Effects of neuroblastoma tumor gangliosides on platelet adhesion to collagen

Gangliosides, sialic acid-containing glycosphingolipids, enhance tumor formation in experimental animals and are associated with tumor progression and metastasis in humans. The mechanism(s) for this activity is (are) unknown. One possibility is enhanced platelet activation, since the interaction of platelets with tumor cells contributes to tumor cell arrest in the vascular compartment. We have previously shown that neuroblastoma tumor gangliosides (NBTG) enhance platelet adenosine triphosphate (ATP) secretion, aggregation, and adhesion. We determined that these NBTG effects are specific for collagen and are mediated through an (( integrin-dependent mechanism. This report describes the effects of NBTG on a physiologically relevant model of collagen-( interaction. Platelet adhesion to immobilized native collagen fibers similar to those found in the extracellular matrix of blood vessels was determined. Platelet adhesion is enhanced by NBTG in a concentration-dependent manner. Incubation with concentrations of 1 and 10 m NBTG increased platelet adhesion by 9% and 52%, respectively, compared to less than 1% in controls not incubated with gangliosides (P = 0.001 and P < 0.0001, respectively). In addition to increasing the number of adherent platelets, NBTG promoted more rapid attachment. In NBTG-incubated platelets, platelet adhesion began after a 5-min lag phase and was maximal at 30 min compared to a 20-min lag phase and maximal adhesion at 60 min for control platelets. At 30 min this difference was significant (P = 0.017); however, by 120 min there was no difference between NBTG and controls (P = 0.259). NBTG also induces platelet adhesion at collagen concentrations (0.1 g) that failed to support adhesion of control platelets. These effects of NBTG require Mg or Mn ions but are not supported by Zn or Ca ions. Furthermore, preincubation of platelets with a blocking antibody (6F1) to the integrin collagen receptor ( abrogates all of the effects of NBTG. These results indicate that tumor gangliosides enhance platelet adhesion to extracellular matrix collagen and promote rapid stabilization of the collagen-(( interaction, the initial steps in platelet activation.     

Human monoclonal IgM with autoantibody activity against two gangliosides (GM1 and GD1b) in a patient with motor neuron syndrome.

Small amounts of oligoclonal immunoglobulins were detected by Western blotting in the serum from a patient with motor neuron syndrome. The prominent one, a monoclonal IgM lambda, reacted strongly with the gangliosides GM1 and GD1b and more weakly with asialo GM1, as shown by immunoenzymatic staining of thin-layer chromatograms of gangliosides, ELISA on purified glycolipid coats and immunoadsorption with purified GM1. Affinity-chromatography with purified GM1 resulted in the purification of monoclonal IgM lambda. This purified IgM and its Fab fragments showed the same pattern of reactivity with gangliosides as that observed with whole serum. Such monoclonal IgM could be responsible for motor neuron diseases in some patients with overt or barely detectable monoclonal gammopathies.     

Reversibility of ganglioside effects on astrocyte morphology

The B-subunit of cholera toxin (BCT) induces a morphological change in cultured rat cerebral astrocytes from flat (epithelioid) to stellate (process-bearing). This stellation is reversed by the gangliosides GM1 and GD1a at concentrations of 10 microM or higher. Upon changing to a ganglioside-free medium, the flat astrocytes reacquire the stellate morphology within 3 hr, indicating that the antistellation effect of gangliosides is reversible. The possibility that this reversibility was due to a loss of exogenously acquired gangliosides from the cell membrane can be ruled out since pretreatment with GM1, but not GD1a, which does not bind BCT, results in an increased responsiveness to BCT, which was identical whether measured immediately after withdrawal of the ganglioside or 3 hr later. Asialo-GM1, which neither binds BCT nor reverses BCT-induced stellation by itself, prevents the return to stellation after withdrawal of the gangliosides. These data suggest that while gangliosides remain associated with the cell, their effect on astrocytes can change from opposing to permitting the stellate morphology.     

Expression of GD3 ganglioside by developing rat cerebellar Purkinje cells in situ

GD3 is a major ganglioside of the immature vertebrate CNS, and its expression is suggested to be characteristic of immature neuroectodermal cells. Using immunocytochemistry on cryostat sections of developing rat cerebellum with a monoclonal antibody specific for GD3, we have found that GD3 begins to be expressed on the plasma membrane of Purkinje cell bodies and dendrites beginning at postnatal day 7. Staining became brighter as the dendritic tree of the cells enlarged. As the Purkinje cells began to mature in different folia, they became GD3+, until by 15 days postnatal all Purkinje cells were GD3+. Positive staining of the dendritic tree was still present in the adult cerebellum. Using a monoclonal antibody 7-8D2, which recognizes cerebellar granule cells and their axons (the parallel fibres), and polyclonal antibodies against a synaptic vesicle component synaptophysin, double-immunofluorescence staining together with anti-GD3 antibodies suggested that the appearance of GD3 immunoreactivity did not correlate either with the ingrowth of parallel fibres or the presence of their synapses on Purkinje cell dendrites. However, comparison with earlier morphological studies showed that the appearance of GD3 immunoreactivity correlated well with the formation of climbing fibre synapses on Purkinje cell dendrites and the onset of the rapid expansion of the dendritic tree. These results are in keeping with the idea that elevated GD3 concentrations are found in certain cell types during periods of rapid growth or high metabolic activity but also show that this is not only restricted to immature cells.     

