Regulation of Myelin P0 Glycoprotein Synthesis in Cultured Rat Schwann Cells and Continuous Rat PNS Cell Lines

We studied the effects of agents that raise intracellular cyclic AMP on synthesis of myelin components by cultured neonatal rat sciatic nerve Schwann cells and by continuous PNS cell lines derived from the fusion of neonatal rat sciatic nerve Schwann cells with rat RN22 Schwannoma. Treatment with N6,2'-O-dibutyryl cyclic AMP (dibutyryl cyclic AMP) caused a fourfold increase in Schwann cell incorporation of 35SO4 into sulfogalactosylceramide (sulfatide), and elicited a 10- to 20-fold increase in such incorporation by the continuous PNS cell lines; a similar effect on PNS cell line sulfatide radiolabelling was obtained with forskolin. Cultured Schwann cells expressed barely detectable levels of myelin P0 glycoprotein (P0) mRNA and myelin basic protein (MBP) mRNA. Treatment of the Schwann cells with axolemmal fragments or with dibutyryl cyclic AMP did not elicit a detectable increase in the levels of these mRNAs. The PNS cell lines constitutively expressed much higher levels of P0 mRNA than did the Schwann cells, and synthesized immunochemically demonstrable P0 glycoprotein, but did not express MBP. Treatment of the PNS cell lines with dibutyryl cyclic AMP markedly reduced expression of P0 mRNA and also diminished immunoreactive P0 glycoprotein. These PNS cell lines should prove useful for further studies of the control of Schwann cell differentiation.     

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.     

Expression of Myelin Components in Mouse Schwann Cells in Culture

Mouse Schwann cells cultured in vitro are capable of expressing basal levels of the major myelin components P1, P2, P0, and galactocerebroside. Numerical counts of immunostained cultures indicated that between 22 and 40% of the cells are positive up to 21 days for all of the components indicated. Electrophoretic analysis of Schwann cells labeled with a 14C-amino acid mixture revealed the presence of proteins with relative mobilities identical to those of P0 and P1. Positive identification of the two proteins was indicated by immunoprecipitation of P1 and immunoblotting of P0. These data show that in the absence of neurites, Schwann cells in culture can express low levels of myelin characteristic components even in the absence of myelin assembly.     

Role of basal lamina in Schwann cell glycolipid biosynthesis

Schwann cells that are deprived of axonal contact switch their glycolipid metabolic pathway from primarily galactocerebroside (GalCe) synthesis to the formation of glucocerebroside (GlcCe) and its homologs. The removal of axonal influence has a dual effect on Schwann cell phenotype; they lose the ability to assemble both myelin and basement membrane. To determine whether a loss of basement membrane directly affects glycolipid expression, we have examined lipid biosynthesis in Schwann cells which were allowed to interact with axons of dorsal root ganglion neurons but which were deprived of the ability to assemble basal lamina. These Schwann cells resemble those from myelinating nerve in that they synthesize a large amount of galactohydroxycerebroside. This suggests that axon contact, even in the absence of basement membrane, is sufficient to induce the GalCe metabolic pathway.Abbreviations DRG dorsal root ganglia - GalCe galactocerebroside - GalCe-OH galactohydroxycerebroside - GlcCe glucocerebroside - GL-2 lactosylceramide - GL-3 trihexosylceramide - GL-4 tetrahexosylceramide - HPTLC high-performance thin-layer chromatography - MGDG monogalactosyl diacylglycerol - NL non-polar lipids - PC phosphatidylcholine - Su sulfatide - Su-OH hydroxysulfatide     

Axons regulate Schwann cell expression of the major myelin and NGF receptor genes

The elaboration of myelin by Schwann cells is triggered by contact with appropriate peripheral axons. Among the most prominent features of this interaction is the activation and high-level expression of the genes encoding the major myelin proteins P0 and Myelin Basic Protein (MBP). Although the initial induction of these genes is thought to be dependent upon contact with axons, neither the inductive signal of the axon nor the receptor and associated second messenger system of the Schwann cell that transduces this signal has been identified. In this report, we demonstrate that expression of the P0 and MBP genes in rapidly myelinating Schwann cells is sharply reduced upon withdrawal of axons, but that this expression can be substantially restored by agents that raise the intracellular concentration of cyclic AMP. We further show that Schwann cell expression of a third gene, i.e. that encoding the Nerve Growth Factor receptor, is strongly activated by the withdrawal of axons, and that this activation is largely independent of cAMP.     

