Hyaluronectin is produced by oligodendrocytes and Schwann cells in vitro

A hyaluronectin (HN)-like antigen was found in rat O-2A progenitors and oligodendrocytes, as well as in Schwann cells and in their culture medium. The HN-like antigen secreted in culture supernatants had a higher molecular mass than HN extracted from rat brain at acidic pH. In vitro the secreted HN-like antigen was spontaneously and slowly degraded into species whose Mr was close to that of HN found in acidic brain extract. In brain or nerve neutral pH extracts, both HN-like antigen and HN were present. The high Mr of the secreted antigen, the homology in amino acid sequences between HN and N-terminal domain of PG-M/versican, in addition to a positive hybridization between Schwann cell RNAs and a probe obtained with primers derived from HN sequences also found in versican suggested that HN is closely related to the large proteoglycan PG-M/versican. The presence in Schwann cell extract of a HN mRNA whose Mr was compatible with the size expected for HN showed that HN may be directly secreted by cells and not only the consequence of a proteolytic cleavage. The similarity of HN with PG-M (V3) suggested that HN found in vivo could be the result of an alternative splicing of a single gene. We conclude that HN as other members of the PG-M/versican family is a marker of oligodendrocytes and Schwann cells in culture.     

NGF controls axonal receptivity to myelination by Schwann cells or oligodendrocytes

Axons dictate whether or not they will become myelinated in both the central and peripheral nervous systems by providing signals that direct the development of myelinating glia. Here we identify the neurotrophin nerve growth factor (NGF) as a potent regulator of the axonal signals that control myelination of TrkA-expressing dorsal root ganglion neurons (DRGs). Unexpectedly, these NGF-regulated axonal signals have opposite effects on peripheral and central myelination, promoting myelination by Schwann cells but reducing myelination by oligodendrocytes. These findings indicate a novel role for growth factors in regulating the receptivity of axons to myelination and reveal that different axonal signals control central and peripheral myelination.     

Binding constants of soluble NGF-receptors in rat oligodendrocytes and astrocytes in culture

The neuronotrophic factor NGF binds to peripheral neurons of the dorsal root ganglion and the sympathetic nervous system. NGF binds to a cell surface receptor, NGFR, on these cells and displays Kd's of 10(-9) and 10(-11)M. NGF receptors have also been reported for basal forebrain magnocellular neurons. In addition, NGF specifically binds to NGFR on Schwann cells although the biological significance of this binding is not known. Here we report that NGF binds in a saturable and specific fashion to receptors on cultured isolated populations of rat astrocytes but not to oligodendrocytes. The binding to astrocytes in culture displayed a Kd of 2.7 +/- 1.0 nM with 36,000 receptors per cell.     

Precursor cells of oligodendrocytes in rat primary cultures

In monolayer primary cultures of brain from newborn rats, which contain astrocytes and oligodendrocytes, a new morphological cell type (flat black cells) was observed. Microphotographs of different areas of the monolayer, taken every 30 min, showed that these flat black cells can divide and that they undergo morphological transformation in vitro. They give rise to oligodendrocytes which were identified by their characteristics morphology but also by their content of W1 Wolfgram protein. These findings suggest that the flat black cells are precursors for oligodendrocytes, in culture.     

An efficient system for selection and culture of Schwann cells from adult rat peripheral nerves

Schwann cells (SCs), the supporting cells of the peripheral nerves, are indispensable for regenerating the peripheral and central nervous system. Copious preparation of these cells in a well-defined manner is to be a privileged position. SCs cultivation is overwhelmed by contaminating fibroblasts which are often outgrowing as the predominant cell type in an in vitro culture. This study introduces a technically simple and efficient procedure for SCs isolation and enrichment based on implementing recombinant and defined supplements. Collected adult rat sciatic nerves were cultured for 10 days as in vitro predegeneration. After dissociation and plating, the medium changed to knockout serum replacement supplemented DMDM/F12 medium containing various growth factors. The whole procedure took 3 weeks and SCs purity was then evaluated through implementing specific cytoplasmic and membranous markers. The viability of enriched SCs were evaluated by MTT assay. Within 10 days, over 99 % homogenous SCs were achieved and confirmed through immunofluorescence staining and flow-cytometry for P75^NTR and S100 markers, respectively. MTT data revealed that the viability and metabolic activities of purified SCs were increased in expansion medium. This study provides a technically easy and efficient method with the benefits of not utilizing bovine serum or other animal products for SCs isolation and enrichment.     

