RESUMEN
Despite being a major health concern, little is known about the pathophysiological changes that underly concussion. Nonetheless, emerging evidence suggests that selective damage to white matter axons, or diffuse axonal injury (DAI), disrupts brain network connectivity and function. While voltage-gated sodium channels (NaChs) and their anchoring proteins at the nodes of Ranvier (NOR) on axons are key elements of the brain's network signaling machinery, changes in their integrity have not been studied in context with DAI. Here, we utilized a clinically relevant swine model of concussion that induces evolving axonal pathology, demonstrated by accumulation of amyloid precursor protein (APP) across the white matter. Over a two-week follow-up post-concussion with this model, we found widespread loss of NaCh isoform 1.6 (Nav1.6), progressive increases in NOR length, the appearance of void and heminodes and loss of ßIV-spectrin, ankyrin G, and neurofascin 186 or their collective diffusion into the paranode. Notably, these changes were in close proximity, yet distinct from APP-immunoreactive swollen axonal profiles, potentially representing a unique, newfound phenotype of axonal pathology in DAI. Since concussion in humans is non-fatal, the clinical relevance of these findings was determined through examination of post-mortem brain tissue from humans with higher levels of acute traumatic brain injury. Here, a similar loss of Nav1.6 and changes in NOR structures in brain white matter were observed as found in the swine model of concussion. Collectively, this widespread and progressive disruption of NaChs and NOR appears to be a form of sodium channelopathy, which may represent an important substrate underlying brain network dysfunction after concussion.
Asunto(s)
Conmoción Encefálica , Lesiones Encefálicas , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Ancirinas/análisis , Ancirinas/metabolismo , Axones/patología , Conmoción Encefálica/patología , Lesiones Encefálicas/patología , Humanos , Isoformas de Proteínas/metabolismo , Nódulos de Ranvier/química , Nódulos de Ranvier/metabolismo , Nódulos de Ranvier/patología , Sodio/metabolismo , Canales de Sodio/análisis , Canales de Sodio/metabolismo , Espectrina/análisis , Espectrina/metabolismo , PorcinosRESUMEN
The acquired chronic demyelinating neuropathies include a growing number of disease entities that have characteristic, often overlapping, clinical presentations, mediated by distinct immune mechanisms, and responding to different therapies. After the discovery in the early 1980s, that the myelin associated glycoprotein (MAG) is a target antigen in an autoimmune demyelinating neuropathy, assays to measure the presence of anti-MAG antibodies were used as the basis to diagnose the anti-MAG neuropathy. The route was open for describing the clinical characteristics of this new entity as a chronic distal large fiber sensorimotor neuropathy, for studying its pathogenesis and devising specific treatment strategies. The initial use of chemotherapeutic agents was replaced by the introduction in the late 1990s of rituximab, a monoclonal antibody against CD20+ B-cells. Since then, other anti-B cells agents have been introduced. Recently a novel antigen-specific immunotherapy neutralizing the anti-MAG antibodies with a carbohydrate-based ligand mimicking the natural HNK-1 glycoepitope has been described.
