ABOUT

The XIV Sphingolipid Club Meeting has been held at Pozzilli, Italy, from Sept 7 to 11, 2022, chaired by Vittorio Maglione and Alba Di Pardo, from Neuromed, a private research and clinical institution, that provided a substantial support, in both the Meeting organization and financing.

The Meeting has been a great opportunity for young PhD students and post-docs scientists to share their researches with more senior experienced scientists, in a friendly athmosphere and a top-level science.

Approximately 100 researchers from 15 different countries, from all Europe, North and South America and Australia, participated at the event, which hosted more than 70 presentations  and posters. Among them the eminent keynote lecture by Prof. Timothy Cox, from the University of Cambridge, titled “Sphingolipids – classical matters in the ancient and modern world”.

All the sessions have been passionately participated, and the discussions intense and fruitful. A minisimposium, supported by the International Society of Neurochemistry and titled “Sphingolipids and brain: pathophysiology and therapeutics”, was a basic part of the program.

All young participants, who gave a talk or presented a poster, have been supported with travel grants, just to remark and confirm a primary mission of the Sphingolipid Club, namely that to promote the career of young trainees and acknowledge their interest in the Sphingolipid field.

The social events, including the amazing visit to Agnone, the city of artistic bells, and the gala dinner, were the occasion to widen personal contacts, promote reciprocal knowledge and open fruitful collaborations.

This Meeting celebrated the 20th anniversary of our Club, and posed the basis for further activities, aimed to enlarge our association, and grow the interest of the scientific community in the fascinating issue of sphingolipids.

We are now already at work to organize the 2024 Sphingolipid Club Meeting !

PROGRAM

KEYNOTE LECTURE

NEW ASPECTS OF SPHINGOSINE-1-PHOSPHATE IN INFLAMMATION AND CANCER

Sarah Spiegel and Sheldon Milstien
Department of Biochemistry and Molecular Biology, Virginia Commonwealth University School of Medicine and the Massey Cancer Center, Richmond, VA, USA.

Sphingosine 1-phosphate (S1P) is a pleiotropic bioactive sphingolipid metabolite that regulates numerous processes important for inflammation and cancer. S1P is generated intracellularly by two sphingosine kinases (SphK1, SphK2) and is exported out of cells by Spinster 2 (Spns2) to exert its effects through activation of five specific cell surface S1PRs in autocrine or paracrine manners. In this lecture, I will focus on several new roles of SphKs and Spns2 in regulation of immune cell trafficking, cancer progression, and pulmonary metastasis. I will highlight critical roles for circulating S1P produced by tumors and the SphKs/S1P/S1PR axis in obesity-promoted inflammation, metastatic niche formation and breast cancer metastasis. I will also summarize the role of Spns2 in autoimmune diseases and metastatic pulmonary colonization. In addition, I will discuss recent studies demonstrating that SphK1 is recruited to sphingosine-enriched endocytic vesicles and that phosphorylation of sphingosine to S1P by SphK1 is involved in endocytic membrane trafficking and autophagy and in the crosstalk between endocytosis and autophagy. Collectively, our work suggests that targeting Spns2 and/or SphKs/S1P/S1PR axis would be a useful therapeutic for several human diseases including metastatic cancers. Supported by the DOD BCRP program award W81XWH-14-1- 0086 and NIH grant 2R01GM043880.

TALKS

INDUCTION OF “ENDOMITOSIS” BY PSYCHOSINE IS CONTROLLED BY MEMBRANE GLYCOSPHINGOLIPIDS AND CLUSTER OF SPHINGOMYELIN

Hiroshi Watanabe, Yuki Itani, Toshihide Kobayashi*, Shogo Oka, Hiromu Takematsu
Kyoto Univ, Japan and * Strasbourg Univ, France

Background

Psychosine (Galactosyl-sphingosine) is a lyso-form of galactosyl-ceramide (GalCer), accumulated in the genetic disorder of galactosylceramidase, Krabbe’s disease. In Krabbe’s disease patient cells, GalCer is the primary lipid species accumulated, its de-N-acylated psychosine is also accumulated and psychosine was suggested as a cause of toxicity. To date, however, it is still elusive how psychosine could cause disease-related biological effect(s).

Results

When psychosine was supplied to proliferating cells, cells underwent special type of mitosis, endomitosis, which sustains cellular content duplication without segregation, resulted in the giant and multiploid cell formation. Endomitosis seemed to be achieved by the defect in the cytokinesis, a final step of mitosis. Here, psychosine’s effect was cell-type specific. Psychosine susceptibility to produce multiploid cells was thus further explored. Membrane glycosphingolipid (GSL) expression positively and sphingomyelin (SM) negatively correlated with multiploid cell formation, indicating the importance of lipid environment in the psychosine-mediated endomitosis. It was reported that SM could form cluster in outer leaflet of the membrane, where juxtapose inner leaflet region could recruit phosphatidylinositol-5-kinase to form PIP₂. Psychosine disrupted SM cluster, visualized with lysenin probe, which could be inhibitory for the cleavage furrow maintenance, required for the proper mitosis. Psychosine therefore shifted mitotic cells into endomitotic cells.

Conclusion

These data indicated that membrane lipid environment could be important for the proper mitotic event(s). Moreover, endomitotic cell cycles could be modulated by the lipid composition of the membrane among GSL, SM and phosphoinositides.

IDENTIFICATION OF A FUNCTIONAL SPHINGOLIPID BINDING MOTIF IN LAPTM4B (LYSOSOME ASSOCIATED PROTEIN TRANSMEMBRANE 4B)

Kecheng Zhou^1,2, Andrea Dichlberger^1,2, Hector Martinez-Seara³, Thomas Nyholm⁴, Shiqian Li^1,2, Young Ah Kim⁵, Ilpo Vattulainen¹, Elina Ikonen^1,2, Tomas Blom^1,2
1Department of Anatomy, Faculty of Medicine, University of Helsinki, Helsinki, Finland;² Minerva Foundation Institute for Medical Research, Helsinki, Finland; ³ Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic; ⁴ Biochemistry, Faculty of Science and Engineering, Åbo Akademi University, Turku, Finland; ⁵ Department of Chemistry and Biochemistry, Queens College, City University of New York, Flushing, NY 11367, USA.

Background

Membrane embedded proteins are functionally regulated by the lipid composition of the surrounding bilayer. Ceramide generated in late endosomal compartments is recognized as a potent regulator of cell signaling, but its molecular interactions with late endosomal transmembrane proteins have not been studied in depth. Here we uncover structural motifs required for ceramide interaction with the four membrane spanning Lysosome Associated Protein Transmembrane 4B (LAPTM4B).

Results

By using a combination of in vitro lipid transfer assays and cell biological methods we find that the third transmembrane domain of LAPTM4B is critical for its interaction with ceramide. Atomistic simulations suggest that a centrally located aspartate residue (D202) in combination with an adjacent sphingolipid binding motif (SLB) are essential for the ceramide interaction. The aspartate residue confers flexibility to the transmembrane helix, while the sphingolipid binding motif are the preferred points of contact for ceramide. LAPTM4B proteins with mutated D202 or SLB display reduced interaction with a crosslinkable ceramide, suggesting that the sphingolipid interaction motif is functional in cells. Ceramide facilitates the heterodimerization of LAPTM4B and its protein interaction partner 4F2hc (Leucine transporter heavy chain) in late endosomes. Mutating either D202 or the sphingolipid binding motif leads to dysregulated heterodimerization of LAPTM4B and 4F2hc and altered downstream mTORC1 signaling. This suggests a role for ceramide binding to LAPTM4B in regulating nutrient sensing.

Conclusions

We identify a ceramide interaction motif in LAPTM4B necessary for promoting its interaction with the leucine transporter heavy chain 4F2hc. The findings give key insight into the mechanisms of ceramide-protein interaction and establishes LAPTM4B as a novel late endosomal sphingolipid sensor.

A NOVEL SPHINGOMYELIN/CHOLESTEROL DOMAIN-SPECIFIC PROBE REVEALS THE DYNAMICS OF THE MEMBRANE DOMAINS DURING VIRUS RELEASE AND IN NIEMANN-PICK TYPE C

Makino A¹, Abe M¹, Ishitsuka R¹, Murate M¹, Kishimoto T¹, Sakai S¹, Hullin-Matsuda F¹, Inaba T¹, Greimel P¹ and Kobayashi T^1,2
1RIKEN, Wako Saitama, Japan; ²UMR7213 CNRS, Univ Strasbourg, France

(Background) Plasma membrane lipid domains enriched with sphingolipids and cholesterol (Chol) are known as lipid rafts and have been proposed to play crucial roles in various physiologic events as diverse as signal transduction and viral and bacterial infection. However, the molecular organization and dynamics of sphingolipid/Chol domains are not well understood, mainly because of the lack of appropriate probes.

(Results) We identified a novel, nontoxicmushroomprotein that specifically binds to a complex of sphingomyelin (SM), a major sphingolipid in mammalian cells, and cholesterol (Chol). The purified protein, termed nakanori, labeled cell surface domains in an SM- and Chol-dependent manner and decorated specific lipid domains that colocalized with inner leaflet small GTPase H-Ras, but not K- Ras. The use of nakanori as a lipid-domain–specific probe revealed altered distribution and dynamics of SM/Chol on the cell surface of Niemann-Pick type C fibroblasts, possibly explaining some of the disease phenotype. In addition, that nakanori treatment of epithelial cells after influenza virus infection potently inhibited virus release demonstrates the therapeutic value of targeting specific lipid domains for anti-viral treatment.

(Conclusion) We showed that nakanori is a novel, nontoxic protein that specifically binds to SM/Chol lipid domains and thus is a unique probe for SM/Chol-rich lipid raft microdomains.

Makino A et al. FASEB J 31.1301-1322 (2017)

DEVELOPMENT OF LYSOSOME-MIMICKING VESICLES TO STUDY THE EFFECT OF ABNORMAL ACCUMULATION OF SPHINGOSINE ON MEMBRANE PROPERTIES

Ana C. Carreira, ^§,‡ Rodrigo F. M. de Almeida,^‡ and Liana C. Silva^§,†,*
§Med.ULisboa –Research Institute for Medicines, Faculdade de Farmácia, Universidade de Lisboa,1649-003 Lisboa, Portugal
^‡Centro de Química e Bioquímica, DQB, Faculdade de Ciências, Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal
^†Centro de Química-Física Molecular and Institute of Nanoscience and Nanotechnology, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal.

Synthetic systems are widely used to unveil the molecular mechanisms of complex cellular events. Artificial membranes are key examples of models employed to address lipid-lipid and lipid-protein interactions. In this work, we developed a new synthetic system that more closely resembles the lysosome – the lysosome-mimicking vesicles (LMVs) – displaying stable acid-to-neutral pH gradient across the membrane. To evaluate the advantages of this synthetic system, we assessed the distinct effects of sphingosine (Sph) accumulation in membrane structure and biophysical properties of standard liposomes (no pH gradient) and in LMVs with lipid composition tuned to mimic physiological- or NPC1-like lysosomes. Ternary 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/Sphingomyelin (SM)/Cholesterol (Chol) mixtures with, respectively, low and high Chol/SM levels were prepared. The effect of Sph on membrane permeability and biophysical properties was evaluated by fluorescence spectroscopy, electrophoretic and dynamic light scattering. The results showed that overall Sph has the ability to cause a shift in vesicle surface charge, increase membrane order and promote a rapid increase in membrane permeability. These effects are enhanced in NPC1- LMVs. The results suggest that lysosomal accumulation of these lipids, as observed under pathological conditions, might significantly affect lysosomal membrane structure and integrity, and therefore contribute to the impairment of cell function.

Funding: Fundação para a Ciência e Tecnologia (FCT), Portugal: PTDC/BBB-BQB/0506/2012, PTDC/BBB-BQB/3710/2014, UID/00612/2013, SFRH/BD/88194/2012 to ACC and Investigador FCT to RFMA (IF/00317/2012) and LCS (IF/00437/2014).

A FLUORESCENT PROBE FOR MONITORING ACTIVITY OF ACID SPHINGOMYELINASE IN VITRO AND IN LIFE CELLS

Thomas Pinkert, Zain Ahmed, Christoph Arenz
Humboldt Universität zu Berlin, Institut für Chemie, Brook-Taylor-Str. 2, 12489 Berlin, Germany

Background

The analysis of membrane lipid dynamics usually requires destruction of membranes, which is at the cost of spatial and temporal resolution. FRET probes allow for investigating individual enzyme activities in a non-destructive manner in real-time. The design of FRET probes, encompassing two individually addressable fluorescent dyes allows for ratio-imaging and thus quantification of their cleavage rate in situ.

Results

In order to monitor acid sphingomyelinase activities in vitro and in life cells, we synthesized a first generation FRET probe consisting of a NBD donor and a Nile red acceptor fluorophore. Upon cleavage of the probe, the expected increase in NBD fluorescence was elusive, due to quenching of the latter dye upon its liberation into the aqueous phase. In a second generation FRET probe, we decided to make use of the NBD quenching in aqueous environment and turned NBD into a FRET acceptor, which was excited by a coumarine as the new FRET donor. Upon cleavage of the probe, the NBD fluorescence decreased strongly, while the coumarine fluorescence significantly increased, leading to a total ratio change of about 80fold. The probe was selective for acid sphingomyelinase in vitro. In life cells, the probe indicated cleavage by acid sphingomyelinase and allowed differentiation between normal cells and acid sphingomyelinase overexpressing cells.

Conclusion

Our second generation FRET probe allows for the first time life cell investigation of acid sphingomyelinase activity. As the dyes of the probe require the use of a microscope capable of 2 photon excitation (2PE), we are currently investigating a third generation probe that should display higher sensitivity to 2PE and thus higher brightness and better signal to noise ratios.

NOVEL DOUBLY-LABELLED FLUORESCENT (FRET) CERAMIDE ANALOGUES FOR THE REAL- TIME DETERMINATION OF THE CERAMIDASE ACTIVITY

Essa M. Saied, Linh-Stella Le, Stephanie Diederich, Christoph Arenz
Humboldt Universität zu Berlin, Institute for Chemistry, Brook-Taylor-Str. 2, 12489 Berlin, Germany.

The topic of ceramidases has experienced an enormous boost during the last few years. Ceramidases catalyze the degradation of ceramide to sphingosine and fatty acids. Ceramide is not only the central hub of sphingolipid biosynthesis and degradation, it is also a key molecule in sphingolipid signaling, promoting differentiation or apoptosis. The different ceramidases play key roles in cell fate and might offer attractive targets for pharmacological intervention. The quest for novel ceramidase inhibitors with improved potency and selectivity requires a powerful activity-screenebing assay, which allows the simultaneous testing of large number of compounds. In the present study, we have synthesized novel doubly-labelled fluorogenic ceramide analogues as substrates for the real-time determination of ceramidase activity in vitro.

SEMIQUANTITATIVE DISCOVERY OF CERAMIDES BY ISOENERGETIC PRECURSOR ION SCAN IN THE TRIPLE QUADRUPOLE MASS SPECTROMETER: FUNDAMENTAL ISSUES AND PROOF- OF-PRINCIPLE APPLICATION

Federico Maria Rubino, Giuseppe Matteo Campisi, Rita Paroni
Università degli Studi di Milano, Dipartimento di Scienze della Salute.

The triple quadrupole mass spectrometer (TSQ) has boosted discovery and quantification of trace components in biological samples using class-selective scan modes. The use of suitable molecular precursors and of characteristic fragment ions allows identifying and measuring ceramides with micro- heterogeneous fatty acid or sphingosine. Collision energy in the TSQ is a key parameter to extract information from MS-MS experiments for compounds of which authentic standards are not available.

We describe the theoretical basis and proof-of-principle application of a novel scan mode of a commercial TSQ by which the molecular precursor ions of all ceramides in a sample experiment the same Center-of-Mass Collision Energy (CoM-CE), irrespective of their different m/z values over a range of homologous or chemical analogs. At this value of CoM-CE, the different precursor ions have, for the same fragmentation channel, essentially the same fragment ion cross section, thus the same response factor for quantification. This scan mode can be applied to Precursor and Neutral Loss scans, and is performed by modulating the voltage drop of a scanning mass filter according to a specific linear function of precursor ion mass. Under this instrumental condition, all precursor ions collide under the same conditions and fragment to a common charged (in Precursor Ion scan) or neutral (in Neutral Loss scan) sub-structure, and response factors are thus intrinsically homogeneous, especially for first-generation fragments. As a proof-of-principle application in the sphingolipid field, isoenergetic fast scanning during LC separation of lipid extracts highlights the presence of unanticipated ceramides that can be quantified as equivalents of known analogs without resorting to the construction of individual calibration curves. The use of a scan mode coupled to the LC separation allows improving the amount of information on the sphingolipidome that can be extracted from complex samples, and discovering components with unusual or modified fatty acids. Among displayed proof-of principle examples are the sphingolipid fractions of organs and tissues of experimental animals, and of edible seeds and nuts.

NOVEL ACTIVITY-BASED PROBES FOR DETECTION OF ACID CERAMIDASE IN PROTEIN EXTRACTS AND INTACT CELLS

José Luís Abad,^a Yadira F. Ordóñez,^a Josefina Casas,^a Alexandre Garcia,^a Jordi Guasch,^a Mazen Aseeri,^a Virginie Garcia,^b Thierry Levade,^b Antonio Delgado,^a,c Edward Schuchman,^d Gemma Triola^e and Gemma Fabrias^a
aResearch Unit on Bioactive Molecules (RUBAM), Department of Biomedicinal Chemistry, Institute for Advanced Chemistry of Catalonia (IQAC-CSIC), Barcelona, Spain; ^bLaboratoire de Biochimie Métabolique, Institut Fédératif de Biologie, CHU Purpan, and INSERM UMR1037 CRCT, Toulouse, France; ^cUnit of Pharmaceutical Chemistry (Associated Unit to CSIC), Department of Pharmacology, Toxicology and Medicinal Chemistry, Faculty of Pharmacy and Food Sciences, University of Barcelona (UB), Spain; ^dDepartment of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA; ^eChemical Biology group, Department of Biological Chemistry and Molecular Modelling, Institute for Advanced Chemistry of Catalonia (IQAC-CSIC), Barcelona, Spain.

A few irreversible inhibitors of acid ceramidase (AC) have been reported. SABRAC was designed and synthesized by our group as an AC irreversible inhibitor able to react with the catalytic cysteine-143 (human protein). Irreversibility of inhibition was demonstrated, which opened the door to using SABRAC for the development of activity-based probes (ABP) for AC. These probes will allow the detection of the enzymatically active protein, in contrast to antibodies, which detect both active and mutated inactive forms.

Different α-haloacetamides of long chain bases, containing either a fluorophore or an azide unit for further introduction of the fluorophore via click chemistry, have been synthesized and tested as AC irreversible inhibitors. Incubation of recombinant human AC with the probes and further trypsin digestion allowed the identification of the expected lipid modified-CTSIVAEDK peptide by mass spectrometry. Both the fluorescent and the azido-containing probes were able to label AC in cell lysates, the latter after a click reaction with a suitably functionalized fluorophore, Thus, in-gel fluorescence analysis showed the presence of a band with the same mobility than that detected by Western blot using an antibody against AC. Importantly, this band was absent in lysates from cells lacking AC or containing mutated inactive AC (Farber cells). The fluorescent probe was also found to label AC in intact cells, showing the expected co-localization with lysotracker. Of note, lysosomes were labeled only in cells with active AC, but not in cells not expressing AC or expressing an inactive mutated enzyme (Farber cells).

These probes should be useful to discover AC small-molecule chaperones, by monitoring the capacity of the compounds found to increase the activity of mutant AC at promoting its trafficking to the lysosome. This endeavor might lead to treatments for Farber disease.

ROLE OF ACID SPHINGOMYELINASE IN DRUG-INDUCED LIVER INJURY AND STEATOHEPATITIS

Carmen Garcia-Ruiz^1, 2, 3, Sandra Torres^1, 2, Anna Baulies^1, 2, Jose C. Fernandez-Checa^1, 2, 3
1 Department of Cell Death and Proliferation, Instituto Investigaciones Biomedicas de Barcelona, CSIC, Barcelona, and Liver Unit-Hospital Clinic-IDIBAPS; ²Centro de Investigación Biomédica en Red (CIBERehd), Barcelona, Spain, ³University of Southern California Research Center for Alcohol Liver and Pancreatic Diseases and Cirrhosis, Keck School of Medicine, USC, Los Angeles, CA, USA.

Acid sphingomyelinase (ASMase) functions as a mechanism of transient ceramide generation through sphingomyelin hydrolysis, which regulates a wide range of cellular effects, most notably apoptosis. Indeed ASMase has been shown to mediate TNF/Fas and radiation-mediated apoptosis in multiple cell types. Recently, we have addressed the role of ASMase in drug-induced liver injury. Quite intriguingly, however, genetic ASMase deletion promotes acetaminophen (APAP)-mediated liver injury and liver failure. In addition, pharmacological ASMase inhibition in wild type mice reproduced the sensitization to APAP-induced hepatocellular apoptosis and mitochondrial injury of ASMase null mice and these effects were mediated by accumulation of cholesterol in lysosomes. Moreover, the induction of lysosomal cholesterol accumulation by targeting NPC1 with U186669A mimicked the effect of ASMase deletion in the sensitization to APAP-mediated hepatocellular death. These effects were accompanied by impaired mitophagy induced by APAP due to defective autophagosome fusion with cholesterol-loaded lysosomes. Despite the role of autophagy in the progression of fatty liver disease towards steatohepatitis (NASH), ASMase null mice were protected against high fat diet (HFD)-induced NASH with decreased hepatic steatosis, inflammation and liver injury. Although autophagy was impaired in HFD fed ASMase^-/- mice, lysosomal membrane permeabilization (LMP) also was defective. Indeed, primary hepatocytes from ASMase null mice were highly resistant to palmitic acid-induced apoptosis and LMP. Therefore, these findings indicate that ASMase regulate LMP and autophagy whose interplay and impact in liver disease depends on the context.

CERAMIDE METABOLISM IN HYPOTHALAMUS CAUSED INSULIN CENTRAL RESISTANCE AND DYSREGULATION OF GLUCOSE HOMEOSTASIS DURING OBESITY

Hervé Le Stunff¹, Mélanie Campana¹, Lara Bellini¹, Claude Rouch¹, Nadim Kassis¹, Nicolas Coant¹, Benoit Colsch², Eric Hajduch³, Christophe Magnan¹
1Unité Biologie Fonctionnelle et Adaptative – UMR CNRS 8251, Équipe Régulation Centrale de la Glycémie (RéGlys), Université PARIS DIDEROT (7), Paris Cedex 13, France. ²CEA-Centre d’Etude de Saclay, Laboratoire d’étude du Métabolisme des Médicaments, Gif-sur-Yvette, France. ³INSERM UMR_S 1138, Paris, France.

Background: Accumulation of lipids in hypothalamus has been proposed to be responsible for the establishment of central lipotoxicity, a phenomenon that could contribute to the development of peripheral insulin resistance and type 2 diabetes by deregulating the nervous control of glucose homeostasis. Here, we tested the hypothesis that ceramide metabolism could play a role in the installation of a central insulin resistance and the deregulation of obesity-induced glucose homeostasis.

Results: In this study, we showed that an insulin resistance state appears in hypothalamic GT1-7 cells treated with palmitate, which was associated with an accumulation of ceramides. Using a lipidomic analysis using palmitate deutered, we found that ceramide increases is coming from de novo synthesis. In the presence of myriocin, ceramides were no longer accumulated and the insulin resistance induced by palmitate were reversed. Inhibition of PKC ζ also counteract palmitate induced insulin resistance despite the presence of an accumulation of ceramides. In obese Zucker rats, we found an increase of hypothalamic ceramides compared to lean rats, which is counteract when they are perfused intra-cerebroventricularly with myriocin. Importantly, this is associated with an improvement of insulin sensitivity in hypothalamus. Interestingly, treatment with myriocin, improve glucose tolerance of obese Zucker rats, which is associated with an increase on insulin secretion and sensitivity. Finally, we found that islets of Langerhans isolated from obese Zucker rats restore an in vitro glucose-induced insulin secretion when they are treated with myriocin.

Conclusions: Our study reveal that hypothalamic lipotoxicity is associated with an accumulation of ceramides, responsible for the installation of insulin resistance. They also highlight the key role of central ceramide metabolism in the dysregulation of glucose homeostasis induced by obesity.

SECRETORY SPHINGOMYELINASE AND MACROPHAGE FUNCTIONS DURING OBESITY

Deevska, G, and Mariana Nikolova-Karakashian
University of Kentucky College of Medicine, Department of Physiology, Lexington KY.

Acid sphingomyelinase gene encodes two forms of acid sphingomyelinase, the lysosomal (L-SMase) and the secretory (S-SMase). Elevated S-SMase activity has been found in patients with heart failure, sepsis, Hepatitis C, and fatty liver disease, as well as in many animal models. Our earlier studies have shown that obesity is also associated with elevated serum S-SMase. The goal of the current study is to evaluate the mechanism and consequences of these obesity-associated increases in S- SMase.

C57Bl6 mice fed high fat (12 weeks) and ob/ob mice were used in these experiments. Diet-induced obesity was associated with elevated serum S-SMase activity. Ex vivo studies with peritoneal macrophages and aortic discs from obese and lean mice suggested that increased macrophage secretion is likely responsible for the elevated serum S-SMase activity. The increased in S-SMase release was accompanied by a reciprocal decline in the activity of L-SMase, suggesting a shift in the post-translational processing of the ASMase precursor. To test whether this shift has an impact on macrophage function, wound-healing model was used. Obese and slim C57Bl6 mice received 4 dorsal wounds. Activated macrophages, isolated from the wounds, exhibited 6-8 fold higher-S-SMase activities as compared to peritoneal macrophages. Obesity was associated with even greater increases in S-SMase and decline in L-SMase. Wound macrophages isolated from obese mice also exhibited higher macrophage TNF release. Loss- and gain- of function studies indicated that both, the decline in L-SMase and the increase in S-SMase seen in obese animals, influence TNF secretion in response to wounding or direct LPS stimulation.

These results suggest that obesity is associated with a shift in the processing of acid SMase in activated macrophages, which impacts the capacity to secrete TNF. Earlier studies have indicated that the underlying mechanism likely involves regulation of the activity of TNF converting enzyme, TACE.

Acknowledgements. This work was supported by CDMRP Discovery Award PR152294

THE CERAMIDE TRANSPORTER CERT IS A REGULATOR OF LIPID-INDUCED INSULIN RESISTANCE IN MUSCLES

Cécile Bandet¹, Rana Mahfouz¹, Julien Véret², Athanassia Sotiropoulos³, Maxime Poirier¹, Agnieszka Blachnio-Zabielska⁴, Raphaëlle Ballaire¹, Xavier Le Liepvre¹, Olivier Bourron¹, Dušan Berkeš⁵, Pascal Ferré¹, Fabienne Foufelle¹, Hervé Le Stunff² and Eric Hajduch¹
INSERM UMR_S 1138, Paris, France; ² Université Paris-Diderot, Paris, France; ³ INSERM U1016, Paris, France; ⁴ Medical University of Bialystok, Bialystok, Poland; ⁵ Slovak University of Technology, Bratislava, Slovakia.

Background – Ceramides produced from saturated fatty acids (FA) play a central role in the development of lipotoxicity-induced insulin resistance (IR) in muscles. Ceramides are produced in the endoplasmic reticulum (ER) and transported to the Golgi to be converted into sphingomyelin. The ceramide transporter CERT has been identified as a key factor for the ER-to-Golgi trafficking of ceramide. We tested the hypothesis that changes in CERT expression could contribute to the development of IR in muscle cells.

Results – We assessed CERT expression in various insulin resistance models in vitro in C2C12 and human myotubes treated with palmitate and in diabetic patient myotubes, and in vivo in mice fed a high fat diet. In all insulin resistance models studied, CERT protein expression is decreased. We also demonstrate that the decrease in CERT expression observed in presence of FA excess is not related to the activation of ER stress or degradation via the proteasome, but through caspase cleavage. Then, to figure out whether CERT plays an important role in maintaining insulin sensitivity in muscle cells, we overexpressed CERT or inhibited its expression using siRNA or CERT chemical inhibitors (HPA-12 and gedunin) in muscle cells in vitro and in vivo. Inhibiting CERT expression in vitro or in vivo potentiates the deleterious action of lipotoxicity on insulin signaling whereas overexpression of CERT in vitro (in C2C12 cells) or in vivo (in mouse tibialis muscle) counteracts the deleterious action of lipotoxicity on insulin signaling (associated with a 20% decrease in muscle ceramide content). In addition, inhibition of caspase activity prevents ceramide-induced IR in C2C12 muscle cells.

Conclusion – Altogether, these results demonstrate the importance of a normal traffic of ceramide from the ER to the Golgi to preserve muscle cell insulin sensitivity and the importance of CERT in this process.

MYRIOCIN AS POST-CONDITIONING THERAPEUTIC IN MYOCARDIAL ISCHEMIA/REPERFUSION DAMAGE: REGULATION OF SPHINGOLIPID METABOLISM FOR THE RECOVERY FROM INFARCTION

Fabiola Tecla Bonezzi¹, Marco Piccoli², Giuseppina Milano^3,4, Rita Paroni¹, Riccardo Ghidoni¹, Anna Caretti¹, Luigi Anastasia², Paola Signorelli¹
1 Department of Health Sciences, University of Milan, San Paolo Hospital, Milan, Italy.
2 Laboratory of Stem Cells for Tissue Engineering, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Policlinico San Donato, Milan, Italy.
3 University Hospital Centre Vaudois (CHUV), Cardiovascular Research Center, Lausanne, Switzerland.
4 Vascular Biology and Regenerative Medicine Unit, Centro Cardiologico Monzino–IRCCS, Milan, Italy.

Background.

Myocardial infarction is the irreversible death of heart muscle secondary to lack of oxygen supply (ischemia). Reperfusion following ischemia is necessary for tissue recovery, but leads to additional damage that causes increasing infarct size. The sphingolipid ceramide was found accumulated in myocardium upon ischemia/reperfusion and its modulation exhibited therapeutic effect in animal studies. We here demonstrate that ceramide modulation by the orphan drug myriocin can be considered a promising post-conditioning approach after myocardial ischemia.

