Ganglioside GM1 Research Articles

Oral MucoRice-CTB vaccine is safe and immunogenic in healthy US adults

MucoRice-CTB is a promising cold-chain-free oral cholera vaccine candidate. Here, we report a double-blind, randomized, placebo-controlled, phase I study conducted in the USA in which vaccination with the 6-g dose of MucoRice-CTB induced cross-reactive antigen-specific antibodies against the B subunit of cholera toxin (CTB) and enterotoxigenic Escherichia coli heat-labile enterotoxin without inducing serious adverse events. This dosage was acceptably safe and tolerable in healthy men and women. In addition, it induced a CTB-specific IgA response in the saliva of two of the nine treated subjects; in one subject, the immunological kinetics of the salivary IgA were similar to those of the serum CTB-specific IgA. Antibodies from three of the five responders to the vaccine prevented CTB from binding its GM1 ganglioside receptor. These results are consistent with those of the phase I study in Japan, suggesting that oral MucoRice-CTB induces neutralizing antibodies against diarrheal toxins regardless of ethnicity.

Gangliosides in nervous system development, regeneration, and pathologies

Gangliosides, sialic acid-containing sphingolipids, are major constituents of neuronal membranes. According to the number of sialic acids and the structure of the oligosaccharide chain, gangliosides can be classified as simple or complex and grouped in different ganglio-series. Hundreds of gangliosides have been identified in vertebrate cells, with different expression patterns during development and related to several physiological processes, especially in the nervous system. While GD3 and its O-acetylated form, 9acGD3, are highly expressed in early developmental stages, GM1, GD1a, GD1b, and GT1b are the most abundant ganglioside species in the mature nervous system. Mutations in enzymes involved in ganglioside metabolism can lead to the accumulation of specific species, a condition termed gangliosidosis and usually marked by severe neurological impairment. Changes in ganglioside levels have also been described in several neurodegenerative diseases, such as Alzheimer’s and Parkinson’s. In this review, we summarized recent information about the roles of GD3, 9acGD3, GM1, GD1a, GD1b, GT1b, and other ganglioside species in nervous system development and regeneration, as well as clinical trials evaluating possible therapeutic applications of these molecules.

GM1 ganglioside modifies microglial and neuroinflammatory responses to α-synuclein in the rat AAV-A53T α-synuclein model of Parkinson's disease

Among the pathological events associated with the dopaminergic neurodegeneration characteristic of Parkinson's disease (PD) are the accumulation of toxic forms of α-synuclein and microglial activation associated with neuroinflammation. Although numerous other processes may participate in the pathogenesis of PD, the two factors mentioned above may play critical roles in the initiation and progression of dopamine neuron degeneration in PD. In this study, we employed a slowly progressing model of PD using adeno-associated virus-mediated expression of human A53T α-synuclein into the substantia nigra on one side of the brain and examined the microglial response in the striatum on the injected side compared to the non-injected (control) side. We further examined the extent to which administration of the neuroprotective ganglioside GM1 influenced α-synuclein-induced glial responses. Changes in a number of microglial morphological measures (i.e., process length, number of endpoints, fractal dimension, lacunarity, density, and cell perimeter) were indicative of the presence of activated microglial and an inflammatory response on the injected side of the brain, compared to the control side. In GM1-treated animals, no significant differences in microglial morphology were observed between the injected and control striata. Follow-up studies showed that mRNA expression for several inflammation-related genes was increased on the A53T α-synuclein injected side vs. the non-injected side in saline-treated animals and that such changes were not observed in GM1-treated animals. These data show that inhibition of microglial activation and potentially damaging neuroinflammation by GM1 ganglioside administration may be among the many factors that contribute to the neuroprotective effects of GM1 in this model and possibly in human PD.

