Human adenovirus binding to host cell receptors: a structural view.

Human Adenoviruses (HAdVs) are a family of clinically and therapeutically relevant viruses. A precise understanding of their host cell attachment and entry mechanisms can be applied in inhibitor design and the construction of targeted gene delivery vectors. In this article, structural data on adenovirus attachment and entry are reviewed. HAdVs engage two types of receptors: first, an attachment receptor that is bound by the fibre knob protein protruding from the icosahedral capsid, and next, an integrin entry receptor bound by the pentameric penton base at the capsid vertices. Adenoviruses use remarkably diverse attachment receptors, five of which have been studied structurally in the context of HAdV binding: Coxsackie and Adenovirus Receptor, CD46, the glycans GD1a and polysialic acid, and desmoglein-2. Together with the integrin entry receptors, they display both symmetrical and asymmetrical modes of binding to the virus as demonstrated by the structural analyses reviewed here. The diversity of HAdV receptors contributes to the broad tropism of these viruses, and structural studies are thus an important source of information on HAdV-host cell interactions. The imbalance in structural data between the more and less extensively studied receptors remains to be addressed by future research.

GD3 ganglioside-enriched extracellular vesicles stimulate melanocyte migration.

Melanomas often accumulate gangliosides, sialic acid-containing glycosphingolipids found in the outer leaflet of plasma membranes, as disialoganglioside GD3 and its derivatives. Here, we have transfected the GD3 synthase gene (ST8Sia I) in a normal melanocyte cell line in order to evaluate changes in the biological behavior of non-transformed cells. GD3-synthase expressing cells converted GM3 into GD3 and accumulated both GD3 and its acetylated form, 9-O-acetyl-GD3. Melanocytes were rendered more migratory on laminin-1 surfaces. Cell migration studies using the different transfectants, either treated or not with the glucosylceramide synthase inhibitor d-1-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (PPPP), allowed us to show that while GM3 is a negative regulator of melanocyte migration, GD3 increases it. We showed that gangliosides were shed to the matrix by migrating cells and that GD3 synthase transfected cells shed extracellular vesicles (EVs) enriched in GD3. EVs enriched in GD3 stimulated cell migration of GD3 negative cells, as observed in time lapse microscopy studies. Otherwise, EVs shed by GM3+veGD3-ve cells impaired migration and diminished cell velocity in cells overexpressing GD3. The balance of antimigratory GM3 and promigratory GD3 gangliosides in melanocytes could be altered not only by the overexpression of enzymes such as ST8Sia I, but also by the horizontal transfer of ganglioside enriched extracellular vesicles. This study highlights that extracellular vesicles transfer biological information also through their membrane components, which include a variety of glycosphingolipids remodeled in disease states such as cancer.

Coordinated existence of multiple gangliosides is required for cartilage metabolism.

OBJECTIVE: Gangliosides, ubiquitously existing membrane components that modulate transmembrane signaling and mediate cell-to-cell and cell-to-matrix interactions, are key molecules of inflammatory and neurological disorders. However, the functions of gangliosides in the cartilage degradation process remain unclear. We investigated the functional role of gangliosides in cartilage metabolism related to osteoarthritis (OA) pathogenesis. DESIGN: We generated knockout (KO) mice by targeting the ß1, 4-N-acetylgalactosaminyltransferase (GalNAcT) gene, which encodes an enzyme of major gangliosides synthesis, and the GD3 synthase (GD3S) gene, which encodes an enzyme of partial gangliosides synthesis. In vivo OA and in vitro cartilage degradation models were used to evaluate the effect of gangliosides on the cartilage degradation process. RESULTS: The GalNAcT and GD3S KO mice developed and grew normally; nevertheless, OA changes in these mice were enhanced with aging. The GalNAcT KO mice showed significantly enhanced OA progression compared to GD3S mice in vivo. Both GalNAcT and GD3S KO mice showed severe IL-1α-induced cartilage degradation ex vivo. Phosphorylation of MAPKs was enhanced in both GalNAcT and GD3S KOs after IL-1α stimulation. Gangliosides modulated by GalNAcT or GD3S rescued an increase of MMP-13 induced by IL-1α in mice lacking GalNAcT or GD3S after exogenous replenishment in vitro. CONCLUSION: These data show that the deletion of gangliosides in mice enhanced OA development. Moreover, the gangliosides modulated by GalNAcT are important for cartilage metabolism, suggesting that GalNAcT is a potential target molecule for the development of novel OA treatments.

