Dissecting the abilities of murine Siglecs to interact with gangliosides.

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.

Neural-specific alterations in glycosphingolipid biosynthesis and cell signaling associated with two human ganglioside GM3 synthase deficiency variants.

GM3 Synthase Deficiency (GM3SD) is a neurodevelopmental disorder resulting from pathogenic variants in the ST3GAL5 gene, which encodes GM3 synthase, a glycosphingolipid (GSL)-specific sialyltransferase. This enzyme adds a sialic acid to the terminal galactose of lactosylceramide (LacCer) to produce the monosialylated ganglioside GM3. In turn, GM3 is extended by other glycosyltransferases to generate nearly all the complex gangliosides enriched in neural tissue. Pathogenic mechanisms underlying the neural phenotypes associated with GM3SD are unknown. To explore how loss of GM3 impacts neural-specific glycolipid glycosylation and cell signaling, GM3SD patient fibroblasts bearing one of two different ST3GAL5 variants were reprogrammed to induced pluripotent stem cells (iPSCs) and then differentiated to neural crest cells (NCCs). GM3 and GM3-derived gangliosides were undetectable in cells carrying either variant, while LacCer precursor levels were elevated compared to wildtype (WT). NCCs of both variants synthesized elevated levels of neutral lacto- and globo-series, as well as minor alternatively sialylated GSLs compared to WT. Ceramide profiles were also shifted in GM3SD variant cells. Altered GSL profiles in GM3SD cells were accompanied by dynamic changes in the cell surface proteome, protein O-GlcNAcylation, and receptor tyrosine kinase abundance. GM3SD cells also exhibited increased apoptosis and sensitivity to erlotinib-induced inhibition of epidermal growth factor receptor signaling. Pharmacologic inhibition of O-GlcNAcase rescued baseline and erlotinib-induced apoptosis. Collectively, these findings indicate aberrant cell signaling during differentiation of GM3SD iPSCs and also underscore the challenge of distinguishing between variant effect and genetic background effect on specific phenotypic consequences.

Exosomes derived from human umbilical cord mesenchymal stem cells pretreated by monosialoteterahexosyl ganglioside alleviate intracerebral hemorrhage by down-regulating autophagy.

PURPOSE: Intracerebral hemorrhage (ICH) results in substantial morbidity, mortality, and disability. Depleting neural cells in advanced stages of ICH poses a significant challenge to recovery. The objective of our research is to investigate the potential advantages and underlying mechanism of exosomes obtained from human umbilical cord mesenchymal stem cells (hUMSCs) pretreated with monosialoteterahexosyl ganglioside (GM1) in the prevention of secondary brain injury (SBI) resulting from ICH. PATIENTS AND METHODS: In vitro, hUMSCs were cultured and induced to differentiate into neuron-like cells after they were pretreated with 150 µg/mL GM1. The exosomes extracted from the culture medium following a 6-h pretreatment with 150 µg/mL GM1 were used as the treatment group. Striatal infusion of collagenase and hemoglobin (Hemin) was used to establish in vivo and in vitro models of ICH. RESULTS: After being exposed to 150 µg/mL GM1 for 6 h, specific cells displayed typical neuron-like cell morphology and expressed neuron-specific enolase (NSE). The rate of differentiation into neuron-like cells was up to (15.9 ± 5.8) %, and the synthesis of N-Acetylgalactosaminyltransferase (GalNAcT), which is upstream of GM1, was detected by Western blot. This study presented an increase in the synthesis of GalNAcT. Compared with the ICH group, apoptosis in the treatment group was remarkably reduced, as detected by TUNEL, and mitochondrial membrane potential was restored by JC-1. Additionally, Western blot revealed the restoration of up-regulated autophagy markers Beclin-1 and LC3 and the down-regulation of autophagy marker p62 after ICH. CONCLUSION: These findings suggest that GM1 is an effective agent to induce the differentiation of hUMSCs into neuron-like cells. GM1 can potentially increase GalNAcT production through "positive feedback", which generates more GM1 and promotes the differentiation of hUMSCs. After pretreatment with GM1, exosomes derived from hUMSCs (hUMSCs-Exos) demonstrate a neuroprotective effect by inhibiting autophagy in the ICH model. This study reveals the potential mechanism by which GM1 induces differentiation of hUMSCs into neuron-like cells and confirms the therapeutic effect of hUMSCs-Exos pretreated by GM1 (GM1-Exos) on an ICH model, potentially offering a new direction for stem cell therapy in ICH.

