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1.
Anal Bioanal Chem ; 411(22): 5659-5668, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31254056

RESUMO

Mass spectrometry imaging (MSI) is a powerful tool to perform untargeted mapping of biomolecules in situ. In the current study, we performed matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) to evaluate lipid changes during disease progression (asymptomatic to symptomatic time points) in Niemann-Pick disease, type C1 (NPC1), a cerebellar neurodegenerative, lipid storage disorder. Our data show that gangliosides GM2 and GM3 are elevated in NPC1 disease and localize in the posterior lobules of the cerebellum, which is enhanced over a time-course analysis of the disease. Further analysis of sphingolipids in negative ion mode indicated reduction of sulfatides in white matter of the cerebellum and patterned distribution and co-localization of ceramide species Cer(d36:1), HexCer(d36:1), and the ganglioside GM1(d36:1) during disease progression. Finally, a putative lipid of unknown structure demonstrated similar patterning during NPC1 cerebellar degeneration. These studies provide insight into lipid markers of neurodegeneration in NPC1 and link lipid alterations to altered pathways that lead to cell death.


Assuntos
Ceramidas/metabolismo , Cerebelo/patologia , Gangliosídeos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espectrometria de Massas em Tandem/métodos , Animais , Cromatografia Líquida/métodos , Camundongos , Camundongos Knockout
2.
Nat Microbiol ; 4(9): 1558-1570, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31160823

RESUMO

Several Ebola viruses cause outbreaks of lethal haemorrhagic fever in humans, but developing therapies tackle only Zaire Ebola virus. Dendritic cells (DCs) are targets of this infection in vivo. Here, we found that Ebola virus entry into activated DCs requires the sialic acid-binding Ig-like lectin 1 (Siglec-1/CD169), which recognizes sialylated gangliosides anchored to viral membranes. Blockage of the Siglec-1 receptor by anti-Siglec-1 monoclonal antibodies halted Ebola viral uptake and cytoplasmic entry, offering cross-protection against other ganglioside-containing viruses such as human immunodeficiency virus type 1.


Assuntos
Anticorpos Monoclonais/farmacologia , Citoplasma/virologia , Ebolavirus/fisiologia , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/antagonistas & inibidores , Ligação Viral/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Células Dendríticas/virologia , Gangliosídeos/metabolismo , HIV-1/fisiologia , Doença pelo Vírus Ebola/virologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Interferon-alfa/farmacologia , Lipopolissacarídeos/farmacologia , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Vírion/metabolismo
3.
Biol Pharm Bull ; 42(6): 867-872, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31155585

RESUMO

Dynamic conformational transitions and molecular assemblies are essential properties of proteins, and relevant to their biological and pathological functions. Neurodegenerative diseases are known to be caused by abnormal, toxic assemblies of related proteins, e.g., amyloid ß (Aß) in Alzheimer's disease. Growing evidence indicates that the aggregation of various amyloidogenic proteins, including Aß, can be highly enhanced at glycolipid membranes, suggesting that dynamic glycolipid-dependent conformational changes of proteins constitute crucial steps for their subsequent pathogenic amyloid fibril formation. It has also been proposed that several proteins, including molecular chaperones, can capture amyloidogenic proteins and thereby suppress their fibrillization. NMR spectroscopy provides a powerful tool for characterizing the conformational dynamics and intermolecular interactions of proteins, as well as for exploring transiently formed weak interactions among proteins in solution with various biomolecules, such as glycolipids. Our research group therefore attempted to elucidate the structural basis of protein-glycolipid and protein-protein interactions that either promote or suppress molecular assemblies of amyloidogenic proteins, using both solution and solid-state NMR methods in conjunction with other biophysical techniques. Our findings provide structural views of molecular processes involving amyloidogenic proteins of clinical and pathological interest and offer clues for the development of drugs to prevent and treat neurodegenerative diseases.


