Brain reaggregate cultures: Biochemical evidence for myelin membrane synthesis

Myelin membrane synthesis was studied using mechanically dissociated fetal rodent CNS which formed spherical reaggregates while being maintained in rotating culture flasks. These reaggregate cultures exhibited myelinogenesis in vitro after precisely the same period of time needed for myelin synthesis to commence in vivo. The myelin membrane related enzymes, 2′,3′ cyclic nucleotide phosphohydrolase (CNP) and cerebroside sulfotransferase (CST), appear similar in their specific activities and follow the same developmental patterns that these enzymes exhibit in vivo. In addition, phosphorylation of myelin basic protein occurs by the third week in vitro which agrees with previously published in vivo studies. These experiments indicate that this nerve-cell culture system may be an appropriate model for studying the biological regulation of myelinogenesis as well as a variety of other nervous-system functions.     

Galactolipids are molecular determinants of myelin development and axo-glial organization

Myelination is a developmentally regulated process whereby myelinating glial cells elaborate large quantities of a specialized plasma membrane that ensheaths axons. The myelin sheath contains an unusual lipid composition in that the glycolipid galactosylceramide (GalC) and its sulfated form sulfatide constitute a large proportion of the total lipid mass. These glycolipids have been implicated in a range of developmental processes such as cell differentiation and myelination initiation, but analyses of mice lacking UDP-galactose:ceramide galactosyltransferase (CGT), the enzyme required for myelin galactolipid synthesis, have more recently demonstrated that the galactolipids more subtly regulate myelin formation. The CGT mutants display a delay in myelin maturation and axo-glial interactions develop abnormally. By interbreeding the CGT mutants with mice that lack myelin-associated glycoprotein, it has been shown that these specialized myelin lipids and proteins act in concert to promote axo-glial adhesion during myelinogenesis. The analysis of the CGT mutants is helping to clarify the roles myelin galactolipids play in regulating the development, and ultimately the function of the myelin sheath.     

Lipid-metabolizing enzymes of myelin and their relation to the axon

The old concept of myelin as a metabolically inert membrane has been considerably revised as a result of the discovery of numerous enzyme activities in the isolated membrane. The high degree of purification and low levels of contamination markers leave little doubt that the measured activities are intrinsic to myelin itself. Slightly more than half of the discovered activities involve lipid metabolism. One such enzyme, neutral cholesterol esterase, is myelin-specific, while the rest occur in other subcellular fractions as well as myelin. These include activities involved in synthesis of cerebrosides, phospholipids, and cholesteryl esters; only a few degradative enzymes are presently known. In vivo studies have shown that various substrates utilized by lipid-synthesizing enzymes of myelin can originate in the axon. Six such substrates have been characterized. The possibility exists that these enzymes may be wholly or partially dependent on the axon as the primary source of substrate, thereby suggesting a possible form of metabolic dependency of myelin on the axon.     

Phosphorylation of Myelin-Associated Glycoprotein In Vivo and In Vitro Occurs Only in the Cytoplasmic Domain of the Large Isoform

Myelin-associated glycoprotein (MAG) was radioactively labelled with 32P both in intact brain and in myelin membrane preparations. Chemical deglycosylation of the phosphorylated products revealed that only one of the MAG isoforms (L-MAG) is labelled in vitro. Furthermore, the phosphorylation events in vivo and in vitro are confined to the cytoplasmic portion of the L-MAG isoform. Tryptic mapping of L-MAG labelled both in vivo and in vitro revealed that the majority of the sites phosphorylated in intact brain are also phosphorylated in myelin membrane preparations; however, the extent of phosphorylation at individual sites is variable. The results demonstrate that partially purified myelin membrane preparations can be used to study the enzymes responsible for MAG phosphorylation and dephosphorylation events in vivo.     

