Coexpression of lactosyl and gangliotetraosyl gangliosides in rat cerebellar radial glial cells in culture

The expression of gangliosides of the lactosylceramide (LC) and of the gangliotetraosylceramide (GTC) series on the surface of cells from rat embryonic cerebellar tissue was investigated by double-color indirect immunofluorescence. GD3 was assumed to be representative of LC and was detected using a specific monoclonal antibody. GM1 was assumed to be representative of GTC and was detected using the binding of cholera toxin followed by the binding of cholera toxin antibodies. The expression of polysialosylated GTC (polysialosyl-GTC) was detected using the cholera toxin-cholera toxin antibody experimental approach after conversion of polysialosyl-GTC to GM1 by treatment of the cells with neuraminidase. To distinguish the major neural cell types present in the cultures the expression of the following cell type-specific markers was investigated: neuron-specific enolase and microtubule-associated protein-2 (MAP-2) as probes for neuronal cells and the intermediate filament protein glial fibrillar acidic protein (GFAP) as a probe for astroglial cells. More than 80% of cells dissociated from cerebellar tissue of 15-day-old rat embryos (E15) are positive for the expression of GD3 and about 50% for the expression of GM1 and polysialosyl-GTC, but most are negative for the expression of neuron-specific enolase, MAP-2, and GFAP. After culturing for 4 days (E15 + 4) most cells that show characteristics of neuronal cells are positive for the expression of polysialosyl-GTC and "inactivate" the expression of GD3. Most cells with characteristics of radial and stellate glial cells are also positive for the expression of polysialosyl-GTC, but unlike neuron-like cells, they do not "inactivate" the expression of GD3.     

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.     

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.     

Changes in ganglioside profile in chick embryo retina: Studies on tissue and cell cultures

The developmental profile of gangliosides in the neural retina of the chick embryo is characterized by a progressive decrease in the concentration of GD3 complex from a high level on day 6; by a continuous increase in GD1a concentration; and by less striking increases in GD1b and GT1b concentrations during the growth phase; GM1 increases in the post-mitotic retina. Gangliosides were analyzed by thin layer chromatography and by densitometry of the TLC plates. (Ganglioside nomenclature is according to Svennerholm.³⁷)We have examined comparatively ganglioside changes in organ cultures of retina tissue from 6 day embryos (R³⁶), in cell aggregates and in primary monolayer cultures of R²⁶ cells, all maintained for 6 days in vitro. In all cases, the pattern of ganglioside changes was qualitatively similar to that in the retina in vivo. These results suggest that, unlike some other aspects of retina differentiation, the progression of ganglioside changes in the 6–12 day embryonic retina is not critically dependent on histotypic cell organization or on specific contact-dependent cell interactions; these changes appear to be largely preprogrammed in the cells at some earlier phase of development.     

Uniform distribution and similar turnover rates of individual gangliosides along axons of retinal ganglion cells in the chicken

In 5-month-old chickens, an intracranial injection of N-[³H]acetylmannosamine led to a labelling of all optic lobe ganglioside species in a fashion parallelling the relative ganglioside distribution. In contrast, after an intraocular injection of the same precursor, the optic nerve and the optic lobe connected to the injected eye, possessed an exceptionally high labelling of GD1a (in comparison with GD1a-sialic acid), and only negligible incorporation of radioactivity into the myelin-specific GM4 and into a fraction migrating close to GM1. Subtracting both these very low labelling fractions from the total gave a percentage distribution of ganglioside sialic acid which now corresponded well to the distribution of radioactivity along the whole optic nerve, including the region of nerve terminals in the optic lobe. This pattern of ganglioside labelling, which indicates that GD1a carries about 60% of total ganglioside sialic acid of retinal ganglion cell axons, did not change remarkably during post-hatching development up to 5 months. Long-time incorporation studies revealed similar turnover rates of the main retinal ganglion cell gangliosides. The average half-lives were 34 (GD1a), 35 (GQ1b), 36.3 (GT1b) and 38.5 days (GD3). The findings suggest that the retinal ganglion cell axons and their presynaptic terminals possess a similar ganglioside pattern, characterized by a high content of GD1a.     