Assuntos
Amiloide/química , Amiloide/metabolismo , Membrana Celular/metabolismo , Gangliosídeos/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Chaperonas Moleculares , Conformação Proteica
4.
Int J Mol Sci ; 20(11)2019 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-31185614

RESUMO

Gangliosides are widely expressed in almost all tissues and cells and are also considered to be essential in the development and maintenance of various organs and tissues. However, little is known about their roles in bone metabolism. In this study, we investigated the effects of genetic deletion of ganglioside D3 (GD3) synthase, which is responsible for the generation of all b-series gangliosides, on bone metabolism. Although b-series gangliosides were not expressed in osteoblasts, these gangliosides were expressed in pre-osteoclasts. However, the expression of these gangliosides was decreased after induction of osteoclastogenesis by receptor activator of nuclear factor kappa-B ligand (RANKL). Three-dimensional micro-computed tomography (3D-µCT) analysis revealed that femoral cancellous bone mass in GD3 synthase-knockout (GD3S KO) mice was higher than that in wild type (WT) mice at the age of 40 weeks, although there were no differences in that between GD3S KO and WT mice at 15 weeks old. Whereas bone formation parameters (osteoblast numbers/bone surface and osteoblast surface/bone surface) in GD3S KO mice did not differ from WT mice, bone resorption parameters (osteoclast numbers/bone surface and osteoclast surface/bone surface) in GD3S KO mice became significantly lower than those in WT mice at 40 weeks of age. Collectively, this study demonstrates that deletion of GD3 synthase attenuates bone loss that emerges with aging.


Assuntos
Envelhecimento/patologia , Reabsorção Óssea/genética , Sialiltransferases/genética , Animais , Células Cultivadas , Gangliosídeos/metabolismo , Camundongos , Osteoclastos/metabolismo , Osteoclastos/patologia , Osteogênese , Ligante RANK/metabolismo , Células RAW 264.7 , Sialiltransferases/deficiência
5.
Talanta ; 201: 364-372, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31122436

RESUMO

The selection of a suitable matrix and deposition technique constitutes a critical step in successful matrix-assisted laser desorption/ionization mass spectrometry imaging measurement. In the present work, we compared three techniques of matrix deposition, specifically, sublimation and spraying of 1,5-diaminonaphthalene with two automatic sprayers, ImagePrep and iMatrixSpray. The studied methods were evaluated in experiments for the analysis of lipid composition in the brains of two mouse models of neurodegeneration: APP/PS1 mice with plaques of amyloid ß (Aß) peptides and THY-Tau22 mice with pathologically hyperphosphorylated Tau protein, two hallmarks of Alzheimer's disease-like pathology. The sublimation method provided irreproducible results because of significant matrix loss due to the high vacuum in the ion source and laser irradiation. In contrast, the ImagePrep and iMatrixSpray provided stable film of the matrix. The deposited matrix was stable during the measurement, and highly reproducible datasets were obtained. Both spraying methods yielded similar results with approximately the same number of detected lipids and comparable signal intensity. However, iMatrixSpray has two main advantages: a faster matrix deposition and the formation of smaller matrix crystals leading to better spatial resolution. In the APP/PS1 mouse model at an age of 6 months, we found colocalization of Aß plaques with different phospholipids, sphingolipids and lysophospholipids. We did not find a difference in lipid composition between the THY-Tau22 mice and the wild-type controls. The results indicate that hyperphosphorylation of tau protein in the THY-Tau22 mouse model at the age of 6 months is not accompanied with a significant change in lipid content in the brain. However, considering limitations of the used method, a definitive conclusion in this respect will need further research.


Assuntos
2-Naftilamina/análogos & derivados , Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Gangliosídeos/análise , Glicerofosfolipídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , 2-Naftilamina/química , Peptídeos beta-Amiloides/metabolismo , Animais , Modelos Animais de Doenças , Gangliosídeos/metabolismo , Glicerofosfolipídeos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Reprodutibilidade dos Testes , Proteínas tau/metabolismo
6.
Int J Mol Sci ; 20(8)2019 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-31010057

RESUMO

The interactions between neuronal, glial, and vascular cells play a key role in regulating blood flow in the retina. In the present study, we examined the role of the interactions between neuronal and glial cells in regulating the retinal vascular tone in rats upon stimulation of retinal neuronal cells by intravitreal injection of N-methyl-d-aspartic acid (NMDA). The retinal vascular response was assessed by measuring the diameter of the retinal arterioles in the in vivo fundus images. Intravitreal injection of NMDA produced retinal vasodilation that was significantly diminished following the pharmacological inhibition of nitric oxide (NO) synthase (nNOS), loss of inner retinal neurons, or intravitreal injection of glial toxins. Immunohistochemistry revealed the expression of nNOS in ganglion and calretinin-positive amacrine cells. Moreover, glial toxins significantly prevented the retinal vasodilator response induced by intravitreal injection of NOR3, an NO donor. Mechanistic analysis revealed that NO enhanced the production of vasodilatory prostanoids and epoxyeicosatrienoic acids in glial cells in a ryanodine receptor type 1-dependent manner, subsequently inducing the retinal vasodilator response. These results suggest that the NO released from stimulated neuronal cells acts as a key messenger in neuron-glia signaling, thereby causing neuronal activity-dependent and glial cell-mediated vasodilation in the retina.


