Essential Roles of Gangliosides in the Formation and Maintenance of Membrane Microdomains in Brain Tissues

Gangliosides are considered to be involved in the maintenance and repair of nervous tissues. Recently, novel roles of gangliosides in the regulation of complement system were reported. Here we summarized roles of gangliosides in the formation and maintenance of membrane microdomains in brain tissues by comparing complement activation, inflammatory reaction and disruption of glycolipid-enriched microdomain (GEM)/rafts among several mutant mice of ganglioside synthases. Depending on the defects in ganglioside compositions, corresponding up-regulation of complement-related genes, proliferation of astrocytes and infiltration of microglia were found with gradual severity. Immunoblotting of fractions separated by sucrose density gradient ultracentrifugation revealed that DAF and NCAM having GPI-anchors tended to disappear from the raft fraction with intensities of DKO > GM2/GD2 synthase KO > GD3 synthase KO > WT. The lipid raft markers tended to disperse from the raft fractions with similar intensities. Phospholipids and cholesterol also tended to decrease in GEM/rafts in GM2/GD2 synthase KO and DKO, although total amounts were almost equivalent. All these results indicate that GEM/rafts architecture is destroyed by ganglioside deficiency with gradual intensity depending on the degree of defects of their compositions. Implication of inflammation caused by deficiency of gangliosides in various neurodegenerative diseases was discussed.     

Alterations of membrane lipids and in gene expression of ganglioside metabolism in different brain structures in a mouse model of mucopolysaccharidosis type I (MPS I)

Mucopolysaccharidosis I (MPS I) is a congenital disorder caused by the deficiency of α-l-iduronidase (IDUA), with the accumulation of glycosaminoglycans (GAGs) in the CNS. Although GAG toxicity is not fully understood, previous works suggest a GAG-induced alteration in neuronal membrane composition. This study is aimed to evaluate the levels and distribution of gangliosides and cholesterol in different brain regions (cortex, cerebellum, hippocampus and hypothalamus) in a model using IDUA knockout (KO) mice (C57BL/6). Lipids were extracted with chloroform–methanol and then total gangliosides and cholesterol were determined, followed by ganglioside profile analyses. While no changes in cholesterol content were observed, the results showed a tissue dependent ganglioside alteration in KO mice: a total ganglioside increase in cortex and cerebellum, and a selective presence of GM3, GM2 and GD3 gangliosides in the hippocampus and hypothalamus. To elucidate this, we evaluated gene expression of ganglioside synthesis (GM3, GD3 and GM2/GD2 synthases) and degradation of (Neuraminidase1) enzymes in the cerebellum and hippocampus by RT-sq-PCR. The results obtained with KO mice showed a reduced expression of GD3 and GM2/GD2 synthases and Neuraminidase1 in cerebellum; and a decrease in GM2/GD2 synthase and Neuraminidase1 in the hippocampus. These data suggest that the observed ganglioside changes result from a combined effect of GAGs on ganglioside biosynthesis and degradation.     

Metastatic potential of mouse Lewis lung cancer cells is regulated via ganglioside GM1 by modulating the matrix metalloprotease-9 localization in lipid rafts

To analyze mechanisms for cancer metastasis, we established high metastatic sublines from mouse Lewis lung cancer (P29) by repeated injection. Sublines established from the two subclones H7 and C4 commonly exhibited increased proliferation and invasion activity and reduced expression of ganglioside GM1, although they showed different preferences in their target organs of metastasis. The high metastatic sublines secreted higher levels of activated matrix metalloprotease (MMP)-9 as well as pro-MMP-9 in the culture medium than the parent lines. Furthermore, they contained MMP-9 at the glycolipid-enriched microdomain (GEM)/rafts fractionated by the sucrose density gradient ultracentrifugation of Triton X-100 extracts, whereas the parent cells showed faint bands at the fraction. When high metastatic sublines were treated with methyl-beta-cyclodextrin, their invasion activities were dramatically suppressed, and the MMP-9 secretion was also suppressed. All these results indicated that GEM/rafts play crucial roles in the increased invasion and high metastatic potential. To clarify the implication of reduced GM1 expression, low GM1-expressing cell lines were established using an RNA interference-expression vector of the GM1 synthase. Low GM1-expressing cell lines showed increased proliferation and invasion, enrichment in the GEM/rafts, and increased secretion of MMP-9. Among adhesion molecules, only integrin beta1 was detected in GEM/rafts with stronger intensity in high metastatic lines and low GM1-expressing cells. Taken together, integrins seemed to be enriched in the GEM/rafts by reduced GM1 levels, and subsequently MMP-9 was recruited to the GEM/rafts, resulting in its efficient secretion and activation, and eventually in the increased invasion and metastatic potentials.     

