CEREBRAL LIPIDS IN A CASE OF SYSTEMIC GM2-GANGLIOSIDOSIS OF A LATE INFANTILE TYPE1

—Quantitative analyses performed on the lipids of cerebral grey matter from brains of a normal child and a child with Tay-Sachs (T-S) disease were compared with such analyses on the brain of a 6-year-old, non-Jewish male with systemic G_M2-gangliosidosis of a late infantile type (G_M2-LI). Analysis of gangliosides showed a 3·5-fold increase of total gangliosides in the G_M2-LI brain and a six-fold increase in the T-S brain, compared to normal brain. Both pathological brains had similar distribution patterns for gangliosides, with the G_M2-ganglioside component constituting more than 80 per cent of the total. Lipid components in the T-S brain were below normal values except for lecithin and cholesterol, while in the G_M2-LI brain there were increases in sulphatides, cerebrosides, sphingomyelin and cholesterol. Approximately twice as much ceramide trihexoside was present in the T-S brain as in the G_M2-LI brain, and none could be detected in the normal brain. The clinical, pathological and biochemical data support the conclusion that this case represents a new variant of systemic late-infantile gangliosidosis in which there is an accumulation of the G_M2-ganglioside like that in Tay-Sachs disease.     

Thermotropic behavior of binary mixtures of diplamitoylphosphatidylcholine and glycosphingolipids in aqueous dispersions

The thermotropic behavior of mixtures of dipalmitoylphosphatidylcholine (DPPC) with natural glycosphingolipids (galactosylceramide, phrenosine, kerasine, glucosylceramide, lactosylceramide, asialo-G_M1, sulfatide, G_M3, G_M1, G_D1a, G_T1b) in dilute aqueous dispersions were studied by high sensitivity differential scanning calorimetry over the entire composition range. The pretransition of DPPC is abolished and the cooperativity of the main transition decreases sharply at mole fractions of glycosphingolipids below 0.2. All systems exhibit non-ideal temperature-composition phase diagrams. The mono- and di-hexosylceramides are easily miscible with DPPC when the proportion of glycosphingolipids in the system is high. A limited quantity (1–6 molecules of DPPC per molecule of glycosphingolipid (GSL) can be incorporated into a homogeneously mixed lipid phase. Domains of DPPC, immiscible with the rest of a mixed GSL-DPPC phase that shows no cooperative phase transition, are established as DPPC exceeds a certain proportion in the system. One negative charge (sulfatide) or four neutral carbohydrate residues (asialo-G_M1) in the oligosaccharide chain of the glycosphingolipids results in phase diagrams exhibiting coexistence of gel and liquid phases over a broad temperature-composition range. Systems containing gangliosides show complex phase diagrams, with more than one phase transition. However, no evidence for phase-separated domains of pure ganglioside species is found. The thermotropic behavior of systems containing DPPC and glycosphingolipids correlates well with their interactions in mixed monolayers at the air/water interface.     

THE DISTRIBUTION OF LIPIDS IN THE HUMAN NERVOUS SYSTEM-V. GANGLIOSIDES AND ALLIED NEUTRAL GLYCOLIPIDS OF INFANT BRAIN

—Gangliosides and allied neutral glycosylceramides were isolated from human infant (2-24 months of age) cerebral cortex and white matter. The individual glycolipids were separated quantitatively by a combination of column and thin-layer chromatographic methods on silica gel, DEAE-cellulose and Sephadex G-25. In cerebral cortex G_D1a and G_M1 were the major fractions and constituted more than 70 per cent of the total gangliosides. The concentrations of neutral glycolipids, except for galactosylceramides, were very low: lactosylceramide and glucosylceramide comprised 30 and 5 nmol/g wet weight, respectively. In white matter their concentrations were 10 times higher. The ganglioside concentration was only 50 per cent of that in cerebral cortex: the difference was accounted for mainly by the much lower content of the major di- and trisialogangliosides. Stearic acid was the predominant fatty acid of all brain gangliosides. G_M3, and G_D3 had a considerable content of the very long-chain fatty acids, C₂₂-C₂₄, particularly in the white matter. Glucosylceramide and lactosylceramide had almost identical fatty acid patterns between each other in cerebral cortex and white matter. In the cerebral cortex stearic acid and in the white matter the very long-chain acids predominated. d20:1 Sphingosine comprised more than 20 per cent of total sphingosine in all the gangliosides of the G_l- and G₂-series. G_M3, and G_D3 like lactosylceramide contained significantly less of d20:1 sphingosine. The findings suggest the existence of separate compartments for the biosynthesis of the gangliosides. Glucosylceramides and lactosylceramides of white matter have the same ceramide composition as the galactosylceramides with normal fatty acids and are thus unlikely to be intermediates in the metabolism of the major brain gangliosides which have a completely different fatty acid composition.     