Results.

Intra-ventricular nanocarriers, loaded with the inhibitor of ceramide synthesis myriocin, were injected intraventricular at the beginning of reperfusion, after 30 minutes of left anterior descending (LAD) coronary ligation in mice. We observed ceramide increase after ischemia/reperfusion, both in infarct and in the risk areas; myriocin treatment lowered this increase in the risk area.

Echocardiography, performed to evaluate cardiac functions, and histological analysis on cardiac tissues, confirmed that inhibition of ceramide synthesis is involved in the ischemic/reperfusion injury and it can mediate tissue recovery. In the risk area, transcript and protein levels of inflammatory markers were measured as well as oxidative stress responses were studied. Myriocin post conditioning was able to significantly reduce expression and transcription of the pro-inflammatory cytokines IL-1, IL-6 and TNF-. Moreover, myriocin triggers anti-oxidant responses by lowering peroxide concentration and by the activation of Nrf2/HO-1 axis, which plays a pivotal role in both intrinsic resistance and cellular adaptation to reactive oxygen species (ROS).

Conclusions

We demonstrated that enhanced ceramide synthesis takes part to ischemic/reperfusion injury and that is modulation by myriocin can be considered a useful candidate in the research for post conditioning mimetics.

CERS 6 IS A KEY MEDIATOR OF THE CERAMIDE RESPONSE TO DIETARY FOLATE IN MOUSE LIVER

Keri Barron and Natalia Krupenko
Department of Nutrition UNC Chapel Hill, Nutrition Research Institute

Ceramides are increasingly recognized as key bio-effector molecules in cellular adaptation to stress, including response to nutrient deprivation. Recent work from our lab has demonstrated that folate stress elicits ceramide elevation response in cultured cells, with CerS6 being the central player in this mechanism. We hypothesized that CerS6 regulation is a general element of adaptation to dietary folate but the studies linking folate and ceramide metabolism in a whole organism are missing.

Here we evaluated the CerS6-dependent sphingolipid response to dietary folate in mouse liver. Ten- week-old CerS6 KO mice and their WT siblings were fed diets containing 0, 2, or 12 ppm of folic acid (FA) for four weeks. Animal weight and body composition (MRI) were assessed immediately before the start of dietary regiments and four weeks later. Our experiments revealed a significant difference in both the body weight gain and fat mass increase over the course of experiment between the WT and KO mice. Measurements of sphingomyelins (SM) and ceramides have demonstrated that changes in dietary folate evoked sphingolipid response in the mouse livers. FA deficient diet (0 ppm) reduced levels of SMs with all acyl chain lengths in both genotypes. The total SM levels were lowered by ~28% and 24% in WT and KO mice, respectively. On all diets, CerS6 KO mice showed higher levels of SMs than WT mice, with the exception of C₁₆-SM, which was higher in WT mice on all diets. The total ceramide levels were ~ 60% higher in CerS6 KO than in WT mice, and did not change in response to diet in either genotype. However, the folate-deficient diet induced acyl chain length-specific ceramide changes in the WT animals. C₁₆-ceramide increased by ~75%; C₁₈– and C₂₄– ceramides were also elevated. Levels of other ceramides were either not changed, or slightly reduced. In CerS6 KO mice changes for any ceramide species did not exceed 10%.

Our experiments demonstrate that dietary alterations in FA induce the sphingolipid response in mice. Significant changes of C₁₆-ceramide in WT livers, but not in CerS6 KO livers in response to folate deficient diet underscore the key role of CerS6 enzyme in mediation of dietary folate effects.

SPHINGOSINE KINASE-2 DEFICIENCY AMELIORATES KIDNEY FIBROSIS IN A MOUSE MODEL OF UNILATERAL URETERAL OBSTRUCTION BY UPREGULATING SMAD7

Stephanie Schwalm¹, Sandra Beyer¹, Helena Frey¹, Riad Haceni¹, Liliana Schaefer¹, Josef Pfeilschifter¹ and Andrea Huwiler²
1Pharmazentrum Frankfurt/ZAFES, University Hospital, Goethe University Frankfurt am Main, Germany; ²Institute of Pharmacology, University of Bern, Switzerland.

Kidney fibrosis is a hallmark of chronic kidney disease and leads to extracellular matrix accumulation, organ scarring and loss of kidney function. In this study, we investigated the role of the sphingosine kinase-2 (SPHK2) on the progression of tubular fibrosis by utilizing a unilateral ureteral obstruction (UUO) model in the mouse. We found that SPHK2 protein and activity were upregulated in fibrotic renal tissue. Functionally, Sphk2 deficient (Sphk2^-/-) mice showed an attenuated fibrotic response to UUO compared to WT mice as demonstrated by reduced collagen abundance and decreased expression of fibronectin-1, collagen I, α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF) and plasminogen activator inhibitor (PAI-1) and a reduced infiltration of macrophages. Importantly, this was associated with increased expression of the antifibrotic protein Smad7 and higher levels of sphingosine, which further increased upon UUO in Sphk2^-/- kidneys. Notably, Smad7 was also increased in isolated kidney fibroblasts of SphK2^-/- mice and in SPHK2 downregulated human proximal tubular epithelial cells. In a complementary approach, in human Sphk2- overexpressing mice, UUO resulted in exacerbated signs of fibrosis with increased collagen accumulation, higher expression levels of fibronectin-1, collagen I, α-SMA CTGF, PAI-1 and macrophage infiltration but decreased Smad7 expression.

In summary, these data reveal that SPHK2 plays an important role in kidney fibrogenesis by

modulating TGFβ signaling. Thus, SPHK2 might be an attractive new target for the treatment of fibrosis in chronic kidney disease.

THE ROLE OF SPHINGOSINE-1-PHOSPATE IN SEPSIS

Jefri Jeyapaul, Xin He, Cynthia Weigel, and Markus H. Gräler
Department of Anesthesiology and Intensive Care Medicine, Center for Sepsis Control and Care (CSCC), and Center for Molecular Biomedicine (CMB), Jena University Hospital, Hans-Knöll-Str. 2, 07745 Jena, Germany.

Sepsis is defined as a dysregulated host response to infection leading to life-threatening organ dysfunction. Despite many attempts to develop specific drugs for sepsis treatment, the only therapeutic options today consist of antibiosis and supportive treatment including volume resuscitation and vasoactive drug therapy. In order to identify new therapeutic options for sepsis treatment we are focusing on the lipid signaling molecule sphingosine 1-phosphate (S1P) and its receptors. We found that mice treated with the S1P-lyase inhibitor 4-deoxypyridoxine (DOP) showed increased survival, decreased expression of pro-inflammatory cytokines and decreased bacterial load in liver tissue. Besides the immune modulatory effects of increased S1P in tissues of DOP-treated mice, S1P is additionally known to stabilize the vascular endothelial barrier, which we could also demonstrate in DOP-treated septic mice by measuring vascular leakage of intravenously injected Evans Blue dye in lung and liver tissues. Surprisingly this stabilizing effect on the endothelial barrier was also observed after treating septic mice with the sphingosine analogue FTY720, which is phosphorylated in vivo to FTY-P and induces prolonged internalization and inhibition of S1P₁, the main receptor transducing the stabilizing effect. In order to define the mechanism of S1P and FTY-P mediated stabilization of the vascular endothelial barrier, we performed electric cell-substrate impedance sensing (ECIS) at 6 kHz and transendothelial electrical resistance (TEER) measurements in primary human umbilical vein endothelial cells (HUVEC) and in the endothelial cell line EA.hy926 under static and dynamic conditions in cell culture dishes and in biochip assays at a flow rate of 3 dyn/cm². Under these conditions, the barrier-stabilizing effect of FTY-P was not inhibited by the S1P₁ antagonist W146, pointing to a S1P₁ independent mechanism for FTY-P mediated membrane stabilization. Current data indicate that S1P₃ may play a role, and S1P₃ deficient septic mice do not show a protective effect of FTY-P on the vascular endothelial barrier anymore. We thus conclude that endothelial barrier protection in septic mice by FTY-P is mainly transduced via S1P₃. The exact mechanism is currently under investigation.

EXPLOITING SPHINGOLIPIDS AGAINST FUNGAL INFECTIONS

Del Poeta, Maurizio
Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY and Veterans Administration Medical Center, Northport, NY.

Among invasive fungal infections, cryptococcosis, candidiasis, aspergillosis and pneumocystosis are the most life-threatening. The incidence of these infections has risen dramatically over the last 20 years, more than 14-fold. The CDC estimates that more than 1 million new cases per year of cryptococcosis will occur worldwide in patients with AIDS. Disseminated candidiasis is the 4^th most common hospital-acquired sepsis. while disseminated aspergillosis is steadily increasing in immunocompromised patients. and is responsible for severe asthma by fungal sensitization (SAFS) disease. Based on these reports, over 1,300,000 people are estimated to die every year due to invasive fungal infections, which is most likely underestimated. Even with the current antifungal treatments, the associated mortality rate of invasive fungal infections is similar to malaria (1,240,000/year) and tuberculosis (1,400,000/year). However, the therapy of human systemic fungal infection has not received a commensurate level of R&D attention.

A number of recent studies have established that sphingolipids play a key role in fungal growth and pathogenesis of these fungal infections. The fungal sphingolipid glucosylceramide (GlcCer) allows growth at neutral/alkaline conditions whereas inositol-containing sphingolipids (e.g. IPC) allow fungal growth at acidic conditions. Fungi possess three ceramide synthases producing ceramides and phytoceramides which are then used to make GlcCer and IPC, respectively.

I will discuss the synthesis and metabolism of these fungal sphingolipids, highlighting the similarities and differences with the synthesis and metabolism of sphingolipids in mammalian cells. I will then provide a comprehensive outline of the past and current effort in the R&D of antimicrobials targeting these pathways focusing on the studies performed in my laboratory.

Dr. Maurizio Del Poeta is a Co-Founder and Chief Scientific Officer (CSO) of MicroRid Technologies Inc.

CIRCULATING ACID SPHINGOMYELINASE AND CERAMIDE CONTRIBUTE TO RADIATION- INDUCED INTESTINAL TOXICITY

D.Leonetti ¹, H. Estephan H^1,2, M-H Gaugler¹, I. Corre¹, F. Paris¹
1Endothelium Radiobiology and Targeting, UMR Inserm 1232, Cancer Research Center, Nantes, France ²Anti ‑ Tumor Therapeutic Targeting Laboratory, Faculty of Sciences, Lebanese University, Hadath

Background: The response of endothelial cells (EC) to high doses of irradiation leads to clonogenic cell death and damage of normal tissue. Paracrine factors are essential mediators of pathophysiological processes; their involvement in endothelial-intestinal tissue linkage during acute radiation toxicity has not been assessed. In the present study we evaluated whether acid sphingomyelinase (ASM) and ceramide represent paracrine factors enhancing radiation-induced toxicity in the small intestine. Results: We showed that 15 Gy-irradiation induced ASM and ceramide secretion in primary endothelial cells, 24 and 72h after exposure. Furthermore, the exogenous ASM and ceramide (C2-ceramide and C-16 ceramide) induces intestinal epithelial T84 cell dysfunction in term of cell growth, cell death, and IL-8 production, a marker of intestinal injury. The in vivo regulation of ASM activity and ceramide levels was next assessed in a model of radiation-induced intestinal toxicity. Irradiation of WT mice at 15 Gy resulted in a significant increase of both serum secretory ASM (S-ASM) and lysosomal (L-ASM) activities at 30 min after exposure and remained elevated up to 96h. Radiation-induced activation of circulating ASM resulted in a sustained increase in serum ceramide levels within 30 min of irradiation, and remained elevated up to 96 h. Intestinal L-ASM and S-ASM exhibited activity peaked 24h after irradiation and ceramide increased between 24h and 96h. In irradiated ASM-knockout mice, where circulating ASM is absent, circulating ceramide was not increased and intestinal injury was limited. Intestinal damage was also reduced in irradiated mice treated with an ASM inhibitor, D609. Conclusions: This study highlights the role of ASM and ceramide as paracrine factors in radiation-induced intestinal toxicity. Endothelial cells may be responsible for their increased secretion suggesting the need to protect these cells from the toxic effects of radiation in order to limit intestinal failure.

SPHINGOMYELIN SYNTHASE 1 (SMS1) DOWNREGULATION CONTRIBUTES TO CD95L AND TRAIL RESISTANCE IN MELANOMA

Bilal Fatima^1,2,3,4, Constantinescu Andrei Alexandru^5,6, Lafont Elodie, Dufour Florent^5,6, Céline Colacios^1,2,4, Levade Thierry^1,2,4,7, Badran Bassam³, Daher Ahmad³, Andrieu-Abadie Nathalie^1,2, Micheau Olivier^5,6 and Ségui Bruno^1,2,4,#
1INSERM UMR 1037, CRCT, 31037 Toulouse, France.
2Equipe Labellisée Ligue Contre Le Cancer, 31037 Toulouse, France.
3Université Libanaise, Ecole Doctorale de Sciences et Technologies, Campus Universitaire de Rafic Hariri, Hadath, Lebanon.
4Université Toulouse III – Paul Sabatier, 118 route de Narbonne, 31062 Toulouse, France.
5INSERM UMR 1231, LNC, UFR Science de Santé, 21078 Dijon, France.
6Université Bourgogne Franche-Comté, UFR Science de Santé, 21078 Dijon, France.
7Laboratoire de Biochimie, Institut Fédératif de Biologie, CHU Purpan, 31059 Toulouse, France.

Background

Melanoma cells display sphingolipid (SL) metabolism alterations that are likely to contribute to melanoma progression.

Results

While in non-cancer cells, de novo synthesized ceramide is mainly converted to sphingomyelin (SM) and, to a lesser extent, glucosylceramide (GlcCer), we provide evidence herein that melanoma cell lines express higher content of GlcCer than SM. Moreover, both SM synthases 1 (SMS1) and 2 (SMS2) are expressed at low levels in human melanoma biopsies and low SMS1 expression is associated with a worse prognosis. Importantly, loss of SMS1 and/or SMS2 in mouse embryonic fibroblasts (MEFs) increased GlcCer content and impaired death receptor (DR) ligands-induced cell death. However, restoring the SM-GlcCer homeostasis in human melanoma cell lines, either by expressing SMS1 or by inhibiting GCS, overcame melanoma resistance to DR ligands.

Conclusion

Our study indicates that SMS1 downregulation in melanoma enhances GlcCer synthesis, triggering SM/GlcCer homeostasis alteration, and confers resistance to DR ligands. Reprogramming SL metabolism is thus likely to represent an original approach to sensitize melanoma cells to CD95L or TRAIL.

THE PHYSIOLOGICAL ROLE OF SIALIDASE NEU3

Luigi Anastasia^1,2
1IRCCS Policlinico San Donato, San Donato, Italy. ²Department of Medical Chemistry, Biochemistry and Biotechnology, University of Milan, Milan, Italy.

NEU3 sialidase is a glycohydrolytic enzyme often referred to as the “ganglioside sialidase”, because it preferentially removes sialic acid residues from gangliosides. While the pathological role of the enzyme in cancerogenesis has been extensively studied, its physiological role has only recently been revealed. In particular, NEU3 has been shown to participate in various cellular processes, including the differentiation of skeletal myoblasts, where its activity is needed for myotube formation and apoptosis protection. Moreover, NEU3 was shown to be up-regulated under hypoxic conditions, and the overexpression of the enzyme greatly increases cell resistance to oxygen deprivation opposing cell death. Along this line, we recently reported a novel mechanism of HIF activation, the master regulator of cell response to hypoxia, mediated by NEU3. Interestingly, we discovered that NEU3 is up-regulated in the myocardium of chronic cyanotic congenital patients, as compared to acyanotic congenital hearts that were used as controls. We found that endogenous NEU3 expression and activity of NEU3 is up-regulated in the heart of cyanotic congenital heart pediatric patients, and that this caused an up-regulation of the EGFR signaling pathway. This caused the activation of a signaling cascade, down-stream of EGFR, ultimately leading to an activation of HIF-1alpha and down-stream pro-survival signaling pathways. These results support that NEU3 sialidase plays a critical role in the response of the myocardium to hypoxia and the preservation of cell viability. Pharmacologically mimicking NEU3 sialidase activation was also tested, and it may result in the development of new drug candidates for hypoxic diseases of the cardiovascular system.

CERAMIDE SYNTHASE 4 COORDINATES STEM CELL MAINTENANCE AND CELL FATE DECISIONS

Franziska Peters^1,2, Susanne Brodesser³, Jens C. Brüning^3,4,6,7, Carien M. Niessen^2,3,4, Martin Krönke1,3,4,5
1Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Germany, 2Department of Dermatology, University of Cologne, Germany, ³Cologne Excellence Cluster on Cellular Stress Responses in Aging Associated Diseases (CECAD), ⁴Center for Molecular Medicine Cologne (CMMC),⁵German Center for Infection Research (DZIF), ⁶Institute for Genetics, University of Cologne, Cologne, Germany, ⁷Max Planck Institute for Metabolism Research Cologne, Germany.

Aging affects the homeostasis of multiple organs of the body and is associated with an overall reduction in fitness. Skin aging involves impaired epidermal barrier maintenance, an increased susceptibility to injury and infection, reduced wound healing, a loss of dermal elasticity, wrinkling, hair loss and increased cancer risk. In addition, there is increasing evidence that skin aging goes in line with a reduction in hair follicle stem cells. We have previously shown that ceramide synthase 4 (CerS4)-deficiency leads to a reduction in quiescent hair follicle bulge stem cells. We now addressed, if this is due to a stem cell intrinsic phenotype or due to a potential niche function of CerS4.

Our study shows that CerS4 is highly expressed in adult murine epidermis where it is localized in the interfollicular epidermis and specific compartments of the hair follicle where CerS4⁺ cells co-localize with bulge hair follicle stem cell markers. Inactivation of CerS4 led to precocious activation of hair follicle bulge stem cells. This was manifested in a loss of quiescent label retaining cells and in a continuous growth state of CerS4^-/- hair follicles at a time point where CerS4^+/+ hair follicles had entered the resting phase. This ultimately led to an almost complete depletion of bulge stem cells in one-year old mice. At the time point where CerS4^+/+ hair follicles had entered the resting phase a reduction in BMP target gene mRNA expression was identified in CerS4^-/- epidermis, indicating a decrease in BMP signaling. As BMP activity promotes entry into the resting phase and quiescence of bulge stem cells this may explain the inability of CerS4^-/- hair follicle stem cells to properly enter the resting phase. Further whole body CerS4-deficiency alters cell fate decisions. These changes in cell fate decisions are not due to a stem cell intrinsic function of CerS4, thus highlighting a niche function of CerS4.

Thus our data reveal an essential role of CerS4 in the regulation of the regenerative capacity of the hair follicle stem cell compartment.

CHANGE IN SPHINGOLIPIDS PROFILES IN ENDOPLASMIC RETICULUM, MITOCHONDRIA ASSOCIATED MEMBRANES AND MITOCHONDRION UPON INDUCTION OF APOPTOSIS

Vincent Mignard¹, Nolwenn Dubois^1,2, Lisa Oliver^1,2, Claire Pecqueur^1,2, François M. Vallette^1,2 & François Paris^1,2
1CRCINA, INSERM 1232, Université de Nantes. Nantes, France. ²Institut de Cancérologie de l’Ouest, St Herblain, France.

Background: The implication of sphingolipids in mitochondrial functions, especially cell death induction, is acknowledged as an essential step. However, little is known about the precise sphingolipid composition of mitochondria and related organelles and their modulation during apoptosis.

Results: We have analyzed the presence of key lipids of the ceramide pathway (ceramide, sphingomyelin, sphinganine, sphingosine, sphingosine 1-Phosphate, glucosylceramide, and lactosyl- ceramide) by LC-MS/MS in the reticulum endoplasmic (ER), the mitochondria associated membranes (MAM) and the mitochondria (PM) purified from mouse liver and brain and U251 glioblastoma cells. We found that tissues and cell line express similar sphingolipid profiles although differences in chain lengths were observed. We then analyzed the distribution of sphingolipids during staurosporin (STS)- induced early apoptosis in U251 cells. We observed an important increase of ceramides in the PM fraction, 4h after STS treatment when early apoptosis is observed. Quite surprisingly, a similar increase for sphinganine was observed in mitochondria, although the cellular global concentrations was decreased. On the contrary, the content in sphingomyelin was specifically decreased in MAM. Finally, no change in the distribution of sphingosine, glucosyl- and lactosyl-ceramides was observed.

Conclusion: Our results show that the sphingolipid distribution in the organelles was regulated differently from total cellular contents during early apoptosis. Further studies should better explain those dynamic and their links to the early stage of apoptosis.

SPHINGOSINE-1-PHOSPHATE LYASE AND NEURODEGENERATION

Gerhild van Echten-Deckert
LIMES Institute, Membrane Biology & Lipid Biochemistry, University of Bonn, Bonn, Germany.

Autophagy defects have been identified as critical factors underlying the pathogenesis of neurodegenerative diseases. We recently reported a previously unidentified route through which sphingosine 1-phosphate (S1P) lyase (SPL) modulates autophagy in neurons [1]. SPL cleaves S1P into ethanolamine phosphate, which is directed towards the synthesis of phosphatidylethanolamine (PE) that anchors LC3-I to phagophore membranes in the form of LC3-II. In the brains of SPLfl/fl/Nes mice with developmental neural specific SPL ablation, we observed significantly reduced PE levels. Accordingly, alterations in basal and stimulated autophagy involving decreased conversion of LC3-I to LC3-II and increased beclin-1 and p62 levels were apparent. Alterations were also noticed in downstream events of the autophagiclysosomal pathway such as increased levels of lysosomal markers and aggregate-prone proteins such as amyloid precursor protein (APP) and α-synuclein. In vivo profound deficits in cognitive skills were observed. Genetic and pharmacological inhibition of SPL in cultured neurons promoted these alterations, whereas addition of PE was sufficient to restore LC3-I to LC3-II conversion, and control levels of p62, APP and α-synuclein. Electron and immunofluorescence microscopy showed accumulation of unclosed phagophore-like structures, reduction of autolysosomes and altered distribution of LC3 in SPLfl/fl/Nes brains. In addition to our recent data that connect S1P and presynaptic architecture [2], and earlier data indicating its neurotoxicity [3], we herein provide an additional route that connects SPL deficiency and neurodegeneration via a PE-mediated defective autophagy mechanism.

• Mitroi DN et al, SGPL1 (sphingosine phosphate lyase 1) modulates neuronal autophagy via phosphatidylethanolamine Autophagy 2017, 13:885‑899.
• Mitroi DN et al. Sphingosine 1-phosphate lyase ablation disrupts   presynaptic lyase ablation disrupts architecture and function via an ubiquitin‑ proteasome mediated Scientific Reports 2016, 6:37064.
• Hagen N et al, Sphingosine‑1-phosphate links glycosphingolipid metabolism to neurodegeneration via a calpainmediated Cell Death Differ 2011, 18:1356‑1365.

SPHINGOLIPIDS IN MAJOR DEPRESSION

Erich Gulbins
Dept. of Molecular Biology, University of Duisburg-Essen, Hufelandstrasse 55, 45122 Essen, Germany

Major depressive disorder is a severe and chronic disease with a lifetime prevalence of more than 10%. With suicide as cause of death for an estimated 10% of patients with severe major depressive disorder, this disease is also often a life-threatening illness. Major depressive disorder is treated with antidepressants, sleep deprivation or electroconvulsive therapy. The previously held monoamine hypothesis for the actions of antidepressants was recently revised because the antidepressant effect is not clearly correlated with the monoaminergic effect of the drugs; in fact, the antidepressant tianeptine is even a serotonin reuptake enhancer. We have shown that therapeutic concentrations of the antidepressants amitriptyline and fluoxetine reduce acid sphingomyelinase activity and ceramide concentrations in the hippocampus; increase neuronal proliferation, maturation, and survival; and improve behaviour in models of stress-induced depression. A genetic deficiency in acid sphingomyelinase mimics the effects of antidepressants and abrogates any further effect of antidepressants on neurogenesis and behaviour. Further, we have identified that ceramide-containing vesicles released by endothelial cells in the hippocampus are critically involved in the regulation of stem cell proliferation. Finally, I will discuss novel findings on the signalling of antidepressants. Collectively, these data show that many antidepressants target the acid sphingomyelinase/ceramide system.

SPHINGOLIPIDS AND VITAMIN D₃ IN NEURODEGENERATIVE DISEASES

Mercedes Garcia-Gil ^a,b
a) Department of Biology, University of Pisa, Via San Zeno, 31, 56127 Pisa, Italy; b) Interdepartmental Research Center Nutrafood “Nutraceuticals and Food for Health”, University of Pisa, Via del Borghetto 80, 56124 Pisa, Italy.

Alterations in the signaling and concentrations of sphingolipids as well as deficits of vitamin D₃ levels and polymorphisms in vitamin D₃ receptor have been found in neurodegenerative diseases such as Alzheimer disease, Parkinson disease and multiple sclerosis. Besides its classical function of bone metabolism regulation, vitamin D₃ acts on a variety of tissues including nervous system, where it modulates neuroprotection, proliferation and differentiation. Since these processes are also regulated by sphingolipids, these bioactive lipids could be mediators of vitamin D₃ action in neural cells. Indeed, we have found that ceramide kinase is involved in its antiproliferative effect in human neuroblastoma cells. In addition, vitamin D₃ decreases amyloid-β peptide-induced cytotoxicity both in differentiated human neuroblastoma cells and in vivo. It prevents the reduction of S1P1 receptor expression promoted by the peptide, modulating the signaling pathway leading to endoplasmic reticulum stress.

LIPIDS UNDER STRESS – A LIPIDOMIC APPROACH FOR THE STUDY OF MOOD DISORDERS

Tiago Gil Oliveira
University of Minho, Portugal

The emerging field of lipidomics has identified lipids as key players in disease physiology. Their physicochemical diversity allows precise control of cell structure and signaling events through modulation of membrane properties and trafficking of proteins. As such, lipids are important regulators of brain function and have been implicated in neurodegenerative and mood disorders.

Importantly, environmental chronic stress has been associated with anxiety and depression and its exposure in rodents has been extensively used as a model to study these diseases. With the accessibility to modern mass-spectrometry lipidomic platforms, it is now possible to snapshot the extensively interconnected lipid network. We previously used a lipidomic approach to evaluate the impact of a chronic unpredictable stress paradigm on the rat brain in a region-specific manner. We found that the prefrontal cortex was the area with the highest degree of changes induced by chronic stress. Although the hippocampus presented relevant lipidomic changes, the amygdala and to a more extent, the cerebellum, presented few lipid changes upon chronic stress exposure. The sphingolipid and phospholipid metabolism were profoundly affected, showing an increase in ceramide and a decrease in sphingomyelin and dihydrosphingomyelin levels, and decreased phosphatidylethanolamine and ether phosphatidylcholine and increased lysophosphatidylethanolamine levels, respectively. Moreover, we have expanded our lipidomic analysis to further understand the functioning of the hippocampus along its longitudinal axis, which is differentially affected in mood disorders. Overall, our work with lipid profiling provides an exciting avenue for the identification of disease signatures with important implications for diagnosis and treatment of mood disorders.

SPHINGOLIPID CONTROL OF BEHAVIOURAL EXTINCTION, DEPRESSION AND ALCOHOL ABUSE

Christian P. Müller
Department of Psychiatry and Psychotherapy, University Clinic, Friedrich-Alexander-University of Erlangen-Nuremberg, Erlangen, Germany

Alcohol is a major psychoactive drug in western societies involved in many cultural activities. It was shown that alcohol can be instrumentalized, i.e. used to achieve goals that would be impossible to achieve or require more work load without alcohol use. Alcohol use can serve numerous instrumentalization goals, one of the most important goals being the self-medication for innate or induced psychiatric problems, like for depression and/or anxiety disorders. There is a high comorbidity of depression and alcohol use disorder with bi-directional trajectories. While the neuropharmacology of alcohol is well known, neurobiological mechanisms for alcohol instrumentalization are poorly understood. Together with cholesterol and glycerophospholipids, sphingolipids are the most common lipids in brain membranes. Sphingolipids form lipid rafts and signaling platforms, which are membrane compartments enriched in G-protein-coupled receptors. Acid sphingomyelinase (ASM) hydrolyses sphingomyelin to ceramide and phosphorylcholine and, thus, represents a major regulator of sphingolipid metabolism, which was shown to be involved in emotional behaviour. While a temporary decline in brain ASM activity and ceramide levels are associated with the effective extinction of no longer rewarded behaviour in rats, ASM overexpression of ASM in mice (tgASM) induces depression- like behaviour. We found that it also enhanced consumption of alcohol and the alcohol-deprivation- effects after repeated withdrawal in a free-choice drinking paradigm. ASM hyperactivity facilitates the establishment of the rewarding effects of alcohol. Furthermore, we found that free-choice alcohol drinking, but not forced alcohol exposure, reduces depression-like behaviour selectively in depressed animals by normalization of ASM activity. Using SolariX MALDI-MS slice imaging, we showed that ASM hyperactivity induces sphingolipid and subsequent monoamine transmitter allostasis in the nucleus accumbens. Alcohol drinking restores sphingolipid and monoamine homeostasis selectively in depressed mice. These findings provide the first biological evidence for alcohol instrumentalization with the goal to self-medicate and ameliorate behavioural symptoms of a genetically-induced innate depression. We show that alcohol drinking normalises ASM function and re-establishes sphingolipid- and monoamine homeostasis in the nucleus accumbens of depressed mice. Thus, sphingolipid homeostasis emerges as a new mechanism to control depression-alcohol addiction comorbidity.

CHARACTERIZATION AND PREVENTION OF SPHINGOMYELIN-INDUCED NEURONAL AND GLIAL ALTERATIONS IN NIEMANN PICK DISEASE TYPE A

Azucena Pérez-Cañamás, Enrique Gabandé-Rodriguez, Beatriz Soto-Huelin, María Dolores Ledesma
Centro Biología Molecular Severo Ochoa (CSIC-UAM), Madrid, Spain

BACKGROUND: Niemann Pick disease type A (NPA) is an inherited lysosomal storage disorder caused by loss of function mutations in the gene encoding for the acid sphingomyelinase (ASM). Sphingomyelin (SM) accumulates in the cells of NPA patients and ASMko mice, which mimic the disease, leading to motor and cognitive impairment, neurodegeneration and early death.

