Lipidome profiling of neutrophil-derived extracellular vesicles unveils their contribution to the ensemble of synovial fluid-derived extracellular vesicles during joint inflammation.

The molecular signature of cell-derived extracellular vesicles (EVs) from synovial fluid (SF) offers insights into the cells and molecular processes associated with joint disorders and can be exploited to define biomarkers. The EV-signature is determined by cargo molecules and the lesser-studied lipid bilayer. We here investigated the lipidome of SF-EVs in inflamed joints derived from Rheumatoid Arthritis (RA) and Spondyloarthritis (SpA) patients, two autoimmune-driven joint diseases, and compared these signatures to the lipid profile of equine SF-EVs obtained during induced acute synovitis. Since neutrophils are primary SF-infiltrating cells during these inflammatory joint diseases, we also analyzed how inflammatory stimuli alter the lipidomic profile of human and equine neutrophil-derived EVs (nEVs) in vitro and how these signatures relate to the lipidome signatures of SF-EVs from inflamed joints. We identified neutrophil stimulation intensity-dependent changes in the lipidomic profile of nEVs with elevated presence of dihexosylceramide (lactosylceramide), phosphatidylserine, and phosphatidylethanolamine ether-linked lipid classes in human nEVs upon full neutrophil activation. In horses, levels of monohexosylceramide (glucosylceramide) increased instead of dihexosylceramide, indicating species-specific differences. The lipid profiles of RA and SpA SF-EVs were relatively similar and showed a relative resemblance with stimulated human nEVs. Similarly, the lipidome of equine synovitis-derived SF-EVs closer resembled the one of stimulated equine nEVs. Hence, lipidome profiling can provide insights into the contribution of nEVs to the heterogeneous pool of SF-EVs, deepening our understanding of inflammatory joint diseases and revealing molecular changes in joint homeostasis, which can lead to the development of more precise disease diagnosis and treatment strategies.

Glycosphingolipids in Diabetes, Oxidative Stress, and Cardiovascular Disease: Prevention in Experimental Animal Models.

Diabetes contributes to about 30% morbidity and mortality world-wide and has tidal wave increases in several countries in Asia. Diabetes is a multi-factorial disease compounded by inflammation, dyslipidemia, atherosclerosis, and is sometimes accompanied with gains in body weight. Sphingolipid pathways that interplay in the enhancement of the pathology of this disease may be potential therapeutic targets. Thus, the application of advanced sphingolipidomics may help predict the progression of this disease and therapeutic outcomes in man. Pre-clinical studies using various experimental animal models of diabetes provide valuable information on the role of sphingolipid signaling networks in diabetes and the efficacy of drugs to determine the translatability of innovative discoveries to man. In this review, we discuss three major concepts regarding sphingolipids and diabetes. First, we discuss a possible involvement of a monosialodihexosylceramide (GM3) in insulin-insulin receptor interactions. Second, a potential role for ceramide (Cer) and lactosylceramide (LacCer) in apoptosis and mitochondrial dysfunction is proposed. Third, a larger role of LacCer in antioxidant status and inflammation is discussed. We also discuss how inhibitors of glycosphingolipid synthesis can ameliorate diabetes in experimental animal models.

Lactosylceramide-enriched microdomains mediate human neutrophil immunological functions via carbohydrate-carbohydrate interaction.

The innate immune system of mammalian cells is the first line of defense against pathogenic microorganisms. Phagocytes, which play the central role in this system, engulf microorganisms by a mechanism that involves pattern recognition receptors on their own surface and pathogen-associated molecular patterns (PAMPs) expressed by the microorganism. Components of PAMPs include glycans (polysaccharides) and glycoconjugates (carbohydrates covalently linked to other biological molecules). Pathogenic microorganisms display specific binding affinity to various types of glycosphingolipids (sphingosine-containing glycolipids; GSLs), and GSLs are involved in host-pathogen interactions. We observed that lactosylceramide (LacCer), a neutral GSL, binds directly to certain pathogen-specific molecules (e.g., Candida albicans-derived ß-glucans, mycobacterial lipoarabinomannan) via carbohydrate-carbohydrate interaction. LacCer is expressed highly on human neutrophils, and forms membrane microdomains. Such LacCer-enriched microdomains mediate several important neutrophil functions, including chemotaxis, phagocytosis, and superoxide generation. Human neutrophils phagocytose pathogenic mycobacteria (including Mycobacterium tuberculosis) through carbohydrate-carbohydrate interaction between LacCer on their own surface and mannose-capped lipoarabinomannan on the bacterium. During recognition of pathogen-specific glycans, direct association of LacCer-containing C24 fatty acid chain with Lyn (a Src family kinase) is necessary for signal transduction from the neutrophil exterior to interior. Pathogenic mycobacteria utilize a similar interaction to avoid killing by neutrophils. We describe here the mechanisms whereby LacCer mediates neutrophil immune systems via carbohydrate-carbohydrate interaction.