Expressing antisense P0 RNA in Schwann cells perturbs myelination.

Primary Schwann cells were infected in vitro with a recombinant retrovirus expressing a dominant selectable marker, neomycin phosphotransferase (conferring resistance to the drug G418), and antisense P0 RNA under the control of the human beta-actin promoter. A proportion of the G418-resistant cells failed to form myelin when cocultured with dorsal root ganglion neurons under conditions that promote Schwann cell differentiation. These cells expressed high levels of P0 antisense RNA. Among the impaired cells, the majority had segregated and ensheathed individual axon but had not differentiated further. They did not express P0 but did express myelin- associated glycoprotein and galactocerebroside. A minority of partially inhibited Schwann cells were also observed that elaborated thin myelin sheaths containing variable numbers of compacted and noncompacted lamellae. These data indicate that restricting the level of P0 expression inhibits spiralling of the Schwann cell membrane and subsequent compaction.     

Studies on cultured rat Schwann cells. II. Comparison with a rat Schwann cell line.

Cultured rat Schwann cells do not exhibit the ring-like changes in cell shape previously reported to be induced in the Schwann cell line RN22 by elevation of intracellular cyclic AMP. They do, however, undergo different shape changes on treatment with cholera toxin or low serum concentration. Furthermore, DNA synthesis in the cell line is inhibited by treatment with cholera toxin and unaffected by bovine pituitary extract, though both of these agents stimulate DNA synthesis in normal Schwann cells. Our results, therefore, do not support the hypothesis that elevation of intracellular cyclic AMP is a positive signal for myelination by the Schwann cell. Moreover, they illustrate the need for caution in drawing conclusions about normal cells of the nervous system from studies on neural cell lines.     

Myelin-specific proteins and glycolipids in rat Schwann cells and oligodendrocytes in culture

We have used antibodies to identify Schwann cells and oligodendrocytes and to study the expression of myelin-specific glycolipids and proteins in these cells isolated from perinatal rats. Our findings suggest that only Schwann cells which have been induced to myelinate make detectable amounts of galactocerebroside (GC), sulfatide, myelin basic protein (BP), or the major peripheral myelin glycoprotein (P0). When rat Schwann cells were cultured, they stopped making detectable amounts of these myelin molecules, even when the cells were associated with neurites in short-term explant cultures of dorsal root ganglion. In contrast, oligodendrocytes in dissociated cell cultures of neonatal optic nerve, corpus callosum, or cerebellum continued to make GC, sulfatide and BP for many weeks, even in the absence of neurons. These findings suggest that while rat Schwann cells require a continuing signal from appropriate axons to make detectable amounts of myelin- specific glycolipids and proteins, oligodendrocytes do not. Schwann cells and oligodendrocytes also displayed very different morphologies in vitro which appeared to reflect their known differences in myelinating properties in vivo. Since these characteristic morphologies are maintained when Schwann cells and oligodendrocytes were grown together in mixed cultures and in the absence of neurons, we concluded that they are intrinsic properties of these two different myelin- forming cells.     