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.     

Overexpression of ganglioside GM1 results in the dispersion of platelet-derived growth factor receptor from glycolipid-enriched microdomains and in the suppression of cell growth signals.

To investigate the molecular mechanisms of gangliosides for the regulation of cell proliferation, Swiss 3T3 cells were transfected with GM2/GD2 synthase and GM1 synthase cDNAs, resulting in the establishment of GM1-expressing (GM1(+)) lines. Compared with the vector control (GM1(-)) cell lines, GM1(+) cells exhibited reduced cell proliferation by stimulation with platelet-derived growth factor (PDGF). In accordance with the reduced cell growth, GM1(+) cells showed earlier decreases in the phosphorylation levels of PDGF receptor and less activation of MAP kinases than GM1(-) cells. To analyze the effects of GM1 expression on the PDGF/PDGF receptor (PDGFR) signals, the glycolipid-enriched microdomain (GEM) was isolated and the following results were obtained. (i) PDGFR predominantly distributed in the non-GEM fraction in GM1(+) cells, while it was present in both GEM and non-GEM fractions in GM1(-) cells. (ii) Activation of PDGFR as detected by anti-phosphotyrosine antibody occurred almost in parallel with existing amounts of PDGFR in each fraction. (iii) GM1 binds with PDGFR in GEM fractions. These findings suggested that GM1 regulates signals via PDGF/PDGFR by controlling the distribution of PDGFR in- and outside of GEM, and also interacting with PDGFR in the GEM fraction as a functional constituent of the microdomain.     

Fractionation of Primary Cultured Cerebellar Neurons: Distribution of Sialyltransferases Involved in Ganglioside Biosynthesis

Primary cultured neurons were fractionated using sucrose density gradients. The activities of four sialyltransferases (GM3, GD3, GD1a, and GT1a synthase) involved in ganglioside biosynthesis were assayed in the collected fractions. The distribution of GM3 synthase coincided with that of mannosidase II, an enzyme assumed to be a cis-Golgi marker. Both enzymes were mainly associated with the more dense fraction. GD1a and GT1a synthase activities, on the other hand, were mainly recovered in the less dense fraction. Moreover, they were colocalized with thiamine pyrophosphatase, an enzyme assumed to be a marker of the late Golgi (trans-Golgi and trans-Golgi network). GD3 synthase activity was equally distributed between both fractions. These results are integrated in a model of ganglioside biosynthesis.     

Development of new ganglioside probes and unraveling of raft domain structure by single-molecule imaging

Gangliosides are involved in a variety of biological roles and are a component of lipid rafts found in cell plasma membranes (PMs). Gangliosides are especially abundant in neuronal PMs and are essential to their physiological functions. However, the dynamic behaviors of gangliosides have not been investigated in living cells due to a lack of fluorescent probes that behave like their parental molecules. We have recently developed, using an entirely chemical method, four new ganglioside probes (GM1, GM2, GM3, and GD1b) that act similarly to their parental molecules in terms of raft partitioning and binding affinity. Using single fluorescent-molecule imaging, we have found that ganglioside probes dynamically enter and leave rafts featuring CD59, a GPI-anchored protein. This occurs both before and after stimulation. The residency time of our ganglioside probes in rafts with CD59 oligomers was 48 ms, after stimulation. The residency times in CD59 homodimer and monomer rafts were 40 ms and 12 ms, respectively. In this review, we introduce an entirely chemical-based ganglioside analog synthesis method and describe its application in single-molecule imaging and for the study of the dynamic behavior of gangliosides in cell PMs. Finally, we discuss how raft domains are formed, both before and after receptor engagement. This article is part of a Special Issue entitled Neuro-glycoscience, edited by Kenji Kadomatsu and Hiroshi Kitagawa.     

