Metabolism of sulfogalactosyl glycerolipids in the myelinating mouse brain.

The in vivo metabolism of sulfogalactosyl glycerolipids (SGG) was studied in the cerebrum and cerebellum of developing mice after intraperitoneal injection of [35S]sulfate. After correction for the specific radioactivity changes of blood sulfate the quantitative rates of biosynthesis and biodegradation of this lipid could be determined. In addition, the net accumulation of SGG was measured. Throughout development the rates of SGG biosynthesis and net accumulation were higher in the cerebellum than in the cerebrum. The developmental patterns of SGG net synthesis in both parts of the brain were closely related to those observed earlier for sulfatide. During development the rate of SGG biosynthesis in both parts of the brain showed a peak earlier than that of sulfatide (at 14 days versus 20 days). The in vivo patterns of SGG degradation followed those of biosynthesis in the cerebrum and cerebellum. During postnatal development 40 to 80% of the daily synthesized SGG disappeared within 24 hr, suggesting that degradation may also be involved in the regulation of SGG net synthesis during myelination, as previously indicated for sulfatide.

Acid mucopolysaccharide (AMPS) abnormality in multiple sulfatase deficiency: chemical compositions of AMPS in urine and liver.

Extensive chemical analyses of acid mucopolysaccharides (AMPS) were carried out in the urine and tissue (liver and brain) from a Japanese patient and two European patients with multiple sulfatase deficiency (MSD). The Japanese patient with MSD contained excessive quantities of heparan sulfate and moderately increased chondroitin sulfate A/C. Urinary excretion of AMPS in MSD heterozygotes was increased 2-fold compared to our controls. The urinary pattern of AMPS in the mother of the MSD patient showed an increase of 18% heparan sulfate and 36% dermatan sulfate whereas the urinary excretion pattern in the father was increased 21% for heparan sulfate as contrasted to controls (chondroitin sulfate A, 50-52%; chondroitin sulfate C, 38-46%; and heparan sulfate, 3-10%). Seventy-five % of the AMPS and the MSD liver was heparan sulfate rather than dermatan sulfate. The degree of accumulation of AMPS in the MSD liver was 30-50 times that of the control. Cerebral gray matter from the MSD patient contained 30-40 times that of control (relative increase of heparan and dermatan sulfate) whereas only a 5-fold increase was observed in white matter. It seems that a major site of accumulated AMPS appears to be in the gray matter. Carbohydrate analysis of the AMPS obtained from MSD urine and tissues was performed by: enzyme digestion with testicular hyaluronidase, heparitinase and chondroitinase ABC, cellulose acetate electrophoresis, Dowex-1 column chromatography and amino sugar analysis by amino acid analyzer. These findings indicate that the major accumulated AMPS in MSD urine and liver is heparan sulfate and thus, the predominant AMPS metabolic defect in MSD is heparan sulfate degradation.

Vesicular transport of sulfatide in the myelinating mouse brain. Functional association with lysosomes?

Sulfatide synthesis and its subcellular distribution kinetics was followed in the myelinating brain of 17-day-old mice. Pulse-labeling-chasing conditions were achieved by an intraperitoneal injection of (35S)sulfate followed 2 h later by a second injection of a high dose of unlabeled sulfate. At 1, 2, 3, 4, and 6 h after the (35S)sulfate injection, the brains were removed, homogenized, and subcellular fractions were obtained by differential and discontinuous sucrose gradient centrifugation (Eichberg, J., Whittaker, V. P., and Dawson, R. M. (1964) Biochem. J. 92, 91-100). The microsomal membranes were further subfractionated (Siegrist, H. P., Burkart, T., Wiesmann, U. N., Herschkowitz, N. N., and Spycher, M. A. (1979) J. Neurochem. 33, 497-504) into light myelin, plasma membranes, Golgi vesicles, endoplasmic reticulum membranes, and heavy vesicles associated with acid hydrolase activities. The [35S]sulfatide-labeling kinetics was measured in all subcellular fractions. The results indicate that sulfatides are synthesized in the Golgi-endoplasmic reticulum complex and transferred in vesicles at least partially associated with lysosomes to the myelin membranes. The association of sulfatides with lysosomes could explain the existence of the previously described labile pool of newly synthesized sulfatides (Burkart, T., Hofmann, K., Siegrist, H. P., Herschkowitz, N. N., and Wiesmann, U. N. (1981) Dev. Biol. 83, 42-48) and also could be a form of vesicular transport to the myelin.

