Axoglial Contacts Research Articles

Axoglial Adhesion by Cadm4 Regulates CNS Myelination

The initiation of axoglial contact is considered a prerequisite for myelination, yet the role cell adhesion molecules (CAMs) play in mediating such interactions remains unclear. To examine the function of axoglial CAMs, we tested whether enhanced CAM-mediated adhesion between OLs and neurons could affect myelination. Here we show that increased expression of a membrane-bound extracellular domain of Cadm4 (Cadm4dCT) in cultured oligodendrocytes results in the production of numerous axoglial contact sites that fail to elongate and generate mature myelin. Transgenic mice expressing Cadm4dCT were hypomyelinated and exhibit multiple myelin abnormalities, including myelination of neuronal somata. These abnormalities depend on specific neuron-glial interaction as they were not observed when these OLs were cultured alone, on nanofibers, or on neurons isolated from mice lacking the axonal receptors of Cadm4. Our results demonstrate that tightly regulated axon-glia adhesion is essential for proper myelin targeting and subsequent membrane wrapping and lateral extension.

Molecular Disorganization of Axons Adjacent to Human Cortical Microinfarcts.

Cortical microinfarcts (CMIs) are microscopically identified wedge-shaped ischemic lesions that occur at or near the cortical surface and result from occlusion of penetrating arterioles. These microscopic lesions can be observed with high-resolution magnetic resonance imaging in aging brains and in patients with cerebrovascular disease. Recent studies have suggested that strategically located microinfarcts strongly correlate with cognitive deficits, which can contribute to Alzheimer’s disease as well as other forms of dementia. We have recently shown that the molecular organization of axons into functional microdomains is altered in areas adjacent to white matter lacunar and microinfarcts, creating a peri-infarct penumbral injury in surviving axons. Whether similar changes in nodal, adjacent paranodal, and proximal axon initial segment molecular organization occur in the cortex adjacent to human CMIs is not known. Paraffin-embedded sections of autopsy brain tissue from five patients with CMIs were immunofluorescently labeled for nodal and paranodal markers including beta-IV spectrin, ankyrin-G, and contactin-associated protein. High magnification images from the peri-infarct cortical tissue were generated using confocal microscopy. In surviving cortical tissue adjacent to microinfarcts, we observed a dramatic loss of axon initial segments, suggesting that neuronal firing capacity in adjacent cortical tissue is likely compromised. The number of identifiable nodal/paranodal complexes in surviving cortical tissue is reduced adjacent to microinfarcts, while the average paranodal length is increased indicating a breakdown of axoglial contact. This axonal microdomain disorganization occurs in the relative absence of changes in the structural integrity of myelinated axons as measured by myelin basic protein and neurofilament staining. These findings indicate that the molecular organization of surviving axons adjacent to human CMIs is abnormal, reflecting lost axoglial contact and the functional elements necessary for neural transmission. This study provides support for the concept of a microinfarct penumbral injury that may account for the cumulative cognitive effect of these tiny strokes.

The Kv1-associated molecules TAG-1 and Caspr2 are selectively targeted to the axon initial segment in hippocampal neurons.

Caspr2 and TAG-1 (also known as CNTNAP2 and CNTN2, respectively) are cell adhesion molecules (CAMs) associated with the voltage-gated potassium channels Kv1.1 and Kv1.2 (also known as KCNA1 and KCNA2, respectively) at regions controlling axonal excitability, namely, the axon initial segment (AIS) and juxtaparanodes of myelinated axons. The distribution of Kv1 at juxtaparanodes requires axo-glial contacts mediated by Caspr2 and TAG-1. In the present study, we found that TAG-1 strongly colocalizes with Kv1.2 at the AIS of cultured hippocampal neurons, whereas Caspr2 is uniformly expressed along the axolemma. Live-cell imaging revealed that Caspr2 and TAG-1 are sorted together in axonal transport vesicles. Therefore, their differential distribution may result from diffusion and trapping mechanisms induced by selective partnerships. By using deletion constructs, we identified two molecular determinants of Caspr2 that regulate its axonal positioning. First, the LNG2-EGF1 modules in the ectodomain of Caspr2, which are involved in its axonal distribution. Deletion of these modules promotes AIS localization and association with TAG-1. Second, the cytoplasmic PDZ-binding site of Caspr2, which could elicit AIS enrichment and recruitment of the membrane-associated guanylate kinase (MAGuK) protein MPP2. Hence, the selective distribution of Caspr2 and TAG-1 may be regulated, allowing them to modulate the strategic function of the Kv1 complex along axons.

