Effect of GM1 concentration change on plasma membrane: molecular dynamics simulation and analysis.

Ganglioside GM1 is a class of glycolipids predominantly located in the nervous system. Comprising a ceramide anchor and an oligosaccharide chain containing sialic acid, GM1 plays a pivotal role in various cellular processes, including signal transduction, cell adhesion, and membrane organization. Moreover, GM1 has been implicated in the pathogenesis of several neurological disorders, such as Parkinson's disease, Alzheimer's disease, and stroke. In this study, by creating a neural cell model membrane simulation system and employing rigorous molecular models, we utilize a coarse-grained molecular dynamics approach to explore the structural and dynamic characteristics of multi-component neuronal plasma membranes at varying GM1 ganglioside concentrations. The simulation results reveal that as GM1 concentration increases, a greater number of hydrogen bonds form between GM1 molecules, resulting in the formation of larger clusters, which leads to reduced membrane fluidity, increased lipid ordering, decreased membrane thickness and surface area and higher levels of GM1 dissociation. Through a meticulous analysis, while considering GM1's structural attributes, we offer valuable insights into the structural and dynamic traits of the cell membrane. This study provides a robust methodology for exploring membrane characteristics and enhances our comprehension of GM1 molecules, serving as a resource for both experimental and computational researchers in this field.

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.

Aß peptide enhances GluA1 internalization via lipid rafts in Alzheimer's-related hippocampal LTP dysfunction.

Amyloid ß (Aß) is a central contributor to neuronal damage and cognitive impairment in Alzheimer's disease (AD). Aß disrupts AMPA receptor-mediated synaptic plasticity, a key factor in early AD progression. Numerous studies propose that Aß oligomers hinder synaptic plasticity, particularly long-term potentiation (LTP), by disrupting GluA1 (encoded by GRIA1) function, although the precise mechanism remains unclear. In this study, we demonstrate that Aß mediates the accumulation of GM1 ganglioside in lipid raft domains of cultured cells, and GluA1 exhibits preferential localization in lipid rafts via direct binding to GM1. Aß enhances the raft localization of GluA1 by increasing GM1 in these areas. Additionally, chemical LTP stimulation induces lipid raft-dependent GluA1 internalization in Aß-treated neurons, resulting in reduced cell surface and postsynaptic expression of GluA1. Consistent with this, disrupting lipid rafts and GluA1 localization in rafts rescues Aß-mediated suppression of hippocampal LTP. These findings unveil a novel functional deficit in GluA1 trafficking induced by Aß, providing new insights into the mechanism underlying AD-associated cognitive dysfunction.

The Mutagenic Plasticity of the Cholera Toxin B-Subunit Surface Residues: Stability and Affinity.

Mastering selective molecule trafficking across human cell membranes poses a formidable challenge in healthcare biotechnology while offering the prospect of breakthroughs in drug delivery, gene therapy, and diagnostic imaging. The cholera toxin B-subunit (CTB) has the potential to be a useful cargo transporter for these applications. CTB is a robust protein that is amenable to reengineering for diverse applications; however, protein redesign has mostly focused on modifications of the N- and C-termini of the protein. Exploiting the full power of rational redesign requires a detailed understanding of the contributions of the surface residues to protein stability and binding activity. Here, we employed Rosetta-based computational saturation scans on 58 surface residues of CTB, including the GM1 binding site, to analyze both ligand-bound and ligand-free structures to decipher mutational effects on protein stability and GM1 affinity. Complimentary experimental results from differential scanning fluorimetry and isothermal titration calorimetry provided melting temperatures and GM1 binding affinities for 40 alanine mutants among these positions. The results showed that CTB can accommodate diverse mutations while maintaining its stability and ligand binding affinity. These mutations could potentially allow modification of the oligosaccharide binding specificity to change its cellular targeting, alter the B-subunit intracellular routing, or impact its shelf-life and in vivo half-life through changes to protein stability. We anticipate that the mutational space maps presented here will serve as a cornerstone for future CTB redesigns, paving the way for the development of innovative biotechnological tools.