GD3 ganglioside-enriched extracellular vesicles stimulate melanocyte migration

Melanomas often accumulate gangliosides, sialic acid-containing glycosphingolipids found in the outer leaflet of plasma membranes, as disialoganglioside G_D3 and its derivatives. Here, we have transfected the G_D3 synthase gene (ST8Sia I) in a normal melanocyte cell line in order to evaluate changes in the biological behavior of non-transformed cells. G_D3-synthase expressing cells converted G_M3 into G_D3 and accumulated both G_D3 and its acetylated form, 9-O-acetyl-G_D3. Melanocytes were rendered more migratory on laminin-1 surfaces. Cell migration studies using the different transfectants, either treated or not with the glucosylceramide synthase inhibitor d-1-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (PPPP), allowed us to show that while G_M3 is a negative regulator of melanocyte migration, G_D3 increases it. We showed that gangliosides were shed to the matrix by migrating cells and that G_D3 synthase transfected cells shed extracellular vesicles (EVs) enriched in G_D3. EVs enriched in G_D3 stimulated cell migration of G_D3 negative cells, as observed in time lapse microscopy studies. Otherwise, EVs shed by G_M3^+veG_D3^−ve cells impaired migration and diminished cell velocity in cells overexpressing G_D3. The balance of antimigratory G_M3 and promigratory G_D3 gangliosides in melanocytes could be altered not only by the overexpression of enzymes such as ST8Sia I, but also by the horizontal transfer of ganglioside enriched extracellular vesicles. This study highlights that extracellular vesicles transfer biological information also through their membrane components, which include a variety of glycosphingolipids remodeled in disease states such as cancer.     

The fate of glucosylceramide (glucocerebroside) in genetically impaired (lysosomal beta-glucosidase deficient) Gaucher disease diploid human fibroblasts

Diploid human infant skin fibroblasts cultured from normal infants and Gaucher disease infants, with genetically defective lysosomal glucosylceramide:beta-glucohydrolase activity, had a full range of homologous glycosphingolipids from the simplest (glucosylceramide) to higher neutral derivatives (lactosyl-, trihexosyl- and tetrahexosylceramide) and anionic sialo derivatives (gangliosides) (sialosyllactosyl-, disialosyllactosyl-, sialosylgangliotriaosyl-, and mono- and disialosylgangliotetraosylceramide). Although excessive storage of glucosylceramide in histiocytes is pathognomonic for Gaucher disease, we found that Gaucher disease fibroblasts contained 1.23 +/- 0.08 nmol of glucosylceramide/mg cell protein; normal infant cells, 1.11 +/- 0.48. When we aged infantile Gaucher disease fibroblasts for 20 days beyond their confluency state, we found no increased accumulation of glucosylceramide, but a 1.5-2-fold increase in trihexosylceramide, sialosylgangliotetraosylceramide, and disialosyllactosylceramide. Gaucher disease fibroblasts took up and could not degrade but, instead, effectively converted pulse-chase 3-O-[3H]glucosylceramide supplied in the growth medium in liposomes into higher glycosphingolipids, especially the plasma membrane ganglioside, sialosyllactosylceramide. When grown with extracellular particulate [3H]glucosylceramide, infantile Gaucher fibroblasts localized it and higher labeled homologues in the plasma membrane; glucosylceramide did not accumulate in the lysosomes. These findings indicate that fibroblasts that are genetically deficient in lysosomal glucosylceramide:beta-glucosidase avoid pathological lysosomal accumulation by relegating undegradable glucosylceramide to an anabolic compartment where glucosylceramide is converted into more highly glycosylated glycosphingolipids.     