Studies on cultured rat Schwann cells

Summary Cultured rat Schwann cells do not exhibit the ring-like changes in cell shape previously reported to be induced in the Schwann cell line RN22 by elevation of intracellular cyclic AMP. They do, however, undergo different shape changes on treatment with cholera toxin or low serum concentration. Furthermore, DNA synthesis in the cell line is inhibited by treatment with cholera toxin and unaffected by bovine pituitary extract, though both of these agents stimulate DNA synthesis in normal Schwann cells. Our results, therefore, do not support the hypothesis that elevation of intracellular cyclic AMP is a positive signal for myelination by the Schwann cell. Moreover, they illustrate the need for caution in drawing conclusions about normal cells of the nervous system from studies on neural cell lines. Paper I in this series is reference 2.     

Regulated Galactolipid Synthesis and Cell Surface Expression in Schwann Cell Line D6P2T

Clonal cell line D6P2T, subcloned from an ethylnitrosourea-induced tumor line D6 of the rat peripheral nervous system, has been characterized with particular attention to galactolipid metabolism. Galactosylcerebroside and sulfatide synthesis and expression on the cell surface are highly regulated in D6P2T cells by mechanisms involving serum- and cyclic AMP-mediated pathways. These cells also express 2',3'-cyclic nucleotide 3'-phosphohydrolase (Wolfgram protein W1a) and laminin. In contrast, myelin basic protein and antigen HNK-1 were not detected. Line D6P2T appears to be a semi-differentiated Schwann cell model, which offers interesting possibilities for studies of galactolipid synthesis, transport, and sorting.     

Induction of Myelin Components: Cyclic AMP Increases the Synthesis Rate of 2'',3''-Cyclic Nucleotide 3''-Phosphohydrolase in C6 Glioma Cells

In an effort to determine the factors that stimulate myelin synthesis, we investigated the mechanism by which dibutyryl cyclic AMP induces the activity of the myelin enzyme, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP; EC 3.1.4.37), in C6 glioma cells. Immunotitration experiments and measurements of the accumulation of [35S]methionine-labeled CNP showed that dibutyryl cyclic AMP increased the amount of CNP in the cells but not the catalytic activity per molecule of the enzyme. Moreover, inhibition of protein synthesis with cycloheximide abolished induction of enzyme activity. Dibutyryl cyclic AMP doubled the rate of CNP synthesis but had no effect on the half-life of the enzyme (approximately 33 h). The induction was partially blocked by the inhibitors of mRNA synthesis, cordycepin or alpha-amanitin. Thus, cyclic AMP induces the synthesis of CNP.     

FA2H is responsible for the formation of 2-hydroxy galactolipids in peripheral nervous system myelin

Myelin in the mammalian nervous system has a high concentration of galactolipids [galactosylceramide (GalCer) and sulfatide] with 2-hydroxy fatty acids. We recently reported that fatty acid 2-hydroxylase (FA2H), encoded by the FA2H gene, is the major fatty acid 2-hydroxylase in the mouse brain. In this report, we show that FA2H also plays a major role in the formation of 2-hydroxy galactolipids in the peripheral nervous system. FA2H mRNA and FA2H activity in the neonatal rat sciatic nerve increased rapidly during developmental myelination. The contents of 2-hydroxy fatty acids were approximately 5% of total galactolipid fatty acids at 4 days of age and increased to 60% in GalCer and to 35% in sulfatides at 60 days of age. The chain length of galactolipid fatty acids also increased significantly during myelination. FA2H expression in cultured rat Schwann cells was highly increased in response to dibutyryl cyclic AMP, which stimulates Schwann cell differentiation and upregulates myelin genes, such as UDP-galactose:ceramide galactosyltransferase and protein zero. These observations indicate that FA2H is a myelination-associated gene. FA2H-directed RNA interference (RNAi) by short-hairpin RNA expression resulted in a reduction of cellular 2-hydroxy fatty acids and 2-hydroxy GalCer in D6P2T Schwannoma cells, providing direct evidence that FA2H-dependent fatty acid 2-hydroxylation is required for the formation of 2-hydroxy galactolipids in peripheral nerve myelin. Interestingly, FA2H-directed RNAi enhanced the migration of D6P2T cells, suggesting that, in addition to their structural role in myelin, 2-hydroxy lipids may greatly influence the migratory properties of Schwann cells.     

