Exogenous GM1 ganglioside caused G1-arrest of human diploid fibroblasts. Flow cytometric studies

Exogenous GM1 ganglioside (II3 NeuAc-Gg0se4-Cer) inhibited growth and DNA synthesis of human diploid fibroblasts, TIG-1 cells. We examined the effect of exogenous GM1 on their cell cycle traverse by flow cytometry. When the cells were partially synchronized by serum deprivation, addition of GM1 at the time of refeeding caused about 70% reduction of their reentry into S phase from the level observed in the control culture untreated with the ganglioside. However, the addition of GM1 6 h later caused only about 30% reduction of the reentry from the control level. These results suggest that the exogenous ganglioside blocks the cell cycle traverse in an early G1 period. This is consistent with the fact that GM1-treated cells showed a high level of histone H1(0) similar to that observed in G1-arrested cells in confluent culture.     

Ganglioside control over IL-4 priming and cytokine production in activated T cells

Our previous studies have shown that the enzymatic activities of Neu-1, an endogenous sialidase encoded in the murine MHC, are involved in promoting IL-4 synthesis by naive CD4(+)T cells. Our present studies have characterized responsible sialoconjugate targets of Neu-1 and questioned possible biochemical mechanisms responsible for their regulatory influences on IL-4 gene expression. These studies determined that treatment of T cells with the naturally occurring ganglioside GM3 inhibited the production of IL-4 without affecting the production of IL-2. An analysis of IL-4-primed CD4(+)T cells further demonstrated that GM3 treatment specifically inhibited the restimulated production of IL-4, IL-5 and IL-13, without inhibiting the production of IL-2 and IFN-gamma. The inhibitory effects of GM3 could be overcome by treatment with thapsigargin or ionomycin, suggesting ganglioside regulation occurs upstream of activation-induced calcium mobilization. GM3 treatment attenuated the level of calcium influx following CD3epsilon crosslinking, and CD4(+)T cells from Neu-1-deficient B10.SM strain mice (neu-1(a)and IL-4-deficient) expressed reduced levels of intracellular calcium following activation. Our results indicate that activities by membrane gangliosides can influence the cytokine programs in CD4(+)T cells, possibly through the modulation of calcium responses induced by T cell activation.     

Distribution of ganglioside GM3 in the rat ovary after gonadotropin stimulation.

Gangliosides are ubiquitous membrane components in mammalian cells and are suggested to play important roles in various cell functions, such as cell-cell recognition, differentiation and transmembrane signalling. Ovaries have been shown to contain GM3 as a major ganglioside. To study GM3 distribution during gonadotropin stimulation in the hypophysectomized rat ovary, ovarian sections and cultured granulosa cells were stained with specific monoclonal antibody against GM3. Interstitial cells of follicles of immature hypophysectomized rat ovary expressed ganglioside GM3. Theca cells of early antral follicles but not primary follicles expressed GM3. No granulosa cells of these follicles expressed GM3. When a surge dose of FSH/LH was injected, Graafian follicles were formed and GM3 expression was detected in granulosa cells of these follicles. After ovulation, cumulus cells kept expressing GM3 in the ampulla region of ovulated oviduct. The follicles did not show GM3 expression in their granulosa cells after an ovulatory dose of FSH/LH. At 48 h after in vitro culture with FSH/LH of granulosa cells from preantral follicles, GM3 was expressed to a detectable extent on the outer part of the granulosa layer. Finally, at 72 h after culture, all granulosa cells became positive to anti-GM3 antibody. These data suggest that the expression of ganglioside GM3 in the hypophysectomized rat ovary is spatiotemporally regulated by FSH/LH during follicular development and ovulation.     