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Autoantígenos/inmunología , Enfermedades Autoinmunes Desmielinizantes SNC/inmunología , Glicoproteína Asociada a Mielina/inmunología , Polirradiculoneuropatía/inmunología , Adenina/análogos & derivados , Adenina/uso terapéutico , Animales , Autoanticuerpos/sangre , Autoanticuerpos/inmunología , Subgrupos de Linfocitos B/inmunología , Antígenos CD57/inmunología , Enfermedades Autoinmunes Desmielinizantes SNC/diagnóstico , Enfermedades Autoinmunes Desmielinizantes SNC/terapia , Epítopos/inmunología , Trastornos Neurológicos de la Marcha/inmunología , Humanos , Inmunosupresores/uso terapéutico , Inmunoterapia , Lenalidomida/uso terapéutico , Mamíferos , Ratones , Imitación Molecular , Vaina de Mielina/química , Vaina de Mielina/inmunología , Vaina de Mielina/ultraestructura , Fibras Nerviosas Mielínicas/inmunología , Fibras Nerviosas Mielínicas/patología , Enfermedad Autoinmune Experimental del Sistema Nervioso/inmunología , Paraproteinemias/inmunología , Paraproteínas/inmunología , Piperidinas/uso terapéutico , Intercambio Plasmático , Polirradiculoneuropatía/diagnóstico , Polirradiculoneuropatía/terapia , Nódulos de Ranvier/química , Nódulos de Ranvier/inmunología , Ratas , Rituximab/uso terapéuticoRESUMEN
The paranodal junctions flank mature nodes of Ranvier and provide a barrier between ion channels at the nodes and juxtaparanodes. These junctions also promote node assembly and maintenance by mechanisms that are poorly understood. Here, we examine their role in the accumulation of NF186, a key adhesion molecule of PNS and CNS nodes. We previously showed that NF186 is initially targeted/accumulates via its ectodomain to forming PNS (hemi)nodes by diffusion trapping, whereas it is later targeted to mature nodes by a transport-dependent mechanism mediated by its cytoplasmic segment. To address the role of the paranodes in this switch, we compared accumulation of NF186 ectodomain and cytoplasmic domain constructs in WT versus paranode defective (i.e., Caspr-null) mice. Both pathways are affected in the paranodal mutants. In the PNS of Caspr-null mice, diffusion trapping mediated by the NF186 ectodomain aberrantly persists into adulthood, whereas the cytoplasmic domain/transport-dependent targeting is impaired. In contrast, accumulation of NF186 at CNS nodes does not undergo a switch; it is predominantly targeted to both forming and mature CNS nodes via its cytoplasmic domain and requires intact paranodes. Fluorescence recovery after photobleaching analysis indicates that the paranodes provide a membrane diffusion barrier that normally precludes diffusion of NF186 to nodes. Linkage of paranodal proteins to the underlying cytoskeleton likely contributes to this diffusion barrier based on 4.1B and ßII spectrin expression in Caspr-null mice. Together, these results implicate the paranodes as membrane diffusion barriers that regulate targeting to nodes and highlight differences in the assembly of PNS and CNS nodes.SIGNIFICANCE STATEMENT Nodes of Ranvier are essential for effective saltatory conduction along myelinated axons. A major question is how the various axonal proteins that comprise the multimeric nodal complex accumulate at this site. Here we examine how targeting of NF186, a key nodal adhesion molecule, is regulated by the flanking paranodal junctions. We show that the transition from diffusion-trapping to transport-dependent accumulation of NF186 requires the paranodal junctions. We also demonstrate that these junctions are a barrier to diffusion of axonal proteins into the node and highlight differences in PNS and CNS node assembly. These results provide new insights into the mechanism of node assembly and the pathophysiology of neurologic disorders in which impaired paranodal function contributes to clinical disability.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Ganglios Espinales/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Nódulos de Ranvier/metabolismo , Animales , Moléculas de Adhesión Celular/análisis , Células Cultivadas , Femenino , Ganglios Espinales/química , Ganglios Espinales/citología , Uniones Intercelulares/química , Uniones Intercelulares/metabolismo , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Factores de Crecimiento Nervioso/análisis , Nódulos de Ranvier/químicaRESUMEN
A high density of Na+ channels at nodes of Ranvier is necessary for rapid and efficient action potential propagation in myelinated axons. Na+ channel clustering is thought to depend on two axonal cell adhesion molecules that mediate interactions between the axon and myelinating glia at the nodal gap (i.e., NF186) and the paranodal junction (i.e., Caspr). Here we show that while Na+ channels cluster at nodes in the absence of NF186, they fail to do so in double conditional knockout mice lacking both NF186 and the paranodal cell adhesion molecule Caspr, demonstrating that a paranodal junction-dependent mechanism can cluster Na+ channels at nodes. Furthermore, we show that paranode-dependent clustering of nodal Na+ channels requires axonal ßII spectrin which is concentrated at paranodes. Our results reveal that the paranodal junction-dependent mechanism of Na+channel clustering is mediated by the spectrin-based paranodal axonal cytoskeleton.