Inhibition of sialyltransferase activity in cultured cells by a natural inhibitor from rat brain

Summary Brain from mature rats has been shown previously to contain a natural inhibitor of rat brain sialyltransferase I (CMP-sialic acid: lactosylceramide sialyltransferase activity). This same inhibitor preparation was effective against sialyltransferase I and a second sialyltransferase activity from different lines of cultured cells. Cardiolipin which stimulates sialyltransferase I activity in cultured cells apparently was not required for inhibition. Inhibition was specific for sialyltransferase activities while a third ganglioside biosynthetic enzyme, UDP-gal: glucosyl-ceramide galactosyltransferase activity, was not inhibited. Inhibition of sialyltransferase I was linear with time, heat-resistant, and increased with inhibitor concentration.Gangliosides are sialic-acid containing glycosphingolipids found in brain as well as extraneural tissues and cultured cells t,2. Although the functions of gangliosides are unknown, they appear to play a role in morphological differentiation³ sensory and visual stimulation 4 and as receptor for cholera toxin^5–8 and possibly thyroid stimulating hormone⁹. The monosaccharide units are added to the elongating oligosaccharide chain of the gangliosides in step-wise fashion and different glycosyltransferases catalyze each addition^10,11. The activities of these enzymes have been observed to change during development^12,14 malignant transformation¹³ and morphological differentiation³.Although little is known about the regulation of ganglioside synthesis, a natural inhibitor of CMP-AcNeu: GL-2 sialyltransferase (sialyltransferase I) from rat brain has been described^14,15. As the inhibitor activity increased with the age of the animal, the same authors suggested that it may regulate the biosynthetic pathway of gangliosides. In this paper, the effects of the rat brain inhibitor on the activities of ganglioside biosynthetic enzymes from several cultured cell lines are described.Abbreviations AcNeu N-acetylneuraminic acid - GL1 glucosylceramide - GL-2 lactosylceramide - G_M3 Nacetylneuraminylgalactosylglucosylceramide - G_M1 gal actosyl-N-acetylgalactosaminyl-(N-acetylneuraminylgalactosylglucosylceramide - G_D1a Nacetylneuraminylgalactosyl-N-acetylgalactosaminyl-(Nacetylneuraminyl)-galactosylglucosylceramide. DR. DUFFARD was a recipient of a Fogarty Center Fellowship.     

Glycosphingolipid patterns of fetal and adult small intestinal mucosa in the sheep

The ganglioside and neutral glycosphingolipid composition of fetal and adult sheep small intestinal mucosa were characterized and compared. Mono- and tetrahexosylceramide were the major neutral glycolipids of both fetal and adult tissue. Fetal mucosa, however, possessed a higher content of monohexosylceramide than its adult counterpart. Similarly, G_D1a, G_M3 and G_M2 were found to be the principal gangliosides in fetal and adult tissue. Adult intestinal mucosa possessed significant amounts of G_T1a not present in fetal tissue. Analysis of the hydroxy and nonhydroxy fatty acids as well as the long-chain bases of the major glycosphingolipids revealed differences between these lipophilic components of glycolipids in fetal and adult intestinal mucosa. The results, therefore, indicate that both quantitative and qualitative differences in glycosphingolipid composition exist between fetal and adult sheep small intestinal mucosa.     

Immunochemical studies of isolated human brain ganglioside components

Abstract— Gangliosides G₁ to G₅ were isolated from human brain by means of TLC and tested with respect to their specificity to antisera against normal brain and Tay-Sachs brain gangliosides by agar double diffusion analysis. Gangliosides G₂ and G₄ gave precipitation reactions with antisera to normal human gangliosides (NHG) while only ganglioside G₆ reacted with antisera to Tay-Sachs gangliosides (TSG). Additional specificity information was also obtained by use of the enzyme neuraminidase for the removal of specific sialic acid (NANA) residues. It was concluded from these data that the specificity of the anti-NHG antibodies is determined by the presence of a galactose (β1, 3) N-acetyl galactos-amine–while that of anti-TSG antibodies is due to a N-acetyl galactosamine (β1, 4) galactose-end sequence. By means of natural compounds of known structure it was found that both the sequence of carbohydrate residues and position of NANA residues in the molecule played a critical role in the formation of precipitation bands with NHG-antisera. This information was utilized to distinguish one isomeric form of disialoganglioside from another, i.e. G₂ from G₃ and to confirm the structure of the trisialoganglioside, G₁. The immunochemical method appears to be a useful one for elucidating structural differences in ganglioside molecules.     