Chemical validation of molecular mimicry: interaction of cholera toxin with Campylobacter lipooligosaccharides

It is generally believed that molecular mimicry between bacterial lipooligosaccharide (LOS) and nerve glycolipids may play an important pathogenic role in immune-mediated peripheral neuropathy. One of the putative infectious agents is Campylobacter jejuni (C. jejuni). To elucidate the structural basis for the molecular mimicry, we investigated the structure of the lipooligosaccharide (LOS) fraction of C. jejuni, strain HS19, and found that it includes at least two components, characterized as fast-and slow-moving bands (LF and LS) by thin-layer chromatography as revealed by cholera toxin B subunit (Ctxb) overlay. Structural analysis of the oligosaccharide portion of LS established that it had the following structure: Gal-GalNAc-(NeuAc)Gal-Hep-(Glc;PO₃H)Hep-Kdo. The GM1-like epitope was validated by a terminal tetrasaccharide unit within this structure. On the other hand, analysis of LF revealed an entirely different structure: 1, 4′-bisphosphoryl glucosamine disaccharide N, N’-acylated by 3-(2-hydroxytetracosanoyloxy)octadecanoic acid at 2- and 2′-positions, which is consistent with that of lipid A. No GM1-like epitope was observed in LF. Both LS and LF interacted with Ctxb as demonstrated by TLC-overlay and sucrose density gradient centrifugation. Surprisingly, LF does not have the basic GM1 structure for interacting with Ctxb. Instead, the affinity of LF to Ctxb required that one or both of the phosphate groups be present in the glucosamine disaccharide residue because after alkaline phosphatase treatment the dephosphorylated LF was unable to bind to Ctxb. We conclude that LS is likely the component contributing to GM1-mimicry in autoimmune peripheral neuropathy and that the role of LF is not clear but may be associated with the initial activation of autoreactive T cells. Ganglioside nomenclature is based on that of Svennerholm [50]     

Expression of glycosphingolipids in lymph nodes of mice lacking TNF receptor 1: biochemical and flow cytometry analysis

The expression of gangliosides and neutral glycosphingolipids (GSLs) in the lymph nodes of mice lacking the gene for the tumour necrosis factor-alpha receptor p55 (TNFR1) has been investigated. GSL expression in the tissues of mice homozygous (TNFR1-/-) or heterozygous (TNFR1+/-) for the gene deletion was analysed by flow cytometry and high-performance thin-layer chromatography (HPTLC) followed by immunostaining with specific antibodies. HPTLC immunostaining revealed that lymph nodes from TNFR1-/- mice had reduced expression of ganglioside GM1b and GalNAc-GM1b, neolacto-series gangliosides, as well as the globo- (Gb3, Gb4 and Gb5) and ganglio-series (Gg3 and Gg4) neutral GSLs. Flow cytometry of freshly isolated lymph node cells showed no significant differences in GSL expression, except for the GalNAc-GM1b ganglioside, which was less abundant on T lymphocytes from TNFR1-/- lymph nodes. In TNFR1-/- mice, GalNAc-GM1b+/CD4+ T cells were twofold less abundant (3.8% vs 7.6% in the control mice), whereas GalNAc-GM1b+/CD8+ T cells were fourfold less abundant (5.0% vs 20.2% in the control mice). This study provides in vivo evidence that TNF signalling via the TNFR1 is important for the activation of GM1b-type ganglioside biosynthetic pathway in CD8 T lymphocytes, suggesting its possible role in the effector T lymphocyte function.     

Ganglioside GM3 inhibits the high glucose- and TGF-beta1-induced proliferation of rat glomerular mesangial cells

