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In pediatric and intensive care units, propofol is widely used for general anesthesia and sedation procedures as a short-acting anesthetic. Multiple studies have revealed that propofol causes hippocampal injury and cognitive dysfunction in developing animals. As is known, GM1, a type of ganglioside, plays a crucial role in promoting nervous system development. Consequently, this study explored whether GM1 mitigated neurological injury caused by propofol during developmental stages and investigated its underlying mechanisms. Seven-day-old SD rats or PC12 cells were used in this study for histopathological analyses, a Morris water maze test, a lactate dehydrogenase release assay, Western blotting, and an ELISA. Furthermore, LY294002 was employed to explore the potential neuroprotective effect of GM1 via the PI3K/AKT signaling cascade. The results indicated that GM1 exerted a protective effect against hippocampal morphological damage and pyroptosis as well as behavioral abnormalities following propofol exposure by increasing p-PI3K and p-AKT expression while decreasing p-p65 expression in developing rats. Nevertheless, the inhibitor LY294002, which targets the PI3K/AKT cascade, attenuated the beneficial effects of GM1. Our study provides evidence that GM1 confers neuroprotection and attenuates propofol-induced developmental neurotoxicity, potentially involving the PI3K/AKT/NF-κB signaling cascade.

Monosialoganglioside GM1 (GM1) has long been used as a therapeutic agent for neurological diseases in the clinical treatment of ischemic stroke. However, the mechanism underlying the neuroprotective function of GM1 is still obscure until now. In this study, we investigated the effects of GM1 in ischemia and reperfusion (I/R) brain injury models. Middle cerebral artery occlusion and reperfusion (MCAO/R) rats were treated with GM1 (60 mg·kg -1 ·d -1 , tail vein injection) for 2 weeks. The results showed that GM1 substantially attenuated the MCAO/R-induced neurological dysfunction and inhibited the inflammatory responses and cell apoptosis in ischemic parietal cortex. We further revealed that GM1 inhibited the activation of NFκB/MAPK signaling pathway induced by MCAO/R injury. To explore its underlying mechanism of the neuroprotective effect, transcriptome sequencing was introduced to screen the differentially expressed genes (DEGs). By function enrichment and PPI network analyses, Sptbn1 was identified as a node gene in the network regulated by GM1 treatment. In the MCAO/R model of rats and oxygen-glucose deprivation and reperfusion (OGD/R) model of primary culture of rat cortical neurons, we first found that SPTBN1 was involved in the attenuation of I/R induced neuronal injury after GM1 administration. In SPTBN1-knockdown SH-SY5Y cells, the treatment with GM1 (20 μM) significantly increased SPTBN1 level. Moreover, OGD/R decreased SPTBN1 level in SPTBN1-overexpressed SH-SY5Y cells. These results indicated that GM1 might achieve its potent neuroprotective effects by regulating inflammatory response, cell apoptosis, and cytomembrane and cytoskeleton signals through SPTBN1. Therefore, SPTBN1 may be a potential target for the treatment of ischemic stroke., (Copyright © 2024 IBRO. Published by Elsevier Inc. All rights reserved.)

TNF receptor-associated factor 2 (TRAF2) is involved in different cellular processes including signal transduction and transcription regulation. We here provide evidence of a direct interaction between the TRAF domain of TRAF2 and the monosialotetrahexosylganglioside (GM1). Previously, we showed that the TRAF domain occurs mainly in a trimeric form in solution, but it can also exist as a stable monomer when in the nanomolar concentration range. Here, we report that the quaternary structure of the TRAF domain is also affected by pH changes, since a weakly acidic pH (5.5) favors the dissociation of the trimeric TRAF domain into stable monomers, as previously observed at neutral pH (7.6) with the diluted protein. The TRAF domain-GM1 binding was similar at pH 5.5 and 7.6, suggesting that GM1 interacts with both the trimeric and monomeric forms of the protein. However, only the monomeric protein appeared to cause membrane deformation and inward vesiculation in GM1-containing giant unilamellar vesicles (GUVs). The formation of complexes between GM1 and TRAF2, or its TRAF domain, was also observed in cultured human leukemic HAP1 cells expressing either the truncated TRAF domain or the endogenous full length TRAF2. The GM1-protein complexes were observed after treatment with tunicamycin and were more concentrated in cells undergoing apoptosis, a condition which is known to cause cytoplasm acidification. These findings open the avenue for future studies aimed at deciphering the physiopathological relevance of the TRAF domain-GM1 interaction., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Gaucher disease (GD) is a rare genetic metabolic disorder characterized by a dysfunction of the lysosomal glycoside hydrolase glucocerebrosidase (GCase) due to mutations in the gene GBA1, leading to the cellular accumulation of glucosylceramide (GlcCer). While most of the current research focuses on the primary accumulated material, lesser attention has been paid to secondary storage materials and their reciprocal intertwining. By using a novel approach based on flow cytometry and fluorescent labelling, we monitored changes in storage materials directly in fibroblasts derived from GD patients carrying N370S/RecNcil and homozygous L444P or R131C mutations with respect to wild type. In L444P and R131C fibroblasts, we detected not only the primary accumulation of GlcCer accumulation but also a considerable secondary increase in GM1 storage, comparable with the one observed in infantile patients affected by GM1 gangliosidosis. In addition, the ability of a trivalent trihydroxypiperidine iminosugar compound (CV82), which previously showed good pharmacological chaperone activity on GCase enzyme, to reduce the levels of storage materials in L444P and R131C fibroblasts was tested. Interestingly, treatment with different concentrations of CV82 led to a significant reduction in GM1 accumulation only in L444P fibroblasts, without significantly affecting GlcCer levels. The compound CV82 was selective against the GCase enzyme with respect to the β-Galactosidase enzyme, which was responsible for the catabolism of GM1 ganglioside. The reduction in GM1-ganglioside level cannot be therefore ascribed to a direct action of CV82 on β-Galactosidase enzyme, suggesting that GM1 decrease is rather related to other unknown mechanisms that follow the direct action of CV82 on GCase. In conclusion, this work indicates that the tracking of secondary storages can represent a key step for a better understanding of the pathways involved in the severity of GD, also underlying the importance of developing drugs able to reduce both primary and secondary storage-material accumulations in GD.