The human anti-ganglioside GM1 autoantibody response following traumatic and surgical central nervous system insults

Predictors of the central nervous system (CNS) directed autoantibody response after acute CNS injury are poorly understood. We analyzed titers of IgG and IgM autoantibodies to ganglioside GM1 in serial serum specimens collected from human patients following acute spinal cord injury (SCI), traumatic brain injury (TBI) and brain tumor resection. We also assessed putative predictors of the autoantibody titers. We enrolled 19 patients with acute SCI, 14 patients with acute severe TBI, and 19 patients undergoing brain tumor resection. We also enrolled 25 control subjects. Some SCI, TBI and tumor patients exhibited elevated IgG titers as compared with control values; some SCI and TBI patients exhibited an acute peak in IgG titers, most commonly 14 days after insult. Some clinical and radiographic measures of injury severity correlated with IgG titer elevation in SCI and TBI patients but not tumor patients. Our study demonstrates that diverse CNS insults are followed by increased IgG autoimmune antibody titers to the CNS antigen ganglioside GM1, however the response inherent to each insult type is unique. IgG autoimmune antibody titers to GM1 merit further study as a biomarker of traumatic injury severity that can be measured in delayed fashion after CNS insult. These human data help to inform which patients with CNS insults are at risk for CNS-directed autoimmunity as well as the time course of the response.

C. elegans BLMP-1 controls apical epidermal cell morphology by repressing expression of mannosyltransferase bus-8 and molting signal mlt-8

Skin epidermis secretes apical extracellular matrix (aECM) as a protective barrier from the external environment. The aECM is highly dynamic and constantly undergoes remodeling during animal development. How aECM dynamics is temporally regulated during development, and whether and how its mis-regulation may impact epidermal cell morphology or function remains to be fully elucidated. Here, we report that the conserved Zn-finger transcription factor BLMP-1/Blimp1, which regulates epidermal development in C. elegans, controls apical cell shape of the epidermis by downregulation of aECM remodeling. Loss of blmp-1 causes upregulation of genes essential for molting, including bus-8 and mlt-8, in adult, leading to an abnormal shape in the apical region of adult epidermal cells. The apical epidermal morphological defect is suppressed by reduction of bus-8 or mlt-8. BUS-8 is a key mannosyltransferase, which functions in glycosylation of N-linked glycoproteins; MLT-8 has a ganglioside GM2 lipid-binding domain and is implicated in signaling during molting, a process where the old cuticle is shed and synthesized anew. Overexpression of bus-8 or mlt-8 induces an apical epidermal cell defect as observed in blmp-1 mutants. MLT-8::GFP fusion protein is localized to lysosomes and secreted to aECM. BUS-8 is important for MLT-8 stability and lysosomal targeting, which may be regulated by BUS-8-mediated glycosylation of MLT-8 and function as a molting signaling cue in aECM remodeling. We propose that BLMP-1 represses MLT-8 expression and glycosylation in the epidermis to prevent inappropriate aECM remodeling, which is essential for maintenance of apical epidermal cell morphology during larva-to-adult transition.

Therapeutic advantages of combined gene/cell therapy strategies in a murine model of GM2 gangliosidosis

Genetic deficiency of β-N-acetylhexosaminidase (Hex) functionality leads to accumulation of GM2 ganglioside in Tay-Sachs disease and Sandhoff disease (SD), which presently lack approved therapies. Current experimental gene therapy (GT) approaches with adeno-associated viral vectors (AAVs) still pose safety and efficacy issues, supporting the search for alternative therapeutic strategies. Here we leveraged the lentiviral vector (LV)-mediated intracerebral (IC) GT platform to deliver Hex genes to the CNS and combined this strategy with bone marrow transplantation (BMT) to provide a timely, pervasive, and long-lasting source of the Hex enzyme in the CNS and periphery of SD mice. Combined therapy outperformed individual treatments in terms of lifespan extension and normalization of the neuroinflammatory/neurodegenerative phenotypes of SD mice. These benefits correlated with a time-dependent increase in Hex activity and a remarkable reduction in GM2 storage in brain tissues that single treatments failed to achieve. Our results highlight the synergic mode of action of LV-mediated IC GT and BMT, clarify the contribution of treatments to the therapeutic outcome, and inform on the realistic threshold of corrective enzymatic activity. These results have important implications for interpretation of ongoing experimental therapies and for design of more effective treatment strategies for GM2 gangliosidosis.

