The evolutionary demand for rapid nerve impulse conduction led to the process of myelinationdependent organization of axons into distinct molecular domains. These domains include the node of Ranvier flanked by highly specialized paranodal domains where myelin loops and axolemma orchestrate the axoglial septate junctions. These junctions are formed by interactions between a glial isoform of neurofascin (Nfasc NF155 ) and axonal Caspr and Cont. Here we report the generation of myelinating glia-specific Nfasc NF155 null mouse mutants. These mice exhibit severe ataxia, motor paresis, and death before the third postnatal week. In the absence of glial Nfasc NF155 , paranodal axoglial junctions fail to form, axonal domains fail to segregate, and myelinated axons undergo degeneration. Electrophysiological measurements of peripheral nerves from Nfasc NF155 mutants revealed dramatic reductions in nerve conduction velocities. By using inducible PLP-CreER recombinase to ablate Nfasc NF155 in adult myelinating glia, we demonstrate that paranodal axoglial junctions disorganize gradually as the levels of Nfasc NF155 protein at the paranodes begin to drop. This coincides with the loss of the paranodal region and concomitant disorganization of the axonal domains. Our results provide the first direct evidence that the maintenance of axonal domains requires the fence function of the paranodal axoglial junctions. Together, our studies establish a central role for paranodal axoglial junctions in both the organization and the maintenance of axonal domains in myelinated axons. The anatomical organization of myelinated axons into distinct molecular domains is the basis for rapid propagation of action potentials in a saltatory manner (Hartline and Colman, 2007). Although the signal transduction mechanisms that underlie the axonal organization into specific domains (i.e., the node, the paranode, the juxtaparanode, and the internode) are poorly understood, considerable progress has been made in identifying key molecular components within these axonal domains. The paranodal region is unique in its organization and ultrastructural characteristics and contains specialized axoglial junctions referred to as the paranodal axoglial septate junctions, which resemble the ladder-like invertebrate septate junctions (Einheber et al., 1997;Pedraza et al., 2001; Banerjee et al., 2006a, b). Three major paranodal proteins have been identified: Caspr or paranodin (Einheber et al., 1997;Menegoz et al., 1997;Peles et al., 1997;Bhat et al., 2001), and a GPI-anchored neural cell adhesion molecule Contactin (Cont;Berglund et al., 1999;Boyle et al., 2001) on the axonal side, and the 155-kDa neurofascin isoform (Nfasc NF155 ) on the glial side (Tait et al., 2000;Charles et al., 2002). Although Nfasc NF155 is the only known glial paranodal protein, many proteins expressed in myelinating glia are required at some level in the formation and/or stability of the paranodal junctions (Coetzee et al., 1996;Griffiths et al., 1998;Ishibashi et al., 2002; LappeSie...

Axonal insulation is critical for efficient action potential propagation and normal functioning of the nervous system. In Drosophila, the underlying basis of nerve ensheathment is the axonal insulation by glial cells and the establishment of septate junctions (SJs) between glial cell membranes. However, the details of the cellular and molecular mechanisms underlying axonal insulation and SJ formation are still obscure. Here, we report the characterization of axonal insulation in the Drosophila peripheral nervous system (PNS). Targeted expression of tau-green fluorescent protein in the glial cells and ultrastructural analysis of the peripheral nerves allowed us to visualize the glial ensheathment of axons. We show that individual or a group of axons are ensheathed by inner glial processes, which in turn are ensheathed by the outer perineurial glial cells. SJs are formed between the inner and outer glial membranes. We also show that Neurexin IV, Contactin, and Neuroglian are coexpressed in the peripheral glial membranes and that these proteins exist as a complex in the Drosophila nervous system. Mutations in neurexin IV, contactin, and neuroglian result in the disruption of blood-nerve barrier function in the PNS, and ultrastructural analyses of the mutant embryonic peripheral nerves show loss of glial SJs. Interestingly, the murine homologs of Neurexin IV, Contactin, and Neuroglian are expressed at the paranodal SJs and play a key role in axon-glial interactions of myelinated axons. Together, our data suggest that the molecular machinery underlying axonal insulation and axon-glial interactions may be conserved across species.

