g(m3)-ganglioside and Disease-Models--Animal

Numerous molecules have been considered as targets for cancer immunotherapy because of their levels of expression on tumor cells, their putative importance for tumor biology, and relative immunogenicity. In this review we focus on the ganglioside GM3(Neu5Gc), a glycosphingolipid present on the outer side of the plasma membrane of vertebrate cells. The reasons for selecting GM3(Neu5Gc) as a tumor-specific antigen and its use as a target for cancer immunotherapy are discussed, together with the development of antitumor therapies focused on this target by the Center of Molecular Immunology (CIM, Cuba).

Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Murine-Derived; Antigens, Neoplasm; Carbohydrate Sequence; Disease Models, Animal; G(M3) Ganglioside; Humans; Immunotherapy; Neoplasms

Gangliosides are highly enriched in the brain and are critical for its normal development and function. However, in some rare neurometabolic diseases, a deficiency in lysosomal ganglioside hydrolysis is pathogenic and leads to early-onset neurodegeneration, neuroinflammation, demyelination, and dementia. Increasing evidence also suggests that more subtle ganglioside accumulation contributes to the pathogenesis of more common neurological disorders including Alzheimer's disease (AD). Notably, ganglioside GM3 levels are elevated in the brains of AD patients and in several mouse models of AD, and plasma GM3 levels positively correlate with disease severity in AD patients.. Tg2576 AD model mice were fed chow formulated with a small molecule inhibitor of glucosylceramide synthase (GCSi) to determine whether reducing glycosphingolipid synthesis affected aberrant GM3 accumulation, amyloid burden, and disease manifestations in cognitive impairment. GM3 was measured with LC-MS, amyloid burden with ELISA and amyloid red staining, and memory was assessed using the contextual fear chamber test.. GCSi mitigated soluble Aβ42 accumulation in the brains of AD model mice when treatment was started prophylactically. Remarkably, GCSi treatment also reduced soluble Aβ42 levels and amyloid plaque burden in aged (i.e., 70 weeks old) AD mice with preexisting neuropathology. Our analysis of contextual memory in Tg2576 mice showed that impairments in remote (cortical-dependent) memory consolidation preceded deficits in short-term (hippocampal-dependent) contextual memory, which was consistent with soluble Aβ42 accumulation occurring more rapidly in the cortex of AD mice compared to the hippocampus. Notably, GCSi treatment significantly stabilized remote memory consolidation in AD mice-especially in mice with enhanced cognitive training. This finding was consistent with GCSi treatment lowering aberrant GM3 accumulation in the cortex of AD mice.. Collectively, our results indicate that glycosphingolipids regulated by GCS are important modulators of Aβ neuropathology and that glycosphingolipid homeostasis plays a critical role in the consolidation of remote memories.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Disease Models, Animal; G(M3) Ganglioside; Glucosyltransferases; Memory, Long-Term; Mice; Mice, Transgenic; Plaque, Amyloid

Glycosphingolipids (GSLs) are amphipathic lipids composed of a sphingoid base and a fatty acyl attached to a saccharide moiety. GSLs play an important role in signal transduction, directing proteins within the membrane, cell recognition, and modulation of cell adhesion. Gangliosides and sulfatides belong to a group of acidic GSLs, and numerous studies report their involvement in neurodevelopment, aging, and neurodegeneration. In this study, we used an approach based on hydrophilic interaction liquid chromatography (HILIC) coupled to high-resolution tandem mass spectrometry (HRMS/MS) to characterize the glycosphingolipid profile in rat brain tissue. Then, we screened characterized lipids aiming to identify changes in glycosphingolipid profiles in the normal aging process and tau pathology. Thorough screening of acidic glycosphingolipids in rat brain tissue revealed 117 ganglioside and 36 sulfatide species. Moreover, we found two ganglioside subclasses that were not previously characterized-GT1b-Ac2 and GQ1b-Ac2. The semi-targeted screening revealed significant changes in the levels of sulfatides and GM1a gangliosides during the aging process. In the transgenic SHR24 rat model for tauopathies, we found elevated levels of GM3 gangliosides which may indicate a higher rate of apoptotic processes.

Topics: Acidic Glycosphingolipids; Aging; Animals; Animals, Genetically Modified; Brain; Chromatography, Liquid; Disease Models, Animal; G(M3) Ganglioside; Humans; Hydrophobic and Hydrophilic Interactions; Neurofibrils; Rats; Sulfoglycosphingolipids; tau Proteins; Tauopathies

Juvenile neuronal ceroid lipofuscinosis (JNCL) is caused by mutations in the

Topics: Animals; Cerebellum; Cholera Toxin; Disease Models, Animal; G(M3) Ganglioside; Lysosomes; Membrane Glycoproteins; Mice; Molecular Chaperones; Neuronal Ceroid-Lipofuscinoses

Elucidation of the healing mechanisms in damaged tissues is a critical step for establishing breakthroughs in tissue engineering. Articular cartilage is clinically one of the most successful tissues to be repaired with regenerative medicine because of its homogeneous extracellular matrix and few cell types. However, we only poorly understand cartilage repair mechanisms, and hence, regenerated cartilage remains inferior to the native tissues. Here, we show that glycosylation is an important process for hypertrophic differentiation during articular cartilage repair. GM3, which is a precursor molecule for most gangliosides, was transiently expressed in surrounding damaged tissue, and depletion of GM3 synthase enhanced cartilage repair. Gangliosides also regulated chondrocyte hypertrophy via the Indian hedgehog pathway. These results identify a novel mechanism of cartilage healing through chondrocyte hypertrophy that is regulated by glycosylation. Manipulation of gangliosides and their synthases may have beneficial effects on articular cartilage repair.

