GM1 ganglioside-independent intoxication by Cholera toxin.

Cholera toxin (CT) enters and intoxicates host cells after binding cell surface receptors via its B subunit (CTB). We have recently shown that in addition to the previously described binding partner ganglioside GM1, CTB binds to fucosylated proteins. Using flow cytometric analysis of primary human jejunal epithelial cells and granulocytes, we now show that CTB binding correlates with expression of the fucosylated Lewis X (LeX) glycan. This binding is competitively blocked by fucosylated oligosaccharides and fucose-binding lectins. CTB binds the LeX glycan in vitro when this moiety is linked to proteins but not to ceramides, and this binding can be blocked by mAb to LeX. Inhibition of glycosphingolipid synthesis or sialylation in GM1-deficient C6 rat glioma cells results in sensitization to CT-mediated intoxication. Finally, CT gavage produces an intact diarrheal response in knockout mice lacking GM1 even after additional reduction of glycosphingolipids. Hence our results show that CT can induce toxicity in the absence of GM1 and support a role for host glycoproteins in CT intoxication. These findings open up new avenues for therapies to block CT action and for design of detoxified enterotoxin-based adjuvants.

The multi-tasked life of GM1 ganglioside, a true factotum of nature.

GM1 ganglioside occurs widely in vertebrate tissues, where it exhibits many essential functions, both in the plasma membrane and intracellular loci. Its essentiality is revealed in the dire consequences resulting from genetic deletion. This derives from its key roles in several signalosome systems, characteristically located in membrane rafts, where it associates with specific proteins that have glycolipid-binding domains. Thus, GM1 interacts with proteins that modulate mechanisms such as ion transport, neuronal differentiation, G protein-coupled receptors (GPCRs), immune system reactivities, and neuroprotective signaling. The latter occurs through intimate association with neurotrophin receptors, which has relevance to the etiopathogenesis of neurodegenerative diseases and potential therapies. Here, we review the current state of knowledge of these GM1-associated mechanisms.

Ganglioside GM1 Contributes to the State of Insulin Resistance in Senescent Human Arterial Endothelial Cells.

Vascular endothelial cells (ECs) play central roles in physiologically important functions of blood vessels and contribute to the maintenance of vascular integrity. Therefore, it is considered that the impairment of EC functions leads to the development of vascular diseases. However, the molecular mechanisms of the EC dysfunctions that accompany senescence and aging have not yet been clarified. The carbohydrate antigens carried by glycoconjugates (e.g. glycoproteins, glycosphingolipids, and proteoglycans) mainly present on the cell surface serve not only as marker molecules but also as functional molecules. In this study, we have investigated the abundance and functional roles of glycosphingolipids in human ECs during senescence and aging. Among glycosphingolipids, ganglioside GM1 was highly expressed in abundance on the surface of replicatively and prematurely senescent ECs and also of ECs derived from an elderly subject. Insulin signaling, which regulates important functions of ECs, is impaired in senescent and aged ECs. Actually, by down-regulating GM1 on senescent ECs and overloading exogenous GM1 onto non-senescent ECs, we showed that an increased abundance of GM1 functionally contributes to the impairment of insulin signaling in ECs. Taken together, these findings provide the first evidence that GM1 increases in abundance on the cell surface of ECs under the conditions of cellular senescence and aging and causes insulin resistance in ECs. GM1 may be an attractive target for the detection, prevention, and therapy of insulin resistance and related vascular diseases, particularly in older people.

GM1 Ganglioside is Involved in Epigenetic Activation Loci of Neuronal Cells.