[Neuronal glycolipids regulate glial cell division negatively during development and following a lesion]. / Glicolipidos neuronales regulan negativamente la division glial durante el desarrollo y tras una lesion.

Glial cells in the central nervous system of adult mammals outnumber neurons 10-fold. Their number remains stationary throughout adulthood, controlled by the concomitant presence of mitogens and mitogen inhibitors. The most abundant inhibitor, neurostatin, is ganglioside GD1b O-acetylated on hydroxyl 9 of its outermost sialic acid. Neurostatin inhibited the proliferation of primary microglia and astroblasts in culture (cytostatic) as well as both rodent and human glioma cells (cytotoxic) at nanomolar concentrations. At those concentrations neurostatin had no effect on non-glial lineage cells or differentiated glia. Neurostatin shows direct antimitotic activity on tumoral cells, interfering with multiple signals regulating cell cycle progression. But it also promotes indirectly total destruction of experimental rat brain glioma, presumably by making it visible to the host immune system and activating CD4+ and CD8+ lymphocytes. Neurostatin could be a new anti-inflammatory agent, with multiple convergent direct and indirect actions on glioma growth, a pathology without satisfactory clinical treatment. Neurostatin is produced by neurons but its expression is up-regulated by neuron-astrocyte contact. The action of neurostatin could be mediated by a number of receptor proteins, including integrins, Toll-like receptors and siglecs.

TITLE: Glicolipidos neuronales regulan negativamente la division glial durante el desarrollo y tras una lesion.En el sistema nervioso central de los mamiferos, las celulas gliales superan diez veces en numero a las neuronas. Su numero permanente estacionario durante la edad adulta, controlado por la presencia simultanea de mitogenos gliales e inhibidores de esos mitogenos. El inhibidor mas abundante, la neurostatina, es el gangliosido GD1b O-acetilado en el grupo 9 del acido sialico mas externo. La neurostatina y los oligosacaridos sinteticos inhiben la proliferacion de astroblastos en cultivo primario (citostaticos) y de celulas de gliomas (citotoxicos), tanto de roedores como de humanos, en concentracion nanomolar. A esas concentraciones, la neurostatina no tuvo efecto sobre celulas de linaje no glial ni sobre glia madura. La neurostatina y sus analogos mostraron actividad antimitotica directa sobre las celulas tumorales, interfiriendo con la progresion del ciclo celular en multiples sitios, pero tambien actuaron indirectamente, haciendo visibles las celulas tumorales al sistema inmune del huesped y activando linfocitos CD4+ y CD8+. Analogos de neurostatina podrian generar nuevos farmacos antiinflamatorios, con multiples acciones directas e indirectas contra el crecimiento de gliomas, una patologia todavia sin tratamiento clinico satisfactorio. La neurostatina es producida por las neuronas, pero el contacto de estas con astrocitos estimula notablemente su expresion. La accion de la neurostatina puede estar mediada por numerosas proteinas receptoras, incluyendo integrinas, siglecs y receptores Toll-like.

Common molecular mechanism of amyloid pore formation by Alzheimer's ß-amyloid peptide and α-synuclein.