Transbilayer Coupling of Lipids in Cells Investigated by Imaging Fluorescence Correlation Spectroscopy.

Plasma membranes host numerous receptors, sensors, and ion channels involved in cellular signaling. Phase separation within the plasma membrane has emerged as a key biophysical regulator of signaling reactions in multiple physiological and pathological contexts. There is much evidence that plasma membrane composition supports the coexistence of liquid-ordered (Lo) and liquid-disordered (Ld) phases or domains at physiological conditions. However, this phase/domain separation is nanoscopic and transient in live cells. It has been recently proposed that transbilayer coupling between the inner and outer leaflets of the plasma membrane is driven by their asymmetric lipid distribution and by dynamic cytoskeleton-lipid composites that contribute to the formation and transience of Lo/Ld phase separation in live cells. In this Perspective, we highlight new approaches to investigate how transbilayer coupling may influence phase separation. For quantitative evaluation of the impact of these interactions, we introduce an experimental strategy centered around Imaging Fluorescence Correlation Spectroscopy (ImFCS), which measures membrane diffusion with very high precision. To demonstrate this strategy, we choose two well-established model systems for transbilayer interactions: cross-linking by multivalent antigen of immunoglobulin E bound to receptor FcεRI and cross-linking by cholera toxin B of GM1 gangliosides. We discuss emerging methods to systematically perturb membrane lipid composition, particularly exchange of outer leaflet lipids with exogenous lipids using methyl alpha cyclodextrin. These selective perturbations may be quantitatively evaluated with ImFCS and other high-resolution biophysical tools to discover novel principles of lipid-mediated phase separation in live cells in the context of their pathophysiological relevance.

α/β-Peptides as Nanomolar Triggers of Lipid Raft-Mediated Endocytosis through GM1 Ganglioside Recognition

Cell delivery of therapeutic macromolecules and nanoparticles is a critical drug development challenge. Translocation through lipid raft-mediated endocytic mechanisms is being sought, as it can avoid rapid lysosomal degradation. Here, we present a set of short α/β-peptide tags with high affinity to the lipid raft-associated ganglioside GM1. These sequences induce effective internalization of the attached immunoglobulin cargo. The structural requirements of the GM1-peptide interaction are presented, and the importance of the membrane components are shown. The results contribute to the development of a receptor-based cell delivery platform.

Collaborative studies of U.S.-China neurologists on acute motor axonal neuropathy.

Collaborative studies of U.S.-China neurologists on acute motor axonal neuropathy.

487 The Human Anti-Ganglioside GM1 Autoantibody Response Following Traumatic and Surgical Central Nervous System Insults

INTRODUCTION: The immune response to central nervous system (CNS) injuries has long been considered a significant contributor to secondary injury following neurotrauma however the autoimmune response to the normal immune-privileged CNS has been insufficiently considered. It has been identified as a possible pathomechanism underlying delayed dementias known to occur after TBI and SCI however the CNS-directed autoantibody response following CNS injury remains poorly understood and the autoimmune response following intra-axial brain surgery is unknown. METHODS: Nine serum samples were collected serially from 17 acute severe TBI, 22 acute SCI, and 24 intra-axial brain tumor resection patients for 30 days; 25 healthy subjects served as a control group. An ELISA assay quantitated IgG and IgM autoantibodies to ganglioside GM-1. The maximum autoantibody titers and their correlation with putative predictors such as age, gender, clinical and radiological measurements were investigated. RESULTS: Anti-GM1 IgG autoantibody titers were increased following SCI, TBI, and brain tumor resection as compared with baseline values and control patients. By contrast, anti-GM1 IgM autoantibody titers did not significantly change following insult nor show promise as a biomarker. The maximum IgG titers measured in the first 30 days were significantly increased in TBI patients (p<0.01), in SCI patients (p<0.01), and in brain tumor resection patients (p<0.05). The highest IgG titers (or peaks) were most commonly seen at day 14 post-insult in TBI and SCI patients while brain tumor patients did not demonstrate acute increases consistent with a peak. Higher GM1 IgG autoantibodies were associated with more severe insults. The Rotterdam score strongly correlated with increased IgG titers in TBI patients (p<0.05). Age and gender did not correlate with GM1 autoantibody titers in any of the types of insult we studied. CONCLUSION: These findings suggest that patients with diverse CNS insults are at risk for CNS-directed autoimmune responses and in particular those with more severe insults. These findings highlight the need to better understand the CNS-directed autoimmune response to insult, its clinical relevance and potential as a therapeutic target.

