Ganglioside metabolism in the hippocampus after septal lesion in rats

The changes in ganglioside composition and metabolism of deafferentiated rat hippocampus were estimated after septal lesion. A significant decrease in total ganglioside concentration was found 7 days after the lesion. The reduced level of total gangliosides persisted at 17 and 25 days. Relative increase in the proportion of GD1b and GX (O-acetylated GT1b) and decrease in GM1 were found in hippocampus only at 25 days post-lesion. The incorporation of 3H-N-acetylmannoseamine into gangliosides was examined in rats whose hippocampi were lesioned 25 days prior to radioprecursor injection. Differences in the labeling pattern of total and individual gangliosides were found. Increases in the label in GM1, GD3, and GD1a and decreases in GT1b and GQ1b were found 10 hr after isotope injection. However, decreases in the specific activity of all gangliosides except GT1b and GQ1b were observed 24 hr after 3H-N-acetylomannosamine injection, suggesting the activated turnover of gangliosides in postlesioned hippocampus. The significance of these changes has been discussed in terms of cellular damage and repair in the hippocampal tissue.     

Cerebellar ganglioside abnormalities in pcd mutant mice

The distribution of cerebellar gangliosides was studied in Purkinje cell degeneration (pcd/pcd) mutant mice at postnatal days 25, 30, 50, and 150. These mutants lose the majority of Purkinje cells between 18 and 50 days of age. A reactive gliosis accompanies Purkinje cell loss and a partial loss of granule cells occurs in pcd/pcd mice older than p50. Purkinje cell loss is associated with significant reductions in cerebellar weight and ganglioside concentration. This neuronal loss was also developmentally correlated with reductions of gangliosides (GT1a/LD1 and GT1b and with elevations of GD3. These results agree with previous findings in other cerebellar mutants that GT1a/LD1 and GT1b are concentrated in Purkinje cells and that GD3 is enriched in reactive glial cells. A slight, but significant, reduction in GD1a concentration occurred only in older pcd/pcd mice, consistent with previous findings in weaver and staggerer mice that GD1a is enriched in mature granule cells. The findings with pcd/pcd and other neurological mutants indicate that certain gangliosides can serve as cell-surface markers for monitoring changes in cerebellar cytoarchitecture that accompany development or disease.     

Gangliosides in lesion-induced synaptogenesis: studies in the hippocampus of the rat brain

Changes in ganglioside composition, biosynthesis and individual distribution were studied in hippocampal regions after unilateral destruction of the entorhinal cortex. After 1 and 3 days postlesion (dpl), a decrease in ganglioside content was detected in area dentata (AD) and pyramidal cell regions CA1-CA3 (CA), both ipsilateral and contralateral to the lesion. By 5 dpl all the values had returned to control values, except in AD which showed a dramatic increase in total ganglioside content reaching a maximum at 12 dpl. By 30 dpl this area also showed control content. A significant increase in biosynthesis of gangliosides was observed at 5 and 8 dpl in the hippocampus ipsilateral to the lesion without changes in the contralateral counterpart. Individual ganglioside distribution showed a pronounced change in GM1 and GQ1b with small changes in the other major gangliosides. Significant differences were observed in the distribution of gangliosides between the two hippocampal regions studied in unoperated control animals. GD1a was more concentrated in AD, whereas GQ1b, GT1b and GD1b predominated in CA. The data presented here indicate that important modifications in ganglioside content as well as pattern occur in the deafferented hippocampus a phenomenon that could be related with the known effect of gangliosides on neuritogenesis observed in cell culture studies.     