Assuntos
Neuroglia/metabolismo , Neurônios/metabolismo , Vasos Retinianos/metabolismo , Transdução de Sinais , Animais , Gangliosídeos/metabolismo , Hidroxilaminas , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Masculino , Modelos Biológicos , N-Metilaspartato/metabolismo , N-Metilaspartato/farmacologia , Neuroglia/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Óxido Nítrico Sintase Tipo I/metabolismo , Prostaglandinas/metabolismo , Ratos Wistar , Vasos Retinianos/patologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos
7.
Int J Mol Sci ; 20(8)2019 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-31013778

RESUMO

Insulin in physiological concentrations is important to maintain vascular function. Moreover, vascular insulin resistance contributes to vascular impairment. In the elderly, other factors including hypertension, dyslipidemia, and chronic inflammation amplify senescence of vascular endothelial and smooth muscle cells. In turn, senescence increases the risk for vascular-related diseases such as arteriosclerosis, diabetes, and Alzheimer's disease. Recently, it was found that GM1 ganglioside, one of the glycolipids localized on the cell membrane, mediates vascular insulin resistance by promoting senescence and/or inflammatory stimulation. First, it was shown that increased GM1 levels associated with aging/senescence contribute to insulin resistance in human aortic endothelial cells (HAECs). Second, the expression levels of gangliosides were monitored in HAECs treated with different concentrations of tumor necrosis factor-alpha (TNFα) for different time intervals to mimic in vivo acute or chronic inflammatory conditions. Third, the levels of insulin signaling-related molecules were monitored in HAECs after TNFα treatment with or without inhibitors of ganglioside synthesis. In this review, we summarize the molecular mechanisms of insulin resistance in aged/senescent and TNFα-stimulated endothelial cells mediated by gangliosides and highlight the possible roles of gangliosides in vascular insulin resistance-related diseases.


Assuntos
Vasos Sanguíneos/metabolismo , Gangliosídeos/metabolismo , Resistência à Insulina , Insulina/metabolismo , Animais , Senescência Celular , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Humanos , Transdução de Sinais , Vasculite/etiologia , Vasculite/metabolismo
8.
PLoS Biol ; 17(3): e3000169, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30822302

RESUMO

CD1d-restricted invariant natural killer T (iNKT) cells represent a heterogeneous population of lipid-reactive T cells that are involved in many immune responses, mediated through T-cell receptor (TCR)-dependent and/or independent activation. Although numerous microbial lipid antigens (Ags) have been identified, several lines of evidence have suggested the existence of relevant Ags of endogenous origin. However, the identification of their precise nature as well as the molecular mechanisms involved in their generation are still highly controversial and ill defined. Here, we identified two mammalian gangliosides-namely monosialoganglioside GM3 and disialoganglioside GD3-as endogenous activators for mouse iNKT cells. These glycosphingolipids are found in Toll-like receptor-stimulated dendritic cells (DC) as several species varying in their N-acyl fatty chain composition. Interestingly, their ability to activate iNKT cells is highly dependent on the ceramide backbone structure. Thus, both synthetic GM3 and GD3 comprising a d18:1-C24:1 ceramide backbone were able to activate iNKT cells in a CD1d-dependent manner. GM3 and GD3 are not directly recognized by the iNKT TCR and required the Ag presenting cell intracellular machinery to reveal their antigenicity. We propose a new concept in which iNKT cells can rapidly respond to pre-existing self-molecules after stress-induced structural changes in CD1d-expressing cells. Moreover, these gangliosides conferred partial protection in the context of bacterial infection. Thus, this report identified new biologically relevant lipid self-Ags for iNKT cells.