Gangliosides are sialic acid-containing glycosphingolipids that are most abundant in the nerve tissues. The quantity and expression pattern of gangliosides in brain change drastically throughout development and are mainly regulated through stage-specific expression of glycosyltransferase (ganglioside synthase) genes. We previously demonstrated that acetylation of histones H3 and H4 on the N-acetylgalactosaminyltransferase I (GalNAcT, GA2/GM2/GD2/GT2-synthase) gene promoter resulted in recruitment of trans-activation factors. In addition, we reported that epigenetic activation of the GalNAcT gene was also detected as accompanied by an apparent induction of neuronal differentiation in neural stem cells responding to an exogenous supplement of ganglioside GM1. Here, we present evidence supporting the concept that nuclear GM1 is associated with gene regulation in neuronal cells. We found that nuclear GM1 binds acetylated histones on the promoters of the GalNAcT and NeuroD1 genes in differentiated neurons. Our study demonstrates for the first time that GM1 interacts with chromatin via acetylated histones at the nuclear periphery of neuronal cells.

Ganglioside GD1a promotes oocyte maturation, furthers preimplantation development, and increases blastocyst quality in pigs.

Gangliosides are key lipid molecules required for the regulation of cellular processes such as proliferation, differentiation, and cell signaling, including signaling of epidermal growth factor receptor (EGFR). Epidermal growth factor (EGF) has long been considered a potential regulator of meiotic and cytoplasmic maturation in mammalian oocytes. However, there is no report on the direct effect of ganglioside GD1a in porcine oocyte maturation. In this study, we first investigated a functional link between GD1a and meiotic maturation during in vitro maturation (IVM) of porcine embryos. Moreover, we confirmed the effect of exogenous GD1a treatment on blastocyst development, quality, and fertilization rate in early embryonic development. First, we observed that the protein level of ST3GAL2, a GD1a synthesizing enzyme, significantly increased (P < 0.01) in cumulus-oocyte-complexes (COCs) during IVM progress. The proportion of arrested germinal vesicles (GV) increased in oocytes treated with EGF+GD1a (41.6 ± 1.5%) at the IVM I stage. Upon completion of meiotic maturation, the proportion of metaphase II (M II) was significantly higher (P < 0.05) in the EGF+GD1a (89.9 ± 3.6%) treated group. After IVF, the percentage of penetrated oocytes was significantly higher (P < 0.05) in the EGF+GD1a (89.1 ± 2.3%) treated group than in the control group. Furthermore, exogenous GD1a treatment improved the developmental competence and quality of blastocysts during preimplantation embryo development stage. These results suggest that ganglioside GD1a may play an important role in IVM mechanisms of porcine maturation capacity. Furthermore, our findings will be helpful for better promoting the embryo development and blastocyst quality in pigs.

Capability of ganglioside GM1 in modulating interactions, structure, location and dynamics of peptides/proteins: biophysical approaches: interaction of ganglioside GM1 with peptides/proteins.

Gangliosides, are glycosphingolipids, present in all vertebrate plasma membranes with particular abundance in nerve cell membrane. Gangliosides can act as portals for antimicrobial peptides, hormones, viruses, lectins, toxins and pathogens. They are strategically positioned on the outer membrane and hence can participate in a large number of recognition processes. Their abundance in nerve cell membrane makes them "likely" receptor candidates for neuropeptides. In this review we outline our work in the area of GM1-peptide/protein interaction. We have explored the effect of GM1 containing micelles/bicelles on structures of peptides, proteins as well as on denatured proteins. It has been observed that the peptides that are disordered or having random coil structure in aqueous solution, attained an ordered three-dimensional structure when interact with GM1. It is also observed that denatured proteins undergo refolding in presence of ganglioside. Peptides/proteins show stronger interaction with membrane lipid bilayer in presence of ganglioside than that without ganglioside. This review mainly focuses on capability of ganglioside GM1 in modulating interaction, structural, location and dynamics of peptides/proteins using a number of biophysical techniques-solution NMR, DOSY, CD, fluorescence etc.

Clinical trials in spinal cord injury: lessons learned on the path to translation. The 2011 International Spinal Cord Society Sir Ludwig Guttmann Lecture.

After three decades of clinical research on interventions to improve neurological outcomes in persons with spinal cord injury (SCI), the promise of preclinical discovery has yet to be translated into a consensus standard of care treatment. Nonetheless, SCI researchers remain hopeful that advances in preclinical discovery coupled with improved clinical trial performance will yield effective restorative treatment. This historical review of key studies in SCI over the past 30 years illustrates the progress that has been achieved in establishing a high standard in the conduct of clinical research while providing important lessons for improving trial design, conduct and reporting. Through application of these lessons, the performance of SCI trials can be improved, thereby shortening the pathway to successful translation and the development of effective therapies.

