Structure-function analysis of protein complexes involved in the molecular architecture of juxtaparanodal regions of myelinated fibers

Demyelinating disorders, including multiple sclerosis (MS), are common causes of neurological disability. One critical step towards the management and therapy of demyelinating diseases is to understand the basic functions of myelinating glia and their relationship with axons. Axons and myelinating glia, oligodendrocytes in the central (CNS) and Schwann cells in the peripheral (PNS) nervous systems, reciprocally influence each other's development and trophism. These interactions are critical for the formation of distinct axonal domains in myelinated fibers that ensure the rapid propagation of action potentials. Macromolecular complexes mediating axo-glial interactions in these domains have been identified, consisting of members of the immunoglobulin superfamily (IgSF) of adhesion molecules and the neurexin/NCP superfamily as well as other proteins. We have investigated the molecular details of axo-glial interactions in the juxtaparanodal region of myelinated fibers by utilizing domain-specific GFP constructs and immunoprecipitation assays on transfected cells. We have shown that the immunoglobulin domains of the IgSF member TAG-1/Cnt-2 are necessary and sufficient for the direct, cis interaction of this protein with Caspr2 and potassium channels.     

A molecular view on paranodal junctions of myelinated fibers.

The axoglial paranodal junctions, flanking the Ranvier nodes, are specialized adhesion sites between the axolemma and myelinating glial cells. Unraveling the molecular composition of paranodal junctions is crucial for understanding the mechanisms involved in the regulation of myelination, and positioning and segregation of the voltage-gated Na+ and K+ channels, essential for the generation and conduction of action potentials. Paranodin/Caspr was the first neuronal transmembrane glycoprotein identified at the paranodal junctions. Paranodin/Caspr is associated on the axonal membrane with contactin/F3, a glycosylphosphatidylinositol-anchored protein, essential for its correct targeting. The extra and intracellular regions of paranodin encompass multiple domains which can be involved in protein-protein interactions with other axonal proteins and glial proteins. Thus, paranodin plays a central role in the assembly of multiprotein complexes necessary for the formation and maintenance of paranodal junctions.     

Cellular contacts in myelinated fibers of the peripheral nervous system

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.     

Cellular contacts in myelinated fibers of the peripheral nervous system

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. Here we review current knowledge in this field.     

Association of TAG-1 with Caspr2 is essential for the molecular organization of juxtaparanodal regions of myelinated fibers

Myelination results in a highly segregated distribution of axonal membrane proteins at nodes of Ranvier. Here, we show the role in this process of TAG-1, a glycosyl-phosphatidyl-inositol-anchored cell adhesion molecule. In the absence of TAG-1, axonal Caspr2 did not accumulate at juxtaparanodes, and the normal enrichment of shaker-type K+ channels in these regions was severely disrupted, in the central and peripheral nervous systems. In contrast, the localization of protein 4.1B, an axoplasmic partner of Caspr2, was only moderately altered. TAG-1, which is expressed in both neurons and glia, was able to associate in cis with Caspr2 and in trans with itself. Thus, a tripartite intercellular protein complex, comprised of these two proteins, appears critical for axo-glial contacts at juxtaparanodes. This complex is analogous to that described previously at paranodes, suggesting that similar molecules are crucial for different types of axo-glial interactions.     

Continuous conduction of impulses in peripheral myelinated nerve fibers

1. Conduction of impulses in peripheral myelinated fibers of a nerve trunk is a continuous process, since with uninjured nerve fibers: (a) within each internodal segment the conduction time increases continuously and linearly with increasing conduction distance; (b) the presence of nodes of Ranvier does not result in any detectable discontinuity in the conduction of the impulse; (c) the ascending phase of the spike always has an S shape and never presents signs of fractionation; (d) the shape and magnitude of the spike are constant at all points of each internodal segment. 2. Records have been presented of the external logitudinal current that flows during propagation of an impulse in undissected single nerve fiber (Fig. 6). 3. Propagation of impulses across a conduction block occurs with a readily demonstrable discontinuity.     

Rapid conduction and the evolution of giant axons and myelinated fibers

Nervous systems have evolved two basic mechanisms for increasing the conduction speed of the electrical impulse. The first is through axon gigantism: using axons several times larger in diameter than the norm for other large axons, as for example in the well-known case of the squid giant axon. The second is through encasing axons in helical or concentrically wrapped multilamellar sheets of insulating plasma membrane--the myelin sheath. Each mechanism, alone or in combination, is employed in nervous systems of many taxa, both vertebrate and invertebrate. Myelin is a unique way to increase conduction speeds along axons of relatively small caliber. It seems to have arisen independently in evolution several times in vertebrates, annelids and crustacea. Myelinated nerves, regardless of their source, have in common a multilamellar membrane wrapping, and long myelinated segments interspersed with 'nodal' loci where the myelin terminates and the nerve impulse propagates along the axon by 'saltatory' conduction. For all of the differences in detail among the morphologies and biochemistries of the sheath in the different myelinated animal classes, the function is remarkably universal.     

