Altered levels of α-synuclein and sphingolipids in Batten disease lymphoblast cells

Batten disease (juvenile neuronal ceroid lipofuscinosis) is a neurodegenerative disorder characterized by blindness, seizures, cognitive decline, and early death due to the inherited mutation of the CLN3 gene. Although α-synuclein and sphingolipids are relevant for the pathogenesis of some neuronal disorders, little attention has been paid to their role in Batten disease. To identify the molecular factors linked to autophagy and apoptotic cell death in Batten disease, the levels of α-synuclein, sphingomyelin, and gangliosides were examined. We observed enhanced levels of α-synuclein oligomers and gangliosides GM1, GM2, and GM3 and reduced levels of sphingomyelin and autophagy in Batten disease lymphoblast cells compared with normal lymphoblast cells, possibly resulting in a higher rate of apoptosis typically found in Batten disease lymphoblast cells.     

Ganglioside molecular species containing C18- and C20-sphingosine in mammalian nervous tissues and neuronal cell cultures

Gangliosides exist as a very complex mixture of species differing in both the hydrophilic and hydrophobic moieties. They are particularly abundant in the central nervous system (CNS), where they have been associated with development and maturation of the brain, neuritogenesis, synaptic transmission, memory formation and synaptic aging. Today, many data suggest that some of the effects exerted by gangliosides are due to interactions with proteins that participate in the transduction of signals through the membrane in membrane microdomains. A specific characteristic of CNS gangliosides is the structure of their long-chain base (LCB). In fact, considering all the mammalian cell sphingolipids, gangliosides, sulphatides, neutral glycosphingolipids, sphingomyelin and ceramides, it would seem that while the LCB with 18 carbons is the main component of all sphingolipids, only CNS gangliosides contain significant amounts of LCB with 20 carbons. C18-Sphingosine is always present in cell gangliosides; the individual ganglioside species containing C18-sphingosine increase during cell differentiation then remain constant during cell aging. Gangliosides containing C20-sphingosine are absent, or present only in traces, in undifferentiated cells but with the onset of cell differentiation they appear, their content slowly but continuously increasing throughout the life span. In this review we discuss the chemistry, physico-chemistry and metabolism of ganglioside species differing in LCB length and introduce the hypothesis that the varying ratio between C18- and C20-gangliosides during CNS development and aging can be instrumental in modulating membrane domain organisation and cell properties.     

Sphingolipids and gangliosides of the nervous system in membrane function and dysfunction

Simple sphingolipids such as ceramide and sphingomyelin (SM) as well as more complex glycosphingolipids play very important roles in cell function under physiological conditions and during disease development and progression. Sphingolipids are particularly abundant in the nervous system. Due to their amphiphilic nature they localize to cellular membranes and many of their roles in health and disease result from membrane reorganization and from lipid interaction with proteins within cellular membranes. In this review we discuss some of the functions of sphingolipids in processes that entail cellular membranes and their role in neurodegenerative diseases, with an emphasis on SM, ceramide and gangliosides.     

A simple and novel method for tritium labeling of gangliosides and other sphingolipids

A very simple method for introducing tritium specifically into the ceramide portion of gangliosides, neutral glycosphingolipids and sphingomyelin has been developed using potassium boro [3H]hydride and palladium as catalyst. In this way six different gangliosides, five different neutral glycosphingolipids and sphinomyelin with high specific radioactivity were prepared for the first time. This simple procedure which does not require sophisticated apparatuses is suitable for large scale preparation of tritium-labeled sphingolipids as well as for nanogram quantities of individual sphingolipids so as to serve for analytical purposes. During the course of the labeling procedure no degradation of even the most labile trisialosyl-gangliotetraosylceramide has been observed. The yield of tritiated compounds is almost quantitative. The specific radioactivity depends on the unsaturation of the ceramide moiety and the specific activity of the boro[3H]hydride employed.     

Sphingolipids as modulators of membrane proteins

The diversity of the transmembranome of higher eukaryotes is matched by an enormous diversity of sphingolipid classes and molecular species. The intrinsic properties of sphingolipids are not only suited for orchestrating lateral architectures of biological membranes, but their molecular distinctions also allow for the evolution of protein motifs specifically recognising and interacting with individual lipids. Although various reports suggest a role of sphingolipids in membrane protein function, only a few cases have determined the specificity of these interactions. In this review we discuss examples of specific protein–sphingolipid interactions for which a modulator-like dependency on sphingolipids was assigned to specific proteins. These novel functions of sphingolipids in specific protein–lipid assemblies contribute to the complexity of the sphingolipid classes and other molecular species observed in animal cells. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.     