Galactocerebrosidase deficiency induces an increase in lactosylceramide content: A new hallmark of Krabbe disease?

Galactocerebrosidase (GALC) hydrolyses galactose residues from various substrates, including galactosylceramide, psychosine (galactosylsphingosine), and lactosylceramide. Its severe deficiency has been associated with the accumulation of psychosine, a toxic molecule with detergent-like features, which alters membrane structures and signalling pathways, inducing the death of oligodendrocytes and a sequence of events in the nervous system that explain the appearance of many clinical signs typical of Krabbe disease. Nevertheless, new evidence suggests the existence of other possible links among GALC action, myelination, and myelin stability, apart from psychosine release. In this study, we demonstrated that lactosylceramide metabolism is impaired in fibroblasts isolated from patients with Krabbe disease in the absence of psychosine accumulation. This event is responsible for the aberrant and constitutive activation of the AKT/prolin-rich AKT substrate of 40 kDa (PRAS40) signalling axis, inducing B cell lymphoma 2 (BCL2) overexpression and glycogen synthase kinase 3 beta (GSK-3ß) inhibition. In addition, nuclear factor E2-related factor 2 (NRF2) showed increased nuclear translocation. Due to the relevance of these molecular alterations in neurodegeneration, lactosylceramide increase should be evaluated as a novel marker of Krabbe disease, and because of its significant connections with signalling pathways.

Empagliflozin Improves Metabolic and Hepatic Outcomes in a Non-Diabetic Obese Biopsy-Proven Mouse Model of Advanced NASH.

Empagliflozin, an established treatment for type 2 diabetes (T2DM), has shown beneficial effects on liver steatosis and fibrosis in animals and in humans with T2DM, non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH). However, little is known about the effects of empagliflozin on liver function in advanced NASH with liver fibrosis and without diabetes. This study aimed to assess the effects of empagliflozin on hepatic and metabolic outcomes in a diet-induced obese (DIO) and insulin-resistant but non-diabetic biopsy-confirmed mouse model of advanced NASH. Male C57BL/6JRj mice with a biopsy-confirmed steatosis and fibrosis on AMLN diet (high fat, fructose and cholesterol) for 36-weeks were randomized to receive for 12 weeks: (a) Empagliflozin (10 mg/kg/d p.o.), or (b) vehicle. Metabolic outcomes, liver pathology, markers of Kupffer and stellate cell activation and lipidomics were assessed at the treatment completion. Empagliflozin did not affect the body weight, body composition or insulin sensitivity (assessed by intraperitoneal insulin tolerance test), but significantly improved glucose homeostasis as assessed by oral glucose tolerance test in DIO-NASH mice. Empagliflozin improved modestly the NAFLD activity score compared with the vehicle, mainly by improving inflammation and without affecting steatosis, the fibrosis stage and markers of Kupffer and stellate cell activation. Empagliflozin reduced the hepatic concentrations of pro-inflammatory lactosylceramides and increased the concentrations of anti-inflammatory polyunsaturated triglycerides. Empagliflozin exerts beneficial metabolic and hepatic (mainly anti-inflammatory) effects in non-diabetic DIO-NASH mice and thus may be effective against NASH even in non-diabetic conditions.

Lipidomics analysis of monocytes from patients with acute myocardial infarction reveals lactosylceramide as a new player in monocyte migration.