Hexosaminidases and ganglioside catabolism in the GM2-gangliosidoses

The G_M2-gangliosidoses are a set of neurological diseases whose common features include the storage of the ganglioside G_M2, N-acetyl galactosaminyl (N-acetylneuraminyl-) galactosylglucosylceramide and related neutral glycosphingolipids in various organs (particularly brain) of affected individuals and the inability of such individuals' hexosaminidases to catalyze the hydrolysis of G_M2. Associated with this finding has been the demonstration of a deficiency in none, one or both major forms (A and B) of hexosaminidase which can be measured with artificial flurogenic or chromogenic substrates. Additionally, a deficiency in the A form of hexosaminidase which is usually associated with Tay-Sachs disease has been demonstrated in certain clinically normal adults.Recent advances in the purification of the two forms of hexosaminidase have allowed their catalytic, immunological, physical and genetic characteristics to be examined in great detail. This examination has resulted in the proposal of several models for the relationship of the hexosaminidases and their involvment in the G_M2-gangliosidoses. I discuss the evidence for these models and the implications which can be drawn from them in this review.     

High-performance thin-layer chromatography and densitometric determination of brain ganglioside compositions of several species.

Improved resolution of complex brain ganglioside mixtures was achieved by high-performance thin-layer chromatography. The percentage distribution of individual gangliosides was then determined by direct densitometric seanning, employing a transmittance mode, of the resorcinol-positive spots on the plate. As little as 90 pmol (29 ng) of lipid-bound sialic acid could be detected with a good signal-to-noise ratio. A linear detector response was observed up to 3.0 μg of lipid-bound sialic acid. The brain white matter ganglioside patterns of eight animal species, including human, chimpanzee, monkey, chicken, bovine, sheep, and pig, were examined in detail. In addition, human brain gray matter, rat cerebral, rat brain gray matter, and rat cerebellar ganglioside patterns were also studied. Ganglioside G_M4 (G₇) was found to be one of the major components in primate and chicken brain white matter, but it represented only a minor ganglioside in other species. Other major gangliosides in all brain samples studied were G_M1, G_D1a, G_D1b, and G_T1b. G_M1 was more abundant in white matter than in gray matter. G_T1a, a recently discovered ganglioside species, was found in all species examined, but was most abundant in the rat cerebellum. The latter source also contained high proportions of G_T1b and G_Q1b.     

Effects of Divalent Cations on the Glycolipids from Cultured Mouse Neuroblastoma Cells

Abstract: The influence of divalent cations on glycosphingolipid metabolism was examined in the NB41A mouse neuroblastoma clonal cell line. HPLC methods were utilized to quantitate the effects on neutral glycolipids and monosialogangliosides. NB41A cells were shown to contain G_M3, G_M2, G_M1, G_D3, and G_D1a by HPLC and TLC. The neutral glycosphingolipids consisted of glucosylceramide (GlcCer), lactosylceramide (LacCer), GaINAc(β1→4) Gal(β1→4)Glc(β1→1)Cer (GgOse₃Cer), and GaINAc(β1→3)Gal(α1→4) Gal-(β1→4)Glc(β1→1)Cer (GbOse₃Cer) according to their HPLC behavior. Cells grown in the presence of 1.85 mm -EGTA showed a two- to threefold increase in G_M3 whereas other glycosphingolipids were only slightly affected. When cells were grown in the presence of 1.45 mm -EGTA plus 0.4 mm -EDTA a similar increase in G_M3 was observed but this change was now accompanied by decreases in G_M2, G_M1 GgOse₃Cer, and GbOse₄Cer. The EGTA-EDTA effects were reversed when growth was in the presence of Ca²⁺ sufficient to bind all chelator. Mn²⁺ replacement reversed the chelator effects differentially; G_M2 and G_M1 levels were the most sensitive to increases in Mn²⁺ concentration; GgOse₃Cer and GbOse₄Cer were also sensitive, whereas G_M3 was the least affected. These results suggest calcium serves an important regulatory role on G_M3 levels and that manganese concentration may regulate the levels of galactosamine-containing glycolipids in mouse NB41A neuroblastoma cells.     