A mechanism of self-lipid endocytosis mediated by the receptor Mincle.

Cellular lipid uptake (through endocytosis) is a basic physiological process. Dysregulation of this process underlies the pathogenesis of diseases such as atherosclerosis, obesity, diabetes, and cancer. However, to date, only some mechanisms of lipid endocytosis have been discovered. Here, we show a previously unknown mechanism of lipid cargo uptake into cells mediated by the receptor Mincle. We found that the receptor Mincle, previously shown to be a pattern recognition receptor of the innate immune system, tightly binds a range of self-lipids. Moreover, we revealed the minimal molecular motif in lipids that is sufficient for Mincle recognition. Superresolution microscopy showed that Mincle forms vesicles in cytoplasm and colocalizes with added fluorescent lipids in endothelial cells but does not colocalize with either clathrin or caveolin-1, and the added lipids were predominantly incorporated in vesicles that expressed Mincle. Using a model of ganglioside GM3 uptake in brain vessel endothelial cells, we show that the knockout of Mincle led to a dramatic decrease in lipid endocytosis. Taken together, our results have revealed a fundamental lipid endocytosis pathway, which we call Mincle-mediated endocytosis (MiME), and indicate a prospective target for the treatment of disorders of lipid metabolism, which are rapidly increasing in prevalence.

Ganglioside GD3 regulates neural stem cell quiescence and controls postnatal neurogenesis.

The postnatal neural stem cell (NSC) pool hosts quiescent and activated radial glia-like NSCs contributing to neurogenesis throughout adulthood. However, the underlying regulatory mechanism during the transition from quiescent NSCs to activated NSCs in the postnatal NSC niche is not fully understood. Lipid metabolism and lipid composition play important roles in regulating NSC fate determination. Biological lipid membranes define the individual cellular shape and help maintain cellular organization and are highly heterogeneous in structure and there exist diverse microdomains (also known as lipid rafts), which are enriched with sugar molecules, such as glycosphingolipids. An often overlooked but key aspect is that the functional activities of proteins and genes are highly dependent on their molecular environments. We previously reported that ganglioside GD3 is the predominant species in NSCs and that the reduced postnatal NSC pools are observed in global GD3-synthase knockout (GD3S-KO) mouse brains. The specific roles of GD3 in determining the stage and cell-lineage determination of NSCs remain unclear, since global GD3S-KO mice cannot distinguish if GD3 regulates postnatal neurogenesis or developmental impacts. Here, we show that inducible GD3 deletion in postnatal radial glia-like NSCs promotes NSC activation, resulting in the loss of the long-term maintenance of the adult NSC pools. The reduced neurogenesis in the subventricular zone (SVZ) and the dentate gyrus (DG) of GD3S-conditional-knockout mice led to the impaired olfactory and memory functions. Thus, our results provide convincing evidence that postnatal GD3 maintains the quiescent state of radial glia-like NSCs in the adult NSC niche.

GM1 ganglioside protects against LPS-induced neuroinflammatory and oxidative responses by inhibiting the activation of Akt, TAK1 and NADPH oxidase in MG6 microglial cells.

GM1 is a major brain ganglioside that exerts neurotrophic, neuroprotective and antineuroinflammatory effects. The aim of this study was to obtain insights into the antineuroinflammatory mechanisms of exogenous GM1 in lipopolysaccharide (LPS)-stimulated MG6 mouse transformed microglial cell line. First, we found that GM1 prevented the LPS-induced transformation of microglia into an amoeboid-like shape. GM1 treatment inhibited LPS-induced expression of inducible nitric oxide synthase, cyclooxygenase-2 (COX-2), and proinflammatory cytokines such as TNF-α, IL-1ß and IL-6 in MG6 cells. In LPS-treated mice, GM1 also reduced striatal microglia activation and attenuated COX-2 expression. Subsequent mechanistic studies showed that GM1 suppressed LPS-induced nuclear translocation of nuclear factor κB (NF-κB) and activator protein-1 (AP-1), two critical transcription factors responsible for the production of proinflammatory mediators. GM1 exhibited antineuroinflammatory properties by suppressing Akt/NF-κB signaling and the activation of mitogen-activated protein kinases (MAPKs), including p38 MAPK, extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK). Furthermore, GM1 suppressed LPS-induced activation of transforming growth factor-ß-activated kinase 1 (TAK1) and NADPH oxidase 2 (NOX2), upstream regulators of the IκBα/NF-κB and MAPK/AP-1 signaling pathways. GM1 also inhibited NOX-mediated reactive oxygen species (ROS) production and protected against LPS-induced MG6 cell death, suggesting an antioxidant role of GM1. In conclusion, GM1 exerts both antineuroinflammatory and antioxidative effects by inhibiting Akt, TAK1 and NOX2 activation.

