Linking glycosphingolipids to Alzheimer's amyloid-ß: extracellular vesicles and functional plant materials.

Glycosphingolipids (GSLs) are a specialized class of membrane lipids composed of a ceramide and a carbohydrate head group. GSLs are localized in cell membranes and were recently found to be enriched in the membrane of neuron-derived exosomes, which are a type of extracellular vesicle. Our studies demonstrated that exosomal GSLs may be associated with the amyloid-ß (Aß) peptide, a principal agent of Alzheimer's disease (AD), and act to clear Aß by transporting Aß into brain phagocytic microglia. In this review, we summarize and discuss the function of exosomal GSLs in Aß homeostasis in AD pathology. Improvement in Aß clearance is a potent strategy for AD prevention and therapy. Dietary glucosylceramides (GlcCer) isolated from plants are absorbed into the body as various metabolites, including ceramides. Our recent work demonstrated that dietary GlcCer accelerates neuronal exosome production, which facilitates Aß clearance in mice. Furthermore, studies of AD model mice and human clinical trials have found that oral administration of plant-type GlcCer attenuates the Aß burden in the brain. We also introduce the development of plant-type GlcCer as functional food materials to prevent AD.

Evaluation of chiral N,N-dimethyl-sphingosine for the interaction between nerve growth factor and tropomyosin receptor kinase A.

Neuropathic pain is an unbearable condition caused by nervous system damage. As distinct acute pain, neuropathic pain is chronic, and it severely influences quality of life. N,N-Dimethyl-d-erythro-sphingosine (DMS), a neuropathic pain inducer, is metabolited de novo from sphingosine. In a recent study, metabolomics showed an increased concentration level of DMS in the spinal cord in mice with neuropathic pain. Nerve growth factor (NGF) is one of the peripheral nervous system targeted pain factors that interact with tropomyosin receptor kinase A (trkA). On the basis of this information, we were interested in the possibility that DMS may induce neuropathic pain-like behavior through an increase of NGF activity. In this study, we showed that DMS can enhance the binding of NGF to trkA, followed by neurite outgrowth of epidermal nerve fibers and phosphorylation of trkA. In addition, a stereoisomer, N,N-dimethyl-l-erythro-sphingosine, did not any show such biological activities. The results suggest that DMS can enhance the binding of NGF to trkA and that its stereochemistry is an essential factor for exhibiting its activity.

Ceramide Metabolism Regulated by Sphingomyelin Synthase 2 Is Associated with Acquisition of Chemoresistance via Exosomes in Human Leukemia Cells.

Ceramide levels controlled by the sphingomyelin (SM) cycle have essential roles in cancer cell fate through the regulation of cell proliferation, death, metastasis, and drug resistance. Recent studies suggest that exosomes confer cancer malignancy. However, the relationship between ceramide metabolism and exosome-mediated cancer malignancy is unclear. In this study, we elucidated the role of ceramide metabolism via the SM cycle in exosomes and drug resistance in human leukemia HL-60 and adriamycin-resistant HL-60/ADR cells. HL-60/ADR cells showed significantly increased exosome production and release compared with parental chemosensitive HL-60 cells. In HL-60/ADR cells, increased SM synthase (SMS) activity reduced ceramide levels, although released exosomes exhibited a high ceramide ratio in both HL-60- and HL-60/ADR-derived exosomes. Overexpression of SMS2 but not SMS1 suppressed intracellular ceramide levels and accelerated exosome production and release in HL-60 cells. Notably, HL-60/ADR exosomes conferred cell proliferation and doxorubicin resistance properties to HL-60 cells. Finally, microRNA analysis in HL-60 and HL-60/ADR cells and exosomes showed that miR-484 elevation in HL-60/ADR cells and exosomes was associated with exosome-mediated cell proliferation. This suggests that intracellular ceramide metabolism by SMS2 regulates exosome production and release, leading to acquisition of drug resistance and enhanced cell proliferation in leukemia cells.

