A Phase I Study of the Anti-Idiotype Vaccine Racotumomab in Neuroblastoma and Other Pediatric Refractory Malignancies.

BACKGROUND: Pediatric neuroectodermal malignancies express N-glycolylated gangliosides including N-glycolyl GM3 (NeuGcGM3) as targets for immunotherapy. PROCEDURE: We evaluated the toxicity and maximum tolerated dose and immunological response of racotumomab, an anti-idiotype vaccine targeting NeuGcGM3 through a Phase I study enrolling children with relapsed or resistant tumors expressing NeuGcGM3. MATERIALS AND METHODS: Drug dose was escalated to three levels (0.15-0.25-0.4 mg) of racotumomab administered intradermally. Each drug level included three patients receiving a total of three doses, every 14 days. A confirmation cohort was added to the highest dose level. Antibody response was assessed upon study entry and at 4-week intervals for at least three immunological determinations for each patient. RESULTS: Fourteen patients were enrolled (10 with neuroblastoma, one with retinoblastoma, one with Wilms' tumor, and two with brainstem glioma). Three patients completed the three drug levels and three were enrolled in the confirmation cohort. One patient died of tumor progression before completing the three applications. Racotumomab was well tolerated. The only side effect observed was grade 1-2 toxicity at the injection site. Racotumomab elicited an IgM and/or IgG antibody response directed against NGcGM3 in nine patients and IgM against racotumomab in 11 of 13 evaluable patients. The maximum tolerated dose was not reached and no dose-limiting toxicity was seen. CONCLUSIONS: Racotumomab vaccination has a favorable toxicity profile up to a dose of 0.4 mg, and most patients elicited an immune response. Its activity as immunotherapy for neuroectodermal malignancies will be tested in further clinical trials.

Alteration of ganglioside biosynthesis responsible for complex hereditary spastic paraplegia.

Hereditary spastic paraplegias (HSPs) form a heterogeneous group of neurological disorders. A whole-genome linkage mapping effort was made with three HSP-affected families from Spain, Portugal, and Tunisia and it allowed us to reduce the SPG26 locus interval from 34 to 9 Mb. Subsequently, a targeted capture was made to sequence the entire exome of affected individuals from these three families, as well as from two additional autosomal-recessive HSP-affected families of German and Brazilian origins. Five homozygous truncating (n = 3) and missense (n = 2) mutations were identified in B4GALNT1. After this finding, we analyzed the entire coding region of this gene in 65 additional cases, and three mutations were identified in two subjects. All mutated cases presented an early-onset spastic paraplegia, with frequent intellectual disability, cerebellar ataxia, and peripheral neuropathy as well as cortical atrophy and white matter hyperintensities on brain imaging. B4GALNT1 encodes ß-1,4-N-acetyl-galactosaminyl transferase 1 (B4GALNT1), involved in ganglioside biosynthesis. These findings confirm the increasing interest of lipid metabolism in HSPs. Interestingly, although the catabolism of gangliosides is implicated in a variety of neurological diseases, SPG26 is only the second human disease involving defects of their biosynthesis.

Role of the 9-O-acetyl GD3 in subventricular zone neuroblast migration.

In the mammalian central nervous system the subventricular zone (SVZ) is one of the few neurogenic regions that persist postnatally. Neuroblasts generated in the SVZ migrate from this region tangentially towards the olfactory bulbs via the rostral migratory stream (RMS) and give rise to interneurons. In previous studies, an important role in radial migration of cerebellar granule neurons has been attributed to the 9-O-acetylated GD3 ganglioside. Previous data demonstrated the expression of 9-O-acetyl GD3 in the rostral migratory stream in vivo as well as in chains of neuroblasts that migrate from SVZ explants in vitro. Herein, using the Jones monoclonal antibody (Jones mAb), we combined SVZ explant migration measurements and time-lapse videomicroscopy of migrating neuroblasts to show that SVZ neuroblast migration is inhibited by the antibody that recognizes 9-O-acetyl GD3 but not by A2B5, an antibody that recognizes c-series gangliosides. In addition, inhibition of ganglioside synthesis results in reduction of migratory halos around SVZ explants. Coherently, we show that most migratory neuroblasts which express the embryonic form of NCAM co-express 9acGD3. Also, we observe that some of the ganglioside positive neuroblasts also express nestin consistent with their maintained proliferative capacity. These results strongly support that the 9-O-acetyl GD3 has a pivotal role in neuroblast migration from SVZ, being fundamental for cell-cell and cell-substrate interactions in this region.

