Effects of Monensin and Colchicine on Myelin Galactolipids

Monensin and colchicine have been used in a variety of systems to disrupt functioning of the Golgi apparatus and transport of Golgi-derived vesicles to the plasma membrane. In this study the effects of monensin and colchicine on the synthesis of cerebroside and sulfatide and their appearance in myelin were examined to determine whether these myelin components are processed through the Golgi apparatus. Brain slices from rats 17 days old were incubated with [3H]galactose and [35S]-sulfate to label cerebroside and sulfatide. Myelin was isolated on sucrose density gradients. Fractions highly enriched in cerebroside and sulfatide were prepared from homogenates and myelin fractions by lipid extraction, alkaline methanolysis, and in some cases TLC. Monensin at 0.1 microM had no significant effect on synthesis of these galactolipids as measured by incorporation of [3H]-galactose into cerebroside or [35S]sulfate into sulfatide in homogenates. However, appearance of [35S]sulfatide in the myelin fraction was reduced to 49% of control, while appearance of [3H]cerebroside was not significantly reduced. Colchicine from 1 mM to 0.1 microM had effects similar to monensin, that is, appearance of [35S]sulfatide in myelin was depressed, but again [3H]cerebroside was not affected. Incorporation of [35S]sulfate into sulfatide in homogenate was 93% of control, while appearance of [35S]sulfatide in the myelin fraction was depressed to 58% of control. The inhibition of appearance of sulfatide in myelin by colchicine and monensin is consistent with the view that sulfation of cerebroside occurs in the Golgi and that sulfatide is transported via Golgi-derived vesicles to the forming myelin membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

MUCOPOLYSACCHARIDES SYNTHESIZED BY CULTURED GLIAL CELLS DERIVED FROM A PATIENT WITH SANFILIPPO A SYNDROME

—Glial cells were cultured from brain tissue obtained at autopsy of a patient with Sanfilippo A syndrome. Mucopolysaccharides were labeled by culturing the cells in the presence of [³⁵S]sulfate. After proteolysis, intracellular and media-elaborated mucopolysaccharides were fractionated by Dowex 1 chromatography. One fraction, identified as heparan sulfate by chromatographic, electrophoretic, and enzyme susceptibility properties, accumulated in Sanfilippo glial cells in greater amounts than in controls. Heparan sulfate was also excreted into the culture media by both Sanfilippo and normal cultures, and it constituted a major fraction of the sulfated mucopolysaccharides synthesized by glial cells. Sanfilippo and normal fibroblasts were also included in these studies for comparative purposes. Sanfilippo fibroblasts accumulated significantly increased amounts of heparan sulfate as compared to normal fibroblasts. Heparan sulfate was excreted into the culture media by Sanfilippo and normal fibroblasts in equivalent amounts, but in contrast to glial cells, it was only a minor component of the sulfated mucopolysaccharides produced. Cultured glial cells should provide a useful system for investigating the role of heparan sulfate in glial cell function.     

Incorporation of [35S] sulfate into chondroitin sulfates by organized cultures of murine peripheral,sympathetic and central nervous tissues

Organized cultures of mouse dorsal root ganglia (PNS), cerebellum (CNS), and sympathetic chain ganglia (ANS) were exposed to feeding media containing radioactive Na₂³⁵SO₄ for 10 day periods beginning either at the onset of myelination or during myelin maintenance. During this period, the used medium was collected at each of three feedings and frozen. Some cultures were frozen and together with the collected medium were analyzed for mucopolysaccharides (MPS). Sister cultures were fixed in ruthenium red-glutaraldehyde and processed for [³⁵S] radioautography by light microscopy, and cellular localization of MPS by electron microscopy. [³⁵S] MPS were isolated from both cultures and medium (by alkali treatment, proteolytic digestion, TCA treatment, and dialysis, followed by precipitation with cetylpyridinium chloride and ethanol). Isolated MPS were analysed by paper chromatography after digestion with chondroitinase-ABC and testicular hyarulonidase. Fifty-five to seventy-five percent of the total sulfated MPS formed in all types of cultures were chondroitin sulfates (Ch-S) A, B, and C,

1 ChS-A, ChS-B, and ChS-C are used throughout to indicate chondroitin -4- sulfate, dermatan sulfate, and chondroitin -6- sulfate, respectively

chondroitin sulfate A accounting for 50 to 60% of the total MPS. PNS and ANS exceeded CNS cultures in total sulfated MPS formed by 10:1. Qualitatively, CNS cultures produced a higher proportion of ChS-C and a lower proportion of ChS-B compared to PNS and ANS. Rutheniumred positive material covered all types of cell surfaces, collagen fibers, and the surfaces of enveloped axons; the layers of compact myelin and its underlying axon-Schwann cell interface showed no such staining, though it appeared regularly in the external mesaxon.     

