Crucial roles of exosomes secreted from ganglioside GD3/GD2-positive glioma cells in enhancement of the malignant phenotypes and signals of GD3/GD2-negative glioma cells.

Neuroectoderm-derived tumors characteristically express gangliosides such as GD3 and GD2. Many studies have reported that gangliosides GD3/GD2 enhance malignant phenotypes of cancers. Recently, we reported that human gliomas expressing GD3/GD2 exhibited enhanced malignant phenotypes. Here, we investigated the function of GD3/GD2 in glioma cells and GD3/GD2-expressing glioma-derived exosomes. As reported previously, transfectant cells of human glioma U251 MG expressing GD3/GD2 showed enhanced cancer phenotypes compared with GD3/GD2-negative controls. When GD3/GD2-negative cells were treated with exosomes secreted from GD3/GD2-positive cells, clearly increased malignant properties were observed. Furthermore, increased phosphorylation of signaling molecules was detected after 5-15 min of exosome treatment, ie, higher tyrosine phosphorylation of platelet-derived growth factor receptor, focal adhesion kinase, and paxillin was found in treated cells than in controls. Phosphorylation of extracellular signal-regulated kinase-1/2 was also enhanced. Consequently, it is suggested that exosomes secreted from GD3/GD2-positive gliomas play important roles in enhancement of the malignant properties of glioma cells, leading to total aggravation of heterogenous cancer tissues, and also in the regulation of tumor microenvironments.

Cholesterol-Dependent Serotonin Insertion Controlled by Gangliosides in Model Lipid Membranes.

Serotonin is distinct among synaptic neurotransmitters because it is amphipathic and released from synaptic vesicles at concentrations superior to its water solubility limit (270 mM in synaptic vesicles for a solubility limit of 110 mM). Hence, serotonin is mostly aggregated in the synaptic cleft, due to extensive aromatic stacking. This important characteristic has received scant attention, as most representations of the serotonergic synapse take as warranted that serotonin molecules are present as monomers after synaptic vesicle exocytosis. Using a combination of in silico and physicochemical approaches and a new experimental device mimicking synaptic conditions, we show that serotonin aggregates are efficiently dissolved by gangliosides (especially GM1) present in postsynaptic membranes. This initial interaction, driven by electrostatic forces, attracts serotonin from insoluble aggregates and resolves micelles into monomers. Serotonin also interacts with cholesterol via a set of CH-π and van der Waals interactions. Thus, gangliosides and cholesterol act together as a functional serotonin-collecting funnel on brain cell membranes. Based on this unique mode of interaction with postsynaptic membranes, we propose a new model of serotonergic transmission that takes into account the post-exocytosis solubilizing effect of gangliosides and cholesterol on serotonin aggregates.

Omalizumab for Treatment of Anti-GD2 Antibody-related Urticaria.

Outcomes for high-risk neuroblastoma have improved with the addition of antidisialoganglioside (GD2) antibody-mediated immunotherapy to multimodality therapy. Urticaria is an expected side effect of anti-GD2 immunotherapy. Rarely, despite maximal use of antihistamines and H2 receptor antagonists, refractory urticaria can result in impaired quality of life, and delays or discontinuation of immunotherapy. The anti-IgE monoclonal antibody, omalizumab, is approved for the treatment of asthma and chronic spontaneous urticaria. We successfully managed grade 3, naxitamab-related urticaria refractory to standard management in 2 patients using omalizumab, allowing for continued anti-GD2 immunotherapy. Omalizumab did not impact antitumor activity or immunogenicity of naxitamab.

Glutamate, Gangliosides, and the Synapse: Electrostatics at Work in the Brain.

The synapse is a piece of information transfer machinery replacing the electrical conduction of nerve impulses at the end of the neuron. Like many biological mechanisms, its functioning is heavily affected by time constraints. The solution selected by evolution is based on chemical communication that, in theory, cannot compete with the speed of nerve conduction. Nevertheless, biochemical and biophysical compensation mechanisms mitigate this intrinsic weakness: (i) through the high concentrations of neurotransmitters inside the synaptic vesicles; (ii) through the concentration of neurotransmitter receptors in lipid rafts, which are signaling platforms; indeed, the presence of raft lipids, such as gangliosides and cholesterol, allows a fine tuning of synaptic receptors by these lipids; (iii) through the negative electrical charges of the gangliosides, which generate an attractive (for cationic neurotransmitters, such as serotonin) or repulsive (for anionic neurotransmitters, such as glutamate) electric field. This electric field controls the flow of glutamate in the tripartite synapse involving pre- and post-synaptic neurons and the astrocyte. Changes in the expression of brain gangliosides can disrupt the functioning of the glutamatergic synapse, causing fatal diseases, such as Rett syndrome. In this review, we propose an in-depth analysis of the role of gangliosides in the glutamatergic synapse, highlighting the primordial and generally overlooked role played by the electric field of synaptic gangliosides.