Expression of GM1 and GD1a in liver of wild mice

Wild mice are divided into two groups with different ganglioside compositions in the liver. Most Japanese and a few Chinese wild mice have GM2(NeuGc) as a major ganglioside, whereas all wild mice caught at other places distributed all over the world other than Japan and China express GM1(NeuGc) and GD1a(NeuGc) in addition to GM2(NeuGC). We recently reported that inbred strains of laboratory mice were also grouped into the same two types based on the ganglioside composition in the liver, and that the expression of GM1(NeuGc) and GD1a(NeuGc) was regulated by a gene located at the left outside the H-2 complex on chromosome 17 (Hashimoto, Y., Suzuki, A., Yamakawa, T., Miyashita, N., & Moriwaki, K. (1983) J. Biochem. 94, 2049-2054). The present study suggests that oriental wild mice would be a donor of a defective gene for expression of GM1(NeuGc) and GD1a(NeuGc) in mice of laboratory stocks which are commonly used for biochemical and immunological studies, such as C57BL/6, C57BL/10, BALB/c, DBA/2, C3H/He, and CBA mice.     

Modulation of phospholipase A2 activity by neutral and anionic glycosphingolipids in monolayers.

The effect of neutral (galactocerebroside and asialo-ganglioside GM1) or anionic (sulphatide and gangliosides GM1, GD1a and GT1b) glycosphingolipids on the activity of phospholipase A2 from pig pancreas was studied in mixed monolayers of dilauroyl phosphatidylcholine with the glycosphingolipids in different molar fractions at various constant surface pressures. The activity of the enzyme depends on the proportion and type of glycosphingolipid in the interface. Sulphatide activates the enzyme at all proportions, whereas galactocerebroside shows inhibition or activation depending on its proportion in the film. Asialo-ganglioside GM1 and gangliosides GM1, GD1a and GT1b can strongly inhibit the enzyme at relatively low molar fractions in the film in the following order: asialo-ganglioside GM1 less than ganglioside GM1 less than ganglioside GT1b less than ganglioside GD1a. The changes of activity are not due to a direct action of the lipids on the active centre or interfacial recognition region of the enzyme.     

UVB-irradiated keratinocytes induce melanoma-associated ganglioside GD3 synthase gene in melanocytes via secretion of tumor necrosis factor α and interleukin 6

Although expression of gangliosides and their synthetic enzyme genes in malignant melanomas has been well studied, that in normal melanocytes has been scarcely analyzed. In particular, changes in expression levels of glycosyltransferase genes responsible for ganglioside synthesis during evolution of melanomas from melanocytes are very important to understand roles of gangliosides in melanomas. Here, expression of glycosyltransferase genes related to the ganglioside synthesis was analyzed using RNAs from cultured melanocytes and melanoma cell lines. Quantitative RT-PCR revealed that melanomas expressed high levels of mRNA of GD3 synthase and GM2/GD2 synthase genes and low levels of GM1/GD1b synthase genes compared with melanocytes. As a representative exogenous stimulation, effects of ultraviolet B (UVB) on the expression levels of 3 major ganglioside synthase genes in melanocytes were analyzed. Although direct UVB irradiation of melanocytes caused no marked changes, culture supernatants of UVB-irradiated keratinocytes (HaCaT cells) induced definite up-regulation of GD3 synthase and GM2/GD2 synthase genes. Detailed examination of the supernatants revealed that inflammatory cytokines such as TNFα and IL-6 enhanced GD3 synthase gene expression. These results suggest that inflammatory cytokines secreted from UVB-irradiated keratinocytes induced melanoma-associated ganglioside synthase genes, proposing roles of skin microenvironment in the promotion of melanoma-like ganglioside profiles in melanocytes.     

