Emerging concepts of ganglioside metabolism.

Gangliosides (GGs) are sialic acid-containing glycosphingolipids (GSLs) and major membrane components enriched on cellular surfaces. Biosynthesis of mammalian GGs starts at the cytosolic leaflet of endoplasmic reticulum (ER) membranes with the formation of their hydrophobic ceramide anchors. After intracellular ceramide transfer to Golgi and trans-Golgi network (TGN) membranes, anabolism of GGs, as well as of other GSLs, is catalyzed by membrane-spanning glycosyltransferases (GTs) along the secretory pathway. Combined activity of only a few promiscuous GTs allows for the formation of cell-type-specific glycolipid patterns. Following an exocytotic vesicle flow to the cellular plasma membranes, GGs can be modified by metabolic reactions at or near the cellular surface. For degradation, GGs are endocytosed to reach late endosomes and lysosomes. Whereas membrane-spanning enzymes of the secretory pathway catalyze GSL and GG formation, a cooperation of soluble glycosidases, lipases and lipid-binding cofactors, namely the sphingolipid activator proteins (SAPs), act as the main players of GG and GSL catabolism at intralysosomal luminal vesicles (ILVs).

Neuronal sphingolipidoses: Membrane lipids and sphingolipid activator proteins regulate lysosomal sphingolipid catabolism.

Glycosphingolipids and sphingolipids of cellular plasma membranes (PMs) reach luminal intra-lysosomal vesicles (LVs) for degradation mainly by pathways of endocytosis. After a sorting and maturation process (e.g. degradation of sphingomyelin (SM) and secretion of cholesterol), sphingolipids of the LVs are digested by soluble enzymes with the help of activator (lipid binding and transfer) proteins. Inherited defects of lipid-cleaving enzymes and lipid binding and transfer proteins cause manifold and fatal, often neurodegenerative diseases. The review summarizes recent findings on the regulation of sphingolipid catabolism and cholesterol secretion from the endosomal compartment by lipid modifiers, an essential stimulation by anionic membrane lipids and an inhibition of crucial steps by cholesterol and SM. Reconstitution experiments in the presence of all proteins needed, hydrolase and activator proteins, reveal an up to 10-fold increase of ganglioside catabolism just by the incorporation of anionic lipids into the ganglioside carrying membranes, whereas an additional incorporation of cholesterol inhibits GM2 catabolism substantially. It is suggested that lipid and other low molecular modifiers affect the genotype-phenotype relationship observed in patients with lysosomal diseases.

Sphingolipids and lysosomal pathologies.

Endocytosed (glyco)sphingolipids are degraded, together with other membrane lipids in a stepwise fashion by endolysosomal enzymes with the help of small lipid binding proteins, the sphingolipid activator proteins (SAPs), at the surface of intraluminal lysosomal vesicles. Inherited defects in a sphingolipid-degrading enzyme or SAP cause the accumulation of the corresponding lipid substrates, including cytotoxic lysosphingolipids, such as galactosylsphingosine and glucosylsphingosine, and lead to a sphingolipidosis. Analysis of patients with prosaposin deficiency revealed the accumulation of intra-endolysosmal vesicles and membrane structures (IM). Feeding of prosaposin reverses the storage, suggesting inner membrane structures as platforms of sphingolipid degradation. Water soluble enzymes can hardly attack sphingolipids embedded in the membrane of inner endolysosomal vesicles. The degradation of sphingolipids with few sugar residues therefore requires the help of the SAPs, and is strongly stimulated by anionic membrane lipids. IMs are rich in anionic bis(monoacylglycero)phosphate (BMP). This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.

Flotillin microdomains stabilize cadherins at cell-cell junctions.