Synthesis of sulfated glycoproteins by glial precursor cells

Populations of enriched glial precursor cells and astrocytes isolated from primary cultures of newborn rat brain were used to study the synthesis of sulfated glycoproteins. Both cell types incorporated [³H]glucosamine and [³⁵S]sulfate into carbohydrate side chains of proteoglycans and glycoproteins. The rate of incorporation of [³H]glucosamine into the oligosaccharides and the pattern of distribution of the label into high mannose and complex glycopeptides recovered from the glycoproteins appeared to be similar for the two glial cell types. However, clear differences were noted in the rate of oligosaccharide sulfation activities. Thus the cultures of precursor glia were about four times more active than cultures enriched in astroglia in their ability to incorporate [³⁵S]sulfate into glycoproteins.     

Synthesis and turnover of cerebroside sulfate of myelin in adult and developing rat brain

The turnover of cerebroside sulfate (sulfatide) was followed in both microsomal and myelin fractions of developing and adult rat brains after an intracerebral injection of Na(2)(35)SO(4). In the adult rats, the specific radioactivity of sulfatide of the microsomal fraction reached a maximum 12 hr after the injection, and after 3 days it was reduced to less than 30% of the maximum. In contrast, the specific radioactivity of the myelin sulfatide did not reach a peak until 3 days after the injection and remained essentially at the same level for as long as 6 months. In the case of 17-day-old rats, the specific radioactivity of myelin sulfatide reached a maximum level around 12 hr after the injection and then appeared to decline. The decline was most marked 2-6 days after the injection, suggesting an apparently rapid turnover of myelin sulfatide. When a correction was made for deposition of newly formed sulfatide, the results indicated that the turnover of myelin in the developing animals was also relatively slow. In vitro experiments with purified myelin and 3'-phosphoadenosine-5'-[(35)S]phosphosulfate showed that myelin does not catalyze the galactocerebroside sulfotransferase reaction. This enzyme was found mainly in the microsomal fraction. In vivo studies suggested that a transfer of sulfatide molecules from the endoplasmic reticulum to myelin might occur. In order to obtain direct evidence for such a transfer, rat brain slices after pulse labeling with Na(2)(35)SO(4) were washed free of the isotope and reincubated with nonlabeled Na(2)SO(4). The specific radioactivity of the microsomal sulfatide declined, with a concomitant rise in the specific radioactivity of the myelin sulfatide. These observations are therefore consistent with the postulate that myelin sulfatide is probably synthesized in the endoplasmic reticulum.     

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.     

[3 5S]Sulfate incorporation into myelin glycoproteins I. Central nervous system

The in vivo incorporation of [^{3 5}S]sulfate and [³H]fucose into rat brain myelin was investigated. Most of the ^{3 5}S in the myelin was in sulfatide, but about 4% was associated with the residual proteins after chloroform/methanol extraction. Polyacrylamide gel electrophoresis of these proteins indicated that the major ^{3 5}S-labeled component corresponded to the major fucose-labeled glycoprotein. The labeling of this predominant glycoprotein with sulfate was more selective than with fucose, since there was relatively little incorporation of sulfate into some of the minor fucose-labeled glycoproteins. There was little or no ^{3 5}S associated with proteolipid or basic protein on polyacrylamide gels. The fucose-labeled glycoproteins were converted to glycopeptides by pronase digestion and separated into two major classes by gel filtration on Sephadex-G50. Only the higher molecular weight class contained significant amounts of ^{3 5}S. The association of ^{3 5}S with the glycopeptides was not due to binding of sulfatide or free inorganic sulfate. The results indicate that the predominant myelin-associated glycoprotein in rat brain is sulfated.     

