Variability in brain ganglioside content and composition of endothermic mammals, heterothermic hibernators and ectothermic fishes

Content and composition of brain gangliosides were compared among endothermic mammals, heterothermic hibernators and ectothermic fishes from habitats with extreme ambient temperatures (tropic vs. antarctic waters). In general the content of brain gangliosides in fishes is significantly lower and exhibits a greater variability than in mammals. The composition of brain gangliosides was investigated using both one- and two-dimensional High Performance Thin Layer Chromatography (HPTLC). Both techniques showed a remarkable increase in the number of individual ganglioside fractions and an additional increase of higher polar fractions in fishes as compared with mammals. The 2D-HPTLC revealed a significant decrease in the relative proportion of alkali-labile gangliosides in the course of evolution from fish to mammals. Moreover this decrease in alkali-lability is correlated with the state of thermal adaptation (antarctic fishes, 53–66%; tropical cichlid fish, 35%). These results provide additional evidence for the notion that the extremely high polarity of brain gangliosides, especially of cold-blooded vertebrates, reflects a very efficient mechanism on the molecular level to keep the neuronal membrane functional under low temperature conditions.     

Brain gangliosides and cold-adaptation in high-antarctic fish

The concentration and composition of gangliosides from the brain of eight species of Antarctic Notothenioid fishes belonging to the class of perciformes and two species of boreal fishes (tropic cichlid fish Oreochromis mossambicus; Codfish Gadus morhua) were investigated. The concentration of whole brain gangliosides in Notothenioid fishes (between 1622 and 2183 μg NeuAc/g dry wt.) was slightly lower than that in the brains of fish species, which live in warm, temperate habitats (2483 μg NeuAc/g dry wt.). The composition of brain gangliosides was completely different from that of warm adapted fish species (e.g. the tropic cichlid fish Oreochromis mossambicus). The relative concentration of polysialogangliosides (GT1b-GH) is strongly increased in all the investigated Antarctic species. They were found to have the most complex and most polar brain ganglioside pattern (high degree of sialylation and alkali-lability) within the teleosts. This may be one of the mechanisms, beside antifreeze proteins, to keep the neuronal membranes functional even below the freezing point.     

Variability in brain ganglioside composition: A further molecular mechanism beside serum antifreeze-glykoproteins for adaptation to cold in Antarctic and Arctic-boreal fishes

Summary Gangliosides and sialoglycoproteins from brain, liver and muscle have been isolated from 6 Antarctic fish species from the suborder Notothenoids and from 4 Arctic-boreal fish species. In addition freezing and melting points from serum of both groups were examined in order to determine the presence of protein antifreezes. In comparison with eurythermic fishes of temperate climates in both groups the phylogenetical adaptation to cold is correlated with a significantly higher concentration of gangliosides in the brain. The ganglioside concentration of liver in Antarctic fish, but not in Arctic species, is 3 to 5fold higher than in mammals (rat); in muscle the ganglioside content is increased only in red-blooded Antarctic fish as compared with mammals. The concentration of neuronal sialo-glycoproteins generally is lower in Antarctic fish than in other marine teleosts; in muscles the content is 2 to 3fold higher than in mammals. The molecular composition of brain gangliosides is characterized by an extreme high polarity which is due to an equipment with highly sialylated fractions (40 to 50% higher sialylated than tetrasialogangliosides). There are distinct differences between the freezing and melting point of blood serum, especially in the Antarctic species in favour of the existence of protein antifreezes. The results are discussed with regard to the fact that the extremely high polarity of brain gangliosides reflects a very efficient mechanism on molecular level to keep the neuronal membrane functional under low temperature conditions.     

Brain gangliosides in monotremes, marsupials and placentals: phylogenetic and thermoregulatory aspects

The concentration and composition of brain gangliosides of 17 mammalian species belonging to the subclasses of Prototheria (monotremes), Metatheria (marsupials), and Eutheria (placentals) were investigated. The mean concentration of brain gangliosides ranges from 525 to 610 micrograms NeuAc/g wet wt in monotremes, 445-900 micrograms in marsupials and from 630 to 1130 micrograms in the placentals. In the phylogenetic series of mammals, a decrease in the complexity of brain ganglioside composition becomes obvious: a drastic reduction in the number of individual ganglioside fractions particularly those of the c-pathway of biosynthesis, took place from the level of monotremes to that of the marsupials and placentals. In monotremes, marsupials and "lower" placentals (insectivores) the percentage of alkali-labile gangliosides is relatively low (between traces and 5%), whereas in the higher evolved mammals it amounts to about 20% of all gangliosides. The ratio of the contents of the two major mammalian ganglioside fractions GD1a and GT1b is generally in the range of 1.0 and even higher; in the heterothermic platypus from the monotremes and in hibernators among the placental mammals, however, it is much lower (about 0.8). These data support the hypothesis that the brain ganglioside composition not only depends on the phylogenetic level of nervous organization (cephalization) but is additionally correlated with the state of thermal adaptation.     

