Monoclonal antibody selectively reactive with myelin sheaths of the peripheral nervous system in paraffin-embedded material

Summary We have produced a monoclonal antibody (P_M43) selectively reactive with formalin-fixed paraffin-embedded peripheral nervous system (PNS) myelin. The hybridomas were generated by fusion of mouse myeloma cell line Sp2/0 with spleen cells of BALB/c mice immunized with cultured human melanocytes. Hybridomas were screened by the indirect immunoperoxidase assay. Ouchterlony analysis showed the immunoclass of P_M43 to be IgM. By the immunoaffinity chromatography technique, among others a 43-kDa protein was isolated from PNS myelin. The antigenic determinant of P_M43 in the mouse is expressed with a similar tissue distribution as observed in man. Expression of the antigenic determinant does not become visible until after birth in mice. P_M43 opens further possibilities for the use of anti-myelin antibodies in the study of myelination and demyelination processes in the PNS and remyelination processes in the central nervous system.Supported by the Dutch Ministry of Housing, Physical Planning and Environmental Management (M. J. van Haperen)     

Laminin overrides the inhibitory effects of peripheral nervous system and central nervous system myelin-derived inhibitors of neurite growth

Axon growth inhibitory proteins associated with central nervous system (CNS) myelin are responsible in part for the absence of long distance axon regeneration in the adult mammalian CNS. We have recently reported that myelin-associated glycoprotein (MAG), which is also present in peripheral nerves, is a potent inhibitor of neurite growth. This was surprising given the robust regenerative capacity of peripheral nerves. We now provide evidence that myelin purified from peripheral nerve also has neurite growth inhibitory activity. However, this activity can be masked by laminin, which is a constituent of the Schwann cell basal lamina. We also report that laminin, which is largely absent from the normal adult mammalian CNS, when added to purified CNS myelin, can override the neurite growth inhibitory activity in CNS myelin. These results have important implications for the development of strategies to foster axon regeneration in the adult mammalian CNS where multiple growth inhibitors exist. © 1995 Wiley-Liss, Inc.     

Rapid assessment of internodal myelin integrity in central nervous system tissue

Monitoring pathology/regeneration in experimental models of de‐/remyelination requires an accurate measure not only of functional changes but also of the amount of myelin. We tested whether X‐ray diffraction (XRD), which measures periodicity in unfixed myelin, can assess the structural integrity of myelin in fixed tissue. From laboratories involved in spinal cord injury research and in studying the aging primate brain, we solicited “blind” samples and used an electronic detector to record rapidly the diffraction patterns (30 min each pattern) from them. We assessed myelin integrity by measuring its periodicity and relative amount. Fixation of tissue itself introduced ±10% variation in periodicity and ±40% variation in relative amount of myelin. For samples having the most native‐like periods, the relative amounts of myelin detected allowed distinctions to be made between normal and demyelinating segments, between motor and sensory tracts within the spinal cord, and between aged and young primate CNS. Different periodicities also allowed distinctions to be made between samples from spinal cord and nerve roots and between well‐fixed and poorly fixed samples. Our findings suggest that, in addition to evaluating the effectiveness of different fixatives, XRD could also be used as a robust and rapid technique for quantitating the relative amount of myelin among spinal cords and other CNS tissue samples from experimental models of de‐ and remyelination. © 2009 Wiley‐Liss, Inc.     

Genes coding for proteins in central nervous system myelin

There has been considerable interest recently in a genetic component as a causative factor in multiple sclerosis, but the identity of putative susceptibility genes is unknown. In the past decade, the primary amino acid sequences of the four proteins making up 90% of the protein content of central nervous system myelin (proteolipid protein, myelin basic protein, 2',3'-cyclic nucleotide-3'-phosphohydrolase, and myelin-associated glycoprotein) have been determined in several species. Additionally, the structural genes coding for these proteins have been analysed and their human chromosomal localization determined. We have been analysing these genes for possible variants conferring susceptibility to multiple sclerosis. Recent results have shown that cholera and pertussis toxin substrates and low molecular-weight GTP-binding proteins are also present in central nervous system myelin. This implies the presence of signal transducing systems whose purpose is currently obscure. The emerging picture of central nervous system myelin is of a complex dynamic structure composed of many more proteins than was previously thought.     

