Neuroimmunology of gangliosides in human neurons and glial cells in culture

Gangliosides (sialic-acid-bearing glycolipids) have received attention in recent years because of their role in cell recognition phenomena, synaptic transmission, memory generation, and nerve regeneration in the fields of neurosciences. It is suggested that each brain region or each neural cell type may contain a specific and characteristic set of gangliosides. We have investigated the immunocytochemical localization of several classes of gangliosides that include GM1, GM4, GD3, and GQ gangliosides on the cell surface of various cell types found in human neural cell cultures with antibodies specific for these gangliosides. Cell cultures were obtained from adult human brains and fetal human dorsal root ganglia and spinal cord and cultured in vitro for the period up to 6 months and utilized for the ganglioside immunocytochemistry. It was demonstrated that GM1 ganglioside was present in all galactocerebroside-positive oligodendrocytes and most of glial fibrillary acid protein (GFAP)-positive astrocytes (80%), most of neurofilament-positive neurons (80%), 50-70% of Schwann cells, and 5-10% of fibronectin-positive fibroblasts; GM4 ganglioside could be detected in all oligodendrocytes, 80% of astrocytes, and 50% of Schwann cells, while no staining was found in neurons or fibroblasts; GD3 ganglioside was present in all oligodendrocytes and 5-10% of astrocytes but not in neurons, Schwann cells, or fibroblasts; and all of fetal CNS neurons and approximately 80-90% of fetal dorsal root ganglia (DRG) neurons and a small percentage of astrocytes (10-20% in fetal and less than 1% in adult astrocytes) was labeled by A2B5 antibody which is specific for GQ ganglioside, while this antibody did not stain cell surface of oligodendrocytes, Schwann cells, or fibroblasts. Three classes of gangliosides, GM1, GM4, and GD3 were found to be definite components of fetal and adult human oligodendroglial plasma membrane, while GM1 and GM4 gangliosides were detected on the surface of most astrocytes. Only a minor population of astrocytes from both fetal and adult human CNS contained GD3 and GQ gangliosides. Two classes of gangliosides, GM1 and GQ, were detected on the surface of fetal human neurons. More than half of fetal Schwann cells reacted to GM1 and GM4 antibodies but did not to GD3 or GQ antibodies. We recognized the presence of a specific and characteristic set of gangliosides on the cell surface of different human neural cell types and these findings should facilitate further investigation of the precise biological activity of these gangliosides.     

Increased circulating T cell reactivity to GM3 and GQ1b gangliosides in primary progressive multiple sclerosis.

We have previously shown that patients with primary progressive multiple sclerosis (MS) have significantly elevated plasma levels of antibody to GM3 ganglioside compared to patients with relapsing-remitting MS, healthy subjects and patients with other neurological diseases. Anti-GM3 antibody levels were elevated also in patients with secondary progressive MS but to a lesser extent than in primary progressive MS. As gangliosides are particularly enriched in the axonal membrane, these findings suggested that antiganglioside immune responses might contribute to the axonal damage in progressive forms of MS. The present study was performed to determine whether peripheral blood T cell responses to GM3 are also increased in progressive MS. Blood was collected from 98 untreated patients with MS (40 with relapsing-remitting, 27 with secondary progressive and 31 with primary progressive MS), 50 healthy subjects and 24 patients with other disorders of the CNS, and reactivity to GM1, GM3, GD1a, GD1b, GD3, GT1b, GQ1b and sulphatide was assessed by 6-day T cell proliferation assays. Increased T cell reactivity to GM3 and GQ1b occurred significantly more often in patients with primary progressive MS than in healthy subjects and patients with other CNS diseases. These findings suggest that ganglioside-specific T cells may contribute to the axonal damage in primary progressive MS.     

IgM M-protein in a patient with sensory-dominant neuropathy binds preferentially to polysialogangliosides

A 77-year-old man presented sensory-dominant neuropathy associated with IgM M-protein reacting with various gangliosides. The M-protein bound to gangliosides with polysialosyl residue, such as GD1b, GD3, GT1b, GT3, GQ1b, and GQ1c. In addition, GD1a, GM3 and LM1, having a terminal monosialosyl epitope, were also recognized. Previously, Ilyas et al. described a similar case in which sensory symptoms were associated with IgM M-protein reacting with gangliosides containing a disialosyl group, such as GD3, GD1b, and GT1b, but not GM3 and GD1a. It is suggested that the reactivity of IgM M-protein with polysialogangliosides may be associated with the pathogenesis of sensory-dominant neuropathy.     

