Lipid content in brain and spinal cord during experimental allergic encephalomyelitis in rats

Abstract— The content of cerebrosides, sulphatides, gangliosides, cholesterol and phospholipids was evaluated in the brain and spinal cord of rats during the acute and recovery stages of experimental allergic encephalomyelitis (EAE). During the acute stage there was a significant decrease of sulphatides and gangliosides in spinal cord; in brain, only sulphatides were diminished. In the recovery stage, cerebrosides and gangliosides were decreased in the brain, whereas the lipid content of the spinal cord was similar to that in control animals. Cholesterol esters were detected in the brain and spinal cord during both periods. The results show that the changes are not the same for brain and spinal cord during the acute and recovery stages and that glycosphingolipids from either white or grey matter seem to be preferentially altered.     

Nervous system ganglioside composition of normothermic and hibernating dormice (Glis glis)

The ganglioside pattern of seven different regions, olfactory bulb, forebrain cortex, midbrain (corpora quadrigemina), cerebellum, brain stem, pons and spinal cord, of nervous system of normothermic and hibernating dormice (Glis glis) were investigated by two dimensional thin layer chromatography and densitometric quantification. Up to thirty different ganglioside spots were resolved, fifteen of which belonging to alkali labile species. Alkali labile gangliosides were present in all the regions obtained from normothermic animals, and their content, expressed as percentage of total ganglioside-bound sialic acid, ranged from a minimum of 10.2% in olfactory bulb, to a maximum of 30.1% in spinal cord. The most abundant alkali labile gangliosides were O-Ac-GT1b, O-Ac-GQ1b and an unidentified one, we coded I3. Alkali labile gangliosides were practically undetectable in hibernating dormice. They could be recognized only in brain stem, 3.3% and olfactory bulb, 0.6%.     

Effect of Maternal Alcohol Consumption on the Lipid Composition of CNS in the Offspring

Maternal alcohol consumption at a level that does not affect calorie intake increases cholesterol concentration and content as well as incorporation of labeled glucose into cholesterol in the brain and spinal cord of newborn rat pups. Continued consumption of alcohol during lactation also affects the galactolipid concentration in the brain and spinal cord of pups at 21 days of age, and this increase seems mainly to be due to an increase in content of myelin lipids. Analysis of myelin shows that the concentration of phospholipids also increases in this fraction. The increase in incorporation of labeled glucose into these membrane lipids suggests an increase in the synthesis of these lipids, which prevents fluidization of the membrane by alcohol. That in the brainstem the increase in levels of cholesterol and galactolipids is higher than in other regions and that there is also an increase in content of sphingomyelin, phosphatidylcholine, and phosphatidylethanolamine suggest that the brainstem needs better protection against fluidization.     

Involvement of the GHSR in the developmental programming and metabolic disturbances induced by maternal undernutrition

The mismatch between maternal undernutrition and adequate nutrition after birth increases the risk of developing metabolic diseases. We aimed to investigate whether the hyperghrelinemia during maternal undernourishment rewires the hypothalamic development of the offspring and contributes to the conversion to an obese phenotype when fed a high-fat diet (HFD). Pregnant C57BL/6 J, wild type (WT) and ghrelin receptor (GHSR)^−/− mice were assigned to either a normal nourished (NN) group, or an undernutrition (UN) (30% food restricted) group. All pups were fostered by NN Swiss mice. After weaning, pups were fed a normal diet, followed by a HFD from week 9. Plasma ghrelin levels peaked at postnatal day 15 (P15) in both C57BL/6 J UN and NN pups. Hypothalamic Ghsr mRNA expression was upregulated at P15 in UN pups compared to NN pups and inhibited agouti-related peptide (AgRP) projections. Adequate lactation increased body weight of UN WT but not of GHSR^−/− pups compared to NN littermates. After weaning with a HFD, body weight and food intake was higher in WT UN pups but lower in GHSR^−/− UN pups than in NN controls. The GHSR prevented a decrease in ambulatory activity and oxygen consumption in UN offspring during ad libitum feeding. Maternal undernutrition triggers developmental changes in the hypothalamus in utero which were further affected by adequate feeding after birth during the postnatal period by affecting GHSR signaling. The GHSR contributes to the hyperphagia and the increase in body weight when maternal undernutrition is followed by an obesity prone life environment.     