Lipids revert inert Abeta amyloid fibrils to neurotoxic protofibrils that affect learning in mice.

Although soluble oligomeric and protofibrillar assemblies of Abeta-amyloid peptide cause synaptotoxicity and potentially contribute to Alzheimer's disease (AD), the role of mature Abeta-fibrils in the amyloid plaques remains controversial. A widely held view in the field suggests that the fibrillization reaction proceeds 'forward' in a near-irreversible manner from the monomeric Abeta peptide through toxic protofibrillar intermediates, which subsequently mature into biologically inert amyloid fibrils that are found in plaques. Here, we show that natural lipids destabilize and rapidly resolubilize mature Abeta amyloid fibers. Interestingly, the equilibrium is not reversed toward monomeric Abeta but rather toward soluble amyloid protofibrils. We characterized these 'backward' Abeta protofibrils generated from mature Abeta fibers and compared them with previously identified 'forward' Abeta protofibrils obtained from the aggregation of fresh Abeta monomers. We find that backward protofibrils are biochemically and biophysically very similar to forward protofibrils: they consist of a wide range of molecular masses, are toxic to primary neurons and cause memory impairment and tau phosphorylation in mouse. In addition, they diffuse rapidly through the brain into areas relevant to AD. Our findings imply that amyloid plaques are potentially major sources of soluble toxic Abeta-aggregates that could readily be activated by exposure to biological lipids.

Gangliosides, NGF, brain aging and disease: a mini-review with personal reflections.

In this mini-review I summarize our research efforts in ascertaining the possible neuro-reparative properties of the GM1 ganglioside and its cooperative effects with NGF in stroke-lesion models. We also review aspects of our NGF investigations which have recently led to the discovery that NGF is released in an activity-dependent manner in the form of its precursor molecule, proNGF. These studies support the notion that in the CNS NGF metabolism conversion and degradation occur in the extracellular milieu. We have also validated this pathway in vivo demonstrating that the pharmacological inhibition of the pro-to mature NGF conversion results in the brain accumulation of proNGF and loss and atrophy of cortical cholinergic synapses. Furthermore, we have gathered neurochemical evidence for a compromise of this newly discovered NGF metabolic pathway in Alzheimer's disease, explaining the vulnerability of NGF-dependent forebrain cholinergic neurons in this disease despite normal NGF synthesis and abundance of NGF precursor.

Impaired ganglioside metabolism in Huntington's disease and neuroprotective role of GM1.

Huntington's disease (HD) is a neurodegenerative disorder caused by the expansion of a polyglutamine stretch in the protein huntingtin (Htt). HD neurons are dysfunctional at multiple levels and have increased susceptibility to stress and apoptotic stimuli. We have discovered that synthesis of the ganglioside GM1 is reduced in fibroblasts from HD patients and in cell and animal models of HD, and that decreased GM1 levels contribute to heighten HD cell susceptibility to apoptosis. The apoptotic susceptibility is recapitulated through inhibition of ganglioside synthesis in wild-type striatal cells, suggesting that decreased GM1 levels might be one of the key events leading to HD pathogenesis and progression. Administration of GM1 restores ganglioside levels in HD cells and promotes activation of AKT and phosphorylation of mutant Htt, leading to decreased mutant Htt toxicity and increased survival of HD cells. Our data identify GM1 as a potential treatment for HD.

Cholesterol in Alzheimer's disease and other amyloidogenic disorders.