[3 5S]Sulfate incorporation into myelin glycoproteins II. Peripheral nervous tissue

The in vivo incorporation of [^{3 5}S]sulfate, [³H]fucose and [³H]leucine into sciatic nerve myelin was investigated. Polyacrylamide gel electrophoresis of the proteins indicated that the ^{3 5}S-labeling of proteins occurred almost exclusively in the major myelin protein. A smaller myelin glycoprotein migrating just ahead of the major one was labeled with [³H]fucose but did not incorporate ^{3 5}S to a detectable extent. There was little or no ^{3 5}S associated with basic proteins on polyacrylamide gels when the proteins were extracted with chloroform/methanol. Fucose-labeled myelin glycoproteins were converted to glycopeptides by pronase digestion. The glycopeptides gave a single peak on Sephadex G-50 in which the ³H and ^{3 5}S coincided. The association of ^{3 5}S with glycopeptides was not caused by binding of sulfatide or free inorganic sulfate. This study shows that the major myelin protein in the sciatic nerve of the rat is glycosylated and sulfated.     

In vivo and in vitro demonstration of reduced myelin synthesis following early postnatal exposure to ionizing radiation in the rat

Irradiation of the immature central nervous system has been demonstrated histopathologically to result in a reduction in the quantity of myelin seen at later developmental ages [S. A. Gilmore, J. Neuropathol. Exp. Neurol. 22, 294-301 (1963). J. A. Beal and J. L. Hall, J. Neuropathol. Exp. Neurol. 33, 128-143 (1974)]. The results from our investigation indicate that this reduction in myelin content can be attributed to a decrease in sulfatide synthesis. Rats received whole-brain irradiation with 0, 500, 1500, 2000, or 2500 rad at 4 days postnatal (dpn). All of the rats exposed to 2000 or 2500 rad and 70% of those exposed to 1500 rad died within 6 to 10 days. At 17 dpn, animals received single intraperitoneal injections of [35S]sodium sulfate. Myelin synthesis, as indexed by the incorporation of sulfate into total lipids and glycolipids, was reduced in a dose-related fashion. To demonstrate a direct effect of ionizing radiation on myelinogenesis, brain cell reaggregate cultures derived from fetal rats were exposed at 12 days in vitro (div) to 0, 250, 500, 1000, or 1500 rad. A dose-related reduction in [35S]sulfate incorporation through 21 div was demonstrated. Reaggregates exposed to 250 or 500 rad but not 1000 or 1500 rad resumed normal myelin synthesis by 28 div. These changes occurred in the absence of histopathological changes, changes in protein content, and changes in the rate of protein synthesis.     

The Polarity Protein Scribble Regulates Myelination and Remyelination in the Central Nervous System

The development and regeneration of myelin by oligodendrocytes, the myelin-forming cells of the central nervous system (CNS), requires profound changes in cell shape that lead to myelin sheath initiation and formation. Here, we demonstrate a requirement for the basal polarity complex protein Scribble in CNS myelination and remyelination. Scribble is expressed throughout oligodendroglial development and is up-regulated in mature oligodendrocytes where it is localised to both developing and mature CNS myelin sheaths. Knockdown of Scribble expression in cultured oligodendroglia results in disrupted morphology and myelination initiation. When Scribble expression is conditionally eliminated in the myelinating glia of transgenic mice, myelin initiation in CNS is disrupted, both during development and following focal demyelination, and longitudinal extension of the myelin sheath is disrupted. At later stages of myelination, Scribble acts to negatively regulate myelin thickness whilst suppressing the extracellular signal-related kinase (ERK)/mitogen-activated protein kinase (MAP) kinase pathway, and localises to non-compact myelin flanking the node of Ranvier where it is required for paranodal axo-glial adhesion. These findings demonstrate an essential role for the evolutionarily-conserved regulators of intracellular polarity in myelination and remyelination.     