Asunto(s)
Citoesqueleto/metabolismo , Nódulos de Ranvier/química , Canales de Sodio/análisis , Animales , Moléculas de Adhesión Celular Neuronal/genética , Moléculas de Adhesión Celular Neuronal/metabolismo , Ratones NoqueadosRESUMEN
The scaffolding protein ankyrin-G is required for Na(+) channel clustering at axon initial segments. It is also considered essential for Na(+) channel clustering at nodes of Ranvier to facilitate fast and efficient action potential propagation. However, notwithstanding these widely accepted roles, we show here that ankyrin-G is dispensable for nodal Na(+) channel clustering in vivo. Unexpectedly, in the absence of ankyrin-G, erythrocyte ankyrin (ankyrin-R) and its binding partner ßI spectrin substitute for and rescue nodal Na(+) channel clustering. In addition, channel clustering is also rescued after loss of nodal ßIV spectrin by ßI spectrin and ankyrin-R. In mice lacking both ankyrin-G and ankyrin-R, Na(+) channels fail to cluster at nodes. Thus, ankyrin R-ßI spectrin protein complexes function as secondary reserve Na(+) channel clustering machinery, and two independent ankyrin-spectrin protein complexes exist in myelinated axons to cluster Na(+) channels at nodes of Ranvier.
Asunto(s)
Ancirinas/análisis , Nódulos de Ranvier/química , Canales de Sodio/análisis , Espectrina/análisis , Animales , Ancirinas/metabolismo , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Nódulos de Ranvier/metabolismo , Ratas , Canales de Sodio/metabolismo , Espectrina/metabolismoRESUMEN
Neurons are highly polarized cells. They can be subdivided into at least two structurally and functionally distinct domains: somatodendritic and axonal domains. The somatodendritic domain receives and integrates upstream input signals, and the axonal domain generates and relays outputs in the form of action potentials to the downstream target. Demand for quick response to the harsh surroundings prompted evolution to equip vertebrates' neurons with a remarkable glia-derived structure called myelin. Not only Insulating the axon, myelinating glia also rearrange the axonal components and elaborate functional subdomains along the axon. Proper functioning of all theses domains and subdomains is vital for a normal, efficient nervous system.
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Axones/metabolismo , Vaina de Mielina/metabolismo , Nódulos de Ranvier/metabolismo , Animales , Axones/química , Evolución Biológica , Neuronas/química , Neuronas/citología , Neuronas/metabolismo , Canales de Potasio/metabolismo , Nódulos de Ranvier/química , Vertebrados/metabolismoRESUMEN
Active invasion of the dendritic tree by action potentials (APs) generated in the axon is essential for associative synaptic plasticity and neuronal ensemble formation. In cortical pyramidal cells (PCs), this AP back-propagation is supported by dendritic voltage-gated Na+ (Nav) channels, whose molecular identity is unknown. Using a highly sensitive electron microscopic immunogold technique, we revealed the presence of the Nav1.6 subunit in hippocampal CA1 PC proximal and distal dendrites. Here, the subunit density is lower by a factor of 35 to 80 than that found in axon initial segments. A gradual decrease in Nav1.6 density along the proximodistal axis of the dendritic tree was also detected without any labeling in dendritic spines. Our results reveal the characteristic subcellular distribution of the Nav1.6 subunit, identifying this molecule as a key substrate enabling dendritic excitability.
Asunto(s)
Región CA1 Hipocampal/química , Dendritas/química , Canales de Sodio/análisis , Potenciales de Acción , Animales , Axones/química , Axones/fisiología , Región CA1 Hipocampal/fisiología , Región CA1 Hipocampal/ultraestructura , Membrana Celular/química , Dendritas/fisiología , Dendritas/ultraestructura , Espinas Dendríticas/química , Técnica del Anticuerpo Fluorescente , Técnica de Fractura por Congelación , Inmunohistoquímica , Activación del Canal Iónico , Masculino , Microscopía Inmunoelectrónica , Canal de Sodio Activado por Voltaje NAV1.1 , Canal de Sodio Activado por Voltaje NAV1.6 , Proteínas del Tejido Nervioso/análisis , Nódulos de Ranvier/química , Ratas , Ratas WistarRESUMEN
In many mammalian neurons, dense clusters of ion channels at the axonal initial segment and nodes of Ranvier underlie action potential generation and rapid conduction. Axonal clustering of mammalian voltage-gated sodium and KCNQ (Kv7) potassium channels is based on linkage to the actin-spectrin cytoskeleton, which is mediated by the adaptor protein ankyrin-G. We identified key steps in the evolution of this axonal channel clustering. The anchor motif for sodium channel clustering evolved early in the chordate lineage before the divergence of the wormlike cephalochordate, amphioxus. Axons of the lamprey, a very primitive vertebrate, exhibited some invertebrate features (lack of myelin, use of giant diameter to hasten conduction), but possessed narrow initial segments bearing sodium channel clusters like in more recently evolved vertebrates. The KCNQ potassium channel anchor motif evolved after the divergence of lampreys from other vertebrates, in a common ancestor of shark and humans. Thus, clustering of voltage-gated sodium channels was a pivotal early innovation of the chordates. Sodium channel clusters at the axon initial segment serving the generation of action potentials evolved long before the node of Ranvier. KCNQ channels acquired anchors allowing their integration into pre-existing sodium channel complexes at about the same time that ancient vertebrates acquired myelin, saltatory conduction, and hinged jaws. The early chordate refinements in action potential mechanisms we have elucidated appear essential to the complex neural signaling, active behavior, and evolutionary success of vertebrates.
