Expression of antigenic markers during the development of oligodendrocytes in mouse brain cell cultures

The expression of myelin basic protein (MBP) and galactocerebroside (GC), two antigenic markers for oligodendrocytes, was chekced on 7-, 14-, 21- and 28-day-old dissociated mouse brain cell cultures (BCC) by using the indirect immunofluorescence method with double staining.

The number of GC positive cells increased between the 7th and the 14th day culture before a steady state was reached. In contrast to this, the MBP-positive cells appeared only on the 14th day oulture, and their number increased with the age of the culture. In double staining, the serum produced against isolated oligodendrocytes shows the same picture as the anti-GC serum, while only a part of GC-positive cells showed also the presence of MBP. Our data suggest that the GC appears very early on the membrane of the oligodendrocytes during development while cells exhibiting both GC and MBP probably represent a more differentiated oligodendrocytes population.     

The timely expression of myelin basic protein gene in cultured rat brain oligodendrocytes is independent of continuous neuronal influences

The developmental expression of the myelin basic protein (MBP) gene was studied in rat cultured oligodendrocytes using immunofluorescence and in situ hybridization. In newborn rat brain cultures, which contain only glial cells, large amounts of MBP-specific mRNA (as assayed by grain counts in autoradiograms) abruptly accumulated within immature oligodendrocytes 5 to 6 days postnatal. MBP always emerged 6 to 8 days after birth; thus, a week after, galactocerebroside (GC), an early oligodendrocyte marker, had appeared. The percentage of MBP mRNA and MBP-positive cells peaked at about 15 days postnatal and decreased thereafter. The time of emergence of MBP in these cultured oligodendrocytes appears to be determined at a very early stage in their development and independent of continuous neuronal influences. There is a striking correspondence between the times of appearance of MBP in cultured oligodendrocytes and those in the intact animal. Thus, primary cultures made from 5-day prenatal, newborn, and 2-day postnatal animals all express MBP at about the same developmental stage, namely, after 14, 8, and 6 days in culture, respectively. Furthermore, cultured oligodendrocytes obtained from the spinal cord express MBP before those obtained from midbrain or hemispheres, as they would in the intact animal. Thus, the developmental expression of the MBP gene occurs in a similar time frame in vitro and in vivo. In oligodendrocyte-enriched cultures, where 60% to 80% of the cells express MBP, in situ hybridization with the cDNA clone revealed MBP-specific mRNA in the cell body and sometimes in the processes of the differentiated oligodendrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

The development of the transferrin-transferrin receptor system in relation to astrocytes, MBP and galactocerebroside in normal and myelin-deficient rat optic nerves

The factor(s) which control the onset of myelination are unknown. It is now accepted that transferrin (Tf), the major iron transport protein in vertebrates, is found in oligodendrocytes in the adult brain. Because of the importance of iron in basic cell metabolism we have hypothesized that iron (mobilized by Tf) may be a permissive agent in the process of myelination. The present study was designed to determine with immunohistochemistry the relationship of Tf receptor expression, Tf accumulation, and the expression of myelin components myelin basic protein (MBP) and galactocerebroside (GAlC)) in the developing rat optic nerve. In addition to Tf and its receptor, the developmental pattern for GalC reported in this study has not been examined in the rat optic nerve. Furthermore, a myelin mutant strain of rats was used to determine if a lack of myelin production affects the Tf-Tf receptor system. Our study found that Tf receptor was expressed from birth on blood vessels and was first seen in the parenchyma of the nerve at 8 days of age. The expression of the Tf receptor preceded that of Tf, MBP or GalC. The accumulation of Tf by oligodendrocytes occurred about the same time as the intracellular appearance of MBP and GalC which was shortly after the onset of myelination. Tf-positive cells as well as MBP- and GalC-positive cells increased in number and staining intensity with age whereas the expression of the Tf receptor declined after reaching a peak at 15 days of age. In the optic nerves of myelin-deficient rats, the Tf receptor expression and Tf accumulation was confined to the vasculature. The results of this study suggest that the expression of the Tf receptor is an early event in oligodendrocytic maturation and is followed by the intracellular accumulation of myelin components and Tf. The temporal association of Tf and myelin production suggests that further study is warranted regarding the possibility that the Tf-iron system supports or perhaps even permits the initiation of the process of myelination.     