Topics: Animals; Biomarkers; Bone Regeneration; Cartilage, Articular; Cell Differentiation; Chondrocytes; Chondrogenesis; Collagen; Disease Models, Animal; Female; G(M3) Ganglioside; Gangliosides; Gene Expression; Hedgehog Proteins; Mice; Mice, Knockout; Osteoarthritis; Signal Transduction; Tissue Engineering; Wound Healing

To determine the capacity of continual low-dose lysosomal enzyme infusion into the cerebrospinal fluid of mucopolysaccharidosis type IIIA (MPS IIIA) mice to reverse established neurodegenerative disease. The rationale behind the study is that there is only limited animal model-derived evidence supporting treatment of symptomatic patients, principally because few studies have been designed to examine disease reversibility.. Twelve-week old MPS IIIA mice were implanted with indwelling unilateral intra-ventricular cannulae. These were connected to subcutaneous mini-osmotic pumps infusing recombinant human sulphamidase. Pump replacement was carried out in some mice at 16-weeks of age, enabling treatment to continue for a further month. Control affected/unaffected mice received vehicle via the same method. Behavioural, neuropathological and biochemical parameters of disease were assessed.. Improvement in some, but not all, behavioural parameters occurred. Sulphamidase infusion mediated a statistically significant reduction in primary (heparan sulphate) and secondary (gangliosides GM2, GM3) substrate accumulation in the brain, with small reductions in micro- but not astro-gliosis. There was no change in axonal spheroid number. All mice developed a humoural response, however the antibodies were non-neutralising and no adverse clinical effects were observed.. Continual infusion of replacement enzyme partially ameliorates clinical, histological and biochemical aspects of MPS IIIA mice, when treatment begins at an early symptomatic stage.

Topics: Animals; Brain; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Administration Routes; Drug Delivery Systems; Female; G(M3) Ganglioside; Gangliosidoses, GM2; Humans; Hydrolases; Male; Maze Learning; Mice; Mice, Inbred C57BL; Mice, Transgenic; Motor Activity; Mucopolysaccharidosis III; Neurodegenerative Diseases; Sulfatases

Mucopolysaccharidosis type IIIA (MPS IIIA) is predominantly a disorder of the central nervous system, caused by a deficiency of sulfamidase (SGSH) with subsequent storage of heparan sulfate-derived oligosaccharides. No widely available therapy exists, and for this reason, a mouse model has been utilized to carry out a preclinical assessment of the benefit of intraparenchymal administration of a gene vector (AAVrh10-SGSH-IRES-SUMF1) into presymptomatic MPS IIIA mice. The outcome has been assessed with time, measuring primary and secondary storage material, neuroinflammation, and intracellular inclusions, all of which appear as the disease progresses. The vector resulted in predominantly ipsilateral distribution of SGSH, with substantially less detected in the contralateral hemisphere. Vector-derived SGSH enzyme improved heparan sulfate catabolism, reduced microglial activation, and, after a time delay, ameliorated GM3 ganglioside accumulation and halted ubiquitin-positive lesion formation in regions local to, or connected by projections to, the injection site. Improvements were not observed in regions of the brain distant from, or lacking connections with, the injection site. Intraparenchymal gene vector administration therefore has therapeutic potential provided that multiple brain regions are targeted with vector, in order to achieve widespread enzyme distribution and correction of disease pathology.

Topics: Animals; Antibodies, Neutralizing; Autophagy; Biomarkers; Brain; Dependovirus; Disease Models, Animal; DNA-Binding Proteins; Endosomes; Enzyme Activation; Female; G(M3) Ganglioside; Genetic Therapy; Genetic Vectors; Glial Fibrillary Acidic Protein; Heparitin Sulfate; High Mobility Group Proteins; Humans; Hydrolases; Lysosomes; Male; Mice; Mucopolysaccharidosis III; Saccharomyces cerevisiae Proteins; Transduction, Genetic

Interaction between epidermal growth factor receptor (EGFR) signaling with GM3 ganglioside expression has been previously described. However, little is known about EGFR and NeuGcGM3 co-expression in cancer patients and their therapeutic implications. In this paper, we evaluate the co-expression of EGFR and NeuGcGM3 ganglioside in tumors from 92 patients and in two spontaneous lung metastasis models of mice (Lewis lung carcinoma (3LL-D122) in C57BL/6 and mammary carcinoma (4T1) in BALB/c). As results, co-expression of EGFR and NeuGcGM3 ganglioside was frequently observed in 63 of 92 patients (68 %), independently of histological subtype. Moreover, EGFR is co-expressed with NeuGcGM3 ganglioside in the metastasis of 3LL-D122 and 4T1 murine models. Such dual expression appears to be therapeutically relevant, since combined therapy with mAbs against these two molecules synergistically increase the survival of mice treated. Overall, our results suggest that NeuGcGM3 and EGFR may coordinately contribute to the tumor cell biology and that therapeutic combinations against these two targets might be a valid strategy to explore.

Topics: Animals; Antibodies, Monoclonal; Carcinoma, Lewis Lung; Cell Line, Tumor; Disease Models, Animal; ErbB Receptors; Female; G(M3) Ganglioside; Gene Expression Regulation, Neoplastic; Humans; Mammary Neoplasms, Animal; Mice; Neoplasm Metastasis

Small fiber neuropathy is a well-recognized complication of type 2 diabetes and has been shown to be responsible for both neuropathic pain and impaired wound healing. In previous studies, we have demonstrated that ganglioside GM3 depletion by knockdown of GM3 synthase fully reverses impaired wound healing in diabetic mice. However, the role of GM3 in neuropathic pain and small fiber neuropathy in diabetes is unknown.. Determine whether GM3 depletion is able to reverse neuropathic pain and small fibers neuropathy and the mechanism of the reversal.. We demonstrate that GM3 synthase knockout and the resultant GM3 depletion rescues the denervation in mouse footpad skin and fully reverses the neuropathic pain in diet-induced obese diabetic mice. In cultured dorsal root ganglia from diet-induced diabetic mice, GM3 depletion protects against increased intracellular calcium influx in vitro.. These studies establish ganglioside GM3 as a new candidate responsible for neuropathic pain and small fiber neuropathy in diabetes. Moreover, these observations indicate that systemic or topically applied interventions aimed at depleting GM3 may improve both the painful neuropathy and the wound healing impairment in diabetes by protecting against nerve end terminal degeneration, providing a disease-modifying approach to this common, currently intractable medical issue.