Immunocytochemical analysis of gangliosides in rat primary cerebellar cultures using specific monoclonal antibodies

We studied the expression of ganglioside antigens in primary cultures of rat cerebellum using an immunocytochemical technique with mouse monoclonal antibodies (MAbs) specific for various gangliosides. Twelve MAbs that specifically recognize each ganglioside were used. Our study revealed that there is a cell type-specific expression of ganglioside antigens in the primary cultures. A number of b-series gangliosides were detected in the granule cells, whereas a-series gangliosides were not intensely expressed. GD1b was detected in the granule cells. GD2 appeared to be present in a subset of the granule cells or a type of small neurons. GD3 was associated not only with the granule cells, but also with both astrocytes and oligodendrocytes. An O-Ac-disialoganglioside, which was suggested to be O-Ac-LD1, was restrictedly detected in Purkinje cells. The other gangliosides were not detected clearly in these cells. These results suggest that several gangliosides may be useful markers for identifying cells in primary cultures of the rat cerebellum; particularly b-series gangliosides such as GD2 and GD1b for the granule cells and O-Ac-LD1 for Purkinje cells.     

GQ1b-seronegative Fisher syndrome: clinical features and new serological markers

IgG anti-GQ1b antibodies are a powerful serological marker for the diagnosis of Fisher syndrome (FS), but little is known regarding serological markers in FS patients that do not have the autoantibodies. The authors analyzed IgG antibodies against gangliosides other than GQ1b, ganglioside complexes, and ganglioside-like lipo-oligosaccharide (LOS) of Campylobacter jejuni isolates from FS patients. We identified 24 (12%) patients with GQ1b-seronegative FS among 207 FS patients who had been referred to our laboratory for anti-ganglioside antibody testing. Patients with GQ1b-seronegative FS were male and had a history of antecedent gastrointestinal illness more frequently than FS patients with IgG anti-GQ1b antibodies. Other clinical features during the illness were not distinguishing for GQ1b-seronegative FS. Four (17%) of 24 patients with GQ1b-seronegative FS had IgG antibodies against single gangliosides such as GM1b, GD1a, or GT1a. Antibodies against GM1 and GT1a complex were detected in four GQ1b-seronegative FS patients, three of whom did not have antibodies against single gangliosides. Mass spectrometry analysis showed that C. jejuni isolates from FS patients had GD1c-, GalNAc-GM1b-, or GalNAc-GD1c-like LOS, and not GQ1b-like LOS, highlighting the utility of examining serum antibodies against these ganglioside mimics in GQ1b-seronegative FS patients. Seven (29%) had IgG antibodies against the LOS from C. jejuni strains expressing GD1c-, GalNAc-GM1b-, or GalNAc-GD1c-like LOS. These findings suggest that IgG antibodies against GM1b, GD1c, GalNAc-GM1b, and ganglioside complexes are serological markers for GQ1b-seronegative Fisher syndrome.     

Expression and function of ganglioside 9-O-acetyl GD3 in postmitotic granule cell development

We have shown previously that the Jones monoclonal antibody (Jones mAb) recognizes 9-O-acetyl GD3 expressed during periods of neuronal migration and neurite outgrowth in the developing rat nervous system. In the present study we investigated the expression of this ganglioside in the developing cerebellum and correlated this expression with granule cell migration. Electron microscopic immunocytochemistry revealed that around the peak of cerebellar neuronal migration (7-day-old rat), 9-O-acetyl GD3 was localized at the contact sites between migrating granule cells and radial glia in the external granular layer and prospective molecular layer. In addition, using microexplant and slice cultures of the postnatal rat cerebellum, we tested whether the ganglioside detected by our antibody contribute to the regulation of neuronal migration in the cerebellar cortex. We have shown that the Jones mAb blocks the migration of neurons in a dose-dependent manner. These findings suggest strongly that 9-O-acetyl GD3 is involved in granule cell migration in the developing cerebellum.     

Effect of crush lesion on ganglioside radiolabelling patterns in rat sciatic nerve