GD1a modulates GM-CSF-induced cell proliferation.

Gangliosides have been extensively described to be involved in the proliferation and differentiation of various cell types, such including hematopoietic cells. Our previous studies on murine models of stroma-mediated myelopoiesis have shown that gangliosides are required for optimal capacity of stromal cells to support proliferation of myeloid precursor cells, being shed to the supernatant and selectively incorporated into myeloid cell membranes. Here we describe the effect of gangliosides on the specific granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced proliferation. For that, we used the monocytic FDC-P1 cell line, which is dependent upon GM-CSF for survival and proliferation. Cells were cultured in the presence of GM-CSF and exogenous gangliosides (GM3, GD1a or GM1) or in the absence of endogenous ganglioside synthesis by the use of a ceramide-synthase inhibitor, D-PDMP. We observed that exogenous addition of GD1a enhanced the GM-CSF-induced proliferation of the FDC-P1 cells. Also, we detected an increase in the expression of the α isoform of the GM-CSF receptor (GMRα) as well as of the transcription factor C/EBPα. On the contrary, inhibition of glucosylceramide synthesis was accompanied by a decrease in cell proliferation, which was restored upon the addition of exogenous GD1a. We also show a co-localization of GD1a and GMR by immunocytochemistry. Taken together, our results suggest for the first time that ganglioside GD1a play a role on the modulation of GM-CSF-mediated proliferative response, which might be of great interest not only in hematopoiesis, but also in other immunological processes, Alzheimer disease, alveolar proteinosis and wherever GM-CSF exerts its effects.

Amyloid-ß induced toxicity involves ganglioside expression and is sensitive to GM1 neuroprotective action.

The effect of Aß25-35 peptide, in its fibrillar and non-fibrillar forms, on ganglioside expression in organotypic hippocampal slice cultures was investigated. Gangliosides were endogenously labeled with D-[1-C(14)] galactose and results showed that Aß25-35 affected ganglioside expression, depending on the peptide aggregation state, that is, fibrillar Aß25-35 caused an increase in GM3 labeling and a reduction in GD1b labeling, whereas the non-fibrillar form was able to enhance GM1 expression. Interestingly, GM1 exhibited a neuroprotective effect in this organotypic model, since pre-treatment of the hippocampal slices with GM1 10 µM was able to prevent the toxicity triggered by the fibrillar Aß25-35, when measured by propidium iodide uptake protocol. With the purpose of further investigating a possible mechanism of action, we analyzed the effect of GM1 treatment (1, 6, 12 and 24h) upon the Aß-induced alterations on GSK3ß dephosphorylation/activation state. Results demonstrated an important effect after 24-h incubation, with GM1 preventing the Aß-induced dephosphorylation (activation) of GSK3ß, a signaling pathway involved in apoptosis triggering and neuronal death in models of Alzheimer's disease. Taken together, present results provide a new and important support for ganglioside participation in development of Alzheimer's disease experimental models and suggest a protective role for GM1 in Aß-induced toxicity. This may be useful for designing new therapeutic strategies for Alzheimer's treatment.

Ganglioside glycosyltransferases and newly synthesized gangliosides are excluded from detergent-insoluble complexes of Golgi membranes.

GEM (glycosphingolipid-enriched microdomains) are specialized detergent-resistant domains of the plasma membrane in which some gangliosides concentrate. Although genesis of GEM is considered to occur in the Golgi complex, where the synthesis of gangliosides also occurs, the issue concerning the incorporation of ganglioside species into GEM is still poorly understood. In this work, using Chinese hamster ovary K1 cell clones with different glycolipid compositions, we compared the behaviour with cold Triton X-100 solubilization of plasma membrane ganglioside species with the same species newly synthesized in Golgi membranes. We also investigated whether three ganglioside glycosyltransferases (a sialyl-, a N-acetylgalactosaminyl- and a galactosyl-transferase) are included or excluded from GEM in Golgi membranes. Our data show that an important fraction of plasma membrane G(M3), and most G(D3) and G(T3), reside in GEM. Immunocytochemical examination of G(D3)-expressing cells showed G(D3) to be distributed as cold-detergent-resistant patches in the plasma membrane. These patches did not co-localize with a glycosylphosphatidylinositol-anchored protein used as GEM marker, indicating a heterogeneous composition of plasma membrane GEM. In Golgi membranes we were unable to find evidence for GEM localization of either ganglioside glycosyltransferases or newly synthesized gangliosides. Since the same ganglioside species appear in plasma membrane GEM, it was concluded that in vivo nascent G(D3), G(T3) and G(M3) segregate from their synthesizing transferases and then enter GEM. This latter event could have taken place shortly after synthesis in the Golgi cisternae, along the secretory pathway and/or at the cell surface.