Topics: Animals; Blood Glucose; Cells, Cultured; Diabetes Mellitus, Type 2; Diet, High-Fat; Disease Models, Animal; G(M3) Ganglioside; Ganglia, Spinal; Insulin Resistance; Mice; Mice, Inbred C57BL; Mice, Knockout; Neurons; Pain; Pain Measurement; Peripheral Nervous System Diseases; Physical Stimulation; Sciatic Nerve; Sialyltransferases; Skin; Small Fiber Neuropathy

Spherical nucleic acid (SNA) gold nanoparticle conjugates (13-nm-diameter gold cores functionalized with densely packed and highly oriented nucleic acids) dispersed in Aquaphor have been shown to penetrate the epidermal barrier of both intact mouse and human skin, enter keratinocytes, and efficiently down-regulate gene targets. ganglioside-monosialic acid 3 synthase (GM3S) is a known target that is overexpressed in diabetic mice and responsible for causing insulin resistance and impeding wound healing. GM3S SNAs increase keratinocyte migration and proliferation as well as insulin and insulin-like growth factor-1 (IGF1) receptor activation under both normo- and hyperglycemic conditions. The topical application of GM3S SNAs (50 nM) to splinted 6-mm-diameter full-thickness wounds in diet-induced obese diabetic mice decreases local GM3S expression by >80% at the wound edge through an siRNA pathway and fully heals wounds clinically and histologically within 12 d, whereas control-treated wounds are only 50% closed. Granulation tissue area, vascularity, and IGF1 and EGF receptor phosphorylation are increased in GM3S SNA-treated wounds. These data capitalize on the unique ability of SNAs to naturally penetrate the skin and enter keratinocytes without the need for transfection agents. Moreover, the data further validate GM3 as a mediator of the delayed wound healing in type 2 diabetes and support regional GM3 depletion as a promising therapeutic direction.

Topics: Animals; Cell Movement; Cell Proliferation; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 2; Disease Models, Animal; ErbB Receptors; G(M3) Ganglioside; Gold; Humans; Male; Metal Nanoparticles; Mice; Mice, Inbred C57BL; Nucleic Acids; Protein Engineering; Receptor, IGF Type 1; RNA Interference; RNA, Small Interfering; Sialyltransferases; Wound Healing

Recent genetic evidence suggests that aberrant glycosphingolipid metabolism plays an important role in several neuromuscular diseases including hereditary spastic paraplegia, hereditary sensory neuropathy type 1, and non-5q spinal muscular atrophy. Here, we investigated whether altered glycosphingolipid metabolism is a modulator of disease course in amyotrophic lateral sclerosis (ALS). Levels of ceramide, glucosylceramide, galactocerebroside, lactosylceramide, globotriaosylceramide, and the gangliosides GM3 and GM1 were significantly elevated in spinal cords of ALS patients. Moreover, enzyme activities (glucocerebrosidase-1, glucocerebrosidase-2, hexosaminidase, galactosylceramidase, α-galactosidase, and β-galactosidase) mediating glycosphingolipid hydrolysis were also elevated up to threefold. Increased ceramide, glucosylceramide, GM3, and hexosaminidase activity were also found in SOD1(G93A) mice, a familial model of ALS. Inhibition of glucosylceramide synthesis accelerated disease course in SOD1(G93A) mice, whereas infusion of exogenous GM3 significantly slowed the onset of paralysis and increased survival. Our results suggest that glycosphingolipids are likely important participants in pathogenesis of ALS and merit further analysis as potential drug targets.

Topics: Amyotrophic Lateral Sclerosis; Animals; Disease Models, Animal; Disease Progression; G(M3) Ganglioside; Glucosyltransferases; Glycosphingolipids; Humans; Injections, Intraventricular; Male; Mice; Mice, Transgenic; Spinal Cord; Superoxide Dismutase

Gangliosides are sialic acid-bearing glycosphingolipids expressed on all mammalian cell membranes, and participate in several cellular processes. During malignant transformation their expression changes, both at the quantitative and qualitative levels. Of particular interest is the overexpression by tumor cells of Neu5Gc-gangliosides, which are absent, or detected in trace amounts, in human normal cells. The GM3(Neu5Gc) ganglioside in particular has been detected in many human tumors, and it is considered one of the few tumor specific antigen. We previously demonstrated that a humanized antibody specific for this molecule, named 14F7hT, retained the binding and cytotoxic properties of the mouse antibody. In this work, we confirm that 14F7hT exerts a non-apoptotic cell death mechanism in vitro and shows its potent in vivo antitumor activity on a solid mouse myeloma model. Also, we demonstrate, in contrast to the murine counterpart, the capacity of this antibody to induce antibody-dependent cell-mediated cytotoxicity using human effector cells, which increases its potential for the treatment of GM3(Neu5Gc)-expressing human tumors.