Influence of monensin on ganglioside anabolism and neurite stability in cultured chick neurons

We have examined the effects of monensin, a monovalent cationophore that disrupts exo- and endocytosis of membrane vesicles and diminishes Golgi anabolic function, on the incorporation of [3H]-galactose into glycosphingolipids in neurited primary cultures of chick embryo central nervous system neurons. A linear rate of incorporation into all ganglioside species from extracellular-labeled galactose was observed. Specific activity of anabolic labeling was markedly lower in GT1b and GQ1b than in the other major gangliosides of the embryonic neuron (GM3, GD3; GM2, GD2; GM1, GD1a, GD1b). With 100 nM monensin in the extracellular medium, the rate of labeling of GT1b diminished markedly to 20% of control; GD1a, GD1b, and GD2, to 35%; GQ1B to 48%; GD3 to 60%. Vigorous incorporation of label into GM3 was entirely undiminished by monensin. From these findings, it is suggested that ganglioside biosynthesis is compartmentalized in the cytodifferentiating embryonic neuron, with GM3 entirely, and GD3 and GQ1b partially, an extra-Golgi product. Extensive loss of neurites that occurred after several hours of exposure of the neurons to monensin could not be correlated directly with decreased ganglioside anabolism.     

Ganglioside metabolism in the hippocampus after septal lesion in rats

The changes in ganglioside composition and metabolism of deafferentiated rat hippocampus were estimated after septal lesion. A significant decrease in total ganglioside concentration was found 7 days after the lesion. The reduced level of total gangliosides persisted at 17 and 25 days. Relative increase in the proportion of GD1b and GX (O-acetylated GT1b) and decrease in GM1 were found in hippocampus only at 25 days post-lesion. The incorporation of 3H-N-acetylmannoseamine into gangliosides was examined in rats whose hippocampi were lesioned 25 days prior to radioprecursor injection. Differences in the labeling pattern of total and individual gangliosides were found. Increases in the label in GM1, GD3, and GD1a and decreases in GT1b and GQ1b were found 10 hr after isotope injection. However, decreases in the specific activity of all gangliosides except GT1b and GQ1b were observed 24 hr after 3H-N-acetylomannosamine injection, suggesting the activated turnover of gangliosides in postlesioned hippocampus. The significance of these changes has been discussed in terms of cellular damage and repair in the hippocampal tissue.     

GQ1b-seronegative Fisher syndrome: clinical features and new serological markers

IgG anti-GQ1b antibodies are a powerful serological marker for the diagnosis of Fisher syndrome (FS), but little is known regarding serological markers in FS patients that do not have the autoantibodies. The authors analyzed IgG antibodies against gangliosides other than GQ1b, ganglioside complexes, and ganglioside-like lipo-oligosaccharide (LOS) of Campylobacter jejuni isolates from FS patients. We identified 24 (12%) patients with GQ1b-seronegative FS among 207 FS patients who had been referred to our laboratory for anti-ganglioside antibody testing. Patients with GQ1b-seronegative FS were male and had a history of antecedent gastrointestinal illness more frequently than FS patients with IgG anti-GQ1b antibodies. Other clinical features during the illness were not distinguishing for GQ1b-seronegative FS. Four (17%) of 24 patients with GQ1b-seronegative FS had IgG antibodies against single gangliosides such as GM1b, GD1a, or GT1a. Antibodies against GM1 and GT1a complex were detected in four GQ1b-seronegative FS patients, three of whom did not have antibodies against single gangliosides. Mass spectrometry analysis showed that C. jejuni isolates from FS patients had GD1c-, GalNAc-GM1b-, or GalNAc-GD1c-like LOS, and not GQ1b-like LOS, highlighting the utility of examining serum antibodies against these ganglioside mimics in GQ1b-seronegative FS patients. Seven (29%) had IgG antibodies against the LOS from C. jejuni strains expressing GD1c-, GalNAc-GM1b-, or GalNAc-GD1c-like LOS. These findings suggest that IgG antibodies against GM1b, GD1c, GalNAc-GM1b, and ganglioside complexes are serological markers for GQ1b-seronegative Fisher syndrome.     