Seasonal variability of sialo-glycoconjugates in the brain of the Djungarian hamster (Phodopus sungorus)

The influence of season, photoperiod and ambient temperature on the content of proteins, sialo-glycoproteins and gangliosides and on the composition of gangliosides of three different brain regions (cortex, cerebellum and basalbrain) of the Djungarian dwarf hamster (Phodopus sungorus) had been investigated. Concomittantly changes in body wt and fur colouration were recorded. Dwarf hamsters living under natural photoperiod and ambient temperature conditions ("outside") showed a distinct annual cycle in body wt (summer: about 45 g; winter: about 25 g) and fur colouration (summer: dark grey; winter: whitish). Among the three brain regions the mean concentration of proteins ranged between 120 and 155 mg protein/g wet wt. The sialo-glycoprotein content varied between 260 and 410 micrograms NeuAc/g wet wt, and that of gangliosides between 800 and 1650 micrograms NeuAc/g wet wt. Seasonal fluctuations were not found. The composition of brain gangliosides remained uninfluenced throughout the year in the cerebellum, whereas seasonal variations were observed in cortex and basalbrain. Consequently the concentration ratio of the two major mammalian ganglioside fractions GD1a vs GT1b remained almost stable in cerebellum (0.3). In contrast to this the seasonal values of cortex and basalbrain changed from 0.6 and 0.8 in winter to 0.7 and 1.1 in summer. This indicated a higher polarity of the gangliosides in these brain regions during cold adaptation. The results are discussed with regard to modulatory functions of neuronal gangliosides for the process of synaptic transmission during seasonal adaptation.     

GANGLIOSIDE COMPOSITION AND CONTENT OF RAT-BRAIN SUBCELLULAR FRACTIONS

Abstract— The composition and content of gangliosides from rat-brain microsomal, synaptosomal, mitochondrial and myelin fractions were studied. Outer membranes of synaptosomes were also isolated, separated into subfractions and investigated. Of all the fractions studied the outer membranes of synaptosomes are richest in gangliosides, in one of their sub-fractions the concentration of gangliosides per mg of protein is five times higher than in the homogenate. Microsomes are rich in gangliosides as well, but to a lesser degree, whereas the mitochondrial fraction contains considerably smaller amounts of gangliosides per mg of protein than does the homogenate. The ganglioside pattern of outer membranes of synaptosomes and of their subfractions is somewhat different from that of the homogenate; the outer membranes contain approximately one-third less monosialogangliosides. On the contrary a very high content of monosialogangliosides is characteristic of the ganglioside pattern of the myelin fraction. In this fraction monosialoganglioside G_MI (nomenclature of Svennerholm, 1963) constitutes 60–63 per cent of ganglioside sialic acid, or 75–80 molar per cent of gangliosides, the content of di- and trisialogangliosides being much lower than in other fractions. Fatty acid and long chain base composition of gangliosides from synaptosomal and microsomal fractions and homogenate is very similar, almost identical. In gangliosides from myelin fractions the relaitve content of palmitic and monoenoic acids is higher and that of arachinic acid and C₂₀-sphingosine—lower than in other fractions studied. The difference in ganglioside composition of synaptosomes and their outer membranes and on the other hand of myelin appears to reflect the difference in ganglioside composition of neuronal and oligodendroglial plasma membranes.     