Schwann cells and peripheral nervous system myelin in the rat retina

Summary Within the retinal nerve fiber layer of a 6-week-old Sprague-Dawley rat, scattered aggregates of PNS myelinated axons have been found and described. We believe this is likely to represent a normal but rare phenomenon in the rat.     

The logistics of myelin biogenesis in the central nervous system

Rapid nerve conduction depends on myelin, but not all axons in the central nervous system (CNS) are myelinated to the same extent. Here, we review our current understanding of the biology of myelin biogenesis in the CNS. We focus on how the different steps of myelination are interconnected and how distinct patterns of myelin are generated. Possibly, a “basal” mode of myelination is laying the groundwork in areas devoted to basic homeostasis early in development, whereas a “targeted” mode generates myelin in regions controlling more complex tasks throughout adulthood. Such mechanisms may explain why myelination progresses in some areas according to a typical chronological and topographic sequence, while in other regions it is regulated by environmental stimuli contributing to interindividual variability of myelin structure. GLIA 2017;65:1021–1031     

Sequential myelin protein expression during remyelination reveals fast and efficient repair after central nervous system demyelination

To understand the mechanisms of remyelination and the reasons for regeneration failure is one of the major challenges in multiple sclerosis research. This requires a good knowledge and reliable analysis of experimental models. This work was undertaken to characterize the pattern of myelin protein expression during experimental remyelination. Acute demyelination of the corpus callosum was induced by feeding of 0.3% cuprizone for 6 weeks, followed by a 10-week remyelination period. We used a combination of Luxol fast blue (LFB) myelin staining, electron microscopy (EM) and immunohistochemistry for the myelin proteins 2',3'-cyclic nucleotide 3' phosphodiesterase (CNPase), myelin basic protein (MBP), proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG). Early remyelination was detected by the re-expression of CNPase, MBP and PLP as early as 4 days. MOG, as a marker for late differentiation of oligodendrocytes, was not detectable until 2 weeks of remyelination. EM data correlated well with the LFB myelin staining and myelin protein expression, with 50% of the axons being rapidly remyelinated within 2 weeks. While particularly MBP but also PLP and CNPase are re-expressed very early before significant remyelination is observed by EM, the late marker MOG shows a lag behind the remyelination detected by EM. The presented data indicate that immunohistochemistry for various myelin proteins expressed early and late during myelin formation is a suitable and reliable method to follow remyelination in the cuprizone model. Furthermore, investigation of early remyelination confirms that the intrinsic repair programme is very fast and switched on within days.     

Immunohistological analysis of macrophages in the central nervous system of Lewis rats with acute experimental allergic encephalomyelitis

Inflammatory cells were characterized by immunohistochemistry, utilizing monoclonal antibodies specific for T cell subsets, B cells, Ia-positive cells and cells of the mononuclear phagocyte system, in cryostat sections of central nervous system of Lewis rats, sacrificed during the course of actively induced experimental allergic encephalomyelitis. The present study provides interesting information about the presence and distribution of cells of the monocyte-macrophage lineage in this immunologically mediated disease. Using these monoclonal antibodies different subpopulations of macrophages having varying distribution patterns in the central nervous system can be recognized.     

Heterosis for myelin content is limited to the central nervous system

We have recently described a murine model for studying aspects of myelination (Seyfried and Yu, 1980; Ebato et al., 1983; Miskimins et al., 1986). This mouse shows hypermyelination during the period of most active synthesis of myelin, 9 to 21 days post-natal. The myelin parameters showing an increase were all measured in the central nervous system. We investigated here whether this effect extends into the peripheral nervous system. Our results indicate that the hypermyelination is limited to the central nervous system.     

Experimental hyperphenylalaninemia: Effect on central nervous system myelin subfractions