Abrupt proliferation of glomerular mesangial cells (GMCs) is a common feature in the early stage of diabetic glomerulopathy, and ganglioside GM3 (NeuAcalpha3Galbeta4Glcbeta1Cer) is thought to regulate the proliferation of many cell types. Recently, we have reported ganglioside GM3 as a modulator of glomerular hypertrophy in streptozotocin-induced diabetic rats []. This study examined whether modulation of cellular ganglioside GM3 could regulate the high glucose- and transforming growth factor-beta1 (TGF-beta1)-induced proliferation of GMCs. To pharmacologically modulate the cellular ganglioside GM3, GMCs originated from rat kidneys were cultured with exogenous ganglioside GM3 or d-threo-PDMP, an inhibitor of ganglioside synthesis, in the RPMI 1640 media containing normal (5.6 mM, NG) or high (25 mM, HG) glucose. HG, TGF-beta1 (10 ng/ml) and d-threo-PDMP (20 microM) significantly stimulated the mesangial cell proliferation, whereas these increments were remarkable attenuated by exogenous ganglioside mixture (0.1-0.2 mg/ml) or GM3 (20-100 microM) in a dose-dependent manner. The mesangial cell proliferation caused by HG, TGF-beta1 and d-threo-PDMP was closely correlated with decreases in both cellular sialic acid contents and ganglioside GM3 synthase activity. Based upon the mobility on high-performance thin-layer chromatography (HPTLC), GMCs showed a complex pattern of ganglioside expression that consisted, at least, of five different components of gangliosides, mainly ganglioside GM3. HG, TGF-beta1 and d-threo-PDMP induced a significant reduction of ganglioside expression with apparent changes in the composition of ganglioside GM3, and semi-quantitative analysis by HPTLC showed that ganglioside GM3 expression reduced to about 35-54% of control. These results provide a pathophysiological link between mesangial cell proliferation and ganglioside GM3 expression, indicating that exogenously added ganglioside GM3 inhibits the high-ambient glucose- and TGF-beta1-induced proliferation of cultured GMCs.     

Induction of GM1a/GD1b synthase triggers complex ganglioside expression and alters neuroblastoma cell behavior; a new tumor cell model of ganglioside function

Neuroblastoma is the most common extracranial solid tumor in children and tumor ganglioside composition has been linked to its biological and clinical behavior. We recently found that high expression of complex gangliosides that are products of the enzyme GM1a/GD1b synthase predicts a more favorable outcome in human neuroblastoma, and others have shown that complex gangliosides such as GD1a inhibit metastasis of murine tumors. To determine how a switch from structurally simple to structurally complex ganglioside expression affects neuroblastoma cell behavior, we engineered IMR32 human neuroblastoma cells, which contain almost exclusively (89%) the simple gangliosides (SG) GM2, GD2, GM3, and GD3, to overexpress the complex gangliosides (CG) GM1, GD1a, GD1b and GT1b, by stable retroviral-mediated transduction of the cDNA encoding GM1a/GD1b synthase. This strikingly altered cellular ganglioside composition without affecting total ganglioside content: There was a 23-fold increase in the ratio of complex to simple gangliosides in GM1a/GD1b synthase-transduced cells (IMR32-CG) vs. wild type (IMR32) or vector-transfected (IMR32-V) cells with essentially no expression of the clinical neuroblastoma marker, GD2, confirming effectiveness of this molecular switch from simple to complex ganglioside synthesis. Probing for consequences of the switch, we found that among functional properties of IMR32-CG cells, cell migration was inhibited and Rho/Rac1 activities were altered, while proliferation kinetics and cell differentiation were unaffected. These findings further implicate cellular ganglioside composition in determining cell migration characteristics of tumor cells. This IMR32 model system should be useful in delineating the impact of ganglioside composition on tumor cell function.     

The oligosaccharide portion of ganglioside GM1 regulates mitochondrial function in neuroblastoma cells

The crucial role of ganglioside GM1 in the regulation of neural homeostasis has been assessed by several studies. Recently we shed new light on the molecular basis underlying GM1 effects demonstrating that GM1 oligosaccharide directly binds TrkA receptor and triggers MAPK pathway activation leading to neuronal differentiation and protection. Following its exogenous administration, proteomic analysis revealed an increased expression of proteins involved in several biochemical mechanisms, including mitochondrial bioenergetics. Based on these data, we investigated the possible effect of GM1 oligosaccharide administration on mitochondrial function. We show that wild-type Neuro2a cells exposed to GM1 oligosaccharide displayed an increased mitochondrial density and an enhanced mitochondrial activity together with reduced reactive oxygen species levels. Interestingly, using a Neuro2a model of mitochondrial dysfunction, we found an increased mitochondrial oxygen consumption rate as well as increased complex I and II activities upon GM1 oligosaccharide administration. Taken together, our data identify GM1 oligosaccharide as a mitochondrial regulator that by acting at the plasma membrane level triggers biochemical signaling pathway inducing mitochondriogenesis and increasing mitochondrial activity. Although further studies are necessary, the capability to enhance the function of impaired mitochondria points to the therapeutic potential of the GM1 oligosaccharide for the treatment of pathologies where these organelles are compromised, including Parkinson’s disease.     