RGD-containing peptides inhibit the synthesis of myelin-like membrane by cultured oligodendrocytes

A synthetic peptide derived from the fibronectin cell-binding domain, GRGDSP, inhibits the adhesion of rat oligodendrocytes to a number of substrates. However, while GRGDSP inhibited the adhesion of cells in a short term adhesion assay, the presence of the peptide did not prevent cells from adhering and thriving in longer term culture. The morphological characteristics of individual cells cultured with 0.1 mg/ml GRGDSP were similar to untreated cultures; small rounded cell bodies radiating numerous fine processes. Peptide-treated cultures were inhibited in their ability to produce myelin specific components. The characteristic developmental peak in sulfolipid synthesis which occurs both in vivo and in vitro was completely inhibited when cells were cultured with GRGDSP. In addition, the synthesis of myelin basic protein was inhibited. Ultrastructurally, cells treated with GRGDSP showed a greatly reduced number of multilamellar myelin-like membrane figures than cells grown without peptide or those grown with GRADSP. Cultured oligodendrocytes did not become sensitive to inhibition of sulfolipid synthesis by GRGDSP until a period immediately preceding the peak in sulfolipid biosynthesis. The effects of pretreatment with peptide for 5 d before this time were completely reversible. Pretreatment which extended into the time of peak myelin synthesis resulted in permanent impairment in the cell's ability to synthesize sulfolipid. The oligodendrocyte's ability to synthesize a myelin-like membrane in culture is, in part, inherent since it occurs in the absence of neurons. The present results indicate that myelin membrane production is also subject to external control since it appears that occupancy of an RGD-dependent cell surface receptor during a critical period of in vitro development is required for the oligodendrocyte to produce myelin-like membrane.     

Evidence That Secondary Rat Schwann Cells in Culture Maintain Their Differentiated Phenotype

Schwann cells, on receiving the correct signal, will encircle an axon and wrap it with a myelin sheath. To begin examining some of the mechanisms underlying the process of myelination in vitro, we isolated Schwann cells from the sciatic nerves of neonatal rats and generated large cell populations with cholera toxin. The immunological and biochemical properties of these secondary Schwann cells were characterized after five to seven passages in the absence of axonal contact. These cells continued to express antigens found in both myelinating (P0 and 2',3'-cyclic nucleotide phosphohydrolase) and nonmyelinating cells in vivo (A5E3 and glial fibrillary acidic protein) in addition to the markers common to both types of cells (Ran-1, 217c, S-100, and laminin). Biochemical analyses showed that these cells synthesize the very-long-chain fatty acids (22-26 carbon atoms) found in myelin membranes. Moreover, the enzymes required for the synthesis of myelin glycolipids (including sphingosine acyltransferase, UDP-galactose:ceramide galactosyltransferase, and cerebroside sulfotransferase) were still active, and metabolic labeling studies showed that galactocerebroside and sulfatide were synthesized even though the galactocerebroside pool was insufficient to be detected by immunostaining. Secondary Schwann cells also synthesized four species of myelin basic protein and the major structural glycoprotein in myelin, P0. The pathway necessary for glycosylation of P0 protein remained active, and an analysis of the oligosaccharide chain revealed that approximately 70% was processed to a complex form. In summary, we found that secondary Schwann cells still express most of the immunological markers of differentiated cells and continue to synthesize low levels of myelin components. Therefore, Schwann cells do not dedifferentiate in culture, as previously believed.     

THE ULTRASTRUCTURE OF ADULT VERTEBRATE PERIPHERAL MYELINATED NERVE FIBERS IN RELATION TO MYELINOGENESIS

Adult chameleon myelinated peripheral nerve fibers have been studied with the electron microscope in thin sections. The outer lamella of the myelin sheath has been found to be connected as a double membrane to the surface of the Schwann cell. The inner lamella is connected as a similar double membrane with the double axon-Schwann membrane. The relations of these double connecting membranes suggest that the layered myelin structure is composed of a double membrane which is closely wound about the axon as a helix. These findings support the new theory of myelinogenesis proposed recently by Geren. The possible significance of these results with respect to cell surface membranes and cytoplasmic double membranes is discussed.     