Calcium-permeable pores formed by small oligomers of amyloid proteins are the primary pathologic species in Alzheimer's and Parkinson's diseases. However, the molecular mechanisms underlying the assembly of these toxic oligomers in the plasma membrane of brain cells remain unclear. Here we have analyzed and compared the pore-forming capability of a large panel of amyloid proteins including wild-type, variant and truncated forms, as well as synthetic peptides derived from specific domains of Aß1-42 and α-synuclein. We show that amyloid pore formation involves two membrane lipids, ganglioside and cholesterol, that physically interact with amyloid proteins through specific structural motifs. Mutation or deletion of these motifs abolished pore formation. Moreover, α-synuclein (Parkinson) and Aß peptide (Alzheimer) did no longer form Ca(2+)-permeable pores in presence of drugs that target either cholesterol or ganglioside or both membrane lipids. These results indicate that gangliosides and cholesterol cooperate to favor the formation of amyloid pores through a common molecular mechanism that can be jammed at two different steps, suggesting the possibility of a universal therapeutic approach for neurodegenerative diseases. Finally we present the first successful evaluation of such a new therapeutic approach (coined "membrane therapy") targeting amyloid pores formed by Aß1-42 and α-synuclein.

Glycolipid GD3 and GD3 synthase are key drivers for glioblastoma stem cells and tumorigenicity.

The cancer stem cells (CSCs) of glioblastoma multiforme (GBM), a grade IV astrocytoma, have been enriched by the expressed marker CD133. However, recent studies have shown that CD133(-) cells also possess tumor-initiating potential. By analysis of gangliosides on various cells, we show that ganglioside D3 (GD3) is overexpressed on eight neurospheres and tumor cells; in combination with CD133, the sorted cells exhibit a higher expression of stemness genes and self-renewal potential; and as few as six cells will form neurospheres and 20-30 cells will grow tumor in mice. Furthermore, GD3 synthase (GD3S) is increased in neurospheres and human GBM tissues, but not in normal brain tissues, and suppression of GD3S results in decreased GBM stem cell (GSC)-associated properties. In addition, a GD3 antibody is shown to induce complement-dependent cytotoxicity against cells expressing GD3 and inhibition of GBM tumor growth in vivo. Our results demonstrate that GD3 and GD3S are highly expressed in GSCs, play a key role in glioblastoma tumorigenicity, and are potential therapeutic targets against GBM.

Effect of ganglioside GT1b on the in vitro maturation of porcine oocytes and embryonic development.

Ganglioside is an acidic glycosphingolipid with sialic acids residues. This study was performed to investigate the effect and mechanism of ganglioside GT1b in porcine oocytes in the process of in vitro maturation (IVM) and preimplantation development. Metaphase II (MII) rates were significantly (P < 0.05) different between the control group and the 5 nM GT1b treatment group. Intracellular glutathione (GSH) levels in oocytes matured with 5 nM and 20 nM and GT1b decreased significantly (P < 0.05). The 10 nM group showed a significant (P < 0.05) decrease in intracellular reactive oxygen species (ROS) levels compared with the control group. Subsequently, the level of intracellular Ca(2+) in oocytes treated with different concentrations of GT1b was measured. Intracellular Ca(2+) was significantly (P < 0.05) increased with a higher concentration of GT1b in a dose-dependent manner. Real-time PCR was performed and showed that the expression of bradykinin 2 receptor (B2R) and calcium/calmodulin-dependent protein kinase II delta (CaMKIIδ) in cumulus cells was significantly (P < 0.05) decreased in the 20 nM GT1b treatment group. Treatment with 5 nM GT1b significantly (P < 0.05) decreased the expression of CaMKIIδ. In oocytes, treatment with 5 nM GT1b significantly (P < 0.05) decreased CaMKIIγ and POU5F1 (POU domain, class 5, transcription factor 1). However, treatment with 20 nM GT1b significantly (P < 0.05) increased the expression of POU5F1. Finally, embryonic developmental data showed no significant differences in the two experiments (parthenogenesis and in vitro fertilization). In conclusion, the results of the present study indicated that GT1b plays an important role in increasing the nuclear maturation rate and decreasing the intracellular ROS levels during IVM. However, GT1b inhibited maturation of the cytoplasm by maintaining intracellular Ca(2+) in the process of oocyte maturation regardless of the cell cycle stage. Therefore, GT1b is thought to act on another mechanism that controls intracellular Ca(2+).

Gangliosides drive the tumor infiltration and function of myeloid-derived suppressor cells.