Cadherins are essential in many fundamental processes and assemble at regions of cell-cell contact in large macromolecular complexes named adherens junctions. We have identified flotillin 1 and 2 as new partners of the cadherin complexes. We show that flotillins are localised at cell-cell junctions (CCJs) in a cadherin-dependent manner. Flotillins and cadherins are constitutively associated at the plasma membrane and their colocalisation at CCJ increases with CCJ maturation. Using three-dimensional structured illumination super-resolution microscopy, we found that cadherin and flotillin complexes are associated with F-actin bundles at CCJs. The knockdown of flotillins dramatically affected N- and E-cadherin recruitment at CCJs in mesenchymal and epithelial cell types and perturbed CCJ integrity and functionality. Moreover, we determined that flotillins are required for cadherin association with GM1-containing plasma membrane microdomains. This allows p120 catenin binding to the cadherin complex and its stabilization at CCJs. Altogether, these data demonstrate that flotillin microdomains are required for cadherin stabilization at CCJs and for the formation of functional CCJs.

My journey into the world of sphingolipids and sphingolipidoses.

Analysis of lipid storage in postmortem brains of patients with amaurotic idiocy led to the recognition of five lysosomal ganglioside storage diseases and identification of their inherited metabolic blocks. Purification of lysosomal acid sphingomyelinase and ceramidase and analysis of their gene structures were the prerequisites for the clarification of Niemann-Pick and Farber disease. For lipid catabolism, intraendosomal vesicles are formed during the endocytotic pathway. They are subjected to lipid sorting processes and were identified as luminal platforms for cellular lipid and membrane degradation. Lipid binding glycoproteins solubilize lipids from these cholesterol poor membranes and present them to water-soluble hydrolases for digestion. Biosynthesis and intracellular trafficking of lysosomal hydrolases (hexosaminidases, acid sphingomyelinase and ceramidase) and lipid binding and transfer proteins (GM2 activator, saposins) were analyzed to identify the molecular and metabolic basis of several sphingolipidoses. Studies on the biosynthesis of glycosphingolipids yielded the scheme of Combinatorial Ganglioside Biosynthesis involving promiscuous glycosyltransferases. Their defects in mutagenized mice impair brain development and function.

Biochemistry and neurobiology of prosaposin: a potential therapeutic neuro-effector.

Prosaposin, a 66 kDa glycoprotein, was identified initially as the precursor of the sphingolipid activator proteins, saposins A-D, which are required for the enzymatic hydrolysis of certain sphingolipids by lysosomal hydrolases. While mature saposins are distributed to lysosomes, prosaposin exists in secretory body fluids and plasma membranes. In addition to its role as the precursor, prosaposin shows a variety of neurotrophic and myelinotrophic activities through a receptor-mediated mechanism. In studies in vivo, prosaposin was demonstrated to exert a variety of neuro-efficacies capable of preventing neuro-degeneration following neuro-injury and promoting the amelioration of allodynia and hyperalgesia in pain models. Collective findings indicate that prosaposin is not a simple house-keeping precursor protein; instead, it is a protein essentially required for the development and maintenance of the central and peripheral nervous systems. Accumulating evidence over the last decade has attracted interests in exploring and developing new therapeutic approaches using prosaposin for human disorders associated with neuro-degeneration. In this review we detail the structure characteristics, cell biological feature, in vivo efficacy, and neuro-therapeutic potential of prosaposin, thereby providing future prospective in clinical application of this multifunctional protein.

The interactomics of sortilin: an ancient lysosomal receptor evolving new functions.