Left sciatic nerves in rats were crushed and allowed to regenerate for variable periods of time up to 14 days; uncrushed right nerves from the same animals were used as controls. Two days before killing the rats, both L-5 dorsal root ganglia (DRG) were injected with 100 microcuries [3H]glucosamine. Gangliosides were purified separately from sciatic nerve (SN) distal to the crush site, lumbosacral trunk (LST) proximal to the crush site, and the injected DRG. Changes in major glycoconjugate classes were previously reported; in this study total gangliosides were separated by high performance thin layer chromatography, located by autofluorography and radioactivity was measured by liquid scintillography. In control DRG, major radiolabelled gangliosides were GM3 and LM1; in control LST and SN, GD1b and GT1b were the major ones. During day two and four following crush, GM3 and LM1 decreased in DRG, but at one and two weeks were at normal and elevated levels, respectively; there were inverse changes in GD3, GT1b and GQ1b. GD1b, GT1b and GQ1b were lower in crushed than in control LST and SN between days zero and four. In LST, GM3 and LM1 remained constant for four days, but were elevated at one and two weeks, whereas GD1a was elevated at all times. Indeed, GD1a is the major recently synthesized ganglioside that is transported into LST and SN two to four days after trauma, suggesting that it may play an important role in regeneration. Indices of oligosaccharide complexity and degree of sialylation indicated that between two and four days following crush, gangliosides in DRG had more complex oligosaccharides and more sialic acid residues than in either controls or in DRG of crushed nerves at one and two weeks post-crush. The degrees of ganglioside sialylation and oligosaccharide complexity in crushed LST and SN were lower than in control specimens between one and seven days after crush. Changes in the ganglioside composition of peripheral nerve following trauma may be important for axonal regeneration.     

Neuroimmunology of gangliosides in human neurons and glial cells in culture

Gangliosides (sialic-acid-bearing glycolipids) have received attention in recent years because of their role in cell recognition phenomena, synaptic transmission, memory generation, and nerve regeneration in the fields of neurosciences. It is suggested that each brain region or each neural cell type may contain a specific and characteristic set of gangliosides. We have investigated the immunocytochemical localization of several classes of gangliosides that include GM1, GM4, GD3, and GQ gangliosides on the cell surface of various cell types found in human neural cell cultures with antibodies specific for these gangliosides. Cell cultures were obtained from adult human brains and fetal human dorsal root ganglia and spinal cord and cultured in vitro for the period up to 6 months and utilized for the ganglioside immunocytochemistry. It was demonstrated that GM1 ganglioside was present in all galactocerebroside-positive oligodendrocytes and most of glial fibrillary acid protein (GFAP)-positive astrocytes (80%), most of neurofilament-positive neurons (80%), 50-70% of Schwann cells, and 5-10% of fibronectin-positive fibroblasts; GM4 ganglioside could be detected in all oligodendrocytes, 80% of astrocytes, and 50% of Schwann cells, while no staining was found in neurons or fibroblasts; GD3 ganglioside was present in all oligodendrocytes and 5-10% of astrocytes but not in neurons, Schwann cells, or fibroblasts; and all of fetal CNS neurons and approximately 80-90% of fetal dorsal root ganglia (DRG) neurons and a small percentage of astrocytes (10-20% in fetal and less than 1% in adult astrocytes) was labeled by A2B5 antibody which is specific for GQ ganglioside, while this antibody did not stain cell surface of oligodendrocytes, Schwann cells, or fibroblasts. Three classes of gangliosides, GM1, GM4, and GD3 were found to be definite components of fetal and adult human oligodendroglial plasma membrane, while GM1 and GM4 gangliosides were detected on the surface of most astrocytes. Only a minor population of astrocytes from both fetal and adult human CNS contained GD3 and GQ gangliosides. Two classes of gangliosides, GM1 and GQ, were detected on the surface of fetal human neurons. More than half of fetal Schwann cells reacted to GM1 and GM4 antibodies but did not to GD3 or GQ antibodies. We recognized the presence of a specific and characteristic set of gangliosides on the cell surface of different human neural cell types and these findings should facilitate further investigation of the precise biological activity of these gangliosides.     

Gangliosides of human cerebrospinal fluid in various neurologic diseases.