We examined the effects of experimental hyperphenylalaninemia on the formation of subfractions of myelin in the central nervous system of 18- to 110-day-old rats. Hyperphenylalaninemia was induced by giving rats daily subcutaneous injections of α-methylphenylalanine and phenylalanine from the 3rd to the 15th postnatal day. A mixture of [³H]glycine and [¹⁴C]glucose was injected intraventricularly into 17-, 20-, and 24-day-old control and experimental rats 18 h prior to the isolation and subfractionation of CNS myelin. Nonradioactive myelin subfractions were isolated from 43- and 110-day-old rats.Animals that had been hyperphenylalaninemic between the 3rd and 15th postnatal day exhibited a deficiency of total myelin protein throughout the study. With the exception of one age, these rats had a deficit of the classical, mature light myelin. The light and medium myelin subfractions from 18- to 43-day-old experimental rats and heavy myelin from the 25- and 43-day-old experimental rats had a deficiency of myelin basic proteins and an excess of the high molecular weight proteins. Nonetheless, previously hyperphenylalaninemic rats incorporated more [³H]glycine into the basic proteins of all myelin subfractions. The proportion of total brain [³H] glycine and [¹⁴C]glucose that was incorporated into combined (total) myelin subfractions was near normal in rats that were hyperphenylalaninemic between the 3rd and 15th postnatal day. However, these rats demonstrated decreased ³H and ¹⁴C incorporation into light myelin at 21 and 25 days, increased ³H and ¹⁴C incorporation into medium myelin at 25 days, and increased incorporation into heavy myelin at 21 days.     

BDNF overexpression produces a long-term increase in myelin formation in the peripheral nervous system

The neurotrophin brain-derived neurotrophic factor (BDNF) is an endogenous regulator of the myelination process during development in the peripheral nervous system. Enhancement of myelin formation by BDNF is mediated by the neurotrophin receptor p75(NTR). Although this neurotrophin is a positive modulator of myelination during early development, the final effects of BDNF on myelin sheaths after active myelination is completed are largely unknown. Using BDNF transgenic mice, we examined the long-term effects of BDNF on myelination of the peripheral nervous system in vivo. Elevation of BDNF levels in the transgenic mice produced an increase in both the rate and extent of the myelination process. BDNF enhanced and accelerated myelin formation during early development and this increase in myelin content and thickness was maintained in adulthood. Besides enhanced myelination, BDNF also influenced axon caliber size but to a lesser extent. This lagging increase in axon caliber compared to myelin suggests that the axon size is not the only determinant of myelin thickness.     

Leukemia inhibitory factor signaling modulates both central nervous system demyelination and myelin repair

Leukemia inhibitory factor (LIF) receptor signaling limits the severity of inflammatory demyelination in experimental autoimmune encephalomyelitis, a T-cell dependent animal model of multiple sclerosis (MS) [Butzkueven et al. (2002) Nat Med 8:613-619]. To identify whether LIF exerts direct effects within the central nervous system to limit demyelination, we have studied the influence of LIF upon the phenotype of mice challenged with cuprizone, a copper chelator, which produces a toxic oligodendrocytopathy. We find that exogenously administered LIF limits cuprizone-induced demyelination. Knockout mice deficient in LIF exhibit both potentiated demyelination and oligodendrocyte loss after cuprizone challenge, an effect that is ameliorated by exogenous LIF, arguing for a direct beneficial effect of endogenous LIF receptor signaling. Numbers of oligodendrocyte progenitor cells in cuprizone-challenged mice are not influenced by either exogenous LIF or LIF deficiency, arguing for effects directed to the differentiated oligodendrocyte. Studies on the influence of LIF upon remyelination after cuprizone challenge fail to reveal any significant effect of exogenous LIF. The LIF-knockout mice do, however, display impaired remyelination, although oligodendrocyte replenishment, previously identified to occur from the progenitor pool, is not significantly compromised. Thus endogenous LIF receptor signaling is not only protective of oligodendrocytes but can also enhance remyelination, and exogenous LIF has therapeutic potential in limiting the consequences of oligodendrocyte damage.     

Proteolipid protein cannot replace P0 protein as the major structural protein of peripheral nervous system myelin

The central nervous system (CNS) of terrestrial vertebrates underwent a prominent molecular change when proteolipid protein (PLP) replaced P₀ protein as the most abundant protein of CNS myelin. However, PLP did not replace P₀ in peripheral nervous system (PNS) myelin. To investigate the possible consequences of a PLP to P₀ shift in PNS myelin, we engineered mice to express PLP instead of P₀ in PNS myelin (PLP‐PNS mice). PLP‐PNS mice had severe neurological disabilities and died between 3 and 6 months of age. Schwann cells in sciatic nerves from PLP‐PNS mice sorted axons into one‐to‐one relationships but failed to form myelin internodes. Mice with equal amounts of P₀ and PLP had normal PNS myelination and lifespans similar to wild‐type (WT) mice. When PLP was overexpressed with one copy of the P₀ gene, sciatic nerves were hypomyelinated; mice displayed motor deficits, but had normal lifespans. These data support the hypothesis that while PLP can co‐exist with P₀ in PNS myelin, PLP cannot replace P₀ as the major structural protein of PNS myelin. GLIA 2015;63:66–77     