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.     

POLYUNSATURATED FATTY ACID LIPIDOSIS

ABSTRACT: Svennerholm, L., Hagberg, B., Haltia, M., Sourander, P. and Vanier, M.-T. (Departments of Neurochemistry, Paediatrics and Pathology I, University of Gothenburg, Sweden and Pathology II, University of Helsinki, Finland). Polyunsaturated fatty acid lipidosis (PFAL). Acta Paediatr Scand, 64:489, 1975.–Lipid analyses were performed on cerebral tissue from three children who had died of a progressive encephalopathy and from one living child in an early stage of the disease. In the terminal stage of the disease, the cortex and white matter content of all lipid classes, particularly the sphingolipids, were very low. The concentration of gangliosides of the cerebral cortex was 10%, and of cerebrosides in white matter, 2–3 % of the normal values for the age. The porportion of the minor gangliosides with short carbohydrate chains was increased because the reduction affected mainly the four major brain gangliosides G_M1, G_D1a. G_D1b and G_T1. In the child from whom the biopsy specimen was obtained in an early phase of the disease the cerebral lipid pattern appeared to be normal. A patient who had died of neuronal ceroid-lipofuscinosis (Janský-Bielschowsky) did not show any major lipid changes. The fatty acid patterns of the phosphoglycerides showed such changes as have never been observed in any other disease. In the three advanced cases the fatty acid compositions in cerebral cortex and white matter were identical. In ethanolamine phosphoglycerides the proportions of 18:1 and 20:4 ( n -6) were increased, while those of 22:4 ( n -6) and 22:6 ( n -3) were markedly diminished. Similar changes in the fatty acid pat-. terns were found in the other phosphoglycerides. In the early phase of the disease 22:4 (n-6) was decreased and 18:1 increased. We propose that this new disease be termed polyunsaturated fatty acid lipidosis (PFAL).     

Developmental studies of the catabolism of sialocompounds by endogenous neuraminidase in the chick retina

The effect of a “particle bound” neuraminidase acting on endogenous sialocompounds (glycoproteins and gangliosides) in chick retina during development has been investigated. The total “particle bound” N-acetyl neuraminic acid (NeuNAc) per gram wet weight, increased fourfold between 8-day-old embryos and hatching. The ratio of ganglioside to glycoprotein-bound NeuNAc changed during retina ontogenesis: it approached 5 for the 8-day-old embryo, decreased to approximately 2 in the 15-day embryo and was approximately one from hatching to adulthood. The lowest neuraminidase activity was observed in the 8-day-old embryo and the highest was obtained at hatching. For each age of retina and for each incubation time 90% of the NeuNAc released by neuraminidase action was of ganglioside origin; the ganglioside-bound NeuNAc therefore seems to be a more accessible substrate for the chick retinal endogenous neuraminidase than that bound to glyco-proteins. The release of NeuNAc from gangliosides leads to drastic changes in the ganglioside patterns: G_M3, G_D3, G_D1a, G_T1, G_Q1 are affected by the enzyme and disappear almost completely after 4 hr of incubation. In contrast, a ganglioside migrating like G_M1 accumulates and does not seem to be hydrolysed. G_D1b undergoes a transient increase within 1 hr which is consistent with the corresponding decrease of G_T1. By comparing the absolute quantities of gangliosides that disappeared after 30 min of incubation, it is shown that G_D1a is the main glycolipid attacked by the enzyme. The greater hydrolysis of gangliosides compared to the particulate glycoproteins may indicate that gangliosides may play a greater role in mechanisms which require sialic acid movements than that played by the glycoproteins. The possible role of the “particle bound” neuraminidase in the retina is discussed.     

Tay-sachs disease brain cells in culture: Mobilization of stored GM2 after concanavalin A-mediated uptake of hexosaminidase A

A human Tay-Sachs disease (TSD) fetal-brain-cell line is a useful model for the disease since the cells lack hexosaminidase A and accumulate the ganglioside, G_M2. This brain-cell line was used to assess the effect of hexosaminidase A treatment on G_M2 storage material. Entry of placental hexosaminidase A into the cells was obtained by pretreatment of the cultures with concanavalin A. Cells were analyzed periodically during six days. During the course of the experiment, G_M2 in the cells decreased by approximately 50%. A substantial amount of hexosaminidase A was maintained in the cultures throughout the experiment. This strategy was successful in mobilizing stored G_M2 in TSD brain-cell cultures. Therefore, the activating factor needed for hexosaminidase A activity must be present in TSD-cultured brain cells.     

Bacterial toxins: an outlook

An overview of some current issues in bacterial toxinology is presented.