Distinct ankyrin isoforms at neuron cell bodies and nodes of Ranvier resolved using erythrocyte ankyrin-deficient mice

Isoforms of ankyrin (ankyrinsR) immunologically related to erythrocyte ankyrin (ankyrinRo) are associated with distinct neuronal plasma membrane domains of functional importance, such as cell bodies and dendrites, axonal hillock and initial segments, and nodes of Ranvier. AnkyrinRo is expressed in brain, and accounts for at least one of the ankyrinR isoforms. Another ankyrin isoform of brain, ankyrinB, is encoded by a distinct gene and is immunologically distinct from ankyrinsR. Mutant mice with normoblastosis (nb/nb) constitute the first described genetic model of ankyrin deficiency: they display a severe hemolytic anemia due to a significantly reduced expression of the ankyrinRo gene in reticulocytes as well as brain (Peters L. L., C. S. Birkenmeier, R. T. Bronson, R. A. White, S. E. Lux, E. Otto, V. Bennett, A. Higgins, and J. E. Barker. 1991. J. Cell Biol. 114:1233-1241). In the present report, we distinguish between ankyrinRo and other ankyrinR isoforms using immunoblot analysis and immunofluorescence localization of ankyrinsR throughout the nervous system (forebrain, cerebellum, brain stem, spinal cord, and sciatic nerve) of nb/nb and normal mice. This is the first immunocytochemical characterization of the neurological component of the nb mutation and shows the following. (a) The isoform of ankyrin at the nodes of Ranvier and initial axonal segments is present in the nb/nb mice and does not cross-react with an ankyrinRo-specific antibody; this isoform, therefore, is distinct from both ankyrin isoforms identified in brain, ankyrinRo and ankyrinB, and is probably the product of a distinct gene and a unique component of the specialized membrane skeleton associated with nodes of Ranvier. (b) AnkyrinRo missing from nb/nb mice is selectively associated with neuronal cell bodies and dendrites, excluded from myelinated axons, and displays a selective pattern of expression in the nervous system whereby expression is almost ubiquitous in neurons of the cerebellum (Purkinje and granule cells) and spinal cord, and restricted to a very minor subset of neurons in hippocampus and neocortex of forebrain.     

A new 440-kD isoform is the major ankyrin in neonatal rat brain

This report describes initial characterization of a 440-kD isoform of brain ankyrin (ankyrinB) representing an alternatively spliced mRNA product of the gene encoding the major isoform of ankyrin in adult human brain (Otto, E., M. Kunimoto, T. McLaughlin, V. Bennett, J. Cell Biology. 114:241-253). Northern and immunoblot analyses indicate that 440-kD ankyrinB includes the spectrin and membrane-binding domains as well as a regulatory domain of the major 220-kD isoform. 440-kD ankyrinB contains, in addition, a sequence of a predicted size of 220 kD which is inserted between the regulatory domain and spectrin/membrane-binding domains. 440-kD ankyrinB has properties expected of a peripherally associated membrane-skeletal protein: it is exclusively present in the particulate fraction of brain homogenates, is extracted with NaOH, and remains associated with Triton-X-100-resistant structures. Expression of 440-kD ankyrinB in rat brain began at birth before other ankyrins could be detected, peaked 10 d after birth, and then decreased progressively to 30% of the maximum in adults. Expression of the 220-kD ankyrinB and ankyrinR (erythroid ankyrin) began approximately 10 d after the 440-kD isoform, increased rapidly between 10 and 15 d after birth, and finally achieved their maximal levels in adults. 440-kD ankyrinB is present in approximately equivalent amounts in all regions of neonatal brain while in adult brain it is present in highest levels in cerebellum and lowest in brain stem. 440-kD ankyrinB was localized by immunofluorescence in regions of neonatal and adult brain containing primarily dendrites and unmyelinated axons. 440-kD ankyrinB thus may play a specialized role in neuronal processes.     