Galactocerebroside is expressed by non-myelin-forming Schwann cells in situ

Interest in the glycosphingolipid galactocerebroside (GC) is based on the consensus that in the nervous system it is expressed only by myelin-forming Schwann cells and oligodendrocytes, and that it has a specific role in the elaboration of myelin sheaths. We have investigated GC distribution in two rat nerves--the sciatic, containing a mixture of myelinated and non-myelinated axons, and the cervical sympathetic trunk, in which greater than 99% of axons are non-myelinated. Immunohistochemical experiments using mono- and polyclonal GC antibodies were carried out on teased nerves and cultured Schwann cells, and GC synthesis was assayed biochemically. Unexpectedly, we found that mature non-myelin-forming Schwann cells in situ and in short-term cultures express unambiguous GC immunoreactivity, comparable in intensity to that of myelinated fibers or myelin-forming cells in short-term cultures. GC synthesis was also detected in both sympathetic trunks and sciatic nerves. In the developing sympathetic trunk, GC was first seen at day 19 in utero, the number of GC-positive cells rising to approximately 95% at postnatal day 10. In contrast, the time course of GC appearance in the sciatic nerve shows two separate phases of increase, between day 18 in utero and postnatal day 1, and between postnatal days 20 and 35, at which stage approximately 94% of the cells express GC. These time courses suggest that Schwann cells, irrespective of subsequent differentiation pathway, start expressing GC at about the same time as cell division stops. We suggest that GC is a ubiquitous component of mature Schwann cell membranes in situ. Therefore, the role of GC needs to be reevaluated, since its function is clearly not restricted to events involved in myelination.     

Induction of myelin gene expression in Schwann cell cultures by an interleukin-6 receptor-interleukin-6 chimera.

Expression of myelin basic protein (MBP) and Po gene products is induced during the final postnatal maturation of Schwann cells and reinduced during nerve regeneration. We show that a chimeric protein containing interleukin-6 fused to its soluble receptor (IL6RIL6 chimera) induces MBP and Po RNAs and proteins in cultures of dorsal root ganglia (DRG) from 14 day old mouse embryos. Activation of gp130 signaling by IL6RIL6 appears comparable to cyclic AMP elevating agents to induce the myelin gene products in DRG and in pure Schwann cell cultures.     

Fate of oligodendrocytes during retinal axon degeneration and regeneration in the goldfish visual pathway

Retinal axons in goldfish regenerate after optic nerve lesion, restore synaptic connections, and become myelinated by oligodendrocytes. The fate of oligodendrocytes during these events is not known and may require generation of new oligodendrocytes or dedifferentiation and redifferentiation of the existing ones. To determine the reaction of oligodendrocytes to optic nerve lesion, we used the terminal transferase technique to detect apoptosis, bromodeoxyuridine incorporation to reveal mitosis, antibodies to identify myelin and oligodendrocytes, and Lucifer yellow injections to reveal cell morphology. Along with the reappearance of the myelin molecules 36K protein, galactocerebroside, and myelin basic protein, myelinating oligodendrocytes (identified by Lucifer yellow injections) reappear 21 days postlesion. Prior to this time, the dye-filled cells had few processes oriented along the regenerating axons. They resembled oligodendrocytes seen both in vitro and in vivo which express the L1-related E587 antigen and synthesize the 36K myelin protein in coculture with axons. No signs of oligodendrocyte apoptosis were detected after lesion and only few of the oligodendrocytes present had recently arisen. 36K/E587 double-labeled oligodendrocytes which were most likely dedifferentiating oligodendrocytes were identified in 8-day postlesion nerves among E587-positive elongate cells whose numbers increased until 14 days postlesion. These findings suggest that oligodendrocytes dedifferentiate-like Schwann cells-from cells which express myelin molecules to elongate cells which express the L1/E587 antigen. They redifferentiate to myelinate axons from roughly 3 weeks onward. These findings suggest an adaptive plasticity of goldfish oligodendrocytes beneficial to the repair of the visual pathway.     