Transferrin, carbonic anhydrase C and ferritin in dissociated murine brain cell cultures

It is shown here that transferrin (Tf), the iron transport protein and carbonic anhydrase C (CA C) are specifically located within oligodendrocytes in murine brain cell cultures. Ferritin (F), the major iron storage protein, was demonstrated in oligodendrocytes, as well as in astrocytes and microglial cells and was more prominent in the former. CA C and Tf were seen first after 6-7 days in culture. CA C and F positivity increased rapidly and at day 20, 80-85% of galactocerebroside + oligodendrocytes were positive for both proteins. Only a small number of oligodendrocytes was Tf+ up to day 14, after which their numbers increased rapidly until day 20, when 67% of the oligodendrocytes were Tf+. Because of the presence of Tf and F in oligodendrocytes it is suggested that these cells may play an important role in the metabolism of iron within the central nervous system.     

Transferrin gene expression and secretion by rat brain cells in vitro

We have previously shown by immunocytochemistry in rat primary glial cultures that transferrin (Tf) is an early developmental marker for oligodendrocytes. The present work addresses the issue of Tf gene expression and synthesis by neural cells in vitro. For this purpose, we used rat embryonic neuronal cultures and newborn glial cultures of astrocytes and oligodendrocytes. Cultured fibroblasts and C6 glioma cells were used as negative controls. We found that Tf mRNA is present in oligodendrocytes, astrocytes, and neurons. However, oligodendrocytes and astrocytes, but not neurons, were shown to synthesize and secrete Tf. Neither fibroblasts nor C6 glioma cells expressed detectable amounts of Tf mRNA. Tf mRNA levels in astrocyte cultures appeared to be under hormonal control since hydrocortisone markedly reduced message levels. These results show that both astrocytes and oligodendrocytes can synthesize and secrete Tf under cell culture conditions. However, epigenetic factors, such as hydrocortisone, may repress the expression of Tf in astrocytes in vivo.     

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.     

Immunocytochemical characterisation of cell cultures grown from dissociated 1-2-day post-natal rat cerebral tissue. A developmental study

A range of cell-specific markers have been employed with immunocytochemical methods to characterise and quantitate the cell types present in mixed brain cell cultures derived from dissociated 1-2-day post-natal rat cerebral hemispheres and grown in the presence of FCS. Protoplasmic astrocytes (GFAP+, A2B5-) were the major cell type to develop in culture, a confluent monolayer forming in 5-8 days. A population of smaller round cells of oligodendrocyte-like morphology appeared on this astrocyte layer. Greater than 70% of these smaller cells were GC- and thus were not oligodendrocytes. The GC- cells were A2B5+ and, in early cultures, may therefore be progenitor glial cells. Examination of GFAP and A2B5 co-expression by these smaller cells was difficult due to the dense underlying GFAP+ astrocyte layer. In less dense areas of older cultures these smaller cells with processes were GFAP+ and A2B5+: these are Type 2, fibrous astrocytes. GC+ oligodendrocytes, comprising 5-10% of the total identified cell population, were initially distributed over the astrocyte monolayer; in older cultures (after about 8 days) GC+ cells were observed in clumps over places where NF+ cells were identifiable. Such GC+ cells mostly became MBP+. Neurones accounted for about 6% of the identifiable cells in early cultures but a lower percentage in older cultures. Minor populations of ependymal cells and macrophages were present; cells displaying fibronectin, fibroblasts, were rarely identified. Use of horse serum in place of FCS gave lower yields of GC+ cells in cultures, slowed down astrocyte development, and resulted in the formation of trunks of GFAP+ cells throughout cultures. Other sera gave lower numbers of GC+ cells.     

Accelerated differentiation of oligodendrocytes in neuronal-rich embryonic mouse brain cell cultures

The expression of two oligodendroglial markers, galactocerebroside (GC) and myelin basic protein (MBP), was studied in brain cell cultures (BCC) from 14-day-old mouse embryos by immunocytochemical methods. The presence of neurons and astrocytes was also investigated. Results show that oligodendrocytes simultaneously express both GC and MBP already at 7 days in vitro. These cultures are rich in neurons, and the astrocyte layer is also well represented. A comparison is made between these data and those previously obtained by the use of newborn mouse brain cell cultures, which are very poor in neurons. The differentiation of oligodendrocytes, as reflected in the expression of MBP, is accelerated in embryonic mouse BCC when compared to neonatal mouse BCC. We therefore speculate that neurons are involved in the enhancement of the ability of oligodendrocytes to express myelin related components in culture.     