High serum prevalence of autoreactive IgG antibodies against peripheral nerve structures in patients with neurological post-COVID-19 vaccination syndrome.

Background: Patients suffering from neurological symptoms after COVID-19 vaccination (post-COVID-19 vaccination syndrome (PCVS)) have imposed an increasing challenge on medical practice, as diagnostic precision and therapeutic options are lacking. Underlying autoimmune dysfunctions, including autoantibodies, have been discussed in neurological disorders after SARS-CoV-2 infection and vaccination. Here, we describe the frequency and targets of autoantibodies against peripheral nervous system tissues in PCVS. Methods: Sera from 50 PCVS patients with peripheral neurological symptoms after COVID-19 vaccination and 35 vaccinated healthy controls were used in this study. IgG autoreactivity was measured via indirect immunofluorescence assays on mouse sciatic nerve teased fibers. The frequencies of autoantibodies were compared between groups using Fisher's exact test. Serum anti-ganglioside antibodies were measured in ganglioside blots. Autoantibody target identification was performed using immunoprecipitation coupled to mass spectrometry. Subsequent target confirmation was conducted via cell-based assays and ELISA. Results: Compared with controls, PCVS patients had a significantly greater frequency of autoantibodies against peripheral nervous system structures (9/50(18%) vs 1/35(3%); p=0.04). Autoantibodies bound to paranodes (n=5), axons (n=4), Schmidt-Lanterman incisures (n=2) and Schwann cell nuclei (n=1). Conversely, antibodies against gangliosides were absent in PCVS patients. Target identification and subsequent confirmation revealed various subunits of neurofilaments as well as DFS-70 as autoantibody epitopes. Conclusion: Our data suggest that autoantibodies against nervous system tissue could be relevant in PCVS patients. Autoantibodies against neurofilaments and cell nuclei with so far non-established links to this disease spectrum should be further elucidated to determine their biomarker potential.

CAR T-cell-mediated delivery of bispecific innate immune cell engagers for neuroblastoma.

Novel chimeric antigen receptor (CAR) T-cell approaches are needed to improve therapeutic efficacy in solid tumors. High-risk neuroblastoma is an aggressive pediatric solid tumor that expresses cell-surface GPC2 and GD2 with a tumor microenvironment infiltrated by CD16a-expressing innate immune cells. Here we engineer T-cells to express a GPC2-directed CAR and simultaneously secrete a bispecific innate immune cell engager (BiCE) targeting both GD2 and CD16a. In vitro, GPC2.CAR-GD2.BiCE T-cells induce GPC2-dependent cytotoxicity and secrete GD2.BiCE that promotes GD2-dependent activation of antitumor innate immunity. In vivo, GPC2.CAR-GD2.BiCE T-cells locally deliver GD2.BiCE and increase intratumor retention of NK-cells. In mice bearing neuroblastoma patient-derived xenografts and reconstituted with human CD16a-expressing immune cells, GD2.BiCEs enhance GPC2.CAR antitumor efficacy. A CAR.BiCE strategy should be considered for tumor histologies where antigen escape limits CAR efficacy, especially for solid tumors like neuroblastoma that are infiltrated by innate immune cells.

Capillary Electrophoresis-Laser Induced Fluorescence Method Development and Validation for Quantification of Nine Gangliosides-Application to Analysis of Cell Lines of CNS Origin.