Papel de dissialogangliosídeos na progressão tumoral de melanomas / Role of disialoganglioside in melanoma tumor progression

Na progressão tumoral, células de melanoma acumulam derivados de dissialogangliosídeos. A função desses glicoconjugados na progressão do tumor ainda é desconhecida. Tem-se estudado um possível papel de dissialogangliosídeos na migração celular. A linhagem celular de melanócito, melan-a, foi usada como modelo para a expressão do gene da GD3-sintase (ST8Sia I), a qual é a única enzima conhecida na conversão de GM3 no gangliosídio associado a tumor, GD3... Essas células também foram mais migratórias do que as células GM3+/GD3-, sugerindo que GM3 tem um papel negativo na migração celular. Depois foi examinado o efeito da adição de gangliosídeos exógenos. A adição de GM3 em células derivadas de melan-a também inibiu a migração celular; enquanto que a adição de GD3 a promoveu a migração. Juntos, nossos resultados sugerem que a razão entre GM3 e GD3 em melanócitos mostra um papel modulatório na motilidade celular dependente de integrina. Nós também mostramos a papel pró-apoptótico de GD3 em células de melanoma murino (TM1). Uma vez que, o insucesso de transfecção da expressão estável de GD3 sintase em TM1 pode ser devido a sensibilização destas células a morte celular na presença de uma superexpressão de GD3. As células sobreviventes seriam aquelas que adquiriam a capacidade de modificar o seu produto da GD3 sintase para um dos seus derivados, não-apoptogênico. Desta forma, nós propomos que a persistência da expressão de GD3 pode selecionar células resistentes a apoptose, as quais, por sua vez adquiririam um fenótipo mais migratório...(aU)

Upon tumor progression, melanoma cells accumulate disialoganglioside derivatives. The function of these molecules in tumor progression remains unknown. To address possible functions of the disialoganglioside G03 , the murine melanocyte cell line, melan-a, was transfected with the G03 synthase gene (ST8Sia 1). In melan-a transfectants, disialogangliosides modulated melanocyte cell adhesion and migration. Ali transfectants displayed equivalent . . + + leveis of 1ntegnns on the cell surface. GM3 /G03 melanocytes tended to adhere and migrate more towards laminin-1 coated surfaces than GM3 +/G03- cells. Depletion of glycosphingolipids, using phenyl-palmitoylamino-pirrolidinopropanol (PPPP), rendered cells (LacCer-)/GM3- /G03-.These latter cells were also more migratory than GM3 +fG03- cells, suggesting that GM3 plays a negative role in cell migration. We next examined the effect of exogenously added gangliosides. Addition of GM3 to melan-a cells also inhibited cell migration; whereas exogenously added G03 promoted it. Taken together, our results suggest that the ratio between GM3 and G03 in melanocytes modulates migration. We have also uncovered a pro-apoptogenic function of G03 in murine melanomas, as we failed to achieve stable expression of G03 in melanoma cells due to their increased sensitivity to cell death inducing agents. Failure of maintaining high expression of G03 in murine melanoma cells was associated with de novo expression of GM2/Go2 synthase gene. This gene converts G03 in Go2. GM2/Go2 synthase gene expression could warrant survival of G03 synthase expressing cells, by consuming Go3 and therefore releasing cells from the pro-apoptogenic effect of this ganglioside (AU)

Ganglioside glycosyltransferases organize in distinct multienzyme complexes in CHO-K1 cells.

