function

Dystroglycan is a cell-surface matrix receptor that requires LARGE-dependent glycosylation for laminin binding. Although the interaction of dystroglycan with laminin has been well characterized, less is known about the role of dystroglycan glycosylation in the binding and assembly of perlecan. We report reduced perlecan-binding activity and mislocalization of perlecan in the LARGE-deficient Large(myd) mouse. Cell-surface ligand clustering assays show that laminin polymerization promotes perlecan assembly. Solid-phase binding assays provide evidence for the first time of a trimolecular complex formation of dystroglycan, laminin and perlecan. These data suggest functional disruption of the trimolecular complex in glycosylation-deficient muscular dystrophy.     

Brevican distinctively assembles extracellular components at the large diameter nodes of Ranvier in the CNS

Brevican is known to be an abundant extracellular matrix component in the adult brain and a structural constituent of perineuronal nets. We herein show that brevican, tenascin-R (TN-R) and phosphacan are present at the nodes of Ranvier on myelinated axons with a particularly large diameter in the central nervous system. A brevican deficiency resulted in a reorganization of the nodal matrices, which was characterized by the shift of TN-R, and concomitantly phosphacan, from an axonal diameter-dependent association with nodes to an axonal diameter independent association. Supported by the co-immunoprecipitation results, these observations indicate that the presence of TN-R and phosphacan at nodes is normally brevican-dependent, while in the absence of brevican these molecules can also be recruited by versican V2. The versican V2 and Bral1 distribution was not affected, thus indicating a brevican-independent role of these two molecules for establishing hyaluronan-binding matrices at the nodes. Our results revealed that brevican plays a crucial role in determining the specialization of the hyaluronan-binding nodal matrix assemblies in large diameter nodes.     

有髓轴突横断损伤后钠通道聚集状态研究

目的:研究有髓轴突横断损伤后郎飞结区钠通道聚集状态的变化.方法:用雪旺细胞-背根神经元髓鞘化共培养系统复制周围神经髓鞘形成和郎飞结发育,于髓鞘化培养基中共培养第14天用前房角切开刀造成有髓轴突横断损伤,在损伤后1、2、3、4、5、6、7、14天进行髓鞘碱性蛋白和钠通道免疫荧光染色,损伤前共培养作为对照.利用SPOT图像分析软件测量钠通道聚集簇的直径、长度和直径/长度比.结果:损伤前钠通道蛋白在有髓轴突郎飞结区形成直径/长度比略大于1的聚集簇;有髓轴灾横断损伤后钠通道蛋白沿轴突纵向扩散,钠通道聚集簇的直径/长度比逐渐减小,损伤后第14天已无法检测到钠通道表达.损伤区出现节段性脱髓鞘.结论:轴突横断损伤可造成钠通道聚集簇扩散、消失,导致郎飞结结构破坏.     

Identification of dystroglycan as a second laminin receptor in oligodendrocytes, with a role in myelination

Developmental abnormalities of myelination are observed in the brains of laminin-deficient humans and mice. The mechanisms by which these defects occur remain unknown. It has been proposed that, given their central role in mediating extracellular matrix (ECM) interactions, integrin receptors are likely to be involved. However, it is a non-integrin ECM receptor, dystroglycan, that provides the key linkage between the dystrophin-glycoprotein complex (DGC) and laminin in skeletal muscle basal lamina, such that disruption of this bridge results in muscular dystrophy. In addition, the loss of dystroglycan from Schwann cells causes myelin instability and disorganization of the nodes of Ranvier. To date, it is unknown whether dystroglycan plays a role during central nervous system (CNS) myelination. Here, we report that the myelinating glia of the CNS, oligodendrocytes, express and use dystroglycan receptors to regulate myelin formation. In the absence of normal dystroglycan expression, primary oligodendrocytes showed substantial deficits in their ability to differentiate and to produce normal levels of myelin-specific proteins. After blocking the function of dystroglycan receptors, oligodendrocytes failed both to produce complex myelin membrane sheets and to initiate myelinating segments when co-cultured with dorsal root ganglion neurons. By contrast, enhanced oligodendrocyte survival in response to the ECM, in conjunction with growth factors, was dependent on interactions with beta-1 integrins and did not require dystroglycan. Together, these results indicate that laminins are likely to regulate CNS myelination by interacting with both integrin receptors and dystroglycan receptors, and that oligodendrocyte dystroglycan receptors may have a specific role in regulating terminal stages of myelination, such as myelin membrane production, growth, or stability.     