The delivery of soluble lysosomal proteins to the lysosomes is dependent primarily on the mannose 6-phosphate receptor (MPR). The MPR has been demonstrated to attain the early endosomes via a process that requires the interaction of its cytosolic domain with the GGA and AP-1 adaptor proteins. Additionally, the MPR can be recycled back to the trans-Golgi network (TGN) through its interaction with the retromer complex. Interestingly, in I-cell disease (ICD), in which the MPR pathway is non-functional, many soluble lysosomal proteins continue to traffic to the lysosomes. This observation led to the discovery that sortilin is responsible for the MPR-independent targeting of the sphingolipid activator proteins (SAPs) and acid sphingomyelinase (ASM). More recently, our laboratory has tested the hypothesis that sortilin is also capable of sorting a variety of cathepsins that exhibit varying degrees of MPR-independent transport. We have demonstrated that the transport of cathepsin D is partially dependent upon sortilin, that cathepsin H requires sortilin, and that cathepsins K and L attain the lysosomes in a sortilin-independent fashion. As a type-1 receptor, sortilin also has numerous cytosolic binding partners. It has been observed that like the MPR, the anterograde trafficking of sortilin and its cargo require both GGAs and AP-1. Similarly, the retrograde recycling pathway of sortilin also involves an interaction with retromer through a YXXphi site in the cytosolic tail of sortilin. In conclusion, the cytosolic domains of sortilin and MPR possess a high degree of functional homology and both receptors share a conserved trafficking mechanism.

Development of an assay for the intermembrane transfer of cholesterol by Niemann-Pick C2 protein.

Niemann-Pick type C disease is an inherited fatal disorder characterized by the accumulation of unesterified cholesterol and other lipids in the endosomal/lysosomal compartment. Two independent genes responsible for this neurodegenerative disorder have been identified, but the precise functions of the encoded Niemann-Pick C1 (NPC1) and C2 (NPC2) proteins are not yet known. We developed a cell-free assay for measuring intermembrane lipid transport and examined the ability of bovine NPC2 (bNPC2) for intermembrane cholesterol transfer. NPC2 specifically extracts cholesterol from phospholipid bilayers and catalyzes intermembrane transfer to acceptor vesicles in a dose- and time-dependent manner. This transfer activity is dependent on temperature, pH, ionic strength, lipid composition of the model membranes, and the ratio of donor to acceptor vesicles. In model membranes, the presence of the lysosomal anionic phospholipids bis(monooleoylglycero)phosphate and phosphatidyl inositol significantly stimulated cholesterol transfer by NPC2, whereas bis(monomyristoylglycero)phosphate, phosphatidyl serine, and phosphatidic acid had no effect. Moreover, ceramide stimulated cholesterol transfer slightly, whereas sphingomyelin reduced cholesterol transfer rates. With our assay system we identified for the first time the ability of other lysosomal proteins, most notably the GM2-activator protein, to mediate intermembrane cholesterol transfer. This assay system promises to be a valuable tool for further quantitative and mechanistic studies of protein-mediated lipid transfer.

Increased cholesterol in Abeta-positive nerve terminals from Alzheimer's disease cortex.

Synapse loss in Alzheimer's disease (AD) is poorly understood but evidence suggests it is a key pathological event. In order to precisely detect stable synaptic changes, we have developed methods for flow cytometry analysis of synaptosomes prepared from cryopreserved AD samples, and have previously shown that amyloid-beta (Abeta) accumulates in surviving presynaptic terminals in AD cortex. In the present experiments we have examined amyloid-containing terminals in more detail, first dual labeling synaptosomes from AD cortex for Abeta and a series of markers, and then using quadrant analysis to compare amyloid-positive and amyloid-negative terminals. Amyloid-positive synaptosomes were larger in size than amyloid-negatives (p<0.007), and significant increases were observed in mean fluorescence for the lipid raft markers cholesterol (27%; p<0.0005) and GM1 ganglioside (24%; p<0.005). SNAP-25 immunofluorescence was increased by 31% (p<0.0001) in amyloid-bearing terminals, consistent with a sprouting response to amyloid accumulation. These results suggest that Abeta accumulation in synaptic terminals may underly dysfunction prior to or independent of extracellular amyloid deposition.

Oxidative stress generated by hemorrhagic shock recruits Toll-like receptor 4 to the plasma membrane in macrophages.

