Paranodal Membrane Research Articles

Autoantibodies in central nervous system and neuromuscular autoimmune disorders: A narrative review

The discovery of novel autoantibodies in neurological disorders contributes to a better understanding of its pathogenesis, improves the accuracy of diagnosis, and leads to new treatment strategies. Advances in techniques for the screening and detection of autoantibodies have enabled the discovery of new antibodies in the central nervous system (CNS) and neuromuscular diseases. Cell-based assays using live or fixed cells overexpressing target antigens are widely used for autoantibody-based diagnosis in clinical practice. Common pathogenic autoantibodies are unknown in most patients with multiple sclerosis (MS) and chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). Novel pathogenic autoantibodies to aquaporin-4 and myelin oligodendrocyte glycoprotein (MOG) have been identified in neuromyelitis optica spectrum disorder and MOG antibody-associated disease, respectively. These diseases have clinical similarities to MS, but with the discovery of pathogenic autoantibodies, they are now recognized as distinct disease entities. Antibodies to paranodal membrane proteins such as neurofascin-155, contactin‑1, contactin‑associated protein‑1 in CIDP and muscle-specific kinase and low-density lipoprotein receptor–related protein 4 in myasthenia gravis were added to the profiles of autoantibodies in neurological disorders. Despite the relatively low frequency of seropositivity, autoantibody detection is currently essential for the clinical diagnosis of CNS and neuromuscular autoimmune disorders, and differential approaches to seropositive patients will contribute to more personalized medicine. We reviewed recent discoveries of autoantibodies and their clinical implications in CNS and neuromuscular disorders.

ATP-induced lipid membrane reordering in the myelinated nerve fiber identified using Raman spectroscopy

We demonstrate a successful application of Raman spectroscopy to the problem of lipid ordering with microscopic resolution in different regions of the myelinated nerve fiber. Simultaneous collection of Raman spectra of lipids and carotenoids has enabled us to characterize membrane fluidity and the degree of lipid ordering based on intensity ratios for the 1527/1160 and 2940/2885 cm−1 bands. We show that the intensity profiles of the major Raman bands vary significantly between the three major regions of myelinated nerve fiber: internode, paranode and the node of Ranvier. Mapping Raman peak intensities over these areas suggested that the carotenoid molecules are localized in the myelin membranes of nerve cells. Paranodal membranes were sensitive to extracellular ATP. ATP solutions (7 mM) influenced the 1527/1160 and 2940/2885 cm−1 intensity ratios. Changes in both carotenoid and lipid Raman spectra were in accord and indicated an increase in lipid ordering degree and decrease in membrane fluidity under ATP administration. The collected data provide evidence for the existence of a regulatory purinergic signaling pathway in the peripheral nervous system.

Electron tomographic analysis of cytoskeletal cross-bridges in the paranodal region of the node of Ranvier in peripheral nerves

The node of Ranvier is a site for ionic conductances along myelinated nerves and governs the saltatory transmission of action potentials. Defects in the cross-bridging and spacing of the cytoskeleton are a prominent pathological feature in diseases of the peripheral nerve. Electron tomography was used to examine cytoskeletal-cytoskeletal, membrane-cytoskeletal, and heterologous cell connections in the paranodal region of the node of Ranvier in peripheral nerves. Focal attachment of cytoskeletal filaments to each other and to the axolemma and paranodal membranes of the Schwann cell via narrow cross-bridges was visualized in both neuronal and glial cytoplasm. A subset of intermediate filaments associates with the cytoplasmic surfaces of supramolecular complexes of transmembrane structures that are presumed to include known and unknown junctional proteins. Mitochondria were linked to both microtubules and neurofilaments in the axoplasm and to neighboring smooth endoplasmic reticulum by narrow cross-bridges. Tubular cisternae in the glial cytoplasm were also linked to the paranodal glial cytoplasmic loop juxtanodal membrane by short cross-bridges. In the extracellular matrix between axon and Schwann cell, junctional bridges formed long cylinders linking the two membranes. Interactions between cytoskeleton, membranes, and extracellular matrix associations in the paranodal region are likely critical not only for scaffolding, but also for intracellular and extracellular communication.

Organizing principles of the axoglial apparatus.