Node of Ranvier disruption as a cause of neurological diseases

Dysfunction and/or disruption of nodes of Ranvier are now recognized as key contributors to the pathophysiology of various neurological diseases. One reason is that the excitable nodal axolemma contains a high density of Nav (voltage-gated Na⁺ channels) that are required for the rapid and efficient saltatory conduction of action potentials. Nodal physiology is disturbed by altered function, localization, and expression of voltage-gated ion channels clustered at nodes and juxtaparanodes, and by disrupted axon–glial interactions at paranodes. This paper reviews recent discoveries in molecular/cellular neuroscience, genetics, immunology, and neurology that highlight the critical roles of nodes of Ranvier in health and disease.     

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.     

A stoichiometric complex of neurexins and dystroglycan in brain

In nonneuronal cells, the cell surface protein dystroglycan links the intracellular cytoskeleton (via dystrophin or utrophin) to the extracellular matrix (via laminin, agrin, or perlecan). Impairment of this linkage is instrumental in the pathogenesis of muscular dystrophies. In brain, dystroglycan and dystrophin are expressed on neurons and astrocytes, and some muscular dystrophies cause cognitive dysfunction; however, no extracellular binding partner for neuronal dystroglycan is known. Regular components of the extracellular matrix, such as laminin, agrin, and perlecan, are not abundant in brain except in the perivascular space that is contacted by astrocytes but not by neurons, suggesting that other ligands for neuronal dystroglycan must exist. We have now identified alpha- and beta-neurexins, polymorphic neuron-specific cell surface proteins, as neuronal dystroglycan receptors. The extracellular sequences of alpha- and beta-neurexins are largely composed of laminin-neurexin-sex hormone-binding globulin (LNS)/laminin G domains, which are also found in laminin, agrin, and perlecan, that are dystroglycan ligands. Dystroglycan binds specifically to a subset of the LNS domains of neurexins in a tight interaction that requires glycosylation of dystroglycan and is regulated by alternative splicing of neurexins. Neurexins are receptors for the excitatory neurotoxin alpha-latrotoxin; this toxin competes with dystroglycan for binding, suggesting overlapping binding sites on neurexins for dystroglycan and alpha-latrotoxin. Our data indicate that dystroglycan is a physiological ligand for neurexins and that neurexins' tightly regulated interaction could mediate cell adhesion between brain cells.     

Membrane proteins of synaptic vesicles and cytoskeletal specializations at the node of Ranvier in electric ray and rat

Summary Binding sites for antibodies against membrane proteins of synaptic vesicles have been shown to be enhanced at nodes of Ranvier in electromotor axons of the electric ray Torpedo marmorata and sciatic nerve axons of the rat, using indirect immunofluorescence and monoclonal antibodies against the synaptic vesicle transmembrane proteins SV2 and synaptophysin (rat) or SV2 (Torpedo). In the electric lobe of Torpedo, vesicle-membrane constituents occurred at higher density in the proximal axon segments covered by oligodendroglia cells than in the distal axon segments where myelin is formed by Schwann cells. Antibody binding sites were enhanced at nodes forming the borderline of the central and peripheral nervous systems. Filamentous actin was present in the Schwann-cell processes covering both the nodal and the paranodal axon segments as suggested by the pattern of phalloidin labelling. Furthermore, in rat sciatic nerve, Schmidt-Lanterman incisures were intensely labelled by phalloidin. A similar nodal distribution was found for binding sites of antibodies against actin and myosin. Binding of antibodies to tubulin was enhanced at nodes in Torpedo electromotor axons. The apparent nodal accumulation of constituents of synaptic vesicle membranes and the presence of filamentous actin and of myosin are discussed in relation to the substantial constriction of the axoplasm at nodes of Ranvier.