Gangliosides are sialic acid-containing glycosphingolipids that play an essential role in many biological and pathophysiological processes. They are present in high amounts in the central nervous system and their abnormal metabolism or expression has been observed in many diseases. We have developed and validated a sensitive capillary electrophoresis laser-induced fluorescence (CE-LIF) method for the separation and quantification of oligosaccharides digested from nine gangliosides of high biological relevance. APTS was used for the labeling of the glycans. Reverse polarity CE was performed for the separation of the labeled glycans bearing negative charges. The optimized background electrolyte is a 15 mM lithium acetate buffer with pH of 5 containing 5% w/v linear polyacrylamide, which allows for the separation of all nine gangliosides. Validation parameters including linearity, precision, and accuracy were evaluated. LOQ and LOD were in the nM range, comparable to those of LC-MS techniques. The method was used to identify and quantify the ganglioside pattern of glioblastoma and neuroblastoma cell lines. The presented method is a valuable tool for further investigations aiming at understanding the role of gangliosides in various neurological diseases or CNS tumors.

Protocol for assessing GD2 on formalin-fixed paraffin-embedded tissue sections using immunofluorescence staining.

The detection of disialoganglioside GD2 on tumor biopsies, especially in paraffin-embedded tissues, has been challenging due to the glycolipid structure of GD2 and its membrane anchorage. Here, we present an immunofluorescence protocol for the reliable assessment of GD2 on formalin-fixed paraffin-embedded (FFPE) tissues. We describe steps for antigen retrieval with Tris-EDTA buffer and staining with unconjugated anti-GD2 antibody (clone 14.G2a) and horse radish peroxidase (HRP)-conjugated secondary antibody. We then detail procedures for signal amplification using the tyramide signal amplification technique. For complete details on the use and execution of this protocol, please refer to Fischer-Riepe et al.1.

Depletion of b-series ganglioside prevents limb length discrepancy after growth plate injury.

INTRODUCTION: Growth plate damage in long bones often results in progressive skeletal growth imbalance and deformity, leading to significant physical problems. Gangliosides, key glycosphingolipids in cartilage, are notably abundant in articular cartilage and regulate chondrocyte homeostasis. This suggests their significant roles in regulating growth plate cartilage repair. METHODS: Chondrocytes from 3 to 5 day-old C57BL/6 mice underwent glycoblotting and mass spectrometry. Based on the results of the glycoblotting analysis, we employed GD3 synthase knockout mice (GD3-/-), which lack b-series gangliosides. In 3-week-old mice, physeal injuries were induced in the left tibiae, with right tibiae sham operated. Tibiae were analyzed at 5 weeks postoperatively for length and micro-CT for growth plate height and bone volume at injury sites. Tibial shortening ratio and bone mineral density were measured by micro-CT. RESULTS: Glycoblotting analysis indicated that b-series gangliosides were the most prevalent in physeal chondrocytes among ganglioside series. At 3 weeks, GD3-/- exhibited reduced tibial shortening (14.7 ± 0.2 mm) compared to WT (15.0 ± 0.1 mm, P = 0.03). By 5 weeks, the tibial lengths in GD3-/- (16.0 ± 0.4 mm) closely aligned with sham-operated lengths (P = 0.70). Micro-CT showed delayed physeal bridge formation in GD3-/-, with bone volume measuring 168.9 ± 5.8 HU at 3 weeks (WT: 180.2 ± 3.2 HU, P = 0.09), but normalizing by 5 weeks. CONCLUSION: This study highlights that GD3 synthase knockout mice inhibit physeal bridge formation after growth plate injury, proposing a new non-invasive approach for treating skeletal growth disorders.

MALDI TIMS IMS Reveals Ganglioside Molecular Diversity within Murine S. aureus Kidney Tissue Abscesses.

Gangliosides play important roles in innate and adaptive immunity. The high degree of structural heterogeneity results in significant variability in ganglioside expression patterns and greatly complicates linking structure and function. Structural characterization at the site of infection is essential in elucidating host ganglioside function in response to invading pathogens, such as Staphylococcus aureus (S. aureus). Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) enables high-specificity spatial investigation of intact gangliosides. Here, ganglioside structural and spatial heterogeneity within an S. aureus-infected mouse kidney abscess was characterized. Differences in spatial distributions were observed for gangliosides of different classes and those that differ in ceramide chain composition and oligosaccharide-bound sialic acid. Furthermore, integrating trapped ion mobility spectrometry (TIMS) allowed for the gas-phase separation and visualization of monosialylated ganglioside isomers that differ in sialic acid type and position. The isomers differ in spatial distributions within the host-pathogen interface, where molecular patterns revealed new molecular zones in the abscess previously unidentified by traditional histology.