Hans Peter Siegrist

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 checked 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 of culture before a steady state was reached. In contrast to this, the MBP-positive cells appeared only on the 14th day of culture, 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 oligodendrocyte population.

CERAMIDE-GALACTOSYLTRANSFERASE AND CEREBROSIDE-SULPHOTRANSFERASE LOCALISATION IN GOLGI MEMBRANES ISOLATED BY A CONTINUOUS SUCROSE GRADIENT OF MOUSE BRAIN MICROSOMES

Abstract— A 17,000 g supernatant of mouse brain microsomes was subfractionated on a continuous sucrose gradient in order to localise ceramide galactosyltransferase (CGalt, EC 2.4.1.47) and cerebroside sulphotransferase (CST, EC 2.8.2.11), both enzymes involved in the synthesis of myelin lipids. The submicrosomal fractions were analysed for marker enzymes of myelin, plasma membranes, Golgi membranes, endoplasmic reticulum and lysosomes, and their protein distribution was studied. The results and EM studies give evidence that CGalT and CST are located in the Golgi membranes of the brain.

Influence of lipids on the activity of cerebroside- -sulphotransferase in mouse brain. A comparative study of jimpy and normal mouse brains.

Abstract— The effect of lipids other than the substrate cerebroside on the activity of cerebroside‐sulphotransferase (CST) in Jimpy and normal mouse brain was investigated.

Lipid-protein interactions with native and modified myelin basic protein☆

The basic protein of central nervous system myelin has been shown to form complexes with acidic lipids in vitro. We measured the interaction of myelin basic protein with several charged and neutral lipids in a biphasic chloroform/methanol/water system and investigated the effect of decreasing the electrical charge of the basic amino groups of the myelin basic protein by acetylation. The modified myelin basic protein, which has an average of eight acetyl residues incorporated, was characterised by gel electrophoresis and circular dichroism. Complexes formed between the acetylated myelin basic protein and acidic lipids exhibited a reduction in the amount of lipids bound, a value that could be correlated with the number of modified amino groups. The significance of these experiments with reference to protein-lipid interaction in the myelin membrane is discussed.

Quantitative measurement of in vivo sulfatide metabolism during development of the mouse brain: Evidence for a large rapidly degradable sulfatide pool

The in vivo metabolism of sulfatide was studied in the cerebellum and in the cerebrum of developing mice by intraperitoneal injection of [ 35 S]sulfate. After correction for the specific radioactivity of the sulfate, total sulfatide synthesis and degradation could be determined. The developmental patterns of synthesis and degradation of sulfatide in vivo were compared with the developmental activity patterns of the synthesizing enzyme cerebroside sulfotransferase (CST, EC 2.8.2.11), the degrading enzyme cerebroside sulfate sulfatase, measured as arylsulfatase A (ASA, EC 3.1.6.1), and the net sulfatide synthesis in both brain parts. During brain development, sulfatide synthesis per milligram of tissue was higher in the cerebellum than in the cerebrum. Its developmental pattern was similar but not identical to the CST activity in vitro . The in vivo pattern of sulfatide degradation followed that of sulfatide biosynthesis and was similar to the developmental activity pattern of ASA. During myelination 40–70% of the newly synthesized sulfatide was degraded within 24 hr, as measured in two ways: (1) by a 24-hr chase of the [ 25 S]sulfatide and (2) by calculating the difference between the total daily sulfatide synthesis and net daily accumulation of sulfatide. Both methods of determination gave similar results. The data presented show that the synthetic enzyme CST is fully active in vivo , producing more sulfatide than is necessary for incorporation into myelin. The resulting sulfatide excess is rapidly degraded in the lysosomes by the action of ASA. Thus net sulfatide synthesis is partly regulated by lysosomal degradation during myelination of the brain.