Chronic activation and dysfunction of microglia have been implicated in the pathogenesis and progression of many neurodegenerative disorders, including Huntington's disease (HD). HD is a genetic condition caused by a mutation that affects the folding and function of huntingtin (HTT). Signs of microglia activation have been observed in HD patients even before the onset of symptoms. It is unclear, however, whether pro-inflammatory microglia activation in HD results from cell-autonomous expression of mutant HTT, is the response of microglia to a diseased brain environment, or both. In this study, we used primary microglia isolated from HD knock-in (Q140) and wild-type (Q7) mice to investigate their response to inflammatory conditions in vitro in the absence of confounding effects arising from brain pathology. We show that naïve Q140 microglia do not undergo spontaneous pro-inflammatory activation and respond to inflammatory triggers, including stimulation of TLR4 and TLR2 and exposure to necrotic cells, with similar kinetics of pro-inflammatory gene expression as wild-type microglia. Upon termination of the inflammatory insult, the transcription of pro-inflammatory cytokines is tapered off in Q140 and wild-type microglia with similar kinetics. However, the ability of Q140 microglia to develop tolerance in response to repeated inflammatory stimulations is partially impaired in vitro and in vivo, potentially contributing to the establishment of chronic neuroinflammation in HD. We further show that ganglioside GM1, a glycosphingolipid with anti-inflammatory effects on wild-type microglia, not only decreases the production of pro-inflammatory cytokines and nitric oxide in activated Q140 microglia, but also dramatically dampen microglia response to re-stimulation with LPS in an experimental model of tolerance. These effects are independent from the expression of interleukin 1 receptor associated kinase 3 (Irak-3), a strong modulator of LPS signaling involved in the development of innate immune tolerance and previously shown to be upregulated by immune cell treatment with gangliosides. Altogether, our data suggest that external triggers are required for HD microglia activation, but a cell-autonomous dysfunction that affects the ability of HD microglia to acquire tolerance might contribute to the establishment of neuroinflammation in HD. Administration of GM1 might be beneficial to attenuate chronic microglia activation and neuroinflammation., (© 2023. The Author(s).)

Large-scale synthesis of GM1, an important ganglioside in mammalian cells especially those in the nervous system, is needed to explore its therapeutic potential. Biocatalytic production is a promising platform for such a purpose. We report herein the development of process engineering and glycosyltransferase improvement strategies to advance chemoenzymatic total synthesis of GM1. Firstly, a new short route was developed for chemical synthesis of lactosylsphingosine from the commercially available Garner's aldehyde. Secondly, two glycosyltransferases including Campylobacter jejuni β1-4GalNAcT (CjCgtA) and β1-3-galactosyltransferase (CjCgtB) were improved on their soluble expression in E. coli and enzyme stability by fusing with an N-terminal maltose binding protein (MBP). Thirdly, the process for enzymatic synthesis of GM1 sphingosines from lactosylsphingosine was engineered by developing a multistep one-pot multienzyme (MSOPME) strategy without isolating intermediate glycosphingosines and by adding a detergent, sodium cholate, to the later enzymatic glycosylation steps. Installation of a desired fatty acyl chain to GM1 glycosphingosines led to the formation of target GM1 gangliosides. The combination of glycosyltransferase improvement with chemical and enzymatic process engineering represents a significant advance in obtaining GM1 gangliosides containing different sialic acid forms by total chemoenzymatic synthesis in a short route and with high efficiency., (© 2023 Wiley-VCH GmbH.)

GM1-gangliosidosis is a catastrophic, neurodegenerative lysosomal storage disease caused by a deficiency of lysosomal β-galactosidase (β-Gal). The primary substrate of the enzyme is GM1-ganglioside (GM1), a sialylated glycosphingolipid abundant in nervous tissue. Patients with GM1-gangliosidosis present with massive and progressive accumulation of GM1 in the central nervous system (CNS), which leads to mental and motor decline, progressive neurodegeneration, and early death. No therapy is currently available for this lysosomal storage disease. Here, we describe a proof-of-concept preclinical study toward the development of enzyme replacement therapy (ERT) for GM1-gangliosidosis using a recombinant murine β-Gal fused to the plant lectin subunit B of ricin (mβ-Gal:RTB). We show that long-term, bi-weekly systemic injection of mβ-Gal:RTB in the β-Gal mouse model resulted in widespread internalization of the enzyme by cells of visceral organs, with consequent restoration of enzyme activity. Most importantly, β-Gal activity was detected in several brain regions. This was accompanied by a reduction of accumulated GM1, reversal of neuroinflammation, and decrease in the apoptotic marker caspase 3. These results indicate that the RTB lectin delivery module enhances both the CNS-biodistribution pattern and the therapeutic efficacy of the β-Gal ERT, with the potential to translate to a clinical setting for the treatment of GM1-gangliosidosis.-/- mouse model resulted in widespread internalization of the enzyme by cells of visceral organs, with consequent restoration of enzyme activity. Most importantly, β-Gal activity was detected in several brain regions. This was accompanied by a reduction of accumulated GM1, reversal of neuroinflammation, and decrease in the apoptotic marker caspase 3. These results indicate that the RTB lectin delivery module enhances both the CNS-biodistribution pattern and the therapeutic efficacy of the β-Gal ERT, with the potential to translate to a clinical setting for the treatment of GM1-gangliosidosis.

Parkinson's disease (PD) is slowly progressing neurodegenerative disorder that affects millions of patients worldwide. While effective symptomatic therapies for PD exist, there is no currently available disease modifying agent to slow or stop the progression of the disease. Many years of research from various laboratories around the world have provided evidence in favor of the potential ability of GM1 ganglioside to be a disease modifying agent for PD. In this paper, information supporting the use of GM1 as a disease modifying therapeutic for PD is reviewed along with information concerning the role that deficiencies in GM1 ganglioside (and potentially other important brain gangliosides) may play in the pathogenesis of PD., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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.

Background: Gangliosides are glycosphingolipids highly enriched in the brain, with important roles in cell signaling, cell-to-cell communication, and immunomodulation. Genetic defects in the ganglioside biosynthetic pathway result in severe neurodegenerative diseases, while a partial decrease in the levels of specific gangliosides was reported in Parkinson's disease and Huntington's disease. In models of both diseases and other conditions, administration of GM1-one of the most abundant gangliosides in the brain-provides neuroprotection. Most studies have focused on the direct neuroprotective effects of gangliosides on neurons, but their role in other brain cells, in particular microglia, is not known. In this study we investigated the effects of exogenous ganglioside administration and modulation of endogenous ganglioside levels on the response of microglia to inflammatory stimuli, which often contributes to initiation or exacerbation of neurodegeneration., Methods: In vitro studies were performed using BV2 cells, mouse, rat, and human primary microglia cultures. Modulation of microglial ganglioside levels was achieved by administration of exogenous gangliosides, or by treatment with GENZ-123346 and L-t-PDMP, an inhibitor and an activator of glycolipid biosynthesis, respectively. Response of microglia to inflammatory stimuli (LPS, IL-1β, phagocytosis of latex beads) was measured by analysis of gene expression and/or secretion of pro-inflammatory cytokines. The effects of GM1 administration on microglia activation were also assessed in vivo in C57Bl/6 mice, following intraperitoneal injection of LPS., Results: GM1 decreased inflammatory microglia responses in vitro and in vivo, even when administered after microglia activation. These anti-inflammatory effects depended on the presence of the sialic acid residue in the GM1 glycan headgroup and the presence of a lipid tail. Other gangliosides shared similar anti-inflammatory effects in in vitro models, including GD3, GD1a, GD1b, and GT1b. Conversely, GM3 and GQ1b displayed pro-inflammatory activity. The anti-inflammatory effects of GM1 and other gangliosides were partially reproduced by increasing endogenous ganglioside levels with L-t-PDMP, whereas inhibition of glycolipid biosynthesis exacerbated microglial activation in response to LPS stimulation., Conclusions: Our data suggest that gangliosides are important modulators of microglia inflammatory responses and reveal that administration of GM1 and other complex gangliosides exerts anti-inflammatory effects on microglia that could be exploited therapeutically., (© 2022. The Author(s).)