Asunto(s)
Axones/metabolismo , Cordados/metabolismo , Evolución Molecular , Canales Iónicos/metabolismo , Nódulos de Ranvier/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Axones/química , Cordados/clasificación , Cordados/genética , Humanos , Invertebrados/química , Invertebrados/clasificación , Invertebrados/genética , Invertebrados/metabolismo , Canales Iónicos/química , Canales Iónicos/genética , Datos de Secuencia Molecular , Filogenia , Nódulos de Ranvier/química , Nódulos de Ranvier/genética , Alineación de SecuenciaRESUMEN
BACKGROUND: Sodium channel (NaCh) expressions change following nerve and inflammatory lesions and this change may contribute to the activation of pain pathways. In a previous study we found a dramatic increase in the size and density of NaCh accumulations, and a remodeling of NaChs at intact and altered myelinated sites at a location just proximal to a combined partial axotomy and chromic suture lesion of the rat infraorbital nerve (ION) with the use of an antibody that identifies all NaCh isoforms. Here we evaluate the contribution of the major nodal NaCh isoform, Nav1.6, to this remodeling of NaChs following the same lesion. Sections of the ION from normal and ION lesioned subjects were double-stained with antibodies against Nav1.6 and caspr (contactin-associated protein; a paranodal protein to identify nodes of Ranvier) and then z-series of optically sectioned images were captured with a confocal microscope. ImageJ (NIH) software was used to quantify the average size and density of Nav1.6 accumulations, while additional single fiber analyses measured the axial length of the nodal gap, and the immunofluorescence intensity of Nav1.6 in nodes and of caspr in the paranodal region. RESULTS: The findings showed a significant increase in the average size and density of Nav1.6 accumulations in lesioned IONs when compared to normal IONs. The results of the single fiber analyses in caspr-identified typical nodes showed an increased axial length of the nodal gap, an increased immunofluorescence intensity of nodal Nav1.6 and a decreased immunofluorescence intensity of paranodal caspr in lesioned IONs when compared to normal IONs. In the lesioned IONs, Nav1.6 accumulations were also seen in association with altered caspr-relationships, such as heminodes. CONCLUSION: The results of the present study identify Nav1.6 as one isoform involved in the augmentation and remodeling of NaChs at nodal sites following a combined partial axotomy and chromic suture ION lesion. The augmentation of Nav1.6 may result from an alteration in axon-Schwann cell signaling mechanisms as suggested by changes in caspr expression. The changes identified in this study suggest that the participation of Nav1.6 should be considered when examining changes in the excitability of myelinated axons in neuropathic pain models.
Asunto(s)
Nervio Maxilar/lesiones , Nervio Maxilar/metabolismo , Órbita/inervación , Dolor/fisiopatología , Nódulos de Ranvier/metabolismo , Canales de Sodio/metabolismo , Animales , Axones/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Microscopía Confocal , Canal de Sodio Activado por Voltaje NAV1.6 , Dolor/etiología , Isoformas de Proteínas/metabolismo , Nódulos de Ranvier/química , Ratas , Ratas Sprague-DawleyRESUMEN
At axon initial segments and nodes of Ranvier in neurons, the spectrin membrane skeleton plays roles in physically stabilizing the plasma membrane integrity and in clustering voltage-gated sodium channels for proper conduction of the action potential. betaIV-Spectrin, an essential component of the membrane skeleton at these sites, has an N-terminal-truncated isoform, Sigma6, which is expressed at much higher levels than the full-length isoform Sigma1. To investigate the role of betaIV-spectrin Sigma6, we generated Sigma1-deficient mice with a normal level of Sigma6 expression (Sigma1(-/-) mice), and compared their phenotypes with those of previously generated mice lacking both Sigma1 and Sigma6(Sigma1Sigma6(-/-) mice). The gross neurological defects observed in Sigma1Sigma6(-/-) mice, such as hindleg contraction, were apparently ameliorated in Sigma1(-/-) mice. At cellular levels, Sigma1Sigma6(-/-) and Sigma1(-/-) neurons similarly exhibited waving and swelling of the plasma membrane at axon initial segments and nodes of Ranvier. By contrast, the levels of ankyrin G and voltage-gated sodium channels at these sites, which are significantly reduced in Sigma1Sigma6(-/-) mice, were substantially recovered in Sigma1(-/-) mice. We conclude that the truncated betaIV-spectrin isoform Sigma6 plays a specific role in clustering voltage-gated sodium channels, whereas it is dispensable for membrane stabilization at axon initial segments and nodes of Ranvier.