Monocyte recruitment after vascular injury and their migration through the vessel wall represent crucial events in the initiation, progression, and destabilization of atherosclerotic plaque. Circulating monocytes are exposed to stimuli that alter their physiological state, and among them, lipids play a key role. Several studies investigated the mechanisms by which lipids affect monocyte functions promoting coronary atherosclerotic plaque initiation, but information on the relationship between lipid composition and function of monocyte is scant. We aimed at studying the migration of circulating monocytes isolated from patients with acute myocardial infarction (AMI) at hospital presentation and investigating its correlation with cellular lipid profile. The migration of monocytes was tested using both fetal bovine serum (FBS) and autologous serum as chemoattractant stimuli. Monocyte lipid profile was evaluated through an untargeted lipidomics approach, using a liquid chromatography/time-of-flight mass spectrometry platform. We observed that AMI patients' monocytes showed a significant increase in FBS and autologous serum-mediated migration compared to controls. Moreover, a different monocyte lipidomic profile between the two study groups was detected. In particular, AMI patients' monocytes showed an altered composition in ceramides, with an increase in lactosylceramide and in phospholipids (ie, phosphatidylethanolamine and lisophosphatidylethanolamine). Of note, a positive correlation between lactosylceramide levels and monocyte migration was observed. Furthermore, the lactosylceramide synthase inhibition significantly reduced FBS-induced monocyte migration. Our results highlight the influence of lactosylceramide on the monocyte migration capacity, pointing out a new possible mechanism of lipids in the onset of atherothrombosis and, hence, in AMI.

Effect of Fluorofenidone Against Paraquat-Induced Pulmonary Fibrosis Based on Metabolomics and Network Pharmacology.

BACKGROUND Fluorofenidone (AKF-PD) is an anti-fibrotic small-molecule compound. Its mechanism of action on paraquat (PQ)-induced pulmonary fibrosis is still unclear. MATERIAL AND METHODS Forty-eight SD rats were divided into 4 groups: control group, PQ group, PQ+AKF-PD group, and AKF-PD group. The pathological changes of lung tissues were observed by Masson and HE staining. The UPLC-QTOF-MS analysis was performed to detect the differences in metabolites among groups, then the possible mechanisms of the anti-pulmonary fibrosis effects of fluorofenidone were further revealed by network pharmacology analysis. Biological methods were used to verify the results of the network pharmacology analysis. RESULTS The results showed that fluorofenidone treatment significantly alleviated paraquat-induced pulmonary fibrosis. Metabolomics analysis showed that 18 metabolites were disordered in the serum of paraquat-poisoned rats, of which 13 were restored following fluorofenidone treatment. Network pharmacology analysis showed that the drug screened a total of 12 targets and mainly involved multiple signaling pathways and metabolic pathways to jointly exert anti-pulmonary fibrosis effects. Autophagy is the main pathway of fluorofenidone in treatment pulmonary fibrosis. The western blot results showed that fluorofenidone upregulated the expression of LC3-II/I and E-cadherin, and downregulated the expression of p62, alpha-SMA, and TGF-ß1, which validated that fluorofenidone could inhibit the development of paraquat-induced pulmonary fibrosis by increasing autophagy. CONCLUSIONS In conclusion, metabolomics combined with network pharmacology research strategy revealed that fluorofenidone has a multi-target and multi-path mechanism of action in the treatment of pulmonary fibrosis.

Convergence: Lactosylceramide-Centric Signaling Pathways Induce Inflammation, Oxidative Stress, and Other Phenotypic Outcomes.