Mutations in GLDN, Encoding Gliomedin, a Critical Component of the Nodes of Ranvier, Are Responsible for Lethal Arthrogryposis

Arthrogryposis multiplex congenita (AMC) is a developmental condition characterized by multiple joint contractures resulting from reduced or absent fetal movements. Through linkage analysis, homozygosity mapping, and exome sequencing in four unrelated families affected by lethal AMC, we identified biallelic mutations in GLDN in the affected individuals. GLDN encodes gliomedin, a secreted cell adhesion molecule involved in the formation of the nodes of Ranvier. Transmission electron microscopy of the sciatic nerve from one of the affected individuals showed a marked lengthening defect of the nodes. The GLDN mutations found in the affected individuals abolish the cell surface localization of gliomedin and its interaction with its axonal partner, neurofascin-186 (NF186), in a cell-based assay. The axoglial contact between gliomedin and NF186 is essential for the initial clustering of Na+ channels at developing nodes. These results indicate a major role of gliomedin in node formation and the development of the peripheral nervous system in humans. These data indicate that mutations of GLDN or CNTNAP1 (MIM: 616286), encoding essential components of the nodes of Ranvier and paranodes, respectively, lead to inherited nodopathies, a distinct disease entity among peripheral neuropathies.

Spatial mapping of juxtacrine axo-glial interactions identifies novel molecules in peripheral myelination.

Cell–cell interactions promote juxtacrine signals in specific subcellular domains, which are difficult to capture in the complexity of the nervous system. For example, contact between axons and Schwann cells triggers signals required for radial sorting and myelination. Failure in this interaction causes dysmyelination and axonal degeneration. Despite its importance, few molecules at the axo-glial surface are known. To identify novel molecules in axo-glial interactions, we modified the ‘pseudopodia' sub-fractionation system and isolated the projections that glia extend when they receive juxtacrine signals from axons. By proteomics we identified the signalling networks present at the glial-leading edge, and novel proteins, including members of the Prohibitin family. Glial-specific deletion of Prohibitin-2 in mice impairs axo-glial interactions and myelination. We thus validate a novel method to model morphogenesis and juxtacrine signalling, provide insights into the molecular organization of the axo-glial contact, and identify a novel class of molecules in myelination.

The back and forth of axonal injury and repair after stroke.

The axon plays a central role in both the injury and repair phases after stroke. This review highlights emerging principles in the study of axonal injury in stroke and the role of the axon in neural repair after stroke. Ischemic stroke produces a rapid and significant loss of axons in the acute phase. This early loss of axons results from a primary ischemic injury that triggers a wave of calcium signaling, activating proteolytic mechanisms and downstream signaling cascades. A second progressive phase of axonal injury occurs during the subacute period and damages axons that survive the initial ischemic insult but go on to experience a delayed axonal degeneration driven in part by changes in axoglial contact and axonal energy metabolism. Recovery from stroke is dependent on axonal sprouting and reconnection that occurs during a third degenerative/regenerative phase. Despite this central role played by the axon, comparatively little is understood about the molecular pathways that contribute to early and subacute axonal degeneration after stroke. Recent advances in axonal neurobiology and signaling suggest new targets that hold promise as potential molecular therapeutics including axonal calcium signaling, axoglial energy metabolism and cell adhesion as well as retrograde axonal mitogen-activated protein kinase pathways. These novel pathways must be modeled appropriately as the type and severity of axonal injury vary by stroke subtype. Stroke-induced injury to axons occurs in three distinct phases each with a unique molecular underpinning. A wealth of new data about the molecular organization and molecular signaling within axons is available but not yet robustly applied to the study of axonal injury after stroke. Identifying the spatiotemporal patterning of molecular pathways within the axon that contribute to injury and repair may offer new therapeutic strategies for the treatment of stroke.