G-protein-coupled receptors (GPCRs) are commonly pharmacologically modulated due to their ability to translate extracellular events to intracellular changes. Previously, studies have mostly focused on protein-protein interactions, but the focus has now expanded also to protein-lipid connections. GM1, a brain-expressed ganglioside known for neuroprotective effects, and GPR37, an orphan GPCR often reported as a potential drug target for diseases in the central nervous system, have been shown to form a complex. In this study, we looked into the functional effects. Endogenous GM1 was downregulated when stably overexpressing GPR37 in N2a cells (N2a GPR37-eGFP ). However, exogenous GM1 specifically rescued N2a GPR37-eGFP from toxicity induced by the neurotoxin MPP+. The treatment did not alter transcription levels of GPR37 or the enzyme responsible for GM1 production, both potential mechanisms for the effect. However, GM1 treatment inhibited cAMP-dependent signaling from GPR37, here reported as potentially consecutively active, possibly contributing to the protective effects. We propose an interplay between GPR37 and GM1 as one of the many cytoprotective effects reported for GM1.

Ketamine is a widely used analgesic and anesthetic in obstetrics and pediatrics. Ketamine is known to promote neuronal death and cognitive dysfunction in the brains of humans and animals during development. Monosialotetrahexosyl ganglioside (GM1), a promoter of brain development, exerts neuroprotective effects in many neurological disease models. Here, we investigated the neuroprotective effect of GM1 and its potential underlying mechanism against ketamine-induced apoptosis of rats. Seven-day-old Sprague Dawley (SD) rats were randomly divided into the following four groups: (1) group C (control group: normal saline was injected intraperitoneally); (2) group K (ketamine); (3) group GM1 (GM1 was given before normal saline injection); and (4) GM1+K group (received GM1 30 min before continuous exposure to ketamine). Each group contained 15 rats, received six doses of ketamine (20 mg/kg), and was injected with saline every 90 min. The Morris water maze (MWM) test, the number of cortical and hippocampal cells, apoptosis, and AKT/GSK3β pathway were analyzed. To determine whether GM1 exerted its effect via the PI3K/AKT/GSK3β pathway, PC12 cells were incubated with LY294002, a PI3K inhibitor. We found that GM1 protected against ketamine-induced apoptosis in the hippocampus and cortex by reducing the expression of Bcl-2 and Caspase-3, and by increasing the expression of Bax. GM1 treatment increased the expression of p-AKT and p-GSK3β. However, the anti-apoptotic effect of GM1 was eliminated after inhibiting the phosphorylation of AKT. We showed that GM1 lessens ketamine-induced apoptosis in the hippocampus and cortex of young rats by regulating the PI3K/AKT/GSK3β pathway. Taken together, GM1 may be a potential preventive treatment for the neurotoxicity caused by continuous exposure to ketamine.

Research on intravenous emulsions has been ongoing for several decades, and their unique advantages bring many opportunities for insoluble drugs. However, emulsions cannot withstand freezing in practical applications because their quality is severely affected. In this study, we used coenzyme Q10 as a model drug to prepare emulsions. Monosialotetrahexosylganglioside (GM1) was used to modify the emulsion to solve the freeze-thaw intolerance problem. The particle size, sterilization and freeze-thaw stability were affected by the oil content, phospholipid content, drug loading and homogenization conditions, which showed significant effects on the preparation properties. Emulsions prepared with a high oil content (30%, W/V) withstood three freeze-thaw cycles when the GM1 content was 0.2%-1.0% (W/V). In addition, pharmacokinetic studies indicated that emulsions modified with high-density GM1 had a long circulation time. Compared with the coenzyme Q10 solution, the emulsions showed different degrees of heart, liver, spleen and brain targeting. The relative uptake rate of the 0.2% GM1-modified emulsion in the heart was 37.06, while that of the 1.0% GM1-modified emulsion in the brain was 17.43. These results strongly suggest that coenzyme Q10 emulsions coated with GM1 can tolerate freeze-thaw cycles and are excellent for treatment of cardiac and neurodegenerative diseases., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Gangliosides are biologically important glycolipids widely distributed in vertebrate cells. An important member of the ganglioside family is the monosialylganglioside GM1, which has been suggested as a potential therapeutic for Parkinson's disease. In the current study, a late-stage radiofluorination protocol was developed, in which fluorine-18 was introduced by substitution of a terminal tosyl group in the fatty acid backbone of GM1. The radiofluorination procedure was remarkably simple and furnished the radiofluorinated ganglioside, [ 18 F]F-GM1, in sufficient quantity and quality without protection of the glycosyl moiety. A positron emission tomography measurement in cynomolgus monkey revealed high uptake of [ 18 F]F-GM1 in heart, bone marrow, and lungs but low (<0.4% of injected dose) distribution to the brain. Thus, choosing administration route of GM1 for therapy of central nervous system disorders poses further challenges. The present study demonstrates the importance of application of positron emission tomography microdosing studies in guiding early clinical drug development.