RESULTS: We have characterized brain cell anomalies in ASMko mice and NPA patients. Besides previously reported alterations in synaptic function and autophagy we will discuss recently observed anomalies in neuronal calcium homeostasis and microglia activation. We show that high SM levels impair the activity of the plasma membrane calcium ATPase (PMCA) leading to increased intracellular calcium in neurons. SM accumulation also corrupts the protective function of M2 microglia leading to lysosomal exocytosis, Cathepsin B release and neurotoxicity. We will also present pharmacological and genetic strategies to counteract these anomalies.

CONCLUSIONS: Our results unveil the relevant contribution of SM to neuron and microglia physiology by influencing synaptic function, autophagy, calcium homeostasis and inflammation. These findings open therapeutic perspectives for a currently untreatable disease such as NPA.

SLS: NEW INSIGHT FOR A MORE SUITABLE THERAPEUTIC APPROACH FOR HUNTINGTON’S DISEASE

Vittorio Maglione, PhD
Centre for Neurogenetics and Rare Diseases IRCCS Neuromed, Località Camerelle 86077 Pozzilli (IS) Italy

Huntington’s disease (HD), the most common dominantly inherited neurodegenerative disorder is characterized by a progressive striatal and cortical neurodegeneration associated with cognitive and behavioral disturbance. The disease-causing mutation is a polyglutamine stretch expansion in N- terminal region of huntingtin, a ubiquitous protein with multiple functions.

So far, many are the aberrant molecular mechanisms described to be associated with the disease, however much remains to be defined. Over the past years, we have described that sphingolipid (ganglioside) metabolism is perturbed in HD. In particular, we have found a significant reduction of ganglioside GM1 levels in both HD preclinical models and HD patients.

New data indicate that lipid breakdown is not only restricted to ganglioside metabolism, but it also affects the regulation of Sphingosine-1-phosphate (S1P), a potent signaling sphingolipid involved in a number of processes essential to cellular homeostasis and viability. Our findings show that S1P metabolism is significantly disrupted in HD even at early stage of the disease and importantly, reveal that such a dysfunction represents a common denominator among multiple disease models ranging from cells to humans through mouse models. Interestingly, the in vitro anti-apoptotic and the pro- survival actions we found, after modulation of S1P-metabolizing enzymes, allows this axis to emerge as a new “druggable” target and unfolds its promising therapeutic potential for the development of more effective and targeted interventions against this incurable condition.

OXIDATIVE STRESS, LIPOXIN A4 AND BIOACTIVE LIPIDS IN MENIERE’S PATIENTS AFTER ORAL ADMINISTRATION OF CORIOLUS VERSICOLOR: RELEVANCE TO ALZHEIMER DISEASE

Trovato A.¹, Dattilo S¹. Scuto M.¹, Maiolino L.², and V. Calabrese¹
1Dept. Biomedical and Biotechnological Sciences, and ²Department of Medical and Surgery Sciences and Advanced Technologies, School of Medicine, University of Catania, Catania, Italy

Ménière’s disease (MD) is characterized by the triad of fluctuating hearing loss, episodic vertigo and tinnitus, and by endolymphatic hydrops found on post-mortem examination. Increasing evidence suggests that oxidative stress is involved in the development of endolymphatic hydrops and that cellular damage and apoptotic cell death might contribute to the sensorineural hearing loss found in later stages of MD. While excess reactive oxygen species (ROS) are toxic, regulated ROS, however, play an important role in cellular signaling^1-4. Modulation of endogenous cellular defense mechanisms via the stress response signaling represents, thus an innovative approach to therapeutic intervention in diseases causing chronic tissue damage, such as neurodegeneration and cancer². Among the cellular pathways conferring protection against oxidative stress, a key role is played by vitagenes, which include heat shock proteins (Hsps) heme oxygenase-1 and Hsp70, as well as the thioredoxin/thioredoxin reductase and the sirtuin systems. Mushrooms have long been used in traditional medicine for thousands of years, being now increasingly recognized as rich source of polysaccharopeptides endowed with significant antitumor, antioxidant, antiviral, antibacterial and hepatoprotective effects, thereby capable to stimulate host immune responses^5-8. In this study we tested the hypothesis that neurotoxicity is an important primary mediator of injury in Ménière’s disease, a model of neurodegenerative disorder, which is reflected in measurable increases in markers of cellular stress response and oxidative stress in the peripheral blood. This study also explores the hypothesis that changes in the redox status of glutathione, can contribute to increase oxidative stress with disruption of redox homeostasis in vulnerable neurons such as spiral ganglion neurons and consequent cellular degeneration. We evaluated systemic oxidative stress and cellular stress response in 30 patients suffering from Meniere’s disease prior to and after oral administration of nutritional mushroom Coriolus versicolor (MRLs). Here, we provide evidence of protective action of Coriolus versicolor when administered orally to patients with diagnosis of Meniere disease. Increased expression of LXA4, measured in lymphocyte and plasma after oral administration for 2 month of a biomass Coriolus versicolor preparation, was associated with an increased content of redox sensitive proteins involved in cellular stress response, such as Hsp72, Heme oxygenase -1, Thioredoxin and Sirtuin-1, and with restoration of both reduced glutathione (GSH) levels and GSH/GSSG ratio in the plasma. Patients under Coriolus treatment also showed decreased levels of pentosidine, protein carbonyls, HNE and F2-isoprostanes, measured as markers f systemic oxidative stress. Since Caspase-3 in increased in Meniere’s patients, the effect of Coriolus versicolor on plasma levels of proapoptotic ceramide is under investigation. The search for novel and efficient inducers of vitagenes will facilitate the development of pharmacological strategies to increase the intrinsic capacity of vulnerable ganglion cells to maximize antidegenerative mechanisms, such as stress response and thus cytoprotection. Furthermore, this findings are relevant to potential pharmacological interventions in healthy medicine strategy, pointing to maximize cellular stress resistance of the brain thus providing neuroprotection, and will be extended to other systemic oxidant disorders such as diabetes and cancer.

IN VITRO AND IN VIVO PRECLINICAL STUDY OF NEUTRAL SPHINGOMYELINASE ROLE IN PROGENITOR/STEM CELLS OF HIPPOCAMPUS

Samuela Cataldi^1§, Cataldo Arcuri^2§, Carmen Mecca², Stéphane Hunot³, François- Pierre Légeron³, Mercedes Garcia-Gil⁴, Irina Nakashidze¹, Andrea Lazzarini⁵, Michela Codini¹, Tommaso Beccari¹, Bernard Fioretti⁶, Giovanna Traina¹, Francesco Saverio Ambesi- Impiombato⁷, Francesco Curcio⁷, Elisabetta Albi^1
§These authors contributed equally to this work
1Department of Pharmaceutical Science, University of Perugia, Italy.
2Department of Experimental Medicine, University of Perugia, Polo Unico Sant’Andrea delle Fratte, Piazzale Gambuli, 06132 Perugia, Italy.
3Inserm U 1127, CNRS UMR 7225, Sorbonne Universités, UPMC Univ Paris 06 UMR S 1127, Institut du Cerveau et de la Moelleépinière, ICM, F-75013, Paris, France.
4Department of Biology, University of Pisa, Pisa, Italy; Interdepartmental Research Center Nutrafood ‘Nutraceuticals and Food for Health’, University of Pisa, Pisa, Italy.
5CRABiON, Perugia, via Ponchielli 4, 06073 Perugia, Italy.
6Department of Chemistry, Biology and Biotechnology, Via Elce di sotto, 06123, Perugia, Italy,
7 Dipartimento di Area Medica (DAME), University of Udine, p.le M. Kolbe 4, 33100 Udine, Italy.

Background: Neutral sphingomyelinase (nSMase) is known to be implicated in growth arrest, differentiation, proliferation and apoptosis. Although previous studies have reported the involvement of nSMase in hippocampus physiopathology, its behavior in the hippocampus during Parkinson’s disease remains undetected.

Results: The in vitro study of 1,25-dihydroxyvitamin D3 (VD3)-dependent embryonic hippocampal cell differentiation resulted in stimulation of nSMase activity with significant decrease in the levels of saturated molecular species such as 16:0SM, 18:0SM, 20:0SM, 22:0SM, 24:0SM, 26:0SM and significant increase in unsaturated molecular species 16:3SM, 16:4SM, 18:1SM,20:4SM, 24:3SM, 26:2SM and 26:3SM. The in vivo study of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)- induced mouse model of Parkinson disease showed a reduction of nSMase immunofluorescence positivity in the dentate gyrus of hippocampus where iNOS immunoflurescence was increased, by indicating neuroinflammation. A novel finding of our study is that the reduction of saturated/unsaturated fatty acid ratio induced by nSMase, by making SM a less rigid molecule, might influence neurite plasticity during embryonic hippocampal cell differentiation. Since it has long been proven that neurogenesis continues in the adult brains of mammals in the dentatus gyrus of the hippocampus due to the presence of neural stem cells, our data would predict that nSMase stimulation by VD3 treatment could be useful for the health of hippocampus in PD.

Conclusion: Further investigation of the potential roles of nSMase in PD could provide a better understanding of the biological relevance of its level and activation in hippocampus as well as the potential benefits of targeting nSMase with VD3.

PLEIOTROPIC EFFECT OF HUMAN APOE4 ON THE CEREBRAL CERAMIDE PROFILE IN MICE

S.M. den Hoedt¹, C. Janssen², G. Astarita³, D. Piomelli⁴, F.P.J. Leijten¹, S.M. Crivelli⁵, A.J.M. Verhoeven¹, H.E. de Vries⁶, J. Walter⁷, P. Martinez-Martinez⁵, E. Sijbrands¹, A. Kiliaan², M.T. Mulder¹
1Department of Internal Medicine, Cardiovascular Research School COEUR, Erasmus University Medical Centre, Rotterdam, the Netherlands, s.denhoedt@erasmusmc.nl; ²Department of Anatomy, Donders Institute for Brain, Cognition, and Behaviour, Radboud University Medical Centre, Nijmegen, the Netherlands;³Georgetown University, Washington DC, USA; ⁵Department of Pharmacology, University of California Irvine, CA 92617, United States of America; ⁵Department for Neuroscience, School for Mental Health and Neuroscience, Maastricht University, Maastricht, The Netherlands; 6Department of Molecular Cell Biology and Immunology, Neuroscience Campus Amsterdam, VU Medical Center, Amsterdam, the Netherlands; ⁷Department of Neurology, University of Bonn, Bonn, Germany.

Background

Apolipoprotein E (ApoE) is known for its role in lipid trafficking. The ε4 allele of ApoE is the strongest genetic risk factor for late onset Alzheimer’s Disease (AD). Brain lipid trafficking is strictly regulated and imbalances are associated with neuropathology. Recently, aberrant ceramide levels have been implicated in AD. Therefore, we analysed the specific effects of human ApoE4 (hE4) on cerebral ceramide content during chow or a high fat/high cholesterol (HFHC) diet.

Results

Cerebral ceramide profiles were determined by LC-MSMS in 15 month old female wild-type (WT), ApoE-knockout (E0) and hE4-knockin mice fed chow or a HFHC diet for 3 months. Similar to E0, hE4 mice displayed lower cerebral total ceramide, Cer16:0 and Cer24:1 levels than WT mice on both diets. The HFHC diet had only minimal effects on ceramide levels. mRNA levels of genes involved in ceramide metabolism and transport were determined by qPCR. Chow-fed hE4 mice showed lower mRNA levels of ceramide synthase (CerS) 6 and of most ceramide transporters than WT and E0 mice. The HFHC diet downregulated the expression of CerSs in hE4 and WT mice and of ceramide transporters in WT mice, but not in E0 mice.

Conclusions

Cerebral ceramide levels were similar in hE4 and E0 mice, but lower than in WT mice, independent of diet. This was associated with hE4-specific effects on the expression of ceramide transporters. This study suggests that ApoE4 may affect AD development by modulating cerebral ceramide homeostasis.

CERAMIDE TRANSPORTER PROTEINS ALTER AMYLOID-BETA PHAGOCYTOSIS BY HUMAN PRIMARY MICROGLIA VIA THE CLASSICAL COMPLEMENT PATHWAY

Pilar Martinez-Martinez
Maastricht University, Department of Psychiatry and Neuropsychology,School for Mental Health and Neuroscience, Maastricht, the Netherlands

Brain ceramide levels are increased in Alzheimer’s disease (AD) and it has been shown that plasma ceramide levels are a strong predictor for the risk of developing AD. In addition, extensive complement activation has been observed in AD brains. It is thought that this complement activation plays an important role in amyloid-beta phagocytosis and potentially harmful inflammatory processes.

Recently we have shown that ceramide transporter proteins (CERT) binds C1q and activates the classical complement pathway. In this paper, we demonstrate that CERT modulates the uptake of oligomeric and fibrillary amyloid-beta by primary human microglia and BV-2 mouse microglia, resulting in secretion of IL-6 by these microglia. In addition, we observed colocalization of CERT, amyloid-beta and complement pathway components such as C1q and C3 in human AD brain sections. These results show for the first time a novel mechanism linking ceramides to inflammation and phagocytosis by microglia in AD

MODULATIONS OF SPHINGOLIPID METABOLISM BY CHEMICAL TUMOR PROMOTERS

Miroslav Machala¹, Josef Slavik¹, Lucie Svrzkova¹, Katerina Pencikova¹, Pavlina Simeckova¹, Jan Vondracek²
1Veterinary Research Institute, Brno, Czech Republic
2Institute of Biophysics, Czech Academy of Sciences, Brno, Czech Republic

BACKGROUND. Environmental toxicants have been found to affect plasma membrane, cell surface proteins or lipid signaling molecules. However, their impact on a epithelial cell lipidome remains unclear.

RESULTS. In our work, we focused on modulation of sphingolipid, dihydrosphingolipid, GM3 and lysophosphatidic acid levels in the liver and lung epithelial cell models (WB-F344, HepaRG and A549 cells), after exposure to highly lipophilic environmental contaminants 2,2,4,4,5,5-hexachlorobiphenyl (PCB 153) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), or polar oxygenated polycyclic aromatic hydrocarbons. A short-term exposure (up to 3 hours) to PCB 153 or several oxy-PAHs led to a significant increase in dihydroceramide and sphinganine and decrease in ceramide concentrations. Moreover, PCB 153 suppressed dihydroceramide desaturase activity and induced autophagy of plasma membrane proteins. These effects persisted also after 24h exposure, and they were accompanied with an increase of ceramide and hexosylceramide levels. In contrast, effects of TCDD were significant only after 24h exposure; TCDD increased ceramide and sphingosin-1-phosphate levels. Although the biological significance of these lipid modulations remains unknown, it indicates that disruption of cell lipidome could be an integral part of toxic effects of these compounds.

CONCLUSIONS. This study indicates possible interaction of chemical toxicants with sphingolipid metabolism and induction of dihydrosphingolipid levels. Nevertheless, a causal link of these lipid modulations with disruption of cell-to-cell communication or autophagy remains to be identified.

ALTERATIONS IN SPHINGOLIPID METABOLISM ATTRIBUTE TO RESISTANCE TO CORNERSTONE AML THERAPIES

Samy A. F. Morad^a,d , Traci S. Davis^a , Matthew R. MacDougall^a , Noha Abdelmageed^a,c, Su-Fern Tan^d , David J. Feith^d,e , Mark Kester^d ,Thomas P. Loughran Jr.^d,e , David F. Claxton^f and Myles C. Cabot^a
aDepartment of Biochemistry and Molecular Biology, East Carolina University, Brody School of Medicine, East Carolina Diabetes and Obesity Institute, Greenville, NC.
^bDepartment of Pharmacology, Faculty of Veterinary Medicine, South Valley University, Qena Egypt.
^cDepartment of Pharmacology, Faculty of Veterinary Medicine, Sohag University, Sohag Egypt.
^dUniversity of Virginia Cancer Center, University of Virginia, Charlottesville, VA.
^eDepartment of Medicine, Hematology/Oncology, University of Virginia, Charlottesville, VA.
^fPenn State Hershey Cancer Institute, Hershey, PA.

Background – Acute myeloid leukemia (AML) is an aggressive, difficult to treat neoplasm. A regimen consisting of daunorubicin (dnr) and cytarabine (Ara-c) is the current cornerstone therapy. Cells that escape therapy are a major factor in patient relapse. Because cancer cells demonstrate strong metabolic alterations in response to chemotherapy, understanding the alterations associated with drug resistance can provide a basis for development of novel therapeutic intervention. A growing body of evidence shows that modifications in sphingolipid (SL) metabolism are key factors in cancer cell response to chemotherapy.

Results – To gain insight into the impact of chemotherapy selection pressure on SL metabolism in AML, we generated HL-60 Ara-c and HL-60 dnr resistant cell lines (HL-60/Ara; HL-60/dnr). Enzymatic analysis revealed that glucosylceramide synthase (GCS), acid ceramidase (AC), and sphingosine kinase-1 (SK1) activities were elevated in HL-60/Ara and HL-60/dnr cells, compared to wild-type. Consistent with enzyme activity, immunoblot analysis showed that HL-60/Ara and HL-60/dnr cells overexpressed GCS, AC and SK1; however, Ara-c selection pressure had a 4-fold greater impact on AC expression compared to dnr. Interestingly, HL-60/dnr cells were cross-resistant to C6-ceramide; however, resistance was reversed by inclusion of a GCS inhibitor. HL-60/dnr cells expressed high levels of the multidrug resistance protein, P-glycoprotein, as would be expected. HL-60/dnr cells also demonstrated elevated glycolytic activity and doubled at 1.5-fold the rate, compared to wild-type cells.

Conclusion – The enzymes of SL metabolism are known, attractive targets in cancer therapy. This study emphasizes the important relationship between chemotherapy selection pressure and key SL branch-point enzymes that regulate ceramide glycosylation, hydrolysis, and sphingosine phosphorylation, players in the delicate balance directing ceramide-regulated cancer cell fate.

SPHINGOSINE 1-PHOSPHATE REPRESSES E-CADHERIN IN EPIDERMAL KERATINOCYTES AND PROMOTES MELANOMA CELL MIGRATION

Justine Noujarède^1,2, Joëlle Riond ^1,2, Virginie Garcia ^1,2, Thierry Levade ^1,2,3, Nathalie Andrieu Abadie ^1,2
1Inserm UMR1037, Equipe Labellisée Ligue Contre le Cancer 2013, Centre de Recherche en Cancérologie de Toulouse, France; ²Université Toulouse III Paul-Sabatier, Toulouse, France; 3Laboratoire de Biochimie Métabolique, CHU Toulouse, France

Melanocyte growth and behaviour are controlled by keratinocytes through cell–cell adhesion molecules. Melanoma cells escape from this control through the regulation of epithelial and mesenchymal markers. This phenomenon is considered as a major event in the initiation of melanomagenesis but the nature of the molecular mechanisms leading to these modifications remains still unknown.

Our group showed alterations of ceramide metabolism in melanoma cells compared to heathy melanocytes. Sphingosine kinase 1 (SK1) is over-expressed in tumor cells as a direct result of BRAF and NRAS mutations. This stimulation leads to increase sphingosine 1-phosphate (S1P) production and release into the tumor microenvironment. This project aims to analyze whether S1P could modulate the expression of adhesion molecules in melanoma cells and neighboring cells to regulate the first stages of melanomagenesis. Here we show that: i) Exogenous S1P or conditioned medium from melanoma cells which overexpressed SK1 inhibits the expression of E-cadherin at the plasma membrane of keratinocytes; ii) This inhibition is dependent of the S1P receptors S1PR2 and S1PR3 but not S1PR1; iii) S1P inhibits E-cadherin expression in keratinocytes by up-regulating the transcription repressors Snail and Slug; and iv) S1P inhibits adhesion of melanoma cells to keratinocytes and increases of tumor cell migration. Beside the paracrine effect of tumoral S1P on keratinocytes, we also show that the SK1/S1P axis increases the expression of mesenchymal markers at the melanoma cell surface.

Taken together, our findings suggest that S1P produced by melanoma cells could stimulate the first stages of melanomagenesis by reducing the adhesion of melanoma cells to keratinocytes in the epidermis and promoting tumor cell migration to the dermis.

β-GALACTOSYLCERAMIDASE AFFECTS ZEBRAFISH MELANOCYTE DIFFERENTIATION AND MURINE MELANOMA TUMOR GROWTH

Mirella Belleri, Giuseppe Paganini, Daniela Coltrini, Daniela Zizioli, Marco Presta
Department of Molecular and Translational Medicine, University of Brescia, Italy

Background: β-galactosylceramidase (GALC) hydrolyzes the galactose ester bonds of galactosylceramide, galactosylsphingosine, and other sphingolipids. Genetic deficiency of GALC leads to Krabbe disease, a lethal autosomal recessive sphingolipidosis. Two orthologs of GALC (galca and galcb) have been identified in zebrafish. Whole mount in situ hybridization has revealed that galcb, expressed in the nervous system, is transiently expressed also by melanoblasts/melanocytes in zebrafish embryos.

Results: Morpholino-mediated knockdown of galcb results in the transient reduction in the number of mitfa, dct and tyr-positive melanoblasts/melanocytes in zebrafish embryos, pointing to a role for galcb in melanocyte differentiation. Given the well-known relationship between alterations of cell differentiation and neoplastic transformation, GALC expression was investigated in murine B16-BL6 melanoma cells. The results indicate that GALC is highly expressed by B16-BL6 cells in vitro and in B16-BL6 cell-derived tumor grafts in vivo. To shed light on the biological role of GALC in melanoma cells, its expression was down-regulated in B16-BL6 cells by RNA interference. GALC down- regulation causes a significant decrease in B16-BL6 cell proliferation, colony formation, motility, invasive capacity and vascular mimicry in vitro. Accordingly, GALC-silenced cells showed a reduced angiogenic activity when tested in vivo the chick embryo CAM assay and a reduced tumorigenic and metastatic activity when grafted in syngeneic mice or zebrafish embryos.

Conclusions: GALC down-regulation inhibits melanocyte differentiation and melanoma tumor growth. Further experiments are required to assess the molecular determinants of such effects, including the impact of GALC down-regulation on the sphingolipid profile in melanocytic/melanoma cells.

REGULATION OF FOCAL ADHESIONS AND CELL MIGRATION BY THE SPHINGOLIPID INTERACTING PROTEIN LAPTM4B

Kecheng Zhou^1,2, Andrea Dichlberger^1,2, Shiqian Li^1,2, Abel Szkalisity¹, Elina Ikonen^1,2, Tomas Blom1,2 *
1 Faculty of Medicine, University of Helsinki, Finland
2Minerva Foundation Institute for Medical Research, Finland

Background: LAPTM4B interacts with ceramide and controls its subcellular distribution, thereby regulating cellular sensitivity to chemotherapeutics. There are two isoforms of LAPTM4B (35 kDa and

24 kDa) but so far no studies have been conducted to systematically address their functional differences. Here we characterize the effects of the two isoforms on cell migration and cell death pathways.

Results: In this study, we generated cell lines with stable expression of either LAPTM4B-35 or LAPTM4B-24 on a knockout background. Both LAPTM4B isoforms localize mainly to late endosomal compartments. A minor fraction of the LAPTM4B-35 isoform is in addition found on the plasma membrane in association with filopodia. LAPTM4B knockout cells have reduced levels of sphingomyelin on the plasma membrane, altered focal adhesions and display reduced migratory capacity. The migratory phenotype is reversed by reintroducing the LAPTM4B-35 isoform into the cells, but not by LAPTM4B-24. LAPTM4B partially co-localizes with integrin β1 and regulates its internalization. Multivariate analysis of different clinical cancer cohorts suggests that LAPTM4B may be a prognostic biomarker of cancer metastasis and patient survival.

Conclusions: Our data suggests that LAPTM4B-35, but not LAPTM4B-24, is active in sensitizing cells to apoptosis and stimulating cell migration. High expression of LAPTM4B mRNA predicts poor outcome in cancers, and our findings suggest that monitoring specifically the LAPTM4B-35 isoform may be beneficial.

TARGETING SPHINGOSINE KINASE-1 ENHANCES ANTI-MELANOMA IMMUNE RESPONSE AND IMPROVES EFFICACY OF IMMUNE CHECKPOINT BLOCKADE

Caroline Imbert^1,2, Marine Fraisse^1,2, Elie Marcheteau^1,2, Florie Bertrand^1,2, Sophie Tartare-Deckert⁴, Bruno Ségui^1,2, Thierry Levade^1,2,3, Céline Colacios^1,2, Nathalie Andrieu Abadie^1,2
1 Inserm UMR1037, Equipe Labellisée Ligue Contre le Cancer 2013, Centre de Recherches en Cancérologie de Toulouse, France;
2 Université Toulouse III Paul-Sabatier, Toulouse, France;
3 Laboratoire de Biochimie Métabolique, CHU Toulouse, France;
4 Centre Méditerranéen de Medecine Moléculaire de Nice.

Treatment of advanced melanoma has been recently revolutionized thanks to immune checkpoint inhibitors (ICI). However, to extend the proportion of responders and prevent cancer recurrence, these treatments need to be improved.

Our study aims at analyzing whether inhibition of Sphingosine Kinase-1 (SK1), which is over-expressed in melanoma cells as a result of BRAF and NRAS mutations, improves anti-tumor immune response as well as ICI efficacy. Here, we show that the shRNA-based downregulation of SK1 in murine melanoma cells harboring the BRAF^V600E mutation resulted in a reduction of tumor growth in vivo. Importantly, this phenomenom was associated with a significant decrease in the percentage of CD4+FoxP3+ regulatory T cells (Treg) and, conversely, to an increase in CD8+ effector T cells into the tumors. These modifications led to a high CD8+/Treg ratio of tumor-infiltrating lymphocytes, which represents a good prognostic factor for patients with melanoma. Moreover, our data show that, whereas anti-CTLA-4 or anti-PD-1 treatment alone failed to elicit total rejection of the tumors in preclinical melanoma models, inhibition of SK1 has greatly improved the antitumoral effect of the ICI, leading to tumor rejection in all mice or in almost 70%, respectively. Interestingly, was the finding that the combination of SK1 inhibition with ICI greatly enhanced animal survival.

Our results demonstrate that combining ICI with approaches targeting the sphingosine-1-phosphate metabolism may represent the basis for innovative anti-melanoma therapies that could be extended to other cancers.

NEUTRAL SPHINGOMYELINASE 2 HEIGHTENS ANTI-MELANOMA IMMUNE RESPONSE AND SYNERGIZES WITH ANTI-PD-1

Florie Bertrand^1,2,#, Julia Rochotte^1,2,3,#, Anne Montfort^1,2#, Caroline Imbert^1,2, Nathalie Andrieu- Abadie^1,2, Thierry Levade^1,2,3,4, Céline Colacios^1,2,3,## and Bruno Ségui^1,2,3,##,§
1INSERM UMR 1037, CRCT, 31037 Toulouse, France.
2Equipe Labellisée Ligue Contre Le Cancer, 31037 Toulouse, France.
3Université Toulouse III – Paul Sabatier, 118 route de Narbonne, 31062 Toulouse, France.
4Laboratoire de Biochimie, Institut Fédératif de Biologie, CHU Purpan, 31059 Toulouse, France.
#Co-first authorship.
##Co-seniorship.

Background

Neutral sphingomyelinase 2 (nSMase2) is encoded by the SMPD3 gene and catalyzes sphingomyelin breakdown to ceramide.

Results

We show that SMPD3 is frequently downregulated in human metastatic melanoma, likely contributing to immune escape mechanisms. To determine the role of nSMase2 in melanoma, we reconstituted the expression of the gene in mouse melanoma with low levels of expression. Expression of nSMase2 in mouse melanoma reduced tumor growth in syngeneic wild-type but not in CD8-deficient mice. Analysis of tumor content showed that nSMase2 overexpression elicited accumulation of both ceramide and CD8+ tumor-infiltrating lymphocytes (TIL) in wild-type mice. Mechanistically, nSMase2 augmented the immunogenicity of melanoma cell-derived exosomes by increasing their association with miR-155, a major pro-inflammatory miRNA. Overexpression of wild-type but not a catalytic inactive mutant of nSMase2 in mouse melanoma synergized with anti-PD-1.

Conclusion

Increasing SMPD3 expression and/or ceramide in melanoma may serve as an original therapeutic strategy to overcome resistance to anti-PD-1 in melanoma.

POSTERS

CHANGES ON CELL MEMBRANES BIOPHYSICAL PROPERTIES INDUCED BY THE ENDOGENOUS PRODUCTION OF ATYPICAL SPHINGOLIPIDS

Tânia CB Santos^(1,2,3), Ana E Ventura^(1,2), Thorsten Hornemann⁽³⁾, Manuel Prieto⁽²⁾, Liana C Silva(1,2)
(1) iMed.ULisboa – Research Institute for Medicines, Faculdade de Farmácia, Universidade de Lisboa,
Av. Prof. Gama Pinto 1649-003 Lisboa, Portugal
(2) Centro de Química-Física Molecular and IN – Institute of Nanoscience and Nanotechnology, Instituto Superior Técnico, Universidade de Lisboa, Complexo Interdisciplinar Av. Rovisco Pais 1049- 001 Lisboa, Portugal
(3) Institute for Clinical Chemistry, University Hospital Zurich, Zurich 8091, Switzerland

Sphingolipids (SLs) with atypical long chain bases structures (atypical SLs) are recognized as important players in pathology. Elevated levels of SLs with 1-deoxy-sphingoid bases were found to be associated with the development and progression of Hereditary Sensory Neuropathy type 1 (HSAN1), metabolic syndrome, type 2 diabetes mellitus and likely involved in the pathomechanism of the diabetic sensory polyneuropathy. However, the molecular mechanisms underlying their pathological roles remain elusive.

