Myelin basic protein is necessary for the regulation of myelin-associated glycoprotein expression in mouse oligodendroglia.

Myelin-associated glycoprotein (MAG) has currently been proposed to be a possible igniter of myelination in the central nervous system. We propose here that myelin basic protein (MBP) is prerequisite, which is more diffusely expressed than MAG in early stages of myelination. MBP-deficient mice show significant reduction of MAG-positive immature oligodendroglia at early stages of myelination, although significant increase of such cells is noted in the adult. MBP/Fyn-double deficient adult mice also show moderate increase of the same type of immature glia, but to a lesser degree than MBP-deficiency alone. The present study suggests that MBP is required for the regulation of MAG expression in oligodendroglia, involving Fyn therein. We discuss the possibility that hitherto unknown molecule, not MAG, may be in responsible for the ignition of the myelination.

Fyn tyrosine kinase participates in the compact myelin sheath formation in the central nervous system.

The cellular mechanisms for spiral wrapping and compaction of myelin sheaths by oligodendrocytes are not known yet. In this study, we examined the role of fyn tyrosine kinase, which could be responsible for molecular events during the stage of myelination in the CNS. Western blot and immunohistochemical analyses revealed that fyn-deficient mice have significantly lower levels of myelin basic protein (MBP), which is required for intracellular membrane adhesion parts so-called major dense line (MDL) and thought to be essential for the stability of myelin sheath. Electron microscopy verified that the myelin ultrastructure could be used to distinguish fyn-deficient mice from wild-type mice, showing a thin and redundant myelin sheath in the corpus callosum. Further, the electron-dense 'major' line in myelin from the purified myelin fractions remained condensed, and myelin compaction was split opened in fyn-deficient mice. To determine whether there was a change in the microheterogeneity of MBP due to a post-translational event we first investigated peptidylarginine deiminase (PAD), which is an enzyme that converts arginine residues in peptides to citrulline residues. PAD immunoreactivity was observed both in the myelin from fyn-deficient and wild-type mice. By Western blot analysis we found an increase of the citrullined form of MBP. In addition, MBP from fyn-deficient mice did weakly induce vesicle aggregation properties of MBP-mediated adhesion. We concluded that although oligodendrocytes from fyn-deficient mice are able to wrap around the axon, they are unable to form compact myelin due to decreased MBP level and the presence of increased citrullinated MBP.

Monoclonal antibody 14F7, which recognizes a stage-specific immature oligodendrocyte surface molecule, inhibits oligodendrocyte differentiation mediated in co-culture with astrocytes.

Cells at an intermediate stage of oligodendrocyte lineage are not only well characterized by biochemical studies but also are likely to relate to the outcome of physiological events. To elucidate the molecular events leading to the development of oligodendrocyte lineage cells, we have raised monoclonal antibodies against stage-specific immature oligodendrocytes, which have previously been isolated by a novel oligodendrocyte-lineage cell culture technique (Sakurai et al.: J Neurosci Res 52:17-26, 1998). We have isolated a mouse monoclonal antibody termed 14F7 which predominantly labels stage-specific immature oligodendrocytes and have found that the expression of 14F7 immunoreactivity in the developing neonatal rat forebrain is closely associated with cells expressing the oligodendrocyte progenitor marker A2B5 and to immature oligodendrocyte expressing O4 antigen. 14F7+ cells were distributed in the ventricular and subventricular zone and the nearby forming corpus callosum as non-myelinating cells. In contrast to cell culture observations, 14F7+ cells were seen only in oligodendrocyte lineage cells. For instance, dissociated cell culture studies indicated that 14F7 labels a cell surface molecule, and its cellular distribution is coincident with all of O4+ cells and A2B5+ cells, and even A2B5- cells. By contrast, 14F7-positive cells did not label astrocytes and, furthermore, did not label myelin basic protein (MBP)-positive oligodendrocytes. 14F7 recognized a 48-kDa protein on sodium dodecyl sulfate polyacrylamide gel electrophoresis. 14F7 immunoreactivity was detectable in rat brain as early as embryonic day 18. Furthermore, in these cells, the total time for differentiation was extended, and on maturation, these cells subsequently expressed an array of myelin-specific proteins, which normally occurs by direct contact with type-1 astrocytes. However, in the presence of 14F7, stage-specific oligodendrocytes co-cultured with astrocytes completely failed to express MBP. These data suggest that the 14F7 antigen is a novel cell surface molecule that is expressed in the intermediate stage of oligodendrocyte-lineage cells, and it is expected that it regulates the differentiation of oligodendrocyte throughout development.

Differentiation of oligodendrocyte occurs in contact with astrocyte.

We have generated stage-specific oligodendrocytes, which are identified as immature oligodendrocytes by the expression of specific markers. These cells were generated by using 3 types of intrinsic and extrinsic factors for influencing the characteristics of oligodendrocyte-lineage cells. The factors included 10% serum in the medium to induce cell proliferation, bFGF in serum-free medium to increase survival and induce differentiation, and 0.25% trypsin to remove nonoligodendrocyte lineage cells. We have also demonstrated that it is possible to supply a huge amount of homogeneous populations of stage-specific immature oligodendrocytes after 35 days' culture. The maturation of these cells is controlled by interacting with astrocytes in vitro, resulting in the expression of MBP isoforms (14.0 and 18.5 kDa) that may participate in mature oligodendrocyte membrane compaction.