Assuntos
Ceramidas/metabolismo , Gangliosídeos/metabolismo , Células T Matadoras Naturais/metabolismo , Receptor Toll-Like 9/metabolismo , Animais , Antígenos CD1d/metabolismo , Células da Medula Óssea/metabolismo , Células Dendríticas/metabolismo , Gangliosídeo G(M3)/metabolismo , Glicoesfingolipídeos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo Real
9.
Adv Carbohydr Chem Biochem ; 76: 113-148, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30851743

RESUMO

Gangliosides comprise a varied family of glycosphingolipid structures bearing one or more sialic acid residues. They are found in all mammalian tissues but are most abundant in the brain, where they represent the quantitatively major class of sialoglycans. As prominent molecular determinants on cell surfaces, they function as molecular-recognition partners for diverse glycan-binding proteins ranging from bacterial toxins to endogenous cell-cell adhesion molecules. Gangliosides also regulate the activity of plasma membrane proteins, including protein tyrosine kinases, by lateral association in the same membranes in which they reside. Their roles in molecular recognition and membrane protein regulation implicate gangliosides in human physiology and pathology, including infectious diseases, diabetes, cancer, and neurodegeneration. The varied structures and biosynthetic pathways of gangliosides are presented here, along with representative examples of their biological functions in health and disease.


Assuntos
Gangliosídeos/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Doença , Gangliosídeos/química , Gangliosídeos/genética , Saúde , Humanos , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética
11.
J Biol Chem ; 294(12): 4437-4449, 2019 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-30670592

RESUMO

Neuroblastoma cells highly express the disialoganglioside GD2, a tumor-associated carbohydrate antigen, which is only sparsely expressed on healthy tissue. GD2 is a primary target for the development of immunotherapy for neuroblastoma. Immunotherapy with monoclonal anti-GD2 antibodies has proven safety and efficacy in clinical trials and is included in the standard treatment for children with high-risk neuroblastoma. Strategies to modulate GD2 expression in neuroblastoma could further improve anti-GD2-targeted immunotherapy. Here, we report that the cellular sialylation pathway, as well as epigenetic reprogramming, strongly modulates GD2 expression in human and mouse neuroblastoma cell lines. Recognition of GD2 by the 14G2a antibody is sialic acid-dependent and was blocked with the fluorinated sialic acid mimetic Ac53FaxNeu5Ac. Interestingly, sialic acid supplementation using a cell-permeable sialic acid analogue (Ac5Neu5Ac) boosted GD2 expression without or with minor alterations in overall cell surface sialylation. Furthermore, sialic acid supplementation with Ac5Neu5Ac combined with various histone deacetylase (HDAC) inhibitors, including vorinostat, enhanced GD2 expression in neuroblastoma cells beyond their individual effects. Mechanistic studies revealed that Ac5Neu5Ac supplementation increased intracellular CMP-Neu5Ac concentrations, thereby providing higher substrate levels for sialyltransferases. Furthermore, HDAC inhibitor treatment increased mRNA expression of the sialyltransferases GM3 synthase (ST3GAL5) and GD3 synthase (ST8SIA1), both of which are involved in GD2 biosynthesis. Our findings reveal that sialic acid analogues and HDAC inhibitors enhance GD2 expression and could potentially be employed to boost anti-GD2 targeted immunotherapy in neuroblastoma patients.


Assuntos
Antígenos de Neoplasias/metabolismo , Gangliosídeos/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Ácido N-Acetilneuramínico/farmacologia , Neuroblastoma/imunologia , Regulação para Cima/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Imunoterapia , Camundongos , Neuroblastoma/enzimologia , Neuroblastoma/patologia , Neuroblastoma/terapia , Sialiltransferases/metabolismo
12.
Glycoconj J ; 36(1): 79-90, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30612272