Stable Transfection of GM1 Synthase Gene into GM1-Deficient NG108-15 Cells,CR-72 Cells,Rescues the Responsiveness of TRK-Neurotrophin Receptor to Its Ligand,NGF

Previous studies from this laboratory and others have suggested the evidences that acidic glycosphingolipid, ganglioside GM1 (GM1), is an endogenous regulator of high affinity nerve growth factor receptor, Trk, which is an essential factor for the normal development and differentiation of neuronal cells by forming a complex with Trk. The present study was aimed to examine whether Trk expressed in cells that are deficient in endogenous GM1 due to the mutation of GM1 synthase gene (NG-CR72 cells) is responsive to its ligand nerve growth factor and how genetic restoration of GM1 synthase gene by a stable transfection of the gene affects the function of the Trk protein. The data clearly showed that (1) confocal lazor microscopic studies disclosed NG-CR72 cells are really deficient in GM1, (2) stable transfection of GM1 synthase cDNA into these cells (NG-CR72G cells) restores the expression of GM1 in the cells, and (3) Trk protein is expressed in NG-CR72 cells but its location seemed not to be on the plasma membrane, whereas we clearly observed that the Trk protein is expressed on the plasma membrane in NG-CR72G cells. (4) NGF did not elicit the autophosphorylation of the Trk protein in GM1 deficient NG-CR72 cells but did elicit the activation of the Trk protein in NG-CR72G cells with an activation of mitogen activated protein kinase. These studies strongly suggested that GM1 is necessary for the normal expression of the Trk protein function and for normal targeting of the Trk protein to the plasma membrane.     

Stable transfection of DAOY cells with a GM3 synthase antisense construct and transient reduction in ganglioside content

Tumor cell gangliosides are bioactive molecules involved in tumor-host interactions. To investigate their role in tumor formation and angiogenesis, we sought to develop an inhibitory model targeting human GM3 synthase, an essential enzyme in the ganglioside synthesis pathway, by antisense transfection. We prepared a number of transfectants from DAOY human medulloblastoma cells and isolated clones that stably expressed a 560-bp fragment of human GM3 synthase cDNA, in either sense or antisense orientation, as well as clones transfected with an empty vector. Both sense and antisense clones permanently incorporated mammalian expression vectors into their genomes. The DAOY cell clones were screened for ganglioside content using total lipid extraction, ganglioside isolation, and HPTLC. One antisense-transfected clone, 7.2A, in which total ganglioside content was reduced by 70%, was selected for further study. All sense-and sham-transfectants had ganglioside levels not different from that of untransfected DAOY cells. After 10 passages however, while antisense mRNA expression was fully maintained, the ganglioside content of 7.2A cells had reverted to normal levels. Antisense RNA transfection can sometimes have a reversible effect on the expression of a target. Possible regulatory mechanisms of this previously unrecognized process of reversion to wild type phenotype are discussed.     

Coordinate Regulation of Ganglioside Glycosyltransferases in Differentiating NG108-15 Neuroblastoma X Glioma Cells

The enzymatic basis for ganglioside regulation during differentiation of NG108-15 mouse neuroblastoma x rat glioma hybrid cells was studied. This cell line contains four gangliosides that lie along the same biosynthetic pathway: GM3, GM2, GM1, and GD1a. Chemically induced neuronal differentiation of NG108-15 cells led to an 80% drop in the steady-state level of their major ganglioside, GM3, a sixfold increase in the level of a minor ganglioside, GM2 (which became the predominant ganglioside of differentiated cells); and relatively little change in the levels of GM1 and GD1a, which lie further along the same biosynthetic pathway. The enzymatic basis for this selective change in ganglioside expression was investigated by measuring the activity of two glycosyltransferases involved in ganglioside biosynthesis. UDP-N-acetylgalactosamine: GM3 N-acetylgalactosaminyltransferase (GM2-synthetase) activity increased fivefold during butyrate-induced differentiation, whereas UDP-galactose: GM2 galactosyltransferase (GM1-synthetase) activity decreased to 10% of its control level. Coordinate regulation of these two glycosyltransferases appears to be primarily responsible for the selective increase of GM2 expression during NG108-15 differentiation.     