Long-Term Maintenance of Na+Channels at Nodes of Ranvier Depends on Glial Contact Mediated by Gliomedin and NrCAM

Clustering of Na(+) channels at the nodes of Ranvier is coordinated by myelinating glia. In the peripheral nervous system, axoglial contact at the nodes is mediated by the binding of gliomedin and glial NrCAM to axonal neurofascin 186 (NF186). This interaction is crucial for the initial clustering of Na(+) channels at heminodes. As a result, it is not clear whether continued axon-glial contact at nodes of Ranvier is required to maintain these channels at the nodal axolemma. Here, we report that, in contrast to mice that lack either gliomedin or NrCAM, absence of both molecules (and hence the glial clustering signal) resulted in a gradual loss of Na(+) channels and other axonal components from the nodes, the formation of binary nodes, and dysregulation of nodal gap length. Therefore, these mice exhibit neurological abnormalities and slower nerve conduction. Disintegration of the nodes occurred in an orderly manner, starting with the disappearance of neurofascin 186, followed by the loss of Na(+) channels and ankyrin G, and then βIV spectrin, a sequence that reflects the assembly of nodes during development. Finally, the absence of gliomedin and NrCAM led to the invasion of the outermost layer of the Schwann cell membrane beyond the nodal area and the formation of paranodal-like junctions at the nodal gap. Our results reveal that axon-glial contact mediated by gliomedin, NrCAM, and NF186 not only plays a role in Na(+) channel clustering during development, but also contributes to the long-term maintenance of Na(+) channels at nodes of Ranvier.

Axonal spectrins: All-purpose fences

A membrane barrier important for assembly of the nodes of Ranvier is found at the paranodal junction. This junction is comprised of axonal and glial adhesion molecules linked to the axonal actin–spectrin membrane cytoskeleton through specific adaptors. In this issue, Zhang et al. (2013. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201308116) show that axonal βII spectrin maintains the diffusion barrier at the paranodal junction. Thus, βII spectrin serves to compartmentalize the membrane of myelinated axons at specific locations that are determined either intrinsically (i.e., at the axonal initial segment), or by axoglial contacts (i.e., at the paranodal junction).

Organisation and control of neuronal connectivity and myelination by cell adhesion molecule neurofascin.

The neuronal cell adhesion molecule neurofascin is expressed in highly complex temporally and spatially regulated patterns. Accordingly, many different functions have been described including control of neurite outgrowth, clustering of protein complexes at the axon initial segments as well as at the nodes of Ranvier and axoglial contact formation at paranodal segments. At the molecular level, neurofascin provides a link between extracellular interactions of many different interaction partners and cytoskeletal components or signal transduction. Such interactions are subject to intimate regulation by alternative splicing and posttranslational modification. The versatile functional aspects of neurofascin interactions pose it at a central position for the shaping and maintenance of neural circuitry and synaptic contacts which are implicated in nervous system disorders.

Neuro-glial interactions at the nodes of Ranvier: implication in health and diseases

Specific cell adhesion molecules (CAMs) are dedicated to the formation of axo-glial contacts at the nodes of Ranvier of myelinated axons. They play a central role in the organization and maintenance of the axonal domains: the node, paranode, and juxtaparanode. In particular, CAMs are essential for the accumulation of voltage-gated sodium channels at the nodal gap that ensures the rapid and saltatory propagation of the action potentials (APs). The mechanisms regulating node formation are distinct in the central and peripheral nervous systems, and recent studies have highlighted the relative contribution of paranodal junctions and nodal extracellular matrix. In addition, CAMs at the juxtaparanodal domains mediate the clustering of voltage-gated potassium channels which regulate the axonal excitability. In several human pathologies, the axo-glial contacts are altered leading to disruption of the nodes of Ranvier or mis-localization of the ion channels along the axons. Node alterations and the failure of APs to propagate correctly from nodes to nodes along the axons both contribute to the disabilities in demyelinating diseases. This article reviews the mechanisms regulating the association of the axo-glial complexes and the role of CAMs in inherited and acquired neurological diseases.