RESUMO

Mainly restricted to the nervous system in healthy adults, complex gangliosides such as GD3 and GD2 have been shown to be involved in aggressiveness and metastasis of neuro-ectoderm derived tumors such as melanoma and neuroblastoma. Interestingly, O-acetylated forms of GD2, not expressed in human peripheral nerve fibers, are highly expressed in GD2+ tumor cells. Very little information is known regarding the expression of O-acetylated disialogangliosides in breast cancer (BC) cell lines. Here, we analyzed the expression of GD2, GD3 and their O-acetylated forms O-acetyl-GD2 (OAcGD2) and O-acetyl-GD3 (OAcGD3) in BC cells. We used Hs 578T and SUM159PT cell lines, as well as cell clones over-expressing GD3 synthase derived from MDA-MB-231 and MCF-7. Using flow cytometry and immunocytochemistry/confocal microscopy, we report that BC cells express b-series gangliosides GD3 and GD2, as well as significant amounts of OAcGD2. However, OAcGD3 expression was not detected in these cells. O-acetylation of gangliosides isolated from BC cells was examined by LC-MS analysis of sialic acid DMB-derivatives. We report that the main acetylated form of sialic acid expressed in BC gangliosides is 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac2). These results highlight a close interrelationship between Neu5,9Ac2 and OAcGD2 expression, and suggest that OAcGD2 is synthetized from GD2 and not from OAcGD3 in BC cells.


Assuntos
Neoplasias da Mama/metabolismo , Gangliosídeos/química , Ácidos Siálicos/análise , Feminino , Gangliosídeos/metabolismo , Humanos , Células MCF-7 , Ácidos Siálicos/química
13.
J Biol Chem ; 294(6): 1997-2008, 2019 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-30523158

RESUMO

Toll-like receptors (TLRs) are pathogen-recognition receptors that trigger the innate immune response. Recent reports have identified accessory proteins that provide essential support to TLR function through ligand delivery and receptor trafficking. Herein, we introduce leucine-rich repeats (LRRs) and calponin homology containing 4 (Lrch4) as a novel TLR accessory protein. Lrch4 is a membrane protein with nine LRRs in its predicted ectodomain. It is widely expressed across murine tissues and has two expression variants that are both regulated by lipopolysaccharide (LPS). Predictive modeling indicates that Lrch4 LRRs conform to the horseshoe-shaped structure typical of LRRs in pathogen-recognition receptors and that the best structural match in the protein database is to the variable lymphocyte receptor of the jawless vertebrate hagfish. Silencing Lrch4 attenuates cytokine induction by LPS and multiple other TLR ligands and dampens the in vivo innate immune response. Lrch4 promotes proper docking of LPS in lipid raft membrane microdomains. We provide evidence that this is through regulation of lipid rafts as Lrch4 silencing reduces cell surface gangliosides, a metric of raft abundance, as well as expression and surface display of CD14, a raft-resident LPS co-receptor. Taken together, we identify Lrch4 as a broad-spanning regulator of the innate immune response and a potential molecular target in inflammatory disease.


Assuntos
Regulação da Expressão Gênica , Imunidade Inata , Receptores Toll-Like , Animais , Gangliosídeos/metabolismo , Leucina , Ligantes , Receptores de Lipopolissacarídeos , Lipopolissacarídeos/metabolismo , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Camundongos , Conformação Proteica , Domínios Proteicos
14.
Traffic ; 20(1): 39-60, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30328220

RESUMO

Prominin-1 is a cell surface biomarker that allows the identification of stem and cancer stem cells from different organs. It is also expressed in several differentiated epithelial and non-epithelial cells. Irrespective of the cell type, prominin-1 is associated with plasma membrane protrusions. Here, we investigate its impact on the architecture of membrane protrusions using microvilli of Madin-Darby canine kidney cells as the main model. Our high-resolution analysis revealed that upon the overexpression of prominin-1 the number of microvilli and clusters of them increased. Microvilli with branched and/or knob-like morphologies were observed and stimulated by mutations in the ganglioside-binding site of prominin-1. The altered phenotypes were caused by the interaction of prominin-1 with phosphoinositide 3-kinase and Arp2/3 complex. Mutation of tyrosine 828 of prominin-1 impaired its phosphorylation and thereby inhibited the aforementioned interactions abolishing altered microvilli. This suggests that the interplay of prominin-1-ganglioside membrane complexes, phosphoinositide 3-kinase and cytoskeleton components regulates microvillar architecture. Lastly, the expression of prominin-1 and its mutants modified the structure of filopodia emerging from fibroblast-like cells and silencing human prominin-1 in primary hematopoietic stem cells resulted in the loss of uropod-associated microvilli. Altogether, these findings strengthen the role of prominin-1 as an organizer of cellular protrusions.