EGF-induced neuritogenesis and correlated synthesis of plasma membrane gangliosides in cultured embryonic chick CNS neurons

Epidermal growth factor (EGF), over a low range of concentrations (165-825 pM), induced neuritogenesis in post-mitotic chick CNS precursor neurons cultured in a serum-free medium, without the addition of other growth factors. Antibody to EGF blocks the neurite-promoting activity of EGF. Similarly, neuritogenesis of cultured chick CNS neurons in medium supplemented with 20% fetal bovine serum is blocked by antibody to EGF, even though serum may contain other neuronotrophic bioactive proteins and steroids. Quantitatively, the only major gangliosides of the undifferentiated post-mitotic neurons are GD3 and GD2. GD3 as well as its biosynthetic precursor, GM3, undergo active biosynthesis in serum-free medium as evidenced by their vigorous labeling by radioactive galactose supplied in the culture medium. When the undifferentiated neurons in serum-free medium are exposed to EGF, the ensuing generation of neurite plasma membrane coincides with initiation of biosynthesis of the sialosyl gangliotetraosyl ceramide species of gangliosides (GD1A, GD1B, GT1B, GQ1B). Antibody to EGF simultaneously inhibits biosynthesis of these gangliosides as well as inhibition of neuritogenesis. These findings indicate that EGF may be a primary neurite-inducing growth factor for post-mitotic embryonic CNS neurons and that gangliosides, particularly those of the sialosyl gangliotetraosyl ceramide species, characterize the plasma membrane of CNS neurons during neuritogenesis.     

Ganglioside changes in the chicken optic lobes as biochemical indicators of brain development and maturation

The development profiles of 16 different gangliosides of the optic lobes of the chicken were followed from the sixth day of incubation to the tenth posthatching week and correlated to known morphological development. Several, previously undetected novel fractions occurred between the sixth and tenth embryonic days. According to their migration rates on TLC-plates 4 of them may be GT3, GT2, GT1c, GQ1c. Three even more slowly migrating fractions represent penta-, hexa-, and septa-sialogangliosides. At the sixth day of incubation, characterized by maximal proliferation of neuro-epithelial cells, the optic lobes contained predominantly GD3.Up to the eleventh day of incubation, parallel to decreased mitotic activity, maximal cell migration and neuron differentiation, GD3, GD2, and GT3 decreased in favor of newly detected polysialogangliosides. Thereafter, up to hatching, parallel to increased growth and arborization of dendrites and axons as well as synaptogenesis, the newly detected polysialogangliosides decreased in favor of GD1b, GT1b, GQ1b, and GD1a.At hatching the myelin-specific GM4 appeared, reaching about 8% of total ganglioside sialic acid after 10 weeks. Likewise a fraction, migrating somewhat faster than GM1,increased. This band, named GM1', is suggested to be also myelin-associated. The other monosialogangliosides were always minor fractions, none exceeding 4% of total ganglioside sialic acid.     

GD1b-specific antibodies may bind to complex of GQ1b and GM1, causing ataxia

Monospecific IgG antibodies to GD1b ganglioside (GD1b-specific antibodies) have been found in patients with acute ataxic neuropathy and Guillain–Barré syndrome, but the association of the GD1b-specific antibodies with specific neurological conditions has yet to be established. We tested sera from more than 10,000 patients with various neurological disorders, and found six sera, which contained IgG antibodies to GD1b, but not to LM1, GM1, GM1b, GD1a, GalNAc-GD1a, GT1a, GT1b and GQ1b. All six patients who carried GD1b-specific antibodies presented with acute onset of ataxia and monophasic course of the illness, of whom five demonstrated cerebellar-like ataxia. Four patients had antecedent symptoms of upper respiratory tract infection. The six patients demonstrated areflexia, and four complained of distal numbness. All the six patients who had the GD1b-specific antibodies carried IgG antibodies to complex of GQ1b/GM1 and GT1a/GM1. GD1b-specific antibodies were significantly absorbed by GQ1b/GM1 and GT1a/GM1 and anti-GQ1b/GM1 and -GT1a/GM1 antibodies were absorbed by GD1b. In conclusion, the GD1b-specific antibodies, which recognizes GQ1b/GM1 or GT1a/GM1 complex, are associated with acute ataxia.     