Alpha-Synuclein and GM1 Ganglioside Co-Localize in Neuronal Cytosol Leading to Inverse Interaction-Relevance to Parkinson's Disease.

Research on GM1 ganglioside and its neuroprotective role in Parkinson's disease (PD), particularly in mitigating the aggregation of α-Synuclein (aSyn), is well established across various model organisms. This essential molecule, GM1, is intimately linked to preventing aSyn aggregation, and its deficiency is believed to play a key role in the initiation of PD. In our current study, we attempted to shed light on the cytosolic interactions between GM1 and aSyn based on previous reports demonstrating gangliosides and monomeric aSyn to be present in neuronal cytosol. Native-PAGE and Western blot analysis of neuronal cytosol from mouse brains demonstrated the presence of both GM1 and monomeric aSyn in the neuronal cytosol of normal mouse brain. To demonstrate that an adequate level of GM1 prevents the aggregation of aSyn, we used NG108-15 and SH-SY5Y cells with and without treatment of 1-phenyl-2-palmitoyl-3-morpholino-1-propanol (PPMP), which inhibits the synthesis/expression of GM1. Cells treated with PPMP to reduce GM1 expression showed a significant increase in the formation of aggregated aSyn compared to untreated cells. We thus demonstrated that sufficient GM1 prevents the aggregation of aSyn. For this to occur, aSyn and GM1 must show proximity within the neuron. The present study provides evidence for such co-localization in neuronal cytosol, which also facilitates the inverse interaction revealed in studies with the two cell types above. This adds to the explanation of how GM1 prevents the aggregation of aSyn and onset of Parkinson's disease.

Ganglioside GM1 Drives Hemin and Protoporphyrin Adsorption in Phospholipid Membranes: A Structural Study.

Monosialoganglioside (GM1), a ubiquitous component of lipid rafts, and hemin, an integral part of heme proteins such as hemoglobin, are essential to the cell membranes of brain neurons and erythrocyte red blood cells for regulating cellular communication and oxygen transport. Protoporphyrin IX (PPIX) and its derivative hemin, on the contrary, show significant cytotoxic effects when in excess causing hematological diseases, such as thalassemia, anemia, malaria, and neurodegeneration. However, the in-depth molecular etiology of their interactions with the cell membrane has so far been poorly understood. Herein, the structure of the polymer cushion-supported lipid bilayer (SLB) of the binary mixture of phospholipid and GM1 in the presence of PPIX and its derivative hemin has been investigated to predict the molecular interactions in model phospholipid membranes. A high-resolution synchrotron-based X-ray scattering technique has been employed to explore the out-of-plane structure of the assembly at different compositions and concentrations. The structural changes have been complemented with the isobaric changes in the mean molecular area obtained from the Langmuir monolayer isotherm to predict the additive-induced membrane condensation and fluidization. PPIX-induced fluidization of phospholipid SLB without GM1 was witnessed, which was reversed to condensation with 2-fold higher structural changes in the presence of GM1. A hemin concentration-dependent linear condensing effect was observed in the pristine SLB. The effect was significantly reduced, and the linearity was observed to be lost in the mixed SLB containing GM1. Our study shows that GM1 alters the interaction of hemin and PPIX with the membrane, which could be explained with the aid of hydrophobic and electrostatic interactions. Our study indicates favorable and unfavorable interactions of GM1 with PPIX and hemin, respectively, in the membrane. The observed structural changes in both SLB and the underlying polymer cushion layer lead to the proposal of a molecule-specific interaction model that can benefit the pharmaceutical industries specialized for drug designing. Our study potentially enriches our fundamental biophysical understanding of neurodegenerative diseases and drug-membrane interactions.

Multiscale Modeling of Macromolecular Interactions between Tau-Amylin Oligomers and Asymmetric Lipid Nanodomains That Link Alzheimer's and Diabetic Diseases.