The complex association of cholesterol metabolism and Alzheimer's disease is presented in depth, including the possible benefits to be gained from cholesterol-lowering statin therapy. Then follows a survey of the role of neuronal membrane cholesterol in Abeta pore formation and Abeta fibrillogenesis, together with the link with membrane raft domains and gangliosides. The contribution of structural studies to Abeta fibrillogenesis, using TEM and AFM, is given some emphasis. The role of apolipoprotein E and its isoforms, in particular ApoE4, in cholesterol and Abeta binding is presented, in relation to genetic risk factors for Alzheimer's disease. Increasing evidence suggests that cholesterol oxidation products are of importance in generation of Alzheimer's disease, possibly induced by Abeta-produced hydrogen peroxide. The body of evidence for a link between cholesterol in atherosclerosis and Alzheimer's disease is increasing, along with an associated inflammatory response. The possible role of cholesterol in tau fibrillization, tauopathies and in some other non-Abeta amyloidogenic disorders is surveyed.

In search of a solution to the sphinx-like riddle of GM1.

Among the many glycoconjugates contributing to the sugar code, gangliosides have drawn special attention owing to their predominance as the major sialoglycoconjugate category within the nervous system. However, their occurrence, albeit at lower levels, appears ubiquitous in vertebrate cells and even some invertebrate tissues. Now that over 100 gangliosides have been structurally characterized, their diverse physiological functions constitute a remaining enigma. This has been especially true of GM1, for which a surprising array of functions has already been revealed. Our current research has focused on two areas of GM1 function: (a) signaling induced in neural and immune cells by cross-linking of GM1 in the plasma membrane that leads to activation of TRPC5 (transient receptor potiential, canonical form 5) channels, a process important in neuritogenesis and autoimmune suppression; (b) activation by GM1 of a sodium-calcium exchanger (NCX) in the inner membrane of the nuclear envelope (NE) with resulting modulation of nuclear and cellular calcium. The latter has a role in maintaining neuronal viability, loss of which renders neurons vulnerable to Ca(2+) overload. Pathological manifestations in mutant mice and their cultured neurons lacking GM1 have shown dramatic rescue with a membrane permeable derivative of GM1 that enters the nucleus and restores NCX activity. Nuclear function of GM1 is related to the presence of neuraminidase in the NE, an enzyme that generates GM1 through hydrolysis of GD1a. A different isoform of this enzyme was found in each of the two membranes of the NE.

Ganglioside structure dictates signal transduction by cholera toxin and association with caveolae-like membrane domains in polarized epithelia.

In polarized cells, signal transduction by cholera toxin (CT) requires apical endocytosis and retrograde transport into Golgi cisternae and perhaps ER (Lencer, W.I., C. Constable, S. Moe, M. Jobling, H.M. Webb, S. Ruston, J.L. Madara, T. Hirst, and R. Holmes. 1995. J. Cell Biol. 131:951-962). In this study, we tested whether CT's apical membrane receptor ganglioside GM1 acts specifically in toxin action. To do so, we used CT and the related Escherichia coli heat-labile type II enterotoxin LTIIb. CT and LTIIb distinguish between gangliosides GM1 and GD1a at the cell surface by virtue of their dissimilar receptor-binding B subunits. The enzymatically active A subunits, however, are homologous. While both toxins bound specifically to human intestinal T84 cells (Kd approximately 5 nM), only CT elicited a cAMP-dependent Cl- secretory response. LTIIb, however, was more potent than CT in eliciting a cAMP-dependent response from mouse Y1 adrenal cells (toxic dose 10 vs. 300 pg/well). In T84 cells, CT fractionated with caveolae-like detergent-insoluble membranes, but LTIIb did not. To investigate further the relationship between the specificity of ganglioside binding and partitioning into detergent-insoluble membranes and signal transduction, CT and LTIIb chimeric toxins were prepared. Analysis of these chimeric toxins confirmed that toxin-induced signal transduction depended critically on the specificity of ganglioside structure. The mechanism(s) by which ganglioside GM1 functions in signal transduction likely depends on coupling CT with caveolae or caveolae-related membrane domains.

Direct evidence that endogenous GM1 ganglioside can mediate thymocyte proliferation.