Background: Monosialotetrahexosylganglioside (GM1) is a neuroprotective glycosphingolipid that repairs nerves. Oxaliplatin-based chemotherapy is neurotoxic. This study assessed the efficacy of GM1 for preventing oxaliplatin-induced peripheral neurotoxicity (OIPN) in colorectal cancer (CRC) patients receiving oxaliplatin-based chemotherapy., Methods: In total, 196 patients with stage II/III CRC undergoing adjuvant chemotherapy with mFOLFOX6 were randomly assigned to intravenous GM1 or a placebo. The primary endpoint was the rate of grade 2 or worse cumulative neurotoxicity (NCI-CTCAE). The secondary endpoints were chronic cumulative neurotoxicity (EORTC QLQ-CIPN20), time to grade 2 neurotoxicity (NCI-CTCAE or the oxaliplatin-specific neuropathy scale), acute neurotoxicity (analog scale), rates of dose reduction or withdrawal due to OIPN, 3-year disease-free survival (DFS) and adverse events., Results: There were no significant differences between the arms in the rate of NCI-CTCAE grade 2 or worse neurotoxicity (GM1: 33.7% vs placebo: 31.6%; P = .76) or neuropathy measured by the EORTC QLQ-CIPN20 or time to grade 2 neurotoxicity using NCI-CTCAE and the oxaliplatin-specific neuropathy scale. GM1 substantially decreased participant-reported acute neurotoxicity (sensitivity to cold items [P < .01], discomfort swallowing cold liquids [P < .01], throat discomfort [P < .01], muscle cramps [P < .01]). The rates of dose reduction or withdrawal were not significantly different between the arms (P = .08). The 3-year DFS rates were 85% and 83% in the GM1 and placebo arms, respectively (P = .19). There were no differences in toxicity between the arms., Conclusion: Patients receiving GM1 were less troubled by the symptoms of acute neuropathy. However, we do not support the use of GM1 to prevent cumulative neurotoxicity. (ClinicalTrials.gov number, NCT02251977)., (© 2019 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Alpha-Synuclein (AS) is the protein playing the major role in Parkinson's disease (PD), a neurological disorder characterized by the degeneration of dopaminergic neurons and the accumulation of AS into amyloid plaques. The aggregation of AS into intermediate aggregates, called oligomers, and their pathological relation with biological membranes are considered key steps in the development and progression of the disease. Here we propose a multi-technique approach to study the effects of AS in its monomeric and oligomeric forms on artificial lipid membranes containing GM1 ganglioside. GM1 is a component of functional membrane micro-domains, called lipid rafts, and has been demonstrated to bind AS in neurons. With the aim to understand the relation between gangliosides and AS, here we exploit the complementarity of microscopy (Atomic Force Microscopy) and neutron scattering (Small Angle Neutron Scattering and Neutron Reflectometry) techniques to analyze the structural changes of two different membranes (Phosphatidylcholine and Phosphatidylcholine/GM1) upon binding with AS. We observe the monomer- and oligomer-interactions are both limited to the external membrane leaflet and that the presence of ganglioside leads to a stronger interaction of the membranes and AS in its monomeric and oligomeric forms with a stronger aggressiveness in the latter. These results support the hypothesis of the critical role of lipid rafts not only in the biofunctioning of the protein, but even in the development and the progression of the Parkinson's disease., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Monosialotetrahexosylganglioside (GM1) has good activity on brain diseases and was developed to be a drug applied in clinics for neurological disorders and nerve injury. It is difficult to isolate GM1 in industry scale from the brains directly. In this work, a simple and highly efficient method with high yield was developed for the isolation, conversion, and purification of GM1 from a pig brain. Gangliosides (GLS) were first extracted by supercritical CO 2 (SCE). The optimum extraction time of GLS by SCE was 4 h, and the ratio of entrainer to acetone powder from the pig brain was 3:1 ( v/w ). GM1 was then prepared from GLS by immobilized sialidase and purified by reverse-phase silica gel. Sodium alginate embedding was used for the immobilization of sialidase. Under the optimized method, the yield of high-purity GM1 was around 0.056%. This method has the potential to be applied in the production of GM1 in the industry.

Biomembranes are built up from lipid bilayers with two leaflets that typically differ in their lipid composition. Each lipid molecule stays within one leaflet of the bilayer before it undergoes a transition, or flip-flop, to the other leaflet. The corresponding flip-flop times are very different for different lipid species and vary over several orders of magnitude. Here, we use molecular dynamics simulations to elucidate the consequences of this separation of time scales for compositionally asymmetric bilayers. We first study bilayers with two lipid components that do not undergo flip-flops on the accessible time scales. In such a situation, one must distinguish a bilayer state in which both leaflets have the same preferred area from another state in which each leaflet is tensionless. However, when we add a third lipid component that undergoes frequent flip-flops, the bilayer relaxes toward the state with tensionless leaflets, not to the state with equal preferred leaflet areas. Furthermore, we show that bilayers with compositional asymmetry acquire a significant spontaneous curvature even if both leaflets are tensionless. Our results can be extended to lipid bilayers with a large number of lipid components provided at least one of these components undergoes frequent flip-flops. For cellular membranes containing lipid pumps, the leaflet tensions also depend on the rates of protein-induced flip-flops.

Developmental lead (Pb) exposure involves various serious consequences, especially leading to neurotoxicity. In this study, we examined the possible role of monosialoganglioside (GM1) in lead-induced nervous impairment in the developing rat. Newborn male Sprague-Dawley rat pups were exposed to lead from birth for 30 days and then subjected to GM1 administration (0.4, 2, or 10 mg/kg; i.p.) or 0.9% saline. The results showed that developmental lead exposure significantly impaired spatial learning and memory in the Morris water maze test, reduced GM1 content, induced oxidative stress, and weakened the antioxidative systems in the hippocampus. However, co-treatment with GM1 reversed these effects. Moreover, GM1 counteracted lead-induced apoptosis by decreasing the expression of Bax, cleaved caspase-3, and by increasing the level of Bcl-2 in a dose-dependent manner. Furthermore, we found that GM1 upregulated the expression of SIRT1, CREB phosphorylation, and BDNF, which underlie learning and memory in the lead-treated developing rat hippocampus. In conclusion, our study demonstrated that GM1 exerts a protective effect on lead-induced cognitive deficits via antioxidant activity, preventing apoptosis, and activating SIRT1/CREB/BDNF in the developing rat hippocampus, implying a novel potential assistant therapy for lead poisoning.

Recently, we highlighted that the ganglioside GM1 promotes neuroblastoma cells differentiation by activating the TrkA receptor through the formation of a TrkA-GM1 oligosaccharide complex at the cell surface. To study the TrkA-GM1 interaction, we synthesized two radioactive GM1 derivatives presenting a photoactivable nitrophenylazide group at the end of lipid moiety, 1 or at position 6 of external galactose, 2; and a radioactive oligosaccharide portion of GM1 carrying the nitrophenylazide group at position 1 of glucose, 3. The three compounds were singly administered to cultured neuroblastoma Neuro2a cells under established conditions that allow cell surface interactions. After UV activation of photoactivable compounds, the proteins were analyzed by PAGE separation. The formation of cross-linked TrkA-GM1 derivatives complexes was identified by both radioimaging and immunoblotting. Results indicated that the administration of compounds 2 and 3, carrying the photoactivable group on the oligosaccharide, led to the formation of a radioactive TrkA complex, while the administration of compound 1 did not. This underlines that the TrkA-GM1 interaction directly involves the GM1 oligosaccharide, but not the ceramide. To better understand how GM1 relates to the TrkA, we isolated plasma membrane lipid rafts. As expected, GM1 was found in the rigid detergent-resistant fractions, while TrkA was found as a detergent soluble fraction component. These results suggest that TrkA and GM1 belong to separate membrane domains: probably TrkA interacts by 'flopping' down its extracellular portion onto the membrane, approaching its interplay site to the oligosaccharide portion of GM1., (© 2019 International Society for Neurochemistry.)