PROTEIN AND GLYCOPROTEIN COMPOSITION OF MYELIN AND MYELIN SUBFRACTIONS FROM BRAINS OF ''QUAKING'' MICE

Abstract— Quaking mutants in mice are known to be affected by an arrest of myelinogenesis and to have a purified myelin which is more dense than that of controls. Their myelin has been shown to demonstrate a striking decrease in proteolipid protein, a lesser decrease in the small myelin basic protein and changes in glycoproteins comprising reduction in the major peak and shift of this peak towards a higher apparent molecular weight. The possibility that these findings might reflect merely contamination of myelin with other membranes was tested by subfractionation. Light myelin (floats on 0.62 m -sucrose) is generally accepted as more compact and mature than the heavier subfraction (floating on 0.85 m -sucrose). The changes previously found were present in both subfractions and even more marked in the light myelin. These results indicate that the anomalies of myelin proteins and glycoproteins were not caused by contaminants and are present in compact myelin as well as in membranes which are transitional between the glial plasma membrane and the myelin sheath. Therefore, we suggest that the Quaking mutation results in dysmyelination rather than hypomyelination.     

Biochemical Study of Heterosis for Brain Myelin Content in Mice

Heterosis (hybrid vigor) for brain myelin content has been examined in detail in (C57BL/6J x DBA/2J)F1 hybrid mice at 17 days of age. The amount of myelin isolated from the F1 hybrid brain is greater than that isolated from either parental strain. In addition, the total protein content in the myelin of the three genotypes showed the following trend: F1 greater than DBA greater than C57. However, no discernible differences in myelin protein compositions could be detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analysis of the whole brain for several myelin-associated constituents such as GM1 ganglioside, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase), 5'-nucleotidase, and carbonic anhydrase indicated that heterosis exists for these components. No heterosis was found for such nonmyelin constituents as gangliosides GD1a, GT, GQ, RNA, DNA, and choline acetyltransferase. A developmental study of the whole brain CNPase indicated that the heterotic effect was greatest during the most active period of myelination (17-30 days). We conclude that the heterotic effect is specific for myelin content and is probably the result of an accelerated myelin synthesis. The heterotic effect should have great potential as a new model for studying aspects of myelinogenesis.     

Axonal Signals and Oligodendrocyte Differentiation

Axons produce signals that regulate oligodendrocyte proliferation, survival, terminal differentiation, and myelinogenesis. We review here recent in vitro and in vivo experimental approaches that aim to characterize axonal signals to oligodendroglia and to identify molecular mediators that regulate differentiation of oligodendendrocytes. We propose that the promoters of myelin genes, whose activation during terminal differentiation is modulated by axonal signals, can provide a means to identify molecular mediators of axo-oligodendroglial signals.     

Antibodies to Myelin/Oligodendrocyte-Specific Protein and Myelin/Oligodendrocyte Glycoprotein Signal Distinct Changes in the Organization of Cultured Oligodendroglial Membrane Sheets

Abstract: Cultured murine oligodendrocytes elaborate extensive membrane sheets that, unlike multilamellar myelin in vivo, allow the study of interactions between myelin proteins and cytoskeletal elements. This article describes the events that occur due to the interaction of specific antibodies with their respective antigens, myelin/oligodendrocyte-specific protein (MOSP) and myelin/oligodendrocyte glycoprotein (MOG), which are expressed uniquely by oligodendrocytes. After antibody binding, surface anti-MOSP:MOSP complexes redistribute over those cytoplasmic microtubular veins that have 2',3'-cyclic nucleotide 3'-phosphohydrolase colocalized along them. In contrast, surface anti-MOG-MOG complexes redistribute over internal myelin basic protein domains. Long-term anti-MOSP IgM exposure results in an apparent increase in number as well as thickness of microtubular structures in oligodendrocyte membrane sheets, whereas long-term anti-MOG exposure causes depolymerization of microtubular veins in membrane sheets. These data suggest that antibody binding to these two surface proteins elicits signals that have opposite effects on the cytoskeleton in oligodendroglial membrane sheets. Thus, it is possible that signals transduced via antibody binding may contribute to the pathogenesis of diseases affecting CNS myelin.     

