Glia trigger endocytic clearance of axonal proteins to promote rodent myelination.

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.

Dual Color, Live Imaging of Vesicular Transport in Axons of Cultured Sensory Neurons.

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.

Independent anterograde transport and retrograde cotransport of domain components of myelinated axons.

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.

Under the ECM Dome: The Physiological Role of the Perinodal Extracellular Matrix as an Ion Diffusion Barrier.

Enriched Na+ channel clustering allows for rapid saltatory conduction at a specialized structure in myelinated axons, the node of Ranvier, where cations are exchanged across the axon membrane. In the extracellular matrix (ECM), highly negatively charged molecules accumulate and wrap around the nodal gaps creating an ECM dome, called the perinodal ECM. The perinodal ECM has different molecular compositions in the central nervous system (CNS) and peripheral nervous system (PNS). Chondroitin sulfate proteoglycans are abundant in the ECM at the CNS nodes, whereas heparan sulfate proteoglycans are abundant at the PNS nodes. The proteoglycans have glycosaminoglycan chains on their core proteins, which makes them electrostatically negative. They associate with other ECM molecules and form a huge stable ECM complex at the nodal gaps. The polyanionic molecular complexes have high affinity to cations and potentially contribute to preventing cation diffusion at the nodes.In this chapter, we describe the molecular composition of the perinodal ECM in the CNS and PNS, and discuss their physiological role at the node of Ranvier.

A deficiency of the link protein Bral2 affects the size of the extracellular space in the thalamus of aged mice.

Bral2 is a link protein stabilizing the binding between lecticans and hyaluronan in perineuronal nets and axonal coats (ACs) in specific brain regions. Using the real-time iontophoretic method and diffusion-weighted magnetic resonance, we determined the extracellular space (ECS) volume fraction (α), tortuosity (λ), and apparent diffusion coefficient of water (ADCW ) in the thalamic ventral posteromedial nucleus (VPM) and sensorimotor cortex of young adult (3-6 months) and aged (14-20 months) Bral2-deficient (Bral2-/- ) mice and age-matched wild-type (wt) controls. The results were correlated with an analysis of extracellular matrix composition. In the cortex, no changes between wt and Bral2-/- were detected, either in the young or aged mice. In the VPM of aged but not in young Bral2-/- mice, we observed a significant decrease in α and ADCW in comparison with age-matched controls. Bral2 deficiency led to a reduction of both aggrecan- and brevican-associated perineuronal nets and a complete disruption of brevican-based ACs in young as well as aged VPM. Our data suggest that aging is a critical point that reveals the effect of Bral2 deficiency on VPM diffusion. This effect is probably mediated through the enhanced age-related damage of neurons lacking protective ACs, or the exhausting of compensatory mechanisms maintaining unchanged diffusion parameters in young Bral2-/- animals. A decreased ECS volume in aged Bral2-/- mice may influence the diffusion of neuroactive substances, and thus extrasynaptic and also indirectly synaptic transmission in this important nucleus of the somatosensory pathway.

Modifications of perineuronal nets and remodelling of excitatory and inhibitory afferents during vestibular compensation in the adult mouse.

Perineuronal nets (PNNs) are aggregates of extracellular matrix molecules surrounding several types of neurons in the adult CNS, which contribute to stabilising neuronal connections. Interestingly, a reduction of PNN number and staining intensity has been observed in conditions associated with plasticity in the adult brain. However, it is not known whether spontaneous PNN changes are functional to plasticity and repair after injury. To address this issue, we investigated PNN expression in the vestibular nuclei of the adult mouse during vestibular compensation, namely the resolution of motor deficits resulting from a unilateral peripheral vestibular lesion. After unilateral labyrinthectomy, we found that PNN number and staining intensity were strongly attenuated in the lateral vestibular nucleus on both sides, in parallel with remodelling of excitatory and inhibitory afferents. Moreover, PNNs were completely restored when vestibular deficits of the mice were abated. Interestingly, in mice with genetically reduced PNNs, vestibular compensation was accelerated. Overall, these results strongly suggest that temporal tuning of PNN expression may be crucial for vestibular compensation.

The hyaluronan and proteoglycan link proteins: Organizers of the brain extracellular matrix and key molecules for neuronal function and plasticity.