The synthesis of gangliosides is compartmentalized in the Golgi complex. In most cells, glycosylation of LacCer, GM3, and GD3 to form higher order species (GA2, GM2, GD2, GM1, GD1b) is displaced toward the most distal aspects of the Golgi and the trans-Golgi network, where the involved transferases (GalNAcT and GalT2) form physical and functional associations. Glycosylation of the simple species LacCer, GM3, and GD3, on the other hand, is displaced toward more proximal Golgi compartments, and we investigate here whether the involved transferases (GalT1, SialT1, and SialT2) share the property of forming physical associations. Co-immunoprecipitation experiments from membranes of CHO-K1 cells expressing epitope-tagged versions of these enzymes indicate that GalT1, SialT1, and SialT2 associate physically in a SialT1-dependent manner and that their N-terminal domains participate in these interactions. Microscopic fluorescence resonance energy transfer and fluorescence recovery after photobleaching in living cells confirmed the interactions, and in addition to showing a Golgi apparatus localization of the complexes, mapped their formation to the endoplasmic reticulum. Neither co-immunoprecipitation nor fluorescence resonance energy transfer detected interactions between either GalT2 or GalNAcT and GalT1 or SialT1 or SialT2. These results, and triple color imaging of Golgi-derived microvesicles in nocodazole-treated cells, suggest that ganglioside synthesis is organized in distinct units each formed by associations of particular glycosyltransferases, which concentrate in different sub-Golgi compartments.

Understanding the stepwise synthesis of glycolipids.

Glycolipid expression is highly regulated during development and differeniation. The control relies mainly on transcriptional modulation of key glycosyltransferases acting at the branching points of the pathway of biosynthesis. Transferases are Golgi residents that depend on N-glycosylation and oligosaccharide processing for proper folding in the endoplasmic reticulum. The N-terminal domain bears information for their transport to the Golgi, retention in the organelle and differential concentration in sub-Golgi compartments. In the Golgi, some transferases associate forming functional multienzyme complexes. It is envisaged that the machinery for synthesis in the Golgi complex, and its dynamics, constitute a potential target for fine tuning of the control of glycolipid expression according to cell demands.

Expression and function of ganglioside 9-O-acetyl GD3 in postmitotic granule cell development.

We have shown previously that the Jones monoclonal antibody (Jones mAb) recognizes 9-O-acetyl GD3 expressed during periods of neuronal migration and neurite outgrowth in the developing rat nervous system. In the present study we investigated the expression of this ganglioside in the developing cerebellum and correlated this expression with granule cell migration. Electron microscopic immunocytochemistry revealed that around the peak of cerebellar neuronal migration (7-day-old rat), 9-O-acetyl GD3 was localized at the contact sites between migrating granule cells and radial glia in the external granular layer and prospective molecular layer. In addition, using microexplant and slice cultures of the postnatal rat cerebellum, we tested whether the ganglioside detected by our antibody contribute to the regulation of neuronal migration in the cerebellar cortex. We have shown that the Jones mAb blocks the migration of neurons in a dose-dependent manner. These findings suggest strongly that 9-O-acetyl GD3 is involved in granule cell migration in the developing cerebellum.

2,4-Dichlorophenoxyacetic acid disrupts the cytoskeleton and disorganizes the Golgi apparatus of cultured neurons.

2,4-Dichlorophenoxyacetic acid (2,4-D) is a potent neurotoxic herbicide widely used in agriculture. The basic mechanisms by which 2,4-D produces cell damage have not yet been determined. In this study we have examined the effects of 2,4-D in primary cultures of cerebellar granule cells in order to obtain insights into the possible mechanisms underlying the toxic effects of this herbicide. The results obtained indicate that a 24-hour exposure to 2,4-D produces a striking and dose-dependent inhibition of neurite extension. This phenomenon is paralleled by a significant reduction in the cellular content of both dynamic and stable microtubules, a disorganization of the Golgi apparatus, and an inhibition in the synthesis of complex gangliosides. Interestingly, 2,4-D inhibits the in vitro polymerization of purified tubulin. Taken together, the present observations raise the possibility that at least one basic mechanism underlying 2,4-D neurotoxicity involves an inhibition of microtubule assembly. That event may cause a decreased neurite outgrowth response, and could also explain the observed differences in the pattern of ganglioside biosynthesis and/or the disorganization of the Golgi apparatus.

Ganglioside expression during differentiation of chick retinal cells in vitro.

The neural retina has been widely used to study the developmental patterns of ganglioside metabolism. Recent findings about in vitro differentiating chick embryo retina cells showed that: a) GD3 and GD1a ganglioside patterns undergo the most dramatic changes; b) when the cells emit neurites, GD3 ganglioside and a group of complex gangliotetraosylgangliosides (GTOG) are transiently coexpressed; c) synchronized developmental phenomena are dissociated by anti-GM1 antibodies; d) GD3 remains as a major ganglioside in differentiated neurons, though it is almost not immunoexpressed; e) GTOG affect antibody binding to GD3; f) the content of gangliosides involved in neural differentiation modifies their immunostain localization on cell membrane; g) after exogenous GTOG uptake, immature neurons mimic GD3 immunofluorescent localization of mature cells; h) a subset of purified retinal ganglion cells express GTOG characteristic of mature neurons.