Lactosylceramide (LacCer), also known as CD17/CDw17, is a member of a large family of small molecular weight compounds known as glycosphingolipids. It plays a pivotal role in the biosynthesis of glycosphingolipids, primarily by way of serving as a precursor to the majority of its higher homolog sub-families such as gangliosides, sulfatides, fucosylated-glycosphingolipids and complex neutral glycosphingolipids-some of which confer "second-messenger" and receptor functions. LacCer is an integral component of the "lipid rafts," serving as a conduit to transduce external stimuli into multiple phenotypes, which may contribute to mortality and morbidity in man and in mouse models of human disease. LacCer is synthesized by the action of LacCer synthase (ß-1,4 galactosyltransferase), which transfers galactose from uridine diphosphate galactose (UDP-galactose) to glucosylceramide (GlcCer). The convergence of multiple physiologically relevant external stimuli/agonists-platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), stress, cigarette smoke/nicotine, tumor necrosis factor-α (TNF-α), and in particular, oxidized low-density lipoprotein (ox-LDL)-on ß-1,4 galactosyltransferase results in its phosphorylation or activation, via a "turn-key" reaction, generating LacCer. This newly synthesized LacCer activates NADPH (nicotinamide adenine dihydrogen phosphate) oxidase to generate reactive oxygen species (ROS) and a highly "oxidative stress" environment, which trigger a cascade of signaling molecules and pathways and initiate diverse phenotypes like inflammation and atherosclerosis. For instance, LacCer activates an enzyme, cytosolic phospholipase A2 (cPLA2), which cleaves arachidonic acid from phosphatidylcholine. In turn, arachidonic acid serves as a precursor to eicosanoids and prostaglandin, which transduce a cascade of reactions leading to inflammation-a major phenotype underscoring the initiation and progression of several debilitating diseases such as atherosclerosis and cancer. Our aim here is to present an updated account of studies made in the field of LacCer metabolism and signaling using multiple animal models of human disease, human tissue, and cell-based studies. These advancements have led us to propose that previously unrelated phenotypes converge in a LacCer-centric manner. This LacCer synthase/LacCer-induced "oxidative stress" environment contributes to inflammation, atherosclerosis, skin conditions, hair greying, cardiovascular disease, and diabetes due to mitochondrial dysfunction. Thus, targeting LacCer synthase may well be the answer to remedy these pathologies.

Hydrogen sulfide exacerbated periodontal inflammation and induced autophagy in experimental periodontitis.

Hydrogen sulfide (H2S), the metabolite produced by gram-negative bacteria, is present in deep periodontal pockets of periodontitis patients at high concentrations. The harsh conditions in the diseased periodontium may stimulate a local autophagy response. However, how H2S participates in pathogenesis and whether H2S induces autophagy in periodontitis remain partially unknown. In this article, we determined the role of the slow-releasing H2S donor GYY4137 in experimental periodontitis and its possible regulation in autophagy involved. We found that GYY4137 dose-dependently decreased cell viability and increased the level of proinflammatory cytokines in LPS-stimulated human periodontal ligament cells (HPDLCs). Topically applied GYY4137 also exacerbated periodontal inflammation and alveolar bone loss in ligature-induced rats. Moreover, GYY4137 activated autophagy by upregulating the expression levels of the autophagy-related proteins LC3 and Beclin-1 and downregulating P62 in LPS-treated HPDLCs and inflamed periodontal tissues. Blocking autophagy with 3-methyladenine resulted in further increased expression of proinflammatory cytokines in LPS- and GYY4137-induced HPDLCs. Our results indicate that GYY4137 exerted proinflammatory effects and promoted autophagy in periodontitis, and the induced autophagy may function as a cytoprotective mechanism to prevent excessive inflammation.

Oleate-induced aggregation of LC3 at the trans-Golgi network is linked to a protein trafficking blockade.

Oleate, the most abundant endogenous and dietary cis-unsaturated fatty acid, has the atypical property to cause the redistribution of microtubule-associated proteins 1A/1B light chain 3B (referred to as LC3) to the trans-Golgi network (TGN), as shown here. A genome-wide screen identified multiple, mostly Golgi transport-related genes specifically involved in the oleate-induced relocation of LC3 to the Golgi apparatus. Follow-up analyses revealed that oleate also caused the retention of secreted proteins in the TGN, as determined in two assays in which the secretion of proteins was synchronized, (i) an assay involving a thermosensitive vesicular stomatitis virus G (VSVG) protein that is retained in the endoplasmic reticulum (ER) until the temperature is lowered, and (ii) an isothermic assay involving the reversible retention of the protein of interest in the ER lumen and that was used both in vitro and in vivo. A pharmacological screen searching for agents that induce LC3 aggregation at the Golgi apparatus led to the identification of "oleate mimetics" that share the capacity to block conventional protein secretion. In conclusion, oleate represents a class of molecules that act on the Golgi apparatus to cause the recruitment of LC3 and to stall protein secretion.

Glycosphingolipid-associated ß-1,4 galactosyltransferase is elevated in patients with systemic lupus erythematosus.