Receptor ganglioside content of three hosts for Sendai virus. MDBK, HeLa, and MDCK cells

Specific gangliosides GD1a, GT1b and GQ1b isolated from brain have been shown to function as receptors for Sendai virus by conferring susceptibility to infection when they are incorporated into receptor-deficient cells (Markwell, M.A.K., Svennerholm, L. and Paulson, J.C. (1981) Proc. Natl. Acad. Sci. USA 78, 5406-5410). The endogenous gangliosides of three commonly used hosts for Sendai virus: MDBK, HeLa, and MDCK cells were analyzed to determine the amount and type of receptor gangliosides present. In all three cell lines, GM3 was the major ganglioside component. The presence of GM1, GD1a and the more complex homologs of the gangliotetraose series was also established. In cell lines derived from normal tissue, MDBK and MDCK cells, gangliosides contributed 47-65% of the total sialic acid. In HeLa cells, gangliosides contributed substantially less (17% of the total sialic acid). The ganglioside content of each cell line was shown not to be immutable but instead to depend on the state of differentiation, passage number, and surface the cells were grown on. Thus, the ganglioside concentration of undifferentiated MDCK cells was found to be substantially greater than that of MDBK or HeLa cells, but decreased as the MDCK cells underwent differentiation. Changes in culture conditions that were shown to decrease the receptor ganglioside content of the cells resulted in a corresponding decrease in susceptibility to infection. The endogenous oligosialogangliosides present in susceptible host cells were shown to function as receptors for Sendai virus.     

Acetylcholinesterase activity in developing rat brain during undernutrition

The effect of low protein diet on rat brain AChE activity has been studied during gestation, lactation and postweaning periods. There was decrease in enzyme activity of pups undernourished either during gestation and lactation or lactation alone, the decrease being maximum in 18-day-old pups. In postweaning rats, a significant decrease was observed after 2 and 4 weeks of undernutrition compared to the control. However, the effect of undernutrition was annuled by 2-week rehabilitation, thereby indicating that imposed undernutrition only delays the normal level of the enzyme. Moreover, it appears that the enzyme activity depends both on the nutritional status and the development age.     

THE EFFECT OF DEVELOPMENT ON THE GANGLIOSIDES OF RAT AND PIG BRAIN

Abstract— The ganglioside content of the forebrain, brain stem and cerebellum have been studied, in the rat at various ages from 1 day to 27 months, and in the pig at various ages from 93 days gestation to 30 months. Each part of the brain was analysed for total ganglioside NANA and for four major gangliosides (G_Ml, G_D1a, G_Dlb and G_T1 in the nomenclature of S vennerholm , 1963). In the rat forebrain, the concentration of ganglioside NANA rose rapidly between 1 and 21 days after birth, fell to 3 months and subsequently rose to a mature value at 6 months. In the rat cerebellum, the peak concentration was reached at 2 months and the lower adult value at 9 months, whilst in the brain stern, the concentration rose more slowly and had a broad peak from 15 days to 2 months. Values are also given for the changes in the total amounts in each brain part. The changes in the concentrations and total amounts of ganglioside NANA, in the three parts of the pig brain were, on the whole, similar to those in rat brain except that the percentage distribution of the major gangliosides had almost attained the mature pattern at birth. In the forebrain of both species, the disialoganglioside, G_D1a, accounted for the highest percentage of the total gangliosides. The results are discussed with respect to their possible structural significance.     

Effects of neonatal pantothenic acid deficiency on brain lipid composition in rats.

—Studies were made of the effects of pantothenic acid deficiency during the neonatal period on brain lipids in rats. Mothers with 6–8 pups to a litter were fed from soon after birth a diet either normal or deficient in pantothenate. An additional control group (restricted controls) was pair-fed with the deficient group. Significant deficits were found in the pups of the pantothenate-deficient group and in those of the restricted controls with regard to body weight, brain weight and brain concentration of lipids (total lipid, cholesterol, phospholipid, galactolipid and gangliosides) at 21 days of age. The deficits in both these groups were comparable. The results suggest that the effects of pantothenate deficiency may be due to the resulting growth deficit rather than to the deficiency of pantothenate per se.     