The B subunit of cholera toxin, which is multivalent and binds exclusively to a specific ganglioside, GM1, was mitogenic for rat thymocytes. When exposed to the B subunit, the cells proliferated, as measured by 3H-labeled thymidine incorporation. Mitogenesis depended on the direct interaction of the B subunit with GM1 on the surface of the cells. This demonstrates that endogenous plasma membrane gangliosides can mediate proliferation in lymphocytes.

GM1 ganglioside contributes to retain the neuronal conduction and neuronal excitability in visceral and baroreceptor afferents.

GM1 ganglioside has a great impact on the function of nodes of Ranvier on myelinated fiber, suggesting its potential role to maintain the electrical and neuronal excitability of neurons. Here we first demonstrate that visceral afferent conduction velocity of myelinated and unmyelinated fibers are reduced significantly by tetrodotoxin (TTX) or cholera toxin-B subunits (CTX-B), and only the effects mediated by CTX-B are prevented by GM1 pre-treatment. At soma of myelinated A and unmyelinated C-type nodose ganglion neurons (NGNs), the action potential spike frequency reduced by CTX-B is also prevented by GM1. Additionally, the current density of both TTX-sensitive (TTX-S) and TTX-resistant (TTX-R) Na(+) channels were significantly decreased by CTX-B without changing the voltage-dependent property. These data confirm that endogenous GM1 may play a dominant role in maintaining the electrical and neuronal excitability via modulation of sodium (Na(+)) channel around nodes and soma as well, especially TTX-S Na(+) channel, which is also confirmed by the reduction of spike amplitude and depolarization. Similar data are also extended to fluorescently identified and electrophysiologically characterized aortic baroreceptor neurons. These findings suggest that GM1 plays an important role in the neural modulation of electric and neuronal excitability in visceral afferent system.

N-glycolyl GM1 ganglioside as a receptor for simian virus 40.

Carbohydrate microarrays have emerged as powerful tools in analyses of microbe-host interactions. Using a microarray with 190 sequence-defined oligosaccharides in the form of natural glycolipids and neoglycolipids representative of diverse mammalian glycans, we examined interactions of simian virus 40 (SV40) with potential carbohydrate receptors. While the results confirmed the high specificity of SV40 for the ganglioside GM1, they also revealed that N-glycolyl GM1 ganglioside [GM1(Gc)], which is characteristic of simian species and many other nonhuman mammals, is a better ligand than the N-acetyl analog [GM1(Ac)] found in mammals, including humans. After supplementing glycolipid-deficient GM95 cells with GM1(Ac) and GM1(Gc) gangliosides and the corresponding neoglycolipids with phosphatidylethanolamine lipid groups, it was found that GM1(Gc) analogs conferred better virus binding and infectivity. Moreover, we visualized the interaction of NeuGc with VP1 protein of SV40 by molecular modeling and identified a conformation for GM1(Gc) ganglioside in complex with the virus VP1 pentamer that is compatible with its presentation as a membrane receptor. Our results open the way not only to detailed studies of SV40 infection in relation to receptor expression in host cells but also to the monitoring of changes that may occur with time in receptor usage by the virus.

GM1 and GM3 gangliosides highlight distinct lipid microdomains within the apical domain of epithelial cells.

The apical domain of epithelial cells is composed of distinct subdomains such as microvilli, primary cilia and a non-protruding region. Using the cholesterol-binding protein prominin-1 as a specific marker of plasma membrane protrusions we have previously proposed the co-existence of different cholesterol-based lipid microdomains (lipid rafts) within the apical domain [Röper, K., Corbeil, D. and Huttner, W.B. (2000), Retention of prominin in microvilli reveals distinct cholesterol-based lipid microdomains in the apical plasma membrane. Nat. Cell Biol. 2, 582-592]. To substantiate the hypothesis that the microvillar plasma membrane subdomains contain a distinct set of lipids compared to the planar portion we have investigated the distribution of prominin-1 and two raft-associated gangliosides GM(1) and GM(3) by fluorescence microscopy. GM(1) was found to co-localize with prominin-1 on microvilli whereas GM(3) was segregated from there suggesting its localization in the planar region. Regarding the primary cilium, overlapping fluorescent signals of GM(1) or GM(3) and prominin-1 were observed. Thus, our data demonstrate that specific ganglioside-enriched rafts are found in different apical subdomains and reveal that two plasma membrane protrusions with different structural bases (actin for the microvillus and tubulin for the cilium) are composed of distinct types of lipid.