Assuntos
Antígeno AC133/metabolismo , Microvilosidades/metabolismo , Antígeno AC133/química , Antígeno AC133/genética , Animais , Sítios de Ligação , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Cães , Gangliosídeos/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Humanos , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos C57BL , Microvilosidades/ultraestrutura , Mutação , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica
15.
J Neurosci ; 39(1): 63-77, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30446529

RESUMO

Sulfatides and gangliosides are raft-associated glycolipids essential for maintaining myelinated nerve integrity. Mice deficient in sulfatide (cerebroside sulfotransferase knock-out, CST -/-) or complex gangliosides (ß-1,4-N-acetylegalactosaminyltransferase1 knock-out, GalNAc-T -/-) display prominent disorganization of proteins at the node of Ranvier (NoR) in early life and age-dependent neurodegeneration. Loss of neuronal rather than glial complex gangliosides underpins the GalNAc-T -/- phenotype, as shown by neuron- or glial-specific rescue, whereas sulfatide is principally expressed and functional in glial membranes. The similarities in NoR phenotype of CST -/-, GalNAc-T -/-, and axo-glial protein-deficient mice suggests that these glycolipids stabilize membrane proteins including neurofascin155 (NF155) and myelin-associated glycoprotein (MAG) at axo-glial junctions. To assess the functional interactions between sulfatide and gangliosides, CST -/- and GalNAc-T -/- genotypes were interbred. CST -/-× GalNAc-T -/- mice develop normally to postnatal day 10 (P10), but all die between P20 and P25, coinciding with peak myelination. Ultrastructural, immunohistological, and biochemical analysis of either sex revealed widespread axonal degeneration and disruption to the axo-glial junction at the NoR. In addition to sulfatide-dependent loss of NF155, CST -/- × GalNAc-T -/- mice exhibited a major reduction in MAG protein levels in CNS myelin compared with WT and single-lipid-deficient mice. The CST -/- × GalNAc-T -/- phenotype was fully restored to that of CST -/- mice by neuron-specific expression of complex gangliosides, but not by their glial-specific expression nor by the global expression of a-series gangliosides. These data indicate that sulfatide and complex b-series gangliosides on the glial and neuronal membranes, respectively, act in concert to promote NF155 and MAG in maintaining the stable axo-glial interactions essential for normal nerve function.SIGNIFICANCE STATEMENT Sulfatides and complex gangliosides are membrane glycolipids with important roles in maintaining nervous system integrity. Node of Ranvier maintenance in particular requires stable compartmentalization of multiple membrane proteins. The axo-glial adhesion molecules neurofascin155 (NF155) and myelin-associated glycoprotein (MAG) require membrane microdomains containing either sulfatides or complex gangliosides to localize and function effectively. The cooperative roles of these microdomains and associated proteins are unknown. Here, we show vital interdependent roles for sulfatides and complex gangliosides because double (but not single) deficiency causes a rapidly lethal phenotype at an early age. These findings suggest that sulfatides and complex gangliosides on opposing axo-glial membranes are responsible for essential tethering of the axo-glial junction proteins NF155 and MAG, which interact to maintain the nodal complex.


Assuntos
Axônios/fisiologia , Gangliosídeos/metabolismo , Gangliosídeos/fisiologia , Bainha de Mielina/fisiologia , Neuroglia/fisiologia , Neurônios/fisiologia , Sulfoglicoesfingolipídeos/metabolismo , Animais , Moléculas de Adesão Celular/genética , Feminino , Genótipo , Expectativa de Vida , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Glicoproteína Associada a Mielina/genética , Glicoproteína Associada a Mielina/fisiologia , N-Acetilgalactosaminiltransferases/genética , Fatores de Crescimento Neural/genética , Neuroglia/metabolismo , Neurônios/metabolismo , Nós Neurofibrosos/fisiologia , Sulfotransferases/genética , Sulfotransferases/fisiologia
16.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1864(3): 422-432, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-29908366