Antibodies that recognize astrotactin block granule neuron binding to astroglia

To provide a rapid, specific assay for receptor systems involved in the binding of cerebellar granule neurons to astroglia, granule cells, purified from early postnatal mice, or from E15-E16 chicks, were radiolabeled with [35S]methionine and plasma membranes were prepared. The kinetics of binding of radiolabeled material to primary mouse or chick glia or to the mouse G26-24 astrocytoma cell line was measured in the presence or absence of antibodies against astrotactin, neural cell adhesion molecules, cadherins, or integrins. Addition of Fab fragments of astrotactin antibodies reduced the amount of granule cell membrane binding to astroglia by 70%. In contrast, Fab fragments of antibodies against the neural adhesion molecules N-CAM, L1, and N-cadherin and against integrin did not reduce the level of granule cell membrane binding to astroglia. Combinations of antibodies against N-CAM, L1, N-cadherin, and integrin also did not impair neuron binding to glia.     

Sulfated glycosaminoglycans (GAG) in the developing mouse brain : Quantitative aspects on the metabolism of total and individual sulfated GAG in vivo

Sulfation and desulfation of total glycosaminoglycans (GAG) as well as of chondroitin sulfates (A + C), dermatan sulfate, and heparan sulfate were quantified in the developing cerebrum and cerebellum of mice by labeling with [35S]sulfate combined with chases started 24 hr after [35S]sulfate injection. In both the developing cerebrum and cerebellum, the rate of biosynthesis of total sulfated GAG was highest shortly after birth (2 days), decreased sharply thereafter, and reached a plateau after 14 days. The biosynthetic activities of chondroitin sulfates and heparan sulfate decreased sharply up to 14 days and retained constant levels afterward. By contrast, the rates of biosynthesis of dermatan sulfate increased up to 14 days. The biodegradation rates of total sulfated GAG as well as of chondroitin sulfates, heparan sulfate, and dermatan sulfate were strongly correlated with the corresponding rates of biosynthesis during the first 2 postnatal weeks. Total and individual sulfated GAG showed high degradation rates resulting in half-life times of a few hours up to 1 1/2 days. Thus sulfated GAG are synthesized in excess and the actual net content seems to be co-regulated to a high degree by lysosomal degradation. In both brain parts, a proportional increase of the sulfated GAG content vs the total GAG content from 40% at birth to 90% at 28 days was observed. Since during development heparan sulfate and dermatan sulfate manifested a relative increase in their daily net synthesis besides a decrease of chondroitin sulfates, a developmental increase of the sulfate groups linked to GAG is evidenced. This molecular differentiation resulting in microenvironmental changes may be of high functional significance.     

Reduction by lead of hydrocortisone-induced glycerol phosphate dehydrogenase activity in cultured rat oligodendroglia

Summary Time- and dose-dependent toxic effects of lead (Pb) acetate on astroglia, oligodendroglia, and meningeal fibroblasts cultured from immature rat brain were measured. Cultures were exposed for 3 d to Pb (1,10, and 100 μM) and then examined immediately (Day 0) or 3 or 10 d after Pb treatment was discontinued. The percentages of astroglia and fibroblasts excluding dye were unaffected by Pb, whereas the percentage of oligodendroglia excluding dye decrease significantly (P<0.01) at all time points after exposure to 100 μM Pb. Lead (100 μM) also reduced the total cell numbers of astroglia, oligodendroglia, and meningeal fibroblasts. Amino acid incorporation into protein by oligodendroglia was stimulated after exposure to 100 μM Pb at all time points and also by 1 and 10 μM on Day 3. Incorporation was stimulated in astroglia only on Day 0 by 10 and 100 μM. Hydrocortisone-stimulated glycerolphosphate dehydrogenase (GPDH) activity was assayed in oligodendroglia cultures. A significant decrease in specific activity was seen after a 4-d exposure to lead. Because oligodendroglia are responsible for myelin synthesis in the central nervous system, and GPDH may synthesize a precursor for myelin lipid synthesis, it was proposed that the hypomyelination observed in lead-intoxicated neonatal rats may result partially from a primary toxic effect on oligodendroglia. GPDH activity was not inhibited by Pb in mixed glial cultures containing both astroglia and oligodendroglia. This result suggests that astroglia in culture have the ability to delay the lead-induced inhibition of oligodendroglial GPDH activity and supports the hypothesis that astroglia in culture serve a protective function. This work was supported by Environmental Protection Agency Grant R811500 and by U. S. Department of Agriculture Project M-6839 Animal Health Formula Funding Project 6652. This work was carried out by J.-N. Wu in partial fulfillment of the requirements for a Master of Science degree in Veterinary Public Health at Texas A&M University.     