Evaluation of Plant Ceramide Species-Induced Exosome Release from Neuronal Cells and Exosome Loading Using Deuterium Chemistry.

The extracellular accumulation of aggregated amyloid-ß (Aß) in the brain leads to the early pathology of Alzheimer's disease (AD). The administration of exogenous plant-type ceramides into AD model mice can promote the release of neuronal exosomes, a subtype of extracellular vesicles, that can mediate Aß clearance. In vitro studies showed that the length of fatty acids in mammalian-type ceramides is crucial for promoting neuronal exosome release. Therefore, investigating the structures of plant ceramides is important for evaluating the potential in releasing exosomes to remove Aß. In this study, we assessed plant ceramide species with D-erythro-(4E,8Z)-sphingadienine and D-erythro-(8Z)-phytosphingenine as sphingoid bases that differ from mammalian-type species. Some plant ceramides were more effective than mammalian ceramides at stimulating exosome release. In addition, using deuterium chemistry-based lipidomics, most exogenous plant ceramides were confirmed to be derived from exosomes. These results suggest that the ceramide-dependent upregulation of exosome release may promote the release of exogenous ceramides from cells, and plant ceramides with long-chain fatty acids can effectively release neuronal exosomes and prevent AD pathology.

Comprehensive Glycomic Approach Reveals Novel Low-Molecular-Weight Blood Group-Specific Glycans in Serum and Cerebrospinal Fluid.

ABO blood antigens on the human red blood cell membrane as well as different cells in various human tissues have been thoroughly studied. Anti-A and -B antibodies of IgM are present in serum/plasma, but blood group-specific glyco-antigens have not been extensively described. In this study, we performed comprehensive and quantitative serum glycomic analyses of various glycoconjugates and free oligosaccharides in all blood groups. Our comprehensive glycomic approach revealed that blood group-specific antigens in serum/plasma are predominantly present on glycosphingolipids on lipoproteins rather than glycoproteins. Expression of the ABO antigens on glycosphingolipids depends not only on blood type but also on secretor status. Blood group-specific glycans in serum/plasma were classified as type I, whereas those on RBCs had different structures including hexose and hexosamine residues. Analysis of free oligosaccharides revealed that low-molecular-weight blood group-specific glycans, commonly containing lacto-N-difucotetraose, were expressed in serum/plasma according to blood group. Furthermore, comprehensive glycomic analysis in human cerebrospinal fluid showed that many kinds of free oligosaccharides were highly expressed, and low-molecular-weight blood group-specific glycans, which existed in plasma from the same individuals, were present. Our findings provide the first evidence for low-molecular-weight blood group-specific glycans in both serum/plasma and cerebrospinal fluid.

Mevalonate Pathway-mediated ER Homeostasis Is Required for Haploid Stability in Human Somatic Cells.

The somatic haploidy is unstable in diplontic animals, but cellular processes determining haploid stability remain elusive. Here, we found that inhibition of mevalonate pathway by pitavastatin, a widely used cholesterol-lowering drug, drastically destabilized the haploid state in HAP1 cells. Interestingly, cholesterol supplementation did not restore haploid stability in pitavastatin-treated cells, and cholesterol inhibitor U18666A did not phenocopy haploid destabilization. These results ruled out the involvement of cholesterol in haploid stability. Besides cholesterol perturbation, pitavastatin induced endoplasmic reticulum (ER) stress, the suppression of which by a chemical chaperon significantly restored haploid stability in pitavastatin-treated cells. Our data demonstrate the involvement of the mevalonate pathway in the stability of the haploid state in human somatic cells through managing ER stress, highlighting a novel link between ploidy and ER homeostatic control.Key words: haploid, ER stress, Mevalonate pathway.

Lysosomal-associated transmembrane protein 4B regulates ceramide-induced exosome release.