Oxidative stress generated by ischemia/reperfusion is known to prime inflammatory cells for increased responsiveness to subsequent stimuli, such as lipopolysaccharide (LPS). The mechanism(s) underlying this effect remains poorly elucidated. These studies show that alveolar macrophages recovered from rodents subjected to hemorrhagic shock/resuscitation expressed increased surface levels of Toll-like receptor 4 (TLR4), an effect inhibited by adding the antioxidant N-acetylcysteine to the resuscitation fluid. Consistent with a role for oxidative stress in this effect, in vitro H2O2 treatment of RAW 264.7 macrophages similarly caused an increase in surface TLR4. The H2O2-induced increase in surface TLR4 was prevented by depleting intracellular calcium or disrupting the cytoskeleton, suggesting the involvement of receptor exocytosis. Further, fluorescent resonance energy transfer between TLR4 and the raft marker GM1 as well as biochemical analysis of the raft components demonstrated that oxidative stress redistributes TLR4 to lipid rafts in the plasma membrane. Preventing the oxidant-induced movement of TLR4 to lipid rafts using methyl-beta-cyclodextrin precluded the increased responsiveness of cells to LPS after H2O2 treatment. Collectively, these studies suggest a novel mechanism whereby oxidative stress might prime the responsiveness of cells of the innate immune system.

Amphotropic murine leukemia virus is preferentially attached to cholesterol-rich microdomains after binding to mouse fibroblasts.

BACKGROUND: We have recently shown that amphotropic murine leukemia virus (A-MLV) can enter the mouse fibroblast cell line NIH3T3 via caveola-dependent endocytosis. But due to the size and omega-like shape of caveolae it is possible that A-MLV initially binds cells outside of caveolae. Rafts have been suggested to be pre-caveolae and we here investigate whether A-MLV initially binds to its receptor Pit2, a sodium-dependent phosphate transporter, in rafts or caveolae or outside these cholesterol-rich microdomains. RESULTS: Here, we show that a high amount of cell-bound A-MLV was attached to large rafts of NIH3T3 at the time of investigation. These large rafts were not enriched in caveolin-1, a major structural component of caveolae. In addition, they are rather of natural occurrence in NIH3T3 cells than a result of patching of smaller rafts by A-MLV. Thus cells incubated in parallel with vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped MLV particles showed the same pattern of large rafts as cells incubated with A-MLV, but VSV-G pseudotyped MLV particles did not show any preference to attach to these large microdomains. CONCLUSION: The high concentration of A-MLV particles bound to large rafts of NIH3T3 cells suggests a role of these microdomains in early A-MLV binding events.

Principles of lysosomal membrane digestion: stimulation of sphingolipid degradation by sphingolipid activator proteins and anionic lysosomal lipids.

Sphingolipids and glycosphingolipids are membrane components of eukaryotic cell surfaces. Their constitutive degradation takes place on the surface of intra-endosomal and intra-lysosomal membrane structures. During endocytosis, these intra-lysosomal membranes are formed and prepared for digestion by a lipid-sorting process during which their cholesterol content decreases and the concentration of the negatively charged bis(monoacylglycero)phosphate (BMP)--erroneously also called lysobisphosphatidic acid (LBPA)--increases. Glycosphingolipid degradation requires the presence of water-soluble acid exohydrolases, sphingolipid activator proteins, and anionic phospholipids like BMP. The lysosomal degradation of sphingolipids with short hydrophilic head groups requires the presence of sphingolipid activator proteins (SAPs). These are the saposins (Saps) and the GM2 activator protein. Sphingolipid activator proteins are membrane-perturbing and lipid-binding proteins with different specificities for the bound lipid and the activated enzyme-catalyzed reaction. Their inherited deficiency leads to sphingolipid- and membrane-storage diseases. Sphingolipid activator proteins not only facilitate glycolipid digestion but also act as glycolipid transfer proteins facilitating the association of lipid antigens with immunoreceptors of the CD1 family.

