Hereditary spastic paraplegia (HSP) is a heterogeneous group of neurological disorders that are characterized by progressive spasticity and weakness in the lower limbs. SPG26 is a complicated form of HSP, which includes not only weakness in the lower limbs, but also cognitive impairment, developmental delay, cerebellar ataxia, dysarthria, and peripheral neuropathy, and is caused by biallelic mutations in the B4GALNT1 (beta-1,4-N-acetylgalactosaminyltransferase 1) gene. The B4GALNT1 gene encodes ganglioside GM2/GD2 synthase (GM2S), which catalyzes the transfer of N-acetylgalactosamine to lactosylceramide, GM3, and GD3 to generate GA2, GM2, and GD2, respectively. The present study attempted to characterize a novel B4GALNT1 variant (NM_001478.5:c.937G>A p.Asp313Asn) detected in a patient with progressive multi-system neurodegeneration as well as deleterious variants found in the general population in Japan. Peripheral blood T cells from our patient lacked the ability for activation-induced ganglioside expression assessed by cell surface cholera toxin binding. Structural predictions suggested that the amino acid substitution, p.Asp313Asn, impaired binding to the donor substrate UDP-GalNAc. An in vitro enzyme assay demonstrated that the variant protein did not exhibit GM2S activity, leading to the diagnosis of HSP26. This is the first case diagnosed with SPG26 in Japan. We then extracted 10 novel missense variants of B4GALNT1 from the whole-genome reference panel jMorp (8.3KJPN) of the Tohoku medical megabank organization, which were predicted to be deleterious by Polyphen-2 and SIFT programs. We performed a functional evaluation of these variants and demonstrated that many showed perturbed subcellular localization. Five of these variants exhibited no or significantly decreased GM2S activity with less than 10% activity of the wild-type protein, indicating that they are carrier variants for HSP26. These results provide the basis for molecular analyses of B4GALNT1 variants present in the Japanese population and will help improve the molecular diagnosis of patients suspected of having HSP.

Triple-negative breast cancer (TNBC), which constitutes 10-20 percent of all breast cancers, is aggressive, has high metastatic potential, and carries a poor prognosis due to limited treatment options. LT-IIc, a member of the type II subfamily of ADP-ribosylating-heat-labile enterotoxins that bind to a distinctive set of cell-surface ganglioside receptors-is cytotoxic toward TNBC cell lines, but has no cytotoxic activity for non-transformed breast epithelial cells. Here, primary TNBC cells, isolated from resected human tumors, showed an enhanced cytotoxic response specifically toward LT-IIc, in contrast to other enterotoxins that were tested. MDA-MB-231 cells, a model for TNBC, were used to evaluate potential mechanisms of cytotoxicity by LT-IIc, which induced elevated intracellular cAMP and stimulated the cAMP response element-binding protein (CREB) signaling pathway. To dissect the role of ADP-ribosylation, cAMP induction, and ganglioside ligation in the cytotoxic response, MDA-MB-231 cells were exposed to wild-type LT-IIc, the recombinant B-pentamer of LT-IIc that lacks the ADP-ribosylating A polypeptide, or mutants of LT-IIc with an enzymatically inactivated A1-domain. These experiments revealed that the ADP-ribosyltransferase activity of LT-IIc was nonessential for inducing the lethality of MDA-MB-231 cells. In contrast, a mutant LT-IIc with an altered ganglioside binding activity failed to trigger a cytotoxic response in MDA-MB-231 cells. Furthermore, the pharmacological inhibition of ganglioside expression protected MDA-MB-231 cells from the cytotoxic effects of LT-IIc. These data establish that ganglioside ligation, but not the induction of cAMP production nor ADP-ribosyltransferase activity, is essential to initiating the LT-IIc-dependent cell death of MDA-MB-231 cells. These experiments unveiled previously unknown properties of LT-IIc and gangliosides in signal transduction, offering the potential for the targeted treatment of TNBC, an option that is desperately needed.

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.

BACKGROUND: 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.
CONCLUSIONS: 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.