Topics: Animals; Antibodies, Monoclonal, Humanized; Antibody Specificity; Antibody-Dependent Cell Cytotoxicity; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Disease Models, Animal; Female; G(M3) Ganglioside; Humans; Isografts; Mice; Tumor Burden

In several lysosomal storage disorders, including Niemann-Pick disease Type C (NP-C), sphingolipids, including glycosphingolipids, particularly gangliosides, are the predominant storage materials in the brain, raising the possibility that accumulation of these lipids may be involved in the NP-C neurodegenerative process. However, correlation of these accumulations and NP-C neuropathology has not been fully characterized. Here we derived NP-C mice with complete and partial deletion of the Siat9 (encoding GM3 synthase) gene in order to investigate the role of ganglioside in NP-C pathogenesis. According to our results, NPC mice with homozygotic deletion of GM3 synthase exhibited an enhanced neuropathological phenotype and died significantly earlier than NP-C mice. Notably, in contrast to complete depletion, NP-C mice with partial deletion of the GM3 synthase gene showed ameliorated NP-C neuropathology, including motor disability, demyelination, and abnormal accumulation of cholesterol and sphingolipids. These findings indicate the crucial role of GM3 synthesis in the NP-C phenotype and progression of CNS pathologic abnormality, suggesting that well-controlled inhibition of GM3 synthesis could be used as a therapeutic strategy.

Topics: Animals; Cells, Cultured; Cerebellum; Cerebrum; Disease Models, Animal; G(M3) Ganglioside; Gene Deletion; Humans; Mice, Inbred BALB C; Mice, Transgenic; Niemann-Pick Disease, Type C; Sialyltransferases

Heart failure (HF) and atrial fibrillation (AF) share common risk factors, frequently coexist and are associated with high mortality. Treatment of HF with AF represents a major unmet need. Here we show that a small molecule, BGP-15, improves cardiac function and reduces arrhythmic episodes in two independent mouse models, which progressively develop HF and AF. In these models, BGP-15 treatment is associated with increased phosphorylation of the insulin-like growth factor 1 receptor (IGF1R), which is depressed in atrial tissue samples from patients with AF. Cardiac-specific IGF1R transgenic overexpression in mice with HF and AF recapitulates the protection observed with BGP-15. We further demonstrate that BGP-15 and IGF1R can provide protection independent of phosphoinositide 3-kinase-Akt and heat-shock protein 70; signalling mediators often defective in the aged and diseased heart. As BGP-15 is safe and well tolerated in humans, this study uncovers a potential therapeutic approach for HF and AF.

Topics: Animals; Atrial Fibrillation; Caveolin 1; Caveolin 3; Disease Models, Animal; Electrocardiography; G(M3) Ganglioside; Heart Failure; HSP70 Heat-Shock Proteins; Humans; Male; Mice; Mice, Knockout; Mice, Transgenic; Microarray Analysis; Oximes; Phosphatidylinositol 3-Kinases; Phosphorylation; Piperidines; Proto-Oncogene Proteins c-akt; Receptor, IGF Type 1; Receptors, Somatomedin; Risk Factors; Signal Transduction; Transgenes

Sanfilippo syndrome type III B (MPS III B) is an inherited disorder characterized by a deficiency of α-N-acetylglucosaminidase (Naglu) enzyme leading to accumulation of heparan sulfate in lysosomes and severe neurological deficits. We have previously shown that a single administration of human umbilical cord mononuclear cells (hUCB MNCs) into Naglu knockout mice decreased behavioral abnormalities and tissue pathology. In this study, we tested whether repeated doses of hUCB MNCs would be more beneficial than a single dose of cells. Naglu mice at 3 months of age were randomly assigned to either a Media-only group or one of three hUCB MNC treatment groups--single low dose (3 × 10(6) cells), single high dose (1.8 × 10(7) cells), or multiple doses (3 × 10(6) cells monthly for 6 months) delivered intravenously; cyclosporine was injected intraperitoneally to immune suppress the mice for the duration of the study. An additional control group of wild-type mice was also used. We measured anxiety in an open field test and cognition in an active avoidance test prior to treatment and then at monthly intervals for 6 months. hUCB MNCs restored normal anxiety-like behavior in these mice (p < 0.001). The repeated cell administrations also restored hippocampal cytoarchitecture, protected the dendritic tree, decreased GM3 ganglioside accumulation, and decreased microglial activation, particularly in the hippocampus and cortex. These data suggest that the neuroprotective effect of hUCB MNCs can be enhanced by repeated cell administrations.

Topics: Acetylglucosaminidase; Animals; Anxiety; Avoidance Learning; Behavior, Animal; Brain; Cell Count; Cognition; Cord Blood Stem Cell Transplantation; Dendrites; Disease Models, Animal; Female; G(M3) Ganglioside; Humans; Male; Mice, Knockout; Microglia; Mucopolysaccharidosis III; Phenotype; Treatment Outcome; Umbilical Cord; Urine

Ceramide has been suggested to play a role in apoptosis during gastric ulcerogenesis. The present study is designed to investigate whether accumulated ceramide could serve as the effector molecules of ulcer formation in a rat model of acetic acid-induced gastric ulcer.. The effect of fumonisin B1, an inhibitor of ceramide synthase, and of d,l,-threo-1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol (PPMP) and N-butyldeoxynojirimycin (NB-DNJ), both inhibitors of glucosylceramide synthase, on the accumulation of ceramide and formation of gastric ulcer were examined in the rat model of acetic acid-induced gastric ulcer.. Fumonisin B1 attenuated acetic acid-induced gastric ulcer formation, associated with a decrease in the number of apoptotic cells. Our results showed that it is neither the C18- nor the C24-ceramide itself, but the respective metabolites that were ulcerogenic, because PPMP and NB-DNJ attenuated gastric mucosal apoptosis and the consequent mucosal damage in spite of their reducing the degradation of ceramide.. The ceramide pathway, in particular, the metabolites of ceramide, significantly contributes to acetic acid-induced gastric damage, possibly via enhancing apoptosis. On the other hand, PPMP and NB-DNJ treatment attenuated gastric mucosal apoptosis and ulcer formation despite increasing the ceramide accumulation, suggesting that it was not the ceramides themselves, but their metabolites that contributed to the ulcer formation in the acetic acid-induced gastric ulcer model.