Exosomes are extracellular vesicles that mediate the transport of intracellular molecules, including neurodegenerative agents. Exogenously administrated ceramides have been implicated in the acceleration of exosome production by neurons; however, the molecular machinery involved in this process is unknown. Here, we found that ceramides, especially those consisting of long fatty acids, were internalized into the endocytic pathway in neuroblastoma SH-SY5Y cells to induce exosome secretion through lysosome-associated protein transmembrane 4B (LAPTM4B). Knockdown of LAPTM4B inhibited the ceramide-mediated increase in exosome release completely. Fluorescence microscopy observations indicated that exogenous ceramides promote the transport of multivesicular bodies to the plasma membranes in a LAPTM4B-dependent manner. Similarly, inhibition of acid ceramidase, which tends to induce intracellular ceramide accumulation, increased exosome production by SH-SY5Y cells in a LAPTM4B-dependent manner. Furthermore, the level of amyloid-ß protein (Aß) was decreased in neuronal cells following treatment with exogenous ceramide or inhibition of acid ceramidase, and this effect was attributed to the LAPTM4B-dependent efflux of Aß-containing exosomes. Overall, these findings reveal the novel machinery involved in exosome secretion regulated by ceramides and LAPTM4B, and may contribute to efforts to ameliorate the cellular accumulation of neurodegenerative agents such as Aß.

Structure-dependent absorption of atypical sphingoid long-chain bases from digestive tract into lymph.

BACKGROUND: Dietary sphingolipids have various biofunctions, including skin barrier improvement and anti-inflammatory and anti-carcinoma properties. Long-chain bases (LCBs), the essential backbones of sphingolipids, are expected to be important for these bioactivities, and they vary structurally between species. Given these findings, however, the absorption dynamics of each LCB remain unclear. METHODS: In this study, five structurally different LCBs were prepared from glucosylceramides (GlcCers) with LCB 18:2(4E,8Z);2OH and LCB 18:2(4E,8E);2OH moieties derived from konjac tuber (Amorphophallus konjac), from GlcCers with an LCB 18(9Me):2(4E,8E);2OH moiety derived from Tamogi mushroom (Pleurotus cornucopiae var. citrinopileatus), and from ceramide 2-aminoethyphosphonate with LCB 18:3(4E,8E,10E);2OH moiety and LCB 18(9Me):3(4E,8E,10E);2OH moiety derived from giant scallop (Mizuhopecten yessoensis), and their absorption percentages and metabolite levels were analyzed using a lymph-duct-cannulated rat model via liquid chromatography tandem mass spectrometry (LC/MS/MS) with a multistage fragmentation method. RESULTS: The five orally administered LCBs were absorbed and detected in chyle (lipid-containing lymph) as LCBs and several metabolites including ceramides, hexosylceramides, and sphingomyelins. The absorption percentages of LCBs were 0.10-1.17%, depending on their structure. The absorption percentage of LCB 18:2(4E,8Z);2OH was the highest (1.17%), whereas that of LCB 18:3(4E,8E,10E);2OH was the lowest (0.10%). The amount of sphingomyelin with an LCB 18:2(4E,8Z);2OH moiety in chyle was particularly higher than sphingomyelins with other LCB moieties. CONCLUSIONS: Structural differences among LCBs, particularly geometric isomerism at the C8-C9 position, significantly affected the absorption percentages and ratio of metabolites. This is the first report to elucidate that the absorption and metabolism of sphingolipids are dependent on their LCB structure. These results could be used to develop functional foods that are more readily absorbed.

Sphingomyelin synthase 2 loss suppresses steatosis but exacerbates fibrosis in the liver of mice fed with choline-deficient, L-amino acid-defined, high-fat diet.