Dietary ganglioside decreases cholesterol content, caveolin expression and inflammatory mediators in rat intestinal microdomains

Membrane microdomains rich in cholesterol and sphingolipids, including gangliosides (GGs), are known to be important regions for cell signaling and binding sites for various pathogens. Cholesterol depletion inhibits the cellular entry of pathogens and also reduces inflammatory signals by disrupting microdomain structure. Our previous study showed that dietary gangliosides increased total ganglioside incorporation while decreasing cholesterol in the intestinal mucosa. We hypothesized that diet-induced reduction in cholesterol content in the intestinal mucosa disrupts microdomain structure resulting in reduced pro-inflammatory signals. Male weanling Sprague-Dawley rats were fed semipurified diets for 2 weeks. Experimental diets were formulated to include either ganglioside-enriched lipid (GG diet, 0.02% gangliosides [w/w of diet] ) or polyunsaturated fatty acid (PUFA diet, 1% arachidonic acid and 0.5% docosahexaenoic acid, w/w of total fat), in a control diet containing 20% fat. Levels of cholesterol, GG, caveolin, platelet activating factor (PAF), and diglyceride (DG) were measured in the microdomain isolated from the intestinal brush border. The GG diet increased total gangliosides by 50% with a relative increase in GD3 and a relative decrease in GM3. Cholesterol content was also reduced by 23% in the intestinal microdomain. These changes resulted in a significant decrease in the ratio of cholesterol to ganglioside. The GG diet and the PUFA diet were both associated with reduction in caveolin, PAF, and DG content in microdomains, whereas no change occurred in the ganglioside profile of animals fed the PUFA diet. Dietary gangliosides decrease the cholesterol/ganglioside ratio, caveolin, PAF and DG content in microdomains thus exerting a potential anti-inflammatory effect during gut development.     

GANGLIOSIDE1−COMPOSITION OF BRAIN IN TAY-SACHS DISEASE: INCREASED AMOUNTS OF GD2 AND N-ACETYL-β-D-GALACTOSAMINYL GD1a GANGLIOSIDE

Abstract— The ganglioside composition of the brain of a patient with Tay-Sachs disease (TS-brain) was determined by a newly developed ganglioside-mapping procedure and compared with that of an age-matched control brain. GM2 ganglioside was the predominant component in TS-brain and the following gangliosides were also found, GM1, GD1a, GD1b and GT1 (major gangliosides in normal brain), and GM3, GD3, GD2 and GD1a-GAN (minor or undetectable components of normal brain). Individual gangliosides were isolated by column chromatography using a combination of DEAE-Sepharose, Iatrobeads and Silica Gel 60 and their structures were confirmed by comparing them with authentic standards using TLC, analysing their carbohydrate compositions by gas-liquid chromatography and cleaving them sequentially with glycosidases. The amounts of individual components were measured by quantitative densitometric scanning of the thin-layer plates. As a reflection of myelin breakdown, no sialosylgalactosyl ceramide was detectable in TS-brain. Although the total amounts of all gangliosides except GM2 in TS-brain were low, there were normal molar ratios of the main gangliosides in normal brain, that is, GM1, GD1a, GD1b and GT1. In comparison with the amount of GDla ganglioside, the amounts of GM2, GD2 and GD1a-GAN, which contain N-acetylgalactosamine as a terminal carbohydrate residue, were all elevated in TS-brain. The long chain bases of individual gangliosides contained both C-18 and C-20 sphingosine in different ratios and the ratio of C-20 to C-18 increased in the gangliosides in the order: GM2 < GM1 < GD1a < GD1a-GAN < GD1b < GT1 in both normal brain and TS-brain. In contrast, GD2 and GD3 gangliosides consisted mainly of C-18 sphingosine. The C-20 to C-18 ratios of individual gangliosides in the TS-brain were lower than those of age-matched control brain. Hexosaminidase from Turbo cornutus showed the same specific activity and K_m value in catalysing the cleavage of terminal N-acetylgalactosaminyl residues from GM2, GD2 and GD1a-GAN, suggesting that the brain gangliosides that increase in Tay-Sachs disease may be cleaved by the same enzyme.     

Gangliosides in the human brain development and aging.

In this study, brain gangliosides in prenatal and postnatal human life were analyzed. Immunohistochemically, the presence of "c"-pathway of gangliosides (GQ1c) in embryonic brain was only recorded at 5 weeks of gestation. Biochemical results indicated a twofold increase in human cortex ganglioside concentration between 16 and 22 weeks of gestation. The increasing ganglioside concentration was based on an increasing GD1a ganglioside fraction in all regions analyzed except cerebellar cortex, which was characterized by increasing GT1b. In this developmental period, GD3 was found to be localized in the ventricular zone of the cortical wall. After birth, GD1b ganglioside in neuropil of granular cell layer corresponding to growing mossy fibers was expressed in cerebellar cortex. Between birth and 20/30 years of age, a cerebral neocortical difference of ganglioside composition was observed, characterized by lowest GD1a in visual cortex. Analyzing the composition of gangliosides in cortical regions during aging, they were observed to follow region-specific alterations. In frontal cortex, there was a greater decrease in GD1a and GM1 than in GT1b and GD1b, but in occipital (visual) cortex there was no change in individual gangliosides. In hippocampus, GD1a moderately decreased, whereas other fractions were stable. In cerebellar cortex, GD1b and GT1b fractions decreased with aging.     