The molecular events of protein misfolding and self-aggregation of tau and amylin are associated with the progression of Alzheimer's and diabetes, respectively. Recent studies suggest that tau and amylin can form hetero-tau-amylin oligomers. Those hetero-oligomers are more neurotoxic than homo-tau oligomers. So far, the detailed interactions between the hetero-oligomers and the neuronal membrane are unknown. Using multiscale MD simulations, the lipid binding and protein folding behaviors of hetero-oligomers on asymmetric lipid nanodomains or raft membranes were examined. Our raft membranes contain phase-separated phosphatidylcholine (PC), cholesterol, and anionic phosphatidylserine (PS) or ganglioside (GM1) in one leaflet of the lipid bilayer. The hetero-oligomers bound more strongly to the PS and GM1 than other lipids via the hydrophobic and hydrophilic interactions, respectively, in the raft membranes. The hetero-tetramer disrupted the acyl chain orders of both PC and PS in the PS-containing raft membrane, but only the GM1 in the GM1-containing raft membrane as effectively as the homo-tau-tetramer. We discovered that the alpha-helical content in the heterodimer was greater than the sum of alpha-helical contents from isolated tau and amylin monomers on both raft membranes, indicative of a synergetic effect of tau-amylin interactions in surface-induced protein folding. Our results provide new molecular insights into understanding the cross-talk between Alzheimer's and diabetes.

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.

Mechanical activation of TWIK-related potassium channel by nanoscopic movement and rapid second messenger signaling.

Rapid conversion of force into a biological signal enables living cells to respond to mechanical forces in their environment. The force is believed to initially affect the plasma membrane and then alter the behavior of membrane proteins. Phospholipase D2 (PLD2) is a mechanosensitive enzyme that is regulated by a structured membrane-lipid site comprised of cholesterol and saturated ganglioside (GM1). Here we show stretch activation of TWIK-related K+ channel (TREK-1) is mechanically evoked by PLD2 and spatial patterning involving ordered GM1 and 4,5-bisphosphate (PIP2) clusters in mammalian cells. First, mechanical force deforms the ordered lipids, which disrupts the interaction of PLD2 with the GM1 lipids and allows a complex of TREK-1 and PLD2 to associate with PIP2 clusters. The association with PIP2 activates the enzyme, which produces the second messenger phosphatidic acid (PA) that gates the channel. Co-expression of catalytically inactive PLD2 inhibits TREK-1 stretch currents in a biological membrane. Cellular uptake of cholesterol inhibits TREK-1 currents in culture and depletion of cholesterol from astrocytes releases TREK-1 from GM1 lipids in mouse brain. Depletion of the PLD2 ortholog in flies results in hypersensitivity to mechanical force. We conclude PLD2 mechanosensitivity combines with TREK-1 ion permeability to elicit a mechanically evoked response.

"Ouch!": you have just stabbed your little toe on the sharp corner of a coffee table. That painful sensation stems from nerve cells converting information about external forces into electric signals the brain can interpret. Increasingly, new evidence is suggesting that this process may be starting at fat-based structures within the membrane of these cells. The cell membrane is formed of two interconnected, flexible sheets of lipids in which embedded structures or molecules are free to move. This organisation allows the membrane to physically respond to external forces and, in turn, to set in motion chains of molecular events that help fine-tune how cells relay such information to the brain. For instance, an enzyme known as PLD2 is bound to lipid rafts ­ precisely arranged, rigid fatty 'clumps' in the membrane that are partly formed of cholesterol. PLD2 has also been shown to physically interact with and then activate the ion channel TREK-1, a membrane-based protein that helps to prevent nerve cells from relaying pain signals. However, the exact mechanism underpinning these interactions is difficult to study due to the nature and size of the molecules involved. To address this question, Petersen et al. combined a technology called super-resolution imaging with a new approach that allowed them to observe how membrane lipids respond to pressure and fluid shear. The experiments showed that mechanical forces disrupt the careful arrangement of lipid rafts, causing PLD2 and TREK-1 to be released. They can then move through the surrounding membrane where they reach a switch that turns on TREK-1. Further work revealed that the levels of cholesterol available to mouse cells directly influenced how the clumps could form and bind to PLD2, and in turn, dialled up and down the protective signal mediated by TREK-1. Overall, the study by Petersen et al. shows that the membrane of nerve cells can contain cholesterol-based 'fat sensors' that help to detect external forces and participate in pain regulation. By dissecting these processes, it may be possible to better understand and treat conditions such as diabetes and lupus, which are associated with both pain sensitivity and elevated levels of cholesterol in tissues.