Quantitative studies on the regeneration of myelinated nerve fibers. I. Variations in the number and the size of myelinated fibers in the nerves of normal rats]

The number and size of myelinated nerve fibers have been determined at standard levels in the nerve to medial head of right and left gastrocnemius muscles of 24 normal rats (11 males and 13 females). The mean values of all results were comparable on right and left sides. Thus, 271 +/- 5 myelinated nerve fibers were found in the right nerve and 272 +/- 4 in the left; their mean diameter were respectively 8.1 +/- 0.1 and 8.0 +/- 0.1 micron. There were 60.1% of large nerve fibers on the right side and 59,9% on the left, their mean diameters being 10.5 and 10.6 micron. Some variations occured in all these values, depending of the weight and sex of the animals. Nevertheless, the differences between both sides of a same rat were negligible and the histograms of both nerves could be superposed. Accordingly, in the operated animals, the contralateral nerve may be used as control.     

Activity of myelinated fibers under heat and burn influences on the skin

The dynamics of the activity of myelinated fibers of a cutaneous nerve under heat and burn influences on hair-covered skin was investigated in acute experiments on cats using the method of cross-correlation functions. Under heat influence on the skin the total activity of the nerve was reduced chiefly at the expense of the rapidly conducting myelinated fibers, whereas in the case of painful heating of the skin the A fibers were activated. The results obtained make it possible to solve the problem of the heat-information code from the standpoint of the flow "pattern" theory.S. M. Kirov Medical Institute, Ministry of Health of the Russian Federation, Nizhnii Novgorod. Translated from Neirofiziologiya, Vol. 24, No. 5, pp. 567–577, September–October, 1992.     

Ultrastructure of the Ranvier's nodes in myelinated nerve fibers of the shrimp Penaeus

From a comparative point of view the axonal cytology and the ultrastructure of Ranvier nodes in non-giant myelinated fibers of the shrimp Penaeus are described.     

The myelinated parallel fibers of the cerebellar cortex and their regional distribution

Summary In the cerebellar cortex of the Rhesus monkey and the cat, the supraganglionic plexus in the molecular layer exhibits regional differences. The plexus is very well developed in the vermal parts of the anterior lobe, but only poorly developed in the nodulofloccular lobe. Most of the fibers of this plexus are myelinated parallel fibers, which synapse in the typical manner with dendritic thorns of Purkinje cells. Only very few fibers of this plexus are recurrent collaterals of Purkinje cells. Their distribution throughout the cerebellar cortex does not display regional differences. These findings agree with physiological data on the disinhibition of Purkinje cells in different parts of the cerebellar cortex.Dedicated to Professor Dr. med. Drs. h.c. W. Bargmann in honour of his 70th birthday.Supported by the Deutsche Forschungsgemeinschaft (La 184/2).     

Cytochemical demonstration of nucleoside phosphatase activity in myelinated nerve fibers of the rat

Summary The histochemical and cytochemical localization of end product from the Wachstein-Meisel reaction was examined in transverse frozen sections of rat ischiatic nerve and spinal nerve roots. A variety of fixatives, substrates, and inhibitors were used at varying pH. Following glutaraldehyde fixation, nucleoside phosphatase activity was noted in the axon-Schwann cell interface of both unmyelinated and myelinated nerve fibers. The presence of this enzymatic activity at this strategic location in the nerve fiber complex attests to the importance of this space as a metabolically active site.Additional deposits of end product occurred on neurofilaments of myelinated and unmyelinated axons and were observed as diffuse axonal stains by light microscopy. These precipitates had a predilection for tissue fixed in formalin or hydroxyadipaldehyde and were relatively more prominent following acidic incubations. The resemblance between these deposits and spurious axonal acid phosphatase precipitates was discussed.This study was supported by Grant NB 04161 of the National Institute of Neurological Disease and Blindness.     

Unusual particle trajectories and structural arrangements in myelinated nerve fibers

Others have reported that axonally transported particles, which usually travel in a direction roughly parallel to the axis of the nerve fiber, may suddenly shift sideways as though changing tracks. Examples of this rare type of movement are shown for particles undergoing transport in myelinated axons of Xenopus laevis. An examination of the structure of axons from Xenopus showed that some microtubules, neurofilaments, and elements of endoplasmic reticulum may also exhibit marked deviations from the axial direction. It is concluded that it is not necessary to propose any mechanism for changing tracks in order to explain the particle motion.     

Quinidine blocking of sodium and potassium channels in myelinated nerve fibers

Experiments by the voltage clamp method showed that external application of quinidine (5 × 10^–5 M) to the Ranvier node membrane of the frog nerve fiber inhibitis both sodium and potassium currents. Blocking of the sodium current is considerably intensified by repetitive depolarization of the membrane (1–10 Hz); the rate of development of the block increases with an increase in stimulation frequency. After the end of stimulation the sodium current gradually returns to its initial level (with a time constant of the order of 30 sec at 12°C). Unlike repetitive depolarization with short (5 msec) stimuli, a prolonged shift (1 sec) of potential toward depolarization has no significant effect on quinidine blocking of the sodium current. Analysis of the current-voltage characteristic curves showed that quinidine blocks outward sodium current more strongly than inward. Batrachotoxin protects sodium channels against the blocking action of quinidine in a concentration of 10^–5 M. Inhibition of the outward potassium currents by quinidine is distinctly time-dependent in character: Initially the potassium current rises to a maximum, then falls steadily to a new stationary level. The results agree with the view that quinidine, applied externally, penetrates through the membrane in the basic form and blocks open sodium and potassium channels from within in the charged (protonated) form. The similarity in principle between the action of quinidine and local anesthetics on the sodium suggests that these compounds bind with the same receptor, located in the inner mouth of the sodium channel.A. V. Vishnevskii Institute of Surgery, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 14, No. 3, pp. 324–330, May–June, 1982.