Spectrin, a major component of the membrane skeletal meshwork of metazoan cells, is implicated to associate with membrane domains and is known to act as a scaffold for stabilization and activation of different signalling modules. We have studied the effect of GM1 (monosialotetrahexosyl ganglioside), a well-known model ganglioside and a signalling moiety, on the interaction of non-erythroid brain spectrin with both saturated and unsaturated aminophospholipids by spectroscopic methods. We observe that GM1 modulates brain spectrin-aminophospholipid interaction to the greatest degree whereas its effect on erythroid spectrin is not as pronounced. Fluorescence quenching studies show that brain spectrin interacts with DMPC/DMPE-based vesicles with a 10-fold increased affinity in presence of very low amounts of 2% and 5% GM1, and the extent of quenching decreases progressively in presence of increasing amounts of GM1. Interaction of brain spectrin with unsaturated membrane systems of DOPC/DOPE weakens in presence GM1. Increase in the mean lifetime of the Trp residues of brain spectrin in presence of GM1 indicates change in the microenvironment of spectrin, without affecting the secondary structure of the protein significantly. Studies on pressure - area isotherm of Langmuir-Blodgett monolayer and Brewster's angle microscopy show that GM1 has an expanding effect on the aminophospholipid monolayers, and ordered regions in DMPC/DMPE mixed monolayers are formed and are stabilized at higher pressure. GM1-induced fluidization of the phospholipid membranes and probable physical contact between bulky sugar head group of GM1 and spectrin, may explain the modulatory role of GM1 on aminophospholipid interactions with nonerythroid brain spectrin., (Copyright © 2018 Elsevier B.V. All rights reserved.)

High altitude cerebral edema (HACE) is a severe type of acute mountain sickness (AMS) that occurs in response to a high altitude hypobaric hypoxic (HH) environment. GM1 monosialoganglioside can alleviate brain injury under adverse conditions including amyloid-β-peptide, ischemia and trauma. However, its role in HACE-induced brain damage remains poorly elucidated. In this study, GM1 supplementation dose-dependently attenuated increase in rat brain water content (BWC) induced by hypobaric chamber (7600 m) exposurefor 24 h. Compared with the HH-treated group, rats injected with GM1 exhibited less brain vascular leakage, lower aquaporin-4 and higher occludin expression, but they also showed increase in Na+/K+-ATPase pump activities. Importantly, HH-incurred consciousness impairment and coordination loss also were ameliorated following GM1 administration. Furthermore, the increased oxidative stress and decrease in anti-oxidant stress system under the HH condition were also reversely abrogated by GM1 treatment via suppressing accumulation of ROS, MDA and elevating the levels of SOD and GSH. Simultaneously, GM1 administration also counteracted the enhanced inflammation in HH-exposed rats by muting pro-inflammatory cytokines IL-1β, TNF-α, and IL-6 levels in serum and brain tissues. Subsequently, GM1 potentiated the activation of the PI3K/AKT-Nrf2 pathway. Cessation of this pathway by LY294002 reversed GM1-mediated inhibitory effects on oxidative stress and inflammation, and ultimately abrogated the protective role of GM1 in abating brain edema, cognitive and motor dysfunction. Overall, GM1 may afford a protective intervention in HACE by suppressing oxidative stress and inflammatory response via activating the PI3K/AKT-Nrf2 pathway, implying a promising agent for the treatment of HACE., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

We compared the lateral structure of giant unilamellar vesicles (GUVs) composed of three pseudo binary mixtures of different glycosphingolipid (GSL), i.e. sulfatide, asialo-GM1 or GM1, with POPC. These sphingolipids possess similar hydrophobic residues but differ in the size and charge of their polar head group. Fluorescence microscopy experiments using LAURDAN and DiIC 18 show coexistence of micron sized domains in a molar fraction range that depends on the nature of the GSLs. In all cases, experiments with LAURDAN show that the membrane lateral structure resembles the coexistence of solid ordered and liquid disordered phases. Notably, the overall extent of hydration measured by LAURDAN between the solid ordered and liquid disordered membrane regions show marked similarities and are independent of the size of the GSL polar head group. In addition, the maximum amount of GSL incorporated in the POPC bilayer exhibits a strong dependence on the size of the GSL polar head group following the order sulfatide>asialo-GM1>GM1. This observation is in full harmony with previous experiments and theoretical predictions for mixtures of these GSL with glycerophospholipids. Finally, compared with previous results reported in GUVs composed of mixtures of POPC with the sphingolipids cerebroside and ceramide, we observed distinctive curvature effects at particular molar fraction regimes in the different mixtures. This suggests a pronounced effect of these GSL on the spontaneous curvature of the bilayer. This observation may be relevant in a biological context, particularly in connection with the highly curved structures found in neural cells., (Copyright © 2017 Elsevier B.V. All rights reserved.)

For the treatment of glioma and other central nervous system diseases, one of the biggest challenges is that most therapeutic drugs cannot be delivered to the brain tumor tissue due to the blood-brain barrier (BBB). The goal of this study was to construct a nanodelivery vehicle system with capabilities to overcome the BBB for central nervous system administration. Doxorubicin as a model drug encapsulated in ganglioside GM1 micelles was able to achieve up to 9.33% loading efficiency and 97.05% encapsulation efficiency by orthogonal experimental design. The in vitro study demonstrated a slow and sustainable drug release in physiological conditions. In the cellular uptake studies, mixed micelles could effectively transport into both human umbilical vein endothelial cells and C6 cells. Furthermore, biodistribution imaging of mice showed that the DiR/GM1 mixed micelles were accumulated sustainably and distributed centrally in the brain. Experiments on zebrafish confirmed that drug-loaded GM1 micelles can overcome the BBB and enter the brain. Among all the treatment groups, the median survival time of C6-bearing rats after administering DOX/GM1 micelles was significantly prolonged. In conclusion, the ganglioside nanomicelles developed in this work can not only penetrate BBB effectively but also repair nerves and kill tumor cells at the same time., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Nerve growth factor (NGF) is widely used for repairing peripheral nerve injury because of its capability in dominating the survival, migration, proliferation, and differentiation of nerve cells. Monosialoganglioside (GM1), as another kind of nerve growth factor, works for regulating NGF function. In this study, GM1 and NGF were incorporated into the Poly(l-lactic acid-co-ε-caprolactone)/silk fibroin (PLCL/SF) nanofibers by the coaxial electrospinning. The fibers morphology and core–shell structure were characterized by SEM and TEM. The scaffolds demonstrated high tensile stress with good flexibility. In vitro cell viability studies indicated that the scaffolds incorporating both GM1 and NGF played a synergistic effect to enhance Schwann cells (SCs) proliferation and Pheochromocytoma (PC12) cells differentiation, in comparison to the scaffolds only incorporating NGF. Subsequently, the nanofibrous conduit scaffolds (NCSs) were evaluated in vivo in a rabbit sciatic nerve defect model. The NGF/GM1 incorporated NCSs group performed better nerve function recovery than single incorporated group, in consideration of the compound muscle action potential (CMAP) and nerve conduction velocity (NCV) results. Furthermore, hematoxylin and eosin (H&E) staining, toluidine blue (TB) staining, and transmission electron microscope (TEM) analysis displayed better nerve regeneration of NGF/GM1 incorporated NCSs both quantitatively and qualitatively. Therefore, the results indicated the dual neurotrophins-incorporated NCSs had potentials for the application in peripheral nerve repairing.