Chinese hamster ovary cells lacking GM1 and GD1a synthesize gangliosides upon transfection with human GM2 synthase.

GM3-positive Chinese hamster ovary cells (CHO-K1 cells) lack the ability to synthesize GM2 and the complex gangliosides GM1 and GD1a from [3H]Gal added to the culture medium. However, they acquire the ability to synthesize GM2 and to synthesize and immunoexpress complex gangliosides upon transient transfection with a cDNA encoding the human GM3:N-acetylgalactosaminyl transferase (GM2 synthase). The activities of endogenous GM1- and GD1a-synthases in the parental cell line and in cells transfected with the plasmid with or without the GM2 synthase cDNA were essentially identical and comparable in terms of specific activity with the endogenous GM3 synthase. Results indicate that glycosyltransferases acting on GM2 to produce GM1 and GD1a are constitutively present in CHO-K1 cells, and that the expression of their activities depend on the supply of the acceptor GM2. In addition, these results lend support to the notion that GM2 synthase is a key regulatory enzyme influencing the balance between simple and complex gangliosides.

Compartmental organization of the synthesis of GM3, GD3, and GM2 in golgi membranes from neural retina cells.

The relationship among lactosylceramide-(LacCer), GD3- and GM2-synthases and between the two last transferases and their common GM3 acceptor was investigated in intact Golgi membrane from chick embryo neural retina cells at early (8-days) and late (14 days) stages of the embryonic development. [3H]Gal was incorporated into endogenous glucosylceramide by incubation of Golgi membranes with UDP-[3H]Gal. Conversion of the synthesized [3H]Gal-LacCer into GM3, and of the latter into GD3, GM2 and GD2 was examined after a second incubation step with unlabeled CMP-NeuAc and/or UDP-GalNAc. With CMP-NeuAc, most [3H]Gal-LacCer was converted into GM3 in either 8- or 14- day membranes. However, while about 90% of GM3 was converted into GD3 in 8-day membranes, only about 25% followed this route in 14-day membranes. With CMP-NeuAc and UDP-GalNAc, about 90% of GM3 was used for synthesis of GM2 in 14-day membranes, while in 8-day membranes about 80% followed the route to GD3, and a part to GD2. Performing the second incubation step in the presence of increasing detergent concentrations showed that conversion of GM3 to GM2 was inhibited at concentrations lower than those required for inhibition of LacCer to GM3 conversion. Taken together, results indicate that transfer steps leading to synthesis of GM3, GD3, GM2 and GD2 from LacCer are functionally coupled in the Golgi membranes, and that GD3- and GM2-synthases compete in a common compartment for using a fraction of GM3 as substrate. In this competition, the relative activities of the transferases and their relative saturation with the respective donor sugar nucleotides, are important factors influencing conversion of GM3 toward either GD3 or GM2.

Identification of an endogenous inhibitor of the UDP-N-acetylgalactosamine: GM3, N-acetylgalactosaminyl transferase as apolipoprotein A1.

A previously described inhibitor of the UDP-N-acetylgalactosamine: GM3, N-acetylgalactosaminyltransferase (GalNAc-T) (Quiroga et al., 1, 2), was purified from chicken blood serum by a new procedure. When subjected to SDS-PAGE, two major polypeptides of 27 and 70 kDa were observed. When tested in vitro, only the 27 kDa polypeptide inhibited the GalNAc-T. When added to chick cerebral embryonic neurons in culture, both polypeptides inhibited neuritogenesis. Both the 27 kDa and the 70 kDa fractions were present in the cells at 3 h following their addition to the cultures; both polypeptides had aneuritogenic activity and both inhibited the incorporation of [3H]-galactose into the cell gangliosides modifying their labeling pattern to a similar extent. Sequencing of the amino terminal end of the polypeptides showed that 18 and 9 amino acids from, respectively, the 27 and the 70 kDa polypeptides, were 100% homologues with the corresponding region of chick apolipoprotein Al (apo Al). After addition to cells in culture, no interconversion between the two polypeptides was detected after up to 20 h in culture. A monoclonal antibody that recognizes only the 70 kDa polypeptide, blocks its aneuritogenic effect without modifying that of the 27 kDa fraction. It is concluded that the endogenous inhibitor of GalNAc-T is apo Al.