Characterization of a monoclonal antibody specific for human peripheral myelin protein 22 and its use in immunohistochemical studies of the fetal and adult nervous system

We produced a mouse monoclonal antibody using cDNA and peptide immunization against the putative second extra-cellular domain of human peripheral myelin protein 22 (PMP22). It reacted specifically with human PMP22 and not with other human myelin proteins and did not react with bovine, rat, or mouse PMP22. The antibody stained the compact myelin of human peripheral nerve motor and sensory axons and did not stain central nervous system tissue. PMP22 reactivity was detected in the spinal roots of the human fetus at 19-20 weeks of gestation. The staining pattern of the PMP22 antibody resembled that of a monoclonal antibody directed against the myelin protein zero.     

Saxitoxin binding to central and peripheral nervous tissue of the myelin deficiency (md) mutant rat

Tritium-labeled saxitoxin binding was assayed in the CNS and PNS of normal and of 'myelin deficiency' (md) mutant rat pups. This mutant in the Wistar rat is characterized by a virtually complete amyelination of the CNS, the PNS being normal. No significant difference was found in the saxitoxin binding capacity of md and normal brain, spinal cord, or sciatic nerves. This suggests that the presence or absence of myelin does not influence the number of voltage-sensitive sodium channels in the rat.     

Myelination requirements of central nervous system glia in vitro: statistical validation of differences between glia from adult and immature mice

Glia from adult optic nerve, like those from immature nerve, have been shown to colonize and myelinate cerebellar axons in combined cultures. Preliminary experiments suggested, however, that the glia from adults might require the added nutrients and/or chemical signals present in a serum and embryo extract containing medium to produce myelin, whereas glia from immature animals could form myelin in a chemically defined medium described by Hendelman et al. (1985). This observation demanded rigorous confirmation, since it depended on the meaning of a negative result (the absence of myelin), which could be produced by many factors other than the composition of the medium in this complex culture system. Here we report an extensive series of experiments, with controls for all other factors, that provide unquestionable statistical validation of the additional myelination requirements of the glia from adult nerves. The cultures can now be used to identify these required substances, which may be limiting factors for central nervous system myelin regeneration in situ.     

Studies with antisera against peripheral nervous system myelin and myelin basic proteins. I. Effects of antiserum upon living cultures of nervous tissue

We studied the effects of antiserum against rat peripheral nervous system (PNS) myelin, rat or chicken central nervous system myelin basic protein (BP), or rabbit P₂ protein from PNS myelin on myelinated cultures containing only rat dorsal root ganglion neurons and Schwann cells. While anti-PNS myelin serum consistently produced segmental PNS demyelination, anti-BP serum and anti-P₂ serum did not. The culture results suggest that the myelin PNS proteins P₁ (identical to basic protein from central nervous system myelin) and P₂ are not exposed on the extracellular surfaces of myelin-related Schwann cells in tissue culture.     

Quantitation of myelin oligodendrocyte glycoprotein and myelin basic protein in the thymus and central nervous system and its relationship to the clinicopathologic features of autoimmune encephalomyelitis

There is controversy whether the amount of autoantigens expressed in the thymus regulates negative selection of autoreactive T cells and determine susceptibility or resistance to experimental autoimmune encephalomyelitis (EAE). In the present study, we have addressed this issue by quantifying neuroantigens in the thymus of two EAE-susceptible (LEW and LEW.1AV1) and one EAE-resistant (BN) rat strains. We further examined whether amounts of neuroantigens in various parts of the central nervous system (CNS) affect the clinical course and lesion distribution of acute and chronic EAE. Real-time PCR and histologic analyses showed that there was no significant difference in the amount and distribution of myelin oligodendrocyte glycoprotein and myelin basic protein in the thymus and CNS among the three strains and that both acute and chronic EAE lesions in the CNS were preferentially distributed in the area where neuroantigens were abundantly present. These findings suggest that susceptibility or resistance to EAE is not regulated by the amount of the neuroantigens expressed in the thymus. Furthermore, the lesion distribution, but not the clinical course, of EAE is related to the neuroantigen expression in the CNS.