OBJECTIVE: ß-1,4 galactosyltransferase-V (ß-1,4 GalT-V) is an enzyme that synthesises a glycosphingolipid known as lactosylceramide, which has been implicated in general inflammation and atherosclerosis. We asked if ß-1,4 GalT-V was present at elevated levels in patients with SLE, a disease which is associated with increased risk of atherosclerosis. METHODS: In this case-control observational study, serum samples were obtained from patients with SLE who are part of the Johns Hopkins Lupus Cohort. Control serum samples were obtained from healthy adult community members recruited from the Baltimore area. All serum samples (n=50 in the SLE group and n=50 in the healthy control group) were analysed with enzyme-linked immunoassays. These assays used antibodies raised against antigens that enabled us to measure the absorbance of oxidised phosphocholines per apolipoprotein B-100 (ox-PC/apoB) and the concentration of lipoprotein(a) (Lp(a)) and ß-1,4 GalT-V. RESULTS: Absorbance of ox-PC/apoB and concentrations of Lp(a) and ß-1,4 GalT-V were significantly higher in the SLE serum samples as compared with the control serum (p<0.0001). CONCLUSIONS: We conclude that patients with SLE have elevated levels of ß-1,4 GalT-V and ox-PC, which have previously been recognised as risk factors for atherosclerosis.

Lactosylceramide induced by elastin-derived peptides decreases adipocyte differentiation.

Elastin, the major protein of the extracellular matrix, is specially found in cardiovascular tissues and contributing to 30-50% of the dry weight of blood vessels. Elastin regulates cell signalling pathways involved in morphogenesis, injury response and inflammation. The function of elastin is frequently compromised in damaged or aged elastic tissues. Indeed, elastin degradation, observed during ageing, and the resulting production of elastin-derived peptides (EDPs), have crucial impacts on cardiovascular disease (atherosclerosis, thrombosis) or on metabolism disease progressions (type 2 diabetes or non-alcoholic steatohepatitis). In the present study, we analysed the EDP effects on 3T3 preadipocyte cell differentiation. In a first part, we treated 3T3-L1 cells with EDP and visualized the lipid droplet accumulation by the oil red O staining and measured the expression of various transcription factors and adipocyte-specific mRNAs by real-time RT-PCR. We demonstrated that the elastin receptor complex, ERC, is activated by EDPs and decreased adipocyte differentiation by a modulation of crucial adipogenesis transcriptional factor particularly PPARγ. In a second part, we identified the signalling pathway implicated in EDP-reduced cell differentiation. The flow cytometry and immunocytochemistry approaches showed that ERC activated by EDP produced a second messenger, lactosylceramide (Lac-Cer). Moreover, this Lac-Cer production favoured the phosphorylation of ERK1-2 (p-ERK1-2), to decrease adipocyte differentiation by a modulation of adipogenesis transcriptional factor PPARγ. To conclude, the EDP/Lac-Cer/p-ERK1-2 signalling pathway may be studied further as a critical target for treating complications associated with adipocyte dedifferentiation such as obesity and diabetes insulin resistance.

Discrimination of cellular developmental states focusing on glycan transformation and membrane dynamics by using BODIPY-tagged lactosyl ceramides.

Glycosphingolipids (GSLs) are a group of molecules composed of a hydrophilic glycan part and a hydrophobic ceramide creating a diverse family. GSLs are de novo synthesised from ceramides at the endoplasmic reticulum and Golgi apparatus, and transported to the outer surface of the plasma membrane. It has been known that the glycan structures of GSLs change reflecting disease states. We envisioned that analysing the glycan pattern of GSLs enables distinguishing diseases. For this purpose, we utilised a fluorescently tagged compound, LacCerBODIPY (1). At first, compound 1 was taken up by cultured PC12D cells and transformed into various GSLs. As a result, changes in the GSL patterns of differentiation states of the cells were successfully observed by using an analysis platform, nano-liquid chromatography (LC)-fluorescence detection (FLD)-electrospray ionisation (ESI)-mass spectrometry (MS), which could quantify and provide molecular ions simultaneously. We found that compound 1 remained for about 10 min on the plasma membrane before it was converted into other GSLs. We therefore investigated a more rapid way to discriminate different cellular states by fluorescence recovery after photobleaching, which revealed that it is possible to distinguish the differentiation states as well.

Targeting glycosphingolipid metabolism as a potential therapeutic approach for treating disease in female MRL/lpr lupus mice.