Asunto(s)
Axones/química , Nódulos de Ranvier/química , Canales de Sodio/metabolismo , Espectrina/fisiología , Animales , Citoesqueleto , Ratones , Ratones Noqueados , Ratones Mutantes , Neuronas , Fenotipo , Isoformas de Proteínas , Espectrina/análisisRESUMEN
The axon initial segment (AIS) of pyramidal cells is a critical region for the generation of action potentials and for the control of pyramidal cell activity. Here we show that Na+ and K+ voltage-gated channels, together with other molecules involved in the localization of ion channels, are distributed asymmetrically in the AIS of pyramidal cells situated in the human temporal neocortex. There is a high density of Na+ channels distributed along the length of the AIS together with the associated proteins spectrin betaIV and ankyrin G. In contrast, Kv1.2 channels are associated with the adhesion molecule Caspr2, and they are mostly localized to the distal region of the AIS. In general, the distal region of the AIS is targeted by the GABAergic axon terminals of chandelier cells, whereas the proximal region is innervated, mostly by other types of GABAergic interneurons. We suggest that this molecular segregation and the consequent regional specialization of the GABAergic input to the AIS of pyramidal cells may have important functional implications for the control of pyramidal cell activity.
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Axones/química , Corteza Cerebral/citología , Canales de Potasio con Entrada de Voltaje/análisis , Células Piramidales/química , Canales de Sodio/análisis , Adulto , Ancirinas/análisis , Ancirinas/metabolismo , Axones/metabolismo , Células Cultivadas , Corteza Cerebral/metabolismo , Proteínas Transportadoras de GABA en la Membrana Plasmática/análisis , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Humanos , Canal de Potasio Kv.1.2/análisis , Canal de Potasio Kv.1.2/metabolismo , Proteínas de la Membrana/análisis , Proteínas de la Membrana/metabolismo , Persona de Mediana Edad , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/metabolismo , Canales de Potasio con Entrada de Voltaje/metabolismo , Terminales Presinápticos/química , Células Piramidales/metabolismo , Nódulos de Ranvier/química , Canales de Sodio/metabolismo , Espectrina/análisis , Espectrina/metabolismoRESUMEN
Nodes of Ranvier are regularly placed, nonmyelinated axon segments along myelinated nerves. Here we show that nodal membranes isolated from the central nervous system (CNS) of mammals restricted neurite outgrowth of cultured neurons. Proteomic analysis of these membranes revealed several inhibitors of neurite outgrowth, including the oligodendrocyte myelin glycoprotein (OMgp). In rat spinal cord, OMgp was not localized to compact myelin, as previously thought, but to oligodendroglia-like cells, whose processes converge to form a ring that completely encircles the nodes. In OMgp-null mice, CNS nodes were abnormally wide and collateral sprouting was observed. Nodal ensheathment in the CNS may stabilize the node and prevent axonal sprouting.