Topics: 1-Deoxynojirimycin; Acetic Acid; Animals; Apoptosis; Disease Models, Animal; Enzyme Inhibitors; Fumonisins; G(M3) Ganglioside; Gastric Mucosa; Glucosylceramides; Glucosyltransferases; Humans; Male; Morpholines; Rats; Rats, Sprague-Dawley; Sphingolipids; Stomach Ulcer

To investigate the therapeutic effects of exogenous gangliosides GM3 on human bladder cancer cell lines and the severe combined immunodeficiency mouse model of orthotopic bladder cancer.. Human bladder cancer cell lines YTS-1, T24, 5637, and KK47 were used in the study. In vitro cytotoxicity of GM3 was assessed using the cell counting kit-8. Cell adhesion was determined using a spreading assay. Phosphorylation of epidermal growth factor receptor was determined by Western blotting. In vivo, the orthotopic bladder cancer model was established using severe combined immunodeficiency mice and GM3 was administered intravesically by way of a transurethral catheter.. GM3 inhibited the proliferation of all the bladder cancer cell lines tested. The addition of GM3 decreased cell adhesion and epidermal growth factor-dependent phosphorylation of epidermal growth factor receptor. Direct instillation of GM3 into the bladder of the orthotopic model significantly inhibited tumor growth.. Our results suggest exogenous GM3 as a potential therapeutic agent for treating bladder cancer.

Topics: Administration, Intravesical; Animals; Antineoplastic Agents; Cell Adhesion; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; ErbB Receptors; Female; G(M3) Ganglioside; Humans; Mice; Mice, SCID; Neoplasm Transplantation; Phosphorylation; Urinary Bladder Neoplasms

Niemann-Pick disease type C1 (NPC1) is a genetic neurovisceral disorder characterized by abnormalities in intracellular sterol trafficking. A knockout mouse model (NPC1) is an important tool for the study of pathogenesis and treatment strategies. In the present study, NPC1 mice were examined for pathological changes in the cornea.. Fifteen inbred homozygous NPC1 knockout mice (NPC1, 5-10 weeks old), 5 age-matched heterozygous mice (NPC1), and 14 wild-type control mice (NPC1) were examined. In vivo confocal laser scanning microscopy (CLSM) was performed on both eyes of each animal; afterward, the eyes were processed for histology, electron microscopy, and lipid analysis.. In vivo CLSM disclosed hyperreflective intracellular deposits in the intermediate and basal cell layers of corneal epithelium in all NPC1 mice. At the electron microscopy level, however, vacuolated cytoplasmic structures, 200-500 nm in diameter, with electron-dense material appeared in all structures investigated, including all epithelial layers and stromal keratocytes. These deposits were negative for filipin, a marker for unesterified cholesterol. Lipid analysis showed a marked increase in disialotetrahexosylganglioside 2 (GM2) level in NPC1 mice corneas, whereas no changes were detected in free cholesterol and disialotetrahexosylganglioside 3 (GM3) levels when compared with controls.. Morphological changes characteristic for the NPC1 mouse cornea were visualized in all epithelial layers and keratocytes. In vivo CLSM findings were confirmed by other techniques. In vivo detection of ocular manifestations and analysis of ocular tissue have the potential to aid the diagnosis of NPC1 disease and to monitor the efficacy of treatment.

Topics: Animals; Cholesterol; Chromatography, High Pressure Liquid; Corneal Diseases; Disease Models, Animal; G(M2) Ganglioside; G(M3) Ganglioside; Intracellular Signaling Peptides and Proteins; Lipids; Mass Spectrometry; Mice; Mice, Inbred BALB C; Mice, Knockout; Microscopy, Confocal; Niemann-Pick C1 Protein; Niemann-Pick Disease, Type C; Proteins

Sanfilippo syndrome type B (MPS III B) is caused by a deficiency of α-N-acetylglucosaminidase enzyme, leading to accumulation of heparan sulfate within lysosomes and eventual progressive cerebral and systemic multiple organ abnormalities. However, little is known about the competence of the blood-brain barrier (BBB) in MPS III B. BBB dysfunction in this devastating disorder could contribute to neuropathological disease manifestations.. In the present study, we investigated structural (electron microscope) and functional (vascular leakage) integrity of the BBB in a mouse model of MPS III B at different stages of disease, focusing on brain structures known to experience neuropathological changes. Major findings of our study were: (1) endothelial cell damage in capillary ultrastructure, compromising the BBB and resulting in vascular leakage, (2) formation of numerous large vacuoles in endothelial cells and perivascular cells (pericytes and perivascular macrophages) in the large majority of vessels, (3) edematous space around microvessels, (4) microaneurysm adjacent to a ruptured endothelium, (6) Evans Blue and albumin microvascular leakage in various brain structures, (7) GM3 ganglioside accumulation in endothelium of the brain microvasculature.. These new findings of BBB structural and function impairment in MPS III B mice even at early disease stage may have implications for disease pathogenesis and should be considered in current and future development of treatments for MPS III B.