To investigate the biophysical implications of atypical SLs, HEK293 cells overexpressing different subunits and mutant forms of the enzyme serine palmitoyltransferase (SPT) were used. Depending on the expressed SPT variant, the cells form different levels and types of atypical SLs and were studied using complementary fluorescence-based methodologies. Cells with increased levels of 1-deoxy- sphingolipds had a slightly reduced overall membrane order compared to control cells. The opposite was observed for cells with increased levels of C16 or C20 sphingoid bases, where a decrease in the overall membrane fluidity was observed compared to controls. Moreover, increased cellular density, which is associated to overproduction of atypical SLs, resulted in a significant decrease in the overall membrane fluidity but not of the plasma membrane, suggesting that these lipids have a stronger impact at the intracellular membrane compartments. Even though decreased membrane fluidity was observed for all cell lines, the changes were more distinct in cells with increased levels of 1-deoxy-SLs and SLs with shorter sphingoid bases, suggesting that these lipids might function as modulators of membrane biophysical properties and possibly of its function.

In conclusion, the increased production of atypical SLs seems to affect membrane biophysical properties in particular on intracellular membranes.

PERMEABILITY BARRIER AND MICROSTRUCTURE OF SKIN LIPID MEMBRANE MODELS OF IMPAIRED GLUCOSYLCERAMIDE PROCESSING

Michaela Sochorová¹, Klára Staňková¹, Petra Pullmannová¹, Andrej Kováčik¹, Jarmila Zbytovská^2,3, Kateřina Vávrová¹
1 Skin Barrier Research Group, Charles University, Faculty of Pharmacy, Hradec Králové, 500 05, Czech Republic;
2 Department of Pharmaceutical Technology, Faculty of Pharmacy, Hradec Králové, 500 05, Czech Republic
3 Department of Organic Technology, University of Chemistry and Technology Prague, 166 28, Czech Republic

Ceramide (Cer) release from glucosylceramides (GlcCer) is critical for the formation of the skin permeability barrier. Changes in β-glucocerebrosidase (GlcCer’ase) activity lead to diminished Cer, GlcCer accumulation and structural defects in SC lipid lamellae; however, the molecular basis for this impairment is not clear. In our work we studied how the presence of GCer and/or lack of Cer influences permeability barrier properties of model SC lipid membranes simulating GlcCer’ase deficiency.

The SC model membranes were prepared as an equimolar mixture of sphingolipid (Cer and/or GlcCer in different ratios), Chol, FFA and 5% of CholS. We used the full spectrum of isolated human SC Cer (hCer). To determine the permeability of prepared membranes, four permeability markers – water loss through the membrane (TEWL), electrical impedance and steady state fluxes of theophylline and indomethacin, were evaluated. The membrane microstructure was characterized using X-ray powder diffraction and infrared spectroscopy. Minor impairment (5-25%) of the hCer generation from GlcCer decreased the permeability of the model membrane to four markers and altered the membrane microstructure, in agreement with the effects of topical GlcCer in human skin. At these concentrations, the accumulation of GlcCer was a stronger contributor to this disturbance than the lack of hCer. However, replacement of 50-100% hCer by GlcCer led to the formation of a new lamellar phase and the maintenance of a rather good barrier to the four studied permeability markers.

In conclusion, the barrier perturbation by free GlcCer seems to be concentration-dependent. Our results suggest that the major cause of the impaired water permeability barrier in complete GlcCer’ase deficiency is not the accumulation of free GlcCer but other factors.

EFFECTS OF 6-HYDROXYCERAMIDES ON THE THERMOTROPIC PHASE BEHAVIOUR AND PERMEABILITY OF MODEL SKIN LIPID MEMBRANES

Andrej Kovacik,^1,* Michaela Silarova,¹ Petra Pullmannova,¹ Jaroslav Maixner,² and Katerina Vavrova¹
1Skin Barrier Research Group, Charles University, Faculty of Pharmacy in Hradec Kralove, Heyrovskeho 1203, CZ-50005 Hradec Kralove, Czech Republic
2University of Chemistry and Technology Prague, Technicka 5, CZ-16628 Prague, Czech Republic

Ceramides (Cer) play an essential role in the barrier function of human epidermis, because they help prevent water loss from the body and absorption of exogenous substances. Cer based on 6- hydroxysphingosine (H) have only been found in epidermis; however, their role in the skin barrier homeostasis is not fully understood. In this work we focused on the permeability and microstructure of model lipid membranes based on Cer NH in comparison with other non-hydroxylated Cer based on sphingosine, dihydrosphingosine or phytosphingosine, i.e. Cer NS, Cer NdS and Cer NP, respectively. Model skin membranes were composed of Cer/free fatty acids (C₁₆-C₂₄)/cholesterol/cholesteryl sulfate. Their permeability was assessed in Franz-type diffusion cells using the following permeability markers: flux of two model compounds (theophylline and indomethacin), electric impedance and water loss through the membrane. To elucidate the mechanisms of Cer effects on skin permeability, their biophysical properties were investigated by infrared spectroscopy (ATR-FTIR) and X-ray powder diffraction.

The model membrane containing Cer NH attained a comparable water loss value with the model membrane composed of Cer NP, but higher than in the Cer NS-membranes. Interestingly, electric impedance of the Cer NH-containing membrane was 6times higher than of the Cer NS-membrane. In model membrane based on Cer NH, the flux of theophylline was comparable with Cer NS-membrane, but the flux of indomethacin was 2.6times higher compared to Cer NS-membrane. Using X-ray powder diffraction we found the short periodicity phase and crystalline cholesterol in all membranes. In addition, in Cer NH-based membrane we observed also a long periodicity phase. Next, using infrared spectroscopy we showed differences in lipid mixing, packing and thermotropic phase behaviour. In membranes containing hydroxylated Cer, i.e. Cer NP and Cer NH, free fatty acids did not mix well with the Cer chains.

The results showed that the individual skin Cer classes have unique properties and changes in their structure lead to differences in barrier function of model lipid membranes. We hypothesize that this apparent heterogeneity in chemical structure, permeability and biophysical properties helps the skin lipid barrier better resist external stressors.

NOVEL SUBSTRATES FOR DIHYDROCERAMIDE DESATURASE AND SPHINGOSINE 1- PHOSPHATE LYASE

Pol Sanllehí^a,d, Ana Pou,^a Mireia Casasampere,^a José Luís Abad,^a Yadira F. Ordóñez^a, Maria Garrido^a, Olga López,^b Jordi Bujons,^c Gemma Fabriàs,^a Josefina Casas,^a Antonio Delgado^a,d
aResearch Unit on Bioactive Molecules (RUBAM), Department of Biomedicinal Chemistry, ^bGroup of Biophysics of Lipids and Interfaces (BLI), Department of Chemical and Surfactants Technology and cBiotransformations group, Department of Biological Chemistry and Molecular Modelling, Institute for Advanced Chemistry of Catalonia (IQAC-CSIC), ^dUnit of Pharmaceutical Chemistry (Associated Unit to CSIC), Department of Pharmacology, Toxicology and Medicinal Chemistry, Faculty of Pharmacy and Food Sciences, University of Barcelona (UB)

Both dihydroceramide desaturase 1 (Des1) and sphingosine 1-phosphate lyase (Sgpl1) have been identified as therapeutic targets in different diseases and the development of probes for high throughput screening (HTS) of their activity is of biological and therapeutic relevance. None of the reported assays for Des1 is amenable for HTS formats. On the other hand, one HTS assay for Sgpl1 has been reported, but the substrate used (RBM13) exhibits poor kinetic parameters and it is not applicable to intact cells.

In this communication we report that introduction of a double bond at C6 of dihydroceramide has a different outcome on Des1 activity depending on the double bond geometry. Thus, while the Z isomer is desaturated by Des1 to afford the conjugated C4,C6-diene (K_m=7.2 μM; V_max=24.6 pmol/h/mg,), the E isomer is a very potent reversible non-competitive inhibitor (K_i=111.4 nM). The former finding paves the way to the development of HTS methods for Des1 based on Diels-Alder click reaction of the conjugated Des1 diene product with labeled or labelable dienophiles. On the other hand, by applying the principle of vinylogy to RBM13, the incorporation of a vinyl unit between the sphingosine 1-phosphate amino phosphate core and the relatively bulky umbelliferone unit in RBM13 has resulted in two novel fluorogenic Sgpl1 substrates endowed with better kinetic parameters than RBM13. Importantly, these novel substrates have been found to be processed in intact cells upon incorporation into nanoliposomal formulations.

New fluorogenic sensors with suitable kinetic parameters and sensitivity have been developed for the determination of Des1 and Sgpl1 activity in cell lysates and intact cells. These sensors are currently used to develop HTS for screening of libraries and cell panels.

SYNTHESIS, RADIOSYNTHESIS AND PRELIMINARY IN VITRO AND IN VIVO EVALUATION OF THE FLUORINATED CERAMIDE TRAFFICKING INHIBITOR (HPA-12) FOR BRAIN APPLICATIONS

Simone         M.        Crivelli^1†, Andreas        Paulus^2,3,4†, Jozef Markus⁵,       Matthias Bauwens²,   Dusan    Berkes⁵, Helga      E     De     Vries⁶, T.    Mulder⁷,   Jochen Walter⁸, Felix M. Mottaghy^2,3,4, Mario Losen1, Pilar Martinez – Martinez¹
1 Maastricht University, Department of Psychiatry and Neuropsychology,School for Mental Health and Neuroscience, Maastricht, the Netherlands
2 NUTRIM, School for Nutrition and Translational Research in Metabolism, Maastricht University, Maastricht, the Netherlands
3Department of Medical Imaging, Division of Nuclear Medicine, MUMC, Maastricht, the Netherlands
4Division of       Nuclear        Medicine,       Uniklinikum      Aachen,        Aachen,       Germany
5 Department of Organic Chemistry, Slovak University of Technology, Radlinského 9, 81237, Bratislava, Slovak Republic
6 Department of Molecular Cell Biology and Immunology, Neuroscience Campus Amsterdam, VU University Medical Center, Amsterdam, the Netherlands
7 Department of Internal Medicine, Erasmus Medical Center, Rotterdam, the Netherlands
8 Department of Neurology, University of Bonn, Bonn, Germany

Ceramide levels are increased in blood and brain tissue of Alzheimer’s disease (AD) patients. In alignment to this, proteins controlling ceramide metabolism are abnormally concentrated in AD brains. Since the ceramide transporter protein (CERT) is the only known protein able to mediate non-vesicular transfer of ceramide between organelle membranes, the modulation of CERT function may impact on ceramide accumulation. The competitive CERT inhibitor N-(3-Hydroxy-1-hydroxymethyl-3- phenylpropyl) dodecanamide (HPA-12) interferes with ceramide trafficking. To understand the role of ceramide/CERT in AD, HPA-12 can be a useful tool to modulate ceramide trafficking and to study CERT dynamics. Here we first report the synthesis and in vitro properties of HPA-12 radiolabeled with fluorine-18, and present preliminary in vitro and in vivo positron emission tomography (PET) imaging and biodistribution data. In vitro results demonstrated that the fluorination did not alter the biological properties of HPA-12 since the [Fluorine-19]HPA-12, interferes with 5-DMB-ceramide trafficking in HeLa cells. Radiolabeled HPA-12, [Fluorine-18]HPA-12, was obtained with a radiochemical yield of 90% and a specific activity of 73 MBq/µmol. PET imaging on wild-type mice showed hepatobiliary clearance and a brain uptake on the order of 0.3 standard uptake value (SUV) one hour post injection. Furthermore, the biodistribution data showed that after removal of the blood by intracardial perfusion, radioactivity was still measurable in the brain demonstrating that the [Fluorine-18]HPA-12 crosses the blood brain barrier and is retained in the brain.

MEASUREMENT OF CERAMIDE SPECIES IN DIFFERENT BIOLOGICAL MODELS BY LC-MS/MS

Giuseppe Matteo Campisi, Jessica Rizzo, Paola Signorelli, Fabiola Bonezzi, Anna Caretti, Riccardo Ghidoni, Federico Maria Rubino, Rita Paroni
Dept. Of Health Sciences, University of Milan, Milan, Italy

Introduction

Ceramide (Cer) is one of the most important sphingolipids subclasses actively involved in apoptosis and cell death. Recently, it has been proposed as pharmaceutical target in pathologies related to its over productions and, in this optic, its specific involvement within the more complex sphingolipid pathways is needed. Liquid Chromatography coupled to mass spectrometry (LC-MS/MS) is the most important technique to study the Cer metabolism, and the unique able to provide an in-depth profile of all sphingolipid species.

Here we present the optimized LC-MS/MS procedure to quantify Cer species in different biological matrices and in different biological models related to deregulation of Cer metabolism.

Materials and methods

Sphingolipids from different tissues and cells were fortified with internal standard N-lauroyl-D-erythro- sphingosine (0.2 nmol), and extracted with a customized procedure modified from the one described by Merrill et al. (1).

The LC-MS/MS (Dionex Ultimate 3000 tandem AB Sciex 3200 Q Trap), operated in the positive electrospray ionization (ESI) mode. Identification and quantification of all targeted Cer was accomplished by multiple reactions monitoring (MRM) by following the transition from the [MH⁺] species to the common ion fragment 264.4 m/z. The Cer content was expressed as pmol normalized to total protein content (mg). The analysis run to run lasted 22 min at flow rate 0.3 mL/min.

Results and Conclusions

As low as 50 fmoles can be detected on-column and quantified with ±15% precision and within ±20% of target level; the method was linear up to 400 pmoles in a typical 150 microL extract. Examined biological samples (˃50), include mouse retinas and hearts, human Chordoma tumor biopsies, C38 and meningioma cell cultures. The method here presented resulted suitable for all the tested samples provided by in vivo and in vitro studies. In particular, the method was useful as a direct read-out for pharmacological treatments aimed to lower the Cer content in different pathological models related to the Cer overproduction.

STRUCTURAL AND DYNAMICAL ANALYSIS OF BACTERIAL NEUTRAL SPHINGOMYELINASE

Begum Yagci¹, Özkimili Ölmez¹, Kutlu O. Ulgen
Department of Chemical Engineering, Bogazici University, 34342 Bebek, Istanbul, Turkey

Neutral sphingomyelinase 2 (nSMase2) is an enzyme that hydrolyzes sphingomyelin to generate ceramide, a bioactive lipid involved in several cellular processes including inflammation and apoptosis. Its involvement in cancer and Alzheimer’s disease suggests that it can be a potential therapeutic target for these pathologies [1]. In this study, to shed light on the mechanism of nSMase2, B. cereus nSMase (Bc-SMase) that shares a similar catalytic mechanism and protein fold with nSMase2 [1], is analyzed structurally and dynamically by performing molecular dynamics (MD) simulations on the apo- form and ligand-bound forms of this enzyme. Active and inactive forms of Bc-SMase are examined in order to understand which structural and dynamic changes are required for inactive Bc-SMase to become active. Furthermore, the noncompetitive inhibitor, GW4869 is docked into computationally determined possible hotspots on Bc-SMase and analyzed to see what conformational changes that this enzyme undergoes with GW4869.

1. Shamseddine, A. A., Airola, M. V, & Hannun, Y. A. (2015). Roles and regulation of neutral sphingomyelinase-2 in cellular and pathological Advances in Biological Regulation, 57, 24– 41. https://doi.org/https://doi.org/10.1016/j.jbior.2014.10.002

UNDERSTANDING THE IMMUNE SYSTEM AND INFLAMMATION BY NETWORK ANALYSIS

Elif Esvap, Elif Özkimili Ölmez, Kutlu Ülgen
Department of Chemical Engineering, Bogazici University, Istanbul, Turkey

The immune system is an organization of cells and molecules whose primary function is to protect the host from foreign invasion like microbial infection. Inflammation is part of the non-specific immune response and is an active defense reaction of multicellular organisms. Inflammatory response may cause more damage to host than the microbe if the response is not rapid, destructive and specific. Recent works showed that sphingolipids take part in various cellular processes including inflammation.

In this work, network reconstruction of immune system along with inflammation and sphingolipid related processes with an interactomics approach integrated with transcriptomics was conducted. The protein-protein interaction (PPI) network of the immune system proteins produced in the human liver were constructed using databases and then visualized by Cytoscape. Candidate proteins were determined by investigating the KEGG Pathways related to immune system and inflammation. Then, the protein data was integrated with the gene expression profiles obtained from databases like GEO Omnibus or MERAV to identify the active regions of the network under a specific condition such as diseased state of the organism. The network was divided into subnetworks using a heuristic based algorithm, MCODE. For the analysis of the reconstructed network, their topological and functional properties will be estimated. For the topological properties, graph theory will be used. The acquired subnetworks will be then further analyzed for functional modules to identify drug targets. The linear pathway analysis using the netsearch algorithm will be used for extracting the signal transduction pathways.

Network analysis has great potential for identifying the causes of inflammatory disorders or revealing the relationship between diseases and the immune system. Also, additional drug targets or key regulators of immune system can be identified using biological networks for therapeutic purposes.

SPHINGOSINE KINASE INHIBITORS SKI AND ABC294640 INDUCE ENDOPLASMIC RETICULUM STRESS AND PROMOTE POST-TRANSLATIONAL MODIFICATION OF p53 IN HUMAN EMBRYONIC KIDNEY 293T CELLS

Mariam Alsanafi, Nigel J. Pyne, and Susan Pyne
Strathclyde Institute of Pharmacy and Biomedical Science, University of Strathclyde, Glasgow G4 0RE, UK

Background

Sphingosine kinases (SK1 and SK2) are lipid kinases that catalyse the formation of the bioactive lipid, sphingosine 1-phosphate (S1P) by phosphorylation of sphingosine. S1P acting via G-protein coupled receptors (S1P_1-5) or intracellular targets induces cellular responses. We have previously shown that sphingosine kinase inhibitors induce the proteasomal degradation of SK1 in cells to promote growth arrest and/or senescence or apoptosis.

Results

In this study, we have assessed the effect of SKi (2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole)) and ABC294640 (3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide), which inhibit sphingosine kinase and dihydroceramide desaturase (Des1) on ER stress and p53 in HEK293T cells. Both compounds induce the proteasomal degradation of SK1 and post- translational modification of p53 as evidence by the appearance of two proteins with Mr = 63 and 73 kDa that cross-react with anti-p53 antibody. The proteasomal inhibitors, MG132 blocks the accumulation of p63 and p73 suggesting that an unidentified protein, which is normally degraded by the proteasome has an inhibitory role in the post-translational modification of p53. Interestingly, the formation of p63 and p73 was not affected by N-acetylcystin (NAC), suggesting that oxidative stress is not involved. In addition, SKi and ABC294640 induce an ER stress response as evidence by post-translational modification of protein kinase R-like ER kinase (PERK); indicated by mobility shift on SDS-PAGE. The modulation of PERK was not affected by NAC. Both inhibitors induce ‘alarm signal’ activation of p38 MAPK and JNK.

Conclusion

SK/Des1 inhibitors potentially alter the lipid composition of the ER membrane, which directly activates PERK independent of unfolded proteins. Future objectives are to provide a mechanistic link between ER stress and p53 to define the action of SK/Des1 inhibitors on growth arrest/cell death.

HUMAN SERUM LIPIDOME MIRRORS THE LIPID SPECIES LEVELS FOUND IN SYNOVIAL FLUID DURING HEALTH AND OSTEOARTHRITIS

MK Kosinska¹, G Liebisch², J Wilhelm³, B Ishaque¹, M Rickert¹ & J Steinmeyer¹
1Laboratory for Experimental Orthopaedics, Department of Orthopaedics, Justus Liebig University Giessen, Germany
2Department of Clinical Chemistry and Laboratory Medicine, University Hospital Regensburg, Germany
3Medical Clinic II/IV, Justus-Liebig-University Giessen, Germany

Objectives: Biomarkers that reflect the molecular remodeling of early stages of OA may provide a novel diagnostic tool. We recently reported that the human synovial fluid (SF) lipidome often differ between healthy and early and/or late stages of OA. In this line, we hypothesize that blood mirrors at least some of the alterations of SF lipidome during OA. Thus, in the current study we compared the lipidome of serum and SF obtained from healthy donors and patients with early or late stage OA knee joints.

Results: Lipids were extracted from cell- and cellular debris-free knee SF and serum obtained from 16 postmortem (SF) and 33 (serum) joint-healthy donors, 49 patients with early and 43 patients with late OA changes. Extracted lipids were quantified using ESI-MS/MS. The general linear model analysis was applied to investigate possible effects of age, BMI, and gender on the level of lipids. Statistical analysis was performed using program R. The present study was approved by the ethical review committee of our University.

Human SF and serum have a comparable composition of lipid classes and species. The seven different lipid classes present in human SF and serum compromise 107 lipid species with the following species distribution: 28 phosphatidylcholine, 5 lysophosphatidylcholine, 16 sphingomyelin, 24 phosphatidylethanolamine based plasmalogen, 14 phosphatidylethanolamine, 11 phosphatidylinositol, and 9 ceramide species. Interestingly, the lipid levels were 4 to 10-fold elevated in serum and, approximately parallel to those of the SF. Importantly, some of the lipid species being elevated in OA SF are also elevated in OA serum obtained from the same donors.

Conclusions: Our quantitative data reveal firstly that human serum often reflect the alterations in lipid species levels seen in OA SF as compared to normal controls. Further studies with larger well characterized cohorts are needed to verify individual lipid species as novel OA biomarkers.

SPHINGOSINE-1-PHOSPHATE ENHANCES α₁-ADRENORECEPTOR-MEDIATED VASOCONSTRICTION

Panta¹, D. Móré¹, É. Ruisanchez¹, P. Dancs¹, M. Kerék¹, S. Offermanns², Z. Benyó¹
1Institute of Clinical Experimental Research, Semmelweis University, Budapest, Hungary (Funding: OTKA K-112964);
2Department of Pharmacology, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany

Background. We aimed to examine the vasoactive effects of sphingosine-1-phosphate (S1P) by evaluating its capability to alter the basal vascular tone and to influence vasoconstriction mediated by α1-adrenoreceptors. Segments of the thoracic aorta were isolated from adult male wild type (WT) and knockout (KO) mice deficient in S1P₁, S1P₂, or S1P₃ receptors or the α subunits of G_12/13 proteins in the smooth muscle. Vascular reactions were tested under isometric conditions via myogrpahy.

Results. Addition of 5 µM S1P – that is in the concentration range of S1P in human serum – did not cause significant change in the resting vascular tone. In contrast, EC50 of the vasoconstrictor effect of the α1-adrenoreceptor agonist phenylephrine (PE) decreased, whereas E_max increased following 20 min incubation with 5 µM S1P in WT vessels. Similar enhancement of the vascular reactivity was detected in S1P₁– and S1P₃-KO segments. In S1P_2-KO vessels, however, this phenomenon was absent. In addition, the potentiating effect of S1P was also lacking in vessels of G_12/13-KO mice. The modulation of PE-induced vasoconstriction by S1P was abolished after inhibition of the Rho-kinase by Y27632.

In further experiments we aimed to evaluate the duration of the S1P-induced enhancement of α1- adrenoreceptor-mediated vasoconstriction. Therefore, 80 nM PE was added every 20 minutes, repeatedly, following a 20 min long incubation with S1P. Reactivity remained enhanced in the following 3 hours in WT segments, whereas this increase could not be detected in S1P₂-KO vessels.

Conclusions. Although S1P does not directly modify resting vascular tone by itself, yet it significantly enhances α₁-adrenoreceptor-mediated vasoconstriction even at three hours after exposure. The S1P₂ receptor / G_12/13 / Rho-kinase pathway appears to be responsible for this effect of S1P.

TIME-DEPENDENT ALTERATIONS OF SPHINGOLIPID METABOLISM ELICITED BY PCB 153 (2,2‘,4,4‘,5,5‘-HEXACHLOROBIPHENYL) IN LIVER PROGENITOR CELLS

Katerina Pencikova¹, Josef Slavik¹, Lucie Svrzkova¹, Jan Vondracek^1,2, Miroslav Machala¹
1 Veterinary Research Institute, Brno, Czech Republic
2 Institute of Biophysics, Czech Academy of Sciences, Brno, Czech Republic

Background. Significant changes of sphingolipid metabolism were observed in rat liver progenitor- like WB-F344 cells after exposure to PCB 153, a highly abundant PCB congener found both in the environment and in living organisms.

Results. After short-term exposure (1 – 3 h), the intracellular concentrations of ceramide and hexosylceramide decreased significantly, while the levels of dihydroceramide and dihydrosphingomyelin were strongly increased. Using ceramide 12/0, an artificial substrate for dihydroceramide desaturase (DES), we found that DES activity was suppressed after PCB 153 treatment. Fenretinide, a small molecular inhibitor of DES, mimicked the effects of PCB 153. These results suggest that DES activity can be rapidly suppressed by PCB 153, which is a novel mode of action of this important environmental contaminant. In contrast to the short-term exposure, longer incubation of cells with PCB 153 (24 h) increased concentrations of ceramide, hexosylceramide and dihydroceramide, thus suggesting a more complex, dynamic deregulation of sphingolipid metabolism by PCB 153.

Conclusions. The present results suggest that there might exist links between modulation of sphingolipid metabolism and further cellular effects of non-dioxin-like PCB congeners, including disruption of cell-cell communication and modulations of intracellular signaling pathways.

INCREASED 1-DEOXY-SPHINGOLIPIDS LEVELS DURING DEVELOPMENT OF DIABETES, ARE CYTOTOXIC FOR PANCREATIC ACINAR CELLS

Chen R¹, Hornemann T^2,3,5, Yu W², Camargo S⁴, Graf R^1,5 and Sonda S^1,5
1Swiss Hepato-Pancreatico-Biliary(HPB) Centre, Division of Surgical Research, Department of Visceral & Transplantation Surgery, University Hospital Zurich
2Institute for Clinical Chemistry, University Hospital Zurich
3Competence Centre for Systems Physiology and Metabolic Diseases, Zurich
4Institute of Physiology, University of Zurich, 5Centre for Integrative Human Physiology, University   of Zurich, Switzerland

Background: Dysfunctions of pancreatic acinar cells leading to exocrine pancreatic insufficiency are frequently associated with diabetes mellitus (DM). DM is associated with increased plasma levels of 1-deoxy-sphingolipids (dSLs), an atypical class of sphingolipids with demonstrated cytotoxic effects on several cell types. Here we investigated whether elevated dSLs in DM may compromise the function of pancreatic acinar cells, thus increasing the predisposition to develop exocrine pancreatic diseases.

Results:

Induction of DM in mice with streptozotocin recapitulated the increased dSL levels and atrophy of acinar cells in diabetic patient. Reduction of dSL synthesis by oral L-serine supplementation ameliorated acinar cell damage, without restoring insulin production in beta cells. This observation suggests that elevated dSLs rather than insulin deficiency contributed to the acinar cell damage in DM. In vitro studies further confirmed that treatment with the dSL 1-deoxysphinganine (dSA), directly promoted programmed cell death in pancreatic acinar cells in a dose- and time-dependent manner. Analysis of the molecular mechanisms underlying the observed dSL-mediated cytotoxicity showed that dSL induced mitochondrial dysfunction. This was evidenced by increased production of reactive oxygen species (ROS) and reduction of redox activity. Importantly, ROS formation was a key inducer of cytotoxicity as ROS scavenging upon antioxidant treatment abrogated dSL- induced cell death. Finally, we identified the metabolic conversion of dSA into ceramide as a critical step in dSLmediated ROS production and consequent acinar cell cytotoxicity.

Conclusion: Our work revealed that dSLs are cytotoxic for pancreatic acinar cells, suggesting a role for these lipids in exocrine dysfunction observed following DM. Oral L-serine supplementation could be a therapeutic strategy to ameliorate exocrine pancreatic diseases in diabetic patients.

LYSOPHOSPHATIDIC ACID, A JANUS-FACED MEDIATOR OF THE VASCULAR TONE

Péter Dancs¹, Éva Ruisanchez¹, Andrea Balogh¹, Cecília Rita Panta¹, Zsuzsanna Miklós¹, Rolf M. Nüsing², Junken Aoki³, Jerold Chun⁴, Stefan Offermanns⁵, Gábor Tigyi⁶, Zoltán Benyó¹
1Institute of Clinical Experimental Research, Semmelweis University, Budapest, Hungary (Funding: OTKA K-112964)
2Institute of Clinical Pharmacology, JW Goethe-University, Frankfurt, Germany
3Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan
4Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California, USA
5Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany
6Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee, USA

Background. Lysophosphatidic acid (LPA) is a bioactive lysophospholipid having a multitude of actions in the immune-, nervous-, and cardiovascular systems. We previously reported that LPA elicits endothelium-dependent vasorelaxation mediated by LPA₁ receptors, phospholipase C and nitric oxide. Removal of the endothelium not only abolished this effect but converted it to vasoconstriction. Here we describe a novel paracrine/autocrine lipid mediator cascade mediating this constrictor action of LPA.

Results. LPA₁, LPA₂, LPA₄ and LPA₆ transcripts were abundantly detectable with qPCR in the aortic vascular smooth muscle (VSM) of wild type (WT) mice. Isometric tension of endothelium- denuded abdominal aortic segments was measured via myography, where LPA elicited dose- dependent vasoconstriction in endothelium-denuded WT segments. The LPA_1-3 agonist VPC31143 mimicked, whereas the LPA_1&3 antagonist Ki16425 inhibited the constrictor effect. The VPC31143- induced vasoconstriction was abolished in the vessels of LPA₁ knockout (KO) mice but not in that of LPA₂ KO mice. Genetic deletion of Gα_q/11 or Gα_12/13 as well as inhibition of Gα_i/o by pertussis toxin (PTX) also attenuated the VPC31143-elicited vasoconstriction. Gα_i/o signaling can be coupled to phospholipase A₂ and COX1 activation, thus we investigated the potential involvement of autocrine/paracrine thromboxane A₂ (TXA₂) release in the LPA₁-mediated vasoconstriction. Vessels deficient in COX1 or TP showed diminished vasoconstrictor responses to VPC31143. Furthermore, VPC31143 increased TXA₂ production in WT and TP KO vessels measured with TXB₂ ELISA, but failed to do so in LPA₁ KO, COX1 KO or PTX-treated WT vessels.

Conclusions. Acting on VSMC, LPA elicits vasoconstriction, which counteracts the endothelium- dependent vasodilation. We propose that in case of endothelial injury the LPA/LPA₁ and TXA₂/TP pathways may interact as a vicious circle between platelet activation and vasoconstriction.