Chloroquine Intoxication Induces Ganglioside Storage in Nervous Tissue: A Chemical and Histopathological Study of Brain, Spinal Cord, Dorsal Root Ganglia, and Retina in the Miniature Pig

Abstract :
The effect of chronic chloroquine intoxication on lipid composition, particularly the gangliosides, was studied in the nervous system of miniature pigs, type Göttingen. The tissues examined were cerebrum, spinal cord, dorsal root ganglia and retina. Chloroquine was given in the diet in doses of 2.0-3.5 g/kg food. The intoxication of the pigs was started at the age of 100–240 days and continued for 177-219 days. The control pigs received the same diet without chloroquine. The ganglioside concentration was increased in all the tissues examined. Dorsal root ganglia and retina were the tissues affected most and showed a twofold increase. This corresponded to the light and electron microscopically demonstrated extensive storage process in the perikarya of dorsal root ganglion cells and inner ganglion cells of the retina. Under light microscopy the storage material was granular, intensely PAS-positive and dissolved by paraffin embedding. The electron microscopical equivalent consisted of conglomerates of membranous lysosomal residual bodies. In cerebrum the ganglioside concentration was increased by 12%. Storage in the brain varied widely between different systems and types of cells. The allocortex was much more affected than the isocortex. Certain inhibitory ganglion cell types, such as the basket cells, exhibited the most massive storage of all. The spinal medulla was morphologically less involved but showed approximately the same ganglioside increase, though not statistically significant. With the exception of cerebrum the increase in the tissues examined involved all the individual gangliosides, most severely ganglioside GM2 and three fucogangliosides. In cerebrum only the ganglioside GM2 was increased more than the other gangliosides. Chloroquine intoxication did not affect the concentration of phospholipids or cholesterol in the cerebrum, spinal cord or dorsal root ganglia, but in retina the acidic phospholipids were significantly increased.     

Neuronal perikarya and astroglia of rat brain: chemical composition during myelination

Cells isolated by a new technique from 10-, 20-, and 30-day-old rat brains have been analyzed for total lipid, cholesterol, galactolipid, individual phospholipids, gangliosides, DNA, and RNA. The lipid composition does not vary appreciably in either neurons or astrocytes during this period of rapid myelination. Moreover, the lipid compositions of the two cell types are surprisingly similar, both having very low galactolipid concentrations, high phospholipid content, and cholesterol concentrations lower than whole brain. Astrocytes have a higher ganglioside content than neuronal perikarya, a finding ascribed to the higher ratio of surface membrane to mass in the astrocytes, and considered as evidence that gangliosides are normal glial constituents. Compared with an average astrocyte, the individual neuron soma has less mass, a lower total lipid content, and a much higher RNA content.     

1H NMR Ganglioside Ceramide Resonance Region on the Differential Diagnosis of Low and High Malignancy of Brain Gliomas

1. The high-resolution ¹H NMR (MRS) spectra of human brain tumor homogenates revealed a broad resonance at 5.3–5.4 ppm in glioblastoma multiforme (N = 16) and brain metastases (N = 2). The broad resonance was identified as ceramide, a sphingosine–fatty acid combination portion of ganglioside, indicating an elevated abundance of monounsaturated fatty acids. GLC analysis of gangliosides in the highly malignant glioblastoma multiforme revealed that the elevated monounsaturated fatty acid is oleic acid (C18:1). The resonance at 5.3–5.4 ppm region was not detectable in normal human brain (N = 2), in meningiomas (N = 2), or in low-grade astrocytomas (N = 12). In normal human brain the abundance of monounsaturated fatty acid is minimal.2. This investigation was made possible because the method of producing homogenate resulted in (i) no loss of lipids during the process and (ii) a well-homogenised sample, with (iii) no loss in chemical integrity.3. The properties of tumor gangliosides include antigenic specificity and immunosuppresive activity and the ceramide, a sphingosine–fatty acid combination, noticeably influences the ganglioside immunosuppressive activity.4. The observation of ¹H NMR ceramide resonance in high-malignant brain tumors emphasizes the dramatic role of aberrant gangliosides and ceramide precursors on the grade of malignancy and invasiveness.5. Further insight into the specific nature of the ceramide portion of gangliosides in grading the malignancy of brain tumors should be investigated further.     