The UDP-Galactose:Ceramide Galactosyltransferase: Expression Pattern in Oligodendrocytes and Schwann Cells During Myelination and Substrate Preference for Hydroxyceramide

Abstract: Galactosylceramide ("galactocerebroside"; GalC) is a major glycolipid in the myelin sheath of the CNS and the PNS. The enzyme UDP-galactose:ceramide galactosyltransferase (CGalT) catalyzes the final step of the synthesis of GalC: the transfer of galactose to ceramide. By a differential screening approach, we have isolated a cDNA, the sequence of which is identical to the recently isolated cDNA clones for CGalT. By northern analysis and in situ hybridization we demonstrated that CGalT mRNA is expressed at birth in oligodendrocytes and Schwann cells, an expression pattern corresponding to the onset of myelination. In addition to the high expression levels of CGalT in oligodendrocytes and Schwann cells, in situ hybridization also showed expression in subtypes of neurons in spinal cord, cerebellum, and brainstem in the adult CNS, but at a much lower level than in oligodendrocytes. Expression of CGalT in COS cells demonstrated that CGalT has a preference for hydroxyceramide as a substrate. CGalT-expressing COS cells synthesize and transport GalC to their cell surface as shown by immunofluorescence and by lipid analysis of living cells. Our results suggested that the CGalT specifically uses hydroxyceramide for the synthesis of GalC and that separate (co)enzymes are not needed.     

PC12 cells as a source of neurite-derived cell surface mitogen, which stimulates Schwann cell division

Primary cultures of rat dorsal root ganglion Schwann cells were used to assay the efficacy of PC12 cells in stimulating Schwann cell proliferation. Co-cultures of PC12 cells and Schwann cells assayed by [3H]thymidine labeling followed by autoradiography showed proliferation of Schwann cells only where contact occurred between PC12 neurites and Schwann cells. Membranes derived from PC12 cells were shown to have many characteristics similar to membranes derived from sensory neurons; both could mimic whole cells in stimulating Schwann cell division; both were inactivated by mild heat treatment and by trypsinization, and both elevated intracellular cyclic AMP concentrations in Schwann cells 16 h after addition of membranes. We conclude that PC12 cells will be a valuable source for the isolation of the neuronal cell surface component which controls proliferation of Schwann cells during development of the peripheral nervous system.     

Peripheral Nervous System Myelin and Schwann Cell Glycoproteins: Identification by Lectin Binding and Partial Purification of a Peripheral Nervous System Myelin-Specific 170,000 Molecular Weight Glycoprotein

Radioiodinated lectins were used to detect glycoproteins of peripheral nervous system (PNS) myelin (rat, human, bovine) and cultured rat Schwann cells. Proteins were resolved by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and transferred to nitrocellulose filters. The filters were overlaid with radioiodinated lectins of known saccharide affinities. These included concanavalin A, Helix pomatia, Limulus polyphemus, Maclura pomifera, peanut, soybean, Ulex europaeus, and wheat germ agglutinins. Inclusion of the appropriate monosaccharide in the overlay solution (0.2 M) inhibited lectin binding to the nitrocellulose-fixed proteins. Fluorography permitted identification of 26 myelin glycoproteins and many more in Schwann cells. All lectins labeled a band present in myelin, but not Schwann cells, corresponding to the major PNS myelin protein, P0. Our attention focused on a high-molecular-weight myelin glycoprotein [apparent molecular weight (Mr) 170,000], which appeared abundant by Coomassie Blue staining and which was heavily labeled by all lectins except concanavalin A. A protein with approximately this Mr and lectin-binding pattern was present in human and bovine PNS myelin as well, but not detected in rat Schwann cells, CNS myelin, liver and fibroblast homogenates, or cultured bovine oligodendroglia. Hence this 170,000 Mr glycoprotein is apparently unique to PNS myelin.