Ganglioside Function in Calcium Homeostasis and Signaling

Ganglioside function in eukaryotic cells encompasses a variety of modulatory interactions related to both development and mature cellular behavior. In relation to the nervous system this includes induction of neurite outgrowth and trophic/neuroprotective phenomena; more generally this applies to ganglioside effects on receptor function, adhesion reactions, and signal transduction mechanisms in neural and extraneural systems. Underlying many of these trophic effects are ganglioside-induced changes in cellular calcium, accomplished through modulation of Ca²⁺ influx channels, Ca²⁺ exchange proteins, and various Ca²⁺-dependent enzymes that are altered through association with gangliosides. A clear distinction needs to be drawn between intrinsic functions of gangliosides as naturally expressed by the cell and activities created by application of exogenous ganglioside(s) that may or may not reflect natural function. This review attempts to summarize findings in this area and point to possible future directions of research.     

Stimulation of Neurite Outgrowth in Neuroblastoma Cells by Neuraminidase: Putative Role of GM1 Ganglioside in Differentiation

Treatment of three neuroblastoma cell types in culture with neuraminidase resulted in enhanced neurite outgrowth. These included the mouse Neuro-2A and rat B104 and B50 lines. The morphological changes depended on the presence of exogenous Ca2+ and were accompanied by modest but statistically significant increases in 45Ca2+ influx. Neuraminidase-stimulated neuritogenesis was blocked by the B subunit of cholera toxin (cholera B) and anti-GM1 antibody, a finding suggesting the effect was due to an increased amount of GM1 on the cell surface. Cholera B also blocked the increase in 45Ca2+ influx. The mouse N1A-103 line, previously characterized as "neurite minus," did not respond to neuraminidase with either neurite outgrowth or enhanced Ca2+ influx. These results point to an influence of GM1 on neuritogenesis in cells with differentiation potential and suggest a mechanism involving modulation of Ca2+ flux.     

Endogenous GM1 Ganglioside of the Plasma Membrane Promotes Neuritogenesis by Two Mechanisms

The influence of GM1 on the neuritogenic phase of neuronal differentiation has been highlighted in recent reports showing upregulation of this ganglioside in the plasma and nuclear membranes concomitant with axonogenesis. These changes are accompanied by alterations in Ca²⁺ flux which constitute an essential component of the signaling mechanism for axon outgrowth. This study examines 2 distinct mechanisms of induced neurite outgrowth involving plasma membrane GM1, as expressed in 3 neuroblastoma cell lines. Growth of Neuro-2a and NG108-15 cells in the presence of neuraminidase (N'ase), an enzyme that increases the cell surface content of GM1, caused prolific outgrowth of neurites which, in the case of Neuro-2a, could be blocked by the B subunit of cholera toxin (Ctx B) which binds specifically to GM1; however, the latter agent applied to NG108-15 cells proved neuritogenic and potentiated the effect of N'ase. With N18 cells, the combination was also neuritogenic as was Ctx B alone, whereas N'ase by itself had no effect. Neurite outgrowth correlated with influx of extracellular Ca²⁺, determined with fura-2. Treatment of NG108-15 and N18 cells with Ctx B alone caused modest but persistent elevation of intracellular Ca²⁺ while a more pronounced increase occurred with the combination Ctx B + N'ase. Treatment with N'ase alone also caused modest but prolonged elevation of intracellular Ca²⁺ in NG108-15 and Neuro-2a but not N18; in the case of Neuro-2a this effect was blocked by Ctx B. Neuro-2a and N18 thus possess 2 distinctly different mechanisms for neuritogenesis based on Ca²⁺ modulation by plasma membrane GM1, while NG108-15 cells show both capabilities. The neurites stimulated by N'ase + Ctx B treatment of N18 cells were shown to have axonal character, as previously demonstrated for NG108-15 cells stimulated in this manner and for Neuro-2a cells stimulated by N'ase alone.     

Regulation of Transmembrane Signaling by Ganglioside GM1 :

Interaction of antibodies to ganglioside GM1 with Neuro2a cells was studied to investigate the role of GM1 in cell signaling. Binding of anti-GM1 to Neuro2a cells induced the formation of 3H-inositol phosphates (3H-IPs) and elevated the intracellular Ca2+ concentration [Ca2+]i. The rise in [Ca2+]i was due to the influx of Ca2+ from the extracellular medium and release from intracellular Ca2+ pools. The Ca2+ influx pathway did not allow the permeation of Na+ or K+. The influx was inhibited by amiloride, a specific blocker of T-type Ca2+ channels, whereas nifedipine and diltiazem, blockers of L-type Ca2+ channels, did not have any effect. Thus, anti-GM1 appears to activate a T-type Ca2+ channel in Neuro2a cells. The intracellular Ca2+ release was inhibited by pretreatment of cells with neomycin sulfate, phorbol dibutyrate, and pertussis toxin (PTx), which also inhibited the 3H-IP formation in Neuro2a cells. Addition of caffeine neither elevated the [Ca2+]i nor affected the anti-GM1-induced [Ca2+]i rise. The data reveal that the binding of anti-GM1 to Neuro2a cells activates phospholipase C via a PTx-sensitive G protein, which leads to formation of IPs and release of Ca2+ from inositol trisphosphate-sensitive pool of endoplasmic reticulum. Anti-GM1 also arrested the differentiation of Neuro2a cells in culture and significantly stimulated their proliferation. This stimulatory effect of anti-GM1 on cell proliferation was blocked by amiloride but not by PTx, suggesting that the influx of Ca2+ was essentially required for cell proliferation. Our data suggest a role for GM1 in the regulation of transmembrane signaling events and cell growth.     