Mechanism study of cross presentation of exogenous antigen induced by cholera toxin-like chimeric protein.

Tumor subunit vaccines have great potential in personalized cancer immunotherapy. They are usually administered with adjuvant owing to their low immunogenicity. Cholera toxin (CT) is a biological adjuvant with diverse biological functions and a long history of use. Our earlier study revealed that a CT-like chimeric protein co-delivered with murine granulocyte-macrophage colony stimulating factor (mGM-CSF) and prostate cancer antigen epitope could co-stimulate dendritic cells (DCs) and enhance cross presentation of tumor epitope. To further study the molecular mechanism of CT-like chimeric protein in cross presentation, major histocompatibility complex class I (MHC I)-restricted epitope 257-264 of ovalbumin (OVAT) was used as a model antigen peptide in this study. Recombinant A subunit and pentameric B subunit of CT protein were respectively genetically constructed and purified. Then both assembled into AB5 chimeric protein in vitro. Three different chimeric biomacromolecules containing mGM-CSF and OVAT were constructed according to the different fusion sites and whether the endoplasmic reticulum (ER) retention sequence was included. It was found that A2 domain and B subunit of CT were both available for loading epitopes and retaining GM1 affinity. The binding activity of GM1 was positively correlated with antigen endocytosis. Once internalized, DCs became mature and cross-presented antigen. KDEL helped the whole molecule to be retained in the ER, and this improved the cross presentation of antigen on MHC I molecules. In conclusion, hexameric CT-like chimeric protein with dual effects of GM1 affinity and ER retention sequence were potential in improvement of cross presentation. The results laid a foundation for designing personalized tumor vaccine based on CT-like chimeric protein molecular structure.

Identification of GM1-Ganglioside Secondary Accumulation in Fibroblasts from Neuropathic Gaucher Patients and Effect of a Trivalent Trihydroxypiperidine Iminosugar Compound on Its Storage Reduction.

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 ß-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 ß-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.

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.

Molecular and cellular evidence of a direct interaction between the TRAF2 C-terminal domain and ganglioside GM1.

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.

Comprehensive evaluation of fruit quality for premium Wangmo Castanea mollissima plants.

In this study, the fruit phenotype and quality of 32 superior Wangmo Castanea mollissima plants (designated GM1 to GM32) were examined to identify the trait characteristics of different cluster groups and germplasms with excellent comprehensive performance. The goal was to provide a theoretical basis and research foundation for collecting high-quality germplasm resources and breeding superior cultivars of Wangmo C. mollissima. Ten fruit phenotypic traits and 13 quality traits were measured and analyzed in these 32 superior Wangmo C. mollissima plants. Cluster analysis and principal component analysis (PCA) were used to perform a comprehensive evaluation. Extremely significant positive correlations (P<0.01) were observed for 15 pairs of fruit phenotypic and quality traits, and significant positive correlations (P<0.05) were observed for 16 pairs of traits. Highly significant negative correlations (P<0.01) were observed for 4 pairs of fruit phenotypic and quality traits, and significant negative correlations (P<0.05) were observed for 15 pairs. The plants were divided into three groups by cluster analysis: the first group had large fruits and good fruit quality, the second group had small fruits and poor fruit quality, and the third group had medium-sized fruits with a high starch content. Four principal components were extracted from the 23 traits by PCA, contributing 76.23% of the variance. The ten plants with the highest comprehensive quality were GM32, GM31, GM29, GM1, GM8, GM17, GM10, GM30, GM3 and GM28. The results of this study provide a reference for the development and utilization of Wangmo C. mollissima germplasm resources.