The hyaluronan and proteoglycanbinding link protein (Hapln) is a key molecule in the formation and control of hyaluronan-based condensed perineuronal matrix in the adult brain. This review summarizes the recent advances in understanding the role of Haplns in the formation and control of two distinct types of perineuronal matrices, one for "classical" PNN and the other for the specialized extracellular matrix (ECM) at the node of Ranvier in the central nervous system (CNS). We introduce the structural components of each ECM organization including the basic concept of supramolecular structure named "HLT model". We furthermore summarize the developmental and physiological role of perineuronal ECMs from the studies of Haplns and related molecules. Finally, we also discuss the potential mechanism modulating PNNs in the adult CNS. This layer of organized matrices may exert a direct effect via core protein or sugar moiety from the structure or by acting as a binding site for biologically active molecules, which are important for neuronal plasticity and saltatory conduction.

Assembly and maintenance of nodes of ranvier rely on distinct sources of proteins and targeting mechanisms.

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.

Bral2 is indispensable for the proper localization of brevican and the structural integrity of the perineuronal net in the brainstem and cerebellum.

Perineuronal nets (PNNs) are pericellular coats of condensed matrix that enwrap the cell bodies and dendrites of many adult central nervous system (CNS) neurons. These extracellular matrices (ECMs) play a structural role as well as instructive roles in the control of CNS plasticity and the termination of critical periods. The cartilage link protein Crtl1/Hapln1 was reported to be a trigger for the formation of PNNs in the visual cortex. Bral2/Hapln4 is another link protein that is expressed in PNNs, mainly in the brainstem and cerebellum. To assess the role of Bral2 in PNN formation, we examined the expression of PNN components in targeted mouse mutants lacking Bral2. We show here that Bral2-deficient mice have attenuated PNNs, but the overall levels of chondroitin sulfate proteoglycans, lecticans, are unchanged with the exception of neurocan. Bral2 deficiency markedly affected the localization of brevican in all of the nuclei tested, and neurocan concomitant with Crtl1 in some of the nuclei, whereas no effect was seen on aggrecan even with the attenuation of Crtl1. Bral2 may have a role in the organization of the PNN, in association with brevican, that is independent of aggrecan binding. There was a heterogenous attenuation of PNN components, including glycosaminoglycans, indicating the elaborate molecular organization of the PNN components. Strikingly, a slight decrease in the number of synapses in deep cerebellar nuclei neurons was found. Taken together, these results imply that Bral2-brevican interaction may play a key role in synaptic stabilization and the structural integrity of the PNN.

Bral1: its role in diffusion barrier formation and conduction velocity in the CNS.

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 contributes to the molecular heterogeneity of the perinodal ECM.

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.

Brevican distinctively assembles extracellular components at the large diameter nodes of Ranvier in the CNS.

Brevican is known to be an abundant extracellular matrix component in the adult brain and a structural constituent of perineuronal nets. We herein show that brevican, tenascin-R (TN-R) and phosphacan are present at the nodes of Ranvier on myelinated axons with a particularly large diameter in the central nervous system. A brevican deficiency resulted in a reorganization of the nodal matrices, which was characterized by the shift of TN-R, and concomitantly phosphacan, from an axonal diameter-dependent association with nodes to an axonal diameter independent association. Supported by the co-immunoprecipitation results, these observations indicate that the presence of TN-R and phosphacan at nodes is normally brevican-dependent, while in the absence of brevican these molecules can also be recruited by versican V2. The versican V2 and Bral1 distribution was not affected, thus indicating a brevican-independent role of these two molecules for establishing hyaluronan-binding matrices at the nodes. Our results revealed that brevican plays a crucial role in determining the specialization of the hyaluronan-binding nodal matrix assemblies in large diameter nodes.

Molecular cloning and developmental expression of a hyaluronan and proteoglycan link protein gene, crtl1/hapln1, in zebrafish.