What does anisotropy measure? Insights from increased and decreased anisotropy in selective fiber tracts in schizophrenia

Schizophrenia is a common, severe, and chronically disabling mental illness of unknown cause. Recent MRI studies have focused attention on white matter abnormalities in schizophrenia using diffusion tensor imaging (DTI). Indices commonly derived from DTI include (1) mean diffusivity, independent of direction, (2) fractional anisotropy (FA) or relative anisotropy (RA), (3) axial diffusivity, and (4) radial diffusivity. In cerebral white matter, contributions to these indices come from fiber arrangements, degree of myelination, and axonal integrity. Relatively pure deficits in myelin result in a modest increase in radial diffusivity, without affecting axial diffusivity and with preservation of anisotropy. Although schizophrenia is not characterized by gross abnormalities of white matter, it does involve a profound dysregulation of myelin-associated gene expression, reductions in oligodendrocyte numbers, and marked abnormalities in the ultrastructure of myelin sheaths. Since each oligodendrocyte myelinates as many as 40 axon segments, changes in the number of oligodendrocytes (OLG), and/or in the integrity of myelin sheaths, and/or axoglial contacts can have a profound impact on signal propagation and the integrity of neuronal circuits. Whereas a number of studies have revealed inconsistent decreases in anisotropy in schizophrenia, we and others have found increased FA in key subcortical tracts associated with the circuits underlying symptom generation in schizophrenia. We review data revealing increased anisotropy in dopaminergic tracts in the mesencephalon of schizophrenics and their unaffected relatives, and discuss the possible biological underpinnings and physiological significance of this finding.

Disposition of axonal caspr with respect to glial cell membranes: Implications for the process of myelination

Neurofascin-155 (NF155) and caspr are transmembrane proteins found at discrete locations early during development of the nervous system. NF155 is present in the oligodendrocyte cell body and processes, whereas caspr is on the axonal surface. In mature nerves, these proteins are clustered at paranodes, flanking the node of Ranvier. To understand how NF155 and caspr become localized to the paranodal regions of myelinated nerves, we have studied their distribution over time in myelinating cultures. Our observations indicate that these two proteins are recruited to the cell surface at the contact zone between axons and oligodendrocytes, where they trans-interact. This association explains the early pattern of caspr distribution, a helical coil that winds around the axon, resembling the turns of the myelin sheath. Caspr, an axonal membrane protein, therefore seems to move in register with the overlying myelinating cell via its interactions with myelin proteins. We suggest that NF155 is the glial cell membrane protein responsible for caspr distribution. The pair act as interacting partners on either side of the axoglial contact area. Most likely, there are other proteins on the axonal surface whose distribution is equally influenced by interaction with the nascent myelin sheath. The fact that caspr follows the movement of the spiraling membrane has a direct affect on the interpretation of the way in which myelin is formed.

Variations in oligodendrocyte-related gene expression across multiple cortical regions: implications for the pathophysiology of schizophrenia

The disconnectivity syndrome hypothesis of schizophrenia suggests that communication between multiple brain circuits and regions may be disrupted. Microarray studies analysed gene expression in 15 different brain regions derived from 13 persons with schizophrenia and controls. The superior temporal gyrus, cingulate gyrus and hippocampus evidence the greatest numbers of abnormally expressed genes. Gene ontology categorization suggested that gene classes associated with oligodendrocytes and myelin function were among the most profoundly affected. qPCR and additional microarray studies have validated these oligodendrocyte- and myelin-associated findings in independent cohorts. At least some of the affected genes are associated with the regulation of axoglial contacts, axon calibre and the integrity of functional elements involved in signal propagation. The confluence of emerging evidence shows that myelination abnormalities are major components of the neurobiology of schizophrenia and suggest that re-evaluation of some long-held hypotheses and beliefs regarding the biological substrates of schizophrenia may be warranted.