Decreasing ganglioside synthesis delays motor and cognitive symptom onset in Spg11 knockout mice.

Biallelic variants in the SPG11 gene account for the most common form of autosomal recessive hereditary spastic paraplegia characterized by motor and cognitive impairment, with currently no therapeutic option. We previously observed in a Spg11 knockout mouse that neurodegeneration is associated with accumulation of gangliosides in lysosomes. To test whether a substrate reduction therapy could be a therapeutic option, we downregulated the key enzyme involved in ganglioside biosynthesis using an AAV-PHP.eB viral vector expressing a miRNA targeting St3gal5. Downregulation of St3gal5 in Spg11 knockout mice prevented the accumulation of gangliosides, delayed the onset of motor and cognitive symptoms, and prevented the upregulation of serum levels of neurofilament light chain, a biomarker widely used in neurodegenerative diseases. Importantly, similar results were observed when Spg11 knockout mice were administrated venglustat, a pharmacological inhibitor of glucosylceramide synthase expected to decrease ganglioside synthesis. Downregulation of St3gal5 or venglustat administration in Spg11 knockout mice strongly decreased the formation of axonal spheroids, previously associated with impaired trafficking. Venglustat had similar effect on cultured human SPG11 neurons. In conclusion, this work identifies the first disease-modifying therapeutic strategy in SPG11, and provides data supporting its relevance for therapeutic testing in SPG11 patients.

Homeostatic and pathogenic roles of GM3 ganglioside molecular species in TLR4 signaling in obesity.

Innate immune signaling via TLR4 plays critical roles in pathogenesis of metabolic disorders, but the contribution of different lipid species to metabolic disorders and inflammatory diseases is less clear. GM3 ganglioside in human serum is composed of a variety of fatty acids, including long-chain (LCFA) and very-long-chain (VLCFA). Analysis of circulating levels of human serum GM3 species from patients at different stages of insulin resistance and chronic inflammation reveals that levels of VLCFA-GM3 increase significantly in metabolic disorders, while LCFA-GM3 serum levels decrease. Specific GM3 species also correlates with disease symptoms. VLCFA-GM3 levels increase in the adipose tissue of obese mice, and this is blocked in TLR4-mutant mice. In cultured monocytes, GM3 by itself has no effect on TLR4 activation; however, VLCFA-GM3 synergistically and selectively enhances TLR4 activation by LPS/HMGB1, while LCFA-GM3 and unsaturated VLCFA-GM3 suppresses TLR4 activation. GM3 interacts with the extracellular region of TLR4/MD2 complex to modulate dimerization/oligomerization. Ligand-molecular docking analysis supports that VLCFA-GM3 and LCFA-GM3 act as agonist and antagonist of TLR4 activity, respectively, by differentially binding to the hydrophobic pocket of MD2. Our findings suggest that VLCFA-GM3 is a risk factor for TLR4-mediated disease progression.

GT1b functions as a novel endogenous agonist of toll-like receptor 2 inducing neuropathic pain.

Spinal cord microglia contribute to nerve injury-induced neuropathic pain. We have previously demonstrated that toll-like receptor 2 (TLR2) signaling is critical for nerve injury-induced activation of spinal cord microglia, but the responsible endogenous TLR2 agonist has not been identified. Here, we show that nerve injury-induced upregulation of sialyltransferase St3gal2 in sensory neurons leads to an increase in expression of the sialylated glycosphingolipid, GT1b. GT1b ganglioside is axonally transported to the spinal cord dorsal horn and contributes to characteristics of neuropathic pain such as mechanical and thermal hypersensitivity. Spinal cord GT1b functions as an TLR2 agonist and induces proinflammatory microglia activation and central sensitization. Pharmacological inhibition of GT1b synthesis attenuates nerve injury-induced spinal cord microglia activation and pain hypersensitivity. Thus, the St3gal2-GT1b-TLR2 axis may offer a novel therapeutic target for the treatment of neuropathic pain.

IgM anti-GM2 antibodies in patients with multifocal motor neuropathy target Schwann cells and are associated with early onset.