Ganglioside Micelles Affect Amyloid ß Aggregation by Coassembly.

Amyloid ß peptide (Aß) is the crucial protein component of extracellular plaques in Alzheimer's disease. The plaques also contain gangliosides lipids, which are abundant in membranes of neuronal cells and in cell-derived vesicles and exosomes. When present at concentrations above its critical micelle concentration (cmc), gangliosides can occur as mixed micelles. Here, we study the coassembly of the ganglioside GM1 and the Aß peptides Aß40 and 42 by means of microfluidic diffusional sizing, confocal microscopy, and cryogenic transmission electron microscopy. We also study the effects of lipid-peptide interactions on the amyloid aggregation process by fluorescence spectroscopy. Our results reveal coassembly of GM1 lipids with both Aß monomers and Aß fibrils. The results of the nonseeded kinetics experiments show that Aß40 aggregation is delayed with increasing GM1 concentration, while that of Aß42 is accelerated. In seeded aggregation reactions, the addition of GM1 leads to a retardation of the aggregation process of both peptides. Thus, while the effect on nucleation differs between the two peptides, GM1 may inhibit the elongation of both types of fibrils. These results shed light on glycolipid-peptide interactions that may play an important role in Alzheimer's pathology.

Piezo1 Is Required for Myoblast Migration and Involves Polarized Clustering in Association with Cholesterol and GM1 Ganglioside.

A specific plasma membrane distribution of the mechanosensitive ion channel Piezo1 is required for cell migration, but the mechanism remains elusive. Here, we addressed this question using WT and Piezo1-silenced C2C12 mouse myoblasts and WT and Piezo1-KO human kidney HEK293T cells. We showed that cell migration in a cell-free area and through a porous membrane decreased upon Piezo1 silencing or deletion, but increased upon Piezo1 activation by Yoda1, whereas migration towards a chemoattractant gradient was reduced by Yoda1. Piezo1 organized into clusters, which were preferentially enriched at the front. This polarization was stimulated by Yoda1, accompanied by Ca2+ polarization, and abrogated by partial cholesterol depletion. Piezo1 clusters partially colocalized with cholesterol- and GM1 ganglioside-enriched domains, the proportion of which was increased by Yoda1. Mechanistically, Piezo1 activation induced a differential mobile fraction of GM1 associated with domains and the bulk membrane. Conversely, cholesterol depletion abrogated the differential mobile fraction of Piezo1 associated with clusters and the bulk membrane. In conclusion, we revealed, for the first time, the differential implication of Piezo1 depending on the migration mode and the interplay between GM1/cholesterol-enriched domains at the front during migration in a cell-free area. These domains could provide the optimal biophysical properties for Piezo1 activity and/or spatial dissociation from the PMCA calcium efflux pump.

GM1 structural requirements to mediate neuronal functions.