LYSOPHOSPHATIDIC ACID INDUCED VASOCONSTRICTION IN THE CORONARY VASCULAR BED

Zsuzsanna Miklós¹, Dina Wafa¹, Eszter Hobot¹, Éva Ruisanchez¹, Margit Kerék¹, Andrea Balogh¹, Péter Dancs¹, Stefan Offermanns², Gábor Tigyi³, Zoltán Benyó¹
1Institute of Clinical Experimental Research, Semmelweis University, Budapest, Hungary (Funding: OTKA K112964)
2Max-Planck-Institute for Heart and Lung Research, Bad Nauheim, Germany
3Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee, USA

Background: Lysophosphatidic acid (LPA) is known to act on 6 documented G protein-coupled receptors (LPA1-6). It has diverse effects in the cardiovascular system including its major influence on vascular tone. Although several LPA species are released in acute coronary syndrome, the effects of LPA on coronary vascular tone remain to be elucidated. Our aim was to describe the effects of LPA on the coronary flow of isolated murine hearts and to identify the signaling pathways mediating the effect.

Results: RT-PCR analysis of LPA1-6 mRNA abundance in segments of mouse coronaries verified the expression of each LPA receptors in the vessels. Administration of 1-oleoyl-LPA (10^-6 M) to the perfusion line of Langendorff perfused hearts of wild type male mice reduced the coronary flow by a maximum of 35% which resulted in the drop of left ventricular developed pressure by 30%. The coronary flow reducing effect of LPA also developed in LPA1 and LPA2 deficient mice and in the presence of antagonists which block LPA3 receptors (Ki16425 10^-5 M and VPC32183 10^-6 M). However, the LPA4 antagonist BrP-LPA (10^-8 M) abolished LPA-induced coronary flow reduction. In mouse experimental models used for the identification of intracellular signaling pathways, the smooth muscle specific deletion of Gα_q/11 did not influence, whereas smooth muscle specific deletion of Gα_12/13 diminished by 50 % and Rho-kinase (RhoK) inhibition (Y27632 10^-6 M) abolished the effect of LPA on coronary flow.

Conclusion: LPA is a strong vasoconstrictor in the coronaries. Its effect is mediated via LPA4 receptor – Gα_12/13 protein – RhoK signaling pathways. This mechanism might have relevance in acute coronary events when LPA is released in the coronary system due to platelet activation and plaque rupture.

SPHINGOSINE INHIBITS CALMODULIN ACTION: IMPLICATION FOR REGULATION OF ENDOTHELIAL NITRIC OXIDE SYNTHASE-MEDIATED VASORELAXATION

Éva Ruisanchez^a, Tünde Juhász^b, Veronika Harmat^c, Viktor Erdősi^a, Zoltán Benyó^a, Károly Liliom^d
aInstitute of Clinical Experimental Research, Semmelweis University, Budapest, Hungary (Supported by OTKA K-112964)
^bInstitute of Enzymology, Research Center for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary
^cLaboratory of Structural Chemistry and Biology, Institute of Chemistry, Eötvös Loránd University, Budapest, Hungary
^dDepartment of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary

Background: Calmodulin (CaM), the main modulator in calcium signaling, regulates the function of a great number of proteins. The signaling lipid sphingosine (SPH) has been reported to inhibit several CaM-dependent enzymes, including endothelial nitric oxide synthase (eNOS). We hypothesized that Sph and its structural derivatives can directly bind to CaM. Here we characterize the interaction of CaM with Sph and its functional effects on the vascular tone.

Results: In vitro binding assays utilizing fluorescence spectroscopy, isothermal titration calorimetry and quartz crystal microbalance revealed that SPH, sphinganine (DHS), and N,N- dimethylsphingosine (DMS) binds to both apo and Ca²⁺-saturated CaM in a concentration and stoichiometry dependent manner. We observed high affinity, sub-micromolar binding to SPH- containing surfaces. We demonstrated in vitro that SPH was able to displace the model CaM- binding domain melittin from CaM. The crystal structure of the SPH-Ca²⁺CaM complex revealed a closed, inhibitory conformation of CaM. eNOS activity in vitro was also inhibited by SPH, DHS, and DMS. We examined eNOS mediated vasorelaxation in phenylephrine precontracted, isolated mouse thoracic aorta segments by myography. 30 min incubation with 10-30 μM SPH, DHS, or DMS dose-dependently shifted the acetylcholine dose-response curves to the right compared to vehicle treated vessels. However, vasorelaxation elicited by sodium-nitroprusside remained unaltered, indicating that the sensitivity of the vascular smooth muscle to NO was not affected by these compounds.

Conclusions: SPH, DHS, and DMS directly bind to CaM, inhibiting its activity on eNOS. In vitro, we observed sub-micromolar affinity when lipids were in associated forms. Ex vivo, SPH and analogues inhibited eNOS mediated vasorelaxation revealing a new regulatory mechanism of the vascular tone by sphingolipids.

ENHANCED ENDOTHELIAL NITRIC OXIDE SYNTHASE MEDIATED VASORELAXATION BY SPHINGOMYELINASE IN DB/DB MICE

Éva Ruisanchez^a, Levente Kiss^b, Éva Zsuzsa Straky^a, Dávid Korda^a, Adrienn Párkányi^a, Károly Liliom^c, Gábor Tigyi^d, Zoltán Benyó^a
aInstitute of Clinical Experimental Research, Semmelweis University, Budapest, Hungary (Supported by OTKA K-112964)
^bDepartment of Physiology, Semmelweis University, Budapest, Hungary
^cDepartment of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary
^dDepartment of Physiology, University of Tennessee Health Science Center, Memphis, US

Background: Sphingolipids, derived from sphingomyelin metabolism, have been implicated as important mediators of inflammatory processes in diabetes. The first step of sphingolipid biosynthesis is catalyzed by sphingomyelinase (SMase) enzymes which are reportedly upregulated in diabetes. In the present study we aimed to analyze the vascular effects of SMase in a mouse model of type 2 diabetes and to gain understanding on the signaling pathways involved.

Results: Db/db mice developed signs of the metabolic syndrome as indicated by their markedly increased body weight and nonfasting blood glucose level compared to control littermates. Interestingly, serum level of phosphorylcholine – the byproduct of the conversion of sphingomyelin to ceramide by SMase – was also significantly increased in the diabetic group. In phenylephrine precontracted aortic segments, the endothelium-dependent vasorelaxation by acetylcholine was diminished in vessels of db/db animals whereas the effect of the NO donor sodium nitroprusside remained unchanged. Surprisingly, in contrast to the transient contraction and subsequent weak relaxation in control vessels, SMase evoked marked relaxation in vessels of diabetic mice. In the presence of the thromboxane receptor antagonist SQ 29,548, SMase induced enhanced relaxation in both groups, but the effect was 3-fold stronger in vessels of db/db mice as compared to controls. Co-administration of the NO synthase inhibitor L-NAME abolished the vasorelaxation in both groups.

Conclusions: SMase induces biphasic changes in the tone of db/db mice derived aorta and these effects are mediated by thromboxane A₂ and endothelial nitric oxide. In spite of the general endothelial dysfunction in type 2 diabetes, the eNOS-mediated vasorelaxant effect of SMase is markedly enhanced.

PROTEIN-(SPHINGO)LIPID INTERACTIONS OF INFLUENZA A WITH HOST CELLS

Laura-Christin Förster*, Dr. Susann Kummer**, Prof. Dr. Hans-Georg Kräusslich** and Prof. Dr. Britta Brügger*
* Heidelberg University Biochemistry Centre (BZH), Im Neuenheimer Feld 328, 69120 Heidelberg
** Department of Infectious Diseases, Virology, University Hospital Heidelberg, Im Neuenheimer   Feld 324, 69120 Heidelberg

Viral budding of Influenza A virus (IAV) occurs at specific apical plasma membrane regions enriched in cholesterol, glyco- and sphingolipids (SLs). SLs seem to play a crucial role in IAV propagation since total cellular SL amounts increase during viral infection and defects in SL biosynthesis largely affect cell-surface localization of viral glycoproteins.

The aim of this study is to shed light on the role of SLs for the IAV life cycle. One goal was to identify SL-binding host proteins which might play a key role in IAV infection. To this end, a SILAC- based proteomic approach using a photoactivatable and clickable (pac) SL precursor, pac- sphingosine (pacSph), was performed. The proteomic screen was conducted with A549 cells possessing a stable knock-out (KO) in Sphingosine-1-phosphate lyase (S1PL) to prevent that pacSph derivatives are irreversibly channeled into the glycerophospholipid metabolic pathway.

Furthermore, effects of SL metabolizing enzymes on viral infection were investigated with the help of CRISPR-mediated KO cells. In initial experiments, HeLa and A549 cells deficient in S1PL (as described in the proteomic experiment) and CerS2, an enzyme catalyzing the synthesis of very- long chain ceramides, were used. Indeed, viral particle load comparison showed differences between WT and KO cells. Interestingly, lipidomic analysis revealed that the different enzymatic KOs directly affect the amount of sphingomyelin species with a sphinganine backbone which presumably influences infection, not the enzymatic KO itself.

Future objectives shall include to assess the role of specific SL-binding proteins as well as the balance between SLs with a sphingosine or sphinganine backbone, the latter referred to as dihydro-SLs, for the viral life cycle.

REGULATION OF LC3-ASSOCIATED PHAGOCYTOSIS BY ACID SPHINGOMYELINASE

Alexander Gluschko¹, Marc Herb¹, Katja Wiegmann¹, Martin Krönke^1,2,3,4, Michael Schramm¹
1Institute for Medical Microbiology, Immunology and Hygiene, ²Center of Molecular Medicine Cologne, ³Cologne Cluster of Excellence on Cellular Stress Responses in Aging-associated Diseases (CECAD), ⁴German Center for Infection Research, Cologne, Germany

Background: LC3-associated phagocytosis (LAP) is a recently discovered form of non-canonical autophagy that, by recruiting LC3 to single-membrane phagosomes, promotes fusion of these phagosomes with lysosomes. As acid sphingomyelinase (ASMase) is required for efficient fusion of phagosomes with lysosomes and thereby for immunity to infection with pathogenic bacteria such as Listeria monocytogenes, we hypothesized that ASMase might play a role in LAP.

Results: Infection of macrophages with Listeria resulted in recruitment of LC3 to a subpopulation of Listeria-containing phagosomes. These LC3-positive phagosomes fused more efficiently with lysosomes leading to enhanced killing of the pathogen. LC3 recruitment to Listeria-containing phagosomes was by LAP as it depended on production of reactive oxygen species (ROS) by the phagocyte NADPH oxidase Nox2 and occurred onto single-membrane phagosomes, which are both hallmarks of LAP. In ASMase-KO macrophages, Listeria-containing phagosomes were less often decorated with LC3 indicating impaired LAP. Furthermore, ROS production in response to Listeria-infection was impaired in ASMase-KO macrophages. As ROS production by Nox2 is a prerequisite for induction of LAP, these data indicate that ASMase regulates LAP by influencing Nox2 activation. Activation of ASMase after Listeria-infection in turn was amplified by Nox2-derived ROS in a positive feedback loop. Moreover, activation of ASMase after Listeria infection was impaired in TNF-KO macrophages and when TNF converting enzyme (TACE) was inhibited.

Conclusions: Together, these data indicate that Listeria infection of macrophages results in TACE- mediated TNF release which activates ASMase. ASMase activation then allows activation of Nox2 and thereby induction of LAP. As LC3 recruitment to phagosomes by LAP promotes phago- lysosomal fusion our data indicate that LAP is one of the mechanisms via which ASMase enhances phago-lysosomal fusion.

MOLECULAR MECHANISMS OF SPHINGOSINE-1-PHOSPHATE INDUCED CHLORIDE CONDUCTANCE IN SENSORY NEURONS

Yanmei Qi, Natalia Schiefermeier, Michiel Langeslag, Michaela Kress
Division Physiology, DPMP, Medical University Innsbruck, Schöpftraße 41/EG, 6020 Innsbruck, Austria

Sphingosine-1-phosphate (S1P) is a small bioactive lipid that mediates diverse cellular responses, such as proliferation, apoptosis, migration and morphogenesis. S1P recently is emerging as an important modulator in regulating nociception. We previously have shown S1P evoked depolarizing ionic current in dorsal root ganglion (DRG) sensory neurons, resulting in the excitation of primary afferent neurons. However, the underlying molecular basis for the S1P-evoked depolarization remains elusive.

The depolarizing chloride currents elicited by S1P have been reported to be correlated with the cytoskeletal remodeling in N1E-115 neuronal cells. Using a siRNA knock-down strategy, we screened several chloride channels involved in S1P-induced cell shape change and assessed whether these chloride channels were involved in the S1P-evoked depolarization in DRG neurons. Combining an adenovirus-based gene silencing technique, RT-PCR and single cell electrophysiological recordings, we showed that the down-regulation of two chloride channels significantly reduced S1P-induced currents in DRG sensory neurons, suggesting that these two chloride channels contributed to the S1P-induced excitatory conductance in sensory neurons.

In summary, we identified two chloride channels that are important for S1P induced inward currents in sensory neurons. Activation of chloride channels by S1P signaling would lead to the influx of Cl^–, resulting in the depolarization and excitation of DRG neurons. The present study provides novel insight into the mechanism by which bioactive sphingolipids regulate neuron function.

DEFECTIVE DE NOVO SPHINGOLIPID SYNTHESIS IN HUNTINGTON’S DISEASE: A NEWPATHOGENIC MOLECULAR MECHANISM?

Di Pardo A¹, Basit A², Armirotti A², Castaldo S¹, Amico E¹, Pepe G¹, Marracino F¹, Digilio A³ and Maglione V¹.
¹IRCCS Neuromed, Pozzilli, Italy
²Department of Drug Discovery and Development, Fondazione Istituto Italiano di Tecnologia, Genova, Italy
³Institute of Biosciences and Bioresources (IBBR), National Research Council (CNR), Naples, Italy

Background:

Alterations of lipid metabolism have been frequently associated with Huntington’s disease (HD) over the past years. HD is the most common neurodegenerative disorder, with a complex pathogenetic profile, typically characterized by progressive striatal and cortical degeneration and associated motor, cognitive and behavioral disturbances. The disease results from a single gene mutation that endows the encoded protein huntingtin (Htt) with toxic properties leading to a broad array of molecular dysfunctions including an overall perturbation of brain lipid homeostasis with significant alteration of sphingolipid metabolism.

Results:

Previous studies form our group demonstrate that disturbed sphingolipid metabolism is an early event in the disease progression. Although such a defect represents a common biological denominator that may underlie the pathology among multiple disease models ranging from cells to humans through mouse models, more efforts are required to be made for deeper investigation of the underlying cause.

Results from our current research provide the first evidence that alteration of sphingolipid metabolism in HD appears to be attributable, at least in part, to a defective de novo synthesis of these specific lipids. In particular, quantitative analysis by Mass spectrometry indicates that levels of the de novo sphingolipids sphinganine and dihydroceramide are significantly reduced in HD.

Conclusions:

Our findings consolidate the evidence of disturbed sphingolipid metabolism in HD and support the hypothesis that it may represent a crucial factor in the pathogenesis of the disease and a novel therapeutic target for the development of more effective treatments.

DISTINCTIVE SPATIOTEMPORAL PATTERN OF COLITIS-ASSOCIATED CANCER DEVELOPMENT DEPENDENT ON S1P LYASE DEFICIENCY IN COLON VERSUS BONE MARROW DERIVED CELLS

Anja Schwiebs^1*, Martina Herrero San Juan¹, Eliza Wiercinska², Martin Anlauf³, Florian Ottenlinger¹, Katrin G Schmidt¹, Dominique Thomas⁴, Eiman Elwakeel⁵, Andreas Weigert⁵, Halvard Bönig^2,6, Klaus Scholich⁴, Gerd Geisslinger⁴, Josef M. Pfeilschifter¹, Heinfried H. Radeke^1*
1 Institute of General Pharmacology and Toxicology, pharmazentrum frankfurt /ZAFES, Hospital of the Goethe University, Frankfurt, Germany
2 German Red Cross Blood Service Institution, Frankfurt, Germany and Institute for Transfusion Medicine and Immunohematology, Goethe University, Frankfurt, Germany
3 Institute of Pathology and Cytology, St. Vincenz Hospital, Limburg, Germany
4 Institute of Clinical Pharmacology, pharmazentrum frankfurt, Hospital of the Goethe University, Frankfurt, Germany
5 Institute of Biochemistry I, pharmazentrum frankfurt, Hospital of the Goethe University, Frankfurt, Germany
6 Department of Medicine/Hematology, University of Washington, Seattle, Washington, USA

Background: Colorectal cancer develops in a highly active immunological environment and has shown considerable resistance to recent cancer immune therapies. Sphingolipids, especially S1P, are involved in both tumor cell survival and immune cell differentiation, migration and cytokine pattern. In this investigation we sought to separately define the contributions of colon vs. immune cells to carcinogenesis, employing inducible SGPL1 knockout bone marrow-chimeric mice in a colitis colon cancer model.

Results: We found that, independent of the compartment of SGPL1 deficiency, hematopoietic or stromal, DSS/AOM-treated mice demonstrated tumors with s1pr1, stat3 and IL-6 elevation. However, compartment-specific actions of the SGPL1-S1P axis contributed both morphologically and genetically to a very distinct development of inflammation and carcinogenesis. Surprisingly, mice with hematopoietic SGPL1 knockout developed severe inflammatory colitis with postponing carcinogenesis specifically characterized by cox2- and pdcd4-driven tumorigenesis. In contrast, stromal SGPL1 deficiency triggered a much more rapid occurrence of epithelial-driven sphingosine kinase 1-positive tumors, exhibiting colon cancer typical egfr and IL23-p19 signatures and a weak but Th-2 driven immune microenvironment. Moreover, we found that S1P lyase derived from hematopoietic cells is sufficient for S1P-driven lymphocyte sequestration.

Conclusions: Our results demonstrate that compartment-specific sphingolipid modulation uniquely clarifies the dichotomy of inflammation-induced cancer versus cancer-induced inflammation.

CERAMIDE SYNTHASE 5 INFLUENCES THE SENSITIVITY OF HCT116 COLON CARCINOMA CELLS TO OXALIPLATIN AND 5-FU IN A P53-DEPENDENT MANNER

Sebastian Brachtendorf, Kerstin Birod, Nerea Ferreirós, Sandra Trautmann, Gerd Geisslinger, Sabine Grösch
Institute of Clinical Pharmacology, Faculty of Medicine, Goethe-University Frankfurt, Theodor- Stern-Kai 7, 60590 Frankfurt am Main, Germany

The sensitivity of colorectal cancer cells to chemotherapy decisively depends on the expression of functional p53 leading across cells into apoptosis upon drug-induced DNA damage. Chemotherapeutic treatment also induces ceramide synthase (CerS) expression and accumulation of pro-apoptotic ceramides. Here we investigated the p53-dependent regulation of CerS expression in oxaliplatin/5-fluorouracil (5-FU)-treated HCT116 cells, and how this affects cellular sensitivity to these genotoxic drugs.

First, we verified a significantly higher sensitivity of wild-type (wt) p53-expressing HCT116 cells to oxaliplatin and 5-FU as compared to isogenic cells lacking p53 expression (HCT116 p53^-/-). After 24 hours treatment with oxaliplatin or 5-FU, we observed a strong increase in CerS5 mRNA in the HCT116 p53^wt cells, but not in the p53^-/- cells, which was accompanied by significantly higher C_16:0– ceramide levels in the WT cells. By contrast, CerS2 expression and CerS2-related C_24:0– and C_24:1– ceramide levels were elevated in the p53-deficient cells only. To elucidate the contribution of p53- dependent CerS2 and CerS5 expression to the cells’ chemosensitivity, we transduced both cell lines with CerS2-/CerS5-targeting shRNA to reach a stable knockdown. CerS5 knockdown led to increased sensitivity of HCT116 p53^wt cells, but not of p53^-/- cells, to oxaliplatin and 5-FU. However, knockdown of CerS2 had no significant effects on chemosensitivity of both cell lines.

These data indicate a p53-dependent protective role of CerS5 in oxaliplatin/5-FU-induced cell death of colon cancer cells. Selective targeting of CerS5 expression might thus be a potential approach to influence the sensitivity of colon cancer cells to chemotherapeutic agents.

VITAMIN D RECEPTOR ACTIVATION INDUCES SPHINGOSINE 1-PHOSPHATE RECEPTOR 1 (S1PR1) EXPRESSION IN RENAL MESANGIAL CELLS

A. Koch¹, M. Grau¹, M. Gebert¹, G. Grammatikos^1,2, R. Brunkhorst^1,3, D. Meyer zu Heringdorf¹, A. Schutkowski⁴, G.I. Stangl⁴ and J. Pfeilschifter^1
1Pharmazentrum Frankfurt/ZAFES, ²Department of Medicine I, ³Department of Neurology, Goethe University Hospital, 60590 Frankfurt am Main, Germany;
⁴Institute of Agricultural and Nutritional Sciences, Martin Luther University Halle-Wittenberg, 06120 Halle (Saale), Germany

Background: Sphingosine 1-phosphate (S1P) acts mainly as an extracellular ligand at five specific G protein-coupled receptors, denoted S1PR1-5. After activation, S1PRs regulate several important cell responses, e.g. intracellular free Ca²⁺ concentration ([Ca²⁺]_i). We investigated whether 1,25(OH)₂ Vitamin D₃ (VitD₃) and the vitamin D analogue EB1089 alter S1PR1 expression levels in renal cells by activation of vitamin D receptor (VDR).

Results: We demonstrate that VitD₃ and EB1089 treatment increased the mRNA abundance of VDR and S1PR1 in rat mesangial cells in a time- and concentration-dependent manner. Further, stimulation with 100 nM VitD₃ and EB1089 for 6 h elevated the VDR and S1PR1 protein expression levels in rat mesangial cells. In addition, in vivo studies showed that feeding of mice for 16 weeks with adequate amounts of VitD₃ (1000 IU/kg/d) enhances S1PR1 expression in kidney lysates compared to the group receiving a vitamin D deficient diet (50 IU/kg/d). In line, S1PR1 levels were upregulated in mouse mesangial cells treated with VitD₃ and EB1089. This effect was not seen in mesangial cells isolated from VDR knockout mice. Functionally, VitD₃ and EB1089 treatment inhibits S1P-induced [Ca²⁺]_i increases, possibly via upregulation of S1PR1, which was previously established as a negative regulator of S1P-induced [Ca²⁺]_i increases in renal mesangial cells (Koch et al. (2013) Biochim. Biophys. Acta 1831: 1634-43).

Conclusions: Activation of VDR by VitD₃ and EB1089 induces S1PR1 expression, which in turn inhibits S1P-induced [Ca²⁺]_i increases in renal mesangial cells. These data show that vitamin D influences sphingolipid metabolism in the kidney.

DOWN-EXPRESSION OF VITAMIN D RECEPTOR IN HUMAN CARCINOMA GASTRIC CELLS: ACID SPHINGOMYELINASE AS POSSIBLE ALTERNATIVE TARGET OF 1 ALPHA,25- DIHYDROXYVITAMIN D3

Elisabetta Albi^1 §, Samuela Cataldi^1§, Irina Nakashidze¹, Ivana Ferri², Angelo Sidoni², Cataldo Arcuri^2§, Carmen Mecca², Giovanna Traina¹, Francesco Saverio Ambesi-Impiombato⁴, Andrea Lazzarini⁵, Francesco Curcio⁴, Maria Rachele Ceccarini ¹, Tommaso Beccari¹, Michela Codini^1
§These authors contributed equally to this work
1 Department of Pharmaceutical Sciences, University of Perugia, Italy
2 Institute of Pathologic Anatomy and Histology, University of Perugia, Italy
3 Department of Experimental Medicine, University of Perugia, Italy
4 Department of Clinical and Biological Sciences, University of Udine, Italy
5 Laboratory of Nuclear Lipid BioPathology, CRABiON, Perugia, Italy

Background: It has been demonstrated that 1 alpha,25-dihydroxyvitamin D3 (VD3) is not a risk factor for gastric cancer but rather exerts chemopreventive effects when given during the post- initiation phase of glandular stomach carcinogenesis. The aim of the work was to test the effect of VD3 in human gastric cancer cells (NCI-N87 cell line) on acid sphingomyelinase (aSMase).

Results. Herein, we showed surprisingly that VD3 strongly down-expressed VD3 receptor. Upregulation of aSMase and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) without changes of neutral sphingomyelinase (nSMase) were dependent on VD3. aSMase activity early increased. VD3 induced overexpression caspase 8, CDKN2B, MAP3K5, cytocrome C apoptotic genes. Morphological analysis highlighted some very large round or oval cells and small cells with angular or fusiform extensions, confirmed by MIB-1 immunodetection and Hercep test. The early activation of aSMase after VD3 treatment raises an important question: it is possible that VD3 early stimulates aSMase activity and this is responsible for the inhibition of VDR expression and for the activation of PTEN and other genes involved in the apoptotic pathway? I will require further studies aimed at solving this problem to clarify the possibility to identify the aSMase as specific target of VD3.

Conclusions. Taken together our results indicated that the action of VD3 in gastric cancer cells was independent on VD3 receptor and suggested the aSMase as a possible target to induce molecular events.

ROLE OF CERAMIDE/PP2A SIGNALING IN NON-DIOXIN-LIKE ORGANIC TOXICANT PCB153 MODULATION OF GAP JUNCTION INTERCELLULAR COMMUNICATION IN LIVER PROGENITOR CELLS

Meacci E., Pierucci F.¹ Squecco R.², Frati A¹, Vestri A. ¹, Vicenti C¹., Matteini F.¹, Machala M.³, Francini F²
1 Dept of Experimental and Clinical Biomedical Sciences “Mario Serio”, Research unit of Molecular and Applied Biology, Viale GB Morgagni 50, University of Firenze, 50134 Firenze Italy;
2 Dept of Experimental and Clinical Medicine, Section of Physiological Sciences, Viale GB Morgagni 63, University of Firenze, Firenze 50134 Italy;
3 Veterinary Research Institute, Hudcova 70, Brno, 62100 Czech Republic

Background. Abundant non-dioxin-like toxicant PCB153, member of a group of pollutants distributed throughout the environment, induces gap junction intercellular communication (GJIC) impairment. Recently, we have shown that PCB153 determines time-dependent changes in sphingomyelin metabolism in WB-F344 cells.

Results. Here, by electrophysiological and biomolecular determinations we provide evidence of the existence of heterotypic GJs, mainly formed by Connexin (Cx) 43, Cx32 and Cx26, and the ability of PCB153 to affect both voltage-dependent and -independent channels. PCB153 can transiently modulate protein phosphatase PP2A activity and promote significant changes in ceramide/Ceramide 1-phosphate (Cer/C1P) level. Interestingly, pharmacological inhibition and gene silencing of PP2A prevent PCB153 effects on GJIC and other membrane biophysical properties. A role for Cer/C1P and PP2A on the expression/function of specific Cx isoform-formed GJ will be also discuss.

Conclusions. Here, we provide experimental evidence for novel mechanisms by which PCB153 promotes GJIC impairment in liver stem-like cells, opening new windows for the identification of potential targets (PP2A and Cer/C1P axis) in the prevention of the tumorigenic action of environmental toxicants.

Membrane lipids regulate sphingolipid catabolism, their enzymes and lipid binding proteins.

Konrad SANDHOFF,
Lymes Institut, University Bonn

Cholesterol and sphingolipids (SLs) stabilize eukaryotic plasma membranes. Together with phospholipids (PLs) they reach luminal vesicles in the late endosomes as platforms for membrane degradation. A maturation process of the luminal vesicles removes lipids inhibiting lysosomal catabolism. Sphingomyelin (SM) is hydrolyzed by acid sphingomyelinase, facilitating cholesterol export to the cytosol by NPC2 and NPC1. SM and cholesterol poor luminal vesicles then serve as platforms for glycosphingolipid degradation employing soluble hydrolases, SAPs (sphingolipid activator proteins) and anionic PLs as stimulators. We reconstituted the catabolic proteins on liposomal surfaces, mimicking luminal vesicles of the lysosomes as platforms for SL degradation. Liposomes with no net surface charge generated only negligible and physiologically irrelevant catabolic rates even at lysosomal pH values. Incorporation of anionic PLs into the SL-carrying liposomes, however, stimulated the catabolic rate by up to more than an order of magnitude. However, the incorporation of cholesterol or SM into the SL carrying liposomal membranes generated a strong inhibition of SL hydrolysis and the transfer of membrane lipids between liposomal vesicles by SAPs, even in the presence of anionic phospholipids. Ongoing in vitro studies indicate that PM-stabilizing lipids, i.e. SM and cholesterol, inhibit several steps of lysosomal SL and glycosphingolipid catabolism, and also lipid solubilisation as studied by Plasmon Resonance (Biacore) and intervesicular lipid transfer activities of several SAPs and NPC2, even in the presence of activating anionic PLs.

The sphinx, bioactive sphingolipids, and sphingomyelinases.

Yusuf HANNUN
Stony Brook

Sphingolipids were initially discovered by J. Thudicum, who identified them as unusual and perplexing lipidic substances from the brain. Their enigmatic nature led Thudicum to naming them after the riddle of the Greek Sphinx. Our studies initially resulted in identification of bioactivities for sphingosine, and intense research over 3 decades has now resulted in appreciation of sphingolipids as an important class of cell regulatory molecules that include sphingosine, sphingosine 1-phosphate, ceramide, ceramide 1-phosphate, and several others. The most recent studies on bioactive sphingolipids have been accelerated by the molecular identification of most/all known enzymes of sphingolipid metabolism, the deployment of model organisms (e.g. yeast and mouse), the development of mass spectrometry to analyze the complexity of lipid structure and levels, and the development of informatics approaches to study the ‘sphingolipidome’. Several attributes are emerging from the study of bioactive sphingolipids. First, metabolism of bioactive sphingolipids constitutes a highly regulated network involving the operation of more than 30 distinct enzymes. Second, most enzymes of sphingolipid metabolism show very specific sub cellular localization, suggesting local metabolism and action of their substrates and products. Third, the study of ceramide in particular shows that this is indeed a family of closely related molecules that show structural specificity, are generated in a combinatorial fashion, and appear to participate in distinct functions. I will illustrate these concepts through the study of the neutral sphingomyelinase family.