Ganglioside reactive antibodies in the neuropathy associated with celiac disease

We tested patients with celiac disease (CD) for the presence of serum anti-ganglioside antibodies. Six of twenty-seven patient sera were reactive against brain gangliosides by an agglutination immunoassay. Neurological examination in all six revealed the presence of distal sensory loss, consistent with the diagnosis of peripheral neuropathy. When tested by ELISA for antibodies to isolated GM1, GM2, GD1a, GD1b, GT1b, and GQ1b gangliosides, all six were positive for IgG antibodies to at least one. The neuropathy of celiac disease may be autoimmune and associated with anti-ganglioside antibodies. The presence of IgG reactivity furthermore implicates a T cell-mediated response to ganglioside antigens.     

A case of pharyngeal-cervical-brachial variant of Guillain-Barré syndrome with positive anti-galactocerebroside (Gal-C) IgM antibody]

A 49-year-old man presented with hoarseness, dysphagia, muscle atrophy and weakness of deltoid, trapezius, sternocleidomastoid, rhomboid, anterior serratus, infraspinatus and supraspinatus. Anti-Gal-C IgM antibody was positive in the serum. The other antiganglioside antibodies (GM1, GM2, GM3, GD1a, GD1b, GD3, GT1a, GT1b, GQ1b, GA1, GalNAc-GD1a, GM1b) were negative. Patient contracted pneumonia but whether it was due to mycoplasma was not evident. Plasmapheresis improved his clinical state including a decrease of the antibody. This case was diagnosed pharyngeal-cervical-brachial variant of Guillain-Barré syndrome, and anti-Gal-C antibody seemed to be correlated with the pathogenesis of this syndrome. Gal-C is a major glycolipid of myelin and the cell membrane of the myelin-forming cell (oligodendrocytes and Schwann cells) and is free of specific localization and distribution. The mechanism how the anti-Gal-C IgM antibody induced bulbar paralysis and the symptoms localizing neck and upper limbs remains to be known.     

Neutral glycolipid and ganglioside composition of type-1 and type-2 astrocytes from rat cerebral hemisphere.

We reported previously that the major gangliosides in primary mixed-type astrocyte cultures are GM3 and GD3. To obtain more information regarding the exact distribution of glycosphingolipids in different types of astrocytes, we established a line of type-1 astrocytes that are characterized by a Ran-2 positive, broad flat morphology, and by the absence of binding to A2B5 antibodies. We also purified O-2A progenitor cells by immunopanning and cultured them in the presence of 10% newborn calf serum. They differentiated into type-2 astrocytes that were identified by immunostaining for each of GD3, A2B5, and GFAP. Using these cell cultures, we demonstrate that the major gangliosides were GM3 in type-1 astrocytes and GM3 and GD3 in type-2 astrocytes. In addition, a set of neutral glycolipids was identified based on the HP-TLC migration properties of CMH, CDH, CTH, and Glob, but the component distribution of these glycolipids is related to that of glycolipids of astrocytes. A marked increase in the expression of CTH and Glob was shown in type-2 astrocytes. The amount of neutral glycolipid-sugar was higher in the type-2 astrocytes than in the type-1 astrocytes. These results suggest that the increase in the total glycosphingolipid content and the change in the neutral glycolipid composition produced by type-2 astrocytes may be related to their biological functions and the cellular compositions.     