Topics: Acetylglucosaminidase; Albumins; Animals; Blood-Brain Barrier; Disease Models, Animal; Evans Blue; G(M3) Ganglioside; Immunohistochemistry; Mice; Mice, Mutant Strains; Microvessels; Mucopolysaccharidosis III

HIBM (Hereditary Inclusion Body Myopathy) is a recessive hereditary disease characterized by adult-onset, slowly progressive muscle weakness sparing the quadriceps. It is caused by a single missense mutation of each allele of the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) gene, a bifunctional enzyme catalyzing the first two steps of sialic acid synthesis in mammals. However, the mechanisms and cellular pathways affected by the GNE mutation and causing the muscle weakness could not be identified so far. Based on recent evidence in literature, we investigated a new hypothesis, i.e. the involvement in the disease of the GM3 ganglioside, a specific glycolipid implicated in muscle cell proliferation and differentiation.. qRT-PCR analysis of St3gal5 (GM3 synthase) gene expression and HPLC quantification of GM3 ganglioside were conducted on muscle tissue from a mouse model of HIBM harboring the M712T mutation of GNE (Gne(M712T/M712T) mouse) vs control mice (Gne(+/+) mouse).. St3gal5 mRNA levels were significantly lower in Gne(M712T/M712T) mouse muscles vs Gne(+/+) mouse muscles (64.41%+/-10% of Gne(+/+) levels). GM3 ganglioside levels showed also a significant decrease in Gne(M712T/M712T) mouse muscle compared to Gne(+/+) mouse muscle (18.09%+/-5.33% of Gne(+/+) levels). Although these Gne(M712T/M712T) mice were described to suffer severe glomerular proteinuria, no GM3 alterations were noted in kidneys, highlighting a tissue specific alteration of gangliosides.. The M712T mutation of GNE hampers the muscle ability to synthesize normal levels of GM3. This is the first time that a mutation of GNE can be related to the molecular pathological mechanism of HIBM.

Topics: Animals; Biomarkers; Carbohydrate Epimerases; Disease Models, Animal; G(M3) Ganglioside; Mice; Multienzyme Complexes; Muscle, Skeletal; Mutation, Missense; Myositis, Inclusion Body; Phosphotransferases (Alcohol Group Acceptor); RNA, Messenger; Sialyltransferases

Polycystic kidney disease (PKD) represents a family of genetic disorders characterized by renal cystic growth and progression to kidney failure. No treatment is currently available for people with PKD, although possible therapeutic interventions are emerging. Despite genetic and clinical heterogeneity, PKDs have in common defects of cystic epithelia, including increased proliferation, apoptosis and activation of growth regulatory pathways. Sphingolipids and glycosphingolipids are emerging as major regulators of these cellular processes. We sought to evaluate the therapeutic potential for glycosphingolipid modulation as a new approach to treat PKD. Here we demonstrate that kidney glucosylceramide (GlcCer) and ganglioside GM3 levels are higher in human and mouse PKD tissue as compared to normal tissue, regardless of the causative mutation. Blockade of GlcCer accumulation with the GlcCer synthase inhibitor Genz-123346 effectively inhibits cystogenesis in mouse models orthologous to human autosomal dominant PKD (Pkd1 conditional knockout mice) and nephronophthisis (jck and pcy mice). Molecular analysis in vitro and in vivo indicates that Genz-123346 acts through inhibition of the two key pathways dysregulated in PKD: Akt protein kinase-mammalian target of rapamycin signaling and cell cycle machinery. Taken together, our data suggest that inhibition of GlcCer synthesis represents a new and effective treatment option for PKD.

Topics: Animals; Cell Cycle; Dioxanes; Disease Models, Animal; G(M3) Ganglioside; Glucosylceramides; Glucosyltransferases; Glycosphingolipids; Humans; Mice; Mice, Knockout; Polycystic Kidney Diseases; Pyrrolidines; Rats

This study describes the preparation and the characterization of poly[ N-(2-hydroxypropyl methacrylamide)] hydrogel with bulk-modified saccharidic portion of ganglioside GM 3 (Neu5Ac-alpha2,3-Gal-beta1,4-Glc). The 3'-sialyllactose is a bioactive epitope recognized by many cell surface receptors on viruses, bacteria, and human cells such as growth factor receptors. Acrylated 3'-sialyllactose was synthesized and incorporated into the macromolecular network of hydrogels by free radical cross-linking copolymerization. Fluorescence techniques coupled to confocal laser scanning microscopy was employed to characterize the binding and accessibility of the sialyl group in the polymer network by using a monoclonal antibody against GM 3 and the lectin wheat germ agglutinin. The morphology of the network was examined by scanning electron microscopy and confocal microscopy to image the gel morphology. The water content of sialyllactosyl-HPMA hydrogel compared to unmodified gel was characterized by swelling measurements and thermogravimetry. A preliminary implantation study in rat brain was performed to examine the biofunctionality of the sialyllactosyl hydrogel using an experimental model of Parkinson's disease.

Topics: Animals; Antibodies, Monoclonal; Axons; Biocompatible Materials; Corpus Striatum; Disease Models, Animal; Dopamine; Fluorescence; G(M3) Ganglioside; Hydrogels; Immunohistochemistry; Implants, Experimental; Materials Testing; Microscopy, Confocal; Microscopy, Electron, Scanning; Oligosaccharides; Oxidopamine; Particle Size; Plant Lectins; Polymethacrylic Acids; Rats; Surface Properties; Temperature; Time Factors; Wheat Germ Agglutinins

We have previously shown GM3 to positively regulate TNF-alpha expression via a PI3K/Akt pathway in mouse melanoma B16 cells [Wang et al.: Biochem Biophys Res Commun 2007;356:438-443]. The GM3 signal was shown to be located upstream of Akt, but whether it is located upstream of PI3K and which molecule is the effector of PI3K remain to be clarified.. We used inhibitors of PI3K and mTOR, and siRNA directed to Rictor, Raptor and Rho-GDP dissociation inhibitor beta (Arhgdib).. PI3K inhibitors LY294002 and LY303511 were shown to suppress TNF-alpha expression that is stimulated by GM3 in B16 cells, suggesting that the GM3 signal is located upstream of the PI3K-Akt pathway. Rapamycin suppressed TNF-alpha expression, indicating mTOR to be involved in the pathway. Either siRNA Raptor or siRNA Rictor suppressed TNF-alpha expression, but the latter suppressed the effects of GM3 on TNF-alpha expression and Akt phosphorylation at Ser(473), indicating the GM3 signal to be transduced via Rictor/mTOR and Akt (Ser(473)), leading to TNF-alpha stimulation. Finally, Arhgdib, the tumor suppressor gene whose expression is associated with GM3, was shown to be upstream of TNF-alpha.. The GM3 signal is thus transduced in B16 cells through a PI3K, Rictor/mTOR, Akt, Arhgdib pathway, leading to stimulated expression of TNF-alpha.