Sphingomyelin synthase 2 (SMS2) regulates sphingomyelin synthesis and contributes to obesity and hepatic steatosis. Here, we investigated the effect of SMS2 deficiency on liver fibrosis in mice fed with choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) or injected with carbon tetrachloride (CCl4), respectively. SMS2 deficiency suppressed hepatic steatosis, but exacerbated fibrosis induced by CDAHFD feeding. Sphingosine 1-phosphate (S1P), which is a key lipid mediator induces fibrosis in various organs, was increased in the liver of mice fed with CDAHFD. The increase of S1P became prominent by SMS2 deficiency. Meanwhile, SMS2 deficiency had no impact on CCl4-induced liver injury, fibrosis and S1P levels. Our findings demonstrated that SMS2 deficiency suppresses steatosis but worsens fibrosis in the liver in a specific condition with CDAHFD feeding.

Neurite Outgrowth and Morphological Changes Induced by 8-trans Unsaturation of Sphingadienine in kCer Molecular Species.

Konjac ceramide (kCer), which consists of plant-type molecular species of characteristic shingoid bases and fatty acids, is prepared from konjac glucosylceramide GlcCer by chemoenzymatical deglucosylation. kCer activates the semaphorin 3A (Sema3A) signaling pathway, inducing collapsin response mediator protein 2 (CRMP2) phosphorylation. This results in neurite outgrowth inhibition and morphological changes in remaining long neurites in PC12 cells. Whether a specific molecular species of kCer can bind to the Sema3A receptor (Neuropilin1, Nrp1) and activate the Sema3A signaling pathway remains unknown. Here, we prepared kCer molecular species using endoglycoceramidase I-mediated deglucosylation and examined neurite outgrowth and phosphorylation of collapsin response mediator protein 2 in nerve growth factor (NGF)-primed cells. The 8-trans unsaturation of sphingadienine of kCer was essential for Sema3A-like signaling pathway activation. Conversely, 8-cis unsaturation of kCer molecular species had no effect on Sema3A-like activation, and neurite outgrowth inhibition resulted in remaining short neurites. In addition, α-hydroxylation of fatty acids was not associated with the Sema3A-like activity of the kCer molecular species. These results suggest that 8-trans or 8-cis isomerization of sphingadienine determines the specific interactions at the ligand-binding site of Nrp1.

Sphingomyelin in microdomains of the plasma membrane regulates amino acid-stimulated mTOR signal activation.

Sphingomyelin (SM) is required for cells to proliferate, but the reason is not fully understood. In order to asses this question, we employed a cell line, ZS, which lacks both SMS1 and SMS2, isolated from mouse embryonic fibroblasts in SMS1 and 2 double knockout mouse, and SMS1 or SMS2 re-expressing cells, ZS/SMS1 or ZS/SMS2, respectively. We investigated regulation of SM in activating the mammalian target of rapamycin (mTOR) signal induced by essential amino acids (EAA), using these cells. EAA-stimulated mTOR signal was more activated in ZS/SMS1 and ZS/SMS2 cells than in controls. Treatment with methyl-b-cyclodextrin dramatically inhibited the activation. Interestingly, we found that the expression of CD98, LAT-1 and ASCT-2, amino acid transporters concerned with mTOR activation, was down-regulated in ZS cells. Transporters localized in microdomains and formed a functional complex. Our results indicate that SM affect proliferation through the transport of amino acids via SM-enriched microdomains.

Characterization of the role of sphingomyelin synthase 2 in glucose metabolism in whole-body and peripheral tissues in mice.