BACKGROUND: Multifocal motor neuropathy (MMN) is a rare, chronic immune-mediated polyneuropathy characterized by asymmetric distal limb weakness. An important feature of MMN is the presence of IgM antibodies against gangliosides, in particular GM1 and less often GM2. Antibodies against GM1 bind to motor neurons (MNs) and cause damage through complement activation. The involvement of Schwann cells (SCs), expressing GM1 and GM2, in the pathogenesis of MMN is unknown. METHODS: Combining the data of our 2007 and 2015 combined cross-sectional and follow-up studies in Dutch patients with MMN, we evaluated the presence of IgM antibodies against GM1 and GM2 in serum from 124 patients with MMN and investigated their binding to SCs and complement-activating properties. We also assessed the relation of IgM binding and complement deposition with clinical characteristics. RESULTS: Thirteen out of 124 patients (10%) had a positive ELISA titer for IgM anti-GM2. Age at onset of symptoms was significantly lower in MMN patients with anti-GM2 IgM. IgM binding to SCs correlated with IgM anti-GM2 titers. We found no correlation between IgM anti-GM2 titers and MN binding or with IgM anti-GM1 titers. IgM binding to SCs decreased upon pre-incubation of serum with soluble GM2, but not with soluble GM1. IgM anti-GM2 binding to SCs correlated with complement activation, as reflected by increased C3 fixation on SCs and C5a formation in the supernatant. CONCLUSION: Circulating IgM anti-GM2 antibodies define a subgroup of patients with MMN that has an earlier onset of disease. These antibodies probably target SCs specifically and activate complement, similarly as IgM anti-GM1 on MNs. Our data indicate that complement activation by IgM antibodies bound to SCs and MNs underlies MMN pathology.

Lipid raft interacting galectin 8 regulates primary ciliogenesis.

Primary cilium is a specialized sensory organelle that transmits environmental information into cells. Its length is tightly controlled by various mechanisms such as the frequency or the cargo size of the intraflagellar transport trains which deliver the building materials such as tubulin subunits essential for the growing cilia. Here, we show the sialoglycan interacting galectin 8 regulates the process of primary ciliogenesis. As the epithelia become polarized, there are more galectin 8 being apically secreted and these extracellular galectin 8 molecules apparently bind to a lipid raft enriched domain at the base of the primary cilia through interacting with lipid raft components, such as GD3 ganglioside and scaffold protein caveolin 1. Furthermore, the binding of galectin 8 at this critical region triggers rapid growth of primary cilia by perturbing the barrier function of the transition zone (TZ). Our study also demonstrates the functionality of this barrier depends on intact organization of lipid rafts at the cilia as genetically knockout of Cav1 and pharmacologically inhibition of lipid raft both phenocopy the effect of apical addition of recombinant galectin 8; that is, rapid elongation of primary cilia and redistribution of cilia proteins from TZ to the growing axoneme. Indeed, as cilia elongated, endogenous galectin 8, caveolin 1, and TZ component, TMEM231, also transited from the TZ to the growing axoneme. We also noted that the interaction between caveolin 1 and TMEM231 could be perturbed by exogenous galectin 8. Taken together, we proposed that galectin 8 promoted primary cilia elongation through impeding the barrier function of the TZ by interfering with the interaction between caveolin 1 and TMEM231.

Antiganglioside antibody frequency in routine clinical care settings.

BACKGROUND AND PURPOSE: Antiganglioside antibodies (AGAs) might be involved in the etiopathogenesis of many neurological diseases, such as Miller-Fisher syndrome (MFS) and Guillain-Barré syndrome (GBS). Available comprehensive reference data regarding AGA positivity rates and cross-responsiveness among AGAs (where one line immunoblot is positive for ≥1 AGA) during routine clinical care are scant. METHODS: In this 10-year monocentric retrospective study, 3560 immunoglobulin (Ig) G and IgM line blots (GA Generic Assays' Anti-Ganglioside Dot kit) obtained using cerebrospinal fluid (CSF) and serum samples from 1342 patients were analyzed for AGA positivity in terms of 14 diagnosis categories and AGA cross-responsiveness. RESULTS: Of all 3560 line blots 158 (4.4%) and of all CSF samples 0.4% (4/924) CSF line blots were AGA positive. For serum IgG, blots with positivity rates higher than the standard deviation of 15.6% were associated with MFS (GD3, GD1a, GT1a and GQ1b) and acute motor axonal neuropathy (AMAN) (GM1, GD1a and GT1a). For serum IgM, blots with positivity rates higher than the standard deviation of 8.1% were associated with AMAN (GM2, GT1a and GQ1b), MFS (GM1, GT1a and GQ1b), multifocal motor neuropathy (MMN) (GM1, GM2 and GQ1b) and chronic inflammatory demyelinating polyneuropathy (CIDP) (GM1). Cross-responsiveness was observed in 39.6% of all positive serum AGA. CONCLUSIONS: Testing for AGAs during routine clinical care rarely led to positive findings, both in serum and even less in CSF, except for the diagnoses AMAN, MFS, MMN and CIDP. Nonspecific findings found as cross-responsiveness between different AGA samples occur frequently, impacting the positivity of most AGA subtypes.