Topics: Animals; Carrier Proteins; Cattle; Chromones; Disease Models, Animal; DNA Primers; G(M3) Ganglioside; Gene Expression Regulation, Neoplastic; GTP Phosphohydrolases; Melanoma, Experimental; Mice; Piperazines; Rapamycin-Insensitive Companion of mTOR Protein; Reverse Transcriptase Polymerase Chain Reaction; rho GTP-Binding Proteins; RNA, Small Interfering; Signal Transduction; Tumor Necrosis Factor-alpha

The neurodegenerative disease MPS III B (Sanfilippo syndrome type B) is caused by mutations in the gene encoding the lysosomal enzyme alpha-N-acetylglucosaminidase, with a resulting block in heparan sulfate degradation. A mouse model with disruption of the Naglu gene allows detailed study of brain pathology. In contrast to somatic cells, which accumulate primarily heparan sulfate, neurons accumulate a number of apparently unrelated metabolites, including subunit c of mitochondrial ATP synthase (SCMAS). SCMAS accumulated from 1 month of age, primarily in the medial entorhinal cortex and layer V of the somatosensory cortex. Its accumulation was not due to the absence of specific proteases. Light microscopy of brain sections of 6-months-old mice showed SCMAS to accumulate in the same areas as glycosaminoglycan and unesterified cholesterol, in the same cells as ubiquitin and GM3 ganglioside, and in the same organelles as Lamp 1 and Lamp 2. Cryo-immuno electron microscopy showed SCMAS to be present in Lamp positive vesicles bounded by a single membrane (lysosomes), in fingerprint-like layered arrays. GM3 ganglioside was found in the same lysosomes, but was not associated with the SCMAS arrays. GM3 ganglioside was also seen in lysosomes of microglia, suggesting phagocytosis of neuronal membranes. Samples used for cryo-EM and further processed by standard EM procedures (osmium tetroxide fixation and plastic embedding) showed the disappearance of the SCMAS fingerprint arrays and appearance in the same location of "zebra bodies", well known but little understood inclusions in the brain of patients with mucopolysaccharidoses.

Topics: Aging; Animals; Cryoelectron Microscopy; Disease Models, Animal; G(M3) Ganglioside; Glycosaminoglycans; Lysosomal Membrane Proteins; Lysosomal-Associated Membrane Protein 2; Lysosomes; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitochondrial Proton-Translocating ATPases; Mucopolysaccharidosis III; Protein Subunits; Pyramidal Cells; Somatosensory Cortex

The lysosomal storage disease alpha-mannosidosis is due to absence or defective function of lysosomal alpha-mannosidase, resulting in primary storage of undegraded mannose-rich oligosaccharides. Disease has been described in humans, cattle, cats, mice, and guinea pigs and is characterized in all species by progressive neurologic deterioration and premature death. We analyzed the neurodegenerative processes relative to clinical disease in alpha-mannosidosis guinea pigs as a human disease model, from birth to end-stage disease. Before the onset of obvious neurologic abnormalities at 2 months, we observed widespread neuronal lysosomal vacuolation including secondary accumulation of GM3 ganglioside, widespread axonal spheroids, and reduced myelination of white matter. Histopathologic changes subsequently showed rapid progression in severity in a pattern common to a number of different lysosomal storage disorders, with additional abnormalities including accumulation of GM2 ganglioside and cholesterol, astrogliosis, neuron loss particularly in the cerebellum, and activation and infiltration of the CNS with microglia/macrophages. End-stage clinical disease was seen at 10 to 14 months of age. Our findings show that complex neuropathologic changes in alpha-mannosidosis guinea pigs are already present at birth, before clinical changes are evident, and similar events are likely to occur in patients with this disorder.

Topics: Age Factors; alpha-Mannosidase; Animals; Animals, Newborn; Central Nervous System; Disease Models, Animal; Disease Progression; Filipin; G(M3) Ganglioside; Gangliosidoses, GM2; Glial Fibrillary Acidic Protein; Guinea Pigs; Lysosomal Storage Diseases, Nervous System; Microscopy, Electron, Transmission; Silver Staining

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.

Topics: Animals; Cattle; Cell Division; Cells, Cultured; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Diabetic Retinopathy; Disease Models, Animal; G(M1) Ganglioside; G(M3) Ganglioside; Gangliosides; Glomerular Mesangium; Glycation End Products, Advanced; Kidney Cortex; Mice; Microcirculation; Pericytes; Rats; Retinal Vessels; RNA, Small Interfering; Sialyltransferases

CD40 expression by dendritic cells (DCs) critically regulates their maturation/antitumor activity. CD40-CD40 ligand (CD40L) signaling stimulates DC-mediated IL-12 production/cytotoxicity. Recent studies suggest that neuroblastoma (NB)-derived gangliosides impair DC maturation, IL-12 secretion, and NK/T-cell activity. Neuroblastoma ganglioside-mediated abrogation of CD40 expression by DC and tumor-induced tolerance has not been studied. The purpose of this study is to determine if NB inhibits DC IL-12 production via CD40. The contributory role of the NB-derived ganglioside GM3 in this process is also examined.. Dendritic cells were generated from bone marrow of mice injected with saline (control) or murine NB. Control DCs were matured with or without GM3. Dendritic cells were cocultured with NB cells treated with or without a ganglioside synthesis inhibitor. Dendritic cell groups were analyzed for maturation/costimulatory markers. Control and tumor-derived DC were stimulated with CD40L or Staphylococcus aureus and studied for IL-12 expression.. CD40 expression on DC generated from NB bearing mice decreased by 64% (P < .001). GM3 down-regulated DC maturation and CD40 expression. Only CD40-dependent IL-12 production was abrogated (60%, P < .01) in DC derived from NB-bearing mice. Dendritic cell capacity to synthesize IL-12 remained intact.. Neuroblastoma-induced inhibition of DC function may result from ganglioside-mediated CD40 signaling deficiency. Strategies to bypass/augment CD40-CD40L signaling may improve current NB immunotherapies.