Transport of sulfatides towards myelin. Effect of colchicine,monensin and calcium on their intracellular translocation

The in vitro effect of colchicine or monensin upon sulfatide delivery from microsomes and perikarya of oligodendroglial cells and its further incorporation into myelin was studied using brain slices obtained from 18-day-old rats incubated during 20 min with [³⁵S]sulfuric acid and reincubated for different times with unlabeled precursor. Labeled sulfatides were measured in a total homogenate, myelin, microsomes and perikarya of oligodendroglial cells. Neither colchicine nor monensin depressed the incorporation of [³⁵S]sulfate into sulfatides of the total homogenate. However, these drugs inhibited by 50% the incorporation of labeled sulfatides into myelin. Furthermore, while the specific radioactivity present in microsomes and perikarya of oligodendroglial cells isolated from controls at 120 min decreased to about 40% of the value at 20 min, no decrease was observed in fractions obtained from slices incubated with colchicine or monensin. Similar results were obtained when the slices were incubated in “Ca²⁺ free” medium. The perturbed delivery of [³⁵S]sulfatides from microsomes and perikarya of oligodendroglial cells and the diminished incorporation into myelin, in the presence of monensin and colchicine, are consistent with a possible involvement of the Golgi complex and of the cytoplasmic microtubules in the transport of sulfatides towards myelin. Moreover, the transport of these galactolipids appears to require calcium.     

S100 immunoreactive glial cells in the forebrain and midbrain of the lizard Gallotia galloti during ontogeny

We identified S100 immunoreactive cells in the brain of the lizard Gallotia galloti during ontogeny using immunohistochemical techniques for light and electron microscopy. In double labeling experiments with antibodies specific for S100A1 and S100B (anti-S100) and proliferative cell nuclear antigen (anti-PCNA), myelin basic protein (anti-MBP), phosphorylated neurofilaments (SMI-31), glial fibrillary acidic protein (anti-GFAP), or glutamine synthetase (anti-GS), we detected S100-like immunoreactivity in glial cells but never in neurons. Restricted areas of the ventricular zone were stained in the hypothalamus from E32 to postnatal stages, and in the telencephalon at E35, E36, and in adults. S100 immunoreactivity was observed predominantly in scattered PCNA-negative cells that increased in number from E35 to the adult stage in the myelinated tracts of the brain and had the appearance of oligodendrocytes. Quantitative analysis revealed that all of the S100-positive glial cells were GFAP-negative, whereas most of the S100-positive glial cells were GS-positive. Ultrastructurally, most of these S100-positive/GS-positive glial cells resembled oligodendrocytes of light and medium electron density. In adult lizards, a small subpopulation of astrocyte-like cells was also stained in the pretectum. We conclude that in the lizard S100 can be considered a marker of a subpopulation of oligodendrocytes rather than of astrocytes, as is the case in mammals. The S100-positive subpopulation of oligodendrocytes in the lizard could represent cells actively involved in the process of myelination during development and in the maintenance of myelin sheaths in the adult.     

Specific inhibition of binding of antistasin and [A103,106,108] antistasin 93-119 to sulfatide (Gal(3-SO4)beta 1-1Cer) by glycosaminoglycans.

Leech-derived antistasin is a potent anticoagulant and antimetastatic protein that binds sulfatide (Gal(3-SO4)beta 1-1Cer) and sulfated polysaccharides. In this study, the synthetic fragment [A103,106,108] antistasin 93-119, which corresponds to the carboxyl terminus, showed specific and saturable binding to sulfatide. Binding was competitively blocked by glycosaminoglycans (GAGs) in the order: dextran sulfate 5000 congruent to dextran sulfate 500,000 greater than heparin greater than dermatan sulfate much greater than chondroitin sulfates A and C. This rank order of inhibitory potency was identical to that observed with whole antistasin. We suggest that residues 93-119 of antistasin represent a critical domain for binding GAGs and sulfated glycolipids.     