Glycosphingolipids are minor yet essential components of eukaryotic cell membranes and are involved in a variety of cellular processes. Although glycosphingolipids such as GM1 have been previously reported to influence the function of G protein-coupled receptors (GPCRs), the molecular mechanism remains elusive. In this paper, we have explored the interaction of GM1 with the serotonin 1A receptor, an important neurotransmitter receptor that belongs to the GPCR family. To examine the molecular basis of the interaction of GM1 with the serotonin 1A receptor, we performed a series of coarse-grain molecular dynamics simulations of the receptor embedded in membrane bilayers containing GM1. Our results show that GM1 interacts with the serotonin 1A receptor predominantly at the extracellular loop 1 and specifically at the sphingolipid binding domain (SBD). The SBD motif consists of a characteristic combination of aromatic, basic and turn-inducing residues, and is evolutionarily conserved in case of the serotonin 1A receptor. The interaction of the SBD site with GM1 appears to stabilize a 'flip-out' conformation in which W102 of the extracellular loop 1 flips out from the central lumen of the receptor toward the membrane. The population of the 'flip-out' conformation is increased in the presence of cholesterol. Our data strongly suggest that a direct interaction between GM1 and the SBD site of the serotonin 1A receptor may occur in vivo. In view of the reported role of GM1 and the serotonin 1A receptor in neurodegenerative diseases, GM1-receptor interaction assumes significance in the context of malfunctioning of neuronal GPCRs under such conditions., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The oolemma of the second metaphase mouse oocyte has a relatively large domain in the peri-polar body region that is non-permissive for sperm attachment. In this study, certain biochemical components of this non-permissive domain were examined and compared with permissive regions to investigate the molecular basis of an oolemma that becomes functionally polarized during fertilization; an attempt was also made to determine whether similarities exist in the formation of abnormally occurring non-permissive domains in human oocytes. In the present study, microdomains composed of lipid rafts enriched in the ganglioside GM1 provided a platform for sperm docking at the initial stage of fertilization and their disruption led to sperm attachment defects resembling those associated with fertilization failure in human IVF. The regulation of GM1 microdomain density is suggested to involve the oolemmal protein annexin 2 and the magnitude of the inner mitochondrial membrane potential (ΔΨm) in the adjacent subplasmalemmal cytoplasm. These studies also showed that a rapid focal reduction in GM1, f-actin, annexin 2 and cholesterol occurs at the site of sperm docking and attachment whereas the distribution of fertilization-associated proteins CD9 and FOLR4 remains unchanged, suggesting that they may not participate in the earliest phases of fertilization., (Copyright © 2016. Published by Elsevier Ltd.)

Gangliosides (sialic acid-containing glycosphingolipids) are abundant in neurons of all animal species and play important roles in many cell physiological processes, including differentiation, memory control, cell signaling, neuronal protection, neuronal recovery, and apoptosis. Gangliosides also function as anchors or entry points for various toxins, bacteria, viruses, and autoantibodies. GM1, a ganglioside component of mammalian brains, is present mainly in neurons. GM1 is one of the best studied gangliosides, and our understanding of its properties is extensive. Simple and rapid procedures are available for preparation of GM1 as a natural compound on a large scale, or as a derivative containing an isotopic radionuclide or a specific probe. Great research interest in the properties of GM1 arose from the discovery in the early 1970s of its role as receptor for the bacterial toxin responsible for cholera pathogenesis.

Objective: To observe the effects of GM1 on apomorphine (APO)-induced rotational behavior and the expression of inflammatory factors in 6-hydroxydopamine-induced Parkinson's disease (PD) rat models., Methods: Mature and healthy Wistar rats of either sex with body weight of 150-200 g were randomly divided into control group, PD+APO group and PD+APO+GM1 group (10 mg/kg of GM1, intraperitoneally, once a day, for 14 days; each group with 15 rats). PD rat models were prepared by injecting 6-hydroxydopamine into rat's right striatum, and then rotational behavior was induced by intraperitoneal injection of APO 7 days after operation. Rat rotational behavior was observed, and mRNA and protein levels of interleukin-1β (IL-1β) and interleukin-1Ra (IL-1Ra) were determined, respectively, by RT-PCR and Western blot., Results: Compared with PD+APO group, the rotational behavior was significantly relieved in PD+APO+GM1 group (p < 0.05). Compared with control group, mRNA and protein expressions of IL-1β in the striatum significantly increased in PD+APO group (p < 0.05). However, mRNA and protein expressions of IL-1β significantly decreased in PD+APO+GM1 group compared with PD+APO group (p < 0.05), but mRNA and protein expressions of IL-1β were also higher in PD+APO+GM1 group than in control group (p < 0.05). mRNA and protein expressions of IL-1Ra in the striatum were significantly higher in PD+APO+GM1 group than in PD+APO group (p < 0.05)., Conclusion: GM1 can inhibit inflammatory reaction through decreasing mRNA and protein expressions of IL-1β and increasing mRNA and protein expressions of IL-1Ra with the therapeutic effects on PD.