The new sphingolipid vascular biology of single dose radiotherapy.

Richard KOLESNICK
Sloan-Kettering Institute – NYC, NY USA

Single dose radiotherapy (SDRT), facilitated by image guidance and intensity modulation technologies that improve precision in tumor targeting to reduce risk of normal tissue toxicity, has revolutionized cancer treatment with local control rates .90%, even in tumors resistant to conventional fractionation. While classic radiobiology focuses on response of tumor cells rather than non-tumor microenvironmental cells, initial pre-clinical studies in our lab found disruption of tumor vasculature obligate for SDRT cure. This endothelial cell dysfunction results from activation of acid sphingomyelinase (ASMase), converting sphingomyelin to the second messenger ceramide in endothelial plasma membranes, events inhibitable by the angiogenic factors bFGF, VEGF-121 or VEGF-165. Conversely, anti-angiogenic agents, such as anti-VEGFR2 Ab DC101 (Imclone), de-repress ASMase activity, synergistically increasing SDRT-induced ceramide elevation, enhancing endothelial dysfunction. That ceramide is critical for anti–ceramide Ab inhibition of DC101-enhanced endothelial damage. These results translate in vivo, as anti-VEGFR2 DC101 or anti-VEGF G6-31 synergistically increase SDRT-induced endothelial injury in MCA/129 sarcomas, and enhance tumor response. Critically, anti-angiogenic Ab delivery must be administered immediately prior (0.5-2h) to SDRT, but not earlier or after, to de-repress ASMase effectively. At longer intervals between drug delivery and SDRT the system appears to counter-regulate, re-setting the ceramide-generating capability of ASMase at or near the original setting. In contrast, tumors in asmase-/- mice, which provide damage-resistant vasculature, are unaffected by either anti-angiogenic agent. This lecture will review fundamentals of this new biology and present unpublished data that define mechanism of coupling of endothelial dysfunction to DNA repair in tumor cells.

Ceramide-enriched compartments in neural stem cell differentiation and neurodegeneration.

Erhard BIEBERICH
Georgia Regents University

Objective: Ceramide is a remarkable sphingolipid in that it organizes membrane microdomains and interacts with protein kinases and phosphatases, thereby eliciting cell signaling pathways regulating neural cell apoptosis, differentiation, cell polarity, and neurodegeneration. We hypothesized that these two functions of ceramide, structural and regulatory, combine the organization of structural cell signaling platforms by ceramide with its function as a specific cell signaling lipid. Using polyclonal antibodies, for the first time generated in our laboratory against specific ceramides, we found that C24:0/C24:1 ceramide was enriched in primary cilia of neural progenitors. Another ceramide species, C18:0 ceramide, was found to induce exosome formation and secretion in astrocytes exposed to Aβ1-42, a neurotoxic amyloid peptide in Alzheimer’s disease. C24:1 as well as C18:0 ceramide bound to atypical PKCζ (aPKCζ), a protein kinase C isoform regulating cell polarity. This data suggests that the interaction of C24:0/C24:1 ceramide and C18:0 ceramide with aPKCζ (and other regulatory proteins in a ceramide-induced protein complex) is critical for the formation of cilia in neural stem cell differentiation and exosomes in neurodegeneration, respectively. Therefore, cilia and exosomes may constitute two compartments that are critically regulated by distinct ceramide species. Supported by NSF grant 112157 and NIH grant R01AG034389.

Ceramide nano-liposomes: road to the clinic.

Mark KESTER Nanostar
Institute, University of Virginia

The ceramide nanoliposome is a non-toxic, non-aggregating 80nm particle that selectively kills cancer cells in multiple solid and non-solid tumor models. Ceramide nanoliposomes are currently being developed for the clinic. Recent pharmacokinetic, pharmacodynamic and toxicology data will be reported from both rodent and canine models. These data are being assembled for a FDA IND submission to support a first in man human trial of the ceramide nanoliposome.
Disclosure: Keystone Nano, Inc. (PA, USA) has licensed ceramide nanotechnology from Penn State Research Foundation. MK is co-founder and CMO of Keystone Nano.

LAPTM4B facilitates late endosomal ceramide export to control sphingolipid mediated cell death pathways.

Tomas BLOM¹, Shiqian LI¹, Andrea DICHLBERGER¹, Nils BÄCK¹, Young Ah KIM², Ursula LOIZIDES-MANGOLD³, Howard RIEZMAN³, Robert BITTMAN², Elina IKONEN^1
1University Of Helsinki / Faculty Of Medicine / Anatomy, ²City University Of New York / Queens College / Department Of Chemistry And Biochemistry, ³University Of Geneva / Department Of Biochemistry

Objectıve: The late endosomal organelles (LE) are a major site for sphingolipid catabolism in eukaryotic cells. The mechanisms by which sphingolipid degradation products exit LE are not well understood. In this study we set out to identify proteins that facilitate the removal of sphingomyelin (SM) degradation product(s) from LE, and to assess their potential role as modulators of cell death pathways.
Methods AND Results: By conducting an siRNA screen of membrane spanning LE proteins using [3H]-SM/LDL as a probe, we identified LAPTM4B as a regulator of endosomal ceramide removal. Using novel crosslinkable and fluorescent ceramide probes we found that LAPTM4B interacts with ceramide and facilitates its removal from LE. LAPTM4B lowers LE ceramide in parallel with, and independent of acid ceramidase-dependent catabolism. In LAPTM4B silenced cells, LE sphingolipid accumulation is accompanied by lysosomal membrane destabilization. However, these cells resist ceramide-driven caspase-3 activation and apoptosis induced by chemotherapeutic agents. Conversely, LAPTM4B overexpression reduces LE ceramide and stabilizes lysosomes but sensitizes to drug-induced caspase-3 activation.
Conclusıon: The data provide evidence for a novel ceramide export route from LE and identify LAPTM4B as its regulator. Moreover, LAPTM4B acts as a gatekeeper between intra- and extra-endosomal ceramide pools, modulating apoptosis sensitivity. By compartmentalizing ceramide, LAPTM4B controls key sphingolipid mediated cell death mechanisms and emerges as a candidate for sphingolipid targeting cancer therapies.

Dihydroceramide accumulation mediates cytotoxic autophagy of cancer cells via autophago-lysosome destabilization.

Sonia HERNÁNDEZ-TIEDRA¹, Gemma FABRIÀS², Josefina CASAS², David DÁVILA¹, L Ruth MONTES3, Israel LÓPEZ-VALERO¹, Kentaro HANADA4, Marja JÄÄTTELÄ5, Alicia ALONSO6, Guillermo VELASCO¹
1Complutense University, 2Institute for Advanced Chemistry of Catalonia , 3Unidad De Biofísica (CSIC, UPV/EHU), 4National Institute of Infectious Diseases, 5Danish Cancer Society Research Center (DCRC), 64Unidad de Biofísica (CSIC, UPV/EHU)

Objective: Autophagy is primarily a cell survival mechanism although depending on the cellular context and duration of the triggering stimuli, this cellular process can also lead to cell death. The molecular bases of this dual role of autophagy in cancer cell survival remains to be clarified. Previous work by our laboratory showed that Δ9-tetrahydrocannabinol (THC, the main active component of marijuana) triggers autophagy-mediated cancer cell death. In this study, we used THC and nutrient deprivation (an autophagic stimuli that triggers protective autophagy) to investigate the specific molecular mechanisms responsible for the activation of autophagy-mediated cancer cell death. Our results show that treatment with THC but not incubation with EBSS enhances the balance between dihydroceramides and ceramides in microsomes and in an autophagosome-enriched fraction. Biophysical experiments with model vesicles revealed that an increase in the dihydroceramide /ceramide ratio similar to that triggered by THC leads to the destabilization and subsequent release of the content of lipidic vesicles. Moreover, treatment with THC induced the release of cathepsins from lysosomes and the subsequent activation of apoptosis in a sphingolipid biosynthesis- and autophagy-dependent manner. Furthermore, pharmacological up-regulation of dihydroceramide levels by inhibiting dihydroceramide desaturase activates autophagy-mediated cancer cell death and exerts a similar anticancer action than THC. Taken together, our findings support that the mechanism of THC-induced autophagy-mediated cancer cell death relies on an increase in the dihydroceramide/ceramide ratio in the endoplasmic reticulum that is transmitted to autophagosomes and leads to autophago-lysosome destabilization, the release of cathepsins and the activation of apoptotic cell death.

Lipid oversupply to cardiomyocytes induces sphingolipid-dependent oxidative stress and induction of mitophagy through ceramide synthase 2.

Brıttany A LAW¹, L. Ashley COWART²
1MUSC, 2MUSC, VA

Objective: Diabetic cardiomyopathy (DbCM) contributes to the high risk of heart failure (HF) in diabetics, but mechanisms underlying DbCM remain unclear. We previously showed that high saturated fat feeding in mice altered cardiomyocyte sphingolipid profiles leading to DbCM and that some of these maladaptations were dependent on autophagy and ceramide synthase 5 (CerS5). In the present study, we sought to further understand the cellular processes in which lipid overload leads to DbCM in in the context of sphingolipids. Mice fed an obesogenic diet and in vitro studies using H9c2 cardiomyocytes were utilized in this study. Increased oxidative stress and apoptosis were identified in the hearts of animals subjected to lipid overload, while animals treated with the sphingolipid synthesis inhibitor myriocin were protected. Similarly, cardiomyocytes treated with palmitate showed a sphingolipid-dependent increase of reactive oxygen species (ROS) and mitophagy. Treatment with mitochondria-targeted ceramide analogs revealed that very long chain ceramides, but not long-chain ceramides, induced cardiomyocyte cell death, which was exacerbated by inhibiting mitophagy. This suggested that lipotoxicity to cardiomyocytes occurs in part through oxidative stress in a sphingolipid-dependent manner, and mitophagy may occur to prevent further damage. In the observation that only very long chain species led to these outcomes suggested involvement of CerS2. Overexpression of CerS2 showed increased mitophagy in cardiomyocytes and knockdown of CerS2 by CRISPR-CAS9 technology decreased mitophagy. Taken together, our data suggest that lipid overload induces mitophagy as a protective measure in defense from CerS2-induced mitochondrial damage, oxidative stress, and cell death in DbCM.

Adjuvant tamoxifen improves effectiveness of ceramide-centric therapy in acute myelogenous leukemia .

Samy MORAD¹, Mark KESTER², Thomas LOUGHRAN², Traci DAVIS¹, Terence RYAN3, Tonya Zeczycki¹, Myles CABOT¹,
1Department Of Biochemistry And Molecular Biology, East Carolina University, Brody School Of Medicine, Greenville, Nc 27834, Usa, 2University Of Virginia Cancer Center, Charlottesville, Va 22908, Usa, 3Department Of Physiology, East Carolina University, Brody School Of Medicine, Greenville, Nc 27834, Usa

Objective: The breast cancer drug tamoxifen is an effective inhibitor of sphingolipid metabolism, blocking ceramide glycosylation as well as inhibiting acid ceramidase activity. These “off-target” actions make tamoxifen an interesting ancillary for improving the apoptosis-inducing potential of ceramide. The purpose of this study was to evaluate the therapeutic properties of single agent C6-ceramide versus a C6-ceramide-tamoxifen regimen in AML, an aggressive leukemia.
Methods: Studies were conducted in AML cells lines (KG-1, HL-60, HL-60/VCR), patients cells, and MLL-AF9 AML mice. Agents were administered in ethanol or as nanoliposomes. Cytotoxicity was assessed by MTS and propidium iodide, apoptosis by Annexin V and DNA fragmentation, therapeutic potential by spleen and marrow leukemia burden, mitochondrial function by respirometry, ATP levels by enzymatic end-point assays, glycolysis by Seahorse, and lipid analysis by mass spectroscopy.
Results: The therapeutic impact (viability, apoptosis) of the C6-ceramide-tamoxifen regimen was superior to single agents. Mitochondrial targeting was evidenced by sharp decreases in Complex I respiration, membrane potential, and ATP levels. In KG-1 cells, a 24 hr exposure reduced glycolytic capacity by 50% and downregulated survivin expression by 60%. Tamoxifen halted conversion of C6-ceramide to C6-glucosylceramide and also synergistically boosted effectiveness of the ceramide-generating retinoid, fenretinide (4-HPR) in drug resistant HL-60/VCR cells, which was accompanied by caspase-3 activation. Nanoliposomal administration of C6-ceramide-tamoxifen lowered leukemia stem cell burden in spleen and marrow in MLL-AF9 AML mice.
Conclusions: Our results demonstrate a multi-hit versatility of tamoxifen with ceramide-centric therapies for magnifying therapeutic potential in AML. Acknowledgment- NCI PO1CA17198

Host sphingolipid-transfer proteins and infectious diseases.

Kentaro HANADA
National Institute of Infectious Diseases

Objective: Intracellular trafficking of lipids are essential events for lipid metabolism and membrane biogenesis [Hanada & Voelker (2014) Traffic ]. Ceramide is transported from the ER to the medial/trans Golgi region for the synthesis of sphingomyelin by the ceramide transfer protein CERT [Hanada et al (2003) Nature; Hanada (2014) BBA]. Ceramide is also transported to the cis Golgi compartment or a sub-region of the ER for the synthesis of glucosylceramide (GlcCer) by a CERT-independent pathway. GlcCer is then transported to the medial/trans Golgi region for the synthesis of lactosylceramide via the glycosphingolipid transfer protein FAPP2-dependent and –independent pathways [D’Angelo et al (2013) Nature]. Recent studies showed that several pathogens exploit the lipid-transfer proteins of host cells to steal host sphingolipids to their own purposes. The obligate intracellular bacterium Chlamydia spp forms the parasitophorous vacuole “inclusion”, and proliferates in it, stealing various host metabolites. The inclusion protein IncD binds the PH domain of CERT, and recruits ceramide from the ER to the inclusion [Derre et al (2011) PLoS Pathog; Elwell et al (2011) ibid]. Although various viruses form membranous structures for their replication in host cells, the mechanisms underlying their formation are largely unknown. Intriguingly, human hepatitis virus type C (HCV) requires host FAPP2 for replication [Khan et al (2014) J Virol]. HCV might exploit FAPP2 to recruit GlcCer from the Golgi complex to the membranous viral factory, and such GlcCer-recruitment would be crucial for the HCV factory to acquire a detergent-resistant “raft-like” characteristics.

A potential function of sphingolipid-dependent secreted exosomes in sequestering alzheimer’s amyloid-ß.

Yasuyuki IGARASHI¹, Kohei YUYAMA²,
1Hokkiado Univ., 2Hokkaido Univ.

Objective: Increased levels of amyloid-ß peptide (Aß) in brain are linked to the pathogenesis of Alzheimer’s disease (AD). Exosome, an extracellular lipid vesicle, contains several proteins related to neurodegenerative disorders, including Aß. However, the role of the exosomes in AD pathogenesis is largely unknown. In the resent study, we demonstrated that the neuronal exosomes were abundant in glycosphingolipids (GSLs) and coupled with Aß in a GSL-dependent manner. The exosome-bound Aß was then incorporated into brain-resident phagocytes, microglia, for degradation, suggesting that the exosomes contribute to Aß clearance. Moreover, we found that the exosome production was modulated by sphingolipid metabolism. Inhibition of sphingomyelin synthase activity enhanced exosome secretion and accelerated Aß clearance in a transwell study. Consistent with the notion of in vitro study, continuous administration of the exosomes into mouse brains resulted in marked reductions in Aß levels, amyloid depositions, and Aß-mediated synaptotoxicity in AD model mice. In addition, we showed that the densities of exosomes in mouse and monkey CSF declines during aging, inversely correlated with intracerebral Aß levels. Our data revealed that the exosomes act as potent scavengers for Aß by carrying it on exosome surface GSLs. Improving Aß clearance by exosome administration or upregulation of endogenous exosome secretion would be a novel strategy for AD therapy. References: Yuyama K., Igarashi Y. et al JBC 287:10977-10989( 2012); Yuyama K, Igarashi Y et al. JBC 289: 24488-24498 (2014); Yuyama K, Igarashi Y. BBA 1841: 793-798 (2014); Yuyama K, Igarashi Y et al, FEBS Lett. 589:84-88 (2015)

Molecular mechanism of the production of acylceramide, the key lipid for skin barrier formation.

Akio KIHARA¹, Yasuyuki IGARASHI¹,
1Hokkaido University

Objective: The epidermal permeability barrier is essential for land-dwelling creatures. Lipid lamellae present in the stratum corneum, the uppermost layer of the epidermis, are responsible for barrier formation. Acylceramide is the most important of the epidermal lipids, both in physiological and pathological terms. Decreases in epidermal acylceramide levels are associated with cutaneous disorders such as ichthyosis and atopic dermatitis. However, the mechanism behind acylceramide production is poorly understood, leaving the broader picture of molecular mechanisms behind epidermal barrier formation still unclear. Here, we identified the cytochrome P450 CYP4F22 as the missing fatty acid ω-hydroxylase required for acylceramide production. Furthermore, we also revealed that PNPLA1 is required for the final step of acylceramide synthesis, ester bond formation between ω-hydroxyceramide and linoleic acid. Both CYP4F22 and PNPLA1 have been identified as causative genes of autosomal recessive congenital ichthyosis (ARCI). ARCI mutant proteins of CYP4F22 and PNPLA1 exhibited reduced enzyme activity. Furthermore, acylceramide levels were greatly reduced in CYP4F22 ARCI patient. Our findings clearly demonstrate a relationship between ARCI pathology, acylceramide levels, and enzyme activities of CYP4F22 and PNPLA1 and provide important new insights into the molecular mechanisms of acylceramide synthesis and skin barrier formation.

Identification of a novel pathway for acylceramide generation in lipid droplets by ceramide synthase, fatty acyl-CoA synthase, and diacylglycerol acyltransferase enzyme complex.

Can SENKAL¹, Yusuf HANNUN², Lina OBEID¹,
1Stony Brook University, 2Stony Brook University

Objective: Fatty acyl-CoA (FA-CoA) dependent ceramide generation is catalyzed by ceramide synthases (CerS) in the de novo pathway. Importantly, six CerS isoforms displaying different preferences to generate ceramides with distinct fatty acyl chains have been identified. Currently, there is little insight into the regulation of the distinct CerS enzymes. In an approach aimed at defining interacting partners of CerS’ and shed light into substrate preference properties of CerS enzymes, we found that that fatty acyl CoA synthase ACSL5 interacts with CerS. Contrary to our initial hypothesis, ACSL5 generated FA-CoA was not a direct substrate utilized by CerS to generate ceramide as measured by LC/MS. Rather, ACSL5 generated FA-CoA was utilized together with de novo ceramide for the generation of acylceramides, poorly studied ceramide metabolites. Moreover, inhibition of ceramide channeling to acylceramide enhanced accumulation of de novo ceramide and resulted in augmentation of ceramide-mediated apoptosis. Mechanistically, we show that acylceramide generation is catalyzed by diacylglycerol acyltransferase 2 (DGAT2) and involves the formation of an ACSL5-CerS-DGAT2 complex on lipid droplets. In summary, this study identifies the molecular mechanism of a novel metabolic pathway of acylceramide generation and implicates the importance of this novel pathway in ceramide-mediated apoptosis.

Epigenetic control of glycosphingolipid metabolic switch.

Giovanni D’ANGELO¹, Domenico RUSSO¹, Serena CAPASSO¹, Lucia STICCO¹, Roberto DE GREGORIO², Riccardo RIZZO¹, Floriana DELLA RAGIONE², Maria MATARAZZO², Gian Carlo BELLENCHI², Maurizio D’ESPOSITO²
1Institute Of Protein Biochemistry , National Research Council Of Italy, Naples, 2Institute Of Genetics And Biophysics, National Research Council Of Italy, Naples

Objective: Glycosphingolipids (GSLs) are membrane lipid constituents characterized by a ceramide backbone linked to a glycan moiety. Hundreds of different glycan sequences can be incorporated into GSLs in a process, which does not depend on a pre-existing template nor is strictly genetically determined. Nevertheless GSLs expression is tightly regulated during embryogenesis and failure to synthesize specific GSLs leads to serious genetic diseases in humans. How individual cells decide which GSLs (among the many possible options) to synthesize under a given condition is not understood. Here we describe a self-contained control circuit regulating the metabolic direction of GSLs glycan elongation through the epigenetic control of GSL synthesizing enzymes expression. We found, indeed, that the globoside Gb3 represses the transcription of the first enzyme involved in the synthesis of gangliosides (i.e. GM3 synthase; GM3S). Gb3 exerts this function by negatively regulating the expression of the epigenetic modulator of neuronal differentiation AUTS2, which in turn activates GM3S promoter by fostering local histone acetylation. Our results provide a mechanistic explanation for the Globo-to-Ganglio GSL switch observed during neuronal differentiation, and ascribe a new role to GSLs in the epigenetic control of neuronogenesis.

Elucidating novel metabolic pathways of the neurotoxic 1-deoxysphingolipids.

Irina ALECU¹, Alaa OTHMAN², Arnold VON ECKARDSTEIN¹ , Thorsten HORNEMANN¹
1Institute Of Clinical Chemistry, University Hospital Zurich, 2Institute Of Experimental And Clinical Pharmacology And Toxicology, University Of Lübeck,

Objective: Serine palmitoyltransferase (SPT), which catalyzes the first step in the de novo synthesis of sphingolipids, typically condenses serine and palmitoyl-CoA. Under certain conditions SPT can also use alanine, resulting in the neurotoxic atypical 1-deoxysphingolipids (1-deoxySLs). Pathologically elevated 1-deoxySLs cause the inherited neuropathy HSAN1. Due to the missing C1-hydroxyl group, 1-deoxySLs are not metabolized to complex sphingolipids nor degraded by the canonical pathways. Here we investigated the metabolic pathways of 1-deoxySLs in order to understand and potentially control their buildup. We used high-resolution high accuracy mass spectrometry and metabolic profiling workflows to identify novel downstream 1-deoxySL-metabolites. The formation of these novel 1-deoxySL metabolites was modulated using specific chemical enzyme inhibitors and inducers. We identified five novel 1-deoxySL downstream metabolites. Blocking the conversion of 1-deoxysphinganine to 1-deoxysphingosine with Fumonisin B1 significantly decreased the levels of these metabolites. When directly treating cells with different synthetic 1-deoxysphingosine isomers, we observed corresponding variations in the newly identified metabolites. Furthermore, the formation of all five metabolites could be modulated by inhibiting or inducing a specific cytochrome P450 subfamily. While neurotoxic 1-deoxySLs are not metabolized by the canonical pathways, we showed for the first time that they are in fact metabolized downstream by a specific subfamily of cytochrome P450 enzymes. We have elucidated the structure of five novel 1-deoxySL metabolites and their metabolic order, which is downstream of 1-deoxysphingosine. This novel metabolic pathway may be further exploited as a novel therapeutic target to reduce 1-deoxySL levels in HSAN1 patients.

Production of 2-N-acyl-amino-14,16-dimethyloctadecan-3-ol (n-acyl-aod) by CHO-LY-B cells in culture: identification of a new family of naturally occurring N-acyl-1-deoxysphinganine analogues using liquid-chromatography electrospray tandem mass spectroscop.

Brandon M. KENWOOD ¹, Samuel KELLY¹, Jingjing DUAN¹, Anita SOLHAUG², Silvio UHLIG², Gunnar Sundstol ERIKSEN², Ronald T. RILEY 3, Alfred H. MERRILL, Jr.¹
1Georgia Institute Of Technology, 2Norwegian Veterinary Institute, 3United States Department Of Agriculture

Objective: Fusarium avenaceum is a fungal contaminate of many starch plants including cereal grains. It produces 2-amino-14,16-dimethyloctadecan-3-ol (AOD), which has structural features of a sphingoid base–more specifically, a 1-deoxysphinganine. Many other categories of sphingoid bases are N-acylated by ceramide synthases, but it is not known if AOD undergoes acylation, or if so, which fatty acyl-chain-length metabolites would be made. To answer these questions, we have used CHO-LY-B cells, which cannot make endogenous sphingolipids due to a defective serine palmitoyltransferase, and culture them under conditions where they contain no detectable sphingoid bases. Thus, the cells can be incubated with AOD and the metabolites analyzed without interference from endogenous sphingoid bases nor ambiguity about the species that are found. When lipid extracts from CHO-LY-B cells incubated with sub-toxic levels of AOD were examined by liquid chromatography electrospray ionization-tandem mass spectrometry, it was evident that the cells produce N-acyl-derivatives of AOD with a wide range of chain lengths (C16 to C24), which suggests that AOD is a substrate for multiple ceramide synthases. The cellular effects and metabolism of N-acyl-derivatives of AOD warrant further investigation due to the prevalence of F. avenaceum in the world food supply. This work was supported by funds from NIH grant R01GM76217, the Smithgall Institute Chair in Molecular Cell Biology, and the Norwegian Veterinary Institute.

Sphingosine‐1‐phosphate lyase deficiency in the brain.

Gerhild VAN ECHTEN1
Limes-Institute For Membrane Biology & Lipid Biochemistry At The Kekulè-Institute Of The University Bonn1

The evolutionary conserved bioactive lipid sphingosine 1‐phosphate (S1P) is essential for brain development but can exert detrimental effects in postmitotic neurons. Its content in the brain is regulated by specific kinases, phosphatases and by S1P‐lyase (SPL).
The potential role of S1P in neurodegenerative disorders, especially in Alzheimer’s disease (AD) is controversially discussed. On the one hand S1P was found to stimulate Abeta formation in neurons by directly interacting with β‐secretase BACE1. On the other hand S1P was described as a neuroprotective factor that is lost early in AD pathogenesis. This raises intriguing questions on the pathophysiological role of S1P metabolism in AD etiology: Do age‐dependent alterations in S1P/sphingosine metabolism trigger or reduce the accumulation of Abeta, hyperphosphorylated tau and ultimately neuronal death in AD? Recent studies in Alzheimer brains revealed that regions most heavily affected by Alzheimer pathology, exhibited the most pronounced decline of S1P levels. To increase S1P amounts in the brain we generated a mouse model with targeted deletion of SPL. Although no neuronal loss was detectable, these animals exhibit age‐dependent deficits of motor coordination, spatial learning and memory. Preliminary studies indicate enhancement of sphingosine and S1P, decrease of phosphatidylethanolamine, impaired autophagy, altered processing of amyloid precursor protein (APP), increased proteasomal activity and decreased expression of presynaptic proteins. Potential molecular links between SPL deficiency and the observed biochemical and physiological changes will be presented.

What have we learnt from the study of ceramide synthase knock-out mice?

Anthony FUTERMAN
Department Of Biological Chemistry, Weizmann Institute Of Science

ABSTRACT NOT PRESENT IN iCC/SLC 2015 MEETING BOOKLET

Lack of ceramide synthase 2 suppresses the development of experimental autoimmune encephalomyelitis by impairing the migratory capacity of neutrophils.

Julia BARTHELMES¹, Anika MANNER DE BAZO², Yael PEWZNER-JUNG3, Max EBERLE¹, Nerea FERREIROS¹, Gerd GEISSLINGER¹, Atnhony FUTERMAN3, Sabine GROESCH¹, Susanne SCHIFFMANN¹
1Instıtute Of Clınıcal Pharmacology; Hospıtal Of The Goethe-Unıversıty Frankfurt, 2Department Of Neurology, Goethe-University Frankfurt, 3Department Of Biological Chemistry, Weizmann Institute Of Science

Objective: Until now, the role of ceramides with specific chain lengths in the development of EAE/MS have not been elucidated. CerS2 null mice which lack very long chain ceramide and sphingolipids, but have increased C16-Cer and -sphingolipid level served as a good tool to study the chain length-specific role of ceramides in the development of EAE.
Methods: EAE was induced in female mice by subcutanously injected with MOG35-55 (myelin oligodendrocyte protein) emulsified in complete Freund`s adjuvant (CFA) and an i.p. injection of pertussis toxin (PTX). Ceramide levels were determined by LC-MS/MS, mRNA levels by quantitative PCR, protein levels of CerS2 by western blot and the expression of the chemokine receptor CXCR2 by FACS.
Results: In EAE mice we observed a significant elevation of CerS2 and its product, C24-ceramide, in CD11b+ cells (monocytes and neutrophils) isolated from blood. Specific genetic deletion of CerS2 significantly delayed the onset of clinical symptoms, due to a reduced infiltration of immune cells, in particular neutrophils, into the central nervous system. Neutrophils isolated from CerS2 null EAE mice, as opposed to WT EAE mice, were characterised by significantly lower CXCR2 receptor mRNA expression resulting in reduced migratory capacity towards CXCL2. Also G-CSF-induced CXCR2 expression was significantly reduced in CerS2 null neutrophils and their migratory capacity was significantly impaired.
Conclusion: Our data strongly indicate that G-CSF-induced CXCR2 expression is regulated in a CerS2-dependent manner and that CerS2 thereby promotes the migration of neutrophils, thus, contributing to inflammation and the development of EAE and MS.

Ceramide transporters: the link between lipid metabolism, inflammation and amyloid-ß aggregation in Alzheimer’s disease.

Pılar Martinez MARTINEZ¹
1Maastricht University

Objective: Alzheimer’s disease (AD) is the most common form of dementia, characterized by neuropathological hallmarks as synaptic loss, aggregates of amyloid-ß peptides (Aβ), neurofibrillary tangles, ceramide accumulation in the brain, gliosis and neuroinflammation. No treatment or intervention options are currently able to modify the pathophysiology of AD. Recently, ceramide transporters (CERTs) have been implicated as possible therapeutic candidates. CERT proteins have the unique function of transporting the lipid ceramide via its (StAR)-related lipid transfer (START) domain. CERTs are known to be responsible for the trafficking of ceramides inside and outside the cells. Interestingly, these ubiquitous proteins bind to proteins that are prone to misfolding and aggregation. And they are essential for the development and homeostasis of the central nervous system (CNS). The knockdown of CERT leads to loss of myelinated tracts and to extensive apoptosis. Recently, we identified CERTL as an Aβ-binding protein (binds Aβ1-42) associated to human and murine amyloid plaques. Our results show that CERTL reduces Aβ aggregation and has a protective effect against Aβ-induced toxicity in neuroblastoma cells. Additionally, we have identified this protein as a receptor for C1q in the surface of damaged cells, which can activate the complement system. This indicates that CERTL takes part in the cellular defensive response against Aβ- induced toxicity and is involved in the pathophysiological mechanisms associated with neurodegenerative processes.