Association of antibodies to ganglioside complexes and conduction blocks in axonal Guillain‐Barré syndrome presenting as acute motor conduction block neuropathy

A close relationship between acute motor conduction block neuropathy and antibodies against the complex of GM1 and GalNAc‐GD1a has been reported. This study investigates the hypothesis that conduction block at the early phase of axonal Guillain‐Barré syndrome (GBS) is also associated with such ganglioside complexes. Sera were obtained from seven French patients with initial evidence of isolated conduction blocks that resolved or progressed to acute motor axonal neuropathy. Serum IgG to asialo‐GM1 and gangliosides of LM1, GM1, GM1b, GD1a, GalNAc‐GD1a, GD1b, GT1a, GT1b, and GQ1b as well as their complexes were measured. Five of seven patients progressed within the first month of disease to AMAN. One patient had IgG antibodies against the complex of asialo‐GM1 and each of the other ganglioside antigens. Another patient carried IgG antibodies against GM1 complex with GM1b, GD1a, and GT1a as well as asialo‐GM1 complex with GD1a and GT1a. None had IgG antibodies against GM1/GalNAc‐GD1a complex. Six patients had IgG against single antigens GM1, GD1a, GalNAc‐GD1a, GD1b, and asialo‐GM1. In three patients, a reduced reaction against GM1/GalNAc‐GD1a complex was observed. The presence of conduction block in axonal GBS is not always associated with anti‐GM1/GalNAc‐GD1a complex antibodies.     

Peripheral neuropathy associated with monoclonal IgM anti-Pr2 cold agglutinins.

A patient with a chronic, large fibre sensory neuropathy had an immunoglobulin M lambda monoclonal paraprotein reactive at titres in excess of 1/10(5) with NeuNAc(alpha 2-8)NeuNAc(alpha 2-3)Gal configured disialosyl groups present on the gangliosides GD1b, GT1b, GQ1b, and GD3. The paraprotein showed weaker reactivity with GD1a, GM3, and LM1 but no reactivity with GM2, GM1, or asialo-GM1. In addition, the paraprotein had cold agglutinating activity with anti-Pr2 specificity, Pr2 being an antigenic determinant on membrane glycoproteins or glycolipids in erythrocytes or both. A large fibre sensory neuropathy with monoclonal anti-disialosyl antibodies is an increasingly recognised form of paraproteinaemic neuropathy.     

Increased circulating antiganglioside antibodies in primary and secondary progressive multiple sclerosis

Plasma samples from 70 patients with multiple sclerosis (MS), 41 patients with other neurological diseases (OND), and 38 healthy subjects were examined for antibodies against gangliosides GM1, GM3, GD1a, GD1b, and GD3 using enzyme-linked immunosorbent assays. The percentages of subjects with increased anti-GM3 responses were significantly higher in the primary progressive MS (56.3%) and secondary progressive MS (42.9%) groups than in the relapsing-remitting MS (2.9%), healthy subject (2.6%), and OND (14.6%) groups. Elevated antiganglioside antibodies may be secondary to axonal damage or may be a cause of axonal damage and accumulating disability in progressive MS. In either case, they may serve as a marker of axonal damage in MS.     

Gangliosides of human cerebrospinal fluid in various neurologic diseases.

Simultaneous profile determination and quantification of human cerebrospinal fluid (CSF) gangliosides in various neurologic diseases (n = 71) was examined. Gangliosides were extracted with methanol/chloroform from clinically available amounts of CSF (4-5 ml), then separated and quantified by high-performance thin-layer chromatography (HPTLC) and direct densitometry. Based on chromatographic comparison with standards, the percentage of lipid-bound NeuAc positive fractions in 'normal' CSF samples were: GM1 (II3 NeuAc-GgOse4Cer) (3%); GD3 (II3 NeuAc2-Lac-Cer) (4%); GD1a (IV3 NeuAc, II3 NeuAc-GgOse4 Cer) (15%); X1 (3%); GD1b (II3(NeuAc)2-GgOse4 Cer) (16%); X2 (4%); GT1b (IV3 NeuAc, II3(NeuAc)2-GgOse4-Cer) (40%); and GQ1b (IV3(NeuAc)2, II3(NeuAc)2-GgOse4-Cer (15%). Similarity between CSF and CSF and human cerebellar cortex, particularly in proportion of "b" series gangliosides (GQ1b, GT1b, GD1b), could be observed. A higher proportion of GD1a ganglioside, with decreased GQ1b was found in infancy. The total ganglioside content (mean +/- 2 SD) varied between 645-894 micrograms/l. Significant alterations of the CSF ganglioside profile, with an increase in less polar gangliosides, GM3 and GD3, correlated with the blood-brain barrier dysfunction (CSF hemorrhages, compressive syndrome), or some malignant processes (metastatic brain melanoma). A statistically significant increase in the content of total CSF gangliosides was found in the following groups of patients as compared to controls: (1) ischemic cerebrovascular accident (CVI) with good outcome (P less than 0.02); (2) peripheral neuropathy and polyneuropathy (P less than 0.001) and (3) intravertebral discopathy (P less than 0.05). A significant decrease in the content of total CSF gangliosides was found in CVI group with lethal outcome (P less than 0.05).     