Despite causing over 1 million deaths annually, Type 2 Diabetes (T2D) currently has no curative treatments. Aggregation of the islet amyloid polypeptide (hIAPP) into amyloid plaques plays an important role in the pathophysiology of T2D and thus presents a target for therapeutic intervention. The mechanism by which hIAPP aggregates contribute to the development of T2D is unclear, but it is proposed to involve disruption of cellular membranes. However, nearly all research on hIAPP-lipid interactions has focused on anionic phospholipids, which are primarily present in the cytosolic face of plasma membranes. We seek here to characterize the effects of three gangliosides, the dominant anionic lipids in the outer leaflet of the plasma membrane, on the aggregation, structure, and toxicity of hIAPP. Our results show a dual behavior that depends on the molar ratio between the gangliosides and hIAPP. For each ganglioside, a low-lipid:peptide ratio enhances hIAPP aggregation and alters the morphology of hIAPP fibrils, while a high ratio eliminates aggregation and stabilizes an α-helix-rich hIAPP conformation. A more negative lipid charge more efficiently promotes aggregation, and a larger lipid headgroup improves inhibition of aggregation. hIAPP also alters the phase transitions of the lipids, favoring spherical micelles over larger tubular micelles. We discuss our results in the context of the available lipid surface area for hIAPP binding and speculate on a role for gangliosides in facilitating toxic hIAPP aggregation.

While much has been learned about sphingolipids, originally named for their sphinx-like enigmatic properties, there are still many unanswered questions about the possible effect(s) of the composition of ceramide on the synthesis and/or behavior of a glycosphingolipid (GSL). Over time, studies of their ceramide component, the sphingoid base containing the lipid moiety of GSLs, were frequently distinct from those performed to ascertain the roles of the carbohydrate moieties. Due to the number of classes of GSLs that can be derived from ceramide, this review focuses on the possible role(s) of ceramide in the synthesis/function of just one GSL class, derived from glucosylceramide (Glc-Cer), namely sialylated ganglio derivatives, initially characterized and named gangliosides (GGs) due to their presence in ganglion cells. While much is known about their synthesis and function, much is still being learned. For example, it is only within the last 15-20 years or so that the mechanism by which the fatty acyl component of ceramide affected its transport to different sites in the Golgi, where it is used for the synthesis of Glu- or galactosyl-Cer (Gal-Cer) and more complex GSLs, was defined. Still to be fully addressed are questions such as (1) whether ceramide composition affects the transport of partially glycosylated GSLs to sites where their carbohydrate chain can be elongated or affects the activity of glycosyl transferases catalyzing that elongation; (2) what controls the differences seen in the ceramide composition of GGs that have identical carbohydrate compositions but vary in that of their ceramide and vice versa; (3) how alterations in ceramide composition affect the function of membrane GGs; and (4) how this knowledge might be applied to the development of therapies for treating diseases that correlate with abnormal expression of GGs. The availability of an updatable data bank of complete structures for individual classes of GSLs found in normal tissues as well as those associated with disease would facilitate research in this area.

Bilateral facial palsy with paresthesia (FDP) is a rare variant of GBS, characterized by simultaneous bilateral facial palsy and paresthesia of the distal limbs. Mounting evidence indicates that the presence of anti-GT1a IgG has a pathogenic role as an effector molecule in the development of cranial nerve palsies in certain patients with GBS, whereas anti-GT1a antibody is rarely presented positive in FDP. Here, we report the case of a 33-year-old male diagnosed with FDP presented with acute onset of bilateral facial palsy and slight paresthesias at the feet as the only neurological manifestation. An antecedent infection with no identifiable reason for the fever or skin eruptions was noted in the patient. He also exhibited cerebrospinal fluid albuminocytologic dissociation and abnormal nerve conduction studies. Notably, the testing of specific serum anti-gangliosides showed positive anti-GT1a IgG/IgM Ab. The patient responded well to intravenous immunoglobulin therapy. This case brings awareness to a rare variant of GBS, and provides the first indication that anti-GT1a antibodies play a causative role in the development of FDP. The case also suggests that prompt management with IVIG should be implemented if FDP is diagnosed.