The cholesterol-dependent cytolysin listeriolysin O aggregates rafts via oligomerization.

The pore-forming toxin listeriolysin O (LLO) is the main virulence factor of Listeria monocytogenes. LLO is known to act as a pseudo cytokine/chemokine, which induces a broad spectrum of host responses that ultimately influences the outcome of listeriosis. In the present study we demonstrate that LLO is a potent aggregator of lipid rafts. LLO was found to aggregate the raft associated molecules GM1, the GPI-anchored proteins CD14 and CD16 as well as the tyrosine kinase Lyn. Abrogation of the cytolytic activity of LLO by cholesterol pretreatment was found not to interfere with LLO's ability to aggregate rafts or trigger tyrosine phosphorylation in cells. However, a monoclonal antibody that blocks the oligomerization of LLO was found to inhibit rafts' aggregation as well as the induction of tyrosine phosphorylation. This implies that rafts aggregation by LLO which is independent of cytolytic activity, is due to the oligomerization of its membrane bound toxin monomers. Thus, LLO most likely induces signalling through the coaggregation of rafts' associated receptors, kinases and adaptors.

Structural membrane alterations in Alzheimer brains found to be associated with regional disease development; increased density of gangliosides GM1 and GM2 and loss of cholesterol in detergent-resistant membrane domains.

The formation of neurotoxic beta-amyloid fibrils in Alzheimer's disease (AD) is suggested to involve membrane rafts and to be promoted, in vitro, by enriched concentrations of gangliosides, particularly GM1, and the cholesterol therein. In our study, the presence of rafts and their content of the major membrane lipids and gangliosides in the temporal cortex, reflecting late stages of AD pathology, and the frontal cortex, presenting earlier stages, has been investigated. Whole tissue and isolated detergent-resistant membrane fractions (DRMs) were analysed from 10 AD and 10 age-matched control autopsy brains. DRMs from the frontal cortex of AD brains contained a significantly higher concentration (micromol/micromol glycerophospholipids), of ganglioside GM1 (22.3 +/- 4.6 compared to 10.3 +/- 6.4, p <0.001) and GM2 (2.5 +/- 1.0 compared to 0.55 +/- 0.3, p <0.001). Similar increases of these gangliosides were also seen in DRMs from the temporal cortex of AD brains, which, in addition, comprised significantly lower proportions of DRMs. Moreover, these remaining rafts were depleted in cholesterol (from 1.5 +/- 0.2 to 0.6 +/- 0.3 micromol/micromol glycerophospholipids, p <0.001). In summary, we found an increased proportion of GM1 and GM2 in DRMs, and accelerating plaque formation at an early stage, which may gradually lead to membrane raft disruptions and thereby affect cellular functions associated with the presence of such membrane domains.

Decrease of P-glycoprotein activity in K562/ADR cells by MbetaCD and filipin and lack of effect induced by cholesterol oxidase indicate that this transporter is not located in rafts.

The effect of low-density membrane domains on function of the plasma membrane transporter P-glycoprotéine (P-gp), involved in multidrug resistance (MDR) phenotype, has been investigated in K562/ADR cells. To this end we reversibly altered the cholesterol content of K562/ADR cells by using methyl-beta-cyclodextrin as a cholesterol chelator and conversely we repleted them through incubation with cholesterol in culture medium. We also used the cholesterol-binding fluorochrome filipin and cholesterol oxidase. Our data show that either cholesterol depletion or complex formation with filipin resulted in a strong decrease of P-gp activity. However, when cells were incubated with cholesterol oxidase that are known to disrupt rafts, no modification of the P-gp activity was observed. In addition, using a free-detergent methodology to separate by ultracentrifugation, "light," "heavy," and "extra heavy" fractions we show that no P-gp is found in the "light" fraction where rafts are usually detected. Altogether, our data strongly suggest that, in this cell line, P-gp is not localized in rafts.