Developmental Changes in Ganglioside Composition and Synthesis in Embryonic Rat Brain

Developmental changes in ganglioside composition and biosynthesis was studied in rat brain between embryonic day (E) 14 and birth. In E14 brains, GM3 and GD3 were predominant. At E16, "b" series gangliosides, such as GD1b, GT1b, and GQ1b, increased in content. After E18, "a" series gangliosides such as GM1, GD1a, and GT1a increased in content, and the content of GM3 and GD3 markedly decreased. Because of these changes in composition, we determined the activities, in homogenates of embryonic brains, of two key enzymes of ganglioside synthesis: sialyltransferase for the synthesis of GD3 from GM3 and N-acetylgalactosaminyltransferase for GM2 synthesis from GM3. The sialyltransferase activity (GM3----GD3) was constant between E14 and E18 but decreased rapidly from E18 to birth. In contrast, the N-acetylgalactosaminyltransferase activity (GM3----GM2) increased between E14 and E18 but was constant from E18 to birth. These changes in ganglioside composition and enzymatic activities indicate that during development there is a shift from synthesis of the simplest gangliosides of the "a" and "b" pathways to synthesis of the more complex gangliosides.     

Changes in sphingosine and fatty acid components of the gangliosides in developing rat and human brain

Rat brain increases in weight after birth in three stages: (I) rapidly for the first 2 weeks, (II) at a lower rate from 2 to 5 weeks, and (III) at a still lower rate from 5 weeks to 5 months. During the succeeding period, designated IV, it maintains constant weight up to 1 year of age. Brain ganglioside content increased linearly during I and II, more slowly during III, and diminished during IV. The appearance of measurable amounts of brain sphingomyelin and cerebroside succeeded that of ganglioside. Ceramide with C18-sphingosine and C18 fatty acid was found in a large proportion of all three sphingolipids upon their first appearance in measurable quantity. C18 fatty acid in cerebroside rapidly declined to a negligible level, while in gangliosides and sphingomyelin it declined slowly but remained the major fatty acid component. Cerebrosides and sphingomyelin contained C18-sphingosine almost exclusively at all stages of rat brain growth. Gangliosides contained C18-sphingosine almost exclusively at birth, but subsequently accumulated C20-sphingosine until they had nearly equal quantities of each base type. Changes in human brain gangliosides resemble those in rat. In Tay-Sachs disease, gangliosides have C18-sphingosine predominantly, and a high content of C18 fatty acid.     

Effects of Embryonic Neural Stem Cell Transplantation on DNA Damage in the Brain and Spinal Cord Following Spinal Cord Injury

Embryonic neural stem cell (ENSC) transplantation is used experimentally for the improvement of spinal cord repair following spinal cord injury (SCI). However, the effects of such intervention on oxidative stress and cell death remain unknown. We used in vivo Comet assay in the acute and chronic SCI groups compared with the SCI+ENSC transplantation groups of experimental rats in order to evaluate DNA damage in the spinal cord. Chronic SCI resulted in the generation of oxidative DNA damage in the spinal cord brain and kidneys, as indicated by high Comet assay parameters, including the percentage of DNA in the tail (T%, or TD), tail moment (TM), and tail length (TL). The DNA damage levels significantly decreased after ENSC transplantation in the spinal cords of acute and chronic SCI groups within the lesion site and rostrally and caudally to the injury, and in the brains and kidneys of the chronic SCI group. Thus, ENSC transplantation is found to be an effective tool for limitation of DNA damage following spinal cord injury.     

Gangliosides GM3 and GD3 in human stomach and breast tumors

Gangliosides of human gastric and mammary tumours and of homologous normal tissues were studied by using biochemical methods and specific antisera. It was found that in most cases GM3, GD3 and GM1 are predominant gangliosides, whereas several polar components are minor ones. A comparison of the relative amount of ganglioside fractions revealed that in gastric tumours the per cent content of polar compounds is higher than in intact tissue; however, the absolute content of all gangliosides is markedly increased. A comparative study of the composition of mammary tumour and normal tissue gangliosides demonstrated two types of changes: i) the absolute content of all gangliosides in tumour tissue was increased and, ii) the increase in the content of total gangliosides was paralleled with the appearance of a new fraction (presumably GM4), the decrease of the GD3 content and the disappearance of polar gangliosides. A possible mechanism of this effect is discussed.     

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.