Studies on the structure and dynamics of interfacial water, emphasizing on the properties of water near the surface of biomolecules, are well reported, but there is a lack of evidence on the behavior of water near a comparatively rough surface containing molecules with a bulky head group like GM1. In this report we comparatively analyze the structure and dynamics of water as a function of distance from the lipid head group in GM1 containing lipid bilayers, with the lipid bilayers where GM1 is not present. This approach effectively demonstrates the behavioral difference and hence delayed convergence from bound water to bulk water in the presence of GM1 compared to a relatively smooth surface., (Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Alzheimer's disease (AD) is a progressive dementia associated with loss of memory and cognitive dysfunction. In a previous study, we demonstrated a decrease in b-series gangliosides along with a change in ganglioside molecular species in the hippocampal grey matter of patients with AD. The present study demonstrates the use of imaging mass spectrometry for analyzing the spatial arrangement of ganglioside GM1 (GM1) molecular species in the hippocampus. In AD patients, we found a decrease in the ratio of GM1(d20:1/C18:0) to GM1 d18:1/C18:0) in the outer molecular layer (ML) of the dentate gyrus. Because the outer ML is the region of main input into the hippocampus, our findings may have a direct relationship to the mechanism of dysfunction in AD., (Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Vascular endothelial cells (ECs) play central roles in physiologically important functions of blood vessels and contribute to the maintenance of vascular integrity. Therefore, it is considered that the impairment of EC functions leads to the development of vascular diseases. However, the molecular mechanisms of the EC dysfunctions that accompany senescence and aging have not yet been clarified. The carbohydrate antigens carried by glycoconjugates (e.g. glycoproteins, glycosphingolipids, and proteoglycans) mainly present on the cell surface serve not only as marker molecules but also as functional molecules. In this study, we have investigated the abundance and functional roles of glycosphingolipids in human ECs during senescence and aging. Among glycosphingolipids, ganglioside GM1 was highly expressed in abundance on the surface of replicatively and prematurely senescent ECs and also of ECs derived from an elderly subject. Insulin signaling, which regulates important functions of ECs, is impaired in senescent and aged ECs. Actually, by down-regulating GM1 on senescent ECs and overloading exogenous GM1 onto non-senescent ECs, we showed that an increased abundance of GM1 functionally contributes to the impairment of insulin signaling in ECs. Taken together, these findings provide the first evidence that GM1 increases in abundance on the cell surface of ECs under the conditions of cellular senescence and aging and causes insulin resistance in ECs. GM1 may be an attractive target for the detection, prevention, and therapy of insulin resistance and related vascular diseases, particularly in older people., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Gangliosides are compounds that are abundant throughout the CNS, participating actively in neuroplasticity. We previously described that exogenous GM1 ganglioside pretreatment enhances the rewarding properties of cocaine, evidenced by a lower number of sessions and/or dosage necessary to induce conditioned place preference (CPP). Since GM1 pretreatment did not modify cocaine's pharmacokinetic parameters, we suspected that the increased rewarding effect found might be mediated by BDNF, a neurotrophic factor closely related to cocaine addiction. This study was performed to investigate the possibility that GM1 may induce changes in BDNF levels in the nucleus accumbens (NAc), a core structure in the brain's reward circuitry, of rats submitted to three conditioning sessions with cocaine (10 mg/kg, i.p.). The results demonstrate that GM1 administration, which showed no rewarding effect by itself in the CPP, induced a significant increase of BDNF protein levels in the NAc, which may account for the increased rewarding effect of cocaine shown in the CPP paradigm., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Gangliosides and the urokinase plasminogen activator receptor (uPAR) tipically partition in specialized membrane microdomains called lipid-rafts. uPAR becomes functionally important in fostering angiogenesis in endothelial progenitor cells (EPCs) upon recruitment in caveolar-lipid rafts. Moreover, cell membrane enrichment with exogenous GM1 ganglioside is pro-angiogenic and opposite to the activity of GM3 ganglioside. On these basis, we first checked the interaction of uPAR with membrane models enriched with GM1 or GM3, relying on the adoption of solid-supported mobile bilayer lipid membranes with raft-like composition formed onto solid hydrophilic surfaces, and evaluated by surface plasmon resonance (SPR) the extent of uPAR recruitment. We estimated the apparent dissociation constants of uPAR-GM1/GM3 complexes. These preliminary observations, indicating that uPAR binds preferentially to GM1-enriched biomimetic membranes, were validated by identifying a pro-angiogenic activity of GM1-enriched EPCs, based on GM1-dependent uPAR recruitment in caveolar rafts. We have observed that addition of GM1 to EPCs culture medium promotes matrigel invasion and capillary morphogenesis, as opposed to the anti-angiogenesis activity of GM3. Moreover, GM1 also stimulates MAPKinases signalling pathways, typically associated with an angiogenesis program. Caveolar-raft isolation and Western blotting of uPAR showed that GM1 promotes caveolar-raft partitioning of uPAR, as opposed to control and GM3-challenged EPCs. By confocal microscopy, we have shown that in EPCs uPAR is present on the surface in at least three compartments, respectively, associated to GM1, GM3 and caveolar rafts. Following GM1 exogenous addition, the GM3 compartment is depleted of uPAR which is recruited within caveolar rafts thereby triggering angiogenesis., (© 2014 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

It has been proposed that GM1 ganglioside promotes neuronal growth, phenotypic expression, and survival by modulating tyrosine kinase receptors for neurotrophic factors. Our studies tested the hypothesis that GM1 exerts its neurotrophic action on dopaminergic neurons, in part, by interacting with the GDNF (glia cell-derived neurotrophic factor) receptor complex, Ret tyrosine kinase and GFRα1 co-receptor. GM1 addition to striatal slices in situ increased Ret activity in a concentration- and time-dependent manner. GM1-induced Ret activation required the whole GM1 molecule and was inhibited by the kinase inhibitors PP2 and PP1. Ret activation was followed by Tyr1062 phosphorylation and PI3 kinase/Akt recruitment. The Src kinase was associated with Ret and GM1 enhanced its phosphorylation. GM1 responses required the presence of GFRα1, and there was a GM1 concentration-dependent increase in the binding of endogenous GDNF which paralleled that of Ret activation. Neutralization of the released GDNF did not influence the Ret response to GM1, and GM1 had no effect on GDNF release. Our in situ studies suggest that GM1 via GFRα1 modulates Ret activation and phosphorylation in the striatum and provide a putative mechanism for its effects on dopaminergic neurons. Indeed, chronic GM1 treatment enhanced Ret activity and phosphorylation in the striatum of the MPTP-mouse and kinase activation was associated with recovery of dopamine and DOPAC deficits. It has been proposed that the ganglioside GM1 promotes neuronal growth, phenotypic expression, and survival by modulating tyrosine kinase receptors for neurotrophic factors. We provide evidence that the GM1 enhances the activity of Ret tyrosine kinase receptor for glia cell-derived neurotrophic factor (GDNF) in the striatum in situ and in vivo, and propose that this might be a mechanism for GM1's neurotrophic actions on dopaminergic neurons. Ret activation is followed by Tyr1062 and Tyr981 phosphorylation and recruitment of PI3-K/Akt, Erk, and Src signaling. GM1 apparently acts by increasing the binding of endogenous GDNF to GFRα1 co-receptor, which is required for the GM1 effect on Ret., (© 2014 International Society for Neurochemistry.)