The pathomechanisms underlying the neurotoxicity of 1-deoxysphingolipids.

Saranya SURIYANARAYANAN¹, Arnold VON ECKARDSTEIN¹, Thorsten HORNEMANN¹
1Institute Of Clinical Chemistry, University Hospital Zürich

Objective: Hereditary sensory and autonomic neuropathy type I (HSAN I) is a slowly progressing neurological disorder characterized by loss of pain and temperature sensation. It has been associated with several mutations in the enzyme serine palmitoyltransferase (SPT) which catalyzes the condensation of palmitoyl-CoA and serine – the first and rate-limiting step in the de novo biosynthesis of sphingolipids. The HSAN1 mutations shift the substrate specificity of SPT from L-serine to L-alanine which results in the formation of atypical and neurotoxic 1-deoxysphingolipids (1-deoxySLs) which are found to be pathologically elevated in HSAN1. However, the pathomechanisms underlying the 1-deoxySLs mediated neurotoxicity is unknown. This study aims to identify and characterize downstream pathways of 1-deoxySLs mediated neurotoxicity. Neurotoxicity of 1-deoxySLs was assessed using immunofluorescence, live cell imaging and by quantifying the neurite outgrowth in a neuronal cell models. Downstream signalling pathways involved in 1-deoxySLs mediated cytotoxicity and neurotoxicity are identified by screening a commercially available kinase inhibitor library using Hek293, SH-SY5Y cells as well as in primary DRG. 1-deoxySLs caused significant cytotoxic and neurotoxic effects in HEK293 and Neuronal cells. Neuronal cells showed changes in cell morphology and reduction in the length and number of neurites. On the basis of these results we developed a high throughput assay to screen a commercial kinase inhibitor library. First results indicate that some of the tested inhibitors are able to decrease 1-deoxySLs mediated toxicity. The 1-deoxySLs were shown to be toxic to neuronal cells. This toxicity appears to be related to specific cellular signalling pathways.

Development of pheochromocytoma in ceramide synthase 2 null mice.

WooJae PARK¹, Ori BRENNER¹, Aviram KOGOT-LEVIN², Ann SAADA², Alfred MERRILL3, Yael PEWZNER-JUNG¹, Anthony FUTERMAN¹
1Weizmann Institute Of Science, 2Hebrew University Medical Center, 3Georgia Institute Of Technology

Objective: Pheochromocytoma (PCC) and paraganglioma are rare neuroendocrine tumors of the adrenal medulla and sympathetic and parasympathetic paraganglia for which mutations in ~15 disease-associated genes have been identified. We now document the role of an additional gene in mice, the ceramide synthase 2 (CerS2) gene. CerS2 is one of six mammalian CerS, which synthesizes ceramide with very-long acyl (C22-C24) chains. The CerS2 null mouse has been well characterized, and displays lesions in several organs including the liver, lung and the brain. We now demonstrate that changes in the acyl chain profile of the adrenal gland leads to the generation of adrenal medullar tumors. Histological analyses revealed that about half of the CerS2 null mice developed PCC by ~13 months, and the rest showed signs of medullary hyperplasia. Norepinephrine and normetanephrine levels in the urine were elevated at 7 months of age consistent with the morphological abnormalities found at later ages. Accumulation of ceroid in the X-zone was observed as early as 2 months of age and as a consequence, older mice displayed elevated levels of lysosomal cathepsins, reduced proteasome activity and reduced activity of mitochondrial complex IV by six months of age. Together, these findings implicate an additional pathway that can lead to PCC formation, which involves alterations in the sphingolipid acyl chain length. Analysis of the role of sphingolipids in PCC may lead to further understanding of the mechanism by which PCC develops, and might implicate the sphingolipid pathway as a possible novel therapeutic target for this rare tumor.

Alkaline ceramidase 3 deficiency results in Purkinje cell degeneration and cerebellar ataxia due to dyshomeostasis of ceramides and their metabolites in the brain.

Kai WANG¹, Ruijuan XU¹, Jennifer SCHRANDT¹, Yong Z. GONG², Demetri D. SPYROPOULOS², Wei SUN ², Ashley J. SNIDER¹, Yusuf A. HANNUN¹, Lina M. OBEID¹, Cungui MAO¹
1Stony Brook University , 2Medical University Of South Carolina

Objective: Dysregulation of the metabolism of ceramides has been implicated in aging and age-related neurodegenerative disorders. However, much remains unknown about how the metabolism of ceramides is controlled in the central nervous system (CNS) during normal aging. Here we report that the mouse alkaline ceramidase 3 (Acer3) is critical for the homeostasis of ceramides in the aging brain and that its deficiency causes degeneration of Purkinje cells (PC) and thereby impairs mouse motor coordination in mice at around 8 months of age. Enzymatic activity assays revealed that, like human ACER3, the mouse Acer3 preferentially catalyzed the hydrolysis of unsaturated long-chain ceramides (ULCCs) such as C18:1-ceramide, a major ceramide species in the brain. Acer3 was found to be upregulated with age in the mouse brain, and its upregulation was associated with a decrease in the levels of C18:1-ceramide and a reciprocal increase in the levels of sphingosine (SPH) and sphingosine-1-phosphate (S1P). Acer3 knockout caused an age-dependent accumulation of various ceramides in the brain while abolishing the age-related increase in the levels of both SPH and S1P in this tissue. Acer3 knockout caused PC degeneration in the cerebellum and impaired the ability in motor coordination and balance in mice at around 8 months of age. Taken together, these results suggest that Acer3 plays critical roles in controlling the homeostasis of ceramides and their metabolites SPH and S1P in the brain as well as protecting PCs from premature degeneration.

Administration of phytoceramide and glucosylceramide ameliorated the memory impairment in mice.

Kyunghwa YUN¹, Ji-yeon YU¹, Yeahyun LEEM¹, Seikwan OH¹
1Ewha Womnas Univ.

Objective: This study was aimed to investigate the possible roles of phyroceramide and glucosylceramide in memory function in mice.
Methods: Phytoceramide was orally administered to ICR mice for 7 days. Memory performances were assessed using the passive avoidance test and Y-maze task.Aged mice were given experimental diet pellets which contains glucosylceramide for 3months. Glucosylceramide (50mg/kg, p.o.) showed memory enhancing activity after 3-month treatment in the aged mice (C56BL/6, 18–20months old) through Y-maze, novel objective test, and Morris water maze test.
Results and Conclusion: Treatment of Pcer enhanced cognitive performances in the passive avoidance test and Y-maze task. Immunoblotting studies revealed that the phosphorylated CREB and BDNF were signifi cantly increased on hippocampus in the Pcer-treated mice.Long-term treatment of glucosylceramide decreased the expression of iNOS and COX-2 in the brain of aged mice. The LPS-induced mRNA level of iNOS, COX-2, IL-1

Structural and functional characterization of serine palmitoyl transferase in mammals.

Museer LONE¹, Thorsten HORNEMANN²
1Institute For Clinical Chemistry, University Hospital Zuric, 2Institute For Clinical Chemistry, University Hospital Zurich

Objective: Regulatory and signaling mechanisms that govern SPT function have been the focus of several recent studies, primarily in yeast cells. However, initial findings from the cultured mammalian cells suggest that mammalian SPT is subject to differential and complex regulations. Moreover, mechanistic changes leading to an increased activity or the shift in substrate preference of the SPT enzyme have not been worked out yet. In particular, the factors which lead to the formation of neurotoxic 1-deoxySLs which are associated with peripheral neuropathies (HSAN1 or diabetic sensory poly neuropathy) is not understood. Here, we aim to identify the protein interactome of the functional SPT complex to determine the regulatory factors of the SPT holoenzyme and to characterize the biological relevance of the interactions in vivo.
Methods: We are using genetically modified KO-cell lines, in combination with fluorescent tagged proteins and FRET as well as co-immunoprecipitation studies followed by mass spectrometry to elucidate SPT subunit interactions. Furthermore, we challenge cells with pharmacologically and biological stimuli to modulate sphingolipid synthesis to investigate the effect on the molecular and enzymatic properties of the SPT complex.
Results: Methods to immuno-purify the SPT enzyme by targeting individual subunits have been established. Preliminary data show that specific mutations in certain SPT subunits influence regulation and alter the overall SPT activity and product spectrum.
Conclusions: SPT activity is subject to complex regulations which might be mediated by direct modifications of subunits and the association and dissociation with the inhibitory and activating proteins.

Targeting sphingolipid signaling in the visual system.

Riccardo GHIDONI
Dept. Health Sciences, Univ. Milan

Current knowledge of mechanisms underlying photoreceptor death in retinal degenerative diseases has lead to the notion that cellular apoptosis is the predominant mechanism, even in genetically heterogeneous disorders such as Retinitis Pigmentosa (RP). We are performing follow up studies based upon pharmacological targeting of the biochemical pathway leading to the ceramide synthesis and sphingosine-1-phosphate (S1P) cleavage, in order to both counteract apoptosis and preserve survival.
Results obtained in vivo on rd10 mutant mice (a model of recessive RP) showed that ceramide levels in the retina increase at the peak of rod degeneration, but can be lowered by ocular administration of myriocin, an inhibitor of Serine Palmitoyl Transferase, the rate-limiting enzyme in ceramide biosynthesis. Noticeably, this treatment also decreases the number of dying photoreceptors. To achieve prolonged treatment (i.e. several weeks), we developed a non-invasive drug-delivery approach based on eye drops of a suspension of solid lipid nanoparticles (SLNs) loaded with myriocin. The nanoparticles function as vectors allowing topical drug administration. Daily supply of SLN-eye drops loaded with myriocin to rd10 mice lowered rod photoreceptor death and promotes survival, preserved their morphology and extended the ability of the retina to respond to light as assessed by ERG recordings. It is important to say that cone photoreceptors and cone-mediated vision were also considerably well preserved over time.
Investigation of the potential role of S1P in photoreceptor survival has been addressed by the use a cone-like 6661W cell line, previously treated with hydrogen peroxide to induce oxidative stress. We wished to increase S1P in cone photoreceptors aiming to enhance their survival and resistance mechanism to oxidative stress. We observed that: i) exogenous S1P, at low doses, recovers 661W cells viability from oxidative stress; b) administration of a S1P lyase inhibitor, THI, recovers 661W cells viability after oxidative stress; c) THI reduces apoptosis of H2O2-stressed 661W cells; d) THI reduces endogenous stress mediators in 661W cells; e) THI enhances anti-oxidant & survival response to H2O2 in 661W cells. Administration of THI to rd10 animals is currently addressed. These data pose promising premises to set up a combination therapy, with both Myriocin and THI, to delay photoreceptor degeneration in Retinitis Pigmentosa.

Complexities of cardiac sphingolipid metabolism.

Ashley COWART
Medical University Of South Carolina

In recent years, numerous studies have revealed important roles of sphingolipids including sphingosine-1-phosphate and ceramide in cell, tissue, and organ responses to nutrient surplus. Our lab has focused on how fatty acids differentially regulate sphingolipid metabolism, and how these bioactive lipids work to exacerbate and/or protect from metabolic insult. In the heart, ceramides serve key functions in various disease contexts. Moreover, data reveal specialized pathways of cardiomyocyte sphingolipid metabolism that make distinct contributions to pathophysiology during cardiac metabolic insult. Our laboratory has investigated two of these pathways: Sptlc3-derived sphingolipids, and CerS5 and CerS2-derived sphingolipids. Each of these pathways are stimulated under conditions of fatty acid surplus, and each mediates a distinct cell process. In addition to teasing out individual contributions of these pathways in cell culture systems, emerging data from mouse models has provided insight into how these pathways may converge to mediate diabetic cardiomyopathy; however, cell type-specific approaches will be vital to better understand cardiomyocyte-specific sphingolipid actions.

Hepatic upregulation of serine palmitoyltransferase subunit 2 by endoplasmic reticulum stress modulates glucose and lipid metabolism.

Su-Yeon LEE¹, Su-Jung KIM², Bo-Rahm KIM¹, Tae-Sik PARK¹
1Gachon University, 2Ulsan University

Objective: The endoplasmic reticulum (ER) is the principal organelle in the cell for protein folding and trafficking, lipid synthesis and cellular calcium homeostasis. Perturbation of ER function by accumulation of misfolded protein results in activation of the unfolded protein response (UPR). Chronic ER stress is reported to have an important role in abnormal lipid and glucose metabolism leading to development of insulin resistance. Here, we reports that transcription of serine palmitoyltransferase (SPT) is regulated by ER stress-mediated UPR pathways. Expression of Sptlc2, a major catalytic subunit of SPT in liver, was upregulated by high fat diet in liver of C57Bl6 mice. Treatment of tunicamycin, an ER stress inducer, elevated Sptlc2 and ceramide levels in primary mouse hepatocytes. Overexpression of the spliced form of X-box binding protein 1 (sXBP1) upregulated Sptlc2 expression, whereas the spliced form of activating transcription factor 4 (ATF4) had no effect on Sptlc2 expression as demonstrated by Sphk2 promoter assays and western blot analyses. Liver-specific overexpression of Sptlc2 (lSptlc2 Tg) elevated fasting glucose levels and inhibited phosphorylation of AKT by insulin. In addition, the response to insulin was diminished and hepatic lipoprotein secretion was increased in lSptlc2 Tg mice. These results demonstrated that Sptlc2 is regulated by ER stress-induced UPR pathways and involved in hepatic secretion of VLDL.

Deoxysphingolipids, novel biomarkers for diabetes, are cytotoxic for insulin-producing cells.

Richard A. ZUELLIG¹, Thorsten HORNEMANN², Alaa OTHMAN², Adrian B. HEHL3, Tanja GÜNTERT4, Omolara O. OGUNSHOLA4, Enrica SAPONARA5, Arnold VON ECKARDSTEIN², Rolf GRAF5, Sabrina SONDA5
1Division Of Endocrinology, Diabetes And Clinical Nutrition, University Hospital Zurich, 2Institute For Clinical Chemistry, University Hospital Zurich, 3Institute Of Parasitology, University Of Zurich,4Institute Of Veterinary Physiology, University Of Zurich, 5Departement Of Visceral & Transplantation Surgery, University Hospital Zurich

Objective: Irreversible failure of pancreatic beta-cells is the main culprit in the pathophysiology of diabetes mellitus, a disease that is now a major global epidemic. Recently, elevated plasma levels of deoxysphingolipids, including 1-deoxysphinganine, have been identified as novel biomarkers for the disease. In this study, we analyzed whether deoxysphingolipids directly compromise the functionality of pancreatic beta-cells.
Methods: Effects of deoxysphingolipids were investigated in insulin-producing Ins-1 cells and primary islets. Cell functionality and signaling pathways were analyzed by biochemical methods and flow cytometry. Cytoskeletal alterations were imaged by confocal microscopy. Levels of mRNA and composition of sphingolipid species were quantified by real-time PCR and mass spectrometry, respectively.
Results: Treatment with 1-deoxysphinganine induced dose-dependent cytotoxicity with senescent, necrotic and apoptotic characteristics and compromised glucose-stimulated insulin secretion of beta-cells. In addition, 1-deoxysphinganine altered cytoskeletal dynamics, resulting in intracellular accumulation of filamentous actin and activation of the RhoGTPase Rac1. Moreover, 1-deoxysphinganine selectively up-regulated ceramide synthase 5 expression and was converted to 1-deoxy-dihydroceramides, without altering normal ceramide levels. Inhibition of intracellular 1-deoxysphinganine trafficking and ceramide synthesis improved the viability of the cells, indicating that the intracellular metabolites of 1-deoxysphinganine contribute to its cytotoxicity. Analyses of signaling pathways identified JNK and p38 MAPK as antagonistic effectors of cellular senescence.
Conclusion: Our results revealed that 1-deoxysphinganine is a cytotoxic lipid for insulin-producing cells, suggesting that the increased levels of this sphingolipid observed in diabetic patients may contribute to the reduced functionality of pancreatic beta-cells. Thus, targeting deoxy-sphingolipid synthesis may complement the currently available therapies of diabetes.

Lysosomal stress drives Sphk1 expression in adipose tissue macrophages.

Tanit GABRIEL¹, Mina MIRZAIAN1, Marc TOL¹, Roelof OTTENHOFF¹, Cindy VAN ROOMEN¹, Johannes AERTS², Marco VAN EIJK¹,
1Medical Biochemsty, AMC, 2LIC, Biochemistry, Leiden University

Objective: During obesity adipose tissue macrophages (ATM) have to handle an increased lipid load. When adipocyte dysfunction occurs large amounts of lipids are spilled, which consequently impacts macrophages and their lysosomes. Recently it has been found that obesity induces a program of lysosome biogenesis in ATM. In addition, we found a novel obese ATM marker Gpnmb to be induced as a consequence of lipid-induced lysosomal stress. Previously we demonstrated that inhibition of GSL synthesis in models of obesity improved aspects of metabolic dysfunction including inflammation. It is not clear if GSL manipulation in adipose tissue impacts macrophages, adipocytes or both. This prompted us to analyze in detail in sorted ATM populations genes involved in (G)SL synthesis, degradation and transport. We observed a striking induction of Sphk1 in obese ATM. These findings are in agreement with literature. Interestingly, also Sphk1 activity and concomitantly formation of S1P was elevated in obese ATM. To get insight in the regulation of Sphk1 expression in ATM we made use of the macrophage cell line RAW 264.7. It was found that Sphk1 induction occurs as a consequence of overfeeding with palmitate,but also with lysosome stressors such as chloroquine. Additional evidence suggests that Sphk1 activity in obese ATM prevents induction of cell death upon lysosomal challenge. We postulate that high lipid load causes lysosomal stress, which triggers induction of sphk1. By controlling the overshoot of ceramide levels, and consequently cell death, it is ensured during obesity that enough ATM lipid buffering capacity persists.

The role of adipose ceramide in metabolic homeostasis.

Scott SUMMERS
Baker Idı Heart And Diabetes Institute

The lipotoxicity posits that the accumulation of fat-derived metabolites in tissues not optimized for lipid storage induces the cellular dysfunction that underlies metabolic disorders. Of the metabolites that accumulate, sphingolipids such as ceramide appear particularly deleterious, as pharmacological inhibition or genetic ablation of enzymes requisite for sphingolipid production ameliorates insulin resistance, diabetes, and cardiovascular disease in rodents. To identify the tissues that produce the sphingolipids that impair metabolic homeostasis, we have studied knockout mice lacking the a requisite biosynthetic enzyme, serine palmitoyltransferase -2, in either liver, muscle, myeloid cells, or adipocytes. The speaker will share data on the metabolic phenotype of these conditional knockout animals, which reveal novel and important roles in adipose biology.

Active phosphorylated FTY720/fingolimod is a potent inhibitor of class i histone deacetylases that reactivates estrogen receptor expression and increases hormonal therapeutic sensitivity of breast cancer.

Sarah SPIEGEL¹, Sheldon MILSTIEN¹,
1VCU School Of Medicine

Objective: Hormonal therapies, including ovarian ablation, ER antagonists, and aromatase inhibitors, are the standards of care for treatment of ERα positive breast cancer. However, development of resistance to hormone therapies in advanced breast cancer is a major obstacle. Moreover, estrogen receptor-α (ERα)-negative breast cancer is clinically aggressive and does not respond to conventional estrogen targeted therapies. Strategies that lead to re-expression of ERα could sensitize breast cancers to selective ER modulators. FTY720/Fingolimod, a sphingosine analog, is an FDA-approved pro-drug for treatment of multiple sclerosis that also has anticancer actions that are not yet well understood. We have now found that FTY720 is phosphorylated in breast cancer cells by nuclear sphingosine kinase 2 and accumulates there. Nuclear FTY720-P in turn is a potent inhibitor of class I histone deacetylases (HDACs) that enhances histone acetylations and gene expression independently of its known effects on canonical signaling through sphingosine-1-phosphate receptors. In ERα negative human and murine breast cancer cells, FTY720 reactivated expression of silenced ERα and sensitized them to tamoxifen. Moreover, oral administration of clinically relevant doses of FTY720 to mice bearing ERα negative syngeneic breast tumors also re-expressed ERα and increased therapeutic sensitivity to tamoxifen in vivo more potently than a known HDAC inhibitor. Our work suggests that FTY720 is a promising strategy for effective treatment of conventional hormonal therapy-resistant breast cancer and triple-negative breast cancer. Supported by NIH grant RO1CA061774 and the Department of Defense BCRP program award W81XWH-14-1-0086 (S. Spiegel).

CerS4/ceramide metabolism in the regulation of TFG-beta receptor signaling and tumor metastasis.

Salih GENCER¹, Can Emre SENKAL², Suriyan PONNUSAMY3, Natalia OLEINIK¹, Mohammed DANY¹, Shanmugam P. SELVAM¹, Joshua OAKS¹, Besim OGRETMEN¹
1Medical University Of South Carolina, 2Stony Brook University, 3University Of Tennessee

Objective: Recent studies indicate that ceramide species play diverse biological functions including, skin barrier function, liver homeostasis, cell death and cancer pathogenesis, highlighting the importance of ceramide synthases (CerS) in these processes. Migration, a part of these processes, also is effected by ceramide metabolism. However, the molecular mechanism of CerS/ceramide involved is unknown. Here, we investigated the effect of CerS on migration and its related signal pathways in situ and in vivo model. Interestingly, our data show that among CerS only CerS4 is related to cell migration. Here, we also have generated CerS4-/- mice, and these mice were viable with no lethal tissue. Interestingly, we observed that loss of CerS4 resulted in irreversible alopecia, which was associated with hyper-proliferation and migration of keratinocytes. Mechanistically, we show that knockout/knockdown of CerS4 enhances cell migration by which ligand-independent signaling of TGFβ receptors in various cell types, including keratinocytes, MEFs, and cancer cells. Additionally, low level of TGFβR1-Smad7 interaction was found in knockdown of CerS4 cells. Moreover, we found that ceramide interact with Smad7 and interaction was decreased by knockdown of CerS4. Thus, ceramide-Smad7 binding modulates plasma membrane association of TGFβR1, and inhibits its signaling through Sonic-Hedgehog (Shh) signaling for migration. In fact, inhibition of TGFβR/Shh signaling using molecular or pharmacologic inhibitors almost completely prevented cell migration in response to CerS4 knockdown. These data suggest that CerS4/ceramide signaling plays key roles in the regulation of cell migration via controlling the TGFβR/Shh axis.

C16-ceramide is a natural regulatory ligand of p53.

Natalia KRUPENKO¹, Baharan FEKRY¹, Amin ESMAEILNIAKOOSHKGHAZI¹, Kevin KNAGGE2, Zdzislaw SZULC3, Yuri PETERSON3, Sergey KRUPENKO¹
1UNC Chapel Hill, 2David H. Murdock Research Institute, 3Medical Unioversity Of South Carolina

Objectives: We have previously observed that a transient elevation of one of the ceramide-generating enzymes, ceramide synthase 6 (CerS6), in cancer cells up-regulates p53 through an obscure mechanism. Since the immediate effect of CerS6 elevation was the raise of C16-ceramide, we hypothesized that the mechanism underlying the p53 elevation was the direct binding of this ceramide to the protein, which leads to the p53 release from its complex with MDM2.
Methods: NMR spectroscopy, membrane-binding assays, bimolecular fluorescence complementation, protein pulldown, titration of tyrosine fluorescence, and LC/MS-MS measurements of ceramides were applied to study the interaction between p53 and ceramide.
Results: We have demonstrated that p53 binds C16-ceramide with high affinity (Kd = 60 ± 20 nM) within its DNA binding domain and that the interaction between p53 and ceramide is highly selective towards the acyl chain length. NMR experiments have demonstrated chemical shifts perturbations upon C16-ceramide binding in the BOX-V sequence of p53, which interacts with the acidic domain of E3 ligase MDM2. In agreement with this finding, we have further demonstrated that the binding of ceramide disrupts the complex between p53 and MDM2, thus preventing the p53 ubiquitination and degradation.
Conclusions: Our study establishes C16-ceramide as the first natural small molecule regulating p53 tumor suppressor through the direct binding. This type of regulation is a novel physiological mechanism of p53 activation, which is fundamentally different from the canonical p53 activation through protein-protein interactions or post-translational modifications.

C stimulating CAMP overproduction accelerate development of cutaneous SCC.

Kyungho PARK¹, Young-Il KIM¹, Anna NIELSEN-SCOTT¹, Kyong-Oh SHIN², Yong-Moon LEE², Walter M. HOLLERAN¹, Sarah ARRON¹, Theodora M. MAURO¹, Peter M. ELIAS¹, Yoshikazu UCHIDA¹
1University Of California, San Francisco, 2College Of Pharmacy Chungbuk National University

Objective: Cutaneous squamous cell carcinoma (cSCC) is a ccomon cancer, often initiated by oxidative stress, particularly ultraviolet irradiation. We demonstrated that oxidative stressors upregulate a key innate immune element, cathelicidin antimicrobial peptide (CAMP) via endoplasmic reticulum (ER)-mediated, sphingosine-1-phosphate signaling (Park K et al., Mol Cell Biol, 2011 & 2014). Prior studies showed that CAMP production also increases in certain cancers. Yet, why CAMP overproduction occurs and how CAMP could stimulate tumorigenesis remains unknown. We assessed whether and how CAMP contributes to the development of cSCC. CAMP mRNA/peptide and sphingosine-1-phosphate productions were significantly higher in cSCC cells than in normal human keratinocytes. Exogenous CAMP significantly stimulated the growth of cSCC (but not normal keratinocytes). We next showed that blockade of the CAMP receptor, formyl peptide receptor-like (FPRL) 1, by a specific receptor antagonist (WRW4) attenuated cSCC gowth. We then demonstrated cSCC invaded into dermis using cultured human skin model consisting with epidermis and dermis and exogenous CAMP further stimulated cSCC invasion into an in vitro dermal equivalent, but WRW4 suppressed cSCC invasion. Finally, elevated CAMP expression and cell growth in SCC were significantly suppressed by specific inhibitors of sphingosine kinase 1. These studies suggest that sphingosine-1-phosphate signaling stimulates CAMP overproduction resulting in a FPRL1-dependent enhancement of cSCC growth and invasion.

Dysregulation of sphingolipid metabolism in melanoma: roles in cell survival and tumor progression.

Nathalie ANDRIEU
Centre de Recherches en Cancérologie de Toulouse

Cutaneous melanoma is a complex disease that arises through the stepwise transformation of melanocytes within the basal epidermal layer of the skin and evolves due to a myriad of genetic aberrations correlated or not with the person’s UV exposure behaviour. Although very promising with meaningful effects on progression-free survival, BRAF-directed therapies are usually short-lived due to the appearance of resistance, which leads to disease progression. This emphasizes the need to develop new therapeutic approaches that could overcome cancer relapse. Interestingly, ceramide metabolism is strongly altered in melanoma and represent an exploitable target for the development of novel therapies. Here, we show that the reduction of sphingosine 1-phosphate levels in resistant metastatic melanoma resulted in sensitization of these cells to apoptosis induced by a BRAF inhibitor. This phenomenon is associated with decreased expression of MITF, a major regulator of melanoma survival which controls the expression of Bcl-2 family members. Moreover, our findings reveal a key role for melanoma sphingosine kinase 1 in macrophage recruitment and polarization within the tumor microenvironment, thereby controling the aggressiveness of this cancer.

Key role of S1P receptors in the action mechanism of TGFβ in myoblasts.

Paola BRUNI
Dipartimento di Scienze Biomediche, Sperimentali e Cliniche, Università di Firenze, Firenze, Italia

Solid experimental evidence is in favor of a key role of sphingosine 1-phosphate (S1P) signaling axis in the action mechanism of multiple extracellular agents, with a role in the onset of their final response.
In our laboratory we have studied the involvement of S1P signaling pathway in skeletal muscle precursor cells, named myoblasts, highlighting its requirement for the full accomplishment of specific biological actions by growth factors and cytokines. In this regard, we have addressed the possible implication of endogenous production of S1P in the action mechanism of TGFβ, a potent cytokine known to hamper skeletal muscle repair. Our study clearly shows that sphingosine kinase-1 is up-regulated by TGFβ. More importantly, the enhanced biosynthetic pathway of S1P is accompanied by a profound modification of S1P receptor expression profile. Overall, our results support the notion that by up-regulation of S1P3 and S1P4, TGFβ is able to readdress S1P pathway in myoblasts. Indeed, in unstimulated cells this signaling axis promotes myogenic differentiation and is physiologically involved in skeletal muscle regeneration. In contrast, via S1P3 induction, TGFβ drives transdifferentiation of myoblasts into myofibroblasts while the induced up-regulation of S1P4 accounts for its apoptotic action, making the S1P-directed signaling detrimental for skeletal muscle repair. Thus, it appears that the biological outcome of S1P signaling axis can be highly flexible depending on the pattern of S1P receptors, that can undergo rapid and critical changes.

Thrombocytopenia induced by sphingomyelin deficiency in SMS1 knock out mice.