Asialo GM1(+) CD8(+) T cells play a critical role in costimulation blockade-resistant allograft rejection.

Simultaneous blockade of the CD40 and CD28 costimulatory pathways is an effective treatment strategy to promote allograft acceptance but does not lead to indefinite allograft survival. The immune mechanisms responsible for costimulation-independent rejection are not defined. Here we have studied the rejection responses of murine C57BL/6 recipients, which we show to be relatively resistant to inhibition by combined CD40/CD28 blockade. We demonstrate that asialo GM1(+) CD8(+) cells play a critical role in this costimulation blockade-resistant rejection. These results provide new insights into the costimulatory requirements for T-cell subsets and demonstrate for the first time that combined blockade of the CD40 and CD28 pathways does not adequately inhibit CD8-mediated skin allograft rejection. Furthermore, we provide evidence that asialo GM1 is a potentially important therapeutic target for CD8-dependent immune responses.

a-Series gangliosides mediate the effects of advanced glycation end products on pericyte and mesangial cell proliferation: a common mediator for retinal and renal microangiopathy?

Advanced glycation end products (AGEs) are involved in the development of microvascular complications, including alterations of retinal pericyte and renal mesangial cell growth occurring during diabetic retinopathy and diabetic nephropathy, respectively. Because gangliosides are implicated in the regulation of cell proliferation, we hypothesized that AGEs could exert cellular effects in part by modulating ganglioside levels. Results of the present study indicate that AGEs caused an inhibition of both bovine retinal pericyte (BRP) and rat renal mesangial cell (RMC) proliferation, associated with an increase of a-series gangliosides consecutive to GM3 synthase activity increase and GD3 synthase activity inhibition. Similar modifications were also found in the renal cortex of diabetic db/db mice compared with controls. Treatment of BRP and RMC with exogenous a-series gangliosides decreased proliferation and blockade of a-series gangliosides with specific antibodies partially protecting the two cell types from the AGE-induced proliferation decrease. Further, inhibition of GM3 synthase using specific SiRNA partially reversed the AGE effects on mesangial cell proliferation. These results suggest that a-series gangliosides are mediators of the adverse AGE effects on BRP and RMC proliferation. They also raise the hypothesis of common mechanisms involved in the development of diabetic retinopathy and diabetic nephropathy.

Glucosamine induces cell-cycle arrest and hypertrophy of mesangial cells: implication of gangliosides.

Alterations in proliferation and hypertrophy of renal mesangial cells are typical features of diabetic nephropathy. The HP (hexosamine pathway) has been proposed as a biochemical hypothesis to explain microvascular alterations due to diabetic nephropathy; however, involvement of HP in the regulation of mesangial cell growth or hypertrophy has been poorly studied. Although gangliosides are known to regulate cell proliferation, their potential role in mesangial cell-growth perturbations has hardly been explored. In the present study, we investigated the effects of the HP activation, mimicked by GlcN (glucosamine) treatment, on mesangial cell growth and hypertrophy and the potential implication of gangliosides in these processes. Our results indicate that GlcN induced hypertrophy of mesangial cells, as measured by an increase in the protein/cell ratio, and it caused cell-cycle arrest by an increase in the expression of cyclin-dependent kinase inhibitor p21(Waf1/Cip1). Furthermore, GlcN treatment resulted in a massive increase in the levels of gangliosides G(M2) and G(M1). Treatment of cells with exogenous G(M2) and G(M1) reproduced the effects of 0.5 mM GlcN on p21(Waf1/Cip1) expression, cell-cycle arrest and hypertrophy, suggesting that gangliosides G(M2) and G(M1) are probably involved in mediating GlcN effects. These results document a new role of the HP in the regulation of mesangial cell growth and hypertrophy. They also suggest a potential new mechanism of action of the HP through modulation of ganglioside levels.