Shirley E. Poduslo

The isolation and characterization of a plasma membrane and a myelin fraction derived from oligodendroglia of calf brain.

—A method is described for the fractionation of bulk isolated oligodendroglial cells from calf brain to produce both a plasma membrane and an attached myelin fraction. The cells are homogenized in a sucrose solution containing Mg2+ and K+ at a pH of 6·5. Crude membrane fractions are obtained from this homogenate by discontinuous sucrose density gradient centrifugation. After being subjected to osmotic shock, these fractions are purified by continuous sucrose density gradient centrifugation. The plasma membrane fraction, which bands at 1·0 m‐sucrose, was identified by its morphology and enzyme content. Electron microscopy showed it to be a homogeneous preparation of vesicles composed, for the most part, of smooth trilaminar membranes. Enzymatic analysis revealed the presence of high specific activities of Na+, K+‐ATPase, 5′‐nucleotidase and 2′,3′‐cyclic AMPase. Lipid analysis showed a higher galactolipid and lower phospholipid content than has been reported for neuronal and synaptic membranes. The attached myelin fraction, which bands at 0·7 m‐sucrose has the typical multilamellar appearance of myelin, but differs considerably from normal myelin in having high concentrations of plasma membrane marker enzymes, and a lipid composition intermediate between normal myelin and the plasma membrane fraction. The ganglioside content and protein patterns of these fractions have also been examined.

The production of a membrane by purified oligodendroglia maintained in culture

Abstract Purified oligodendroglia maintained in culture produce whorls of membrane lamellae, adjacent to the cell soma. When subcellular fractions are prepared from the cells in culture, three membrane fractions are obtained: a glial light fraction which electron micrographs show to be predominantly large vesicles; an intermediate fraction that on electron micrographs consists of whorls of membrane lamellae; and a plasma membrane fraction consisting primarily of small vesicles. In a study on the production of the membrane lamellae, the results show that there is a rapid incorporation of radiolabel into cerebrosides and phosphatidylcholine, with lower incorporation into other phospholipids. There is a delay in incorporation into sulfatides. Incorporation into proteins show a complex heterogeneous pattern of proteins, ranging from high to low molecular weight (MW) bands. The incorporation data may reflect the composition of the subfractions after different times in culture.

Purification of proteolipid protein and production of specific antiserum

Proteolipid protein, the major protein in CNS myelin, was purified using several chromatographic steps. No detergents were used. Chromatography in organic solvents on lipophilic Sephadex (LH-60) for delipidation, on ion exchange chromatography for protein separation, and again on lipophilic Sephadex (LH-20) for desalting, produced homogeneous preparations of proteolipid protein. Rabbit antibodies to proteolipid protein were produced after 2 injections of a total of 500 micrograms of protein. By immunotransfer and absorption experiments, it was found that the antiserum bound to proteolipid protein and a closely related protein, DM-20; it did not bind to basic protein or other myelin proteins.

Synthesis of cerebrosides by intact oligodendroglia maintained in culture.

Oligodendroglia, isolated in bulk from dissected white matter from lamb brain, can now be maintained in culture. The cells actively synthesize both cerebrosides and sulfatides, lipids found concentrated in myelin, as well as other myelin components. Thus this provides a useful model to study myelin assembly in vitro.

Biochemical studies of metachromatic leukodystrophy in three siblings

SummaryBiochemical analyses were performed on cerebral biopsies from three siblings with metachromatic leukodystrophy, and from autopsy tissue obtained 6 months later. The lipids of all gray matter samples were relatively normal, with the exception of an elevated sulfatide: cerebroside ratio. The white matter samples were grossly abnormal and showed a progression in severity of biochemical abnormalities with duration of the discase. Sulfatides were 4–8-fold higher than normal and the ratio of cerebrosides to sulfatides ranged from 0.13 to 0.26, compared to the nommal value of about 4.0.Myelin was isolated in very low yield, but had normal morphology. As others have reported, it had the same chemical defect as whole white matter: sulfatides were 7–8 times higher than normal and cerebrosides were reduced by more than half. The fatty acid compositions of the myelin sphingolipids were found to be of much longer average chain length than those in affected white matter. In myelin, the unsubstituted fatty acids of cerebrosides and sphingomyclin had a higher percentage of short chains than found in normal myelin, but were less abnormal than those in whole white matter, whereas the unsubstituted and α-nydroxy acids of sulfatides and the α-hydroxy acids of cerebrosides were not deficient in long chains. These data indicate that there are at least two compartments of sphingolipids in MLD white matter, each having different average chain lengths.