Asunto(s)
Axones/fisiología , Extensiones de la Superficie Celular/fisiología , Neuritas/fisiología , Neuroglía/fisiología , Neuroglía/ultraestructura , Nódulos de Ranvier/fisiología , Animales , Antígenos/análisis , Axones/ultraestructura , Bovinos , Extensiones de la Superficie Celular/química , Extensiones de la Superficie Celular/ultraestructura , Células Cultivadas , Proteínas Ligadas a GPI , Ganglios Espinales/fisiología , Ganglios Espinales/ultraestructura , Humanos , Ratones , Proteínas de la Mielina , Vaina de Mielina/química , Glicoproteína Asociada a Mielina/análisis , Glicoproteína Mielina-Oligodendrócito , Neuritas/ultraestructura , Neuroglía/química , Oligodendroglía/química , Oligodendroglía/fisiología , Oligodendroglía/ultraestructura , Proteoglicanos/análisis , Proteómica , Nódulos de Ranvier/química , Nódulos de Ranvier/ultraestructura , Ratas , Médula Espinal/citologíaRESUMEN
Molecular mimicry of gangliosides by Campylobacter jejuni lipooligosaccharides (LOSs) in the induction of anti-ganglioside antibodies has been hypothesised to contribute to GBS development. Rabbits were immunised with ganglioside-mimicking C. jejuni LOSs and anti-LOS responses were analysed using passive haemagglutination, and anti-ganglioside responses by enzyme-linked immunosorbent assay and thin-layer chromatography with immunostaining. High titres of anti-LOS antibodies were demonstrated in rabbit antisera that were cross-reactive with a panel of gangliosides. Non-ganglioside-mimicking C. jejuni HS:3 LOS induced a strong anti-LOS response, but no anti-ganglioside antibodies. Control rabbit antisera had no anti-LOS or -ganglioside responses. Moreover, IgG from a patient treated with parenteral gangliosides, who exhibited Guillain-Barré syndrome, had antibodies reactive with C. jejuni LOS. Biotinylated IgG fractions from the rabbit and the patient sera recognised epitopes at the nodes of Ranvier in sectioned human nerves, whereas fractions from controls did not. This study demonstrates that immunisation with ganglioside-mimicking C. jejuni LOS triggers the production of cross-reactive anti-ganglioside antibodies that recognise epitopes at the nodes of Ranvier.
Asunto(s)
Anticuerpos Antibacterianos/biosíntesis , Sitios de Unión de Anticuerpos , Campylobacter jejuni/inmunología , Epítopos/metabolismo , Gangliósido G(M1)/inmunología , Lipopolisacáridos/inmunología , Nódulos de Ranvier/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/metabolismo , Reacciones Cruzadas , Epítopos/inmunología , Síndrome de Guillain-Barré/inmunología , Síndrome de Guillain-Barré/microbiología , Humanos , Inmunoglobulina G/biosíntesis , Inmunohistoquímica , Lipopolisacáridos/aislamiento & purificación , Lipopolisacáridos/metabolismo , Imitación Molecular/inmunología , Conejos , Nódulos de Ranvier/química , Nódulos de Ranvier/metabolismoRESUMEN
A number of studies have investigated electrophysiological and morphological changes of peripheral nerves during gradual elongation. There has been, however, no report on the distribution of sodium channels at Ranvier's nodes during peripheral nerve elongation. We investigated peripheral nerve injury after the gradual elongation of rat sciatic nerves. Indirect nerve elongation was induced by leg lengthening at a rate of 3 mm/day by 15 or 30 mm. At 7 days after the leg lengthening, the electrophysiological properties of sciatic nerves, the ultrastructures of the Ranvier's nodes and axons, and the distribution of voltage-dependent sodium channels were examined. In the control nerves, most sodium channels were localized at Ranvier's nodes in myelinated axons, providing the physiological basis of saltatory conduction. In the elongated nerves, both the amplitude and conduction velocity of compound nerve action potential decreased following leg lengthening. The elongated nerves also showed paranodal demyelination in Ranvier's nodes longer than those in the control group. In addition, the distribution of sodium channels became diffuse or disappeared at Ranvier's nodes of elongated nerves. The diffuse distribution and/or disappearance of sodium channels may underlie the electrophysiological changes in compound nerve action potential induced by nerve elongation.
