Fetal alcohol delays the developmental expression of myelin basic protein and transferrin in rat primary oligodendrocyte cultures.

This study has examined the development of immunoreactive myelin basic protein and transferrin in primary glial cell cultures. Cultures were initiated from control and experimental Sprague-Dawley rats 1-2 days postnatally. Experimental treatment involved exposure to 5% (w/v) ethanol in a liquid diet during the last two weeks of gestation. Prenatal alcohol administration delayed the expression of myelin basic protein and transferrin during the first three weeks postnatally. Other oligodendroglial and astroglial markers were little affected, if at all, by fetal alcohol exposure.     

Culture of rat cerebral oligodendrocytes in a serum-free, chemically defined medium

Oligodendrocytes were isolated from the cerebra of young rats (5-10 days old) by trypsinization of the tissue followed by cell separation on Percoll gradients. The isolation was carried out in physiological, isotonic media. The cell yield was 2-4 X 10(6) cells per brain; the plating efficiency was greater than or equal to 70%. Isolated cells were seeded on poly-L-lysine-coated culture dishes and maintained in a serum-free, chemically defined medium for at least 30 days. After 10 days in culture 67 +/- 10% of the surviving cells were oligodendrocytes, as judged by immunocytochemical and morphological criteria, whereas most of the other cells reacted positively with antiserum against glial fibrillary acidic protein. The expression of typical oligodendrocyte markers (2':3'-cyclic-nucleotide 3'-phosphodiesterase, galactocerebrosides and myelin basic protein) was greatly enhanced under these serum-free conditions as compared with cultures in serum-containing medium. The antigenic markers (galactocerebrosides, myelin basic protein) were absent in the freshly isolated cells but could be detected after 3 days in culture by immunocytochemistry. The activity of 2':3'-cyclic-nucleotide 3'-phosphodiesterase increased from 75 nmol min-1 mg-1 protein on day 4 to 400 nmol min-1 mg-1 protein on day 14 in culture.     

P0 mRNA expression in cultures of Schwann cells and neurons that lack basal lamina and myelin.

Schwann cells of the peripheral nervous system depend on the presence of both axons and basal lamina to achieve a myelinating phenotype. Furthermore, removal of axonal influence results in the cessation of myelination and down-regulation of myelin protein expression by Schwann cells. Here we examine whether both axons and basal lamina are required by Schwann cells for the expression of mRNA encoding the major myelin glycoprotein, P0. Cultures of Schwann cells and neurons obtained from dorsal root ganglia of 15 day embryonic rat pups were grown for up to 20 days in vitro under conditions that either allowed or prohibited basal lamina and myelin formation. These cultures were assayed for the expression of P0 mRNA by using an S1 nuclease-protection assay. After 20 days in vitro, the cultures that did not assemble basal lamina and that were incapable of myelin formation expressed P0 mRNA at a level which was comparable to that seen in identically aged, myelinating cultures. Both the myelinating and nonmyelinating cultures demonstrated an appreciable increase in P0 mRNA when compared to the starting embryonic dorsal root ganglia Schwann cells. The latter had a low, but detectable, level of mRNa for this myelin glycoprotein. The cultures that were devoid of basal lamina and myelin showed a clear increase in P0 mRNA by 11-15 days in culture. This increase in expression depended on the presence of neurons/neurites, since Schwann cells which were grown in neuron-depleted cultures expressed little, if any, P0 mRNA. In contrast to the levels of P0 mRNA, the nonmyelinating cultures had a significantly lower amount of P0 glycoprotein than did the cultures which assemble myelin. This suggests that the nonmyelinating Schwann cells regulate the level of this glycoprotein at the translational and/or the posttranslational level. The data presented here suggest that myelin protein mRNA expression and myelin assembly by Schwann cells are separable events, with the former depending on one or more neuronal/axonal factors.     

Suppressed UDP-galactose: Ceramide galactosyltransferase and myelin protein mRNA in twitcher mouse brain

The developmental changes in expression of steady-state mRNA that encode proteins that are important for myelination (myelin basic protein, myelin-associated glycoprotein, proteolipid protein, UDP-galactose:ceramide galactosyltransferase) and glial fibrillary acidic protein were investigated in the brain of the twitcher mouse, a model of human globoid cell leukodystrophy. This disease is caused by a mutation in the gene encoding the lysosomal enzyme, galactosylceramidase, which catalyzes the degradation of the myelin lipid galactosylceramide. Before postnatal day (PND) 20, the levels of myelin protein mRNA were similar in twitcher and normal mice. With progression of demyelination after PND 25-30, myelin protein mRNA levels gradually decreased. The period of maximum expression of the myelin protein genes in twitcher mice was, however, similar to that of normal control mice. mRNA levels for the gene that encodes the enzyme UDP-galactose:ceramide galactosyltransferase which is responsible for catalyzing the final step in galactosylceramide synthesis, was exceptionally downregulated from the early stages of the disease. The increase of glial fibrillary acidic protein (GFAP) mRNA levels preceded morphological evidence of demyelination. J. Neurosci. Res. 51:536–540, 1998. © 1998 Wiley-Liss, Inc.     