Participation of galactosylceramide and sulfatide in glycosynapses between oligodendrocyte or myelin membranes

The two major glycosphingolipids of myelin, galactosylceramide (GalC) and sulfatide (SGC), interact with each other by trans carbohydrate-carbohydrate interactions. They face each other in the apposed extracellular surfaces of the multilayered myelin sheath produced by oligodendrocytes (OLs). Multivalent galactose and sulfated galactose, in the form of GalC/SGC-containing liposomes or silica nanoparticles conjugated to galactose and galactose-3-sulfate, interact with GalC and SGC in the membrane sheets of OLs in culture. This stimulus results in transmembrane signaling, loss of the cytoskeleton and clustering of membrane domains, suggesting that GalC and SGC could participate in glycosynapses between apposed OL membranes or extracellular surfaces of mature myelin. Such glycosynapses may be important for myelination and/or myelin function.     

Synthesis of Sulfatide by Cultured Rat Schwann Cells

Abstract: The ³⁵S sulfolipids synthesized by purified cultures of rat Schwann cells, fibroblasts, and a rat cell line (RN2) were studied. Schwann cell ³⁵S sulfolipids were almost entirely [³⁵S]sulfatide, as shown by TLC in two different solvent systems with unlabeled authentic sulfatide run in the same track. RN2 and fibroblasts did not synthesize significant amounts of sulfatide, by the same criteria. Previous studies failed to detect any characteristic myelin components, including sulfatide, on Schwann cells after several days in culture (Brockes et al., 1980a; Mirsky et at., 1980). My results show that Schwann cells continue to synthesize some sulfatide in the absence of neurons.     

Biosynthesis of Neutral Glucocerebroside Homologues in the Absence of Myelin Assembly After Nerve Transection

The biosynthesis of myelin-associated glycolipids during various stages of myelination was studied by in vitro incorporation of [3H]Gal, [3H]Glc, or [35S]sulfate into the endoneurium of rat sciatic nerve. In the normal adult nerve, where the level of myelin assembly is substantially reduced and Schwann cells are principally involved in maintaining the existing myelin membrane, [3H]Gal was primarily incorporated into monogalactosyl diacylglycerol (MGDG) and the galactocerebrosides (GalCe) with lower levels of incorporation into the sulfatides. Such incorporation was enhanced 35 days after crush injury of the adult rat sciatic nerve, which is characterized by active myelin assembly. In contrast, at 35 days after permanent nerve transection where there is no axonal regeneration or myelin assembly, the incorporation of [3H]Gal or [3H]Glc into GalCe was nearly undetected whereas the incorporation of [3H]Gal into MGDG was completely inhibited. Instead, the 3H-labeled glycolipids in transected nerve were identified as the glucocerebrosides (GlcCe) and oligohexosylceramide derivatives with tetrahexosylceramide being a major product. In contrast, [35S]sulfate was incorporated into endoneurial sulfatides in the transected nerve, which suggests that endogenous GalCe rather than newly synthesized GalCe served as the substrate for the sulfotransferase reaction. The GlcCe homologues are not considered as constituents of the myelin membrane but are likely plasma membrane components synthesized in the absence of myelin assembly. It is likely that the cells responsible for GlcCe biosynthesis are Schwann cells, since they comprise 90% of the total endoneurial cell area in the distal nerve segment at 35 days after transection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Formation of lysosulfatide, 3',6'-anhydropsychosine, ceramide, and sphingosine by saponification of cerebroside sulfate. Effect of the sulfate group on the hydrolysis.

Saponification of cerebroside sulfate (sulfatide) by refluxing with 1 N KOH in 90% n-butanol for 1 h yielded ceramide, sphingosine, lysosulfatide (psychosine-3'-sulfate ester) and a hitherto unknown compound. The latter compound was identified as 3,6-anhydrogalactosyl sphingosine (3',6'-anhydropsychosine) from its mass spectrum. The structure of lysosulfatide was confirmed by reacylating it to sulfatide by condensing it with lignoceroyl chloride. The resulting sulfatide, which was chromatographically identical to control sulfatides, was not oxidized by periodate. The sulfatide was also permethylated and methanolyzed. The sugar moiety obtained was identified as methyl 2,4,6-tri-O-methylgalactoside by gas-liquid chromatography and thin-layer chromatography. The presence of the sulfate group in lysosulfatide was further confirmed by IR spectroscopy and the presence of radioactivity when it was prepared from [35S]sulfatide. The effect of the sulfate group on cleavage of the galactoside linkage and on the formation of the 3,6-anhydro derivative is discussed.