Molecular composition of the node of Ranvier: identification of ankyrin- binding cell adhesion molecules neurofascin (mucin+/third FNIII domain- ) and NrCAM at nodal axon segments

Neurofascin, NrCAM, L1, and NgCAM are a family of Ig/FNIII cell adhesion molecules that share ankyrin-binding activity in their cytoplasmic domains, and are candidates to form membrane-spanning complexes with members of the ankyrin family of spectrin-binding proteins in a variety of cellular contexts in the nervous system. Specialized forms of ankyrin, 270 kD and/or 480 kD ankyrinG are components of the membrane undercoat of axons at the node of Ranvier. This paper focuses on definition of the isoforms of ankyrin-binding cell adhesion molecules localized with ankyrinG at the nodal axon segment. The exon usage of two major forms of neurofascin was determined by isolation of full-length cDNAs and used to prepare isoform-specific antibodies. An isoform of neurofascin containing a mucin-like domain and lacking the third FNIII domain was concentrated at axon initial segments and colocalized at nodes of Ranvier with ankyrinG and the voltage-dependent sodium channel. An alternative form of neurofascin lacking the mucin-like domain and containing the third FNIII domain was present in unmyelinated axons. The antibody initially raised against neurofascin was used to screen a rat brain cDNA expression library. In addition to neurofascin, this screen yielded a clone with 80% sequence identity to NrCAM from chicken. The sequences of two full-length cDNAs are presented. NrCAM is most closely related to neurofascin among the other members of the L1/neurofascin/NgCAM family, with over 70% identity between cytoplasmic domains. NrCAM, visualized with antibodies specific for the ecto-domain, also was found to be coexpressed with neurofascin at nodes of Ranvier and at axon initial segments. This is the first characterization of defined neuronal cell adhesion molecules localized to axonal membranes at the node of Ranvier of myelinated axons.     

The Fine Anatomy of the Optic Nerve of Anurans—An Electron Microscope Study

In the optic nerve of Anurans numerous myelinated and unmyelinated axons appear under the electron microscope as compact bundles that are closely bounded by one or several glial cells. In these bundles the unmyelinated fibers (0.15 to 0.6 µ in diameter) are many times more numerous than the myelinated fibers, and are separated from each other, from the bounding glial cells, or from adjacent myelin sheaths, by an extracellular gap that is 90 to 250 A wide. This intercellular space is continuous with the extracellular space in the periphery of the nerve through the numerous mesaxons and cell boundaries which reach the surface. Numerous desmosomes reinforce the attachments of adjacent glial membranes. The myelinated axons do not follow any preferential course and, like the unmyelinated ones, have a sinuous path, continuously shifting their relative position and passing from one bundle to another. At the nodes of Ranvier they behave entirely like unmyelinated axons in their relations to the surrounding cells. At the internodes they lie between the unmyelinated axons without showing an obvious myelogenic connection with the surrounding glial cells. In the absence of connective tissue separating individual myelinated fibers and with each glial cell simultaneously related to many axons, this myelogenic connection is highly distorted by other passing fibers and is very difficult to demonstrate. However, the mode of ending of the myelin layers at the nodes of Ranvier and the spiral disposition of the myelin layers indicate that myelination of these fibers occurs by a process similar to that of peripheral nerves. There are no incisures of Schmidt-Lantermann in the optic myelinated fibers.     

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.     

Adenosine triphosphatase activity of cutaneous nerve fibers

Summary The histochemical study of Mg⁺⁺-activated adenosine triphosphatase (Mg⁺⁺-ATPase) activity was carried out on the peripheral nerves of mouse digital skin by light and electron microscopy. Under the light microscope, the ATPase activity was clearly demonstrated on the nerve fibers as a fine network in the subepidermal regions. Under the electron microscope, the reaction product of enzyme activity was located in the interspace between axolemma and the surrounding Schwann cells of the unmyelinated nerve fibers. No reaction product was observed in the space between the axolemma and the Schwann cells associated with myelinated nerve fibers. Demonstrable activity was absent at the nodes of Ranvier as well as on the para- and internodal regions of these myelinated axons. The part of the axolemma lacking a Schwann cell sheath failed to show a reaction product. The perineural epithelial cells surrounding the nerve fibers displayed reaction product in the caveolae. These results suggest a functional difference in the axon-Schwann interface of myelinated as compared to unmyelinated nerve fibers. The function of the perineural epithelial cell would be expected to be a regulatory one in transferring materials across the epithelium to keep the proper humoral environment around nerve fibers.     