Abnormal growth kinetics and 5'-nucleotidase activities in cultured skin fibroblasts from patients with Duchenne muscular dystrophy.

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 less than 0.0001) 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 (McCoy's medium instead of Eagle's minimum essential medium) 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 less than 0.001; nDMD = 5; ncontrols = 4) whereas plating efficiency is equal for both DMD and controls. On day 7 activity of the membrane bound enzyme 5'-nucleotidase either per mg protein or per microgram DNA is significantly elevated in cells from DMD patients (P less than 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, thay could serve as a potential prenatal diagnosis of the disease.

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.

Diminished cerebroside-sulfotransferase activity in the Jimpy mouse mutant due to altered lipid composition in microsomal membranes.

The mouse mutant Jimpy shows a deficient myelination. In the microsomes of the Jimpy brain, the cerebroside-sulfotransferase (EC 2.8.2.11) activity is low. The cerebroside-sulfotransferase activity of Jimpy microsomes could be normalised by delipidating the microsomes with cold acetone and adding to them acetone-extracted lipids from normal microsomes. The lipids extracted from Jimpy membranes did not influence the cerebroside-sulfotransferase activity of neither normal nor Jimpy microsomes. The same results were obtained if artificial cholesterol-phospholipid mixtures in ratios corresponding to the ones found in normal and Jimpy membranes were used for recombination experiments. Therefore the diminished enzyme activities in Jimpy microsomes may be related to the lower cholesterol-phospholipid ratio found in the microsomal membranes of the Jimpy mutant.

3'-phosphoadenylylsulfate:galactosylceramide 3'-sulfotransferase. An optimized assay in homogenates of developing brain.

An optimized in vitro assay of 3'-phosphoadenylysulfate:galactosylceramide 3'-sulfotransferase (EC 2.8.2.11, galactosylceramide sulfotransferase, formerly known as galactocerebroside sulfotransferase) activity is presented, that can be used in crude homogenate of brain tissue of various developmental stages. The enzyme activity is determined by measuring the [35S]sulfatides formed by the enzymic transfer of [35S]sulfate from 3'-phosphoadenoside 5'-phospho [35S]sulfate to galactosylceramides. The sulfatide formation at 30 degrees C is linear up to 30 min and up to a protein concentration of 1 mg per 0.5 ml assay volume. The presence of 0.4% Triton X-100 and 50 micrometer exogenous bovine cerebrosides are optimal for enzyme activity. The pH optimum of the reaction is at pH 6.5 using 0.1 M imidazole buffer. The enzyme reaction is stimulated by NaCl, KCl, MgCl2, CaCl2, MnCl2, ATP and inhibited by ADP. The developmental enzyme activity pattern of mouse brain is the same, if derived from homogenates and microsomes, respectively, under our assay conditions.

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.

Chemical compositions of brain and myelin in two patients with multiple sulphatase deficiency (a variant form of metachromatic leukodystrophy).

The lipid composition of the brain, including myelin, was studied in detail in two cases with a variant form of metachromatic leukodystrophy (multiple sulphatase deficiency type). In the white matter, the sulphatide concentration was 3-4 times higher than the normal level in both cases. There was a significant accumulation of cholesterol sulphate in the brain, liver and kidney of both cases. The ganglioside pattern in the grey and white matter was abnormal, with a higher proportion of GM3, GM2 and GD3-gangliosides. Non-lipid hexosamine contents were increased 1.5-2 times in brain, 8-10 times in liver and 2-3 times in kidney. Increased amounts of glucocerobroside, ceramide lactoside and ceramide trihexoside were present in grey and white matter of both cases. Recovery of purified myelin from two patients' brains was much less than from control (1-2% in case 1 and 20-30% in case 2). The lipid composition of myelin was almost normal except for a higher proportion of sulphatide, with a decreased amount of cerebroside. The fatty acid compositions of myelin sulphatide and sphingomyelin were almost normal, while non-hydroxy fatty acids of cerebroside contained less long-chain fatty acids, as characterized by a significant increase of C16:0 and C18:0 fatty acids. The myelin polypeptide pattern by SDS-disc gel electrophoresis showed a relative decrease of basic protein and of proteolipid protein. A possible mechanism of myelin loss in MSD is discussed.