RESUMO

Melanomas often accumulate gangliosides, sialic acid-containing glycosphingolipids found in the outer leaflet of plasma membranes, as disialoganglioside GD3 and its derivatives. Here, we have transfected the GD3 synthase gene (ST8Sia I) in a normal melanocyte cell line in order to evaluate changes in the biological behavior of non-transformed cells. GD3-synthase expressing cells converted GM3 into GD3 and accumulated both GD3 and its acetylated form, 9-O-acetyl-GD3. Melanocytes were rendered more migratory on laminin-1 surfaces. Cell migration studies using the different transfectants, either treated or not with the glucosylceramide synthase inhibitor d-1-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (PPPP), allowed us to show that while GM3 is a negative regulator of melanocyte migration, GD3 increases it. We showed that gangliosides were shed to the matrix by migrating cells and that GD3 synthase transfected cells shed extracellular vesicles (EVs) enriched in GD3. EVs enriched in GD3 stimulated cell migration of GD3 negative cells, as observed in time lapse microscopy studies. Otherwise, EVs shed by GM3+veGD3-ve cells impaired migration and diminished cell velocity in cells overexpressing GD3. The balance of antimigratory GM3 and promigratory GD3 gangliosides in melanocytes could be altered not only by the overexpression of enzymes such as ST8Sia I, but also by the horizontal transfer of ganglioside enriched extracellular vesicles. This study highlights that extracellular vesicles transfer biological information also through their membrane components, which include a variety of glycosphingolipids remodeled in disease states such as cancer.


Assuntos
Gangliosídeos/metabolismo , Melanócitos/efeitos dos fármacos , Melanócitos/metabolismo , Acetilação , Animais , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/fisiologia , Gangliosídeos/farmacologia , Gangliosídeos/fisiologia , Glicoesfingolipídeos/metabolismo , Camundongos , Transfecção
17.
Mol Neurobiol ; 56(5): 3552-3562, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30145786

RESUMO

Perturbations of ganglioside homeostasis have been observed following stroke whereby toxic simple gangliosides GM2 and GM3 accumulate, while protective complex species GM1 and GD1 are reduced. Thus, there is a need for therapeutic interventions which can prevent ganglioside dysregulation after stroke. A pharmacological intervention using chloroquine was selected for its transient lysosomotropic properties which disrupt the activity of catabolic ganglioside enzymes. Chloroquine was administered both in vitro (0.1 µM), to primary cortical neurons exposed to GM3 toxicity, and in vivo (45 mg/kg i.p.), to 3-month-old male Wistar rats that underwent a severe stroke injury. Chloroquine was administered for seven consecutive days beginning 3 days prior to the stroke injury. Gangliosides were examined using MALDI imaging mass spectrometry at 3 and 21 days after the injury, and motor deficits were examined using the ladder task. Chloroquine treatment prevented ganglioside dysregulation 3 days post-stroke and partially prevented complex ganglioside depletion 21 days post-stroke. Exogenous GM3 was found to be toxic to primary cortical neurons which was protected by chloroquine treatment. Motor deficits were prevented in the forelimbs of stroke-injured rats with chloroquine treatment and was associated with decreased inflammation, neurodegeneration, and an increase in cell survival at the site of injury. Chloroquine administration prevents ganglioside dysregulation acutely, protects against GM3 toxicity in neurons, and is associated with long-term functional and pathological improvements after stroke in the rat. Therefore, targeting lipid dysregulation using lysosomotropic agents such as chloroquine may represent a novel therapeutic avenue for stroke injuries.


Assuntos
Comportamento Animal , Cloroquina/farmacologia , Gangliosídeos/metabolismo , Homeostase/efeitos dos fármacos , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologia , Animais , Comportamento Animal/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Membro Anterior/patologia , Membro Anterior/fisiopatologia , Masculino , Atividade Motora/efeitos dos fármacos , Neurônios/metabolismo , Fármacos Neuroprotetores/farmacologia , Ratos Wistar , Acidente Vascular Cerebral/fisiopatologia
18.
Future Med Chem ; 10(24): 2835-2854, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30539670

RESUMO

Sialic acid is associated with glycoproteins and gangliosides of eukaryotic cells. It regulates various molecular interactions, being implicated in inflammation and cancer, where its expression is regulated by sialyltransferases and sialidases. Angiogenesis, the formation of new capillaries, takes place during inflammation and cancer, and represents the outcome of several interactions occurring at the endothelial surface among angiogenic growth factors, inhibitors, receptors, gangliosides and cell-adhesion molecules. Here, we elaborate on the evidences that many structures involved in angiogenesis are sialylated and that their interactions depend on sialic acid with implications in angiogenesis itself, inflammation and cancer. We also discuss the possibility to exploit sialic acid as a target for the development of novel antiangiogenic drugs.