Isolation and characterization of cDNAs encoding human brain ankyrins reveal a family of alternatively spliced genes

Ankyrins are a family of membrane-associated proteins that can be divided into two immunologically distinct groups: (a) erythrocyte-related isoforms (ankyrinR) that have polarized distributions in particular cell types; and (b) brain-related isoforms (ankyrinB) that display a broader distribution. In this paper, we report the isolation and sequences of cDNAs related to two ankyrinB isoforms, human brain ankyrin 1 and 2, and show that these isoforms are produced from alternatively spliced mRNAs of a single gene. Human brain ankyrin 1 and 2 share a common NH2-terminus that is similar to human erythrocyte ankyrins, with the most striking conservation occurring between areas composed of a repeated 33-amino acid motif and between areas corresponding to the central portion of the spectrin-binding domain. In contrast, COOH-terminal sequences of brain ankyrin 1 and 2 are distinct from one another and from human erythrocyte ankyrins, and thus are candidates to mediate protein interactions that distinguish these isoforms. The brain ankyrin 2 cDNA sequence includes a stop codon and encodes a polypeptide with a predicted molecular mass of 202 kD, which is similar to the Mr of the major form of ankyrin in adult bovine brain membranes. Moreover, an antibody raised against the conserved NH2-terminal domain of brain ankyrin cross-reacts with a single Mr = 220 kD polypeptide in adult human brain. These results strongly suggest that the amino acid sequence of brain ankyrin 2 determined in this report represents the complete coding sequence of the major form of ankyrin in adult human brain. In contrast, the brain ankyrin 1 cDNAs encode only part of a larger isoform. An immunoreactive polypeptide of Mr = 440 kD, which is evident in brain tissue of young rats, is a candidate to be encoded by brain ankyrin 1 mRNA. The COOH-terminal portion of brain ankyrin 1 includes 15 contiguous copies of a novel 12-amino acid repeat. Analysis of DNA from a panel of human/rodent cell hybrids linked this human brain ankyrin gene to chromosome 4. This result, coupled with previous reports assigning the human erythrocyte ankyrin gene to chromosome 8, demonstrates that human brain and erythrocyte ankyrins are encoded by distinct members of a multigene family.     

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.     

Dynamic compartmentalization of the voltage-gated sodium channels in axons

One of the major physiological roles of the neuronal voltage-gated sodium channel is to generate action potentials at the axon hillock/initial segment and to ensure propagation along myelinated or unmyelinated fibers to nerve terminal. These processes require a precise distribution of sodium channels accumulated at high density in discrete subdomains of the nerve membrane. In neurons, information relevant to ion channel trafficking and compartmentalization into sub-domains of the plasma membrane is far from being elucidated. Besides, whereas information on dendritic targeting is beginning to emerge, less is known about the mechanisms leading to the polarized distribution of proteins in axon. To obtain a better understanding of how neurons selectively target sodium channels to discrete subdomains of the nerve, we addressed the question as to whether any of the large intracellular regions of Nav1.2 contain axonal sorting and/or clustering signals. We first obtained evidence showing that addition of the cytoplasmic carboxy-terminal region of Nav1.2 restricted the distribution of a dendritic-axonal reporter protein to axons of hippocampal neurons. The analysis of mutants revealed that a di-leucine-based motif mediates chimera compartmentalization in axons and its elimination in soma and dendrites by endocytosis. The analysis of the others generated chimeras showed that the determinant conferring sodium channel clustering at the axonal initial segment is contained within the cytoplasmic loop connecting domains II-III of Nav1.2. Expression of a soluble Nav1.2 II-III linker protein led to the disorganization of endogenous sodium channels. The motif was sufficient to redirect a somatodendritic potassium channel to the axonal initial segment, a process involving association with ankyrin G. Thus, it is conceivable that concerted action of the two determinants is required for sodium channel compartmentalization in axons.     

The presence of voltage-gated sodium, potassium and chloride channels in rat cultured astrocytes

Patch-clamp recording from the plasmalemma of rat cultured astrocytes reveals the presence of both voltage-dependent sodium and voltage-dependent potassium conductances. These conductances are similar but not identical to the corresponding conductances in the axolemma. Whereas the h infinity relation of the sodium channels has the same voltage dependence as in the nodal axolemma, the peak current-voltage relation is shifted by about 30 mV along the voltage axis in the depolarizing direction. It is speculated that the glial cells synthesize sodium and potassium channels for later insertion into the axolemma of neighbouring axons. The astrocytes also express a plasmalemmal voltage-dependent anion conductance that is turned on at about -40 mV (that is, near the resting potential of the cultured astrocytes). The channels involved are large enough to be just permeable to glutamate but not to ascorbate. It is suggested that the conductance of this channel for chloride plays a physiological role in the spatial buffering of potassium by glial cells.     