Lipid rafts are assumed to undergo biologically important size-modulations from nanorafts to microrafts. Due to the complexity of cellular membranes, model systems become important tools, especially for the investigation of the factors affecting "raft-like" Lo domain size and the search for Lo nanodomains as precursors in Lo microdomain formation. Because lipid compositional change is the primary mechanism by which a cell can alter membrane phase behavior, we studied the effect of the ganglioside GM1 concentration on the Lo/Ld lateral phase separation in PC/SM/Chol/GM1 bilayers. GM1 above 1mol % abolishes the formation of the micrometer-scale Lo domains observed in GUVs. However, the apparently homogeneous phase observed in optical microscopy corresponds in fact, within a certain temperature range, to a Lo/Ld lateral phase separation taking place below the optical resolution. This nanoscale phase separation is revealed by fluorescence spectroscopy, including C12NBD-PC self-quenching and Laurdan GP measurements, and is supported by Gaussian spectral decomposition analysis. The temperature of formation of nanoscale Lo phase domains over an Ld phase is determined, and is shifted to higher values when the GM1 content increases. A "morphological" phase diagram could be made, and it displays three regions corresponding respectively to Lo/Ld micrometric phase separation, Lo/Ld nanometric phase separation, and a homogeneous Ld phase. We therefore show that a lipid only-based mechanism is able to control the existence and the sizes of phase-separated membrane domains. GM1 could act on the line tension, "arresting" domain growth and thereby stabilizing Lo nanodomains., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The incretin hormone Glucagon-like peptide 1 (GLP-1) requires delivery by injection for the treatment of Type 2 diabetes mellitus. Here, we test if the properties of glycosphingolipid trafficking in epithelial cells can be applied to convert GLP-1 into a molecule suitable for mucosal absorption. GLP-1 was coupled to the extracellular oligosaccharide domain of GM1 species containing ceramides with different fatty acids and with minimal loss of incretin bioactivity. When applied to apical surfaces of polarized epithelial cells in monolayer culture, only GLP-1 coupled to GM1-ceramides with short- or cis-unsaturated fatty acids trafficked efficiently across the cell to the basolateral membrane by transcytosis. In vivo studies showed mucosal absorption after nasal administration. The results substantiate our recently reported dependence on ceramide structure for trafficking the GM1 across polarized epithelial cells and support the idea that specific glycosphingolipids can be harnessed as molecular vehicles for mucosal delivery of therapeutic peptides., (Copyright © 2013 Elsevier B.V. All rights reserved.)

One prominent immunoregulatory function of galectin-1 (Gal-1), a β-galactoside binding mammalian lectin, is induction of apoptosis in activated T-cells by a process depending on the activity of Src family tyrosine kinase, Lck. Although the requirement for Lck in Gal-1 induced T-cell death and the ability of Gal-1 to affect the membrane localization of extracellular Gal-1-binding proteins have been well documented, the consequence of the complex and related reorganization of extra- and intracellular signaling components upon Gal-1 treatment of T-cells has not yet been revealed. Therefore, we have analyzed the plasma membrane movement of Lck upon Gal-1 triggered signaling, and the significance of this event in Gal-1 induced T-cell death. Non-receptor tyrosine kinase, Lck primarily localized in the synapse of tumor cell-T-cell during 15 min of the established direct cell contact. Later, after 30 min, a lateral segregation of Lck from the cell synapse was observed. The migration of Lck to the opposite of the cell contact apparently depended on the expression and cell surface presentation of Gal-1 on the effector (tumor) cells and was accompanied by phosphorylation on the negative regulatory tyrosine residue, Tyr505. Receptor tyrosine phosphatase, CD45 played crucial role in this event since CD45 deficiency or inhibition of its phosphatase activity resulted in the failure of Lck membrane movement. Level of the Gal-1-binding glycolipid GM1 ganglioside also essentially regulated Lck localization. Segregation of Lck and Gal-1 induced apoptosis was diminished in T-cells with low GM1 expression compared to T-cells with high GM1. Our results show that spatial regulation of Lck by CD45 and GM1 ganglioside determines the outcome of apoptotic response to Gal-1 and this local regulation may occur only upon intimate effector (Gal-1 expressing) cell-T-cell attachment., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Melittin is a bee venom toxin that can act as antimicrobial peptide. Gangliosides are glycosphingolipids that help maintain membrane structure and organization as well as act as anchors for lectins, toxins, pathogens and antimicrobial peptides. Here we investigate interaction of melittin with fast tumbling isotropic control DMPC/CHAPS bicelles and ganglioside doped DMPC/CHAPS/GM1 bicelles. DOSY result shows that larger percentage of peptide binds to GM1 containing bicelles than that of the control PC bicelles. Bound peptide induces leakage of the bicelles entrapped carboxyfluorescein. Percentage of leakage is higher from control PC bicelles than that of the GM1 containing bicelles. In the presence of control PC bicelles melittin acquired fully α-helical structure. But in the presence of GM1 containing bicelles the peptide is not fully α-helical i.e., some random coil structure is present in this folded form. The present study shows that GM1 has an effect on membrane active antimicrobial peptide melittin., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Large-scale synthesis of GM1, an important ganglioside in mammalian cells especially those in the nervous system, is needed to explore its therapeutic potential. Biocatalytic production is a promising platform for such a purpose. We report herein the development of process engineering and glycosyltransferase improvement strategies to advance chemoenzymatic total synthesis of GM1. Firstly, a new short route was developed for chemical synthesis of lactosylsphingosine from the commercially available Garner's aldehyde. Secondly, two glycosyltransferases including Campylobacter jejuni β1-4GalNAcT (CjCgtA) and β1-3-galactosyltransferase (CjCgtB) were improved on their soluble expression in E. coli and enzyme stability by fusing with an N-terminal maltose binding protein (MBP). Thirdly, the process for enzymatic synthesis of GM1 sphingosines from lactosylsphingosine was engineered by developing a multistep one-pot multienzyme (MSOPME) strategy without isolating intermediate glycosphingosines and by adding a detergent, sodium cholate, to the later enzymatic glycosylation steps. Installation of a desired fatty acyl chain to GM1 glycosphingosines led to the formation of target GM1 gangliosides. The combination of glycosyltransferase improvement with chemical and enzymatic process engineering represents a significant advance in obtaining GM1 gangliosides containing different sialic acid forms by total chemoenzymatic synthesis in a short route and with high efficiency.