Glycosphingolipids (GSLs) hexosylceramides and lactosylceramides are elevated in lupus mice and human patients with nephritis. Whereas other renal diseases characterized by increased GSL levels are thought to be a result of upregulated GSL synthesis, our results suggest elevated hexosylceramides and lactosylceramides in lupus nephritis is a result of increased catabolism of ganglioside GM3 due to significantly increased neuraminidase (NEU) activity. Thus, we hypothesized GM3 would be decreased in lupus nephritis kidneys and blocking NEU activity would reduce GSLs and improve disease in lupus mice. Female MRL/lpr lupus mice were treated with water or the NEU inhibitor oseltamivir phosphate at the onset of proteinuria to block GSL catabolism. Age-matched (non-nephritic) female MRL/MpJ lupus mice served as controls. Renal GM3 levels were significantly higher in the nephritic MRL/lpr water-treated mice compared to non-nephritic MRL/MpJ mice, despite significantly increased renal NEU activity. Blocking GSL catabolism increased, rather than decreased, renal and urine GSL levels and disease was not significantly impacted. A pilot study treating MRL/lpr females with GlcCer synthase inhibitor Genz-667161 to block GSL synthesis resulted in a strong significant negative correlation between Genz-667161 dose and renal GSL hexosylceramide and GM3 levels. Splenomegaly was negatively correlated and serum IgG levels were marginally correlated with increasing Genz-667161 dose. These results suggest accumulation of renal GM3 may be due to dysregulation of one or more of the GSL ganglioside pathways and inhibiting GSL synthesis, but not catabolism, may be a therapeutic approach for treating lupus nephritis.

Colon Cancer and Perturbations of the Sphingolipid Metabolism.

The development and progression of colorectal cancer (CRC), a major cause of cancer-related death in the western world, is accompanied with alterations of sphingolipid (SL) composition in colon tumors. A number of enzymes involved in the SL metabolism have been found to be deregulated in human colon tumors, in experimental rodent studies, and in human colon cancer cells in vitro. Therefore, the enzymatic pathways that modulate SL levels have received a significant attention, due to their possible contribution to CRC development, or as potential therapeutic targets. Many of these enzymes are associated with an increased sphingosine-1-phosphate/ceramide ratio, which is in turn linked with increased colon cancer cell survival, proliferation and cancer progression. Nevertheless, more attention should also be paid to the more complex SLs, including specific glycosphingolipids, such as lactosylceramides, which can be also deregulated during CRC development. In this review, we focus on the potential roles of individual SLs/SL metabolism enzymes in colon cancer, as well as on the pros and cons of employing the current in vitro models of colon cancer cells for lipidomic studies investigating the SL metabolism in CRC.

The lysosomal disease caused by mutant VPS33A.

A rare lysosomal disease resembling a mucopolysaccharidosis with unusual systemic features, including renal disease and platelet dysfunction, caused by the defect in a conserved region of the VPS33A gene on human chromosome 12q24.31, occurs in Yakuts-a nomadic Turkic ethnic group of Southern Siberia. VPS33A is a core component of the class C core vacuole/endosome tethering (CORVET) and the homotypic fusion and protein sorting (HOPS) complexes, which have essential functions in the endocytic pathway. Here we show that cultured fibroblasts from patients with this disorder have morphological changes: vacuolation with disordered endosomal/lysosomal compartments and-common to sphingolipid diseases-abnormal endocytic trafficking of lactosylceramide. Urine glycosaminoglycan studies revealed a pathological excess of sialylated conjugates as well as dermatan and heparan sulphate. Lipidomic screening showed elevated ß-D-galactosylsphingosine with unimpaired activity of cognate lysosomal hydrolases. The 3D crystal structure of human VPS33A predicts that replacement of arginine 498 by tryptophan will de-stabilize VPS33A folding. We observed that the missense mutation reduced the abundance of full-length VPS33A and other components of the HOPS and CORVET complexes. Treatment of HeLa cells stably expressing the mutant VPS33A with a proteasome inhibitor rescued the mutant protein from degradation. We propose that the disease is due to diminished intracellular abundance of intact VPS33A. Exposure of patient-derived fibroblasts to the clinically approved proteasome inhibitor, bortezomib, or inhibition of glucosylceramide synthesis with eliglustat, partially corrected the impaired lactosylceramide trafficking defect and immediately suggest therapeutic avenues to explore in this fatal orphan disease.

Visual Function in Mice Lacking GM3 Synthase.