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.     

A neuron-specific isoform of brain ankyrin, 440-kD ankyrinB, is targeted to the axons of rat cerebellar neurons

Two isoforms of brain ankyrin, 440- and 220- kD ankyrinB, are generated from the same gene by alternative splicing of pre-mRNA. The larger isoform shares the same NH2-terminal and COOH-terminal domains to the smaller isoform and contains, in addition, a unique inserted domain of about 220-kD in size (Kunimoto, M., E. Otto, and V. Bennett. 1991. J. Cell Biol. 115:1319-1331). Both Isoforms were expressed in primary cerebellar cells in a manner similar to that in vivo; the larger isoform appeared first when axogenesis is actively conducted and the smaller isoform came up later. 440-kD ankyrinB was localized in the axons of cerebellar neurons both in vivo and in vitro using an antibody raised against the insert region, while 220-kD isoform was rather localized in the cell bodies and dendrites of neurons by a specific antibody prepared using a synthetic peptide corresponding to the splice site as antigen. Astroglia cells also expressed 220-kD ankyrinB but not the 440-kD isoform. These results indicate that 440-kD ankyrinB is a neuron-specific isoform targeted to the axons and its unique inserted domain is essential for the targeting.     

Molecular structure of the axolemma of developing axons following altered gliogenesis in rat optic nerve

Axonal and axolemmal development of fibers from rat optic nerves in which gliogenesis was severely delayed by systemic injection of 5-azacytidine (5-AZ) was examined by freeze-fracture electron microscopy. In neonatal (0-2 days) rat optic nerves, all fibers lack myelin, whereas in the adult, virtually all axons are myelinated. The axolemma of neonatal premyelinated fibers is relatively undifferentiated. The P-fracture face (P-face) displays a moderate (approximately 550/micron 2) density of intramembranous particles (IMPs), whereas the E-fracture face (E-face) has few IMPs (approximately 125/micron 2) present. By 14 days of age, approximately 25% of the axons within control optic nerves are ensheathed or myelinated, with the remaining axons premyelinated. The ensheathed and myelinated fibers display increased axonal diameter compared to premyelinated axons, and these larger caliber fibers exhibit marked axonal membrane differentiation. Notably, the P-face IMP density of ensheathed and myelinated fibers is substantially increased compared to premyelinated axolemma, and, at nodes of Ranvier, the density of E-face particles is moderately high (approximately 1300/micron 2), in comparison to internodal or premyelinated E-face axolemma. In optic nerves from 14-day-old 5-AZ-treated rats, few oligodendrocytes are present, and the percentage of myelinated fibers is markedly reduced. Despite delayed gliogenesis, some unensheathed axons within 5-AZ-treated optic nerves display an increased axonal diameter compared to premyelinated fibers. Most of these large caliber fibers also exhibit a substantial increase in P-face IMP density. Small (less than 0.4 micron) diameter unensheathed axons within treated optic nerves maintain a P-face IMP density similar to that of control premyelinated fibers. Regions of increased E-face particle density were not observed. The results demonstrate that some aspects of axolemma differentiation continue despite delayed gliogenesis and the absence of glial ensheathment, and suggest that axolemmal ultrastructure is, at least in part, independent of glial cell association.     

Morphometry and ultrastructure of the squirrel monkey (Saimiri sciureus) vestibular nerve

Vestibular nerves of squirrel monkeys (Saimiri sciureus) embedded in plastics and epoxies were examined with light microscopy (LM) and transmission electron microscopy (TEM), and computerized measures were obtained and analyzed statistically. An average of 12,412 perikarya and 12,005 myelinated nerve fibers was obtained. Approximately 0.7% of the perikarya appeared unmyelinated under LM. About 500 unmyelinated fibers were counted. The cross-sectional area of 1,864 perikarya was 200-650 micron 2. The cross-sectional area of 1,346 nerve fibers was 3-11 micron 2 for the axoplasm and 11-12 micron 2 for the myelin sheath of the same fiber. Myelin thickness was directly proportional to the axoplasm cross-sectional area of the nerve fibers. The cross-sectional area of central axons and peripheral dendrites differed significantly (p less than 0.001). The initial segments of peripheral dendrites were usually smaller, but longer than the initial segments of the central axons. Both initial segments increased in diameter after the first node of Ranvier. Schmidt-Lantermann incisures were more abundant in thick and heavily myelinated fibers than in thin and lightly myelinated fibers. Larger perikarya usually had larger fibers and vice versa, within the first 100-200 micron from the first node of Ranvier. No major ultrastructural differences were found between myelinated and unmyelinated perikarya, except at the hillock region. The Nissl substance was preferentially located in the peripheral cytoplasm.     