EP259: A phase 1/2 trial of AXO-AAV-GM1 gene therapy for the treatment of infantile- and juvenile-onset GM1 gangliosidosis

GM1 gangliosidosis is a rare, fatal, neurodegenerative lysosomal storage disorder caused by variants in GLB1 encoding the lysosomal hydrolase β-galactosidase (β-gal). The resulting enzyme deficiency leads to a toxic accumulation of GM1 ganglioside, predominantly in the central nervous system (CNS) where its rate of synthesis is the highest, but also in peripheral tissues. GM1 gangliosidosis is uniformly fatal, and there are no disease-modifying treatments currently available. As this is a monogenic disorder, it is an ideal target for gene therapy to deliver β-gal to the CNS and periphery, with the potential to halt further neurodegeneration, restore function and ameliorate symptoms.

Homeostatic and Pathophysiological Regulation of Toll-like Receptor 4 Signaling by GM3 Ganglioside Molecular Species

Chronic inflammation plays an important role in the pathogenesis of obesity and metabolic disorders. In obesity, pattern-recognition receptors in innate immune system, such as Toll-like receptor 4 (TLR4), cause chronic inflammation through prolonged activation by various endogenous ligands, including fatty acids and its metabolites. Gangliosides and other glycosphingolipids are important metabolites of fatty acids and saccharides. GM3, the simplest ganglioside comprising α2,3-sialyllactose, is expressed in insulin-sensitive peripheral tissues such as liver and adipose tissue, and furthermore secreted abundantly into serum. It has been shown that GM3 regulates the signal transduction of insulin receptor in adipose tissue as a component of membrane microdomains, and elevation in GM3 level causes insulin resistance. However, the homeostatic and pathophysiological functions of extracellularly secreted GM3 are poorly understood. We recently reported that GM3 species with differing fatty acid structures act as pro- and anti-inflammatory endogenous TLR4 ligands. GM3 with very long-chain fatty acid (VLCFA) and α-hydroxyl VLCFA strongly enhanced TLR4 activation. Conversely, GM3 with long-chain fatty acid (LCFA) and ω-9 unsaturated VLCFA inhibited TLR4 activation, counteracting the VLCFA species. GM3 interacted with the extracellular complex of TLR4 and promoted dimerization/oligomerization. In obesity and metabolic disorders, VLCFA species were increased in serum and adipose tissue, whereas LCFA species was relatively decreased; their imbalances were correlated to disease progression. Our findings suggest that GM3 species are disease-related endogenous TLR4 ligands, and "glycosphingolipid sensing" by TLR4 controls the homeostatic and pathological roles of innate immune signaling.

Incorporation of Toll-Like Receptor Ligands and Inflammasome Stimuli in GM3 Liposomes to Induce Dendritic Cell Maturation and T Cell Responses.