Ganglioside accumulation in activated glia in the developing brain: comparison between WT and GalNAcT KO mice

Our previous studies have shown accumulation of GM2 ganglioside during ethanol-induced neurodegeneration in the developing brain, and GM2 elevation has also been reported in other brain injuries and neurodegenerative diseases. Using GM2/GD2 synthase KO mice lacking GM2/GD2 and downstream gangliosides, the current study explored the significance of GM2 elevation in WT mice. Immunohistochemical studies indicated that ethanol-induced acute neurodegeneration in postnatal day 7 (P7) WT mice was associated with GM2 accumulation in the late endosomes/lysosomes of both phagocytic microglia and increased glial fibrillary acidic protein (GFAP)-positive astrocytes. However, in KO mice, although ethanol induced robust neurodegeneration and accumulation of GD3 and GM3 in the late endosomes/lysosomes of phagocytic microglia, it did not increase the number of GFAP-positive astrocytes, and the accumulation of GD3/GM3 in astrocytes was minimal. Not only ethanol, but also DMSO, induced GM2 elevation in activated microglia and astrocytes along with neurodegeneration in P7 WT mice, while lipopolysaccharide, which did not induce significant neurodegeneration, caused GM2 accumulation mainly in lysosomes of activated astrocytes. Thus, GM2 elevation is associated with activation of microglia and astrocytes in the injured developing brain, and GM2, GD2, or other downstream gangliosides may regulate astroglial responses in ethanol-induced neurodegeneration.     

Effect of gangliosides on the distribution of a glycosylphosphatidylinositol-anchored protein in plasma membrane from Chinese hamster ovary-K1 cells

Glycosylphosphatidylinositol (GPI)-anchored proteins are clustered mainly in sphingolipid-cholesterol microdomains of the plasma membrane. The distribution of GPI-anchored fusion yellow fluorescent protein (GPI-YFP) in the plasma membrane of Chinese hamster ovary (CHO)-K1 cells with different glycolipid compositions was investigated. Cells depleted of glycosphingolipids by inhibiting glucosylceramide synthase activity or cell lines expressing different gangliosides caused by stable transfection of appropriate ganglioside glycosyltransferases or exposed to exogenous GM1 were transfected with GPI-YFP cDNA. The distribution of GPI-YFP fusion protein expressed at the plasma membrane was studied using the membrane-impermeable cross-linking agent bis(sulfosuccinimidyl)suberate. Results indicate that GPI-YFP forms clusters at the surface of cells expressing GM3, or cells depleted of glycolipids, or transfected cells expressing mainly GD3 and GT3, or GM1 and GD1a, or mostly GM2, or highly expressing GM1. However, no significant changes in membrane microdomains of GPI-YFP were detected in the different glycolipid environments provided by the membranes of the cell lines under study. On the other hand, wild type CHO-K1 cells exposed to 100 microm GM1 before cross-linking with bis(sulfosuccinimidyl)suberate showed a dramatic reduction in the amount of GPI-YFP clusters. These findings clearly indicate that manipulating the glycolipid content of the cellular membrane, just by changing the ganglioside biosynthetic activity of the cell, did not significantly affect the association of GPI-YFP on the cell surface of CHO-K1 cells. The effect of exogenous GM1 gangliosides on GPI-YFP plasma membrane distribution might be a consequence of the ganglioside level reached in plasma membrane and/or the effect of particular ganglioside species (micelles) that lead to membrane architecture and/or dynamic modifications.     