Temporal expression of myelin-specific components in neonatal mouse brain cultures: evidence that 2',3'-cyclic nucleotide 3'-phosphodiesterase appears prior to galactocerebroside

A range of antibodies to cell-specific markers was employed to characterize and quantify the cell types present in neonatal mouse brain cell cultures. Astrocytes were the major cell type to develop in culture and formed a bed layer by 5-7 days in vitro (DIV). A population of cells appeared on this layer which consisted of oligodendrocytes, progenitor cells and microglia. Some neurons were detected in the cultures up to at least 27 DIV. With time, the number of progenitor cells decreased in the cultures and there was a concomitant increase in the number of oligodendrocytes. Maximal numbers of cells expressing galactocerebroside (GC), 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) and myelin-associated glycoprotein were observed at 18 DIV. The highest number of cells expressing myelin basic protein and proteolipid protein were observed at 25-30 and 35-40 DIV, respectively. Double-label studies with antibodies to A2B5/CNP and A2B5/GC showed that several A2B5+,CNP+ cells were present at 6 DIV. In contrast, no A2B5+,GC+ cells could be seen at this age. Confirming these findings, some CNP+,GC- cells were observed when cells were double-labeled with antibodies to CNP and GC. These findings suggested that the expression of CNP precedes that of GC in oligodendrocytes in these cultures.     

Distribution of proteolipid protein and myelin basic protein in cultured mouse oligodendrocytes: primary vs. secondary cultures.

The distribution of proteolipid protein (PLP) and myelin basic protein (MBP) was examined in differentiating oligodendrocytes of primary and secondary mouse brain cell cultures by single- and double-label indirect immunofluorescence. In primary cultures, MBP and PLP were differentially located in oligodendrocytes. MBP became concentrated as fine punctate dots lining the edges of processes and as coarse grains in flattened sheet-like structures. PLP was distributed diffusely throughout cell bodies and processes but was limited to the perimeter of sheets and some processes within sheets. To compare the detailed distribution of PLP and MBP in the absence of underlying cells, a simple method for the growth of isolated oligodendrocytes in secondary cultures was developed. Cells were maintained in primary culture for 39-41 days, harvested by scraping, enriched for oligodendrocytes, and plated at low cell density. After 1 week, isolated oligodendrocytes had developed long processes and large flattened membranous sheets. MBP and PLP were differentially localized in these cell structures. The sheets contained fine-grained patches of MBP, which were surrounded by networks of MBP- processes. In contrast, PLP was initially seen throughout the cell bodies and processes. In older cultures, PLP became strikingly concentrated in curvilinear membranous profiles. The observations show that PLP and MBP are differentially located in cultured mouse oligodendrocytes. Furthermore, the precise distribution of these myelin-specific antigens is dependent on culture conditions.     

Overexpression of human transferrin in two oligodendroglial cell lines enhances their differentiation

We have previously demonstrated that the addition of apotransferrin (aTf) to oligodendroglial cell (OLGc) primary cultures accelerates their maturation. Cells treated with aTf developed a multipolar morphology and displayed increased expression of mature OLGc markers. In this work, we studied the effect of Tf overexpression in two OLGc lines, N19 and N20.1. The former cells exhibit characteristics of OLGc precursors (O2A), while N20.1 cells express markers of more mature OLGcs. Using the complete cDNA of the human Tf gene, we obtained clones overexpressing Tf in both cell lines. These clones were evaluated for the expression of OLGc differentiation markers. In agreement with our previous results, we found that in the cells overexpressing Tf, there was an increased O(4), GC, and MBP immunoreactivity. To study the myelinogenic potential of these cells, we co-cultured N19 and N20.1 Tf-transfected cells together with cortical neurons. There was a dramatic increase in the morphological differentiation of the OLGcs accompanied by enhanced GC and MBP expression. The OLGcs appeared to establish contact with neurites and extend their processes along them. Only two MBP isoforms were detected in Tf-overexpressing clones, while all the isoforms were present in the co-cultures, suggesting that there was a modulation of MBP expression by neurons. Concomitantly, we found an increase in several proteins involved in axon-glia interaction, such as MAG, N-CAM, and F3/Contactin. This co-culture system represents a potentially powerful tool to study neuron-glia interactions that occur during myelinogenesis and the role of Tf in this process.     