Although it is now widely appreciated that antitumor immunity is critical to impede tumor growth and progression, there remain significant gaps in knowledge about the mechanisms used by tumors to escape immune control. In tumor cells, we hypothesized that one mechanism of immune escape used by tumors involves the synthesis and extracellular shedding of gangliosides, a class of biologically active cell surface glycosphingolipids with known immunosuppressive properties. In this study, we report that tumor cells engineered to be ganglioside deficient exhibit impaired tumorigenicity, supporting a link between ganglioside-dependent immune escape and tumor outgrowth. Notably, we documented a dramatic reduction in the numbers and function of tumor-infiltrating myeloid-derived suppressor cells (MDSC) in ganglioside-deficient tumors, in contrast with the large MDSC infiltrates seen in ganglioside-rich littermate control tumors. Transient ganglioside reconstitution of the tumor cell inoculum was sufficient to increase MDSC infiltration, supporting a direct connection between ganglioside production by tumor cells and the recruitment of immunosuppressive MDSC into the tumor microenvironment. Our results reveal a novel mechanism of immune escape that supports tumor growth, with broad implications given that many human tumors produce and shed high levels of gangliosides.

Removal of GPI-anchored membrane proteins causes clustering of lipid microdomains in the apical head area of porcine sperm.

The release of extracellular proteins is a part of the sperm capacitation process; this allows the sperm surface reorganization that enables the sperm to fertilize an oocyte. Some of the components released are 'decapacitation factors', an uncoordinated or early release of which may cause inappropriate surface destabilization and premature capacitation. We studied the involvement of glycosylphosphatidylinositol-anchored proteins (GPI-APs) in sperm capacitation, and reported that CD52 and CD55 exhibit bicarbonate-dependent release during in vitro sperm capacitation. Treating sperm with phosphatidylinositol-specific phospholipase C (PIPLC) resulted in the enzymatic cleavage of CD55, in both capacitating and noncapacitating conditions. Moreover, PIPLC treatment in noncapacitating conditions caused surface reorganization events that included exposure of the ganglioside GM1, aggregation of flotillin-1, and the swelling of the apical acrosome region; all of which have been reported to be associated with sperm capacitation. The acrosomal swelling was monitored using wet mount atomic force microscopy, a new imaging technique that allows nanometer-level sperm surface measurements in samples hydrated with physiological buffer rather than dried. Despite these surface changes, PIPLC treatment in identical incubation conditions did not stimulate hyperactive sperm motility or protein tyrosine phosphorylation (other hallmarks of sperm capacitation in vitro). In full capacitating conditions (i.e., the presence of bicarbonate and albumin), PIPLC treatment caused sperm deterioration. The possible role of GPI-APs removal from the sperm surface during sperm capacitation is discussed.

Exhaustion of nucleus pulposus progenitor cells with ageing and degeneration of the intervertebral disc.

Despite the high prevalence of intervertebral disc disease, little is known about changes in intervertebral disc cells and their regenerative potential with ageing and intervertebral disc degeneration. Here we identify populations of progenitor cells that are Tie2 positive (Tie2+) and disialoganglioside 2 positive (GD2+), in the nucleus pulposus from mice and humans. These cells form spheroid colonies that express type II collagen and aggrecan. They are clonally multipotent and differentiated into mesenchymal lineages and induced reorganization of nucleus pulposus tissue when transplanted into non-obese diabetic/severe combined immunodeficient mice. The frequency of Tie2+ cells in tissues from patients decreases markedly with age and degeneration of the intervertebral disc, suggesting exhaustion of their capacity for regeneration. However, progenitor cells (Tie2+GD2+) can be induced from their precursor cells (Tie2+GD2-) under simple culture conditions. Moreover, angiopoietin-1, a ligand of Tie2, is crucial for the survival of nucleus pulposus cells. Our results offer insights for regenerative therapy and a new diagnostic standard.

Ganglioside inhibition of CD8+ T cell cytotoxicity: interference with lytic granule trafficking and exocytosis.