Sphingomyelin synthase 2 (SMS2) is a proposed potential therapeutic target for obesity and insulin resistance. However, the contributions of SMS2 to glucose metabolism in tissues and its possible therapeutic mechanisms remain unclear. Thus, to determine whole-body glucose utilization and the contributions of each insulin-targeted tissue to glucose uptake, we performed a glucose kinetics study, using the radiolabeled glucose analog (18)F-2-fluoro-2-deoxy-D-glucose ((18)F-FDG), in wild-type (WT) and SMS2 knockout (KO) mice. Insulin signaling was enhanced in the liver, white adipose tissue and skeletal muscle of SMS2 KO mice compared with those of WT mice. In addition, compared with in WT mice, blood clearance of (18)F-FDG was accelerated in SMS2 KO mice when they were fed either a normal or a high fat diet. (18)F-FDG uptake was also increased in insulin-targeted tissues such as skeletal muscle in the SMS2 KO mice. Whereas skeletal muscle sphingolipid content was not clearly affected, plasma levels of very long-chain fatty acid (VLCFA)-containing ceramides were markedly increased in SMS2 KO mice, compared with in WT mice. We also generated liver-conditional SMS2 KO mice and performed glucose and insulin tolerance tests on mice with a high fat diet. However, no significant effect was observed. Thus, our study provided evidence that genetic inhibition of SMS2 elevated glucose clearance through activation of glucose uptake into insulin-targeted tissues such as skeletal muscle by a mechanism independent of hepatic SMS2. Our findings further indicate that this occurs, at least in part, via indirect mechanisms such as elevation of VLCFA-containing ceramides.

Possible roles of long-chain sphingomyelines and sphingomyelin synthase 2 in mouse macrophage inflammatory response.

To evaluate the precise role of sphingomyelin synthase 2 (SMS2) in sphingomyelin (SM) metabolism and their anti-inflammatory properties, we analyzed species of major SM and ceramide (Cer) (18:1, 18:0 sphingoid backbone, C14 - C26 N-acyl part) in SMS2 knockout and wild-type mouse plasma and liver using HPLC-MS. SMS2 deficiency significantly decreased very long chain SM (SM (d18:1/22:0) and SM (d18:1/24:0 or d18:0/24:1)) and increased very long chain Cer (Cer (d18:1/24:0 or d18:0/24:1) and Cer (d18:1/24:1)), but not long chain SM (SM (d18:1/16:0), SM (d18:1/18:0 or d18:0/18:1) and SM (d18:1/18:1)) in plasma. To examine the effects of SM on inflammation, we studied the role of very long chain SM in macrophage activation. Addition of SM (d18:1/24:0) strongly upregulated several macrophage activation markers, SM (d18:1/6:0) and Cer (d18:1/24:0) however, did not. It was suggested that very long chain SM but not long chain SM were decreased in SMS2-deficient mice liver and plasma. And the exogenously added very long chain SM (d18:1/24:0) could activate macrophages directly, suggesting a novel role of plasma very long chain SM in modulating macrophage activation and resulting inflammation.

Highly efficient preparation of sphingoid bases from glucosylceramides by chemoenzymatic method.

Sphingoid base derivatives have attracted increasing attention as promising chemotherapeutic candidates against lifestyle diseases such as diabetes and cancer. Natural sphingoid bases can be a potential resource instead of those derived by time-consuming total organic synthesis. In particular, glucosylceramides (GlcCers) in food plants are enriched sources of sphingoid bases, differing from those of animals. Several chemical methodologies to transform GlcCers to sphingoid bases have already investigated; however, these conventional methods using acid or alkaline hydrolysis are not efficient due to poor reaction yield, producing complex by-products and resulting in separation problems. In this study, an extremely efficient and practical chemoenzymatic transformation method has been developed using microwave-enhanced butanolysis of GlcCers and a large amount of readily available almond ß-glucosidase for its deglycosylation reaction of lysoGlcCers. The method is superior to conventional acid/base hydrolysis methods in its rapidity and its reaction cleanness (no isomerization, no rearrangement) with excellent overall yield.

Histological analyses by matrix-assisted laser desorption/ionization-imaging mass spectrometry reveal differential localization of sphingomyelin molecular species regulated by particular ceramide synthase in mouse brains.