Spectrins and AnkyrinB Constitute a Specialized Paranodal Cytoskeleton

Paranodal junctions of myelinated nerve fibers are important for saltatory conduction and function as paracellular and membrane protein diffusion barriers flanking nodes of Ranvier. The formation of these specialized axoglial contacts depends on the presence of three cell adhesion molecules: neurofascin 155 on the glial membrane and a complex of Caspr and contactin on the axon. We isolated axonal and glial membranes highly enriched in these paranodal proteins and then used mass spectrometry to identify additional proteins associated with the paranodal axoglial junction. This strategy led to the identification of three novel components of the paranodal cytoskeleton: ankyrinB, alphaII spectrin, and betaII spectrin. Biochemical and immunohistochemical analyses revealed that these proteins associate with protein 4.1B in a macromolecular complex that is concentrated at central and peripheral paranodal junctions in the adult and during early myelination. Furthermore, we show that the paranodal localization of ankyrinB is disrupted in Caspr-null mice with aberrant paranodal junctions, demonstrating that paranodal neuron-glia interactions regulate the organization of the underlying cytoskeleton. In contrast, genetic disruption of the juxtaparanodal protein Caspr2 or the nodal cytoskeletal protein betaIV spectrin did not alter the paranodal cytoskeleton. Our results demonstrate that the paranodal junction contains specialized cytoskeletal components that may be important to stabilize axon-glia interactions and contribute to the membrane protein diffusion barrier found at paranodes.

Age‐related molecular reorganization at the node of Ranvier

In myelinated axons, action potential conduction is dependent on the discrete clustering of ion channels at specialized regions of the axon, termed nodes of Ranvier. This organization is controlled, at least in part, by the adherence of myelin sheaths to the axolemma in the adjacent region of the paranode. Age-related disruption in the integrity of internodal myelin sheaths is well described and includes splitting of myelin sheaths, redundant myelin, and fluctuations in biochemical constituents of myelin. These changes have been proposed to contribute to age-related cognitive decline; in previous studies of monkeys, myelin changes correlate with cognitive performance. In the present study, we hypothesize that age-dependent myelin breakdown results in concomitant disruption at sites of axoglial contact, in particular at the paranode, and that this disruption alters the molecular organization in this region. In aged monkey and rat optic nerves, immunolabeling for voltage-dependent potassium channels of the Shaker family (Kv1.2), normally localizing in the adjacent juxtaparanode, were mislocalized to the paranode. Similarly, immunolabeling for the paranodal marker caspr reveals irregular caspr-labeled paranodal profiles, suggesting that there may be age-related changes in paranodal structure. Ultrastructural analysis of paranodal segments from optic nerve of aged monkeys shows that, in a subset of myelinated axons with thick sheaths, some paranodal loops fail to contact the axolemma. Thus, age-dependent myelin alterations affect axonal protein localization and may be detrimental to maintenance of axonal conduction.

Variations in myelin and oligodendrocyte-related gene expression across multiple brain regions in schizophrenia: A gene ontology study

Large-scale gene expression studies in schizophrenia (SZ) have generally focused on the dorsolateral prefrontal cortex. Studies of other brain regions have been less frequent and have rarely been performed in the same subjects. We analyzed postmortem gene expression in multiple cortical regions (Brodmann areas 8, 10, 44, 46, 23/31, 24/32, 20, 21, 22, 36/28, 7 and 17, respectively) as well as in the hippocampus, caudate nucleus, and putamen of 13 SZ and 13 control subjects using Affymetrix GeneChip® microarrays. The superior temporal cortex (BA22) and cingulate cortices (BA24/32, 23/31) of subjects with SZ demonstrated more profound alterations of gene expression than other brain regions compared to controls [Katsel, P., Davis, K.L., Gorman, J.M., Haroutunian, V., in press. Variations in differential gene expression patterns across multiple brain regions in schizophrenia. Schizophr. Res.]. Functional categorization of genes whose expression was altered revealed multiple gene ontology classes that included oligodendrocyte/myelin-related genes. These myelin-related ontologies were among the top scored categories in temporal and cingulate gyri and in the hippocampus relative to other brain regions. The most altered transcripts in SZ were those encoding for proteins involved in determination of glial differentiation, myelin structure and adhesion proteins participating in axoglial contacts. Hierarchical clustering demonstrated that these myelin-related gene expression abnormalities in SZ were most pronounced in the hippocampus, superior temporal and cingulate cortices. The high representation of abnormally expressed oligodendrocyte/myelin genes in brain regions with the largest numbers of abnormally expressed genes in SZ confirmed their involvement in the disease process and suggested that the integrity of axon–myelin interaction may be impaired in SZ.