Axo-glial junctions (AGJs) play a critical role in the organization and maintenance of molecular domains in myelinated axons. Neurexin IV͞Caspr1͞paranodin (NCP1) is an important player in the formation of AGJs because it recruits a paranodal complex implicated in the tethering of glial proteins to the axonal membrane and cytoskeleton. Mice deficient in either the axonal protein NCP1 or the glial ceramide galactosyltransferase (CGT) display disruptions in AGJs and severe ataxia. In this article, we correlate these two phenotypes and show that both NCP1 and CGT mutants develop large swellings accompanied by cytoskeletal disorganization and degeneration in the axons of cerebellar Purkinje neurons. We also show that ␣II spectrin is part of the paranodal complex and that, although not properly targeted, this complex is still formed in CGT mutants. Together, these findings establish a physiologically relevant link between AGJs and axonal cytoskeleton and raise the possibility that some neurodegenerative disorders arise from disruption of the AGJs.myelin ͉ paranodes ͉ cerebellum ͉ ataxia T he anatomical organization of myelinated fibers into distinct domains is the basis for the saltatory mode of action potential propagation. In the axons, these molecular domains (internode, juxtaparanode, paranode, and node of Ranvier) form as a result of specific polarization driven by signaling between the myelinating glial cells and neurons that has yet to be fully understood. In the paranodal region, closely apposed axon-glial membranes form specialized cell junctions, which resemble the ladder-like invertebrate septate junctions, and are referred to as paranodal septate junctions or paranodal axo-glial junctions (AGJs) (1-4).Three major proteins have been shown to localize to the paranodal AGJs: NCP1 (also known as Caspr1 or paranodin) and contactin (CNTN) on the axonal side and neurofascin (NF155), the 155-kDa isoform on the glial side (5-9). Although NF155 is the only known glial protein at the paranodal membrane, a number of nonparanodal glial proteins are required for proper formation, maintenance, and distribution of AGJs, as in the case of ceramide galactosyltransferase (CGT), proteolipid protein, myelin-basic protein, myelin-associated glycoprotein, 2Ј,3Ј-cyclic nucleotide 3Ј-phosphodiesterase, and the transcription factor Nkx6-2 (10-17).Genetic ablation of NCP1 and CNTN in mice results in the loss of AGJs and a failure to segregate Na ϩ and K ϩ channels at the nodes and juxtaparanodes, respectively (5, 6). Similar phenotypes were observed at the paranodes in CGT mutants (18,19). CGT encodes an enzyme that is needed for the biosynthesis of two important myelin lipids, galactocerebroside and sulfatide (10,(18)(19)(20)(21). Using subcellular fractionation of NF155 in detergents, Rasband and coworkers (22) proposed a model in which myelin lipids assemble in stable lipid rafts to stabilize the clustering of NF155 at the glial side of AGJs. This model is consistent with the phenotype of CGT mutants in which defective biosynthes...

Accumulation of voltage gated sodium (Nav) channels at nodes of Ranvier is paramount for action potential propagation along myelinated fibers, yet the mechanisms governing nodal development, organization and stabilization remain unresolved. Here, we report that genetic ablation of the neuron-specific isoform of Neurofascin (NfascNF186) in vivo results in nodal disorganization, including loss of Nav channel and ankyrin-G (AnkG) enrichment at nodes in the peripheral (PNS) and central (CNS) nervous systems. Interestingly, the presence of paranodal domains failed to rescue nodal organization in the PNS and the CNS. Most importantly, using ultrastructural analysis, we demonstrate that the paranodal domains invade the nodal space in NfascNF186 mutant axons and occlude node formation. Our results suggest that NfascNF186-dependent assembly of the nodal complex acts as a molecular boundary to restrict the movement of flanking paranodal domains into the nodal area, thereby facilitating the stereotypic axonal domain organization and saltatory conduction along myelinated axons.

The formation of paranodal axo-glial junctions is critical for the rapid and efficient propagation of nerve impulses. Genetic ablation of genes encoding the critical paranodal proteins Caspr, contactin (Cont), and the myelinating glia-specific isoform of Neurofascin (Nfasc NF155 ) results in the disruption of the paranodal axo-glial junctions, loss of ion channel segregation, and impaired nerve conduction, but the mechanisms regulating their interactions remain elusive. Here, we report that loss of immunoglobulin (Ig) domains 5 and 6 in Nfasc NF155 in mice phenocopies complete ablation of Nfasc NF155. The mutant mice lack paranodal septate junctions, resulting in the diffusion of Caspr and Cont from the paranodes, and redistribution of the juxtaparanodal potassium channels toward the nodes. Although critical for Nfasc NF155 function, we find that Ig5-6 are dispensable for nodal Nfasc NF186 function. Moreover, in vitro binding assays using Ig5-6 deletion constructs reveal their importance for the association of Nfasc NF155 with Cont. These findings provide the first molecular evidence demonstrating domain-specific requirements controlling the association of the paranodal tripartite complex in vivo. Our studies further emphasize that in vivo structure/function analysis is necessary to define the unique protein-protein interactions that differentially regulate the functions of Neurofascins during axonal domain organization.