Since the 1980s, it has been known that the administration of ganglioside GM1 to cultured cells induced or enhanced neuronal differentiation. GM1 mechanism of action relies on its direct interaction and subsequent activation of the membrane tyrosine kinase receptor, TrkA, which naturally serves as NGF receptor. This process is mediated by the sole oligosaccharide portion of GM1, the pentasaccharide ß-Gal-(1-3)-ß-GalNAc-(1-4)-[α-Neu5Ac-(2-3)]-ß-Gal-(1-4)-ß-Glc. Here we detailed the minimum structural requirements of the oligosaccharide portion of GM1 for mediating the TrkA dependent neuritogenic processing. By in vitro and in silico biochemical approaches, we demonstrated that the minimal portion of GM1 required for the TrkA activation is the inner core of the ganglioside's oligosaccharide ß-Gal-(1-3)-ß-GalNAc-(1-4)-[α-Neu5Ac-(2-3)]-ß-Gal. The addition of a sialic acid residue at position 3 of the outer galactose of the GM1 oligosaccharide, which forms the oligosaccharide of GD1a, prevented the interaction with TrkA and the resulting neuritogenesis. On the contrary, the addition of a fucose residue at position 2 of the outer galactose, forming the Fucosyl-GM1 oligosaccharide, did not prevent the TrkA-mediated neuritogenesis.

Cyclization of Peptides Enhances the Inhibitory Activity against Ganglioside-Induced Aß Fibril Formation.

Alzheimer's disease is a progressive neurodegenerative disease and is the most common cause of dementia. It has been reported that the assembly of amyloid ß-protein (Aß) on the cell membrane is induced by the interaction of the Aß monomer with gangliosides such as GM1. The ganglioside-bound Aß (GAß) complex acts as a seed to promote the toxic assembly of the Aß fibrils. In a previous study, we found that a GM1 cluster-binding peptide (GCBP) specifically recognizes Aß-sensitive ganglioside nanoclusters and inhibits the assembly of Aß on a GM1-containing lipid membrane. In this study, cysteine-substituted double mutants of GCBP were designed and cyclized by intramolecular disulfide bond formation. Affinity assays indicated that one of the cyclic peptides had a higher affinity to a GM1-containing membrane compared to that of GCBP. Furthermore, surface topography analysis indicated that this peptide recognizes GM1 nanoclusters on the lipid membrane. An evaluation of the inhibitory kinetics indicated that the cyclic peptide could inhibit the formation of Aß fibrils with an IC50 value of 1.2 fM, which is 10,000-fold higher than that of GCBP. The cyclic peptide was also shown to have a clearance effect on Aß fibrils deposited on the lipid membrane and suppressed the formation of toxic Aß assemblies. Our results indicate that the cyclic peptide that binds to the Aß-sensitive ganglioside nanocluster is a potential novel inhibitor of ganglioside-induced Aß assembly.

Naïve Huntington's disease microglia mount a normal response to inflammatory stimuli but display a partially impaired development of innate immune tolerance that can be counteracted by ganglioside GM1.

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.

GM1a ganglioside-binding domain peptide inhibits host adhesion and inflammatory response of enterotoxigenic Escherichia coli heat-labile enterotoxin-B in HCT-8 cells.

Enterotoxigenic Escherichia coli (ETEC) is a major cause of illness and death but has no effective therapy. The heat-labile enterotoxin LT is a significant virulence factor produced by ETEC. The heat-labile enterotoxin-B (LT-B) subunit may enter host cells by binding to monosialotetrahexosylganglioside-a (GM1a), a monosialoganglioside found on the plasma membrane surface of animal epithelial cells. This research was conducted to develop conformationally comparable peptides to the carbohydrate epitope of GM1a for the treatment of ETEC. We used the LT-B subunit to select LT-B-binding peptides that structurally resemble GM1a. The ganglioside microarray and docking simulations were used to identify three GM1a ganglioside-binding domain (GBD) peptides based on LT-B recognition. Peptides had an inhibiting effect on the binding of LT-B to GM1a. The binding capacity, functional inhibitory activity, and in vitro effects of the GBD peptides were evaluated using HCT-8 cells, a human intestinal epithelial cell line, to evaluate the feasibility of deploying GBD peptides to combat bacterial infections. KILSYTESMAGKREMVIIT was the most efficient peptide in inhibiting cellular absorption of LT-B in cells. Our findings offer compelling evidence that GM1a GBD-like peptides might act as new therapeutics to inhibit LT-B binding to epithelial cells and avoid the subsequent physiological consequences of LT.