Cancer vaccination aims to activate immunity towards cancer cells and can be achieved by delivery of cancer antigens together with immune stimulatory adjuvants to antigen presenting cells (APC). APC maturation and antigen processing is a subsequent prerequisite for T cell priming and anti-tumor immunity. In order to specifically target APC, nanoparticles, such as liposomes, can be used for the delivery of antigen and adjuvant. We have previously shown that liposomal inclusion of the ganglioside GM3, an endogenous ligand for CD169, led to robust uptake by CD169-expressing APC and resulted in strong immune responses when supplemented with a soluble adjuvant. To minimize the adverse effects related to a soluble adjuvant, immune stimulatory molecules can be incorporated in liposomes to achieve targeted delivery of both antigen and adjuvant. In this study, we incorporated TLR4 (MPLA) or TLR7/8 (3M-052) ligands in combination with inflammasome stimuli, 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC) or muramyl dipeptide (MDP), into GM3 liposomes. Incorporation of TLR and inflammasome ligands did not interfere with the uptake of GM3 liposomes by CD169-expressing cells. GM3 liposomes containing a TLR ligand efficiently matured human and mouse dendritic cells in vitro and in vivo, while inclusion of PGPC or MDP had minor effects on maturation. Immunization with MPLA-containing GM3 liposomes containing an immunogenic synthetic long peptide stimulated CD4+ and CD8+ T cell responses, but additional incorporation of either PGPC or MDP did not translate into stronger immune responses. In conclusion, our study indicates that TLRL-containing GM3 liposomes are effective vectors to induce DC maturation and T cell priming and thus provide guidance for further selection of liposomal components to optimally stimulate anti-cancer immune responses.

Gangliosides in T cell development and function of mice.

The molecular diversity of glycosphingolipids (GSLs) that arouse during thecourseof evolution clearly plays an essential role in maintenance of biological homeostasis. Why is such a wide variety of GSLs necessary, and what gave rise to the expression mechanisms that are selective and specific to individual cells, tissues, or organs? What is the biological significance of these mechanisms? The same questions apply to GSLs involved in T cell development and activation.Primary CD4+ T cells and CD8+ T cells preferentially express differing ganglioside series: a-series and o-series, respectively. Conversely, a-series and o-series ganglioside deficiency results respectively in CD4+ and CD8+ T cell dysfunction. Dynamic changes in ganglioside expression occur during T cell development in thymus. Ganglioside GM3 synthase deficiency, which results in lack of a-series gangliosides, ameliorated CD4+ T cell-mediated airway hypersensitivity in a mouse model of allergic asthma. In this review, we summarize findings from these and many studies to illustrate the key roles of gangliosides in T cell differentiation and function.

Correlation between Sialylation Status and Cell Susceptibility to Amyloid Toxicity.

The interaction between the cell membrane and misfolded protein species plays a crucial role in the development of neurodegeneration. This study was designed to clarify the relationship between plasma membrane composition in terms of the differently linked sialic acid (Sia) content and cell susceptibility to toxic and misfolded Aβ-42 peptides. The sialylation status in different cell lines was investigated by lectin histochemistry and confocal immunofluorescence and then correlated with the different propensities to bind amyloid fibrils and with the relative cell susceptibility to amyloid damage. This study reveals that expressions of Sias α2,3 and α2,6 linked to galactose/N-acetyl-galactosamine, and PolySia are positively correlated with Aβ-42-induced cell toxicity. PolySia shows an early strong interaction with amyloid fibrils, favoring their binding to GM1 ganglioside containing α2,3 galactose-linked Sia and a loss of cell viability. Our findings demonstrate that cell lines with a prevailing plastic neuron-like phenotype and high monoSia and PolySia contents are highly susceptible to amyloid Aβ-42 toxicity. This toxicity may involve a change in neuron metabolism and promote a compensative/protective increase in PolySia, which, in turn, could favor amyloid binding to GM1, thus exacerbating cell dysmetabolism and further amyloid aggregation.

Gangliosides and the Treatment of Neurodegenerative Diseases: A Long Italian Tradition.