GM3与Ca~(2+)-ATP酶的重建及其冷冻断裂电镜观察

应用生物膜的分离与重建技术 ,将GM3、大豆磷脂与肌质网Ca2 + ATP酶共同重建在脂质体上 ,酶活力明显增加 .经负染、冷冻断裂复型后电镜等形态学方法证实形成的脂酶体囊泡封闭性好 ,脂酶体上Ca2 + ATP酶蛋白颗粒均匀、直径增大     

Growth of Ectopic Dendrites on Cortical Pyramidal Neurons in Neuronal Storage Diseases Correlates with Abnormal Accumulation of GM2 Ganglioside

Abstract: Ganglioside analysis and quantitative Golgi studies of the cerebral cortex of cats with ganglioside and nonganglioside lysosomal storage diseases reveal a correlation between the amount of accumulated GM2 ganglioside and the extent of ectopic dendrite growth on cortical pyramidal neurons. This correlation was not observed with any of the other gangliosides assayed for, including GM1 ganglioside. These results suggest a specific role for GM2 ganglioside in the initiation of ectopic neurites on pyramidal cells in vivo and are consistent with the developing hypothesis that different gangliosides have specific roles in different cell types dependent upon the receptor or other effector molecules with which they may interact.     

Ganglioside GM1 Enhances Induction by Nerve Growth Factor of a Putative Dimer of TrkA

Abstract: GM1 enhances nerve growth factor (NGF)-stimulated neuritogenesis and prevents apoptotic death of PC12 cells; both may be due to enhancement of TrkA dimerization. In this study, we examined the effect of GM1 on NGF-induced TrkA dimerization in Trk-PC12 (6–24) cells. NGF increased tyrosine phosphorylation of the 140-kDa protein (TrkA monomer), and preincubation with GM1 potentiated this effect. Adding the protein cross-linker bis(sulfosuccinimidyl) suberate with NGF resulted in the appearance of two major bands (220 and 330 kDa) when probed with antibodies against TrkA or phosphotyrosine, and GM1 also enhanced this effect. We interpret the 330-kDa band as being a homodimer of TrkA. The identity of the 220-kDa band is still not certain but may consist of a posttranslationally modified form of TrkA. Our results suggest that GM1 is augmenting the effects of NGF on PC12 cells by enhancing the dimerization and activation of the TrkA receptor.     

Use of a photoactivable GM1 ganglioside analogue to assess lipid distribution in caveolae bilayer

A new photoactivable, radioactive derivative of ganglioside GM1 has been utilized to assess lipid distribution in the caveolae bilayer, taking advantage of the ability of the glycolipid, endogenous or exogenously added, to concentrate within this membrane compartment and to crosslink neighboring molecules upon illumination. After insertion into A431 plasma membrane and photoactivation, a membrane-enriched and a detergent-resistant fraction, enriched in gangliosides, sphingomyelin and cholesterol, were isolated. While a few radioactive proteins were detected in the membrane-enriched fraction, only radioactive caveolin was detected in the detergent-resistant fraction, indicating at the same time the enrichment of this fraction in caveolae and the presence of ganglioside within this compartment. Among lipids, crosslinked phosphatidylcholine, sphingomyelin and cholesterol were detected in the membrane-enriched fraction, while only crosslinked sphingomyelin was detected in the detergent-resistant fraction. These results suggest the enrichment in sphingomyelin—along with ganglioside—within the outer leaflet, and the preferential localization of cholesterol within the endoplasmic leaflet, of the caveolae bilayer.     

Liposome Fluidity Alters Interactions Between the Ganglioside GM1 and Cholera Toxin B Subunit

Cholera toxin is a complex protein with a biologically active protein (A subunit) and a cell targeting portion (B subunit). The B subunit is responsible for specific cell binding and entry of the A subunit. One way to limit potential toxicity of the toxin after exposure is to introduce cellular decoys to bind the toxin before it can enter cells. In this study the ganglioside GM1, a natural ligand for cholera toxin, was incorporated into liposomes and the interaction between fluorescent B subunit and the liposome determined. Liposome membrane fluidity was determined to play a major role in the binding between liposomes and the cholera toxin B subunit. Liposomes with lower fluidity demonstrated greater binding with the B subunit. The findings from this study could have important implications on formulation strategies for liposome decoys of toxins.     