Role of gangliosides in the differentiation of human mesenchymal-derived stem cells into osteoblasts and neuronal cells

Gangliosides are complex glycosphingolipids that are the major component of cytoplasmic cell membranes, and play a role in the control of biological processes. Human mesenchymal stem cells (hMSCs) have received considerable attention as alternative sources of adult stem cells because of their potential to differentiate into multiple cell lineages. In this study, we focus on various functional roles of gangliosides in the differentiation of hMSCs into osteoblasts or neuronal cells. A relationship between gangliosides and epidermal growth factor receptor (EGFR) activation during osteoblastic differentiation of hMSCs was observed, and the gangliosides may play a major role in the regulation of the differentiation. The roles of gangliosides in osteoblast differentiation are dependent on the origin of hMSCs. The reduction of ganglioside biosynthesis inhibited the neuronal differentiation of hMSCs during an early stage of the differentiation process, and the ganglioside expression can be used as a marker for the identification of neuronal differentiation from hMSCs. [BMB Reports 2013; 46(11): 527-532]     

Roles of l-serine and sphingolipid synthesis in brain development and neuronal survival

Sphingolipids represent a class of membrane lipids that contain a hydrophobic ceramide chain as its common backbone structure. Sphingolipid synthesis requires two simple components: l-serine and palmitoyl CoA. Although l-serine is classified as a non-essential amino acid, an external supply of l-serine is essential for the synthesis of sphingolipids and phosphatidylserine (PS) in particular types of central nervous system (CNS) neurons. l-Serine is also essential for these neurons to undergo neuritogenesis and to survive. Biochemical analysis has shown that l-serine is synthesized from glucose and released by astrocytes but not by neurons, which is the major reason why this amino acid is an essential amino acid for neurons. Biosynthesis of membrane lipids, such as sphingolipids, PS, and phosphatidylethanolamine (PE), in neurons is completely dependent on this astrocytic factor. Recent advances in lipid biology research using transgenic mice have demonstrated that synthesis of endogenous l-serine and neuronal sphingolipids is essential for brain development. In this review, we discuss the metabolic system that coordinates sphingolipid synthesis with the l-serine synthetic pathway between neurons and glia. We also discuss the crucial roles of the metabolic conversion of l-serine to sphingolipids in neuronal development and survival. Human diseases associated with serine and sphingolipid biosynthesis are also discussed.     

Ablation of Neuronal Ceramide Synthase 1 in Mice Decreases Ganglioside Levels and Expression of Myelin-associated Glycoprotein in Oligodendrocytes

Ceramide synthase 1 (CerS1) catalyzes the synthesis of C18 ceramide and is mainly expressed in the brain. Custom-made antibodies to a peptide from the C-terminal region of the mouse CerS1 protein yielded specific immunosignals in neurons but no other cell types of wild type brain, but the CerS1 protein was not detected in CerS1-deficient mouse brains. To elucidate the biological function of CerS1-derived sphingolipids in the brain, we generated CerS1-deficient mice by introducing a targeted mutation into the coding region of the cers1 gene. General deficiency of CerS1 in mice caused a foliation defect, progressive shrinkage, and neuronal apoptosis in the cerebellum. Mass spectrometric analyses revealed up to 60% decreased levels of gangliosides in cerebellum and forebrain. Expression of myelin-associated glycoprotein was also decreased by about 60% in cerebellum and forebrain, suggesting that interaction and stabilization of oligodendrocytic myelin-associated glycoprotein by neuronal gangliosides is due to the C18 acyl membrane anchor of CerS1-derived precursor ceramides. A behavioral analysis of CerS1-deficient mice yielded functional deficits including impaired exploration of novel objects, locomotion, and motor coordination. Our results reveal an essential function of CerS1-derived ceramide in the regulation of cerebellar development and neurodevelopmentally regulated behavior.     