A gradient molecule in developing rat retina: expression of 9-O-acetyl GD3 in relation to cell type, developmental age, and GD3 ganglioside

In the embryonic and postnatal rat retina a cell surface antigen that is detected by monoclonal antibody JONES is distributed in a dorsoventral gradient. Biochemical analysis has determined that the antigen is a modified ganglioside, 9-O-acetyl GD3. In the present study, the distributions of 9-O-acetyl GD3 and its possible precursor GD3 in developing rat retina were compared immunocytochemically using specific monoclonal antibodies JONES and R24. On embryonic day 13 (E13) immunoreactivity to JONES was localized to central retina; however, R24 stained throughout the retinal epithelium. By E20, when JONES binding was distributed in a gradient along the dorsoventral axis, R24 again stained dorsal and ventral regions with uniform intensity. Analysis of freshly dissociated retinal cells further revealed that GD3 and 9-O-acetyl GD3 expressions do not necessarily coincide. At E15 and postnatal day 2 (PN2), the majority of cells (78 and 92%, respectively) were immunolabeled by antibody to GD3, while between E15 and PN2 the percentage of cells immunolabeled by antibody to 9-O-acetyl GD3 rose from 19 to 68%. By PN4, labeling decreased for both molecules; however, the rate of decline in 9-O-acetyl GD3 labeling was more pronounced. Regulation of the numbers of JONES-positive cells does not appear to depend on interaction with the extraretinal environment, for in neural retina explanted at E15 the proportions of 9-O-acetyl GD3-bearing cells was found to be similar to the percentage observed in neural retina developing to an equivalent age in vivo. Experiments to identify the retinal cell types bearing 9-O-acetyl GD3 revealed that it is expressed by both neurons and glia growing in monolayer cultures of rat perinatal neural retina. Examination of freshly dissociated retinal cells following simultaneous labeling for some specific cell types and 9-O-acetyl GD3 demonstrated that the latter determinant is present on photoreceptor, amacrine, and ganglion cells. For each neuronal cell type, however, not all of the cells were immunoreactive with JONES. We conclude that the differences in the percentages of JONES- and R24-positive cells, and in particular the different rates at which JONES and R24 staining declined with age, indicate that the expression of the JONES epitope is regulated with some independence from parent ganglioside GD3.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ganglioside alterations in guinea pig brains at end stages of experimental Creutzfeldt-Jakob disease.

Gangliosides were isolated from guinea pig brains at the end stages of experimental Creutzfeldt-Jakob disease. Quantitative analyses revealed marked decreases of ganglioside levels in pathologically devastated tissues such as cerebral cortex (-21%), basal ganglia and thalamus (-18%), and brain stem (-23%). The cerebellum revealed only minor pathological abnormalities and its ganglioside level remained unchanged. Thin-layer chromatography of the Creutzfeldt-Jakob brain gangliosides showed aberrant ganglioside patterns in all regions studied, including the cerebellum. With some exceptions, a trend in ganglioside pattern changes was detected which consisted of proliferation of GM3, GD3, GD2 and loss of GM1, GD1a, GD1b and GT1b.     

Developmental changes of ganglioside expressions in postnatal rat cerebellar cortex