Asunto(s)
Nervios Periféricos/cirugía , Nódulos de Ranvier/ultraestructura , Canales de Sodio , Animales , Alargamiento Óseo , Masculino , Microscopía Electrónica , Procedimientos Ortopédicos , Nódulos de Ranvier/química , Ratas , Ratas Wistar , Canales de Sodio/análisisRESUMEN
The sensory root entry zone demarcates the transition from the peripheral nervous system (PNS) to the central nervous system (CNS). In this study, we describe the organization of nodes of Ranvier at the trigeminal sensory and dorsal root entry zones of the rat. Caspr immunoreactivity (IR) was used to identify the paranodal region of nodes of Ranvier, while L-MAG-IR was used to identify CNS oligodendrocytes. Immunofluorescence confocal microscopy revealed a dense aggregation of nodes precisely at the PNS to CNS transition with prominent node-depleted zones on either side, while L-MAG-IR was confined to ensheathing fibers on the central side of nodes located in this dense band and identified these as transitional nodes. Morphometric analysis of the PNS and CNS sides of the trigeminal and the PNS side of the dorsal root entry zones confirmed the presence of virtually node-free domains flanking the transitional zone. Further, the reappearance of nodes on the far side of the node-free zones strongly correlated with nodal diameter, with small nodes reappearing first. These findings suggest that the PNS/CNS transition may represent the initial site of myelination of the primary afferent axon within this area.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/análisis , Ganglios Espinales/química , Nódulos de Ranvier/química , Ganglio del Trigémino/química , Animales , Moléculas de Adhesión Celular Neuronal/fisiología , Agregación Celular/fisiología , Femenino , Ganglios Espinales/fisiología , Nódulos de Ranvier/fisiología , Ratas , Ratas Sprague-Dawley , Ganglio del Trigémino/fisiologíaRESUMEN
High densities of sodium channels at nodes of Ranvier permit action potential conduction and depend on betaIV spectrins, a family of scaffolding proteins linked to the cortical actin cytoskeleton. To investigate the molecular organization of nodes, we analyzed qv(3J)"quivering" mice, whose betaIV spectrins have a truncated proline-rich "specific" domain (SD) and lack the pleckstrin homology (PH) domain. Central nodes of qv(3J) mice, which lack betaIV spectrins, are significantly broader and have prominent vesicle-filled nodal membrane protrusions, whereas axon shape and neurofilament density are dramatically altered. PNS qv(3J) nodes, some with detectable betaIV spectrins, are less affected. In contrast, a larger truncation of betaIV spectrins in qv(4J) mice, deleting the SD, PH, and ankyrinG binding domains, causes betaIV spectrins to be undetectable and causes dramatic changes, even in peripheral nodes. These results show that quivering mutations disrupt betaIV spectrin retention and stability at nodes and that distinct protein domains regulate nodal structural integrity and molecular organization.
Asunto(s)
Proteínas del Tejido Nervioso/fisiología , Nódulos de Ranvier/ultraestructura , Espectrina/fisiología , Potenciales de Acción , Animales , Ancirinas/análisis , Ancirinas/metabolismo , Sistema Nervioso Central/citología , Citoesqueleto/ultraestructura , Filamentos Intermedios/ultraestructura , Proteínas de la Membrana/análisis , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes Neurológicos , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Sistema Nervioso Periférico/citología , Fenotipo , Estructura Terciaria de Proteína , Nódulos de Ranvier/química , Espectrina/química , Espectrina/genéticaRESUMEN
Voltage-gated sodium channels interact with cytosolic proteins that regulate channel trafficking and/or modulate the biophysical properties of the channels. Na(v)1.6 is heavily expressed at the nodes of Ranvier along adult CNS and PNS axons and along unmyelinated fibers in the PNS. In an initial yeast two-hybrid screen using the C terminus of Na(v)1.6 as a bait, we identified FHF2B, a member of the FGF homologous factor (FHF) subfamily, as an interacting partner of Na(v)1.6. Members of the FHF subfamily share approximately 70% sequence identity, and individual members demonstrate a cell- and tissue-specific expression pattern. FHF2 is abundantly expressed in the hippocampus and DRG neurons and colocalizes with Na(v)1.6 at mature nodes of Ranvier in myelinated sensory fibers in the dorsal root of the sciatic nerve. However, retinal ganglion cells and spinal ventral horn motor neurons show very low levels of FHF2 expression, and their axons exhibit no nodal FHF2 staining within the optic nerve and ventral root, respectively. Thus, FHF2 is selectively localized at nodes of dorsal root sensory but not ventral root motor axons. The coexpression of FHF2B and Na(v)1.6 in the DRG-derived cell line ND7/23 significantly increases the peak current amplitude and causes a 4 mV depolarizing shift of voltage-dependent inactivation of the channel. The preferential expression of FHF2B in sensory neurons may provide a basis for physiological differences in sodium currents that have been reported at the nodes of Ranvier in sensory versus motor axons.