Exogenous Administration of Gangliosides Displaces GPI-anchored Proteins from Lipid Microdomains in Living Cells

Exogenous application of gangliosides to cells affects many cellular functions. We asked whether these effects could be attributed to the influence of gangliosides on the properties of sphingolipid-cholesterol microdomains on the plasma membrane, also termed rafts. The latter are envisaged as lateral assemblies of sphingolipids (including gangliosides), cholesterol, and a specific set of proteins. Rafts have been implicated in processes such as membrane trafficking, signal transduction, and cell adhesion. Recently, using a chemical cross-linking approach with Madin-Darby canine kidney (MDCK) cells permanently expressing a GPI-anchored form of growth hormone decay accelerating factor (GH-DAF) as a model system, we could show that GPI-anchored proteins are clustered in rafts in living cells. Moreover, this clustering was dependent on the level of cholesterol in the cell. Here we show that incubation of MDCK cells with gangliosides abolished subsequent chemical cross-linking of GH-DAF. Furthermore, insertion of gangliosides into the plasma membrane of MDCK GH-DAF cells renders GH-DAF soluble when subjected to extraction with Triton X-114 at 4 degrees C. Our data suggest that exogenous application of gangliosides displaces GPI-anchored proteins from sphingolipid-cholesterol microdomains in living cells.     

The characterization of sphingolipids from neurons and astroglia of immature rat brain

Abstract— Isolated neuronal cell bodies and astroglia of young (15–20-day-old) rat brains were both found to contain small concentrations of a variety of glycosphingolipids, including glucosylceramide, galactosylceramide, sulphatide, dihexosylceramide and gangliosides. These sphingolipids, plus sphingomyelin, were isolated, quantitated and their fatty acid and long chain base patterns determined. These data were compared to similar data obtained on these lipids isolated from whole brain and myelin of rats of the same age range. Glucosylceramide was found in an amount equal to galactosylceramide in neurons, and accounted for 35 per cent of the total monohexosylceramide in astroglia. Dihexosylceramide was present in nearly the same amount as sulphatide in both cell types. The sphingolipids of each cell type had characteristic fatty acid patterns. Generally the whole brain fatty acid patterns resembled those of astroglial lipids rather than neuronal lipids. In no case did the cell sphingolipid fatty acids resemble those of myelin. However, the galactosylceramide and sulphatides of both cells had unsubstituted and α-hydroxy acids, both of which had appreciable quantities of C₂₄ acids. The ganglioside fatty acids of each cell type were similar and not unusual, but were quite different from those of glucosylceramide and dihexosylceramide; the latter having appreciable quantities of 16:0 and acids longer than 18:0. The ganglioside patterns of these cells were similar and only slightly different from that of whole brain. Long chain bases of sphingolipids were mainly C₁₈-sphingosine in both cell types, and those of ganglioside and sphingomyelin contained small amounts of C₂₀-sphingosine.     

Dynamics of lipid raft components during lymphocyte apoptosis: The paradigmatic role of GD3

Several investigations have been carried out since many years in order to precisely address the function of lipid rafts in cell life and death. On the basis of the biochemical nature of lipid rafts, composed by sphingolipids, including gangliosides, sphingomyelin, cholesterol and signaling proteins, a plethora of possible interactions with various subcellular structures has been suggested. Their structural and functional role at the plasma membrane as well as in cell organelles such as endoplasmic reticulum and Golgi apparatus has been analyzed in detail in several studies. In particular, a specific activity of lipid rafts has been hypothesized to contribute to cell death by apoptosis. Although detected in various cell types, the role of lipid rafts in apoptosis has however been mostly studied in lymphocytes where the physiological apoptotic program occurs after CD95/Fas triggering. In this review, the possible contribution of lipid rafts to the cascade of events leading to T cell apoptosis after CD95/Fas ligation are summarized. Particular attention has been given to the mitochondrial raft-like microdomains, which may represent preferential sites where some key reactions can take place and can be catalyzed, leading to either survival or death of T cells.     