The characterization of sphingolipids from neurons and astroglia of immature rat brain

Abstract— Isolated neuronal cell bodies and astroglia of young (15–20-day-old) rat brains were both found to contain small concentrations of a variety of glycosphingolipids, including glucosylceramide, galactosylceramide, sulphatide, dihexosylceramide and gangliosides. These sphingolipids, plus sphingomyelin, were isolated, quantitated and their fatty acid and long chain base patterns determined. These data were compared to similar data obtained on these lipids isolated from whole brain and myelin of rats of the same age range. Glucosylceramide was found in an amount equal to galactosylceramide in neurons, and accounted for 35 per cent of the total monohexosylceramide in astroglia. Dihexosylceramide was present in nearly the same amount as sulphatide in both cell types. The sphingolipids of each cell type had characteristic fatty acid patterns. Generally the whole brain fatty acid patterns resembled those of astroglial lipids rather than neuronal lipids. In no case did the cell sphingolipid fatty acids resemble those of myelin. However, the galactosylceramide and sulphatides of both cells had unsubstituted and α-hydroxy acids, both of which had appreciable quantities of C₂₄ acids. The ganglioside fatty acids of each cell type were similar and not unusual, but were quite different from those of glucosylceramide and dihexosylceramide; the latter having appreciable quantities of 16:0 and acids longer than 18:0. The ganglioside patterns of these cells were similar and only slightly different from that of whole brain. Long chain bases of sphingolipids were mainly C₁₈-sphingosine in both cell types, and those of ganglioside and sphingomyelin contained small amounts of C₂₀-sphingosine.     

Changes in Rabbit Brain Cytosolic and Membrane-Bound Gangliosides During Prenatal Life

The present study deals with the developmental profile of cytosolic and membrane-bound gangliosides in rabbit whole brain from the 21st day of pregnancy, the time at which brain could be macroscopically recognized and handled, till birth. In this period of prenatal life the content of membrane-bound gangliosides showed a 2.5-fold increase, referred to fresh and dry brain weight and to membrane-bound protein; the content of cytosolic gangliosides reached a maximum at 21-22 days of pregnancy, and then underwent to birth a threefold diminution. The qualitative pattern of membrane-bound gangliosides, in the same period of life, was characterized by an increase of G_D1a and G_M1 (more marked for G_D1a), a decrease of G_T1a, G_T1b and G_Q1b and a constant level of G_D3 and G_D1b. At 21 days of pregnancy the most abundant gangliosides were G_T1b, and G_Q1b, followed by G_D1a and G_D1b; at birth it was G_D1a followed by G_T1b G_D1b, and G_M1 The qualitative pattern of cytosolic gangliosides closely resembled, during the entire period of prenatal life examined, that of membrane-bound gangliosides.     

BIOSYNTHESIS OF LACTOSYLCERAMIDE BY RAT BRAIN PREPARATIONS AND COMPARISON WITH THE FORMATION OF GANGLIOSIDE GM1 AND PSYCHOSINE DURING DEVELOPMENT

—This report deals with some properties of the enzyme from rat brain, which catalyses in vitro the formation of lactosylceramide, a probable precursor of the gangliosides, from UDP-galactose and glucosylceramide. The enzyme is present in microsomes, mitochondria and synaptosomes, the latter having the highest specific activity, and appears to be firmly bound to the membranes of these particles. The enzymic activity is optimal at pH 6·8 and requires the presence of Mn²⁺. Lactosylceramide, the product of the reaction, was tentatively identified by its elution pattern from a Florisil column, its resistance to mild alkaline hydrolysis, thin-layer co-chromatography with authentic standards in six solvent systems, and location of the radioactivity in the galactose portion of the product obtained after incubation of labelled UDP-galactose with unlabelled glucosylceramide and in the glucose portion after incubation of glucose-labelled glucosylceramide with unlabelled UDP-galactose. The activity of this enzyme, per unit of brain weight, was found to be highest at birth and to decrease gradually thereafter. A similar age distribution was observed for another galactosyltransferase, the one which catalyses the formation of ganglioside G_M1 from Tay-Sachs’ganglioside. In contrast, the activity of a third galactosyltransferase, which catalyses the formation of psychosine, and thus possibly is related to the increase of cerebrosides during myelination, is negligible during the first week and maximal about 20 days after birth.     

