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Axons undergo striking changes in their content and distribution of cell adhesion molecules (CAMs) and ion channels during myelination that underlies the switch from continuous to saltatory conduction. These changes include the removal of a large cohort of uniformly distributed CAMs that mediate initial axon-Schwann cell interactions and their replacement by a subset of CAMs that mediate domain-specific interactions of myelinated fibers. Here, using rodent models, we examine the mechanisms and significance of this removal of axonal CAMs. We show that Schwann cells just prior to myelination locally activate clathrin-mediated endocytosis (CME) in axons, thereby driving clearance of a broad array of axonal CAMs. CAMs engineered to resist endocytosis are persistently expressed along the axon and delay both PNS and CNS myelination. Thus, glia non-autonomously activate CME in axons to downregulate axonal CAMs and presumptively axo-glial adhesion. This promotes the transition from ensheathment to myelination while simultaneously sculpting the formation of axonal domains., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The function of neurons in afferent reception, integration, and generation of electrical activity relies on their strikingly polarized organization, characterized by distinct membrane domains. These domains have different compositions resulting from a combination of selective targeting and retention of membrane proteins. In neurons, most proteins are delivered from their site of synthesis in the soma to the axon via anterograde vesicular transport and undergo retrograde transport for redistribution and/or lysosomal degradation. A key question is whether proteins destined for the same domain are transported in separate vesicles for local assembly or whether these proteins are pre-assembled and co-transported in the same vesicles for delivery to their cognate domains. To assess the content of transport vesicles, one strategy relies on staining of sciatic nerves after ligation, which drives the accumulation of anterogradely and retrogradely transported vesicles on the proximal and distal side of the ligature, respectively. This approach may not permit confident assessment of the nature of the intracellular vesicles identified by staining, and analysis is limited to the availability of suitable antibodies. Here, we use dual color live imaging of proteins labeled with different fluorescent tags, visualizing anterograde and retrograde axonal transport of several proteins simultaneously. These proteins were expressed in rat dorsal root ganglion (DRG) neurons cultured alone or with Schwann cells under myelinating conditions to assess whether glial cells modify the patterns of axonal transport. Advantages of this protocol are the dynamic identification of transport vesicles and characterization of their content for various proteins that is not limited by available antibodies., Competing Interests: Competing interestsThe authors declare no competing interests., (Copyright © 2021 The Authors; exclusive licensee Bio-protocol LLC.)

Neurons are highly polarized cells organized into functionally and molecularly distinct domains. A key question is whether the multiprotein complexes that comprise these domains are preassembled, transported, and inserted as a complex or whether their components are transported independently and assemble locally. Here, we have dynamically imaged, in pairwise combinations, the vesicular transport of fluorescently tagged components of the nodes of Ranvier and other myelinated axonal domains in sensory neurons cultured alone or together with Schwann cells at the onset of myelination. In general, most proteins are transported independently in the anterograde direction. In contrast, there is substantial cotransport of proteins from distinct domains in the retrograde direction likely due to coendocytosis along the axon. Early myelination did not substantially change these patterns of transport, although it increased the overall numbers of axonal transport vesicles. Our results indicate domain components are transported in separate vesicles for local assembly, not as preformed complexes, and implicate endocytosis along axons as a mechanism of clearance., (© 2020 Bekku and Salzer.)

Video Abstract: We have investigated the source(s) and targeting of components to PNS nodes of Ranvier. We show adhesion molecules are freely diffusible within the axon membrane and accumulate at forming nodes from local sources, whereas ion channels and cytoskeletal components are largely immobile and require transport to the node. We further characterize targeting of NF186, an adhesion molecule that pioneers node formation. NF186 redistributes to nascent nodes from a mobile, surface pool. Its initial accumulation and clearance from the internode require extracellular interactions, whereas targeting to mature nodes, i.e., those flanked by paranodal junctions, requires intracellular interactions. After incorporation into the node, NF186 is immobile, stable, and promotes node integrity. Thus, nodes assemble from two sources: adhesion molecules, which initiate assembly, accumulate by diffusion trapping via interactions with Schwann cells, whereas ion channels and cytoskeletal components accumulate via subsequent transport. In mature nodes, components turnover slowly and are replenished via transport., (Copyright © 2012 Elsevier Inc. All rights reserved.)

At the nodes of Ranvier, excitable axon membranes are exposed directly to the extracellular fluid. Cations are accumulated and depleted in the local extracellular nodal region during action potential propagation, but the impact of the extranodal micromilieu on signal propagation still remains unclear. Brain-specific hyaluronan-binding link protein, Bral1, colocalizes and forms complexes with negatively charged extracellular matrix (ECM) proteins, such as versican V2 and brevican, at the nodes of Ranvier in the myelinated white matter. The link protein family, including Bral1, appears to be the linchpin of these hyaluronan-bound ECM complexes. Here we report that the hyaluronan-associated ECM no longer shows a nodal pattern and that CNS nerve conduction is markedly decreased in Bral1-deficient mice even though there were no differences between wild-type and mutant mice in the clustering or transition of ion channels at the nodes or in the tissue morphology around the nodes of Ranvier. However, changes in the extracellular space diffusion parameters, measured by the real-time iontophoretic method and diffusion-weighted magnetic resonance imaging (MRI), suggest a reduction in the diffusion hindrances in the white matter of mutant mice. These findings provide a better understanding of the mechanisms underlying the accumulation of cations due to diffusion barriers around the nodes during saltatory conduction, which further implies the importance of the Bral1-based extramilieu for neuronal conductivity.