Simultaneous profile determination and quantification of human cerebrospinal fluid (CSF) gangliosides in various neurologic diseases (n = 71) was examined. Gangliosides were extracted with methanol/chloroform from clinically available amounts of CSF (4-5 ml), then separated and quantified by high-performance thin-layer chromatography (HPTLC) and direct densitometry. Based on chromatographic comparison with standards, the percentage of lipid-bound NeuAc positive fractions in 'normal' CSF samples were: GM1 (II3 NeuAc-GgOse4Cer) (3%); GD3 (II3 NeuAc2-Lac-Cer) (4%); GD1a (IV3 NeuAc, II3 NeuAc-GgOse4 Cer) (15%); X1 (3%); GD1b (II3(NeuAc)2-GgOse4 Cer) (16%); X2 (4%); GT1b (IV3 NeuAc, II3(NeuAc)2-GgOse4-Cer) (40%); and GQ1b (IV3(NeuAc)2, II3(NeuAc)2-GgOse4-Cer (15%). Similarity between CSF and CSF and human cerebellar cortex, particularly in proportion of "b" series gangliosides (GQ1b, GT1b, GD1b), could be observed. A higher proportion of GD1a ganglioside, with decreased GQ1b was found in infancy. The total ganglioside content (mean +/- 2 SD) varied between 645-894 micrograms/l. Significant alterations of the CSF ganglioside profile, with an increase in less polar gangliosides, GM3 and GD3, correlated with the blood-brain barrier dysfunction (CSF hemorrhages, compressive syndrome), or some malignant processes (metastatic brain melanoma). A statistically significant increase in the content of total CSF gangliosides was found in the following groups of patients as compared to controls: (1) ischemic cerebrovascular accident (CVI) with good outcome (P less than 0.02); (2) peripheral neuropathy and polyneuropathy (P less than 0.001) and (3) intravertebral discopathy (P less than 0.05). A significant decrease in the content of total CSF gangliosides was found in CVI group with lethal outcome (P less than 0.05).     

Ganglioside lateralization in the brain of female rats

The ganglioside composition of the cerebral hemispheres of young and adult rats of either sex has been herein assessed for the first time. In females, the total ganglioside content at any age, the content of GM1, GD1a, and GD1b at 8 days, and the content of GM1, GD1b, GT1b, and GQ1b at 60 days were higher in the right than in the left hemisphere. In males, no difference was observed. Concerning the ceramide moiety, a difference was displayed by C18:1 long-chain base in GD1a, whose proportion was higher in the left than in the right hemisphere of females aged 8 days. The comparison between homolateral hemispheres of rats of different sex revealed several differences. On average, in 8-day-old animals, the content of gangliosides was higher in females than in males. At 60 days the amount of gangliosides was on average lower in females than in males, even if with some exception. The data obtained with the current investigation show the existence of a ganglioside lateralization in rat brain, exclusively in females, and almost entirely at charge of the oligosaccharide portion. Moreover, age-dependent changes of ganglioside pattern and content show a dependence on brain lateralization. J. Neurosci. Res. 50:643–648, 1997. © 1997 Wiley-Liss, Inc.     

Sensory neuropathy associated with monoclonal immunoglobulin M to GD1b ganglioside

A 67-year-old woman with a sensory polyneuropathy was shown to have a serum monoclonal immunoglobulin M λ antibody with a titer of 1:10,000 toward GD1b ganglioside. The immunoglobulin M also reacted with some other gangliosides containing disialosyl groups such as GD2, GD3, and GQ1b, but it did not react with GM1, LM1, or GD1a. The principal reactive ganglioside in human cauda equina was GD1b.     

Effect of colchicine on ganglioside composition of rat primary culture neurons.

Developmental changes in gangliosides in the course of neurite outgrowth were examined in dissociated fetal rat cerebral neurons in culture. About a 2-fold increase in ganglioside levels was seen with the progression of neurite formation for up to 24 h in predominantly neuronal cultures. Ganglioside patterns appeared to be unchanged during the first 24 h, subsequently consisted of higher amounts of GD3 and b-series gangliosides (such as GD1b, GT1b, and GQ1b), and lower amounts of a-series gangliosides (GM1 and GD1a). Although the addition of colchicine to the cell growth medium inhibited neurite outgrowth in developing neurons, little if any differences in ganglioside patterns were found between control and colchicine-treated cells. Ganglioside levels decreased slightly in colchicine-treated cells in agreement with the decrease in cell attachment to culture dishes. Although colchicine treatment 8 h after plating caused complete retraction of formed neurites, the ganglioside level of the cells continued to increase during the following 16-hour incubation. Thus, the data suggest that ganglioside synthesis in differentiating neurons does not primarily accompany the expansion in cell surfaces due to neurite formation, and raises the possibility that a large proportion of gangliosides is retained in intracellular compartments.     