Role of membrane gangliosides in the binding and action of bacterial toxins

Summary Gangliosides are complex glycosphingolipids that contain from one to several residues of sialic acid. They are present in the plasma membrane of vertebrate cells with their oligosaccharide chains exposed to the external environment. They have been implicated as cell surface receptors and several bacterial toxins have been shown to interact with them. Cholera toxin, which mediates its effects on cells by activating adenylate cyclase, bind with high affinity and specificity to ganglioside G_M1. Toxin-resistant cells which lack G_M1 can be sensitized to cholera toxin by treating them with G_M1. Cholera toxin specifically protects G_M1 from cell surface labeling procedures and only G_M1 is recovered when toxin-receptor complexes are isolated by immunoadsorption. These results clearly demonstrate that G_M1 is the specific and only receptor for cholera toxin. Although cholera toxin binds to G_M1 on the external side of the plasma membrane, it activates adenylate cyclase on the cytoplasmic side of the membrane by ADP-ribosylation of the regulatory component of the cyclase. G_M1 in addition to functioning as a binding site for the toxin appears to facilitate its transmembrane movement. The heat-labile enterotoxin ofE. coli is very similar to cholera toxin in both form and function and can also use G_M1 as a cell surface receptor. The potent neurotoxin, tetanus toxin, has a high affinity for gangliosides G_D1b and G_T1b and binds to neurons which contain these gangliosides. It is not yet clear whether these gangliosides are the physiological receptors for tetanus toxin. By applying the techniques that established G_M1 as the receptor for cholera toxin, the role of gangliosides as receptors for tetanus toxin as well as physiological effectors may be elucidated.     

Liquid chromatography/electrospray ionisation–tandem mass spectrometry quantification of GM2 gangliosides in human peripheral cells and plasma

G_M2 gangliosidosis is a group of inherited neurodegenerative disorders resulting primarily from the excessive accumulation of G_M2 gangliosides (G_M2) in neuronal cells. As biomarkers for categorising patients and monitoring the effectiveness of developing therapies are lacking for this group of disorders, we sought to develop methodology to quantify G_M2 levels in more readily attainable patient samples such as plasma, leukocytes, and cultured skin fibroblasts. Following organic extraction, gangliosides were partitioned into the aqueous phase and isolated using C18 solid-phase extraction columns. Relative quantification of three species of G_M2 was achieved using LC/ESI–MS/MS with d₃₅G_M1 18:1/18:0 as an internal standard. The assay was linear over the biological range, and all G_M2 gangliosidosis patients were demarcated from controls by elevated G_M2 in cultured skin fibroblast extracts. However, in leukocytes only some molecular species could be used for differentiation and in plasma only one was informative. A reduction in G_M2 was easily detected in patient skin fibroblasts after a short treatment with media from normal cells enriched in secreted β-hexosaminidase. This method may show promise for measuring the effectiveness of experimental therapies for G_M2 gangliosidosis by allowing quantification of a reduction in the primary storage burden.     

Preparation of some labeled glycosphingolipids by catalytic addition of tritium

Three experiments are described in which seven tritium-labeled glycosphingolipids were prepared by catalytic addition of tritium gas to unsaturated centers. These compounds include glucosyl ceramide, galactosyl ceramide, lactosyl ceramide (cytolipin H), N-acetylgalactosaminyl (β1 → 3) galactosyl (α1 → 3) galactosyl (β1 → 4) glucosyl ceramide (cytolipin R), and three species of gangliosides: G_M1 (G₄), G_M3 (G₆), and G_D1a (G₃).     

Accumulation of Glucosylceramide and Glucosylsphingosine (Psychosine) in Cerebrum and Cerebellum in Infantile and Juvenile Gaucher Disease