The role of phospholipases from inflammatory macrophages in demyelination

Activated macrophages harvested from rat peritoneum were shown to contain phospholipase A₁, A₂ and lysophospholipase activities which were defined on a series of radiolabelled phospholipid substrates. During in vitro culture of these elicited macrophage populations, phospholipase enzymes were secreted into the culture medium. Radiolabelled myelin, prepared from young rats after intracerebral injection of¹⁴C acetate, was used as a substrate to analyze the susceptibility of central nervous system (CNS) myelin to attack by cell-associated and secreted macrophage enzymes. Homogenates of peritoneal macrophages degraded the myelin lipids at acid pH; phosphatidyl choline (PC) and ethanolamine phosphatide (EP) were both degraded with liberation of free fatty acid and small amounts of lysolipids. The ethanolamine lipids were most vulnerable; up to 20% of this fraction was degraded in six hours. Selected batches of macrophage culture supernatant similarly degraded the myelin EP at acid pH. These results suggest that phospholipase enzymes, released from activated macrophages in close proximity to the myelin sheath, may participate in primary demyelination in inflammatory CNS lesions.     

The DM20 Protein of Myelin: Intracellular and Surface Expression Patterns in Transfectants

DM20 is an abundant CNS myelin-specific protein whose role in myelinogenesis is unknown. We have cloned the DM20 cDNA from adult mouse brain total RNA using the polymerase chain reaction and expressed it in HeLa cells. DM20, detected by immunofluorescence in stable transfectants, is present in some cells in large, intensely fluorescent intracellular clumps that probably represent elements of the rough endoplasmic reticulum and Golgi apparatus. Frequently, intense DM20 fluorescence could be detected at the plasma membrane. These findings are consistent with previous studies demonstrating that an intracellular "pool" of DM20 and its larger isoform, proteolipid protein, exists and that a substantial lag occurs between synthesis and insertion of these proteins into the expanding myelin membrane. Permanent DM20 expressors in contact with one another do not display any ultrastructural rearrangements at regions of cell-cell contact, in contrast to what we have previously reported for P0, a PNS-specific protein shown to mediate adhesion of the extracellular faces of the Schwann cell during PNS myelinogenesis. We believe that these results indicate that if DM20 is indeed an adhesion molecule, this property is likely to be significantly more subtle than P0-mediated adhesion.     

Turnover of mouse intestinal brush border membrane proteins and enzymes in organ culture: A direct evaluation from studies on the evolution of enzyme activities during the culture

The turnover of mouse intestinal brush border membrane enzymes has been studied by kinetic analysis of the evolution of enzyme activities during organ culture. By comparing the results obtained in these studies with the predictions from a mathematical model of enzyme synthesis and degradation in orgen cultures, it has been possible to reach the following conclusions: (1) There is no degradation of brush border membrane enzymes during culture and the rate of synthesis of each enzyme is directly measurable from the kinetics of total enzyme accumulation (tissue + media). (2)_Brush border membrane enzymes are released in culture media by two complementary processes. The first one involves a differential solubilization of enzymes but its exact nature cannot be exactly stated. The second one involves a microvesiculation of brush border membranes, the importance of which in vivo is seen in the possible conciliation between unitary membrane synthesis and heterogeneous turnover of membrane components.     

Enzyme activity and composition of myelin and subcellular fractions in the developing rat brain

1. Subcellular fractions and myelin were isolated from developing and adult rat brain. 2. Measurements of chemical composition and enzyme activities indicate the presence of a second myelin-like fraction mainly in the brain of developing rats. 3. This membrane fraction has a different lipid composition from myelin, but resembles myelin in its content of phosphohydrolase and aminopeptidase activity. 4. It is suggested that the second myelin-like fraction may be a submicrosomal contaminant or it may be derived from oligodendroglial plasma membrane during myelinogenesis.