Gangliosides and β1‐Integrin Are Required for Caveolae and Membrane Domains

Caveolae are plasma membrane domains involved in the uptake of certain pathogens and toxins. Internalization of some cell surface integrins occurs via caveolae suggesting caveolae may play a crucial role in modulating integrin‐mediated adhesion and cell migration. Here we demonstrate a critical role for gangliosides (sialo‐glycosphingolipids) in regulating caveolar endocytosis in human skin fibroblasts. Pretreatment of cells with endoglycoceramidase (cleaves glycosphingolipids) or sialidase (modifies cell surface gangliosides and glycoproteins) selectively inhibited caveolar endocytosis by >70%, inhibited the formation of plasma membrane domains enriched in sphingolipids and cholesterol (‘lipid rafts'), reduced caveolae and caveolin‐1 at the plasma membrane by approximately 80%, and blunted activation of β1‐integrin, a protein required for caveolar endocytosis in these cells. These effects could be reversed by a brief incubation with gangliosides (but not with asialo‐gangliosides or other sphingolipids) at 10°C, suggesting that sialo‐lipids are critical in supporting caveolar endocytosis. Endoglycoceramidase treatment also caused a redistribution of focal adhesion kinase, paxillin, talin, and PIP Kinase Iγ away from focal adhesions. The effects of sialidase or endoglycoceramidase on membrane domains and the distribution of caveolin‐1 could be recapitulated by β1‐integrin knockdown. These results suggest that both gangliosides and β1‐integrin are required for maintenance of caveolae and plasma membrane domains.     

Life without gangliosides

Gangliosides are sialic acid containing glycosphingolipids that are present on all plasma cell membranes. Although they represent the major sialoconjugates in the central nervous system, their precise functions remain obscure. We have generated a series of knockout mice with gene disruptions in the ganglioside biosynthetic pathway. Individually these mice lack subsets of gangliosides allowing investigations into their specific functions. By breeding mice to contain multiple mutations in the biosynthetic pathway we have now produced mice without neuronal gangliosides. These mice display an extremely severe phenotype and neuronal pathology highlighting the essential function of gangliosides in the central nervous system.     

Membrane domains and polarized trafficking of sphingolipids

The plasma membrane of polarized cells consists of distinct domains, the apical and basolateral membrane, that are characterized by a distinct lipid and protein content. Apical protein transport is largely mediated by (glyco)sphingolipid--cholesterol enriched membrane microdomains, so called rafts. In addition changes in the direction of polarized sphingolipid transport appear instrumental in cell polarity development. Knowledge is therefore required of the mechanisms that mediate sphingolipid sorting and the complexity of the trafficking pathways that are involved in polarized transport of both sphingolipids and proteins. Here we summarize specific biophysical properties that underly mechanisms relevant to sphingolipid sorting, cargo recruitment and polarized trafficking, and discuss the central role of a subapical compartment, SAC or common endosome (CE), as a major intracellular site involved in polarized sorting of sphingolipids, and in development and maintenance of membrane polarity.     

Solving the enigma: Mass spectrometry and small molecule probes to study sphingolipid function

Sphingolipids are highly bioactive lipids. Sphingolipid metabolism produces key membrane components (e.g. sphingomyelin) and a variety of signaling lipids with different biological functions (e.g. ceramide, sphingosine-1-phosphate). The coordinated activity of tens of different enzymes maintains proper levels and localization of these lipids with key roles in cellular processes. In this review, we highlight the signaling roles of sphingolipids in cell death and survival. We discuss recent findings on the role of specific sphingolipids during these processes, enabled by the use of lipidomics to study compositional and spatial regulation of these lipids and synthetic sphingolipid probes to study subcellular localization and interaction partners of sphingolipids to understand the function of these lipids.     

Sphingolipid/cholesterol regulation of neurotransmitter receptor conformation and function

Like all other monomeric or multimeric transmembrane proteins, receptors for neurotransmitters are surrounded by a shell of lipids which form an interfacial boundary between the protein and the bulk membrane. Among these lipids, cholesterol and sphingolipids have attracted much attention because of their well-known propensity to segregate into ordered platform domains commonly referred to as lipid rafts. In this review we present a critical analysis of the molecular mechanisms involved in the interaction of cholesterol/sphingolipids with neurotransmitter receptors, in particular acetylcholine and serotonin receptors, chosen as representative members of ligand-gated ion channels and G protein-coupled receptors. Cholesterol and sphingolipids interact with these receptors through typical binding sites located in both the transmembrane helices and the extracellular loops. By altering the conformation of the receptors (“chaperone-like” effect), these lipids can regulate neurotransmitter binding, signal transducing functions, and, in the case of multimeric receptors, subunit assembly and subsequent receptor trafficking to the cell surface. Several sphingolipids (especially gangliosides) also exhibit low/moderate affinity for neurotransmitters. We suggest that such lipids could facilitate (i) the attachment of neurotransmitters to the post-synaptic membrane and in some cases (ii) their subsequent delivery to specific protein receptors. Overall, various experimental approaches provide converging evidence that the biological functions of neurotransmitters and their receptors are highly dependent upon sphingolipids and cholesterol, which are active partners of synaptic transmission. Several decades of research have been necessary to untangle the skein of a complex network of molecular interactions between neurotransmitters, their receptors, cholesterol and sphingolipids. This sophisticated crosstalk between all four distinctive partners may allow a fine biochemical tuning of synaptic transmission.     