The proteoglycan aggregate of the cartilage is composed of aggrecan, link protein (LP), and hyaluronan, providing resistance to compression in joints and cartilage structures. To further understand the function of LP during the process of chondrogenesis and bone formation in zebrafish, we cloned the zebrafish cDNA for hyaluronan and proteoglycan link protein 1 (crtl1/hapln1) and examined the expression of the gene during embryogenesis using in-situ hybridization. crtl1/hapln1 expression is first observed in the adaxial cells at the bud- stage. Throughout somitogenesis, crtl1/hapln1 is expressed in the sclerotomes, floor plate, and hypochord. In addition, crtl1/hapln1 is expressed in rhombomeres 3 and 5, pharyngeal arches, telecephalon, otic vesicles, and pectral fins. During chondrocranial/skull formation, crtl1/hapln1 expression is highest at around 4 dpf and is colocalized with aggrecan in the cartilaginous arches and with dermacan in the dermal bones.

Lp3/Hapln3, a novel link protein that co-localizes with versican and is coordinately up-regulated by platelet-derived growth factor in arterial smooth muscle cells.

Link proteins (LPs) belong to the link-module superfamily, which can stabilize and enhance the binding of lecticans to hyaluronan. We report here the identification and characterization of a novel rat link protein gene (Lp3/Hapln3). The deduced protein sequence shares the typical modular elements of link proteins and has an estimated mass of 39 kDa. Examination of the rat genomic DNA sequence revealed that Lp3/Hapln3 and aggrecan genes were paired on chromosome 1q31. Another LP gene and the lectican gene were also paired at a different locus, as they are in the human and mouse genomes. Immunohistochemical analysis showed the prominent expression of Lp3/Hapln3 in the smooth muscle tissues of the vascular wall and gastrointestinal tract. Further comparative studies revealed that Lp3/Hapln3 was well co-localized with versican around the smooth muscle cells of blood vessels but not around endothelial cells. In vitro experiments using primary cultured rat arterial smooth muscle cells (ASMCs) demonstrated the coordinated up-regulation of Lp3/Hapln3 and versican by platelet-derived growth factor (PDGF). These data were supported by in vivo studies of a mechanical vascular injury model in mice. Altogether, our results suggest that Lp3/Hapln3 is involved, together with versican and hyaluronan, in the formation of the pericellular matrix of vascular smooth muscle cells.

Characterization of dermacan, a novel zebrafish lectican gene, expressed in dermal bones.

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.

Molecular cloning of Bral2, a novel brain-specific link protein, and immunohistochemical colocalization with brevican in perineuronal nets.

The hyaluronan binding chondroitin sulphate proteoglycans, called lecticans, are the abundant extracellular matrix molecules in the developing and/or adult brain. The link proteins (LPs) are also known to be coordinately present in brain. We report here the molecular cloning and expression analysis of a novel member of LPs: Bral2, predominantly expressed in brain. The Bral2 mRNA expression is first detected at P20 and continued through adulthood, suggesting its functional importance and association with adult-type lecticans. The substantial immunoreactivity of Bral2 is found in several nuclei throughout the midbrain and hindbrain in a perineuronal net pattern. In situ hybridization revealed that Bral2 is synthesized by these neurons themselves, especially by the GABAergic neurons in the cerebellar cortex. Interestingly, the colocalization and synergic importance of Bral2 and brevican in the perineuronal nets is indicated by the comparative immunohistochemical analysis using wild-type and brevican-deficient mouse brain. Our results suggest that Bral2 is involved in the formation of extracellular matrix contributing to perineuronal nets and facilitate the understanding of a functional role of these extracellular matrices.

Requirement of neuropilin 1-mediated Sema3A signals in patterning of the sympathetic nervous system.

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.

Bral1, a brain-specific link protein, colocalizing with the versican V2 isoform at the nodes of Ranvier in developing and adult mouse central nervous systems.

Bral1, a brain-specific hyaluronan-binding protein, has been cloned recently. To gain insight into the role of Bral1, we generated a specific antibody against this protein. We have examined the detailed localization pattern of Bral1 protein and compared it with that of other members of the lectican proteoglycan family, such as brevican and versican, with which Bral1 is predicted to interact. The immunoreactivity of Bral1 antibody was predominantly observed in myelinated fiber tracts in the adult brain and could be detected at P20 in the white matter of the developing cerebellum, suggesting that expression starts when axonal myelination takes place. Furthermore, immunostaining demonstrated that Bral1 colocalized with the versican V2 isoform at the nodes of Ranvier. The present data suggest that Bral1 may play a pivotal role in the formation of the hyaluronan-associated matrix in the CNS that facilitates neuronal conduction by forming an ion diffusion barrier at the nodes.