Gating currents in the node of Ranvier: voltage and time dependence.

Like the axolemma of the giant nerve fibre of the squid, the nodal membrane of frog myelinated nerve fibres after blocking transmembrane ionic currents exhibits asymmetrical displacement currents during and after hyperpolarizing and depolarizing voltage clamp pulses of equal size. The steady-state distribution of charges as a function of membrane potential is consistent with Boltzmanns law (midpoint potential minus 33.7 mV; saturation value 17200 charges/mum-2). The time course of the asymmetry current and the voltage dependence of its time constant are consistent with the notion that due to a sudden change in membrane potential the charges undergo a first order transition between two configurations. Size and voltage dependence of the time constant are similar to those of the activation of the sodium conductance assuming m-2h kinetics. The results suggest that the presence of ten times more sodium channels (5000/mum-2) in the node of Ranvier than in the squid giant axon with similar sodium conductance per channel (2-3 pS).     

Differential expression and functions of neuronal and glial neurofascin isoforms and splice variants during PNS development

The cell adhesion molecule neurofascin (NF) has a major neuronal isoform (NF186) containing a mucin-like domain followed by a fifth fibronectin type III repeat while these domains are absent from glial NF155. Neuronal NF isoforms lacking one or both of these domains are expressed transiently in embryonic dorsal root ganglia (DRG). These two domains are co-expressed in mature NF186, which peaks in expression prior to birth and then persists almost exclusively at nodes of Ranvier on myelinated axons. In contrast, glial NF155 is only detected postnatally with the onset of myelination. All these forms of NF bound homophilically and to Schwann cells but only the mature NF186 isoform inhibits cell adhesion, and this activity may be important in formation of the node of Ranvier. Schwann cells deficient in NF155 myelinated DRG axons in a delayed manner and they showed significantly decreased clustering of both NF and Caspr in regions where paranodes normally form. The combined results suggest that NF186 is expressed prenatally on DRG neurons and it may modulate their adhesive interactions with Schwann cells, which express NF155 postnatally and require it for development of axon-glial paranodal junctions.     

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.     

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.     

nsf is essential for organization of myelinated axons in zebrafish

BACKGROUND: Myelinated axons are essential for rapid conduction of action potentials in the vertebrate nervous system. Of particular importance are the nodes of Ranvier, sites of voltage-gated sodium channel clustering that allow action potentials to be propagated along myelinated axons by saltatory conduction. Despite their critical role in the function of myelinated axons, little is known about the mechanisms that organize the nodes of Ranvier. RESULTS: Starting with a forward genetic screen in zebrafish, we have identified an essential requirement for nsf (N-ethylmaleimide sensitive factor) in the organization of myelinated axons. Previous work has shown that NSF is essential for membrane fusion in eukaryotes and has a critical role in vesicle fusion at chemical synapses. Zebrafish nsf mutants are paralyzed and have impaired response to light, reflecting disrupted nsf function in synaptic transmission and neural activity. In addition, nsf mutants exhibit defects in Myelin basic protein expression and in localization of sodium channel proteins at nodes of Ranvier. Analysis of chimeric larvae indicates that nsf functions autonomously in neurons, such that sodium channel clusters are evident in wild-type neurons transplanted into the nsf mutant hosts. Through pharmacological analyses, we show that neural activity and function of chemical synapses are not required for sodium channel clustering and myelination in the larval nervous system. CONCLUSIONS: Zebrafish nsf mutants provide a novel vertebrate system to investigate Nsf function in vivo. Our results reveal a previously unknown role for nsf, independent of its function in synaptic vesicle fusion, in the formation of the nodes of Ranvier in the vertebrate nervous system.     

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.