Isolation and partial characterization of human erythrocyte membrane NADH: (Acceptor) oxidoreductase

Gengliosides generally provide a small portion of the complex carbohydrate content of cell surfaces. An exception is the central nervous system where they comprise up to 5–10% of the total lipid of some membranes. This tissue is unique in that the quantity of lipid-bound sialic acid exceeds that of the protein-bound fraction. Over 30 different molecular species have been characterized to date. These range in complexity from sialosylgalactosyl ceramide with 2 sugars to the pentasialoganglioside of fish brain with 9 carbohydrate units. Virtually all cellular and subcellular fractions of brain that have been carefully examined contain gangliosides to one degree or another, but the majority of brain ganglioside is located in the neurons. Their mode of distribution within the neuron has not been entirely clarified by subcellular studies. Calculations based on reported values for axon terminal density and synaptosomal ganglioside concentration in the rat reveal that nerve endings contribute less than 12% of total cerebral cortical ganglioside. It is concluded that the plasma membranes of neuronal processes contain most of the neuronal ganglioside. These and other considerations suggest the possibility that gangliosides may be distributed over the entire neuronal surface.     

Changes in brain ganglioside composition of normothermic and hibernating golden hamsters (Mesocricetus auratus).

1. Brain ganglioside patterns of normothermic and hibernating golden hamsters (Mesocricetus auratus) and laboratory mice had been investigated. 2. The ganglioside pattern of normothermic golden hamsters in comparison to that of mice is characterized by an unusual high amount of the polar trisialoganglioside GT1. 3. In the hibernating golden hamster in contrast to normothermic counterparts the brain gangliosides are more polar (polysialization-effect). 4. The results are discussed with regard to the hypothesis that neuronal membranes provided with more polar gangliosides at lower environmental temperatures might be more efficient with respect to the high complexation ability of gangliosides with Ca2+-ions.     

CYTOSOLIC GANGLIOSIDES: OCCURRENCE IN CALF BRAIN AS GANGLIOSIDE-PROTEIN COMPLEXES

—Calf brain was treated in order to prepare separately the cytosol from neuronal bodies and glial cells, and the cytosol from nerve endings. The first cytosol contained 29 μg of ganglioside bound sialic acid/g fresh tissue, the latter 3.1 μg. Upon addition of ammonium sulphate until saturation the gangliosides contained in the two cytosols precipitated and were totally recovered in the pellet. while, under the same conditions, pure gangliosides were completely soluble. After stepwise ammonium sulphate fractionation all the different fractions obtained contained gangliosides and carried an approximately constant ganglioside/protein ratio. Thus cytosolic gangliosides occur in calf brain as ganglioside-protein complexes. The qualitative and quantitative pattern of gangliosides appeared to be similar in the two cytosols and in the different ammonium sulphate fractions obtained from the same cytosols. In addition, the pattern of cytosolic gangliosides was similar to that of membrane bound gangliosides.     

Age-Related Changes in the Ceramide Composition of the Major Gangliosides Present in Rat Brain Subcellular Fractions Enriched in Plasma Membranes of Neuronal and Myelin Origin

Abstract: Age-related changes of the ceramide composition of gangliosides were studied in the synaptosomal and myelin fractions from rat brain, carrying plasma membranes of neuronal and glial origin, respectively. The five major gangliosides (GM1, GD1 a, GD1 b, GT1 b, and GQ1 b) present in these fractions were separated and quantitated by normal-phase HPLC. Each ganglioside was then fractionated by reverse-phase HPLC into the molecular species carrying a single long-chain base (LCB). The largely preponderant LCBs in the synaptosomal and myelin fractions were the C18:1 and C20:1. The content of C20.1 LCB, generally low at 1 month, increased with age in all analyzed gangliosides and in all subcellular fractions and was greater in the "b series" than in the "a series" gangliosides. Remarkably, GM1 was the only ganglioside where the proportion of LCB 20:1 was higher in the synaptosomal fraction than in the myelin fraction. The fatty acid composition of the C18:1 or C20:1 LCB species of the different gangliosides in the synaptosomal and myelin fractions did not undergo appreciable changes with age. Stearic acid was largely predominant in all the gangliosides of the synaptosomal fraction, more in the C18:1 than in the C20:1 LCB species (80–90% vs. 60–70%). The gangliosides of the myelin fraction were characterized by a lower content of 18:0 and a much higher content of 16:0 and 18:1 fatty acids than those of the synaptosomal fraction. Thus, the ceramide composition is different in the gangliosides of neuronal and myelin origin and appears to be subjected to an age-related control.     