Abstract: Three major clinical variants of Gaucher disease have been defined: Type I, chronic nonneuronopathic; Type II, acute neuronopathic; and Type III, subacute neuronopathic. In a search for the underlying molecular basis of the neurological manifestations, the concentration and composition of cholesterol, phospholipids, neutral glycosphingolipids, and gangliosides were examined in cerebral and cerebellar cortices of five cases of Type II, eight cases of Type III, and one case of presumed Type I/III. In Type II the concentration of glucosylceramide was 140-530 μmol/kg in cerebral cortex and 51-450 μmol/kg in cerebellar cortex, the highest values found in the most fulminant cases. These concentrations were 20-80 times greater than normal in cerebral cortex and 5-40 times normal in cerebellar cortex. In Type III the concentration of glucosylceramide was 37-65 and 59-1750 μmol/kg in cerebral and cerebellar cortex, respectively. The highest concentrations were found in the cerebellum of patients who had survived splenectomy for several years. The ceramide composition of the accumulated glucosylceramide suggested that brain gangliosides were the major precursors of the glucosylceramide in brains of Type II but in cerebellar cortex in Type III was partly of extracerebral origin. The levels of lactosylceramide and oligohexaosylceramides were slightly raised in all brain specimens from the Gaucher cases. The ganglioside concentration was normal, whereas there was a certain increase in the proportion of GM2 and GM3 gangliosides. The brain glycosphingolipid changes in the Type I/III case were similar but slightly less than those in Type III cases of corresponding age. Glucosylsphingosine (psychosine), never detected in normal human brain, was demonstrated in brains from all the Gaucher cases. The psychosine concentration was highest in Type II cases, 3.8-8.8 and 3.9-12.3 μmol/kg in cerebral and cerebellar cortex, respectively, with the highest values found in the most fulminant cases. In Type III the psychosine concentration varied more widely, 0.8-4.6 and 1.4-6.3 μmol/kg in cerebral and cerebellar cortex, respectively. The lowest value, 0.7 μmol/kg, was found in the Type I/III case. Our method detected psychosine down to 0.01 μmol/kg, which means that the concentration of psychosine was increased at least 100- to 1000-fold in Gaucher grey matter. We suggest that the accumulation of the cell-toxic substance psychosine is the basis for the extensive neuronal cell loss in Gaucher disease, which is most striking in Type II disease.     

Soluble gangliosides in cultured neurotumor cells

Abstract: The biosynthesis and degradation of glycosphingolipids were studied in cytosolic and membrane fractions obtained from rat glioma C6 cells. Both pools had a similar composition of neutral glycosphingolipids but the soluble pool contained only a few percent of the total. The major ganglioside in C6 cells was GM3, of which only 2% was soluble. Whereas the bulk of the membrane GM3 was accessible to surface labeling procedures, the soluble GM3 was not. Mouse neuroblastoma N18 cells also contained small amounts of cytoplasmic gangliosides corresponding to GM3, GM2, GM1, and GDla. When C6 cells were incubated with medium containing [³H]galactose at 37°C, the specific activity of soluble GM3 initially increased more rapidly than that of membrane GM3; by 4 h, the specific activities in both pools became equal. Total incorporation into the membrane pool, however, was always several-fold greater even at the shortest incubation times examined. The labeling pattern of neutral glycosphingolipids in both soluble and membrane fractions indicated the existence of a precursor-product relationship between glucosylceramide and other glycosphingolipids. When labeled cells were transferred to nonradioactive medium, glucosylceramide disappeared the most rapidly, with a 50% loss within <6 h. The turnover rates of other glycosphingolipids were much slower. Although cytosolic GM3 was degraded more rapidly (t_1/2= 26 h) than membrane-bound GM3 (t_1/2= 44 h), its turnover rate was much slower than the time required for transport of GM3 to the cell surface (20–30 min). Our results are consistent with the existence of a small intracellular pool of soluble gangliosides and neutral glycosphingolipids that is stable and independent of the main membrane-bound pool. Although the role of these cytosolic glycolipids is unknown, they do not appear to represent a transport pool between the site of synthesis and the plasma membrane.     

Shedding of Gangliosides by Human Medulloblastoma Cells

Shedding of immunosuppressive gangliosides is an important characteristic of both experimental and human tumors. Using a medulloblastoma cell line, Daoy, with a very high ganglioside expression (141 ± 13 nmol/10⁸cells) and a well-characterized ganglioside complement, we have now studied ganglioside shedding by human brain tumor cells. Shedding of gangliosides, quantified by metabolic radiolabeling, was significant (169 pmol/10⁸cells/h) and was generalized with respect to the major ganglioside carbohydrate structures (G_M2, G_M3, and G_D1a). For each ganglioside, however, shedding was selective for ceramide structures containing shorter fatty acyl chains. Rapid and ceramide-selective shedding was confirmed in two additional human medulloblastoma cell lines, D341 Med and D283 Med (112 and 59 pmol/10⁸cells/h). Significant ganglioside shedding is therefore a common characteristic of human medulloblastoma cells and may influence the biological behavior of this tumor, in view of immunosuppressive and other biological properties of shed gangliosides.