On axonal surfaces that flank the node of Ranvier and in overlying glial paranodal loops, proteins are arranged within circumscribed microdomains that defy explanation by conventional biosynthetic mechanisms. We postulate that the constraint of proteins to these loci is accomplished in part by discriminative membrane-embedded molecular sieves and diffusion barriers, which serve to organize and redistribute proteins after delivery by vesicular transport to neural cell plasma membranes. One sieve likely comprises a moveable, macromolecular scaffold of axonal and glial cell-derived transmembrane adhesion molecules and their associated cytoplasmic binding partners, located at the ends of each elongating myelin internode; this sieve contributes to restricting the sodium channel complexes to the node. We also anticipate the existence of a passive paranodal diffusion barrier at the myelin/noncompact membrane border, which prohibits protein diffusion out of contiguous paranodal membranes.

Type II brain 4.1 (4.1B/KIAA0987), a member of the protein 4.1 family, is localized to neuronal paranodes

Histochemical analyses of type II brain 4.1/4.1B/KIAA0987, a member of the protein 4.1 family, were carried out in rat brain. In situ hybridization (ISH) showed that type II brain 4.1 mRNA is expressed in a variety of neuronal cells. In particular, type II brain 4.1 mRNA was actively transcribed in the cells of the mesencephalon and the brainstem, which have large myelinated nerve fibers. Expression of type II brain 4.1 mRNA was not observed at least in glial cells distributed in nerve fiber tracts. In immunohistochemical studies using anti-type II brain 4.1-specific antibody, the major immunosignals appeared as brilliant pairs of dots along nerve fibers. Such immunosignals were detected throughout the brain, but were highly concentrated in nerve fiber tracts. These data suggested that type II brain 4.1 is predominantly localized to neuronal paranodes. Detailed analysis concentrating on the nodal region indicated that type II brain 4.1 is present at the paranodal membrane but not in the axoplasm. Weaker type II brain 4.1-specific immunosignals were observed along the internodal membrane of myelinated axons and in the cytoplasm of some neuronal cells. Finally, comparative immunohistochemical studies using antibodies against the other three protein 4.1 family members, type I brain 4.1/4.1N/KIAA0338, erythroid type 4.1 (4.1R) and 4.1G, demonstrated that each of these proteins is distributed in a unique pattern in the cerebellum. Our results are the first to show that type II brain 4.1 is the only member of the protein 4.1 family localized to neuronal paranodes.

On the molecular architecture of myelinated fibers.

Schwann cells and oligodendrocytes make the myelin sheaths of the PNS and CNS, respectively. Their myelin sheaths are structurally similar, consisting of multiple layers of specialized cell membrane that spiral around axons, but there are several differences. (1) CNS myelin has a "radial component" composed of a tight junction protein, claudin-11/oligodendrocyte-specific protein. (2) Schwann cells have a basal lamina and microvilli. (3) Although both CNS and PNS myelin sheaths have incisures, those in the CNS lack the structural as well as the molecular components of "reflexive" adherens junctions and gap junctions. In spite of their structural differences, the axonal membranes of the PNS and CNS are similarly organized. The nodal axolemma contains high concentrations of voltage-dependent sodium channels that are linked to the axonal cytoskeleton by ankyrin(G). The paranodal membrane contains Caspr/paranodin, which may participate in the formation of axoglial junctions. The juxtaparanodal axonal membrane contains the potassium channels Kv1.1 and Kv1.2, their associated beta2 subunit, as well as Caspr2, which is closely related to Caspr. The myelin sheath probably organizes these axonal membrane-related proteins via trans interactions.

Expression of the neurofibromatosis 2 tumor suppressor gene product, merlin, in Schwann cells.

The neurofibromatosis 2 (NF2) tumor suppressor gene encodes a protein with sequence similarity to a family of molecules linking cell membrane proteins to the actin cytoskeleton. Individuals affected with NF2 develop schwannomas at an increased frequency. In this paper, we report that merlin is expressed in Schwann cells, where it is localized in vitro to the cell membrane by immunohistochemistry and subcellular fractionation. Exogenous expression of merlin fragments confirms this subcellular distribution and suggests that both the N-terminal and C-terminal portions of the molecule are required for this localization. In addition, merlin is expressed in rat sciatic nerve Schwann cells at paranodal membranes, where it colocalizes with RhoA. Lastly, expression of the NF2 gene increases during postnatal rat sciatic nerve development, consistent with its role as a negative growth regulator for Schwann cells. These results collectively suggest that merlin may function at the cell surface to modulate cell growth in Schwann cells and to link cell membrane proteins to the cytoskeleton.