Expression of myelin proteolipid and basic protein mRNAs in cultured cells

Studies were undertaken to investigate the regulation of myelin-specific mRNA expression in cultured cells. Three experimental systems were investigated: primary oligodendrocytes grown as enriched cell populations, primary oligodendrocytes grown in the presence of chick spinal cord neurons, and C6 cells. cDNA probes specific for the myelin proteolipid mRNA and the myelin basic protein mRNA were used to quantitate proteolipid and myelin basic protein mRNA levels in cells under different experimental conditions. C6 cells expressed less than 0.2% of the proteolipid mRNA that was expressed in primary oligodendrocytes. Primary oligodendrocytes expressed the myelin-specific mRNAs for at least 104 days in culture, and the level of these mRNAs in cultures was elevated fourfold by coculturing rat oligodendrocytes with chick spinal cord neurons.     

Epidermal growth factor inhibits the expression of myelin basic protein in oligodendrocytes

The major function of the oligodendrocyte is to myelinate axons in the central nervous system (CNS). Two of the components of myelin, galactocerebroside (galc) and myelin basic protein (MBP), have been used as markers of oligodendrocyte maturation in the developing CNS, and it has been found that galc+ cells arise initially, which then mature into MBP+ oligodendrocytes several days later. We have been interested in the control of expression of MBP and have followed its appearance in cultures of brain cells isolated from 4 day-old mice. In low serum (0.5% foetal bovine serum), approximately 330 MBP+ cells arise per 2 x 10(5) brain cells after 3 days incubation. We have examined the ability of several growth factors to influence the expression of MBP in these cultures, including epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and the fibroblast growth factors (acidic and basic FGF). EGF was found to suppress strongly the developmental expression of MBP in these cultures, but the suppression was reversible, since the number of MBP+ cells approached control numbers 3 days after removal of EGF from the cultures. It was also found that MBP could be down-regulated in mature MBP+ oligodendrocytes. The action of EGF in these cultures could be mimicked by transforming growth factor-alpha (TGF alpha). The effects of EGF appear to be associated primarily with MBP production in oligodendrocytes since expression of galc is unaffected by EGF.     

Studies with antisera against peripheral nervous system myelin and myelin basic proteins. II. Immunohistochemical studies in cultures of rat dorsal root ganglion neurons and Schwann cells

Antiserum against rat peripheral nervous system (PNS) myelin contained immunoglobulins which bound preferentially to the extracellular surfaces of myelin-related Schwann cells in intact cultures of dorsal root ganglion (DRG) neurons and Schwann cells, while antiserum against basic protein (BP) from central nervous system myelin or the PNS basic protein P₂ did not. We demonstrate the presence of PNS myelin proteins P₁ (identical to BP) and P₂ by immunoperoxidase techniques in DRG cultures that had been treated to disrupt cellular membranes. These observations suggest that P₁ and P₂ are not exposed on the extracellular surfaces of myelin-related Schwann cells in culture. The results also supported the hypothesis concerning the possible mechanisms by which anti-PNS myelin serum demyelinates DRG cultures, while anti-BP serum and anti-P₂ serum do not.     

Effect of serum deprivation on myelinating mouse cerebellum cultures.

In order to investigate the effect of nutrient deprivation on brain development, mouse cerebellum cultures were grown in nutrient media with reduced serum concentration. After 9--18 days in vitro, experimental cultures grown in media with low serum concentration exhibited a delayed and retarded myelination. Electron microscopic examination of experimental cultures revealed profiles of deficient myelination, but showed normal neuronal and synaptic structures. The amount of myelin basic protein measured by radioimmunoassay was markedly reduced in experimental cultures. Activity of cholinesterase in these experimental cultures was also decreased.     

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.     

Protease activated receptor 2 controls myelin development,resiliency and repair

Oligodendrocytes are essential regulators of axonal energy homeostasis and electrical conduction and emerging target cells for restoration of neurological function. Here we investigate the role of protease activated receptor 2 (PAR2), a unique protease activated G protein‐coupled receptor, in myelin development and repair using the spinal cord as a model. Results demonstrate that genetic deletion of PAR2 accelerates myelin production, including higher proteolipid protein (PLP) levels in the spinal cord at birth and higher levels of myelin basic protein and thickened myelin sheaths in adulthood. Enhancements in spinal cord myelin with PAR2 loss‐of‐function were accompanied by increased numbers of Olig2‐ and CC1‐positive oligodendrocytes, as well as in levels of cyclic adenosine monophosphate (cAMP), and extracellular signal related kinase 1/2 (ERK1/2) signaling. Parallel promyelinating effects were observed after blocking PAR2 expression in purified oligodendrocyte cultures, whereas inhibiting adenylate cyclase reversed these effects. Conversely, PAR2 activation reduced PLP expression and this effect was prevented by brain derived neurotrophic factor (BDNF), a promyelinating growth factor that signals through cAMP. PAR2 knockout mice also showed improved myelin resiliency after traumatic spinal cord injury and an accelerated pattern of myelin regeneration after focal demyelination. These findings suggest that PAR2 is an important controller of myelin production and regeneration, both in the developing and adult spinal cord.