Udpgalactose:Ceramide galactosyltransferase in cultured oligodendrocytes: An enzymological and immunological study

The developmental expression of UDPgalactosexeramide galactosyltransferase (CGalT), an enyme marker of one myelinogenic activity in nervous tissue, was studied in cultured oligodendrocytes. The activity of CGalT in cultures followed a characteristic pattern of developmental changes. In the primary cultures these changes could be represented by a biphasic curve with a maximum of enzymatic activity at about the 25th day in culture. After purifying the oligodendrocytes from the primary cultures and replating them in culture dishes, similar developmental changes of CGalT were observed. In the subultures prepared from 20-day-old primary cultures the activity of CGalT per oligodendrocyte increased from 1.3 × 10⁻⁶ nmol/hr on day 4 to 3.7 × ⁻⁶ nmol/hr on day 21. Immunocytochemical studies with the antiserum against rat brain CGalT showed the presence of CGalT⁺ oligodendrocytes after 7 days in the primary culture (earliest time studied), later on the number of CGalT⁺ oligodendrocytes increased until 28 days (latest time examined). In the subcultures of purified oligodendrocytes the bulk of oligodendrocytes was stained by the anti-CGalT antibodies after 15 days. These results suggest that the initial expression of CGalT in oligodendroglial cultures involves an increase of the number of CGalT⁺ oligodendrocytes and of the amount of enzyme protein per cell.     

Microtubule associated protein (MAP1B) is present in cultured oligodendrocytes and co-localizes with tubulin

Differentiation of oligodendrocytes is accompanied by the extension of processes and the assembly of the myelin membrane. It is likely that the cytoskeleton plays an important role in this process in terms of changes in cell shape, transport of myelin components, and organization of the myelin membrane. Oligodendrocytes contain microtubules (MT) which associate with other components of the cytoskeleton, and microtubule associated proteins (MAPs) may mediate some of these interactions. In this study we have shown the presence of MAP1B in oligodendrocytes grown in primary glial cultures by double-label immunofluorescence using antibodies to galactocerebroside (GC) and MAP1B. The staining of the cultures showed that GC-positive oligodendrocytes were also stained with MAP1B antibodies. However, MAP1B stain was limited to cell bodies and processes, whereas GC stain was also seen in flattened membrane sheets and punctate staining in processes. MAP1B staining was also compared with that of myelin proteolipid (PLP), myelin basic protein (MBP) and beta-tubulin in secondary glial cultures that were enriched for oligodendrocytes. The results showed a typical staining of cell bodies and membranous profiles using PLP antibodies, and the staining of cell bodies and flattened regions of membranous sheets by MBP antibodies. In contrast, both polyclonal and monoclonal antibodies to MAP1B showed a uniform diffuse staining of cell bodies, major processes, and fine interconnected processes. Double-labeling of the cells showed that MAP1B was co-localized with tubulin, but was not present in glial fibrillary acidic protein (GFAP)-positive astrocytes. Western and Northern blot analyses of primary glial cultures showed that MAP1B had a molecular mass of 320 kDa and a mRNA of 10 kb. These values are identical to those previously reported for brain MAP1B (Safaei and Fischer, 1989) and demonstrate the presence of MAP1B in oligodendrocytes.     

Infection of rat brain primary cell cultures with an avirulent A7 strain of Semliki Forest virus

The ability of A7 Semliki Forest Virus (SFV) to infect primary brain cell cultures has been examined using cultures prepared from 1-2-day neonatal rat cerebral hemispheres. These cultures, characterised immunocytochemically using cell-specified markers, contain mainly GFAP+ protoplasmic astrocytes and smaller multiprocessed A2B5+ cells, probably fibrous astrocytes. 10% of the cells are GC+ oligodendrocytes and some neurones are also present. These cultures support virus growth and a cytopathic effect was observed. Using double labelling techniques with the cell-specific markers and anti-SFV antibody A7 has been shown to readily infect cells which carry either the A2B5+ antigen or galactocerebroside marker. Protoplasmic astrocytes (GFAP+/A2B5-) are not readily infected under the conditions used. The protein labelling studies using [35S]methionine show that host cell protein synthesis is not completely shut off and continues in the astrocyte protein region. These results suggest that cells derived from a common A2B5+, GFAP-, GC- progenitor glial cell, i.e. GC+ oligodendrocytes and A2B5+/GFAP+ fibrous astrocytes, are more readily infected than other brain cell types including the protoplasmic astrocytes.     