Induction of cerebroside synthesis in oligodendroglia

Oligodendroglia function to produce myelin membranes which surround axons, enhancing saltatory conduction. Myelin consists of a multitude of condensed membranes which are rich in lipids with the major glycolipids, cerebrosides, being 25% of the total lipid. Thus a fully differentiated oligodendroglial cell that is producing myelin membranes would be actively synthesizing cerebrosides. Our laboratory has prepared and analyzed oligodendroglia from mature bovine brain, from neonatal rat brain, and from actively myelinating rat brain. Our studies suggest that the rat oligodendroglia in our culture systems are less differentiated than bovine cells in that they produce lower levels of cerebrosides. Addition of glucocorticoids, thyroid hormone, or retinoic acid all increased synthesis of cerebrosides in rat oligodendroglia. Ketone bodies were also somewhat stimulatory. Having no effect or causing dedifferentiation of the cells were 5-azacytidine and phorbol esters. Thus induction of cerebroside synthesis in oligodendroglia is complex and may involve many factors.

Ketone body enzyme activities in purified neurons, astrocytes and oligodendroglia

Ketone bodies serve as sources for energy and as precursors for lipid synthesis in developing brain. Whether neurons and neuroglia are equally capable of using ketone bodies during differentiation is not known. Using purified populations of neurons and astrocytes from developing rat brain and purified oligodendroglia from bovine brain, the activities for the three enzymes involved in ketone body metabolism were evaluated. Enzymatic activities were found in all three cell types. Surprisingly, astrocytes had the highest levels of activity for both 3-ketoacid-CoA transferase and acetoacetyl-CoA thiolase; these activites showed dramatic changes during development. Nonetheless, neurons, astrocytes and oligodendroglia are all quite capable of using ketone bodies as metabolic fuels.

Proteins and glycoproteins in plasma membranes and in the membrane lamellae produced by purified oligodendroglia in culture

Oligodendroglial plasma membranes are complex structures composed of a heterogeneous mixture of proteins and glycoproteins. The Coomassie stained gel patterns showed a maximum of 40 proteins with molecular weights ranging from greater than 200,000 to 12,500. Autoradiography was used to detect binding of radioiodinated lectins to glycoproteins. With concanavalin A, 5 major glycoproteins were seen; with wheat germ agglutinin, 2 major glycoproteins with approximate molecular weights of 95,000 and 78,000 were found; with Ulex europaeus, 7 major glycoproteins were observed. Additional minor bands were also seen. The impermeant probe diazodi[125I]iodosulfanilic acid was used to radiolabel intact cells. It was found that 5 major proteins were radiolabeled in the plasma membranes. In all cases, the whorls of membrane lamellae produced in culture by oligodendroglia tend to have a somewhat less complicated pattern with fewer proteins and glycoproteins than the plasma membranes. However, the whorls of membrane lamellae have far more complicated protein patterns than myelin.

Biochemical studies of the late infantile form of metachromatic leukodystrophy

SummaryBiochemical studies from a patient with the late infantile form of metachromatic leukodystrophy are presented. Since the autopsy was performed soon after death, viable cells were isolated from brain. The purified cells had altered densities and unusual appearances. The cells when placed in culture were able to incorporate radiolabeled substrates into cerebrosides, indicating that some of the cells were of oligodendroglial origin. Myelin was isolated using several different methods, and the degree of abnormality appeared to be dependent upon the method of isolation. Nonetheless, MLD myelin, while still retaining its characteristicsmorphology, had increased levels of sulfatides (two to four times that normally found). Other membrane subfractions were isolated that were not present in control tissue and that were more abnormal in composition than myelin. Finally, the glycoproteins in MLD tissue also appeared to be altered. There were losses in MLD myelin glycoproteins that bind to Concanavalin A (Con A) and additional prominent glycoproteins that bind to wheat germ agglutinin.

A sensitive immunotransfer method for the detection of oligodendroglial plasma membrane antigens

Efficient transfer of a wide molecular weight range of plasma membrane proteins from polyacrylamide gels to nitrocellulose paper has been achieved. Using published immunodetection procedures (sensitivity: 1 ng protein), two major antigens of molecular weights of 71,000 and 82,000 have been identified in oligodendroglial plasma membranes. This immunotransfer method will be extremely useful for the detection of low levels of antigen in complex membrane protein mixtures.