A screen for mutations in zebrafish that affect myelin gene expression in Schwann cells and oligodendrocytes

Myelin is the multi-layered glial sheath around axons in the vertebrate nervous system. Myelinating glia develop and function in intimate association with neurons and neuron-glial interactions control much of the life history of these cells. However, many of the factors that regulate key aspects of myelin development and maintenance remain unknown. To discover new molecules that are important for glial development and myelination, we undertook a screen of zebrafish mutants with previously characterized neural defects. We screened for myelin basic protein (mbp) mRNA by in situ hybridization and identified four mutants (neckless, motionless, iguana and doc) that lacked mbp expression in parts of the peripheral and central nervous systems (PNS or CNS), despite the presence of axons. In all four mutants electron microscopy revealed that myelin-forming glia were present and had formed loose wraps around axons but did not form compact myelin. We found that addition of exogenous retinoic acid (RA) rescued mbp expression in neckless mutant embryos, which lack endogenous RA synthesis. Timed application of the RA synthesis inhibitor DEAB to wild type embryos showed that RA signalling is required at least 48 h before the onset of myelin protein synthesis in both CNS and PNS.     

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.     

Spontaneous immortalisation of Schwann cells in culture: short-term cultured Schwann cells secrete growth inhibitory activity.

In the developing peripheral nerve, Schwann cells proliferate rapidly and then become quiescent, an essential step in control of Schwann cell differentiation. Cell proliferation is controlled by growth factors that can exert positive or inhibitory influences on DNA synthesis. It has been well established that neonatal Schwann cells divide very slowly in culture when separated from neurons but here we show that when culture was continued for several months some cells began to proliferate rapidly and non-clonal lines of immortalised Schwann cells were established which could be passaged for over two years. These cells had a similar molecular phenotype to short-term cultured Schwann cells, except that they expressed intracellular and cell surface fibronectin. The difference in proliferation rates between short- and long-term cultured Schwann cells appeared to be due in part to the secretion by short-term cultured Schwann cells of growth inhibitory activity since DNA synthesis of long-term, immortalised Schwann cells was inhibited by conditioned medium from short-term cultures. This conditioned medium also inhibited DNA synthesis in short-term Schwann cells stimulated to divide by glial growth factor or elevation of intracellular cAMP. The growth inhibitory activity was not detected in the medium of long-term immortalised Schwann cells, epineurial fibroblasts, a Schwannoma (33B), astrocytes or a fibroblast-like cell-line (3T3) and it did not inhibit serum-induced DNA synthesis in epineurial fibroblasts, 33B cells or 3T3 cells. The activity was apparently distinct from transforming growth factor-beta, activin, IL6, epidermal growth factor, atrial natriuretic peptide and gamma-interferon and was heat and acid stable, resistant to collagenase and destroyed by trypsin treatment. We raise the possibility that loss of an inhibitory autocrine loop may contribute to the rapid proliferation of long-term cultured Schwann cells and that an autocrine growth inhibitor may have a role in the cessation of Schwann cell division that precedes differentiation in peripheral nerve development.     

Enzyme levels in cultured astrocytes,oligodendrocytes and Schwann cells,and neurons from the cerebral cortex and superior cervical ganglia of the rat

Data are presented for 16 enzymes from 8 metabolic systems in cell cultures consisting of approximately 95% astrocytes and 5% oligodendrocytes. Nine of these enzymes were also measured in cultures of oligodendrocytes, Schwann cells, and neurons prepared from both cerebral cortex and superior cervical ganglia. Activities, in mature astrocyte cultures, expressed as percentage of their activity in brain, ranged from 9% for glycerol-3-phosphate dehydrogenase to over 300% for glucose-6-phosphate dehydrogenase. Creatine phosphokinase activity in astrocytes was about the same as in brain, half as high in oligodendrocytes, but 7% or less of the brain level in Schwann cells and superior cervical ganglion neurons and only 16% of brain in cortical neurons. Three enzymes which generate NADPH, the dehydrogenases for glucose-6-phosphate and 6-phosphogluconate, and the NADP-requiring isocitrate dehydrogenase, were present in astrocytes at levels at least twice that of brain. Oligodendrocytes had enzyme levels only 30% to 70% of those of astrocytes. Schwann cells had much higher lactate dehydrogenase and 6-phosphogluconate dehydrogenase activities than oligodendrocytes, but showed a remarkable similarity in enzyme pattern to those of cortical and superior cervical ganglion neurons.Special issue dedicated to Dr. Lewis Sokoloff.     

Uptake and metabolism of fluorescent ceramide analogs by rat oligodendrocytes in culture.

We studied the metabolism of sphingolipids by oligodendrocytes derived from rat spinal cord by providing lipid vesicles with either N-lissamine-rhodaminyl-ceramide (LRh-Cer) or N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-ceramide (NBD-Cer) to the cells cultured in a chemically-defined medium. With both probes the major fluorescent product turned out to be sphingomyelin (SM). Most of LRh-SM was not cell-associated but recovered from the culture medium, probably due to back-exchange to the lipid vesicles. The accumulation of LRh-SM, both in the cells and in the medium, was inhibited in the presence of monensin or brefeldin A, whereas the production of NBD-SM was much less affected by these Golgi perturbing drugs. With LRh-Cer as substrate, LRh-labelled fatty acid (FA), galactosyl- and sulfogalactosyl-ceramides (GalCer and SGalCer) were also formed. NBD-Cer, however, was metabolized to glucosylceramide (GlcCer) and GalCer but not to SGalCer or NBD-FA. These data demonstrate that chemical modifications of ceramide alter its metabolism in oligodendrocytes and that the metabolites of LRh-Cer reflect the glycolipid composition of myelin more closely than those of NBD-Cer.     