Age-related changes in GM1, GD1a, GT1b components of gangliosides in Wistar albino rats

In this study, age-related changes of GM1, GD1a, GT1b fractions of gangliosides were investigated in whole brain of male Wistar albino rats. Insignificant increases were detected in GM1 values from the third to the 24th month, whereas GD1a and GT1b concentrations of ganglioside in 24-month-old rats decreased significantly as compared to 6-month-old rats. Although there were no significant differences in the GD1a/GT1b ratio of any groups, GM1/GD1a and GM1/GT1b ratios were significantly increased as compared to 6-month-old rats. The increase in the ratios of gangliosides are not due to an increase of GM1 fractions; they result from a decrease of GD1a and GT1b fractions of gangliosides. In conclusion, the concentration of ganglioside decreased with ageing.     

Ganglioside biosynthesis in rat liver: effect of UDP-amino sugars on individual transfer reactions

Several glycosyltransferases participating in ganglioside biosynthesis were measured in Golgi-rich fractions from rat liver. Addition of those UDP-amino sugars to the enzyme assays which accumulate in liver after treatment of rats with D-galactosamine inhibited the transferases to different degrees. The simultaneous presence of UDP-GalN, UDP-GalNAc, UDP-GlcN, and UDP-GlcNAc in concentrations resembling their overall content in livers 6 h after D-galactosamine administration led to an inhibition of the glycolipid galactosyltransferases, GL2 and GM1 synthases of 44 and 64%, respectively. GM2 synthase was moderately inhibited whereas the sialyltransferases (GM3, GD3, and GD1a synthases) were almost unimpaired. Induction of liver cell damage by D-galactosamine did not cause any change of glycosyltransferase activities as determined in rat liver homogenates and Golgi-rich fractions. These results indicate a possible role for UDP-amino sugars in the depression of ganglioside biosynthesis observed in vivo after GalN administration.     

Genetic remodeling of gangliosides resulted in the enhanced reactions to the foreign substances in skin

Several lines of transgenic mice with gangliosides GM2/GD2 synthasegene were established, and the expression levels of the transgenein brain, liver, spleen and thymus were analyzed by comparingwith those in their litter mates. Among four tissues, brainand skin showed markedly high expression levels of the transgenein Northern blotting. Particularly, transgenic mice skin showedabout 10-fold higher expression of GM2/GD2 synthase gene thanthe wild type mice skin. Therefore, alterations in the morphology,glycolipid components, and responses to the exogenous stimulationsin the transgenic mice skin were examined. Gangliosides in thetransgenic skin were dramatically converted from GM3 to GM1,whereas no morphological changes were observed. However, whenskin flap test was performed with insertion of nylon membranesunder the skin flaps, much stronger inflammatory reactions consistingof edema, marked thickness, and cell infiltration were observedin the transgenic mice compared with the wild type. Similarenhanced inflammatory reaction was also observed in the skininjected by silicon gel, and in the peritoneal reaction to theinjected casein. Main cell population in these inflammatoryreactions consisted of neutrophils, suggesting an increasedsensitivity of neutrophils to chemo-tactic factors in the transgenicmice. ganglioside glycosyltransferase GM2 GD2 synthase skin transgenic mouse     

Differential uPAR recruitment in caveolar‐lipid rafts by GM1 and GM3 gangliosides regulates endothelial progenitor cells angiogenesis