Electrodiagnostic methods to verify Guillain-Barré syndrome subtypes in Istanbul: A prospective multicenter study.

BACKGROUND AND AIMS: This study aimed to identify the clinical characteristics and electrodiagnostic subtypes of Guillain-Barré syndrome (GBS) in Istanbul. METHODS: Patients with GBS were prospectively recruited between April 2019 and March 2022 and two electrodiagnostic examinations were performed on each patient. The criteria of Ho et al., Hadden et al., Rajabally et al., and Uncini et al. were compared for the differentiation of demyelinating and axonal subtypes, and their relations with anti-ganglioside antibodies were analyzed. RESULTS: One hundred seventy-seven patients were included, 69 before the coronavirus disease 2019 pandemic (April 2019-February 2020) and 108 during the pandemic (March 2020-March 2022), without substantial changes in monthly frequencies. As compared with the criteria of Uncini et al., demyelinating GBS subtype diagnosis was more frequent according to the Ho et al. and Hadden et al. criteria (95/162, 58.6% vs. 110/174, 63.2% and 121/174, 69.5%, respectively), and less frequent according to Rajabally et al.'s criteria (76/174, 43.7%). Fourteen patients' diagnoses made using Rajabally et al.'s criteria were shifted to the other subtype with the second electrodiagnostic examination. Of the 106 analyzed patients, 22 had immunoglobulin G anti-ganglioside antibodies (14 with the axonal subtype). They had less frequent sensory symptoms (54.5% vs. 83.1%, p = 0.009), a more frequent history of previous gastroenteritis (54.5% vs. 22.9%, p = 0.007), and a more severe disease as compared with those without antibodies. INTERPRETATION: Serial electrodiagnostic examinations are more helpful for accurate subtype diagnosis of GBS because of the dynamic pathophysiology of the disease. We observed no significant increase in GBS frequency during the pandemic in this metropolis.

Cell density-dependent membrane distribution of ganglioside GM3 in melanoma cells.

Monosialoganglioside GM3 is the simplest ganglioside involved in various cellular signaling. Cell surface distribution of GM3 is thought to be crucial for the function of GM3, but little is known about the cell surface GM3 distribution. It was shown that anti-GM3 monoclonal antibody binds to GM3 in sparse but not in confluent melanoma cells. Our model membrane study evidenced that monoclonal anti-GM3 antibodies showed stronger binding when GM3 was in less fluid membrane environment. Studies using fluorescent GM3 analogs suggested that GM3 was clustered in less fluid membrane. Moreover, fluorescent lifetime measurement showed that cell surface of high density melanoma cells is more fluid than that of low density cells. Lipidomics and fatty acid supplementation experiment suggested that monounsaturated fatty acid-containing phosphatidylcholine contributed to the cell density-dependent membrane fluidity. Our results indicate that anti-GM3 antibody senses GM3 clustering and the number and/or size of GM3 cluster differ between sparse and confluent melanoma cells.

The spectrum of anti-GQ1B antibody syndrome: beyond Miller Fisher syndrome and Bickerstaff brainstem encephalitis.