Sphingomyelin (SM) is synthesized by SM synthase (SMS) from ceramide (Cer). SM regulates signaling pathways and maintains organ structure. SM comprises a sphingoid base and differing lengths of acyl-chains, but the importance of its various forms and regulatory synthases is not known. It has been reported that Cer synthase (CerS) has restricted substrate specificity, whereas SMS has no specificity for different lengths of acyl-chains. We hypothesized that the distribution of each SM molecular species was regulated by expression of the CerS family. Thus, we compared the distribution of SM species and CerS mRNA expression using molecular imaging. Spatial distribution of each SM molecular species was investigated using ultra-high-resolution imaging mass spectrometry (IMS). IMS revealed that distribution of SM molecular species varied according to the lengths of acyl-chains found in each brain section. Furthermore, a combination study using in situ hybridization and IMS revealed the spatial expression of CerS1 to be associated with the localization of SM (d18:1/18:0) in cell body-rich gray matter, and CerS2 to be associated with SM (d18:1/24:1) in myelin-rich white matter. Our study is the first comparison of spatial distribution between SM molecular species and CerS isoforms, and revealed their distinct association in the brain. These observations were demonstrated by suppression of CerS2 using siRNA in HepG2 cells; that is, siRNA for CerS2 specifically decreased C22 very long-chain fatty acid (VLCFA)- and C24 VLCFA-containing SMs. Thus, histological analyses of SM species by IMS could be a useful approach to consider their molecular function and regulative mechanism.

Quantitative GSL-glycome analysis of human whole serum based on an EGCase digestion and glycoblotting method.

Glycosphingolipids (GSLs) are lipid molecules linked to carbohydrate units that form the plasma membrane lipid raft, which is clustered with sphingolipids, sterols, and specific proteins, and thereby contributes to membrane physical properties and specific recognition sites for various biological events. These bioactive GSL molecules consequently affect the pathophysiology and pathogenesis of various diseases. Thus, altered expression of GSLs in various diseases may be of importance for disease-related biomarker discovery. However, analysis of GSLs in blood is particularly challenging because GSLs are present at extremely low concentrations in serum/plasma. In this study, we established absolute GSL-glycan analysis of human serum based on endoglycoceramidase digestion and glycoblotting purification. We established two sample preparation protocols, one with and the other without GSL extraction using chloroform/methanol. Similar amounts of GSL-glycans were recovered with the two protocols. Both protocols permitted absolute quantitation of GSL-glycans using as little as 20 µl of serum. Using 10 healthy human serum samples, up to 42 signals corresponding to GSL-glycan compositions could be quantitatively detected, and the total serum GSL-glycan concentration was calculated to be 12.1-21.4 µM. We further applied this method to TLC-prefractionated serum samples. These findings will assist the discovery of disease-related biomarkers by serum GSL-glycomics.

Decreased amyloid-ß pathologies by intracerebral loading of glycosphingolipid-enriched exosomes in Alzheimer model mice.

Elevated levels of amyloid-ß peptide (Aß) in the human brain are linked to the pathogenesis of Alzheimer disease. Recent in vitro studies have demonstrated that extracellular Aß can bind to exosomes, which are cell-secreted nanovesicles with lipid membranes that are known to transport their cargos intercellularly. Such findings suggest that the exosomes are involved in Aß metabolism in brain. Here, we found that neuroblastoma-derived exosomes exogenously injected into mouse brains trapped Aß and with the associated Aß were internalized into brain-resident phagocyte microglia. Accordingly, continuous intracerebral administration of the exosomes into amyloid-ß precursor protein transgenic mice resulted in marked reductions in Aß levels, amyloid depositions, and Aß-mediated synaptotoxicity in the hippocampus. In addition, we determined that glycosphingolipids (GSLs), a group of membrane glycolipids, are highly abundant in the exosomes, and the enriched glycans of the GSLs are essential for Aß binding and assembly on the exosomes both in vitro and in vivo. Our data demonstrate that intracerebrally administered exosomes can act as potent scavengers for Aß by carrying it on the exosome surface GSLs and suggest a role of exosomes in Aß clearance in the central nervous system. Improving Aß clearance by exosome administration would provide a novel therapeutic intervention for Alzheimer disease.