Intranasal administration of total bovine brain gangliosides (6 mg/kg) to rats protected the CA1 hippocampal neurons from the death caused by two-vessel occlusion model (with hypotension) of forebrain ischemia/reperfusion injury. The immunohistochemical reaction of specific antibodies to marker proteins of activated microglia (Iba1) and astrocytes (GFAP) in hippocampal slices revealed the neuroprotective effect of exogenous gangliosides which can be mostly explained by their ability to suppress neuroinflammation and gliosis. The expression of neurotrophic factor BDNF in the CA1 region of hippocampus did not differ in sham-operated rats and animals exposed to ischemia/reperfusion. However, the administration of gangliosides increased the BDNF expression in both control and ischemic groups. The intranasal route of administration allows using lower concentrations of gangliosides preventing the death of hippocampal neurons.

Siglecs are cell surface receptors whose functions are tied to the binding of their sialoglycan ligands. Recently, we developed an optimized liposome formulation and used it to investigate the binding of human Siglecs (hSiglec) against a panel of gangliosides. Animal models, more specifically murine models, are used to understand human biology; however, species-specific differences can complicate the interpretation of the results. Herein, we used our optimized liposome formulation to dissect the interactions between murine Siglecs (mSiglecs) and gangliosides to assess the appropriateness of mSiglecs as a proxy to better understand the biological roles of hSiglec-ganglioside interactions. Using our optimized liposome formulation, we found that ganglioside binding is generally conserved between mice and humans with mSiglec-1, -E, -F, and -15 binding multiple gangliosides like their human counterparts. However, in contrast to the hSiglecs, we observed little to no binding between the mSiglecs and ganglioside GM1a. Detailed analysis of mSiglec-1 interacting with GM1a and its structural isomer, GM1b, suggests that mSiglec-1 preferentially binds α2-3-linked sialic acids presented from the terminal galactose residue. The ability of mSiglecs to interact or not interact with gangliosides, particularly GM1a, has implications for using mice to study neurodegenerative diseases, infections, and cancer, where interactions between Siglecs and glycolipids have been proposed to modulate these human diseases.

Anti-ganglioside antibodies (anti-Gg Abs) have been linked to delayed/poor clinical recovery in both axonal and demyelinating forms of Guillain-Barrè Syndrome (GBS). In many instances, the incomplete recovery is attributed to the peripheral nervous system\'s failure to regenerate. The cross-linking of cell surface gangliosides by anti-Gg Abs triggers inhibition of nerve repair in both in vitro and in vivo axon regeneration paradigms. This mechanism involves the activation of the small GTPase RhoA, which negatively modulates the growth cone cytoskeleton. At present, the identity/es of the receptor/s responsible for transducing the signal that ultimately leads to RhoA activation remains poorly understood. The aim of this work was to identify the transducer molecule responsible for the inhibitory effect of anti-Gg Abs on nerve repair. Putative candidate molecules were identified through proteomic mass spectrometry of ganglioside affinity-captured proteins from rat cerebellar granule neurons (Prendergast et al., 2014). These candidates were evaluated using an in vitro model of neurite outgrowth with primary cultured dorsal root ganglion neurons (DRGn) and an in vivo model of axon regeneration. Using an shRNA-strategy to silence putative candidates on DRGn, we identified tumor necrosis factor receptor 1A protein (TNFR1A) as a transducer molecule for the inhibitory effect on neurite outgrowth from rat/mouse DRGn cultures of a well characterized mAb targeting the related gangliosides GD1a and GT1b. Interestingly, lack of TNFr1A expression on DRGn abolished the inhibitory effect on neurite outgrowth caused by anti-GD1a but not anti-GT1b specific mAbs, suggesting specificity of GD1a/transducer signaling. Similar results were obtained using primary DRGn cultures from TNFR1a-null mice, which did not activate RhoA after exposure to anti-GD1a mAbs. Generation of single point mutants at the stalk region of TNFR1A identified a critical amino acid for transducing GD1a signaling, suggesting a direct interaction. Finally, passive immunization with an anti-GD1a/GT1b mAb in an in vivo model of axon regeneration exhibited reduced inhibitory activity in TNFR1a-null mice compared to wild type mice. In conclusion, these findings identify TNFR1A as a novel transducer receptor for the inhibitory effect exerted by anti-GD1a Abs on nerve repair, representing a significant step forward toward understanding the factors contributing to poor clinical recovery in GBS associated with anti-Gg Abs.