Lack of neurite growth in optic nerve explants in vitro has been suggested to be due to nonpermissive substrate properties of higher vertebrate central nervous system (CNS) white matter. We have searched for surface components in CNS white matter, which would prevent neurite growth. CNS, but not peripheral nervous system (PNS) myelin fractions from rat and chick were highly nonpermissive substrates in vitro. We have used an in vitro spreading assay with 3T3 cells to quantify substrate qualities of membrane fractions and of isolated membrane proteins reconstituted in artificial lipid vesicles. CNS myelin nonpermissiveness was abolished by treatment with proteases and was not associated with myelin lipid. Nonpermissive proteins were found to be membrane bound and yielded highly nonpermissive substrates upon reconstitution into liposomes. Size fractionation of myelin protein by SDS-PAGE revealed two highly nonpermissive minor protein fractions of Mr 35 and 250-kD. Removal of 35- and of 250-kD protein fractions yielded a CNS myelin protein fraction with permissive substrate properties. Supplementation of permissive membrane protein fractions (PNS, liver) with low amounts of 35- or of 250-kD CNS myelin protein was sufficient to generate highly nonpermissive substrates. Inhibitory 35- and 250-kD proteins were found to be enriched in CNS white matter and were found in optic nerve cell cultures which contained highly nonpermissive, differentiated oligodendrocytes. The data presented demonstrate the existence of membrane proteins with potent nonpermissive substrate properties. Distribution and properties suggest that these proteins might play a crucial inhibitory role during development and regeneration in CNS white matter.

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Two membrane protein fractions from rat central myelin with inhibitory properties for neurite growth and fibroblast spreading.

In this study the authors have developed a model with which can be studied directly the influence of circulating anti-myelin antibody on the clinical and pathologic course of inflammatory T-cell-mediated experimental allergic encephalomyelitis (EAE) in the rat. EAE was induced by passive transfer of either myelin basic protein (MBP)-activated spleen cells derived from sensitized donors or long-term-cultured MBP-specific T-cell lines. At the onset of the disease, monoclonal antibodies against a myelin/oligodendrocyte glycoprotein (MOG) were injected intravenously. This antigen is exposed on the surface of central nervous system myelin and oligodendrocytes. Intravenous injection of the antibody in the course of T-cell-mediated transfer EAE augmented the severity and duration of clinical signs and resulted in the formation of large, confluent demyelinated plaques.

Augmentation of demyelination in rat acute allergic encephalomyelitis by circulating mouse monoclonal antibodies directed against a myelin/oligodendrocyte glycoprotein

In the second edition of the book Neurochemistry, Rossiter (1962) stated, “Since pure ‘myelin’ is not available for direct chemical analysis, the neurochemist has been compelled to deduce the constituents of myelin from such analyses as are practicable.” The available analyses were extensive. Qualitative information had been accumulating from histological staining techniques since the latter half of the nineteenth century. Quantitative studies on brain constituents from which information on myelin could be deduced began shortly after the turn of the century. Thus in his chapter on “The Myelin Sheath, Metabolism of Myelin and Experimental Demyelination,” Rossiter was able to discuss myelin composition and metabolism with considerable assurance and accuracy. The direct analysis of myelin constituents was, however, not possible until techniques were developed for isolating myelin membranes essentially free of other subcellular structures. The first of such procedures were being developed and published in the same year Neurochemistry was published. In this chapter, I will discuss isolation procedures and the composition of purified myelin obtained by these procedures.

Isolation and Characterization of Myelin

Myelin-associated glycoprotein (MAG) is a quantitatively minor component in both peripheral and central myelin sheaths that is thought to have a role in cell-cell interactions within the nervous system. We show here that a mouse monoclonal antibody, HNK-1, which is directed against human natural killer cells also recognizes an antigenic determinant of human central and peripheral nervous system white matter by immunoperoxidase staining of tissue sections. Immunoblot analysis of myelin proteins and purified extracted MAG indicates that the antigen recognized is MAG.

Recognition of myelin-associated glycoprotein by the monoclonal antibody HNK-1.

Previous studies have indicated that newly formed oligodendrocytes are dynamic cells whose production, survival, and differentiation depend upon axonal influences. This study has characterized the appearance and fate of newly formed oligodendrocytes in developing rat brain. Oligodendrocytes appear in predictable locations and radially extend DM-20-positive processes that cover 80-microm domains in the cortex and 40-microm domains in the corpus callosum. These premyelinating oligodendrocytes have one of two fates: they myelinate axons or degenerate. Between 7 and 21 d after birth, approximately 20% of premyelinating oligodendrocytes identified in the cerebral cortex were degenerating. Oligodendrocytes that ensheathed axons expressed and selectively targeted proteolipid protein to compact myelin and did not degenerate. These observations support the hypothesis that axonal influences affect oligodendrocyte survival, differentiation, and expression of proteolipid protein gene products.

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Differentiation and death of premyelinating oligodendrocytes in developing rodent brain.

— Myelin was purified from rats which had been injected intracerebrally with radioactive fucose in order to label specifically the glycoproteins. Myelin contained a small amount of fucose-labelled glycoproteins in comparison to that in other subcellular fractions, but polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate revealed a unique pattern of radioactive glycoproteins dominated by a major peak. The same glycoprotein was not prominent in the other subcellular fractions which were examined. This major glycoprotein in the myelin fraction was also labelled after injection with [3H]glucosamine or N-[3H]acetylmannosamine. It was the most intensely staining myelin protein when gels were treated with periodic acid-Schiff reagents, an indication that, in terms of protein-bound carbohydrate, it is the major glycoprotein in the myelin fraction. The glycoprotein was present in myelin purified from rats ranging in age from 14 days to 14 months. Extensive recycling of the myelin through the purification procedures did not significantly reduce the amount of glycoprotein in the myelin. Double label experiments with [3H]fucose and [14C]fucose were used to compare glycoproteins in myelin purified from white and grey matter, respectively, and from mixed homogenates of myelinated and unmyelinated brain. The results obtained from these experiments suggested that the glycoprotein is closely associated with myelin and that it is not in an unrelated contaminating structure. Possible locations of the glycoprotein are discussed. They include the myelin membrane itself, the oligodendroglial plasma membrane, and the axolemma of myelinated axons.

Evidence for the close association of a glycoprotein with myelin in rat brain.

Evidence is presented that the major protein of rat sciatic nerve myelin is a glycoprotein. When myelin proteins were separated by polyacrylamide gel electrophoresis, the major band which was stained with amido black–Coomassie blue was also stained with periodic acid-Schiff reagents for carbohydrate. Radioactive labelling of myelin in vivo with [3H]leucine and [14C]fucose, followed by electrophoresis of the proteins, indicated that with both isotopes the major labelled peak corresponded to the major stained band. In addition, a second smaller peak of [14C]fucose migrated ahead of the major peak. Delipidated myelin contained galactose, mannose, fucose and sialic acid.

John L. Everly

Papers

Evidence for the close association of a glycoprotein with myelin in rat brain.

Evidence that the major protein in rat sciatic nerve myelin is a glycoprotein.

Myelin-associated glycoprotein: a developmental change.

Demonstration of a glycoprotein which is associated with a purified myelin fraction from rat brain.

[3 5S]Sulfate incorporation into myelin glycoproteins II. Peripheral nervous tissue