Pathological roles of ceramide and its metabolites in metabolic syndrome and Alzheimer's disease.

The public health burden of metabolic syndrome (MetS), a multiplex risk factor that arises from insulin resistance accompanying abnormal adipose conditions, and Alzheimer's disease (AD), the most common form of dementia, continues to expand. Current available therapies for these disorders are of limited effectiveness. Recent findings have indicated that alternations in sphingolipid metabolism contribute to the development of these pathologies. Sphingolipids are major constituents of the plasma membrane, where they are known to form several types of microdomains, and are potent regulators for a variety of physiological processes. Many groups, including ours, have demonstrated that membrane sphingolipids, especially ceramide and its metabolites such as ceramide 1-phosphate, have roles in arteriosclerosis, obesity, diabetes, and inflammation associated with MetS. Aberrant sphingolipid profiles have been observed in human AD brains, and accumulated evidence has demonstrated that changes in membrane properties induced by defective sphingolipid metabolism impair generation and degradation of amyloid-ß peptide (Aß), a pathogenic agent of AD. In this review, we summarize current knowledge and pathophysiological implications of the roles of SLs in MetS and AD, to provide insight into the SL metabolic pathways as potential targets for therapy of these diseases. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.

Amelioration of collagen-induced arthritis by a novel S1P1 antagonist with immunomodulatory activities.

Sphingosine 1-phosphate (S1P) regulates lymphocyte trafficking through the type 1 sphingosine 1-phosphate receptor (S1P(1)) and participates in many pathological conditions, including autoimmune diseases. We developed a novel S1P(1)-selective antagonist, TASP0277308, which is structurally unrelated to S1P. This antagonist competitively inhibited S1P-induced cellular responses, such as chemotaxis and receptor internalization. Furthermore, differing from previously reported S1P(1) antagonists, TASP0277308 demonstrated in vivo activities to induce lymphopenia, a block in T cell egress from the thymus, displacement of marginal zone B cells, and upregulation of CD69 expression on both T and B cells, all of which recapitulate phenotypes of S1P(1)-deficient lymphocytes. In a mouse collagen-induced arthritis model, TASP0277308 significantly suppressed the development of arthritis, even after the onset of disease. These findings provide the first chemical evidence to our knowledge that S1P(1) antagonism is responsible for immunosuppression in the treatment of autoimmune diseases and also resolve the discrepancies between genetic and chemical studies on the functions of S1P(1) in lymphocytes.

Sphingolipid-modulated exosome secretion promotes clearance of amyloid-ß by microglia.

Amyloid ß-peptide (Aß), the pathogenic agent of Alzheimer disease, is a physiological metabolite whose levels are constantly controlled in normal brain. Recent studies have demonstrated that a fraction of extracellular Aß is associated with exosomes, small membrane vesicles of endosomal origin, although the fate of Aß in association with exosome is largely unknown. In this study, we identified novel roles for neuron-derived exosomes acting on extracellular Aß, i.e. exosomes drive conformational changes in Aß to form nontoxic amyloid fibrils and promote uptake of Aß by microglia. The Aß internalized together with exosomes was further transported to lysosomes and degraded. We also found that blockade of phosphatidylserine on the surface of exosomes by annexin V not only prevented exosome uptake but also suppressed Aß incorporation into microglia. In addition, we demonstrated that secretion of neuron-derived exosomes was modulated by the activities of sphingolipid-metabolizing enzymes, including neutral sphingomyelinase 2 (nSMase2) and sphingomyelin synthase 2 (SMS2). In transwell experiments, up-regulation of exosome secretion from neuronal cells by treatment with SMS2 siRNA enhanced Aß uptake into microglial cells and significantly decreased extracellular levels of Aß. Our findings indicate a novel mechanism responsible for clearance of Aß through its association with exosomes. The modulation of the vesicle release and/or elimination may alter the risk of AD.