Gangliosides are glycosphingolipids which are particularly abundant in the plasma membrane of mammalian neurons. The knowledge of their presence in the human brain dates back to the end of 19th century, but their structure was determined much later, in the middle of the 1950s. From this time, neurochemical studies suggested that gangliosides, and particularly GM1 ganglioside, display neurotrophic and neuroprotective properties. The involvement of GM1 in modulating neuronal processes has been studied in detail by in vitro experiments, and the results indicated its direct role in modulating the activity of neurotrophin-dependent receptor signaling, the flux of calcium through the plasma membrane, and stabilizing the correct conformation of proteins, such as α-synuclein. Following, in vivo experiments supported the use of ganglioside drugs for the therapy of peripheral neuropathies, obtaining very positive results. However, the clinical use of gangliosides for the treatment of central neurodegeneration has not been followed due to the poor penetrability of these lipids at the central level. This, together with an ambiguous association (later denied) between ganglioside administration and Guillain-Barrè syndrome, led to the suspension of ganglioside drugs. In this critical review, we report on the evolution of research on gangliosides, on the current knowledge on the role played by gangliosides in regulating the biology of neurons, on the past and present use of ganglioside-based drugs used for therapy of peripheral neuropathies or used in human trials for central neurodegenerations, and on the therapeutic potential represented by the oligosaccharide chain of GM1 ganglioside for the treatment of neurodegenerative diseases.

Glucosylceramide synthase inhibition reduces ganglioside GM3 accumulation, alleviates amyloid neuropathology, and stabilizes remote contextual memory in a mouse model of Alzheimer\u2019s disease

BackgroundGangliosides 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.MethodsTg2576 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.ResultsGCSi 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.ConclusionsCollectively, 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.

Storage of GM2 ganglioside and altered gene expression in infantile Tay-Sachs disease during fetal development has implications for therapeutic timing and efficacy

Storage of GM2 ganglioside and altered gene expression in infantile Tay-Sachs disease during fetal development has implications for therapeutic timing and efficacy

One-Pot Enzymatic Synthesis and Biological Evaluation of Ganglioside GM3 Derivatives as Potential Cancer Immunotherapeutics.

Cancer is a leading cause of death worldwide. Recent research studies have revealed that GM3 derivatives have considerable promise as potential therapeutic agents for cancer. To discover novel GM3 derivatives as potential antitumor agents, a one-pot enzymatic synthesis was established, yielding 14 GM3 derivatives in high total yields (22-41%). Subsequently, the inhibitory activities of GM3 derivatives were assessed by wound-healing assays and Transwell assays and tumor-bearing animal models. Among all the GM3 derivatives, N-12 showed excellent migration and invasion inhibitory effects in cells and marked antitumor activity in C57BL/6 mice. The subsequent analysis of cancer tissues and serum samples revealed that N-12 induces tumor inhibition, which was closely related to immune response. Taken together, N-12 can be further developed as an effective therapeutic for the treatment of cancer. An RNA-sequencing (RNA-seq) analysis was then performed and indicated that the antitumor mechanism of N-12 involved focal adhesion and ECM-receptor interaction signaling pathways.

The Key Role of GM1 Ganglioside in Parkinson's Disease.

We have endeavored in this review to summarize our findings, which point to a systemic deficiency of ganglioside GM1 in Parkinson’s disease (PD) tissues. These include neuronal tissues well known to be involved in PD, such as substantia nigra of the brain and those of the peripheral nervous system, such as the colon and heart. Moreover, we included skin and fibroblasts in the study as well as peripheral blood mononuclear cells; these are tissues not directly involved in neuronal signaling. We show similar findings for ganglioside GD1a, which is the metabolic precursor to GM1. We discuss the likely causes of these GM1 deficiencies and the resultant biochemical mechanisms underlying loss of neuronal viability and normal functioning. Strong support for this hypothesis is provided by a mouse PD model involving partial GM1 deficiency based on mono-allelic disruption of the B4galnt1 gene. We point out that progressive loss of GM1/GD1a occurs in the periphery as well as the brain, thus obviating the need to speculate PD symptom transfer between these tissues. Finally, we discuss how these findings point to a potential disease-altering therapy for PD:GM1 replacement, as is strongly implicated in animal studies and clinical trials.