Neurocan is a central nervous tissue-specific chondroitin sulfate proteoglycan of the lectican family. Mainly expressed during modeling and remodeling stages of this tissue, it is thought to play an important role via binding to various extracellular matrix and cellular components. In adults, neurocan expression is associated with the perineuronal net structures. This study shows the neurocan immunolocalization at the node of Ranvier in mouse central nervous tissues. The N-terminal fragment of neurocan (Ncan130) was the predominant form detected in the optic nerve. The expression of neurocan in the white matter of brain tissue and nerve tracts revealed differential expression profiles compared with those of versican V2 and brevican, other perinodal extracellular matrix molecules. Double immunolabeling for neurocan and a nodal marker, Bral1, or a paranodal marker, caspr, demonstrated that neurocan was localized at the node of Ranvier. Neurocan expression was found at many--not all--nodal regions, and neurocan-positive nodes outnumbered brevican-positive nodes. The nodal localization of neurocan was diminished in Bral1-deficient mice. Taken together, these findings indicate that neurocan contributes to the molecular heterogeneity of the perinodal matrix, and its nodal expression is dependent on Bral1.

We report here the isolation and characterization of a cDNA encoding zebrafish dermacan, a novel member of hyaluronan (HA)-binding proteoglycans, which was termed after its characteristic expression in the zebrafish dermal bones. The deduced protein sequence shares the typical modular elements of lecticans. Sequence comparison covering the C-terminal globular domain demonstrated that dermacan shows high homology with zebrafish versican but is distinct from any other identified lecticans. Genomic DNA analysis demonstrated that dermacan and versican were encoded by distinct genes in the zebrafish genome. The expression of dermacan is initiated in the sclerotome and cephalic paraxial mesoderm at 16 h postfertilization. During the pharyngular period, dermacan transcripts were detected in the sclerotome, tail fin bud, pharyngular arch primordial region, and otic vesicle. In the development of craniofacial bones, dermacan expression was detected typically in the opercle and dentary. These regions belong to the craniofacial dermal bones. aggrecan expression, in contrast, was observed in the elements of craniofacial cartilage bones. In the dermacan-morpholino-injected embryos, dermal bones, e.g. opercle, dentary, and branchiostegal rays, as well as axial skeleton in the trunk, showed decreased ossification. We conclude that dermacan is a novel lectican gene, and that zebrafish lectican genes have genetically diverged. In addition, our data suggest the involvement of dermacan in zebrafish dermal bone development.

Neuropilin 1 is the specific receptor for Sema3A and plays a role in nerve fiber guidance. We report that neuropilin 1 and Sema3A mutant mouse embryos, generated by targeted gene disruption, showed displacement of sympathetic neurons and their precursors and abnormal morphogenesis in the sympathetic trunk. We also show that Sema3A suppressed the cell migration activity of sympathetic neurons from wild-type but not neuropilin 1 mutant embryos in vitro and instead promoted their accumulation into compact cell masses and fasciculation of their neurites. These findings suggest that the neuropilin 1-mediated Sema3A signals regulate arrest and aggregation of sympathetic neuron precursors and sympathetic neurons themselves at defined target sites and axon fasciculation to produce the stereotyped sympathetic nerve pattern.

Neuropilin is a neuronal cell surface protein and has been shown to function as a receptor for a secreted protein, semaphorin III/D, that can induce neuronal growth cone collapse and repulsion of neurites in vitro. The roles of neuropilin in vivo, however, are unknown. Here, we report that neuropilin-deficient mutant mice produced by targeted disruption of the neuropilin gene show severe abnormalities in the trajectory of efferent fibers of the PNS. We also describe that neuropilin-deprived dorsal root ganglion neurons are perfectly protected from growth cone collapse elicited by semaphorin III/D. Our results indicate that neuropilin-semaphorin III/D-mediated chemorepulsive signals play a major role in guidance of PNS efferents.

Mizoribine (Mz) is an analogue of azathioprine (Az) with less hepatotoxicity, being extensively used as immunosuppressant in place of the latter agent especially in Japan. However, careful comparative studies of mizoribine (Mz), cyclosporine (Cy), and prednisolone (Pr) versus azathioprine (Az), Cy and Pr or Cy and Pr in renal allotranspalnt patients have not been reported. Retrospectively we compared triple therapy with Mz, Cy, and Pr (group I, n = 50) to triple therapy with Az, Cy and Pr (group II, n = 13) and/or double therapy with Cy and Pr (group III, n = 11) in one-haplotype-identical living related renal transplantations performed between Oct. 1984 through March 1989. Initial and maintenance doses of Cy in groups I and II were largely two thirds of those in group III. Patient and graft survival rates at 3 years in each group are 100% and 92% (group I), 100% and 91% (group II), and 91% and 82% (group III). There were no statistical differences in patient and graft survival rates between these three groups. The incidences of miscellaneous complications were the same in the groups. Bone marrow suppression, however, was significantly less in group I than in group II (P less than 0.005). Cy related nephrotoxicity was apparently less in groups I and II than in group III. Estimated US $5,000 in a year can be saved by immunosuppressive treatment in a patient of group I as compared to a patient in group III. Therefore, we conclude that triple therapy with Mz, Cy and Pr is superior to those with Az, Cy and Pr, and/or double therapy with Cy and Pr.