Progenitors of oligodendrocytes: Limiting dilution analysis in fetal rat brain culture

In this paper, we describe the use of a combination of cell culture techniques and limiting dilution analysis to determine the number of oligodendrocyte progenitor cells and the oligodendrocyte clone size in primary dispersed cultures of 20- to 21-day-old fetal rat brain. Single-cell suspensions (1,2,3 × 10⁶ cells/ml) were plated in either microwell or 100 mm dishes. After 22 days in culture the number of differentiated oligodendrocytes was ascertained by determining the amount of myelin basic protein by radioimmunoassay. The total amount of myelin basic protein was the same in the two types of dish, indicating that proliferation and differentiation were unaffected when oligodendrocytes were grown in microwells. The fraction (F₀) of microwells containing no oligodendrocytes was determined at each cell dilution. F₀ decreased exponentially with increasing total cell concentration. The linearity of the plot of ln F₀ versus cell number indicates that the number of oligodendrocyte progenitor cells is limiting. From the equation describing the Poisson distribution of progenitor cells in microwells we calculated that, at the time of plating, primary cultures of fetal rat brain contain one oligodendrocyte progenitor cell per 1.3 × 10⁵ brain cells, or a total population of 300–500 progenitor cells per brain. The mean oligodendrocyte clone size was determined to be approximately 825 at 22 days and close to 2000 by 35 days in culture. Therefore, each progenitor cell must undergo approximately 11 divisions, on the average, during postnatal development.     

Assays for cholinergic properties in cultured rat Schwann cells

Cultured rat Schwann cells did not contain detectable levels of choline-acetyltransferase (less than 0.5 pmol ACh min-1 mg-1 of protein) or of acetylcholinesterase and nicotinic acetylcholine receptors. After adding Schwann cells to primary rat myotube cultures, the level of cholineacetyl-transferase in the co-cultures increased after three weeks to as high as 5 pmol ACh min-1 mg-1. The activity appearing in co-cultures sedimented at approximately 4S, and was inhibited 50% by 4(1-napthylvinyl)pyridine in the concentration range of 10-50 microM. After treatment of co-cultures with anti rat neural antigen-1 (RAN-1) and complement, 70-80% of the activity was lost, suggesting that it is induced in the Schwann cells. Attempts to obtain the effect by exposure of Schwann cells to medium conditioned by the myotube cultures, or by co-culture with fibroblasts gave levels of activity at or below the limit of detection.     

A calcium-activated cation-selective channel in rat cultured Schwann cells

Calcium-activated channels, in the plasma membrane of rat cultured Schwann cells were studied in isolated 'inside-out' membrane patches. With identical (150 mM NaCl) solutions on either side of the membrane, a single channel conductance of 32 pS was calculated for inward current; the conductance was somewhat less for outward current. The channel is about equally permeable to sodium and potassium ions, but is not detectably permeable to either chloride or calcium. Under our experimental conditions the channel is activated by high (more than 10(-4) M) concentrations of calcium and is sensitive to voltage, channel activity increasing with membrane depolarization.     

Death of oligodendrocytes and microglial phagocytosis of myelin precede immigration of Schwann cells into the spinal cord

Small, circumscribed electrolytic lesions were made in the upper cervical corticospinal tract in adult rats. In the centre of the lesion, the axons and all other tissue elements were totally destroyed. Surrounding this region of destruction is an area of tissue which is only partially damaged. In this area TUNEL positive staining of contiguous rows of tract glial cells indicates massive oligodendrocytic apoptosis at 1–3 days after operation, but axons, astrocytes and blood vessels survive. From around 4 days, the corticospinal axons in this area are demyelinated, and the microglia contain ingested myelin, identified in electron micrographs as characteristic MBP immunoreactive laminar cytoplasmic bodies. After around 3 weeks, large numbers of Schwann cells, continuous with those on the pial surface of the spinal cord, accumulate along the lesion track and selectively infiltrate the perilesional reactive area, where they mingle intimately with the phagocytic microglia. Electron micrographs show that at this time basal lamina-enclosed Schwann cell processes establish non-myelinated ensheathment of axons. From around 4 weeks after operation, prominent Schwann cell myelination is indicated by P0 immunoreactivity, and peripheral type, one-to-one myelination in electron micrographs. Thus the effect of the selective loss of oligodendrocytes is to first activate microglia, and then to induce a replacement of myelin by Schwann cells.