Purpose: Most complex gangliosides in vertebrates are formed from ganglioside GM3. GM3 deficiency in humans can result in epilepsy and visual impairment. To investigate whether a deficiency of GM3 is involved in visual function, ST3GAL5-/- mice with mutations in the ST3GAL5 gene-coded GM3 synthase were employed. Materials and Methods: Sixty mice were employed in this study. The glycosphingolipids of mice retinas were analyzed through high performance thin layer chromatography. The morphology of the optic nerves and retinas were evaluated by hematoxylin and eosin staining and immunohistochemical analysis using an anti-glial fibrillary acidic protein (GFAP) antibody. An electroretinogram (ERG) was applied on the eyes of 4, 9, 12, and 14-month-old mice. Also, visual evoked potential (VEP) was applied on 13-month-old mice. Results: The GM3 in the retinas was detected in ST3GAL5+/+ mice but not ST3GAL5-/- mice. Also, GM1b and GD1α expressions and lactosylceramide accumulation were found in the ST3GAL5-/- mouse retinas. There was no significant difference in GFAP expression in the retinas or optic discs between ST3GAL5+/+ and ST3GAL5-/- mice. Furthermore, the outcome of ERG and VEP analysis showed no disparity between the two strains in 13 and 14-month-old mice. Conclusion: In the eye, neither histopathological abnormalities nor abnormal functions of the retina were found in GM3-deficient mice. Differing from the situation in patients with GM3 deficiency, the lack of GM3 in mice did not lead to optic nerve atrophy.

Role of ceramide synthase 2 in G-CSF signaling and G-CSF-R translocation into detergent-resistant membranes.

Ceramides are sphingolipids with defined acyl chain lengths, which are produced by corresponding ceramide synthases (CerS1-6). In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), the ablation of CerS2 suppresses EAE-pathology by reducing neutrophil migration into the central nervous system. This migration is induced by granulocyte-colony stimulating factor (G-CSF) signaling. G-CSF signaling leads to a signal cascade including the phosphorylation of Lyn kinase and STAT3. This in turn regulates expression of the neutrophil surface receptor chemokine receptor 2 (CXCR2) and causes translocation of the receptor into detergent-resistant membranes (DRMs). In this study we investigated the role of ceramides in G-CSF signaling. We found, that G-CSF treatment of wild type bone marrow cells (BMCs) leads to translocation of G-CSF-receptor (G-CSF-R) into DRMs. G-CSF also induces downregulation of ceramides in WT and CerS2 null BMCs, as well as upregulation of very long chain lactosylceramides. However, in CerS2 null BMCs, G-CSF failed to induce translocation of G-CSF-R into DRMs, leading to reduced phosphorylation of Lyn and reduced CXCR2 expression. Interestingly, G-CSF signaling in CerS6 null BMCs was not affected. In conclusion, very long chain ceramides are important for G-CSF signaling and translocation of G-CSF-R into DRMs.

Induction of selective autophagy in cells replicating hepatitis C virus genome.

Hepatitis C virus (HCV) infection is known to induce autophagy, but the mechanism of autophagy induced by HCV remains controversial. Here, we investigated the characteristics of autophagy induced by HCV infection. First, to examine the involvement of autophagy-related gene (ATG) proteins in HCV-induced LC3 lipidation, we established ATG5, ATG13 or ATG14 knockout (KO) Huh7.5.1 cell lines and confirmed that the accumulation of lipidated LC3 was induced in an ATG13- and ATG14-independent manner. On the other hand, HCV infectivity was not influenced by deficiencies in these genes. We also confirmed that LC3-positive dots were co-localized with ubiquitinated aggregates, and deficiency of ATG5 or ATG14 enhanced the accumulation of ubiquitinated aggregates compared to that in the restored cells, suggesting that HCV infection induces ATG5- and ATG14-dependent selective autophagy. Moreover, LC3-positive ubiquitinated aggregates accumulated near the site of the replication complex. We further examined autophagy flux in cells replicating HCV RNA using bafilomycin or E64d, and found that the increase of LC3 lipidation by treatment with bafilomycin or E64d was impaired in HCV-replicating cells, suggesting that autophagy flux is inhibited by the progress of HCV infection. Our present study suggests that (1) HCV RNA replication induces selective autophagy and (2) the progress of HCV infection impairs autophagy flux.