Asunto(s)
Factores de Crecimiento de Fibroblastos/metabolismo , Ganglios Espinales/química , Hipocampo/química , Proteínas del Tejido Nervioso/metabolismo , Neuronas Aferentes/química , Nódulos de Ranvier/química , Canales de Sodio/metabolismo , Animales , Células del Asta Anterior/química , Axones/química , Axones/ultraestructura , Química Encefálica , Células Cultivadas/química , Factores de Crecimiento de Fibroblastos/análisis , Factores de Crecimiento de Fibroblastos/genética , Ganglios Espinales/citología , Humanos , Ratones , Canal de Sodio Activado por Voltaje NAV1.6 , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/genética , Neuronas Aferentes/fisiología , Especificidad de Órganos , Unión Proteica , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Células Ganglionares de la Retina/química , Nervio Ciático/química , Nervio Ciático/citología , Canales de Sodio/análisis , Canales de Sodio/genética , Transfección , Técnicas del Sistema de Dos HíbridosRESUMEN
The axoglial paranodal junction is essential for the proper localization of ion channels around the node of Ranvier. The integrity of this junction is important for nerve conduction. Although recent studies have made significant progress in understanding the molecular composition of the paranodal junction, it is not known how these membrane components are distributed to the appropriate sites and interact with each other. Here we show that CD9, a member of the tetraspanin family, is present at the paranode. CD9 is concentrated in the paranode as myelination proceeds, but CD9 clusters become diffuse, associated with disruption of the paranode, in cerebroside sulfotransferase-deficient mice. Immunohistochemical and Western blot analysis showed that CD9 is distributed predominantly in the PNS. Ablation of CD9 in mutant mice disrupts junctional attachment at the paranode and alters the paranodal components contactin-associated protein (also known as Paranodin) and neurofascin 155, although the frequency of such abnormalities varies among individuals and individual axons even in the same mouse. Electron micrographs demonstrated that compact myelin sheaths were also affected in the PNS. Therefore, CD9 is a myelin protein important for the formation of paranodal junctions. CD9 also plays a role in the formation of compact myelin in the PNS.
Asunto(s)
Antígenos CD/análisis , Antígenos CD/fisiología , Glicoproteínas de Membrana/análisis , Glicoproteínas de Membrana/fisiología , Nódulos de Ranvier/química , Nódulos de Ranvier/ultraestructura , Animales , Antígenos CD/genética , Glicoproteínas de Membrana/genética , Ratones , Ratones Noqueados , Vaina de Mielina/fisiología , Vaina de Mielina/ultraestructura , Sistema Nervioso Periférico/química , Sistema Nervioso Periférico/citología , Tetraspanina 29RESUMEN
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.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/análisis , Nódulos de Ranvier/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Axones/ultraestructura , Moléculas de Adhesión Celular Neuronal/química , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Neuroglía/ultraestructura , Nódulos de Ranvier/ultraestructura , Alineación de SecuenciaRESUMEN
The rapid, efficient, and faithful propagation of action potentials in myelinated nerve fibers depends on the appropriate complement and localization of ion channels. Recent work has suggested that specific voltage-dependent sodium (Nav) channel isoforms are differentially regulated both spatially and temporally in a myelin-dependent manner. Since the principal site of axoglial contact occurs at the paranode, we postulated that disrupted paranodal structure might result in altered nodal Nav channel isoform localization and clustering. We have used UDP-galactose/ceramide galactosyl transferase (CGT)-deficient mice, which form compact myelin and paranodal loops but lack the transverse bands normally found at the interface of the axon and overlying glial cell, to determine if this structure contributes to the signaling machinery responsible for clustering and localization of distinct Nav channel isoforms. We find that as in control animals, most mutant nodes of Ranvier had Nav1.6 in high-density clusters in the peripheral and central nervous systems; the localization of Nav1.2 and the protein levels of Nav1.2 and Nav1.6 were also normal in the CGT-deficient mouse. However, with increasing age, in the mutant mouse we observed a decrease in the total number of nodal Nav1.6 clusters, a decrease in the density of Nav1.6 channels at nodes, and an increase in the average size of the Nav1.6 clusters. Thus, transverse bands are not required for Nav1.6 clustering and localization at nodes or for exclusion of Nav1.2 from myelinated nerve fibers, but are required for the maintenance of nodal Nav1.6 cluster size and density.