We previously described the differential distribution of gangliosides in adult rat brain as detected by specific antibodies. We report here the distribution of gangliosides during the development of postnatal rat cerebellum by an immunofluorescence technique with mouse monoclonal antibodies (mAbs). Eleven mAbs that specifically recognize each ganglioside were used. Our study revealed that the expression of each ganglioside changed dramatically during the development. GD3 and O-Ac-GD3 were expressed intensely in the external granular layer at 1, 5, and 10 days, whereas GD2 was firstly detected in the internal granular layer at 5 days and GD1b was diffusely detected throughout all layers of the cerebellar cortex at early postnatal days. GD2 and GD1b were more intensely expressed in the granular layer at 20, 30, and 80 days, suggesting that premature granule cells express GD3 and its derivative, O-Ac-GD3, whereas mature granule cells express GD2 and GD1b intensely. On the other hand, GM1 was exclusively detected in the external granular layer and the molecular layer at 1 and 5 days. The staining sites spread gradually from these outer layers into the internal granular layer and the white matter after 10 days. The positive cells in the external granular layer and the molecular layer appeared to be Bergmann glial cells and their radially ascending cytoplasmic processes. The intensity of the staining in these specialized astroglial cells decreased gradually during postnatal days. In contrast, the expression of GQ1b was very faint at birth, but gradually increased during the development and was detected intensely in the internal granular layer, particularly in the cerebellar glomeruli in adulthood, suggesting that GQ1b expression may be associated with synapse-related structures. The developmental changes of the expression of other gangliosides were also recognized in the postnatal rat cerebellum. These results suggest that specific gangliosides may play an important role in regulating the early events responsible for the orderly formation of the cerebellar cortex.     

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.     

Protection of neuro-2a cells against calcium ionophore cytotoxicity by gangliosides.

Gangliosides are known to assert both neuritogenic and neuroprotective effects when applied to a variety of neuroblastoma and primary neuronal cultures. We have developed a model employing Neuro-2a neuroblastoma cells with Ca2+ ionophore A23187 as neurotoxic agent causing neurite retraction and eventual cell death. Gangliosides attenuated the toxicity of this substance, increasing both cell survival and neurite stability. In one series of experiments, cells were exposed to A23187 for 24 hr and then incubated in fresh medium (washout) for 18 hr; gangliosides were present at varying times. The paradigm in which cells were only preincubated (2 hr) with ganglioside provided no benefit, nor did incubation of the cells in both ionophore and ganglioside during the 24-hr exposure period. Significant protection was achieved by exposing the cells to ganglioside after washout of A23187, or continuously throughout the whole period. Bovine brain ganglioside mixture and the four major components (GM1, GD1a, GD1b, GT1b) applied individually were all effective. By contrast, GM3 and GM1-alcohol, a neutral derivative of GM1, provided little or no protection. Dichlorobenzamil, an inhibitor of the Na(+)-Ca2+ exchanger, tended to block the neurite stabilizing effect of gangliosides, suggesting that the mechanism might involve potentiation of this antiporter.     

AII amacrine cells in the mammalian retina show disabled-1 immunoreactivity

Disabled 1 (Dab1) is an adapter molecule in a signaling pathway, stimulated by Reelin, which controls cell positioning in the developing brain. It has been localized to AII amacrine cells in the mouse and guinea pig retinas. This study was conducted to identify whether Dab1 is commonly localized to AII amacrine cells in the retinas of other mammals. We investigated Dab1-labeled cells in human, rat, rabbit, and cat retinas in detail by immunocytochemistry with antisera against Dab1. Dab1 immunoreactivity was found in certain populations of amacrine cells, with lobular appendages in the outer half of the inner plexiform layer (IPL) and a bushy, smooth dendritic tree in the inner half of the IPL. Double-labeling experiments demonstrated that all Dab1-immunoreactive amacrine cells were immunoreactive to antisera against calretinin or parvalbumin (i.e., other markers for AII amacrine cells in the mammalian retina) and that they made contacts with the axon terminals of the rod bipolar cells in the IPL close to the ganglion cell layer. Furthermore, all Dab1-labeled amacrine cells showed glycine transporter-1 immunoreactivity, indicating that they are glycinergic. The peak density was relatively high in the human and rat retinas, moderate in the cat retina, and low in the rabbit retina. Together, these morphological and histochemical observations clearly indicate that Dab1 is commonly localized to AII amacrine cells and that antiserum against Dab1 is a reliable and specific marker for AII amacrine cells of diverse mammals.     

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.     

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).     