Granule exocytosis-mediated cytotoxicity by CD8(+) CTL plays a crucial role in adaptive immunity to tumors and to intracellular pathogens. This T cell effector function has been shown to be defective in various murine tumor models and in human cancer. However, factors and their mechanisms that cause inhibition of CD8(+) T cell lytic function in tumor-bearing hosts remain to be fully defined. We postulate that gangliosides, highly expressed on tumor cell membranes, actively shed into the tumor microenvironment, and having well-established immunosuppressive properties, may be such a factor. We exposed primary mouse CD8(+) CTL to gangliosides derived from three sources (tumors and normal brain). This significantly inhibited cytotoxicity-mediated by granule exocytosis, that is, cytotoxicity of alloantigen-specific and polyclonal CD8(+) CTL in vitro. These molecules did not interfere with the interaction of CD8(+) T cells with their cognate targets. Rather, they inhibited lytic granule release in response both to TCR engagement and to stimuli that induce granule release in a nonpolarized manner. At the subcellular level, confocal microscopic imaging identified inhibition of polarization of lytic granules to the immunological synapse upon target cell recognition. Thus, tumor-shed gangliosides suppress lytic activity of CD8(+) T cells by a novel mechanism, that is, inhibition of trafficking of lytic granules in response to TCR engagement, as well as by interfering with the process of granule exocytosis in CD8(+) T cells.

Molecular identification of GD3 as a suppressor of the innate immune response in ovarian cancer.

Tumors often display mechanisms to avoid or suppress immune recognition. One such mechanism is the shedding of gangliosides into the local tumor microenvironment, and a high concentration of circulating gangliosides is associated with poor prognosis. In this study, we identify ganglioside GD3, which was isolated from the polar lipid fraction of ovarian cancer-associated ascites, as an inhibitory factor that prevents innate immune activation of natural killer T (NKT) cells. Purified GD3 displayed a high affinity for both human and mouse CD1d, a molecule involved in the presentation of lipid antigens to T cells. Purified GD3, as well as substances within the ascites, bound to the CD1d antigenic-binding site and did not require additional processing for its inhibitory effect on NKT cells. Importantly, in vivo administration of GD3 inhibited α-galactosylceramide (α-GalCer)-induced NKT cell activation in a dose-dependent manner. These data therefore indicate that ovarian cancer tumors may use GD3 to inhibit the antitumor NKT cell response as an early mechanism of tumor immune evasion.

Disialogangliosides and TNFα alter gene expression for cytokines and chemokines in primary brain cell cultures.

Gangliosides have long been implicated in multiple pathologies affecting the central nervous system. Empirical studies have suggested the possibility that gangliosides, particularly GD3, work in tandem with pro-inflammatory cytokines, especially tumor necrosis factor alpha (TNFα), to initiate or facilitate cell death in the CNS. As a step toward unraveling the metabolic pathways activated in the pathogenesis of brain cell death, we have surveyed gene expression for a host of cytokines and chemokines in primary brain cell cultures exposed to GD3, GD1b, and TNFα for 24 h. An initial screen of 98 genes on a focused mini-array revealed the expression of at least 28 genes related to cell growth, death, or inflammation in our system of mixed cells cultured from neonatal rat brains. Clear evidence of a differential response to the gangliosides or TNFα was seen in 12 genes. Quantitative PCR was used to validate the response of six of these genes. We found that both GD3 and GD1b, but not TNFα, up-regulated expression of macrophage inflammatory protein 3 (MIP3A) and interleukin-1 receptor 1 (IL1R1), but down-regulated fibroblast growth factor 13 (FGF13). The expression of FGF receptor activating protein 1 (FRAG1) and interleukin-3 receptor alpha (IL3RA) was down-regulated by GD3. Exposure to TNFα resulted in a dramatic up-regulation of IL3RA and chemokine ligand 2 (CCL2), both of which have been implicated in multiple sclerosis. Our results provide strong evidence that the expression of these genes might be critical links in the metabolic cascades leading to cell degeneration and death in the brain.

Identification of host cell factors required for intoxication through use of modified cholera toxin.