Systematic position of fish species and ganglioside composition and content

The ganglioside content in brain of cartilaginous and bony fishes studied varies from 110 to 581 and from 104 to 595 micrograms sialic acid per g of wet weight respectively. A high degree of alkali lability and the predominance of C18-sphingosine and N-acetylneuraminic acid are typical of fish brain gangliosides. A high content of oligosialogangliosides with four and more residues of sialic acid and the predominance of gangliosides with gangliotetraosyl carbohydrate chain are characteristic for teleost brain. No pronounced difference was revealed in ganglioside composition and content of clupeomorphs and percomorphs. Gangliosides with short (lactosyl and gangliotriaosyl) carbohydrate chain predominate in brain of all cartilaginous fishes studied. A statistically significant difference was found in ganglioside content, relative oligosialoganglioside content and ganglioside fatty acid composition of squalomorphs and rajiformes, on one hand, and dasyatiformes and galeomorphs, on the other hand.     

Gangliosides of Active and Inactive Neuroblastoma Clones

It is possible to divide neuroblastoma cells into clones able to synthesize neurotransmitters (active clones) or not (inactive clones).
The analysis of gangliosides of active and inactive clones shows that their total lipid sialic acids is markedly lower than that of neuron-enriched fractions prepared from brain. The ganglioside pattern of the cultured cells also differs notably from those obtained with neuronal fractions from brain. The absence of tri- and tetrasialogangliosides and the presence of appreciable amounts of the simplest monosialogangliosides are particularly noticeable in the neuroblastoma. Morphological differentiation obtained by serum deprivation, dibutyryl cyclic AMP or bromodeoxyuridine does not restore a true neuronal pattern. Gangliosides could not therefore be used as a marker of neuronal differentiation in this type of cell. No correlations can be found between the ganglioside pattern and the ability of cells to synthesize neurotransmitters.     

Gangliosides of active and inactive neuroblastoma clones.

It is possible to divide neuroblastoma cells into clones able to synthesize neurotransmitters (active clones) or not (inactive clones). The analysis of gangliosides of active and inactive clones shows that their total lipid sialic acids is markedly lower than that of neuron-enriched fractions prepared from brain. The ganglioside pattern of the cultured cells also differs notably from those obtained with neuronal fractions from brain. The absence of tri- and tetrasialogangliosides and the presence of appreciable amounts of the simplest monosialogangliosides are particularly noticeable in the neuroblastoma. Morphological differentiation obtained by serum deprivation, dibutyryl cyclic AMP or bromodeoxyuridine does not restore a true neuronal pattern. Gangliosides could not therefore be used as a marker of neuronal differentiation in this type of cell. No correlations can be found between the ganglioside pattern and the ability of cells to synthesize neurotransmitters.     

Gangliosides Affect Membrane-Channel Activities Dependent on Ambient Temperature

1. The functional properties of biological membranes depend on their molecular composition. In regard to this, charged glycosphingolipids play an outstanding role in the functional adaptation of membranes to different temperatures.2. In order to shed some light on the respective functional properties of complex membraneous glycosphingolipids, the effects of altered temperatures (5–40°C) on planar lipid bilayers made from diphytanoylphosphatidylcholine (DPPC) and alamethicin as an ion channel was analyzed in the presence of either a sialoglycosphingolipid (less polar disialoganglioside GD1a or highly polar tetrasialoganglioside GQ1b) or phosphatidylserine (PS; as control).3. Different to the control bilayers made from DPPC or DPPC + PS, the bilayers containing gangliosides had specific maxima in alamethicin conductance and stabile life times. Changes in pore-state conductances indicate structural effects based on an interaction of the large (negatively charged) ganglioside headgroups with the alamethicin pores.4. The results concerning open time and closed time of channels seem to be based on the gangliosides changing the viscosity of the bilayer and possibly introducing phase transitions.5. Thus, the findings suggest that gangliosides (1) directly affect channel molecules via their headgroups and (2) may additionally affect the fluidity of membranes in order to maintain membrane homeoviscosity in areas surrounding ion channels independent from the environmental temperature.6. The effects of gangliosides may be of special interest in describing the ability of neuronal adaptation of vertebrates to temperature and more general regarding the functional adaptation of neurons.     