Bioeffector sphingolipids as stimulators of cell growth and survival

The effects of various bioactive sphingolipids (sphingosine 1-phosphate, sphingosine 1-phosphocholine, ceramide 1-phosphate, ceramide beta-glucoside and beta-lactoside, and gangliosides) on cell proliferation and apoptosis are reviewed. It is concluded that the balance between the bioeffector sphingolipids determines their overall effect on cell. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 3; see also http://www.maik.ru.     

Gangliosides support neural retina cell adhesion

Cell surface carbohydrates and complementary carbohydrate receptors may mediate cell-cell recognition during neuronal development. To model such interactions, we developed a technique to test the ability of cell surface lipids (particularly glycosphingolipids) to mediate specific cell recognition and adhesion (Blackburn, C.C., and Schnaar, R.L. (1983) J. Biol. Chem. 258, 1180-1188). When cells were incubated on plastic microwells adsorbed with various glycolipids, carbohydrate-specific cell adhesion was readily detected. We report here the use of this method to study adhesion of embryonic chick neural retina cells to purified cell surface lipids. Rapid and specific cell adhesion was observed when the neural retina cells were incubated on surfaces adsorbed with gangliosides (an important class of neuronal cell surface glycoconjugates) but not on surfaces adsorbed with various neutral glycosphingolipids, phospholipids, or sulfatide. This suggests that the observed cell adhesion was specific for the carbohydrate moiety of the adsorbed ganglioside and was not due to nonspecific ionic or hydrophobic interactions. Although the surface density of adsorbed lipid required to support cell adhesion was the same for all gangliosides examined, the extent of adhesion varied when different purified gangliosides were used. Ganglioside-specific adhesion was not dependent on the presence of calcium (at 37 degrees C) and was attenuated by pretreatment of the cells with trypsin. The extent of ganglioside-directed neural retinal cell adhesion varied with embryonic age. These results imply that gangliosides may play a role in cell-cell recognition in the developing nervous system.     

Gangliosides play important roles in the nervous system by regulating ion concentrations

Gangliosides are important components of the neuronal cell membrane and play a vital role in the development of neurons and the brain. They participate in neurotransmission and are considered as the structural basis of learning and memory. Gangliosides participate in several and important physiological processes, such as cell differentiation, cell signaling, neuroprotection, nerve regeneration and apoptosis. The stability of ion concentration in excitable cells is particularly important in the maintenance of a steady state of cells and in the regulation of physiological functions. Ion concentration has been found to be related to the ganglioside’s regulation in many neurological diseases, and several studies have found that they can stabilize intracellular ion concentration by regulating ion channels, which highlights their important regulatory role in neuronal excitability and synaptic transmission. Gangliosides can influence some forms of ion transport, by directly binding to ion transporters or through indirect binding and activation of transport proteins via appropriate signaling pathways. Therefore, the important and special role of gangliosides in the homeostasis of ion concentration is becoming a hot topic in the field and a theoretical basis in promoting help gangliosides use as key drugs for the treatment of nervous system diseases.

     

Sphingolipids in cancer: regulation of pathogenesis and therapy

Sphingolipids are known to play important roles in the regulation of cell proliferation, response to chemotherapeutic agents, and/or prevention of cancer. Recently, significant progress has been made in the identification of the enzymes and their biochemical functions involved in sphingolipid metabolism. In addition, development of new techniques for the quantitative analysis of sphingolipids at their physiological levels has facilitated studies to examine distinct functions of these bioactive sphingolipids in cancer pathogenesis and therapy. This review will focus on the recent developments regarding the roles of bioactive sphingolipids in the regulation of cell growth/proliferation, and anti-cancer therapeutics.