We describe a novel labeling strategy to site-specifically attach fluorophores, biotin, and proteins to the C terminus of the A1 subunit (CTA1) of cholera toxin (CTx) in an otherwise correctly assembled and active CTx complex. Using a biotinylated N-linked glycosylation reporter peptide attached to CTA1, we provide direct evidence that ~12% of the internalized CTA1 pool reaches the ER. We also explored the sortase labeling method to attach the catalytic subunit of diphtheria toxin as a toxic warhead to CTA1, thus converting CTx into a cytolethal toxin. This new toxin conjugate enabled us to conduct a genetic screen in human cells, which identified ST3GAL5, SLC35A2, B3GALT4, UGCG, and ELF4 as genes essential for CTx intoxication. The first four encode proteins involved in the synthesis of gangliosides, which are known receptors for CTx. Identification and isolation of the ST3GAL5 and SLC35A2 mutant clonal cells uncover a previously unappreciated differential contribution of gangliosides to intoxication by CTx.

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.

[Visualization of amyloid formation processes on cell membranes: gangliosides as key molecules for the onset of amyloidosis].

Deposition of insoluble amyloid fibrils in tissues is a common hallmark of a wide range of human diseases referred to as amyloidoses, including Alzheimer's disease, type II diabetes mellitus. The amyloid deposits cause cell dysfunction, death, and subsequently severe impairment in tissues. Elucidation of amyloid formation mechanisms is essential for prevention of the onset and development of amyloidoses. Accumulated experimental evidence demonstrates that membrane lipids enhance the fibril formation of amyloidogenic proteins. Our group demonstrated that amyloid formation by amyloid ß-protein (Aß) was facilitated by gangliosides in lipid raft-like model membranes. Phosphatidylserine and phosphatidylglycerol were also reported to trigger fibril formation by human islet amyloid polypeptide (hIAPP). However, it is not verified whether the proposed lipid-protein interactions can occur on plasma membranes of live cells. The author developed a method for visualizing amyloid fibrils on live cell membranes and investigated the roles of gangliosides and cholesterol in lipid rafts for amyloid formation. Congo red, an amyloid-specific dye, was found to be a promising compound for staining amyloids in live cells. Aß was accumulated on cholesterol-dependent ganglioside-rich domains in PC12 neuronal cells in a time- and concentration-dependent manner, leading to cell death. Nerve growth factor-induced differentiation of PC12 cells increased both gangliosides and cholesterol and thereby greatly potentiated the accumulation and cytotoxic effect of Aß. Amyloid formation by hIAPP was also facilitated by gangliosides in lipid rafts. Membrane lipid compositions, in this case, gangliosides in lipid rafts, actually caused striking change in amyloid formation on cell membranes.

Role of GD3-CLIPR-59 association in lymphoblastoid T cell apoptosis triggered by CD95/Fas.

We previously found that a directional movement of the raft component GD3 towards mitochondria, by its association with microtubules, was mandatory to late apoptogenic events triggered by CD95/Fas. Since CLIPR-59, CLIP-170-related protein, has recently been identified as a microtubule binding protein associated with lipid rafts, we analyzed the role of GD3-CLIPR-59 association in lymphoblastoid T cell apoptosis triggered by CD95/Fas. To test whether CLIPR-59 could play a role at the raft-microtubule junction, we performed a series of experiments by using immunoelectron microscopy, static or flow cytometry and biochemical analyses. We first assessed the presence of CLIPR-59 molecule in lymphoblastoid T cells (CEM). Then, we demonstrated that GD3-microtubule interaction occurs via CLIPR-59 and takes place at early time points after CD95/Fas ligation, preceding the association GD3-tubulin. GD3-CLIPR-59 association was demonstrated by fluorescence resonance energy transfer (FRET) analysis. The key role of CLIPR-59 in this dynamic process was clarified by the observation that silencing CLIPR-59 by siRNA affected the kinetics of GD3-tubulin association, spreading of GD3 towards mitochondria and apoptosis execution. We find that CLIPR-59 may act as a typical chaperone, allowing a prompt interaction between tubulin and the raft component GD3 during cell apoptosis triggered by CD95/Fas. On the basis of the suggested role of lipid rafts in conveying pro-apoptotic signals these results disclose new perspectives in the understanding of the mechanisms by which raft-mediated pro-apoptotic signals can directionally reach their target, i.e. the mitochondria, and trigger apoptosis execution.