Analysis of gangliosides from carp intestinal mucosa

The gangliosides of carp intestinal mucosa were isolated and analysed by thin-layer chromatography (TLC), TLC immunostaining test, and TLC/secondary ion mass spectrometry (TLC/SIMS). Four species of gangliosides, designated as G-1, G-2, G-3 and G-4, were separated on TLC. The TLC/SIMS analysis of the G-1 ganglioside of carp intestinal mucosa revealed a series of [M-H](-)ions from m/z 1061 to m/z 1131 representing the molecular mass range of GM4-like ganglioside with NeuAc. G-2, G-3 and G-4 gangliosides were analysed by the TLC immunostaining test. G-2 ganglioside was recognised by the monoclonal antibody specific for ganglioside GM1 (AGM-1 monoclonal antibody). However, G-3 ganglioside migrating on TLC between GM3 and GM1 ganglioside was not recognised by anti-GM3 monoclonal antibody and by AGM-1 monoclonal antibody. Furthermore, G-4 ganglioside with a similar TLC mobility as GD1a ganglioside did not show the reactivity to the anti-GD1a monoclonal antibody. In addition using the AGM-1 monoclonal antibody, the expression of GM1 ganglioside in the carp intestinal tissue was studied. GM1 ganglioside was detected on the epithelial cell surface of carp intestinal mucosa.     

Glycosphingolipid synthesis inhibitor represses cytokine-induced activation of the Ras-MAPK pathway in embryonic neural precursor cells

Neuronal and glial cells in the central nervous system are generated from common neural precursor cells during development. To evaluate the functions of glycosphingolipids (GSLs) in neural precursor cells, neuroepithelial cells (NECs) were prepared from mouse embryos (E14.5), and the effects of an inhibitor of glucosylceramide synthesis, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), on NECs was investigated. In PDMP-treated NECs, the expression of GD3, a major ganglioside of NECs, disappeared. We found that basic fibroblast growth factor (bFGF)-induced proliferation and extracellular signal-regulated kinase (ERK) activation were repressed in PDMP-treated NECs. Leukemia inhibitory factor (LIF)-induced ERK activation was also abolished in PDMP-treated NECs, suggesting that PDMP specifically represses the Ras-MAPK pathway. bFGF-induced activation of the Ras-MAPK pathway in NECs is dependent on GSL-enriched microdomains, lipid rafts. The organization of lipid rafts and the distribution of Ras and Grb2-SOS in the microdomains were not affected. However, Ras activation was repressed in PDMP-treated NECs. In PDMP-treated NECs, some neuronal genes were up-regulated and glial genes were down-regulated. These results suggest that GSLs might be involved in the proliferation, survival, signal transduction and differentiation of NECs.     