The afferent innervation contacting the type I hair cells of the vestibular sensory epithelia form distinct calyceal synapses. The apposed presynaptic and postsynaptic membranes at this large area of synaptic contact are kept at a remarkably regular distance. Here, we show by freeze-fracture electron microscopy that a patterned alignment of proteins at the calyceal membrane resembles a type of intercellular junction that is rare in vertebrates, the septate junction (SJ). We found that a core molecular component of SJs, Caspr, colocalizes with the K ϩ channel KCNQ4 at the postsynaptic membranes of these calyceal synapses. Immunolabeling and ultrastructural analyses of Caspr knock-out mice reveal that, in the absence of Caspr, the separation between the membranes of the hair cells and the afferent neurons is conspicuously irregular and often increased by an order of magnitude. In these mutants, KCNQ4 fails to cluster at the postsynaptic membrane and appears diffused along the entire calyceal membrane. Our results indicate that a septate-like junction provides structural support to calyceal synaptic contact with the vestibular hair cell and that Caspr is required for the recruitment or retention of KCNQ4 at these synapses.

Myelinated axons are endowed with a specialized domain structure that is essential for saltatory action potential conduction. The paranodal domain contains the axoglial junctions and displays a unique ultrastructure that resembles the invertebrate septate junctions (SJs). Biochemical characterizations of the paranodal axoglial SJs have identified several molecular components that include Caspr and contactin (Cont) on the axonal side and neurofascin 155 kDa (NF155) isoform on the glial side. All these proteins are essential for the formation of the axoglial SJs. Based on the interactions between Caspr and Cont and their colocalization in the CA1 synaptic areas, it was proposed that the synaptic function of Cont requires Caspr. Here we have extended the phenotypic analysis of CASPR mutants to address further the role of Caspr at the axoglial SJs and also in axonal orientation and synaptic plasticity. We report that, in CASPR mutants, the smooth endoplasmic reticulum (SER) forms elongated membranous complexes that accumulate at the nodal/ paranodal region and stretch into the juxtaparanodal region, a defect that is consistent with the paranodal disorganization. We show that the cerebellar microorganization is unaffected in CASPR mutants. We also demonstrate that Caspr function is not essential for normal CA1 synaptic transmission and plasticity. Taken together with previous findings, our results highlight that the Caspr/ Cont complex is essential for the formation of axoglial SJs, whereas Cont may regulate axonal orientation and synaptic plasticity independent of its association with Caspr. In vertebrates, myelination provides axonal insulation and organizes axons into unique domains that are distinct in their molecular composition (Bhat, 2003a;Salzer, 2003;Schafer and Rasband, 2006). This domain organization allows myelinated axons to propagate action potentials in a saltatory manner. The molecular mechanisms that underlie the organization of these domains are still poorly understood. Over the past decade, many molecules have been identified that specifically localize to nodes of Ranvier, the paranodes, and the juxtaparanodes (Poliak and Peles, 2003). The paranodal region of the myelinated axons displays specialized structural attributes with ladder-like junctions between the myelin loops and the axolemma (Rosenbluth, 1999;Pedraza et al., 2001;Scherer and Arroyo, 2002). These axoglial junctions resemble the SJs present in invertebrates (Banerjee et al., 2006b). Surprisingly, the molecular components identified at the vertebrate paranodal axoglial SJs are also present at the Drosophila neuronal SJs (Banerjee et al., 2006a). KeywordsThe initial insight into the function of the paranodal axoglial SJs came from the phenotypic analysis of the UDP galactose: ceramide galactosyltransferase (CGT) mutants (Coetzee et al., 1996). CGT mutants are deficient in the production of galactocerebroside and sulfatide and display disruption of the axoglial SJs (Coetzee et al., 1996;Dupree et al., 1998Dupree et al., , 1999. Re...

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