The glioma-associated gangliosides 3'-isoLM1, GD3 and GM2 show selective area expression in human glioblastoma xenografts in nude rat brains

This work describes the in vivo expression and distribution of glioma-associated gangliosides (GD3, GM2, 3'-isoLM1) in a novel human brain tumour nude rat xenograft model. In this model, the tumours, which are established directly from human glioblastoma biopsies, show extensive infiltrative growth within the rat brain. This model therefore provides an opportunity to study ganglioside expression not only within the macroscopic tumour, but also in brain areas with tumour cell infiltration. The ganglioside expression was studied by confocal microscopy of immunostained brain sections using antiganglioside monoclonal antibodies. Xenografts from four human glioblastoma multiformes were established in rats and the brains removed after 3-4 months. Ganglioside GD3 was expressed in the tumour parenchyma while ganglioside 3'-isoLM1 was more abundantly expressed in the periphery of the tumour associated with areas of tumour cell invasion. GM2 expression was only seen in one tumour, where it was located within the main tumour mass. Double staining with a pan antihuman monoclonal antibody (3B4) and the antiganglioside monoclonal antibodies confirmed that the ganglioside expression was associated with tumour cells. This work supports the concept of different biological roles for individual gangliosides and indicates that antibodies or ligands directed against GD3 and 3'-isoLM1 might be complementary when applied in the treatment of human glioblastomas.     

Localization of two calcium binding proteins, calbindin (28 kD) and parvalbumin (12 kD), in the vertebrate retina

We used immunocytochemistry to locate two calcium binding proteins, calbindin (CaB) and parvalbumin (PV), in the retina of goldfish, frog, chick, rat, guinea pig, dog, and man. The location of CaB depended on the type of dominant photoreceptor cells in birds and mammals. In cone-dominant retinas such as those of the chick, CaB-like immunoreactivity was found in the cones, cone bipolars, and ganglion cells. Amacrine cells 5-12 microns across were also labeled. In rod-dominant retinas, such as those of the rat, guinea pig, and dog, horizontal cells, small amacrine cells (about 6 microns across), and cells in the ganglion cell layer were labeled. In the human retina, which has both cones and rods in abundance, cones, cone bipolars, ganglion cells, horizontal cells, and small and large amacrine cells were labeled. In the frog and goldfish, the level of CaB-like immunoreactivity was low. In the frog, a few cones, amacrine cells, and cells in the ganglion cell layer were labeled. No immunoreactive structures were seen in the goldfish retina. PV-like immunoreactivity was found in chicks, rats, and dogs. No such immunoreactive structures were seen in the other species. In the chick, only amacrine cells were labeled. In the rat, amacrine cells and several displaced amacrine cells were labeled. In the dog, in addition to amacrine cells and displaced amacrine cells, horizontal cells were strongly labeled. Thus, PV-like immunoreactivity was found in those elements relating to the modulation of the main pathway of the visual transmission system.     

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.     

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.     

Brain gangliosides in dementia of the Alzheimer type

Gangliosides GM1, GD1a, GD1b, and GT1b were measured in nine brain regions of five patients, clinically and neuropathologically diagnosed as having dementia of the Alzheimer type (DAT), and of three control patients. Analysis of variance revealed that mean concentrations of all gangliosides analyzed were significantly lower in DAT than in control brains. The areas affected in DAT included the nucleus basalis, and entorhinal, posterior cingulate, visual, and prefrontal cortices. A significant interaction between ganglioside type and brain area indicated unequal ganglioside concentrations. Individual gangliosides had significantly different concentrations in the hippocampal, entorhinal, posterior cingulate, visual, and prefrontal cortices. Analysis of ratios of "a"-ganglioside (GM1 and GD1a) and "b"-ganglioside (GD1b and GT1b) subtypes indicated that DAT preferentially affected "b"-gangliosides. Ganglioside concentrations in nucleus basalis did not correlate with age at disease onset, age at death, or postmortem interval. Changes in gangliosides, observed in this study, were not correlated with classic DAT neuropathology.