Biomedical subjects
Publications and source records attributed to S Szuchet.
Evidence is presented for and against the participation of oligodendrocytes in the etiology and pathogenesis of MS,. The structure and properties of these cells are described, and the merits of in-vitro studies to learn of the cells' involvement in MS are argued.
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Isolated lamb oligodendrocytes in tissue culture were tested for their ability to express differenciated functions related to the synthesis of myelin. Galactocerebrosides and sulfatides are highly enriched in myelin and are recognized as markers for myelination. Hence, we followed the synthesis of these glycosphingolipids. At selected times, monolayer cultures of cells were double labeled for 60-72 h with [3H]galactose and H2 35SO4. Glycosphingolipids were separated and identified by thin layer chromatography. During the first week in tissue culture, there was gradual decline in incorporation of both isotopes relative to the values, obtained for freshly isolated cells. However, by 3 weeks high levels of H2 35SO4 incorporation into sulfatide and [3H]galactose into cerebrosides were found. A complex pattern of incorporation ensued after this peak which varied for each particular glycosphingolipid. It is concluded that: (1) cultured oligodendrocytes retain the capacity to synthesize components of myelin; (2) since the isolated cells have already been involved in myelination, these cultures afford a unique model to study remyelination' amd (3) these cells provide a good system to study the properties of oligodendrocytes.
Gangliosides are enriched in the nervous system compared to other tissues. The synthesis of gangliosides by monolayer cultures of isolated oligodendrocytes has not previously been investigated. Cells were labeled with [3H] galactose at preselected times and gangliosides isolated by phase partition, purified, and identified by chromatography. Cultured oligodendrocytes showed selectivity in their synthesis of gangliosides, which was expressed in the type of ganglioside synthesized as well as in the change of incorporation over time in culture. For the first ten days, there was very little incorporation of [3H] galactose in gangliosides, but this was followed by a stimulation of uptake for GM3, GM1/GD3, and GD1 gangliosides, reaching a maximum after approximately 25-30 days in vitro. There was little incorporation into GM2 or trisialogangliosides throughout the life of the cultures. Since oligodendrocytes synthesize extensive membranes during this period, one may speculate that the de novo-synthesized gangliosides are used for membranes.
The changes in the ultrastructure of oligodendrocytes with time in culture were examined by transmission electron microscopy. Oligodendrocytes were isolated from ovine white matter and maintained in Dulbecco's modified Eagle's medium supplemented with 20% horse serum. One day after being placed in plastic culture dishes, most cells aggregate into clusters that do not attach to the surface for several days. Cells within these clusters form junctional complexes resembling gap junctions, their nuclear chromatin becomes uniformly dispersed, they are rich in polyribosomes, and their cytoplasm is less osmiophilic than oligodendrocytes in ovine white matter or cells fixed immediately after isolation. After several days in culture, the cells become attached to the plastic surface, chromatin once again becomes clumped beneath the nuclear membrane, rough endoplasmic reticulum accumulates within the cytoplasm and the cells extend long processes containing cytoplasmic organelles. From these observations, it appears that oligodendrocytes undergo a transient dedifferentiation during their recovery from the trauma of isolation, not unlike the central chromatolysis seen in neurons following axonal injury. After acclimatization to tissue culture conditions, isolated oligodendrocytes show many of the morphologic features seen in vivo and therefore offer a good model to study myelination.