Abnormal Growth Kinetics and 5′-Nucleotidase Activities in Cultured Skin Fibroblasts from Patients with Duchenne Muscular Dystrophy

Summary: The experiments reported herein compare growth kinetics and biochemical properties of cultured skin fibroblasts from patients with Duchenne muscular dystrophy (DMD) and matched normal controls.On day 7 after plating (6000 cells/cm2) cell number and DNA per dish are significantly reduced (P < 0.001) in the cultures from DMD patients (n = 14), compared to those from controls (n = 10). Moreover DMD cells contain less lipids and proteins per dish but more per cell than normal fibroblasts (not significant). Variations of media (McCoys medium instead of Eagles minimum essential medium) and sera (human cord serum instead of fetal calf serum) resulted in the same differences between DMD and control cells.Cell kinetic experiments (plating density: 2000 cells/cm2) show increased doubling times of DMD fibroblasts (P < 0.001; nDMD = 5; ncontrols = 4) whereas plating efficiency is equal for both DMD and controls.On day 7 the activity of the membrane bound enzyme 5‘-nucleotidase either per mg protein or per μg DNA is significantly elevated in cells from DMD patients (P < 0.0005; nDMD = 8; ncontrols = 9) independent of cell density.Thus all findings in cultured DMD fibroblasts: increased doubling time, tendency to more voluminous cells, and elevated 5′-nucleotidase activity per cell suggest, that the DMD cells behave similar to prematurely aging cells. Until now we were not able to check whether any alterations of the plasma membrane are inducing early senescence or, reversely, premature aging is the cause of the postulated membrane alterations.If these findings were to be confirmed in cultured amniotic cells from DMD fetuses, they could serve as a potential prenatal diagnosis of the disease.Speculation: As a systemic disease, Duchenne muscular dystrophy is also expressed in cultured skin fibroblasts by a growth delay of so far unknown origin. Since cultured amniotic cells are metabolically similar to fibroblasts, corresponding growth kinetic alterations might be expected in amniotic cells from DMD fetuses. This would provide a suitable method for prenatal diagnosis.

Influence of reduced cholesterol synthesis on the activity of cerebroside sulfotransferase in cultured glioblastoma cells treated with estradiol.

Cultured glioblastoma cells were inoculated with estradiol in concentrations of 0.5--10 microliter/ml medium in order to check the effect of this hormone on the activity of cerebroside sulfotransferase, an enzyme whose activity is strongly related to myelination. Thereby we could show that the cerebroside-sulfotransferase activity increases to a value of 200% of normal. Concomitant to this effect, the cholesterol content of the membrane bearing cerebroside sulfotransferase activity decreases to 60% of normal. The effect is fully reversible: after 48 h, cholesterol synthesis as well as cerebroside sulfotransferase activity reach normal values again. We suggest that cerebroside sulfotransferase activity is modulated by the changing cholesterol/phospholipid ratio in the cells during the inoculation period.

Effect of Glucose Deprivation on Sulfatide Synthesis and Oligodendrocytes in Cultured Brain Cells of Newborn Mice

Summary: The present work was designed to develop a model for studying the effect of substrates on brain cell metabolism. The synthesis of sulfatide, a myelin lipid, which takes place predominantly in oligodendrocytes was measured in vitro in dissociated cell cultures of newborn mouse brains at 13 days in culture. This corresponds to an active stage of in vivo myelination. The effect of increasing durations of severe glucose deprivation (below 1 mM in the medium) was assessed on morphology, cellular protein, DNA, sulfatide synthesis, cerebroside sulfotransferase activity, and mucopolysaccharide synthesis.Glucose was rapidly consumed by the cultured cells above a concentration of 0.4 mM in the medium. At high and intermediate glucose concentrations, between 43.8 and 63.6% of the glucose consumed was accumulated as lactate on a molar ratio, indicating that the pyruvate dehydrogenase complex was rate limiting. In severe glucose deprivation, lactate consumption of the cultures exceeded accumulation.With increasing duration of severe glucose deprivation (below 1 mM), the following major changes were observed. The number of phase dark cells identified by immunofluorescence as oligoden-drocytes progressively decreased. Immunologically identified astrocytes apparently were unaffected by glucose deprivation. Sulfatide synthesis progressively dropped to 3.8% of control, and cerebroside sulfotransferase dropped to 10.3% of control with prolonged glucose deprivation. Thus, oligodendrocytes and one of their specific functions were damaged by glucose deprivation.Lactate, in spite of its being consumed by the brain cell cultures, was unable to replace glucose as a substrate for sulfatide synthesis.Uninfluenced by glucose deprivation were total cellular phosphocreatinkinase, arylsulfatase A, as well as total mucopolysaccharide synthesis by the brain cell cultures.Speculation: The present study shows that sulfatide synthesis in cultured brain cells of newborn mice at a stage corresponding to active in vivo myelination is dependent upon availability of glucose. Lactate, in spite of its being consumed by the brain cells, is unable to replace glucose with respect to sulfatide synthesis. This system of cultivated brain cells may serve as a model for studying other potential fuels for the maintenance of oligodendrocyte function.