Novel E-cadherin-mediated adhesion in peripheral nerve: Schwann cell architecture is stabilized by autotypic adherens junctions.

Previous studies (Blank, W. F., M. B. Bunge, and R. P. Bunge. 1974. Brain Res. 67:503-518) showed that Schwann cell paranodal membranes were disrupted in calcium free medium suggesting that cadherin mediated mechanisms may operate to maintain the integrity of the paranodal membrane complex. Using antibodies against the fifth extracellular domain of E-cadherin, we now show by confocal laser and electron immunomicroscopy that E-cadherin is a major adhesive glycoprotein in peripheral nervous system Schwann cells. E-Cadherin is not found, however, in compact myelin bilayers. Rather, it is concentrated at the paranodes, in Schmidt-Lanterman incisures, and at the inner and outer loops. At these loci, E-cadherin is associated with subplasmalemmal electron densities that coordinate in register across several cytoplasmic turns of a single Schwann cell. F-Actin and beta-catenin, two proteins implicated in cellular signaling, also co-localize to E-cadherin positive sites. These complexes are autotypic adherens-type junctions that are confined to the plasma membrane synthesized by a single Schwann cell; E-cadherin was never observed between two Schwann cells, nor between Schwann cells and the axon. Our findings demonstrate that E-cadherin and its associated proteins are essential components in the architecture of the Schwann cell cytoplasmic channel network, and suggest that this network has specialized functions in addition to those required for myelinogenesis.

ATP activates cationic and anionic conductances in Schwann cells cultured from dorsal root ganglia of the mouse.

The whole-cell configuration of the patch-clamp technique was used to study membrane responses of mouse Schwann cells in organotypique culture to external application of adenosine 5'-triphosphate (ATP). ATP induced an inward current (IATP) which caused a membrane depolarization. IATP was dose dependent with a Kd of 8.4 mM. ATP analogues had the following relative agonist potency: ATP > ADP approximately alpha-beta methylene ATP. Neither AMP nor adenosine were effective. IATP was reversibly reduced by suramin, a P2 purinoceptor antagonist. Alteration of ionic gradients showed that ATP activated simultaneously cationic (potassium and calcium) and anionic (chloride) conductances. Values of the reversal potentials to ATP suggested the following permeability sequence through the anion channel: SCN- > I- > Br- approximately Cl- > aspartate > isethionate. IATP did not appear to be dependent on intracellular calcium, as inclusion or omission of calcium chelator (BAPTA, 10 mM) in the pipette solution had no effect on IATP. The observed ATP response resembles a P2-mediated response in its kinetics, its relative agonist potency and its blockade by suramin, but differs from known ones by its requirement for high concentrations of ATP and the activation of a cationic as well as an anionic conductance.

Development of nodal and paranodal membrane specializations in amphibian peripheral nerves

Peripheral nerves from the hind legs of frog tadpoles were examined in order to ascertain the pattern of development of nodal and paranodal specializations in myelinated fibers. In thin sections the earliest detectable node-related specializations resemble ‘intermediate’ junctions between axons and Schwann cell processes. These occur in individually ensheathed axons near the edges of the sheath segments and could represent early nodal or paranodal components or transient structures. The characteristic nodal ‘undercoating’ is indistinct and highly variable in thickness in immature fibers and its density is lower in developing nodes than in adult nodes. Corresponding freeze-fracture replicas of developing axons demonstrate aggregates of nodal E face particles whose concentration is lower than that in the adult. Such aggregates usually occur immediately adjacent to Schwann cell indentations, even though early in development the latter may not exhibit the paracrystalline pattern seen in the adult paranodal axolemma. On rare occasions, node-like particle aggregates and presumptive nodal undercoatings have been observed without recognizable paranodal junctions or indentations nearby. However, neither specialization has been found in axons not individually ensheathed by Schwann cells. Paranodal Schwann cell loops are widely separated and irregularly arranged in the developing nodes, and the paranodal regions flanking a node usually mature asymmetrically. Differentiated paranodal junctions appear early in axons ensheathed by only a few loose Schwann cell lamellae. However, such junctions are not formed by all paranodal loops; they consistently appear first in the loops close to the node and only later in those further removed. No junctional specialization has been observed in either the axolemma or the Schwann cell membrane without the close association of the other. Our results indicate that both nodal and paranodal specializations appear early in development and mature gradually together and that the axon and the Schwann cell are consistently in close association where node-related specializations appear. No evidence of ‘independent’ differentiation in either partner was observed.