GM1-ganglioside-induced Abeta assembly on synaptic membranes of cultured neurons

The cell-surface expression of GM1 ganglioside was studied using various cultured cells, including brain-derived endothelial cells, astrocytes, neuroblastoma cells (SH-SY5Y), and pheochromocytoma cells (PC12). GM1 ganglioside was detected only on the surface of native and nerve-growth-factor (NGF)-treated PC12 cells. We investigated whether GM1 ganglioside on the surface of these cells is sufficiently potent to induce the assembly of an exogenous soluble amyloid beta-protein (Abeta). A marked Abeta assembly was observed in the culture of NGF-treated PC12 cells. Notably, immunocytochemical study revealed that, despite the ubiquitous surface expression of GM1 ganglioside throughout cell bodies and neurites, Abeta assembly initially occurred at the terminals of SNAP25-immunopositive neurites. Abeta assembly in the culture was completely suppressed by the coincubation of Abeta with the subunit B of cholera toxin, a natural ligand for GM1 ganglioside, or 4396C, a monoclonal antibody specific to GM1-ganglioside-bound Abeta (GAbeta). In primary neuronal cultures, Abeta assembly initially occurred at synaptophysin-positive sites. These results suggest that the cell-surface expression of GM1 ganglioside is strictly cell-type-specific, and that expression of GM1 ganglioside on synaptic membranes is unique in terms of its high potency to induce Abeta assembly through the generation of GAbeta, which is an endogenous seed for Abeta assembly in Alzheimer brain.     

GM1-ganglioside-induced Aβ assembly on synaptic membranes of cultured neurons

The cell-surface expression of GM1 ganglioside was studied using various cultured cells, including brain-derived endothelial cells, astrocytes, neuroblastoma cells (SH-SY5Y), and pheochromocytoma cells (PC12). GM1 ganglioside was detected only on the surface of native and nerve-growth-factor (NGF)-treated PC12 cells. We investigated whether GM1 ganglioside on the surface of these cells is sufficiently potent to induce the assembly of an exogenous soluble amyloid β-protein (Aβ). A marked Aβ assembly was observed in the culture of NGF-treated PC12 cells. Notably, immunocytochemical study revealed that, despite the ubiquitous surface expression of GM1 ganglioside throughout cell bodies and neurites, Aβ assembly initially occurred at the terminals of SNAP25-immunopositive neurites. Aβ assembly in the culture was completely suppressed by the coincubation of Aβ with the subunit B of cholera toxin, a natural ligand for GM1 ganglioside, or 4396C, a monoclonal antibody specific to GM1-ganglioside-bound Aβ (GAβ). In primary neuronal cultures, Aβ assembly initially occurred at synaptophysin-positive sites. These results suggest that the cell-surface expression of GM1 ganglioside is strictly cell-type-specific, and that expression of GM1 ganglioside on synaptic membranes is unique in terms of its high potency to induce Aβ assembly through the generation of GAβ, which is an endogenous seed for Aβ assembly in Alzheimer brain.     

The plasma membrane-associated sialidase MmNEU3 modifies the ganglioside pattern of adjacent cells supporting its involvement in cell-to-cell interactions

We describe herein the enzyme behavior of MmNEU3, the plasma membrane-associated sialidase from mouse (Mus musculus). MmNEU3 is localized at the plasma membrane as demonstrated directly by confocal microscopy analysis. In addition, administration of the radiolabeled ganglioside GD1a to MmNEU3-transfected cells, under conditions that prevent lysosomal activity, led to its hydrolysis into ganglioside GM1, further indicating the plasma membrane topology of MmNEU3. Metabolic labeling with [1-(3)H]sphingosine allowed the characterization of the ganglioside patterns of COS-7 cells. MmNEU3 expression in COS-7 cells led to an extensive modification of the cell ganglioside pattern, i.e. GM3 and GD1a content was decreased to about one-third compared with mock-transfected cells. At the same time, a 35% increase in ganglioside GM1 content was observed. Mixed culture of MmNEU3-transfected cells with [1-(3)H]sphingosine-labeled cells demonstrates that the enzyme present at the cell surface is able to recognize gangliosides exposed on the membrane of nearby cells. Under these experimental conditions, the extent of ganglioside pattern changes was a function of MmNEU3 transient expression. Overall, the variations in GM3, GD1a, and GM1 content were very similar to those observed in the case of [1-(3)H]sphingosine-labeled MmNEU3-transfected cells, indicating that the enzyme mainly exerted its activity toward ganglioside substrates present at the surface of neighboring cells. These results indicate that the plasma membrane-associated sialidase MmNEU3 is able to hydrolyze ganglioside substrates in intact living cells at a neutral pH, mainly through cell-to-cell interactions.     

Loss of GM1 surface expression precedes annexin V-phycoerythrin binding of neutrophils undergoing spontaneous apoptosis during in vitro aging.