Assuntos
Inibidores da Angiogênese/farmacologia , Descoberta de Drogas , Terapia de Alvo Molecular , Ácido N-Acetilneuramínico/metabolismo , Neovascularização Patológica/tratamento farmacológico , Inibidores da Angiogênese/química , Animais , Descoberta de Drogas/métodos , Gangliosídeos/metabolismo , Glicoproteínas/metabolismo , Humanos , Inflamação/complicações , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Modelos Moleculares , Terapia de Alvo Molecular/métodos , Neoplasias/complicações , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neovascularização Patológica/complicações , Neovascularização Patológica/metabolismo
19.
J Immunol ; 201(12): 3750-3758, 2018 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-30446565

RESUMO

The tumor microenvironment is rendered immunosuppressive by a variety of cellular and acellular factors that represent potential cancer therapeutic targets. Although exosomes isolated from ovarian tumor ascites fluids have been previously reported to induce a rapid and reversible T cell arrest, the factors present on or within exosomes that contribute to immunosuppression have not been fully defined. In this study, we establish that GD3, a ganglioside expressed on the surface of exosomes isolated from human ovarian tumor ascites fluids, is causally linked to the functional arrest of T cells activated through their TCR. This arrest is inhibited by Ab blockade of exosomal GD3 or by the removal of GD3+ exosomes. Empty liposomes expressing GD3 on the surface also inhibit the activation of T cells, establishing that GD3 contributes to the functional arrest of T cells independent of factors present in exosomes. Finally, we demonstrate that the GD3-mediated arrest of the TCR activation is dependent upon sialic acid groups, because their enzymatic removal from exosomes or liposomes results in a loss of inhibitory capacity. Collectively, these data define GD3 as a potential immunotherapeutic target.


Assuntos
Líquido Ascítico/metabolismo , Exossomos/metabolismo , Gangliosídeos/metabolismo , Imunoterapia/métodos , Ácido N-Acetilneuramínico/metabolismo , Neoplasias Ovarianas/metabolismo , Linfócitos T/imunologia , Ascite , Células Cultivadas , Feminino , Humanos , Tolerância Imunológica , NF-kappa B/metabolismo , Estadiamento de Neoplasias , Neoplasias Ovarianas/imunologia , Microambiente Tumoral
20.
PLoS One ; 13(11): e0206881, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30462668

RESUMO

Ganglioside GD3 is widely expressed in human malignant melanomas, and has been reported to be involved in the increased cell proliferation and invasion. In this study, we established GM3-, GM2-, GM1-, GD3-, or GD2-expressing melanoma cell lines by transfecting cDNAs of glyscosyltransferases, and effects of individual gangliosides on the cell phenotypes and signals were examined. The phenotypes of established ganglioside-expressing cells were quite different, i.e. cell growth increased as following order; GD2+, GD3+ > GM1+, GM2+, GM3+ cells. Cell invasion activity increased as GD3+ ≧ GM2+ > GM1+, GM3+, GD2+ cells. Intensity of cell adhesion to collagen I (CL-I) and spreading increased as GD2+ >> GD3+, GM1+ > GM2+, GM3+ cells. In particular, cell adhesion of GD2+ cells was markedly strong. As for cell migration velocity, GD2+ cells were slower than all other cells. The immunocytostaining revealed close localization of gangliosides and F-actin in lamellipodia. Immunoblotting of phosphorylated p130Cas and paxillin by serum treatment reveled that these phosphorylations were more increased in GD3+ cells than in GD2+ or GM3+ cells, while phosphorylation of Akt underwent similarly increased phosphorylation between GD3+ and GD2+ cells compared with GM3+ cells. While GD2 and GD3 enhanced cell growth, GD3 might also contribute in cell invasion. On the other hand, GD2 might contribute in the solid fixation of melanoma cells at metastasized sites. These results suggested that individual gangliosides exert distinct roles in the different aspects of melanomas by differentially regulating cytoskeletons and signaling molecules.


Assuntos
Carcinogênese/patologia , Gangliosídeos/metabolismo , Melanoma/patologia , Neoplasias Cutâneas/patologia , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Humanos
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