INTRODUCTION: Since the initial identification of Miller Fisher syndrome (MFS) and Bickerstaff brainstem encephalitis (BBE),significant milestones have been achieved in understanding these diseases.Discoveries of common serum antibodies (IgG anti-GQ1b), antecedent infections, neurophysiological data, andneuroimaging suggested a shared autoimmune pathogenetic mechanism rather than distinct pathogenesis, leadingto the hypothesis that both diseases are part of a unified syndrome, termed "Fisher-Bickerstaff syndrome". The subsequent identification of atypical anti-GQ1b-positive forms expanded the classification to a broader condition known as "Anti-GQ1b-Antibody syndrome". METHODS: An exhaustive literature review was conducted, analyzing a substantial body of research spanning from the initialdescriptions of the syndrome's components to recent developments in diagnostic classification and researchperspectives. RESULTS: Anti-GQ1b syndrome encompasses a continuous spectrum of conditions defined by a common serological profilewith varying degrees of peripheral (PNS) and central nervous system (CNS) involvement. MFS and BBE represent theopposite ends of this spectrum, with MFS primarily affecting the PNS and BBE predominantly involving the CNS.Recently identified atypical forms, such as acute ophthalmoparesis, acute ataxic neuropathy withoutophthalmoparesis, Guillain-Barré syndrome (GBS) with ophthalmoparesis, MFS-GBS and BBE-GBS overlap syndromes,have broadened this spectrum. CONCLUSION: This work aims to provide an extensive, detailed, and updated overview of all aspects of the anti-GQ1b syndromewith the intention of serving as a stepping stone for further shaping thereof. Special attention was given to therecently identified atypical forms, underscoring their significance in redefining the boundaries of the syndrome.

Recent advances in tumor associated carbohydrate antigen based chimeric antigen receptor T cells and bispecific antibodies for anti-cancer immunotherapy.

Tumor associated carbohydrate antigens (TACAs) are a class of attractive antigens for the development of anti-cancer immunotherapy. Besides monoclonal antibodies and vaccines, chimeric antigen receptor (CAR) T cells and bispecific antibodies (BsAbs) targeting TACA are exciting directions to harness the power of the immune system to fight cancer. In this review, we focus on two TACAs, i.e., the GD2 ganglioside and the mucin-1 (MUC1) protein. The latest advances in CAR T cells and bispecific antibodies targeting these two antigens are presented. The roles of co-stimulatory molecules, structures of the sequences for antigen binding, methods for CAR and antibody construction, as well as strategies to enhance solid tumor penetration and reduce T cell exhaustion and death are discussed. Furthermore, approaches to reduce "on target, off tumor" side effects are introduced. With further development, CAR T cells and BsAbs targeting GD2 and MUC1 can become powerful agents to effectively treat solid tumor.

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.

Sphingolipids: Less Enigmatic but Still Many Questions about the Role(s) of Ceramide in the Synthesis/Function of the Ganglioside Class of Glycosphingolipids.

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.

Quercetin Induces Mitochondrial Apoptosis and Downregulates Ganglioside GD3 Expression in Melanoma Cells.

Malignant melanoma represents a form of skin cancer characterized by a bleak prognosis and heightened resistance to traditional therapies. Quercetin has demonstrated notable anti-carcinogenic, anti-inflammatory, anti-oxidant, and pharmacological effects across various cancer types. However, the intricate relationship between quercetin's anti-cancer properties and ganglioside expression in melanoma remains incompletely understood. In this study, quercetin manifests specific anti-proliferative, anti-migratory, and cell-cycle arrest effects, inducing mitochondrial dysfunction and apoptosis in two melanoma cancer cell lines. This positions quercetin as a promising candidate for treating malignant melanoma. Moreover, our investigation indicates that quercetin significantly reduces the expression levels of ganglioside GD3 and its synthetic enzyme. Notably, this reduction is achieved through the inhibition of the FAK/paxillin/Akt signaling pathway, which plays a crucial role in cancer development. Taken together, our findings suggest that quercetin may be a potent anti-cancer drug candidate for the treatment of malignant melanoma.

Understanding Aß Peptide Binding to Lipid Membranes: A Biophysical Perspective.

Aß peptides are known to bind neural plasma membranes in a process leading to the deposit of Aß-enriched plaques. These extracellular structures are characteristic of Alzheimer's disease, the major cause of late-age dementia. The mechanisms of Aß plaque formation and deposition are far from being understood. A vast number of studies in the literature describe the efforts to analyze those mechanisms using a variety of tools. The present review focuses on biophysical studies mostly carried out with model membranes or with computational tools. This review starts by describing basic physical aspects of lipid phases and commonly used model membranes (monolayers and bilayers). This is followed by a discussion of the biophysical techniques applied to these systems, mainly but not exclusively Langmuir monolayers, isothermal calorimetry, density-gradient ultracentrifugation, and molecular dynamics. The Methodological Section is followed by the core of the review, which includes a summary of important results obtained with each technique. The last section is devoted to an overall reflection and an effort to understand Aß-bilayer binding. Concepts such as Aß peptide membrane binding, adsorption, and insertion are defined and differentiated. The roles of membrane lipid order, nanodomain formation, and electrostatic forces in Aß-membrane interaction are separately identified and discussed.