The nutritional and clinical significance of lipid rafts.

PURPOSE OF REVIEW: Lipid rafts are potentially modifiable by diet, particularly (but not exclusively) by dietary fatty acids. This review examines the potential for dietary modification of raft structure and function in the immune system, brain and retinal tissue, the gut, and in cancer cells. RECENT FINDINGS: In-vitro and ex-vivo studies suggest that dietary n-3 polyunsaturated fatty acids (PUFAs) may exert immunosuppressive and anticancer effects through changes in lipid raft organization. In addition, gangliosides and cholesterol may modulate lipid raft organization in a number of tissues, and recent work has highlighted sphingolipids in membrane microdomains as potential targets for inhibition of tumor growth. The roles of fatty acids and gangliosides, especially in relation to lipid rafts, in cognitive development, age-related cognitive decline, psychiatric disorders, and Alzheimer's disease are poorly understood and require further investigation. The roles of lipid rafts in cancer, in microbial pathogenesis, and in insulin resistance are starting to emerge, and indicate compelling evidence for the growing importance of membrane microdomains in health and disease. SUMMARY: In-vitro and animal studies show that n-3 PUFAs, cholesterol, and gangliosides modulate the structure and composition of lipid rafts, potentially influencing a wide range of biological processes, including immune function, neuronal signaling, cancer cell growth, entry of pathogens through the gut barrier, and insulin resistance in metabolic disorders. The physiological, clinical, and nutritional relevance of these observations remains to be determined.

The effect of ganglioside GQ1b on the NMDA receptor signaling pathway in H19-7 cells and rat hippocampus.

Gangliosides, sialic acid-containing glycosphingolipids, are related to various synaptic functions in the rat brain. Previously, we investigated the behavioral effects of the ganglioside GQ1b on learning and memory using the Y-maze and Morris water maze test. GQ1b-treated rats showed highly increased memory performance on the Y-maze and the Morris water maze test. In this study, we determined the role of GQ1b on the activation of the N-methyl-d-aspartate (NMDA) receptor signaling pathway in H19-7 rat hippocampal cells and the hippocampus of rats. After 12 h of treatment with GQ1b, the expression levels of NMDA receptor subunit 2A and 2B were increased in H19-7 cells and the hippocampus of rats. In addition, treatment of GQ1b increased the tyrosine phosphorylation of NR2B that may enhance NMDA receptor synaptic activation and enhancement of NMDA receptors. Also, following GQ1b treatment, the phosphorylation of extracellular signal-regulated kinases (ERK1/2) and protein kinase A, a cAMP activated protein kinase (PKA) increased in H19-7 cells and the hippocampus of rats. These increases resulted in an increase in the phosphorylation of cAMP response element binding protein (CREB). These results suggest that GQ1b might facilitate the activation of the NMDA receptor signaling pathway in the hippocampus of rats, an effect which is dependent on ERK1/2, PKA and CREB phosphorylation. Also, these data support our previous result that GQ1b improves the learning and memory of rats.

Anchored and soluble gangliosides contribute to myelosupportivity of stromal cells.

Stroma-mediated myelopoiesis depends upon growth factors and an appropriate intercellular microenvironment. Previous studies have demonstrated that gangliosides, produced by hepatic stromal cell types, are required for optimal myelosupportive function. Here, we compared the mielossuportive functions of a bone marrow stroma (S17) and skin fibroblasts (SF) regarding their ganglioside pattern of synthesis and shedding. The survival and proliferation of a myeloid precursor cell (FDC-P1) were used as reporter. Although the ganglioside synthesis of the two stromal cells was similar, their relative content and shedding were distinct. The ganglioside requirement for mielossuportive function was confirmed by the decreased proliferation of FDC-P1 cells in ganglioside synthesis-inhibited cultures and in presence of an antibody to GM3 ganglioside. The distinct mielossuportive activities of the S17 and SF stromata may be related to differences on plasma membrane ganglioside concentrations or to differences on the gangliosides shed and their subsequent uptake by myeloid cells, specially, GM3 ganglioside.