Topics: Animals; CD40 Antigens; CD40 Ligand; Cell Line, Tumor; Dendritic Cells; Disease Models, Animal; Down-Regulation; G(M3) Ganglioside; Interleukin-12; Mice; Mice, Inbred Strains; Nervous System Neoplasms; Neuroblastoma; Signal Transduction

Diabetes and obesity cause abnormal development of reproductive processes in a variety of species, but the mechanisms that underlie this effect have not been fully elucidated. This study examined the expressional changes of ganglioside GM3 during ovarian maturation, in vitro fertilization (IVF) and early embryonic development in diabetic/obese db/db mice. In high-performance thin-layer chromatography studies, GM3 expression was conspicuously low in the ovaries of db/db mice compared to non-diabetic db/+ mice. Signal detected by anti-GM3 monoclonal antibody was greatly reduced in the primary, secondary and graffian follicles of db/db mice compared to control mice. Results from IVF with ova and sperm from db/db mice showed that GM3 expression during early embryonic development was obviously decreased compared to db/+ mice. This study also elucidated the effects of high glucose (20 and 30 mm) on early embryonic development in ICR strain mice. High glucose caused a decrease in GM3 expression during early embryonic development. Taken together, the results of this study indicate decreased GM3 expression during ovarian maturation and embryonic development of db/db mice, suggesting that alteration of ganglioside expression induced by the diabetic condition may be implicated in the abnormal follicular embryonic development.

Topics: Animals; Chromatography, Thin Layer; Diabetes Mellitus, Type 2; Disease Models, Animal; Embryo, Mammalian; Female; Fluorescent Antibody Technique; G(M3) Ganglioside; Mice; Ovary

To examine the role of GM3 monosialoganglioside and sialic acid in the antitumor activity of a vaccine based on GM3, hydrophobically conjugated with the outer-membrane-protein complex from Neisseria meningitidis (GM3/VSSP).. In order to evaluate the relationship between antitumor activity and the presence of GM3 on the surface of tumor cells, we used two murine tumor cell lines with different ganglioside expression. Syngeneic mice were immunized with four i.m. doses of GM3/VSSP (120 micro g) at 14-day intervals and challenged subcutaneously with tumor cells.. B16 melanoma cells showed GM3 on cell surface and GM3-dependent in vitro growth. As expected, preimmunization with the vaccine significantly inhibited tumor formation and prolonged survival in mice challenged with B16 cells. In contrast, no antitumor effect was observed in mice challenged with GM3-negative F3II mammary carcinoma cells. The reactivity of sera from immunized mice against B16 cells was confirmed by flow cytometry and immunoperoxidase staining. Depletion of sialic acid residues from the cell surface completely abolished antibody response against melanoma cells.. These results indicate that the antitumor activity of GM3/VSSP is associated with GM3 expression on tumor cell surface and demonstrate a major role of sialic acid in the humoral response of vaccinated mice.

Topics: Animals; Bacterial Outer Membrane Proteins; Cancer Vaccines; Cell Division; Cell Membrane; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Female; Flow Cytometry; G(M3) Ganglioside; Immunoglobulin G; Immunoglobulin M; Mammary Neoplasms, Experimental; Melanoma, Experimental; Mice; Mice, Inbred C57BL; N-Acetylneuraminic Acid; Neisseria meningitidis

Although gangliosides are abundant molecular determinants on all vertebrate nerve cells (comprising approximately 1.5% of brain dry weight) their functions have remained obscure. We report that mice engineered to lack a key enzyme in complex ganglioside biosynthesis (GM2/GD2 synthase), and which express only the simple ganglioside molecular species GM3 and GD3, develop significant and progressive behavioral neuropathies, including deficits in reflexes, strength, coordination, and balance. Quantitative indices of motor abilities, applied at 8 and 12 months of age, also revealed progressive gait disorders in complex ganglioside knockout mice compared to controls, including reduced stride length, stride width, and increased hindpaw print length as well as a marked reduction in rearing. Compared to controls, null mutant mice tended to walk in small labored movements. Twelve-month-old complex ganglioside knockout mice also displayed significant incidence of tremor and catalepsy. These comprehensive neurobehavioral studies establish an essential role for complex gangliosides in the maintenance of normal neural physiology in mice, consistent with a role in maintaining axons and myelin (Sheikh, K. A. , J. Sun, Y. Liu, H. Kawai, T. O. Crawford, R. L. Proia, J. W. Griffin, and R. L. Schnaar. 1999. Mice lacking complex gangliosides develop Wallerian degeneration and myelination defects. Proc. Natl. Acad. Sci. USA 96: 7532-7537), and may provide insights into the mechanisms underlying certain neural degenerative diseases.

Topics: Animals; Ataxia; Axons; Behavior, Animal; Catalepsy; Demyelinating Diseases; Disease Models, Animal; Exploratory Behavior; G(M2) Ganglioside; G(M3) Ganglioside; Gait; Male; Mice; Mice, Knockout; Mice, Neurologic Mutants; Muscle Contraction; N-Acetylgalactosaminyltransferases; Polypeptide N-acetylgalactosaminyltransferase; Postural Balance; Reflex, Abnormal; Tremor; Walking; Wallerian Degeneration