Molecular Microscopy of Brain Gangliosides: Illustrating their Distribution in Hippocampal Cell Layers

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Antiganglioside antibodies in Guillain-Barré syndrome after a recent cytomegalovirus infection

OBJECTIVE: To study the association between anti-ganglioside antibody responses and Guillan-Barré syndrome (GBS) after a recent cytomegalovirus (CMV) infection. METHODS: Enzyme linked immunosorbant assay (ELISA) was undertaken on serum samples from 14 patients with GBS with recent cytomegalovirus (CMV) infection (CMV+GBS) and 12 without (CMV-GBS), 17 patients with other neurological diseases (OND), 11 patients with a recent CMV infection but without neurological involvement, 11 patients with recent Epstein-Barr virus (EBV) infection but without neurological involvement, and 20 normal control (NC) subjects. RESULTS: IgM antibodies were found at 1:100 serum dilution to gangliosides GM2 (six of 14 patients), GM1 (four of 14), GD1a (three of 14) and GD1b (two of 14) in the serum samples of the CMV+GBS patients, but not in those of any of the CMV-GBS patients. IgM antibodies were also found to gangliosides GM1, GD1a, and GD1b in one of 11 OND patients, to ganglioside GM1 in one of 11 non- neurological CMV patients, and to ganglioside GD1b in one of 20 NC subjects. Some patients with EBV infection had IgM antibodies to gangliosides GM1 (five of 11), GM2 (three of 11), and GD1a (two of 11). However, the antibodies to ganglioside GM2 had a low titre, none being positive at 1:200 dilution, whereas five of the CMV+GBS serum samples remained positive at this dilution. CONCLUSION: Antibodies to ganglioside GM2 are often associated with GBS after CMV infection, but their relevance is not known. It is unlikely that CMV infection and anti-ganglioside GM2 antibodies are solely responsible and an additional factor is required to elicit GBS.     

Derivation of ganglioside-specific T cell lines of suppressor or helper phenotype from cerebrospinal fluid of multiple sclerosis patients

In order to investigate the specificity of activated T cells in the cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS), we have cultured cells in the presence of mitogen-free IL-2 but without any antigen. Two T cell lines have been derived and showed specific reactivity to certain purified gangliosides (GM1, GD1a, GD1b and GQ1b). However, responses to other brain and viral antigens were not seen, and neither were T cell lines from peripheral blood lymphocytes (PBL) of normal, MS or other neurological disease patients stimulated by these gangliosides. Release of IL-2 could be detected after incubation of these CSF lines with specific gangliosides. One line exhibited predominantly helper/inducer (T4+) phenotype whilst the other was suppressor/cytotoxic (T8+), and further analysis indicated it could be of the suppressor phenotype. These data may have implications for T cell-induced demyelination in MS.     

Gangliosides in cerebrospinal fluid in children with autism spectrum disorders

Gangliosides are sialic acid-containing glycolipids found in all cells, especially abundant in nerve cells and mainly situated on outer-membrane surfaces. The aim of this study was to provide data on the concentration of gangliosides in the CSF of children and adolescents with autism spectrum disorders (ASD) - 66 with autistic disorder, and 19 with other autism spectrum disorders. The comparison group consisted of 29 children and adolescents, whose CSF had been sampled to exclude acute infectious CNS disorder. The concentrations of the gangliosides GM1, GD1a, GD1b, and GT1b were determined using a microimmunoaffinity technique. The ASD group had a significantly higher concentration of ganglioside GM1 compared with the comparison group. The GM1 increase could not be explained as secondary to other clinical factors. Mean ganglioside levels did not differentiate subgroups with autistic disorder and those with a more atypical clinical picture, nor subgroups with known medical disorders and those with idiopathic autism. Altered patterns of gangliosides in the CNS might reflect important correlates of pathogenesis in autism.