BACKGROUND: Apoptosis of neutrophil granulocytes is an important determinant of the resolution of inflammation. Apoptotic neutrophils undergo specific alterations in their receptor profiles. These alterations are likely to contribute to the characteristic functional silencing of the dying cells. METHODS: By flow cytometry and fluorescence microscopy, we analyzed the ganglioside GM1, a lipid raft marker, with respect to its surface expression on neutrophil and eosinophil granulocytes. Apoptosis was monitored by morphological changes and by the binding of annexin V-phycoerythrin (AxV-PE). RESULTS: GM1, which was stained by the cholera toxin subunit B, was found only on neutrophil granulocytes; eosinophil granulocytes did not bind cholera toxin subunit B. GM1 was lost from the surfaces of neutrophils before AxV-PE binding (early apoptosis). Surprisingly, GM1 reappeared during the late stages of apoptosis, although without functional consequences. GM1 was found on the cell surface and in intracellular membranes, whereas CD16 was found only at the cell surface. CONCLUSIONS: Loss of surface GM1 is a new marker for the detection of the aging of neutrophils. Its loss precedes the binding of AxV-PE of neutrophils.     

Isolation and characterization of gangliosides from Trypanosoma brucei

Gangliosides were isolated from Trypanosoma brucei and analyzed by thin-layer chromatography (TLC) and TLC immunostaining test. Four species of gangliosides, designated as G-1, G-2, G-3, and G-4, were separated by TLC. G-1 ganglioside had the same TLC migration rate as GM3. In contrast, G-2, G-3, and G-4 gangliosides migrated a little slower than GM1, GD1a, and GD1b, respectively. To characterize the molecular species of gangliosides from T. brucei, G-1, G-2, G-3, and G-4 gangliosides were purified and analyzed by TLC immunostaining test with monoclonal antibodies against gangliosides. G-1 ganglioside showed the reactivity to the monoclonal antibody against ganglioside GM3. G-2 was recognized by the anti-GM1 monoclonal antibody. G-3 showed reaction with the monoclonal antibody to GD1a. G-4 had the reactivity to anti-GD1b monoclonal antibody. Using 4 kinds of monoclonal antibodies, we also studied the expression of GM3, GM1, GD1a, and GD1b in T. brucei parasites. GM3, GM1, GD1a, and GD1b were detected on the cell surface of T. brucei. These results suggest that G-1, G-2, G-3, and G-4 gangliosides are GM3 (NeuAc alpha2-3Gal beta1-4Glc beta1-1Cer), GM1 (Gal beta1-3GalNAc beta1-4[NeuAc alpha2-3]Gal beta1-4Glc beta1-1Cer), GD1a (NeuAc alpha2-3Gal beta1-3GalNAc beta1-4[NeuAc alpha2-3]Gal beta1-4Glc beta1-1Cer), and GD1b (Gal beta1-3GalNAc beta1-4[NeuAc alpha2-8NeuAc alpha2-3]Gal beta1-4Glc beta1-1Cer), respectively, and also that they are expressed on the cell surface of T. brucei.     

GM1 Ganglioside Potentiates Trimethyltin-Induced Expression of Interleukin-1Beta and the Nerve Growth Factor in Reactive Astrocytes in the Rat Hippocampus: An Immunocytochemical Study

This study demonstrates potentiation by GM1 ganglioside treatment of trimethyltin (TMT) induced reactivity of astrocytes, and the expression of astroglial interleukin-lbeta (IL-1beta) and nerve growth factor (NGF) immunoreactivities in the rat hippocampus. GM1 treatment also results in an increase of the number of IL-1beta and NGF immunoreactive astrocytes. Both the intensity of gliosis and stimulation of IL-1beta and NGF expression in astrocytes mostly occurs in the regions of heaviest neurodegeneration in the hippocampus (CA4/CA3c and CA1). It is tempting to assume that enhancement of astroglial NGF expression by GM1 ganglioside may play a role in the protective action of GM1 against neurotoxic insult.