Brain ganglioside patterns of vertebrates

Abstract— The ganglioside content in brains of representatives of six vertebrate classes (lamprey, ray, sheat-fish, carp, frog, triton, tortoise, hen, pigeon, rabbit, rat and monkey) was determined. In most cases a correlation was found between the level of nervous organization and the ganglioside content of brain. In fish and amphibian brain ganglioside concentration is half to one third that in mammalian brain. Ganglioside composition of higher vertebrate brains (mammals, birds and reptiles) has many similar features. Four main gangliosides with 1-3 NANA residues in their molecules–G₁ * * Nomenclature of Korey and Gonatas (1963 ): G₁ trisialyl-hexosaminyl-trihexosyl-ceramide; G₂ and G₃, disialyl-hexosaminyl-trihexosyl-ceramides; G₄ monosialyl-hexosaminyl-trihexosyl-ceramide.
, G₂, G₃ and G₄–constitute 80-90 per cent of total ganglioside NANA. Fractions G_2a ? ? G_o, tetrasialyl-hexosaminyl-trihexosyl-ceramide; G_2a disialyl-hexosaminyl-dihexosyl-ceramide; G₅, monosialyl-hexosaminyl-dihexosyl-ceramide.
G_o and G₅ are present in much lesser amounts. Species peculiarities in distribution of NANA among different fractions were noted. The brain gangliosides of lower vertebrates–fish and amphibia–are unusual in having a high proportion of polysialogangliosides, containing 4 and 5 NANA residues, and a lower content of monosialogangliosides. In ray brain a considerable part of gangliosides has a reduced carbohydrate chain.     

Human brain gangliosides in development, aging and disease.

In this study, brain gangliosides in prenatal and postnatal human life and Alzheimer's disease were analyzed. Immunohistochemically, the presence of the "c"-series of gangliosides (GQ1c) was only registered in the embryonic brain at 5 weeks of gestation. Biochemical results indicated a two-fold increase in ganglioside concentration in the human cortex between 16 and 22 weeks of gestation. The increasing ganglioside concentration was based on an increasing GD1a ganglioside fraction in all regions analyzed except in the cerebellar cortex, which was characterized by increasing GT1b. During prenatal human development, regional differences in ganglioside composition could only be detected between the cerebrum ("a"-pathway) and the cerebellum ("b"-pathway). Between birth and 20-30 years of age, a cerebral neocortical difference of ganglioside composition occurred, characterized by the lowest GD1a in visual cortex. Analyzing the composition of gangliosides in cortical regions during aging, they were observed to follow region-specific alterations. In the frontal cortex, there was a greater decrease in GD1a and GM1 than in GT1b and GD1b, but in the occipital (visual) cortex there was no change in individual gangliosides. In hippocampus, GD1a moderately decreased, whereas other fractions were stable. In the cerebellar cortex, GD1b and GT1b fractions decreased with aging. In Alzheimer's disease, we found all ganglio-series gangliosides (GM1, GD1a, GD1b, GT1b) to be decreased in regions (temporal and frontal cortex and nucleus basalis of Meynert) involved in pathogenesis of disease. In addition, in Alzheimer's disease we found simple gangliosides (GN2, GM3) to be elevated in the frontal and parietal cortex, which might correlate accelerated lysosomal degradation of gangliosides and/or astrogliosis occurring during neuronal death.     

Shedding of gangliosides from tumor cells depends on cell density

The ganglioside composition of mouse ascites hepatoma ( MAH ) cells, the ascites fluid and cell-conditioned media were determined and found to be qualitatively identical, but quantitatively different. The ganglioside content of the ascites fluid and the medium conditioned by MAH -cells at the native cell concentration (10(8) cells/ml) comprised respectively 74.9% and 23% of the cell-associated gangliosides. When incubated at lower cell-density (10(6) cells/ml) the cells were found to be release about three-times higher amounts of ganglioside per cell than during incubation at the native concentration. Centrifugation of the dense-cell-conditioned medium revealed the major part of the released gangliosides to be associated with a 150000 X g pellet that probably contains shed plasma membrane fragments. In the 150000 X g pellet of the extracellular fluids the relative content of the most polar cell ganglioside corresponding chromatographically to GT1b was about ten-times higher than in the cells. The possibility is raised that the more intense shedding of gangliosides from less crowded MAH cells may play a role in the self protection of the tumor from host immune rejection during initial stages of growth.     

Ganglioside composition of normal and leukemic bovine lymphocytes

The ganglioside composition of bovine peripheral lymphocytes was shown to change sharply under lymphoid leukemia. In normal lymph, lymph nodes, spleen and blood lymphocytes the major ganglioside is N-glycolylhematoside, whereas in calf thymus lymphocytes appreciable amounts of more polar components (GM1- and GD1a-like gangliosides) were found. In leukemic lymphocytes isolated from the same tissues the hematoside content is decreased, while the amount of more polar gangliosides is increased. Possible causes of the altered ganglioside pattern in leukemic lymphocytes are discussed.