Theophylline Reduces the Activity of Cerebroside-Sulfotransferase, a Key Enzyme in Myelination, in Cell Cultures from Newborn Mouse Brain

Summary: Theophylline, a drug used in neonatology for the treatment of apnea, affects cholesterol synthesis if administered in concentrations of 10-4 M (a concentration found in serum of treated patients) for 24 hr to dissociated brain cell cultures. The rate-limiting enzyme of cholesterol synthesis, β-hydroxy-β-methylglutaryl-coenzyme A reductase (EC 1.1.1.34), is lowered to 45% 48 hr after removal of theophylline. At the same time, cholesterol content of the cells is lowered to 73%. Inasmuch as the phospholipid content of the cells remains stable, the treatment changes the cholesterol phospholipid ratio. Concomitant to this effect, the activity of cerebroside-sulfotransferase (EC 2.8.2.11) is lowered to 60% of control values. We postulate that these two effects are linked to each other by means of modulation of the cerebroside-sulfotransferase activity by membrane lipids.Speculation: Theophylline reduces cholesterol synthesis and content and by that cerebroside-sulfotransferase activity in dissociated brain cell cultures. This raises the question of whether theophylline could have similar effects in vivo.

Net sulfatide synthesis, galactosylceramide sulfotransferase and arylsulfatase a activity in the developing cerebrum and cerebellum of normal mice and myelin-deficient jimpy mice.

Net sulfatide synthesis, galactosylceramide sulfotransferase (EC 2.8.2.11) and arylsulfatase A (EC 3.1.6.1) activities were measured in two brain regions, cerebrum and cerebellum, of normal and jimpy mice during postnatal development. In normally myelinating mice, two phases of increasing rates of net sulfatide synthesis were observed, the first coinciding with oligodendrocyte proliferation and the second with myelination. Net sulfatide synthesis was quantitatively higher in the cerebellum than in the cerebrum. In both brain regions, the developmental patterns of net sulfatide synthesis were related to the activity patterns of both galactosylceramide sulfotransferase and arylsulfatase A. In jimpy mice, a neurological mutant showing hypomyelination in brain, the first phase of net sulfatide synthesis was preserved in both brain regions and galactosylceramide sulfotransferase and arylsulfatase A activities were normal up to 12 days. However, during the phase in which myelination occurred in controls, the net sulfatide synthesis in both brain regions of jimpy mice was zero or even negative. The sulfatide deficit was larger in the cerebellum than in the cerebrum. In both mutant brain parts, galactosylceramide sulfotransferase activity increased up to 12 days showing about 50% of the maximal activities observed in normal brain regions. Thereafter up to 15 days, enzyme activity decreased to about 25% of that of controls and remained low in both brain regions. The developmental patterns and the activities of arylsulfatase A were, however, normal in the cerebrum and cerebellum of jimpy mice. These results suggest that the enzyme activities and the developmental patterns of galactosylceramide sulfotransferase and arylsulfatase A as measured in vitro reflect to a high degree their functional activity in vivo. Furthermore, sulfatide degradation by arylsulfatase A seems to be important in regulating net sulfatide synthesis during normal and impaired myelination.