Gangliosides and the urokinase plasminogen activator receptor (uPAR) tipically partition in specialized membrane microdomains called lipid‐rafts. uPAR becomes functionally important in fostering angiogenesis in endothelial progenitor cells (EPCs) upon recruitment in caveolar‐lipid rafts. Moreover, cell membrane enrichment with exogenous GM1 ganglioside is pro‐angiogenic and opposite to the activity of GM3 ganglioside. On these basis, we first checked the interaction of uPAR with membrane models enriched with GM1 or GM3, relying on the adoption of solid‐supported mobile bilayer lipid membranes with raft‐like composition formed onto solid hydrophilic surfaces, and evaluated by surface plasmon resonance (SPR) the extent of uPAR recruitment. We estimated the apparent dissociation constants of uPAR‐GM1/GM3 complexes. These preliminary observations, indicating that uPAR binds preferentially to GM1‐enriched biomimetic membranes, were validated by identifying a pro‐angiogenic activity of GM1‐enriched EPCs, based on GM1‐dependent uPAR recruitment in caveolar rafts. We have observed that addition of GM1 to EPCs culture medium promotes matrigel invasion and capillary morphogenesis, as opposed to the anti‐angiogenesis activity of GM3. Moreover, GM1 also stimulates MAPKinases signalling pathways, typically associated with an angiogenesis program. Caveolar‐raft isolation and Western blotting of uPAR showed that GM1 promotes caveolar‐raft partitioning of uPAR, as opposed to control and GM3‐challenged EPCs. By confocal microscopy, we have shown that in EPCs uPAR is present on the surface in at least three compartments, respectively, associated to GM1, GM3 and caveolar rafts. Following GM1 exogenous addition, the GM3 compartment is depleted of uPAR which is recruited within caveolar rafts thereby triggering angiogenesis.     

Sub-Golgi distribution in rat liver of CMP-NeuAc GM3- and CMP-NeuAc:GT1b alpha 2----8sialyltransferases and comparison with the distribution of the other glycosyltransferase activities involved in ganglioside biosynthesis

Using a sucrose density gradient fractionation of a highly purified Golgi apparatus from rat liver, we determined the sub-Golgi distribution of CMP-NeuAc:GM3 ganglioside alpha 2----8sialyltransferase (GM3-SAT) and CMP-NeuAc:GT1b ganglioside alpha 2----8sialyltransferase (GT1b-SAT), in comparison with that of the other glycosyltransferase activities involved in ganglioside biosynthesis. While GM3-SAT was recovered in several density fractions, GT1b-SAT was mainly found on less dense sub-Golgi membranes; this indicates that these two activities are physically separate. Moreover, with regard to the monosialo pathway, CMP-NeuAc:lactosylceramide alpha 2----3sialyltransferase, UDP-GalNAc:GM3 ganglioside beta 1----4N-acetylgalactosaminyltransferase, UDP-Gal:GM2 ganglioside beta 1----3galactosyltransferase, and CMP-NeuAc:GM1 ganglioside alpha 2----3sialyltransferase were resolved from more dense to less dense fractions, respectively. In the disialo pathway, UDP-GalNAc:GD3 ganglioside beta 1----4N-acetylgalactosaminyltransferase, UDP-Gal:GD2 ganglioside beta 1----3galactosyltransferase and CMP-NeuAc:GD1b ganglioside alpha 2----3sialyltransferase co-distributed with the corresponding activities of the monosialo pathway. These last results indicate that many Golgi glycosyltransferases involved in ganglioside biosynthesis are localized in the order in which they act.     

Ganglioside biosynthesis in rat liver: different distribution of ganglioside synthases in hepatocytes, Kupffer cells, and sinusoidal endothelial cells

The activities of five glycolipid-glycosyltransferases, GL2, GM3, GM2, GM1, and GD1a synthase, were determined in a cell-free system with homogenate protein of total rat liver, isolated hepatocytes, Kupffer cells, and sinusoidal endothelial cells. In rat liver parenchymal and nonparenchymal cells ganglioside synthases were distributed differently. Compared to hepatocytes, Kupffer cells expressed a nearly sevenfold greater activity of GM3 synthase, but only 14% of GM2, 19% of GM1, and 67% of GD1a synthase activity. Sinusoidal endothelial cells expressed a pattern of enzyme activities quite similar to that of Kupffer cells with the exception of higher GM2 synthase activity. Activity of GL2 synthase was distributed unifromly in parenchymal and nonparenchymal cells of rat liver, but differed by sex. It was 1 to 2 orders of magnitude below that of all the other ganglioside synthases investigated. The results indicate GL2 synthase regulates the total hepatic ganglioside content, and hepatocytes but not nonparenchymal liver cells have high enzymatic capacities to form a-series gangliosides more complex than GM3.