A new procedure for isolating oligodendrocytes from ovine white matter is described. The method separates oligodendrocytes into two bands on a linear sucrose gradient. Five criteria have been employed to classify the separated cells. It is shown by indirect immunofluorescence with specific antisera that 97% of the cells from both bands carry galactocerebroside, a specific surface marker for oligodendrocytes, on their plasma membranes and 95% of the cells retain myelin basic protein as distinct patches on their surfaces. Isolated cells conform ultrastructurally to current concepts of oligodendrocytes. The cells incorporate [3H]galactose into galactocerebroside and carrier free H2(35)SO4 into sulfatide, specific markers for oligodendrocytes. The specific activity of 2',3'-cyclic nucleotide-3'-phosphodiesterase in the two cell fractions is comparable to that reported for isolated oligodendrocytes by others. It is concluded that conservatively, 95% of the cells in both fractions are oligodendrocytes. Cells from both bands survive in culture for months. In vitro the cells extend two or more processes, contain 'gliosomes', and surround themselves with extensive sheet-like membranes; i.e. they exhibit the morphological characteristics ascribed to oligodendrocytes in explant cultures. Conservatively 90% of cultured cells stain with an antimyelin basic protein serum. The staining is localized in the cytoplasm and processes. The cells also stain with antigalactocerebroside and antioligodendrocyte sera. Cells remain differentiated for up to 70 days in vitro as evidenced by their incorporation of [3H]galactose and H2(35)SO4 into galactosyl and sulfogalactosylceramide, respectively.
A new method for isolation of oligodendrocytes is described. The method was developed to isolate intact, viable cells and to fractionate oligodendrocyte subgroups. Finely minced ovine white matter (WM) is incubated in 0.1% trypsin at 37 degrees C for 3.6 min/g WM. Trypsin inhibitor is added to arrest the action of trypsin. Further disruption of tissue is achieved by passage through a series of screens (350 micrometer down to 30 micrometer pore size) and the crude suspension in 0.9 M sucrose is centrifuged at 2100 rpm (850 g) for 10 min. During this step myelin floats to the top of the tube while the cells form a pellet. The pellet is resuspended in 3-4 ml of 0.9 sucrose and applied to a linear sucrose gradient (1.0-1.2 M), which is then centrifuged at 1200 rpm (277 grams) for 40 min. Oligodendrocytes separate into two distinct bands on this gradient suggesting that two subpopulations have been isolated. There are small differences in size between cells from these bands. Oligodendrocytes isolated by this procedure remain viable and differentiated for months as evidenced by their ability to incorporate labeled precursors into galactocerebrosides and sulfatides and to synthesize myelin basic protein.
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The molecular weight of bovine lactoferrin in a neutral solvent (e.g. 0.2 M NaC1, pH 7.8 20 degrees C) has been determined using high-speed sedimentation equilibrium at various concentrations and speeds. Under these conditions the protein behaved as a single thermodynamic component with a weight-average molecular weight [MW]C=0 = 93000 plus or minus 2000 and a second virial coefficient B = 4.3-10(-5) mol-ml(-1)-g(-2). This value of the molecular weight is in good agreement with values previously reported by Groves (J. Am. Chem. Soc. 82, 3345-3350, 1960) and Szuchet and Johnson (Eur. Polym. J. 2, 115-128, 1966); it is, however, in disagreement with the more recent value given by Castellino et al. (J. Biol. Chem. 245, 4269-4275, 1970). The possible reasons for this discrepancy are discussed.
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We have used a combination of electrophysiological and biochemical approaches to investigate the effects and the mechanisms of action of tumor necrosis factor-alpha (TNF-alpha) on cultured oligodendrocytes (OLGs). Our studies have led to the following conclusions: (1) prolonged exposure of mature ovine OLGs to TNF-alpha leads to inhibition of process extension, membrane depolarization and a decrease in the amplitudes of both inwardly rectifying and outward K+ currents; (2) brief exposure of OLGs to TNF-alpha does not elicit membrane depolarization or consistent changes in cytosolic Ca2+ levels; (3) incubation of OLGs with TNF-alpha for 1 hr results in inhibition of phosphorylation of myelin basic protein and 2',3'-cyclic nucleotide phosphohydrolase. Ceramides, which have been shown to be effectors of TNF-alpha, are ineffective in inhibiting phosphorylation, whereas sphingomyelinase mimics TNF-alpha in this action. These observations suggest that other products of sphingomyelin hydrolysis may be the mediator(s) of TNF-alpha effect on protein phosphorylation. We have thus demonstrated that TNF-alpha can perturb the functions of OLGs via modulation of ion channels and of protein phosphorylation without necessarily inducing cell death. It is conceivable that modulation of ion channels and protein phosphorylation constitutes effective mechanisms for the participation of cytokines in signal transduction during myelination, demyelination and remyelination.