Neurofascin 140 is an embryonic neuronal neurofascin isoform that promotes the assembly of the node of Ranvier.

Rapid nerve conduction in myelinated nerves requires the clustering of voltage-gated sodium channels at nodes of Ranvier. The Neurofascin (Nfasc) gene has a unique role in node formation because it encodes glial and neuronal isoforms of neurofascin (Nfasc155 and Nfasc186, respectively) with key functions in assembling the nodal macromolecular complex. A third neurofascin, Nfasc140, has also been described; however, neither the cellular origin nor function of this isoform was known. Here we show that Nfasc140 is a neuronal protein strongly expressed during mouse embryonic development. Expression of Nfasc140 persists but declines during the initial stages of node formation, in contrast to Nfasc155 and Nfasc186, which increase. Nevertheless, Nfasc140, like Nfasc186, can cluster voltage-gated sodium channels (Nav) at the developing node of Ranvier and can restore electrophysiological function independently of Nfasc155 and Nfasc186. This suggests that Nfasc140 complements the function of Nfasc155 and Nfasc186 in initial stages of the assembly and stabilization of the nodal complex. Further, Nfasc140 is reexpressed in demyelinated white matter lesions of postmortem brain tissue from human subjects with multiple sclerosis. This expands the critical role of the Nfasc gene in the function of myelinated axons and reveals further redundancy in the mechanisms required for the formation of this crucial structure in the vertebrate nervous system.

Identity, developmental restriction and reactivity of extralaminar cells capping mammalian neuromuscular junctions.

Neuromuscular junctions (NMJs) are normally thought to comprise three major cell types: skeletal muscle fibres, motor neuron terminals and perisynaptic terminal Schwann cells. Here we studied a fourth population of junctional cells in mice and rats, revealed using a novel cytoskeletal antibody (2166). These cells lie outside the synaptic basal lamina but form caps over NMJs during postnatal development. NMJ-capping cells also bound rPH, HM-24, CD34 antibodies and cholera toxin B subunit. Bromodeoxyuridine incorporation indicated activation, proliferation and spread of NMJ-capping cells following denervation in adults, in advance of terminal Schwann cell sprouting. The NMJ-capping cell reaction coincided with expression of tenascin-C but was independent of this molecule because capping cells also dispersed after denervation in tenascin-C-null mutant mice. NMJ-capping cells also dispersed after local paralysis with botulinum toxin and in atrophic muscles of transgenic R6/2 mice. We conclude that NMJ-capping cells (proposed name 'kranocytes') represent a neglected, canonical cellular constituent of neuromuscular junctions where they could play a permissive role in synaptic regeneration.

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.

Neurofascins are required to establish axonal domains for saltatory conduction.

Voltage-gated sodium channels are concentrated in myelinated nerves at the nodes of Ranvier flanked by paranodal axoglial junctions. Establishment of these essential nodal and paranodal domains is determined by myelin-forming glia, but the mechanisms are not clear. Here, we show that two isoforms of Neurofascin, Nfasc155 in glia and Nfasc186 in neurons, are required for the assembly of these specialized domains. In Neurofascin-null mice, neither paranodal adhesion junctions nor nodal complexes are formed. Transgenic expression of Nfasc155 in the myelinating glia of Nfasc-/- nerves rescues the axoglial adhesion complex by recruiting the axonal proteins Caspr and Contactin to the paranodes. However, in the absence of Nfasc186, sodium channels remain diffusely distributed along the axon. Our study shows that the two major Neurofascins play essential roles in assembling the nodal and paranodal domains of myelinated axons; therefore, they are essential for the transition to saltatory conduction in developing vertebrate nerves.

The tetraspanin protein, CD9, is expressed by progenitor cells committed to oligodendrogenesis and is linked to beta1 integrin, CD81, and Tspan-2.

Previous studies identified the tetraspanin protein CD9 in myelinating oligodendrocytes. The present report extends these observations by identifying CD9 in a subpopulation of oligodendrocyte progenitor cells (OPCs) and in premyelinating oligodendrocytes in rodents. NG2-positive cells expressed CD9 in a temporal and spatial pattern during development that was consistent with CD9 expression in OPCs just prior to their differentiation into premyelinating oligodendrocytes. NG2-positive cells in mature brains were CD9-negative. CD9 expression during oligodendrocyte development in vitro supported this hypothesis, as all CD9-positive cells became O4-positive when switched to oligodendrocyte differentiating media. CD9 immunoreactivity was enriched in myelinating oligodendrocytes and their processes, and the outer aspects of myelin internodes. Immunoprecipitation of CD9 from postnatal rat cerebrum coprecipitated beta1 integrin, CD81, and Tspan-2, another tetraspanin protein recently identified in oligodendrocytes. Following surface biotinylation of oligodendrocytes in vitro, biotinylated beta1 integrin was identified in a CD9 immunoprecipitate. These data support a molecular link between surface integrins and a CD9, Tspan-2 molecular web during the differentiation of oligodendrocytes. Oligodendrocyte production and myelination appears to be normal in CD9-deficient mice. These data support the hypothesis that CD9 helps form the tetraspanin web beneath the plasma membranes of progenitor cells committed to oligodendrogenesis, but that CD9 is not essential for oligodendrogenesis and myelination.