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 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.     

Treatment with GM1 Ganglioside Restores Striatal Dopamine in the 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine-Treated Mouse

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 30 mg/kg i.p. daily for 7 days, was administered to mice. This dosage regimen resulted in an approximately 50% reduction of striatal dopamine (DA) level. Chronic administration of GM1 ganglioside (II3NeuAc-GgOse Cer), beginning between 1 to 4 days after terminating MPTP dosing, resulted in partial restoration of the striatal DA level. From dose- and time-response studies, it appeared that 30 mg/kg i.p. of GM1 administered daily for approximately 23 days resulted in an approximately 80% restoration of the DA level and complete restoration of the 3,4-dihydroxyphenylacetic acid (DOPAC) content. This dosage of GM1 also restored the turnover rate of DA in the striatum to near normal. Discontinuing GM1 treatment resulted in a fall of DA and DOPAC levels to values found in mice treated with MPTP alone. There was no evidence for regeneration of nerve terminal amine reuptake in the GM1-treated mice as evaluated by DA uptake into synaptosomes. Our biochemical findings in animals suggest that early GM1 ganglioside treatment of individuals with degenerative diseases of dopaminergic nigrostriatal neurons might be fruitful.     

GM1对肌质网Ca~(2+)-ATPase活性及膜流动性的影响

外源性GM1对肌质网Ca2+-ATPase的水解及转运活性都有明显的抑制作用.在GM1浓度为0～8nmol/mg蛋白质范围内抑制作用具有浓度依赖性.当GM1浓度达到8nmol/mg蛋白质时,酶活性受到最大抑制,此时水解活性降低51%,转运活性降低49%.荧光偏振测定结果表明:GM1参入后,肌质网膜流动性降低.     

Differential Effects of GM1 Ganglioside Treatment on Glial Fibrillary Acidic Protein Content in the Rat Septum and Hippocampus After Partial Interruption of Their Connections

Abstract: The glial fibrillary acidic protein (GFAP) content was investigated using immunoblotting techniques in the septum and hippocampus of the rat after bilateral lateral fimbria transection. Seven days after surgery GFAP content increased significantly both in the septum (140% of control) and hippocampus (120% in dorsal, the less denervated, and 145% in the most denervated ventral part), indicating the occurrence of reactive gliosis. The GM1 treatment caused statistically significant attenuation of GFAP increment in all hippocampal parts. In contrast, GM1 treatment has no influence on the increase of GFAP content in the septum. Results suggest a differential effect of GM1 on the two gliotic reactions formed as a consequence of the lesion at the level of the source of innervation (septum) and the target (hippocampus).     

Mechanistic study of modulation of SR Ca2+-ATPase activity by gangliosides GM1 and GM3 through some biophysical measurements

On the basis of confirming the antagonistic effects of GM1 and GM3 on the activity of Ca²⁺-ATPase, we further demonstrated that some of the components of these two gangliosides, including sialic acid (NeuNAc), asialo-GM1, asialo-GM3 and ceramide, failed to show any effects on the activity of Ca²⁺-ATPase. Thus it is apparent that the intact molecules of these two gangliosides with their specific conformations were needed to perform their effects on Ca²⁺-ATPase. From the fluorescence resonance energy transfer measurements, the energy transfer between Cys 670/674 and Lys 515 was decreased by GM1 and increased by GM3, indicating GM1 induced the conformation of the hydrophilic region of Ca²⁺-ATPase to be less compact, while GM3 induced it to be more compact. From the CD spectra measurements, GM1 and GM3 both reduced the content of -helical structures of Ca²⁺-ATPase, but GM1 caused a stronger decrease than that of GM3. Using DPH as the probe, we found that the membrane lipid fluidity of the proteoliposomes containing Ca²⁺-ATPase was decreased by GM1 and tend to increase by GM3.     

Segregation of GM1 and GM3 clusters in the cell membrane depends on the intact actin cytoskeleton

Gangliosides have been implicated in exerting multiple physiological functions, and it is important to understand how their distribution is regulated in the cell membrane. By using freeze-fracture immunolabeling electron microscopy, we showed that GM1 and GM3 make independent clusters that are significantly reduced by cholesterol depletion. In the present study, we examined the effects of actin depolymerization/polymerization and Src-family kinase inhibition on the GM1 and GM3 clusters. Both GM1 and GM3 clustering was reduced when the actin cytoskeleton was perturbed by latrunculin A or jasplakinolide, but the decrease was less significant than that induced by cholesterol depletion. On the other hand, inhibition of Src-family kinases decreased GM3 clustering more drastically than did cholesterol depletion, whereas its effect on GM1 clustering was less significant. GM1 and GM3 were segregated from each other in unperturbed cells, but co-clustering increased significantly after actin depolymerization. Our results indicate that the GM1 and GM3 clusters in the cell membrane are regulated in different ways and that segregation of the two gangliosides depends on the intact actin cytoskeleton.