Ganglioside changes in the chicken optic lobes as biochemical indicators of brain development and maturation

The development profiles of 16 different gangliosides of the optic lobes of the chicken were followed from the sixth day of incubation to the tenth posthatching week and correlated to known morphological development. Several, previously undetected novel fractions occurred between the sixth and tenth embryonic days. According to their migration rates on TLC-plates 4 of them may be GT3, GT2, GT1c, GQ1c. Three even more slowly migrating fractions represent penta-, hexa-, and septa-sialogangliosides. At the sixth day of incubation, characterized by maximal proliferation of neuro-epithelial cells, the optic lobes contained predominantly GD3.Up to the eleventh day of incubation, parallel to decreased mitotic activity, maximal cell migration and neuron differentiation, GD3, GD2, and GT3 decreased in favor of newly detected polysialogangliosides. Thereafter, up to hatching, parallel to increased growth and arborization of dendrites and axons as well as synaptogenesis, the newly detected polysialogangliosides decreased in favor of GD1b, GT1b, GQ1b, and GD1a.At hatching the myelin-specific GM4 appeared, reaching about 8% of total ganglioside sialic acid after 10 weeks. Likewise a fraction, migrating somewhat faster than GM1,increased. This band, named GM1', is suggested to be also myelin-associated. The other monosialogangliosides were always minor fractions, none exceeding 4% of total ganglioside sialic acid.     

Influence of monensin on ganglioside anabolism and neurite stability in cultured chick neurons

We have examined the effects of monensin, a monovalent cationophore that disrupts exo- and endocytosis of membrane vesicles and diminishes Golgi anabolic function, on the incorporation of [3H]-galactose into glycosphingolipids in neurited primary cultures of chick embryo central nervous system neurons. A linear rate of incorporation into all ganglioside species from extracellular-labeled galactose was observed. Specific activity of anabolic labeling was markedly lower in GT1b and GQ1b than in the other major gangliosides of the embryonic neuron (GM3, GD3; GM2, GD2; GM1, GD1a, GD1b). With 100 nM monensin in the extracellular medium, the rate of labeling of GT1b diminished markedly to 20% of control; GD1a, GD1b, and GD2, to 35%; GQ1B to 48%; GD3 to 60%. Vigorous incorporation of label into GM3 was entirely undiminished by monensin. From these findings, it is suggested that ganglioside biosynthesis is compartmentalized in the cytodifferentiating embryonic neuron, with GM3 entirely, and GD3 and GQ1b partially, an extra-Golgi product. Extensive loss of neurites that occurred after several hours of exposure of the neurons to monensin could not be correlated directly with decreased ganglioside anabolism.     

Antibodies to GD3, GT3, and O-acetylated species in Guillain-Barré and Fisher's syndromes: their association with cranial nerve dysfunction.

We examined serum antibodies to the four fetal antigens GD3, O-acetyl GD3, GT3, and O-acetyl GT3 ganglioside in patients with Guillain-Barré syndrome (GBS) or its variant Fisher's syndrome (FS). The patients with FS more often had significant IgG antibodies against GD3, GT3, and O-acetyl GT3 than did the healthy controls. Furthermore, anti-GD3 and anti-GT3 IgG antibodies were more often significantly present in the patients with FS than in those with GBS. IgG antibody to GD3, GT3, and O-acetyl GT3 had a significant association with the presence of ophthalmoparesis. These antibodies, however, cross-reacted with GQ1b and we detected no antibodies which specifically reacted with fetal gangliosides. In addition, oculomotor involvement was more closely related to IgG antibodies to GQ1b than to those to fetal gangliosides. No evidence was obtained that the serum antibodies to these fetal gangliosides are associated with specific neurologic signs of cranial nerves.     