Presence of the myelin-associated glycoprotein correlates with alterations in the periodicity of peripheral myelin.

The myelin-associated glycoprotein (MAG) is an integral membrane protein (congruent to 100,000 mol wt) which is a minor component of purified peripheral nervus system (PNS) myelin. In the present study, MAG was localized immunocytochemically in 1-micrometer thick Epon sections of 7-d and adult rat peripheral nerves, and its localization was compared to that of the major structural protein (Po) of PNS myelin. To determine more precisely the localization of MAG, immunostained areas in 1 micrometer sections were traced on electron micrographs of identical areas from adjacently cut thin sections.l MAG was localized in periaxonal membranes. Schmidt-Lantermann incisures, paranodal membranes, and the outer mesaxon of PNS myelin sheaths. Compact regions of PNS myelin did not react with MAG antiserum. The results demonstrate MAG's presence in "'semi-compact" Schwann cell or myelin membranes that have a gap of 12-14 nm between extracellular leaflets and a spacing of 5 nm or more between cytoplasmic leaflets. In compact regions of the myelin sheath which do not contain MAG, the cytoplasmic leaflets are "fused" and form the major dense line, whereas the extracellular leaflets are separated by a 2.0 nm gap appearing as paired minor dense lines. Thus, it is proposed that MAG plays a role in maintaining the periaxonal space, Schmidt-Lantermann incisures, paranodal myelin loops, and outer mesaxon by preventing "complete" compaction of Schwann cell and myelin membranes. The presence of MAG in these locations also suggests that MAG may serve a function in regulating myelination in the PNS.

Nodal and paranodal membrane structure in complementary freeze-fracture replicas of amphibian peripheral nerves

Complementary freeze-fracture replicas of frog peripheral nerves have revealed new details of membrane structures at the node of Ranvier and paranodal axon-Schwann cell junction. At the node both E and P fracture faces of the axolemma have high particle concentrations (approximately 1350/sq. micron and 1600/sq. micron respectively) and these particles do not overlap when tracings from the respective fracture faces are superimposed. A high proportion of the E face particles are large (> 9.5 nm) and cast long shadows while the proportion of large particles in the P face is much lower. In the paranodal region the diagonal pattern of parallel rows in the junctional axolemma always has the same orientation within a given fracture face. In the E face, the parallel rows form a positive (+ 30 degrees) angle to the groove below and in the P face, a negative (-30 degrees) angle to the ridge above. This implies that the diagonal pattern derives from asymmetric subunits that are able to associate along only one axis and are unable to 'flip over' with respect to the junctional membranes.

Aberrant axon-Schwann cell junctions in dystrophic mouse nerves.

'Amyelinated' axons in the spinal roots of dystrophic mouse nerves lack typical nodal and paranodal membrane specializations. However, at the periphery of the amyelinated bundles some of the naked axons form aberrant junctions with Schwann cells belonging to neighbouring myelinated axons. These junctions are characterized by a narrow intercellular cleft containing regularly-spaced densities that closely resemble the 'transverse bands' found at paranodal axoglial junctions with respect to both configuration and spacing. In addition, the Schwann cells sometimes extend fingerlike projections towards amyelinated axons in regions where the axolemma has a dense cytoplasmic undercoating. Such regions resemble nodes of Ranvier, where Schwann cell processes interlace over the axolemma. Freeze-fracture replicas show no typical nodal or paranodal membrane specializations in the amyelinated fibres where they are apposed to each other. However, isolated paracrystalline patches of membrane occur corresponding to the aberrant junctions between amyelinated axons and Schwann cells at the periphery of the bundles. The observations show that structural differentiation of the axolemma occurs only where axons are in intimate contact with myelinating cells and does not develop independently in the amyelinated regions. Sodium channels, which are normally concentrated in the specialized nodal membrane, are, therefore, probably distributed uniformly along the amyelinated axon segments that show no sign of such regional differentiation. In addition, it is shown that Schwann cells are capable of forming specialized junctions with more than one axon at the same time.