Contacts cellulaires des fibres myélinisées du système nerveux périphérique

Myelination allows the fast propagation of action potentials at a low energetic cost. It provides an insulating myelin sheath regularly interrupted at nodes of Ranvier where voltage-gated Na+ channels are concentrated. In the peripheral nervous system, the normal function of myelinated fibers requires the formation of highly differentiated and organized contacts between the myelinating Schwann cells, the axons and the extracellular matrix. Some of the major molecular complexes that underlie these contacts have been identified. Compact myelin which forms the bulk of the myelin sheath results from the fusion of the Schwann cell membranes through the proteins P0, PMP22 and MBP. The basal lamina of myelinating Schwann cells contains laminin-2 which associates with the glial complex dystroglycan/DPR2/L-periaxin. Non compact myelin, found in paranodal loops, periaxonal and abaxonal regions, and Schmidt-Lanterman incisures, presents reflexive adherens junctions, tight junctions and gap junctions, which contain cadherins, claudins and connexins, respectively. Axo-glial contacts determine the formation of distinct domains on the axon, the node, the paranode, and the juxtaparanode. At the paranodes, the glial membrane is tightly attached to the axolemma by septate-like junctions. Paranodal and juxtaparanodal axoglial complexes comprise an axonal transmembrane protein of the NCP family associated in cis and in trans with cell adhesion molecules of the immunoglobulin superfamily (IgSF-CAM). At nodes, axonal complexes are composed of Na+ channels and IgSF-CAMs. Schwann cell microvilli, which loosely cover the node, contain ERM proteins and the proteoglycans syndecan-3 and -4. The fundamental role of the cellular contacts in the normal function of myelinated fibers has been supported by rodent models and the detection of genetic alterations in patients with peripheral demyelinating neuropathies such as Charcot-Marie-Tooth diseases. Understanding more precisely their molecular basis now appears essential as a requisite step to further examine their involvement in the pathogenesis of peripheral neuropathies in general.

The local differentiation of myelinated axons at nodes of Ranvier.

Efficient and rapid propagation of action potentials in myelinated axons depends on the molecular specialization of the nodes of Ranvier. The nodal region is organized into several distinct domains, each of which contains a unique set of ion channels, cell-adhesion molecules and cytoplasmic adaptor proteins. Voltage-gated Na+ channels - which are concentrated at the nodes - are separated from K+ channels - which are clustered at the juxtaparanodal region - by a specialized axoglial contact that is formed between the axon and the myelinating cell at the paranodes. This local differentiation of myelinated axons is tightly regulated by oligodendrocytes and myelinating Schwann cells, and is achieved through complex mechanisms that are used by another specialized cell-cell contact - the synapse.

Paranodal transverse bands are required for maintenance but not initiation of Nav1.6 sodium channel clustering in CNS optic nerve axons

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.

Nodal sodium channel domain integrity depends on the conformation of the paranodal junction, not on the presence of transverse bands.

Our understanding of the role that axoglial interactions play in node of Ranvier formation and maintenance remains incomplete. Previous studies of CNS myelinated fibers of CGT-null mice showed abnormalities in the arrangement of paranodal myelin loops and absence of a conspicuous component of the paranodal junction, the ridge-like intercellular transverse bands. Axolemmal sodium channel domains were largely preserved at nodes of Ranvier but displayed some abnormalities in form. Using a combination of freeze-fracture and immunocytochemical methods, we have found additional evidence documenting abnormalities in the size, shape, and location of axolemmal sodium channel clusters in CGT-null mice as well as evidence that these nodal abnormalities are complementary to the organization of paranodal myelin loops, despite the absence of transverse bands. We conclude that the differentiated form of the nodal axolemma and the distribution of axolemmal sodium channels depend on the conformation of paranodal axoglial contacts but not on the presence of transverse bands at the sites of contact.