Molecular Microscopy of Brain Gangliosides: Illustrating their Distribution in Hippocampal Cell Layers

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Brain gangliosides in birds with different types of postnatal development (nidifugous and nidicolous type)

Developmental profiles of 14 different brain gangliosides were followed from the first day after hatching to the adult stage in two bird species representing different strategies of posthatch development: the nidifugous type (leaving the nest directly post-hatch, e.g. quail) and the nidicolous type (remaining for longer period in the nest, e.g. finch). In the zebra finch, parallel with a striking increase in ganglioside concentration, two main postnatal changes in the ganglioside composition occurred: after hatching, concomittantly to an increased outgrowth of nerve fibers and synaptogenesis, the polysialogangliosides GQ1b and GP decreased in favour of the less polar fractions GD1b, GD1a and GT1b. The second period of changes started with the onset of myelination and was characterized by an increase of GM1 and GM1'. The results obtained for quails were in close agreement with those of chicken, showing only slight postnatal changes due to the nearly completed morphological differentiation. These data show that gangliosides are useful biochemical markers for brain development, indicating successive periods of brain maturation by means of preferential biosynthesis of specific fractions regardless of the type of development.     

Detection of anti-ganglioside antibodies in Guillain-Barré syndrome and its variants by the agglutination assay

Sera from 40 patients with Guillain-Barré syndrome (GBS), including the subtypes acute inflammatory demyelinating polyneuropathy (AIDP), acute motor axonal neuropathy (AMAN), acute motor and sensory axonal neuropathy (AMSAN), and Miller Fisher syndrome (MFS) were examined for the presence of anti-ganglioside antibodies using the ganglioside agglutination assay, and the enzyme-linked immunosorbent assay (ELISA). In the ELISA system, sera were tested for IgM and IgG antibodies to GM1, GM2, GD1a, GD1b, GT1b, and GQ1b gangliosides. Antibodies to gangliosides were detected in 21 (53%) of the GBS patients by agglutination assay and in 17 (43%) of the patients by ELISA. Some of the sera reacted with more than one ganglioside. Antibodies were not found in the control sera that were studied. The agglutination assay may be useful for rapid screening of GBS sera for antibodies to multiple gangliosides.     

Ganglioside reactive antibodies in the neuropathy associated with celiac disease

We tested patients with celiac disease (CD) for the presence of serum anti-ganglioside antibodies. Six of twenty-seven patient sera were reactive against brain gangliosides by an agglutination immunoassay. Neurological examination in all six revealed the presence of distal sensory loss, consistent with the diagnosis of peripheral neuropathy. When tested by ELISA for antibodies to isolated GM1, GM2, GD1a, GD1b, GT1b, and GQ1b gangliosides, all six were positive for IgG antibodies to at least one. The neuropathy of celiac disease may be autoimmune and associated with anti-ganglioside antibodies. The presence of IgG reactivity furthermore implicates a T cell-mediated response to ganglioside antigens.     

Ethanol-induced increase of sphingosine recycling for ganglioside biosynthesis: a study performed on cerebellar granule cells in culture

The effect of ethanol on ganglioside metabolism was assessed in cultured rat cerebellar granule cells. Cells were incubated in the presence of tritiated serine or galactose, and the synthesis of radioactive gangliosides was followed. The rate of de novo biosynthesis of gangliosides labeled in the oligosaccharide moiety (deriving from tritiated galactose) was not affected by the presence of ethanol. On the contrary, the biosynthesis of gangliosides labeled in the ceramide long chain base moiety (deriving from tritiated serine), dramatically decreased in the presence of alcohol. These results suggest that the gap between the extent of the biosynthesis of lipid and polar portions observed in the presence of ethanol, is filled by an increased recycling of sphingosine produced from ganglioside degradation. This hypothesis was confirmed by pulse-chase experiments with GM1 ganglioside, tritiated in the sphingosine moiety, and following radiolabeled gangliosides deriving from its metabolic processing. In fact, the radioactivity carried by gangliosides whose labeling could derive exclusively (GD1b + GT1b) or partially (GD1a) from the recycling of catabolic radiolabeled sphingosine, dramatically increased in ethanol-treated cells during the chase period. Taken together, these results suggest that ethanol increases ceramide sphingosine recycling for ganglioside biosynthesis.