Direct microculture enzyme-linked immunosorbent assay for studying neural cells: oligodendrocytes

Oligodendrocyte development has been studied in a standardized primary microculture system initiated from day 20-21 fetal rat brain using a solid-phase enzyme-linked immunosorbent assay (ELISA) carried out directly on fixed cells (direct microculture ELISA). A highly reproducible dissociation procedure is described that allows careful control of the number of cells seeded per culture. At a seeding density of 1 x 10(5) cells/culture, up to 250 oligodendrocyte-generating microcultures consisting of 10-12% oligodendrocytes can be prepared from a single fetal rat brain, thereby permitting the simultaneous assay of multiple developmental parameters in sibling cultures. The validity of this method for quantifying myelinogenesis was established by comparing the results obtained by direct microculture ELISA with immunocytochemical counting of cells in parallel cultures. As few as 200 oligodendrocytes could be detected using a biotinylated anti-Ig and an avidin-urease conjugate detection system; CNP immunoreactivity measured by ELISA was linearly proportional to the number of immunolabeled cells between 6 and 34 days in culture; the developmental time courses of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) and myelin basic protein (MBP) expression determined by the two methods were very similar. Finally, cell suspensions were seeded at increasing dilution to determine the number of cells required to generate cultures that tested positive for oligodendrocytes by ELISA. As few as 9,000 cells were sufficient, predicting a minimum of 8,000 oligoprogenitors per 20-21 day fetal rat brain. The application of direct microculture ELISA for studying oligodendrocyte population size and myelinogenesis is discussed.     

Cellular distribution of 04 antigen and galactocerebroside in primary cultures of human foetal spinal cord

Summary The distribution of cell-surface 04 antigen and galactocerebroside (GC) was examined by duallabel indirect immunofluorescence assays on live primary cultures of human spinal cord cells dissociated from 8–12 week-old foetal tissue. Oligodendrocytes expressing GC on their surface were present in the cultures at early time points, and all GC-positive cells were found to also express cell-surface 04 antigen. The 04 antigen was found additionally on a further population of GC-negative cells in the spinal cord cultures, which did not react with antibodies to glial fibrillary acidic protein (GFAP), and were distinct from neuronal cells and cell processes which stained with anti-neurofilament antibody. Previous studies in mouse neural cell cultures have shown that 04 antigen-positive cells are direct precursors to GC-bearing oligodendrocytes (Schachner et al. 1982).In the human spinal cord cultures, a rapid decline in the number of cells expressing GC and/or the 04 antigen to a value below 1% was observed during the first 3 days in vitro.The present studies indicate that synthesis of GC occurs in the human spinal cord many weeks before myelination commences in vivo and that GC-negative oligodendrocyte precursors are present simultaneously with more mature GC-positive cells. In addition, it would appear that complex humoral or cellular ingredients may be required for the long-term in vitro maintenance of viable human foetal oligodendrocytes.     

Astrocytes cultured from mature brain derive from glial precursor cells

We have previously shown that enriched preparations of oligodendrocytes from either mature bovine brain or 30-d-old rat brain, when cultured in serum-free medium, yield mixed cultures of oligodendrocytes and astrocytes even though no GFAP+ cells were present after 24 hr in culture (Norton et al., 1986, 1988). To test the possibility that the astrocytes in these cultures arose from glial precursor cells, we followed the expression of ganglioside GD3, galactosylceramide (GC), glial fibrillary acidic protein (GFAP), and vimentin in the cultures. GD3 has already been shown to be a marker of immature neuroectodermal cells, which in the postnatal brain are glial progenitor cells (Goldman et al., 1984, 1986). The cultures from both species contained at 1 DIV only two populations of cells; 90-95% GC+/GD3- oligodendrocytes and 4-10% GD3+/GC- small, round cells. With time, the oligodendrocytes remained GD3-/GFAP-/vimentin-. The kinetics of antigen expression of the GD3+ cells could best be interpreted by the following sequence: (sequence; see text) We interpret these results to show that the astrocytes arose from a small population of GD3+ glial precursor cells present in the brain that were co-isolated with oligodendroglia. No evidence was obtained that these GD3+ cells could also differentiate into oligodendrocytes.