Toshiro OKAZAKI¹, Yoshibumi UEDA¹, Makoto TANIGUCHI¹,
1Kanazawa Medical University

Objective: The degradation of platelets as well as the production is an important step to maintain the number of platelets. However, it is not fully understood whether sphingolipids and their metabolizing enzymes are involved in the degradation of platelets. Sphingomyelin synthase 1 (SMS1) and SMS2 are in charge of producing sphingomyelin (SM), which is a key sphingolipid consisting of microdomain and occupies around 10 % of all lipids on the plasma membrane. In the present study, we revealed that SMS1 is crucial for maintaining the number of platelets in mice. SMS1-knock out (KO) mice, but not SMS2, exhibit typical thrombocytopenia symptoms including low number of platelets, longer bleeding time and the increase in the number of reticulated platelets. In the complete blood counts the numbers of erythrocytes and leukocytes in SMS1-KO mice were similar to those of the control. SM levels on the plasma membrane of SMS1-KO mice , but not SMS2-KO mice, were massively decreased in platelets and megakaryocytes compared to other cell lineages. In addition, the significant increase of phosphatidylserine (PS) externalization was detected in the platelets and megakaryocytes of SMS1-KO mice as compared to SMS2-KO and wild mice, suggesting that SM deficiency facilitated PS externalization in the plasmamembrane. SMS1-KO mice showed the splenomegaly and exhibited the higher collocalization of CD41-positive platelets and CD-68-positive macrophages in the spleen than SMS2-KO and wild mice, suggesting up-regulation of the phagocytosis of platelets by macrophages in SMS1-KO mice. In the bone marrow SMS1-deficiency induced an increase of megakariocytes as a reactive synthesis to thrombocytopenia like the case of immune thrombocythemia (ITP). When the spelenectomy was performed in SMS1-KO mice the platelets increased after 4 days of operation. These results suggested that SM regulated by SMS1, but not SMS2, in the plasma membrane is involved in the controlling the degradation of platelets through PS externalization.

ApoM+HDLrestrains lymphopoiesis and neuroinflamation via sphingosine 1-phosphate signaling.

Victoria BLAHO¹, Sylvain GALVANI¹, Eric ENGELBRECHT¹, Catherine LIU¹, Steven SWENDEMAN¹, Mari KONO², Richard PROIA², Lawrence STEINMAN3, May HAN3, Timothy HLA ¹
1Weill Cornell Medical College, 2National Institute Of Diabetes And Digestive And Kidney Diseases, – 3Stanford University

Objective: Lipid mediators influence immunity in myriad ways. For example, circulating sphingosine 1-phosphate (S1P) is a key regulator of lymphocyte egress. Although the majority of plasma S1P is bound to apolipoprotein M (ApoM) in the high-density lipoprotein (HDL) particle, how the ApoM-S1P complex regulates immunity is unknown. Here, we show that ApoM-S1P is dispensable for lymphocyte trafficking yet restrains lymphopoiesis by activating the receptor S1P1 on bone marrow (BM) lymphocyte progenitors. Mice that lacked ApoM (Apom-/-) had increased proliferation of Lin-Sca1+cKit+ hematopoietic stem and progenitor cells (LSK) and common lymphoid progenitors (CLP) in BM. Pharmacologic activation or genetic overexpression of S1P1 suppressed CLP proliferation in vivo. Activation of S1P1 on CLP in vivo could be visualized using novel S1P1 GFP signaling mice, and the lack of S1P1 stimulation in Apom-/- mice resulted in decreased ERK1/2 phosphorylation and subsequent increased STAT5 activation. ApoM was stably associated with CLPs in BM and specifically inhibited lymphopoiesis in vitro. Upon immune stimulation, Apom-/- mice developed more severe experimental autoimmune encephalomyelitis, characterized by increased lymphocytes in the central nervous system (CNS) and breakdown of the blood-brain barrier. Thus, the ApoM-S1P-S1P1 signaling axis restrains the lymphocyte compartment and adaptive immune responses. Since plasma HDL levels influence CNS and other inflammatory diseases, this regulatory pathway may represent a novel therapeutic.

Defining the role of acid ceramidase in ulcerative colitis and the inflammatory response.

Mel Pilar ESPAILLA¹, Ashley SNIDER¹, Toshihiko KAWAMORI², Yusuf A. HANNUN¹, Lina M. OBEID¹
1Stony Brook University, 2University Of Hawaii Cancer Center

Objective: The sphingolipid enzyme sphingosine kinase-1 (SK1) and its metabolic product sphingosine-1-phosphate (S1P) are key regulators of disease pathology in inflammatory bowel disease (IBD). Ulcerative colitis, one of the major forms of IBD, is a debilitating condition characterized by chronic colonic inflammation and represents a significant risk factor for the development of colorectal cancer. Unfortunately, the molecular mechanisms that regulate the inflammatory response in ulcerative colitis remain incompletely understood. Our published work showed that SK1 deficient mice are protected from parameters of disease and inflammatory responses in the murine model of dextran sulfate sodium (DSS)-induced colitis. We aimed to investigate the upstream metabolic regulation of the bioactive sphingolipid S1P in ulcerative colitis by identifying the upstream ceramidase enzyme contributing to the inflammatory response. Ceramidases are sphingolipid metabolic enzymes that hydrolyze ceramide to generate sphingosine, the precursor of S1P. Using immunohistochemistry, we determined that lysosomal acid ceramidase is overexpressed in the inflammatory infiltrate of colonic tissue from patients with ulcerative colitis but not in normal tissue. Our preliminary data also shows that acid ceramidase activity is increased in the inflamed colon of DSS-treated mice. In vivo pharmacologic and genetic inhibition of acid ceramidase protects mice from disease pathology and inflammation in the DSS-induced colitis model. Furthermore, ex vivo studies demonstrate acid ceramidase-mediated regulation of macrophage inflammatory responses, including production of inflammatory cytokines. These results begin to implicate a novel and specific role for AC in the regulation of disease pathology in DSS-induced colitis and myeloid inflammatory responses.

Sphingolipid metabolism and signaling in the regulation of drug resistance in CML.

Yusuf BARAN¹
Abdullah Gul University Faculty Of Life And Natural Sciences1

Cellular resistance mechanisms developed by cancer cells and tissues in the beginning or proceeding times to applied anticancer agents is a significant problem preventing succesfull therapy. Trysine Kinase Inhibitors (TKIs) are very effective drugs used for the treatment of chronic myeloid leukemia (CML). TKIs bind to the amino acids of the BCR-ABL tyrosine kinase ATP-binding site and stabilize the non-ATP-binding form of BCR-ABL, thereby preventing phosphorylation of its substrates. Although imatinib has high rates of hematologic and cytogenetic response, after exposure of drug, resistance to TKIs has been recognized as a major problem.Various cellular mechanisms may be involved in the nature of cellular resistance. Increased amount of BCR/ABL, alteration in BCR/ABL structure, inhibition of apoptotic mechanisms, decreased imatinib uptake and increased detoxification are well-known mechanisms of resistance.
Aberrant ceramide metabolism is another one of these inherent or acquired mechanisms that contribute to cellular drug resistance. Stress increases de novo ceramide synthesis by Ceramide Synthase gene family or activate sphingomyelinases and ceramidase and elevate levels of ceramide leading to apoptosis. Many other stimuli, particularly growth and survival factors, convert apoptotic ceramide to antiapoptotic sphingosine-1-phosphate and glucosyleceramide (GlcCer) by Sphingosine Kinase-1 and glucosyle ceramide synthase (GCS).
In this talk, the roles and mechanisms of action of ceramide metabolism, besides all other possible resistance mechanisms, in the regulation of TKIs-induced cell death and resistance in sensitive and imatinib resistant cell lines and CML patient samples will be reviewed. This study was supported by TUBITAK with the Project number 111S391 to Y.B.

Lipid signaling in S1P-lyase-deficient fibroblasts.

Dagmar Meyer ZU HERİNGDORF
Institut für Allgemeine Pharmakologie und Toxikologie, Klinikum der Goethe-Universität Frankfurt am Main, Germany

Sphingosine-1-phosphate (S1P) lyase catalyzes the ultimate step in sphingolipid degradation, i.e., the irreversible cleavage of S1P. Mice lacking S1P lyase have a strongly reduced life span and suffer from multiple organ defects, lymphopenia, and generalized inflammation1,3,4,6. S1P and sphingosine accumulate in cells and tissues of these mice, which furthermore have a hypercholesterolemia and hypertriglyceridemia, despite their strongly reduced body fat4. Similar to the whole organism, both S1P and sphingosine accumulate in embryonic fibroblasts from S1P lyase-deficient mice (Sgpl1-/–MEFs). Therefore, these cells might be used as a model system to study intracellular effects of S1P and sphingosine, and they might help to elucidate the phenotype of the knockout mice. Several research groups have analyzed the properties of Sgpl1-/–MEFs2,5,8,9,10. It has been shown that the cells grow well and are protected from apoptosis2,10. They are furthermore characterized by a disturbed Ca2+ homeostasis, reduced HDAC activity and reduced expression of several HDAC isoforms5,8. S1P lyase deficiency has also been linked to neurodegeneration7, and interestingly, even Sgpl1-/–MEFs have a defect in the processing of the amyloid precursor protein9. Our new data show that not only sphingolipid metabolism but also cholesterol homeostasis is considerably altered in these cells.
Selected References:
1. Schmahl J et al.; Nat Genet (2007)
2. Colié S et al.; Cancer Res (2009)
3. Vogel P et al. PLoS One (2009)
4. Bektas M et al.; J Biol Chem (2010)
5. Claas RF et al.; Cell Signal (2010)
6. Allende ML et al.; J Biol Chem (2011)
7. Hagen-Euteneuer N et al.; J Biol Chem (2012)
8. Ihlefeld K et al.; Biochem J (2012)
9. Karaca I et al.; J Biol Chem (2014)
10. Ihlefeld K et al.; J Lipid Res (2015)

Crosstalk between sphingolipid and glycerophospholipid metabolism in yeast.

Auxiliadora AGUILERA-ROMERO¹, Vladimir GIRIK¹, Aline X.S. SANTOS¹, Isabelle RIEZMAN¹, Fabrice DAVID², Howard RIEZMAN¹,
1University Of Geneva, 2EPFL,

Objective: Systematic lipidomics is a new, rich source of biological information that allows grouping of genes according to function via their lipid profile (1). We have now measured the lipidome of over 600 yeast mutants, including a collection of protein kinases and phosphatases as well as proteins found in the early secretory pathway (2). These studies have revealed new steps in the regulation of ceramide and sphingolipid homeostasis and have also revealed a correlation between a group of mutants with a particular type of defect in sphingolipid homeostasis with a misregulation of glycerophospholipid metabolism. We are currently working on the identification of a potential sphingolipid metabolite that is responsible for this misregulation as well as its mechanism of action. 1. Santos, AXS et al., (2014) Systematic lipidomic analysis of yeast protein kinase and phosphatase mutants reveals novel insights into regulation of lipid homeostasis. Mol. Biol. Cell 25, 3234-46. 2. Schuldiner, M et al., (2005) Exploration of the function and organization of the yeast early secretory pathway through an epistatic miniarray profile. Cell 123, 507-19.

Gycosylceramide supply from ethiopian plants – screening and quantification methods.

Mathias REISBERG¹, Norbert ARNOLD², Reinhard H. H. NEUBERT¹, Birgit DRÄGER¹
1Martin Luther University Halle-wittenberg, Institute Of Pharmacy, 2Leibniz Institute Of Plant Biochemistry, Department Of Bioorganic Chemistry

Objective: Ceramides are widely distributed in nature. They also provide a major lipid class in the outermost layer of mammalian skin – the stratum corneum. A ceramide balance disorder is related to skin diseases as psoriasis or neurodermatitis. A ceramide skin supplementation with plant glycosylceramide resources is therefore economically of high interest. Glycosylceramides occur in Fabaceae species, e.g. Albizia julibrissin. Five Fabaceae species, in part endogenous in Ethiopia, were collected in April 2013 in the Oromia Region. An easy sample preparation was developed. Total lipids were extracted from ground air-dried seeds using a mixture of isopropanol – n-hexane – water 55:20:22 (V/V/V) as solvents [Markham J and Jaworski JG, 2007. Rapid Commun Mass Sp. 21: 1304-1314] and were further partitioned by liquid-liquid-extraction. Qualitative analysis was carried out by TLC on silica gel 60 F254 plates (Merck), quantitative screening by densitometric analysis after an 18-step solvent gradient (Automated Multiple Development-AMD 2 (CAMAG)) was performed on HPTLC silica gel 60 F254 plates (Merck). Structures were identified by LC-MS using glycosylceramides previously isolated as reference compounds by means of intensive HR-FT-ICR-MS, NMR and MS/MS analyses. We demonstrate by using our developed methods and work flow, an intensive screening regarding the occurrence of ceramides in plants and other natural resources is feasible. Our results give first hints on glycosylceramide content in a broader range of plant species. Total amounts ranged from 3 to 13 mg per 100 g dry weight with highest contents in different Albizia species. The plants may serve as ceramide resources.

Systematic lipidomics of mutants in the early secretory pathway.

Isabelle RIEZMAN¹, Auxılıadora AGUILERA-ROMERO¹, Alıne X.S. SANTOS¹, Fabrıce DAVID¹
1Unıversıty Of Geneva

Objective: Systematic lipidomics is a new, rich source of biological information that allows grouping of genes according to function via their lipid profile (1). We have now measured the lipidome of over 450 yeast mutants representing proteins found in the early secretory pathway (2). We have used a statistical analysis of the results to cluster mutants with similar phenotypes in sphingolipid homeostasis. We have also used rankings to determine the mutants with the largest and smallest amount of particular sphingolipids as well as ergosterol, which has a particular relation to sphingolipids (3). Interestingly, apart from mutants in ergosterol biosynthesis, the mutant with the least amount of free ergosterol is sfb3/lst1. This COPII coat protein is specifically involved in the transport of GPI-anchored proteins (4), whose transport also requires ceramide, suggesting a possible coregulation or transport of ceramides and ergosterol. 1. Santos, AXS et al., (2014) Systematic lipidomic analysis of yeast protein kinase and phosphatase mutants reveals novel insights into regulation of lipid homeostasis. Mol. Biol. Cell 25, 3234-46. 2. Schuldiner, M et al., (2005) Exploration of the function and organization of the yeast early secretory pathway through an epistatic miniarray profile. Cell 123, 507-19. 3. Guan, XL et al., (2009) Functional interactions between sphingolipids and sterols in biological membranes regulating cell physiology. Mol. Biol. Cell 20, 2083-95. 4. Manzano-Lopez, J et al., (2014) COPII coat composition is actively regulated by luminal cargo maturation. Curr. Biol. 25, 152-62.

Endosomal sterol, phosphoinositide and sphingolipid signaling integrates cell cycle control with endosomal membrane trafficking.

Vytas BANKAITIS
Texas A&M Health Science Center College Station

ABSTRACT NOT RECEIVED

Reconstruction and analysis of yeast sphingolipid protein interaction network.

F. Betul KAVUN OZBAYRAKTAR¹, Kutlu ULGEN¹
1Bogazici University Department Of Chemical Engineering

Objective: Sphingolipids are both structural and regulatory components of the cell, where they control processes decisive in cell’s fate. The first effort of constructing the protein-protein interaction network of sphingolipids in Saccharomyces cerevisiae enabled us to understand the details of the topological properties of the newly constructed network as well as to assign functions to some of the uncharacterized proteins involving in the network of sphingolipids. The topological analysis of sphingolipid related proteins, especially those under clinical trials for cancer therapy, yielded novel potential drug targets. Novel interactions are predicted using a newly developed integrated methodology employing sequence and structure based computational interaction prediction tools, orthology, expression profiles, co-localization information and Gene Ontology (GO) terms. The sphingolipid network shows topological properties of a scale-free, small-world, and modular structure, as it is the case for biological networks. The function annotation of uncharacterized proteins of the network is performed using a multi-dimensional hybrid method which combines the results from modules and neighbors, and examines them by information gathered from genetic interactions, expression profiles, and sequence similarity. The here constructed sphingolipid network coupled with the newly developed hybrid function annotation method constitutes an efficient platform for function annotation and drug target identification

Sphingolipid analogues and inhibitors: sphingolipid-based therapeutics.

Nigel PYNE¹, Susan PYNE¹
1University Of Strathclyde

Objective: Sphingolipids function as recognition molecules of cellular stress that can result in endoplasmic reticulum stress, unfolded protein responses, autophagy, apoptosis and senescence in cells. Sphingolipids can also represent danger signals to promote host-defense inflammatory reactivity to invading pathogens. Sphingolipid analogues are designed to mimic endogenous sphingolipid metabolites to modulate these cellular stress and inflammatory reactions. For example, sphingolipid analogues can modulate sphingolipid metabolizing enzymes to control the steady state levels of endogenous sphingolipids and thereby induce the death of cancer cells, such as T-cell acute lymphoblastic leukemia cells. In the case of sphingosine kinase inhibitors, there is a close relationship between the targeting these enzymes, sphingolipid metabolism, oxidative stress and the proteasome that when perturbed can result is catastrophic collapse of cell survival signaling networks in solid cancers. Another example is the use of sphingadienes that increase sphingosine 1-phosphate lyase expression to reduce intracellular sphingosine 1-phosphate levels and limit inflammatory driven down-regulation of tumour suppressor genes in colon cancer cells. Sphingosine mimetics also promote activation of the NLRP3 inflammasome to stimulate formation of the pro-inflammatory mediator, IL-1, which could be exploited to produce antagonists that diminish exaggerated inflammation in disease. Alternatively, sphingosine mimetics could be used to promote host-defense against invading pathogens; thereby representing alternative therapeutic strategies to the development of antibiotics. The translation of sphingolipid analogues into therapeutics is challenging because of solubility, bioavailability and ‘low-affinity’ binding to targets. This presentation will highlight these issues and the potential for sphingolipid based therapeutics.

FTY720 induces necroptosis by regulating ceramide signaling at the plasma membrane.

Rose NDETO¹, Besim OGRETMEN¹
1Medical University Of South Carolina

Objective: Sphingolipids are important signaling molecules in cells and have recently been explored as cancer therapy targets. FTY720 (Fingolimod, Gilenya) is an FDA approved sphingosine analogue drug used for the treatment of multiple sclerosis (MS). FTY720 is phosphorylated by sphingosine kinase 2 (SK2), to generate P- FTY720 to exert its immunosuppressive properties through binding to sphingosine -1 phosphate receptors (S1PRs). FTY720 also exhibits anti-cancer properties. Our previous studies indicated that one of the mechanism by which FTY720 induces cell death is through necroptosis. FTY720 directly binds to I2PP2A/SET (Inhibitor 2 of PP2A), consequently activating the tumor suppressor protein phosphatase 2A (PP2A). The activated PP2A then induces cell death by stimulating the activity of Receptor-Interacting Protein kinase-1 (RIPK1), involved in necroptosis signaling. Previous studies have also shown that ceramide also binds I2PP2A and activates PP2A. However, little is known about the roles of FTY720 in ceramide signaling and regulation of necroptosis. We hereby seek to investigate the mechanisms of FTY720 in inducing necroptosis with regard to ceramide signaling. Preliminary data indicate that inhibitors of ceramide generation partially protect cells against FTY720-induced cell death. FTY720 and non-phosphorylated FTY720 analogues do not affect ceramide generation, but lead to the formation of specific ceramide-multi-protein complexes at the plasma membrane involved in plasma membrane disruption for necroptosis. Future mechanistic studies will help us understand the details of how these complexes are formed at the plasma membrane and how they regulate necroptosis in response to cellular stress invoked by FTY720 and other therapeutic agents.

Ceramide-1-phosphate (C1P)-stimulated migration and phospho-ceramide analogue-1 (PCERA-1)-induced il-10 expression are mediated via distinct receptors in macrophages.

Tsaffrir ZOR¹, Sebastián KATZ¹, Dorit AVNI¹, Orna ERNST¹, Lide ARANA2, Alberto OURO², Brian P. GRIFFIN3, Charles E. CHALFANT3, Michael M. MEIJLER4, Antonio GÓMEZ-MUÑOZ²
1Tel-aviv University, 2University Of The Basque Country , 3Virginia Commonwealth University School Of Medicine, 4Ben-Gurion University Of The Negev

Objective: Inflammation is an ensemble of tightly regulated steps, in which macrophages play an essential role. The endogenous phospholipid ceramide 1-phosphate (C1P) stimulates macrophages migration and suppresses LPS-stimulated secretion of the key pro-inflammatory cytokine TNF alpha, while the synthetic C1P mimic, phospho-ceramide analogue-1 (PCERA-1), suppresses TNF alpha secretion and amplifies production of the key anti-inflammatory cytokine IL-10, in LPS-stimulated macrophages. Previous reports suggested that the two compounds act via one or more G-protein coupled receptors. We show that C1P stimulated RAW264.7 macrophages migration via the NFB pathway and MCP-1 induction, while PCERA-1 neither mimicked nor antagonized these activities. Conversely, PCERA-1 synergistically elevated LPS-dependent IL-10 expression in RAW264.7 macrophages via CREB transcriptional activity, while C1P neither mimicked nor antagonized these activities. Both PCERA-1 and C1P inhibited TNF secretion, but while PCERA-1 suppressed TNF alpha transcription, C1P blocked the secretion step itself by inhibiting TNF alpha converting enzyme (TACE). Finally, PCERA-1 failed to interfere with a C1P binding assay. These results thus indicate that the natural sphingolipid C1P and its synthetic analog PCERA-1 bind and activate distinct receptors expressed in RAW264.7 macrophages. Identification of these receptors will be instrumental for elucidation of novel activities of extra-cellular sphingolipids, and may pave the way for the design of new sphingolipid mimics for the treatment of inflammatory diseases, and pathologies which depend on cell migration, as in metastatic tumors.

Uracil and benzoxazolone carboxamides: discovery of potent small-molecule inhibitors of acid ceramidase?

Daniela PIZZIRANI¹, Anders BACH¹, Chiara PAGLIUCA², Natalia REALINI¹, Andrea ARMIROTTI¹, Daniele PIOMELLI3
1Fondazione Istituto Italiano Di Tecnologia, 2Janssen Pharmaceutica, 3Fondazione Istituto Italiano Di Tecnologia, Genova, Italy AND University Of California, Irvine, USA

Objectıve: Herein, we present an overview of our recent work on the discovery and development of the first two classes of potent small-molecule inhibitors of acid ceramidase (AC). As the pharmacology of sphingolipid signaling is still nascent, novel tools are needed to further drive understanding of the roles of ceramide in physiology and pathology.
Results: We identified promising small-molecule hits against AC by screening compound collections towards this target. Medicinal chemistry work around selected hits led to the development of two chemical series of potent AC inhibitors, the uracil- and the benzoxazolone-carboxamide classes. Carmofur and other uracil derivatives were the first identified nanomolar inhibitors of AC. We found that selected uracil carboxamides act synergistically with standard anti-neoplastic drugs in certain types of cancer, suggesting a potential use as chemosensitizers. Prototype members of the benzoxazolone class were found to be metabolically stable and capable of engaging AC both in vitro and in vivo, thus representing the first potent and systemically active inhibitors of this enzyme. We also characterized these compounds in terms of mechanism of action, proving that they inhibit AC by covalent binding to its catalytic cysteine.
Conclusıons: We have developed different chemical classes of potent small-molecule inhibitors of AC which can serve as tools to study the functions of ceramide in health and disease, and may help to validate AC as drug target with potential therapeutic applications in cancer and inflammation. These compounds may also provide promising starting points for the development of novel therapeutic agents.

Sphingolipid analogues as inhibitors of sphingolipid metabolism and trafficking.

Christoph ARENZ
Instıtut für Chemıe / Humboldt Unıversıtaet zu Berlın

ABSTRACT NOT RECEIVED

Targeted LC-tandem-MS analysis and ms imaging reveal new sphingolipid structures and the function of ceramide synthase 3 in vivo.

Roger SANDHOFF¹
1German Cancer Research Center

Objective: Tandem-mass spectrometry (coupled to liquid chromatography) enabled the discovery of tissue-specific sphingolipid structures and of the natural composition of 1-O-acylceramides in mammals. Metabolism and cellular location of the latter remains enigmatic (Sphinx-like), but analysis of knockout mouse models gives first insight. The discovery of germ cell specific sphingolipids led to investigations of ceramide synthase 3 (CerS3) function in vivo. Analyzing systemic and cell-specific knockout mouse models with targeted LC-tandem MS proved CerS3 incorporates in a non-redundant fashion ultra long-acyl chains into sphingolipids of differentiated keratinocytes and of adluminal germ cells. Hence, CerS3 is quintessential for skin barrier/life and for male fertility. Tissue location is a prerequisite to understand the biological function of a molecule, which now can be addressed by mass spectrometry imaging (MSI) to distinguish closely related lipid structures. MSI revealed a highly specific location of such CerS3-dependent sphingomyelins in two different adluminal regions of seminiferous tubules supporting the idea, that these structures are made for more than one purpose in germ cells.

Phenotypic malignant changes and untargeted lipidomic analysis of long-term exposed prostate cancer cells to endocrine disruptors.

Núria DALMAU¹, Joaquim JAUMOT¹, Romà TAULER¹, Carmen BEDIA¹,
1Institute Of Environmental Assessment And Water Research (ydaea-csyc)

Objectıve: Numerous studies involve the endocrine disruptors (EDs) exposure to the initiation and development of cancers, including prostate cancer. Three different EDs (aldrin, aroclor 1254 and chlorpyrifos (CPF)) have been investigated as potential inducers of a malignant phenotype in DU145 prostate cancer cells after a chronic exposure. Then, an untargeted lipidomic analysis has been performed to decipher the lipids involved in the observed transformations.
Methods: Phenotypic malignant changes such as epithelial to mesenchymal transition (EMT) induction, proliferation, migration, colony formation and release of metalloproteinase 2 (MMP-2) have been analyzed in 50-day exposed cells to the selected EDs. Changes in specific sphingolipids and other lipids were assessed by the means of an untargeted lipidomic analysis using chemometric tools on LC-MS data of lipid cell extracts.
Results: Chronic exposure to aldrin and CPF resulted in EMT induction. CPF and aroclor 1254 also increased cell migration, colony formation and MMP-2 release. On one hand, the untargeted lipidomic analysis revealed a global decrease in phospholipids, ceramides glucosylceramides and lactosylceramides in cells treated with aldrin; on the other hand, CPF and aroclor 1254 treatment resulted in an increase of certain phospholipids, glycosphingolipids and a remarkable increase of some cardiolipin species. All treatments resulted in increased levels of triacylglycerides.
Conclusıons: The untargeted lipidomic approach used in this study enabled the identification of some lipid compounds and lipid metabolic pathways which could be involved in the acquisition of a malignant phenotype under ED exposure in prostate cancer cells.

Ceramides in anoxia survival.

J. Thomas HANNICH¹, Augustinus GALIH¹
1Geneva University

Objective: Sphingolipids are major eukaryotic membrane lipids which have been shown to play important signaling roles during cellular stress. Using targeted and non-targeted lipidomics we found a connection between 1‐deoxy sphingolipids and resistance to anoxia in the nematode Caenorhabditis elegans. Animals that produce more 1-deoxy sphingolipids show lower survival during anoxia and suppression of 1-deoxy sphingolipid production can extend survival without oxygen. Genetic screens in yeast and worms to identify the target of toxic 1-deoxysphingolipids might help to not only extend survival of cells during lack of oxygen like in stroke or heart attack but also to reduce lipotoxicity during metabolic syndrome and diabetes where 1-deoxysphingolipids have been found to increase.

Elucidating the double bond position of endogenous 1-deoxysphingosine.

Regula STEINER¹, Alaa OTHMAN², Essa MOSTAFA3, Christoph ARENZ3, Alan MACCARONE4, Stephen BLANKSBY5, Arnold VON ECKARDSTEIN¹, Thorsten HORNEMANN¹
1University Hospital Zurich, 2University Of Lubeck, 3Humboldt University, 4University Of Wollongong, 5Queensland University Of Technology

Objective: 1-Deoxysphinglipids are atypical sphingolipids which are devoid of the 1-OH-group present in canonical sphingolipids. 1-Deoxysphingosine (1-deoxySO) is a downstream metabolite of 1-deoxysphinganine and has been found in mammalian cell lines and human plasma. We observed recently that natural 1-deoxySO shows a deviation in LC retention time when compared to a synthetic 1-deoxySO standard bearing a Δ4-5 trans double bond (DB), as in the canonical sphingosine, albeit the m/z is identical for both compounds. This indicates that the DB positon in native 1-deoxySO is at a different position than in sphingosine. Our objective was therefore to elucidate the double bond position in endogenous 1-deoxySO, which is so far not known. HEK 293 cells were fed with deuterium labelled 1-deoxysphinganine. Whole sphingolipid extract was hydrolyzed and derivatized by the addition of dimethyl disulfide (DMDS) across the double bond in 1-deoxySO. The [M]+ ion of the DMDS adduct of 1-deoxySO was analyzed by direct injection on an MS2. After collision-induced disassociation, we could identify two ions specific for the fragmentation between the two SCH3 adducts at the site of the original double bond position. The double bond position of 1-deoxySO was identified to be Δ14-15. These results were confirmed by ozone-induced dissociation. The DB of native 1-deoxySO is located at Δ14-15 and is therefore distinct to that of canonical sphingosine (Δ4-5). This indicates that 1-deoxySO might not be a substrate of dihydroceramide desaturase 1 and that the DB at Δ14-15 may be introduced by a distinct desaturase.

Increased plasma levels of select deoxy-ceramide and ceramide species are associated with increased odds of diabetic neuropathy in type 1 diabetes.

Samar HAMMAD¹, Richard KLEIN¹, Nathaniel BAKER¹, Jad EL ABIAD¹, Stefanka SPASSIEVA¹, Jason PIERCE¹, Jacek BIELAWSKI¹, Maria LOPES-VIRELLA¹
1Medical University of South Carolina

Objective: The clinical presentation of diabetic neuropathy is similar to neuropathy in hereditary sensory and autonomic neuropathy Type 1 (HSAN1) patients, who exhibit elevated plasma levels of deoxy-sphingolipids (DSL). Supplementation with L-serine reduces plasma DSL levels and decreases neuropathic symptoms in HSAN1 patients. Plasma DSL are elevated in Type-2 diabetes and metabolic syndrome patients but no studies reported in Type-1 diabetes.
Methods: Using mass spectroscopy, plasma levels of DSL and free amino acids in DCCT/EDIC Type-1 diabetes patients (n=80), with and without symptoms of neuropathy were investigated. Patient-determined neuropathy was based on 15-item self-administered questionnaire [Michigan Neuropathy Screening Instrument] developed to assess distal symmetrical peripheral neuropathy in Type-1 diabetes. Patients who scored ≥ 4, or were unable to sense their feet during walking or to distinguish hot from cold water while bathing were considered neuropathic. Levels of ceramide, sphingomyelin, hexosyl- and lactosylceramide species were also measured.
Results: Deoxy-C24-ceramide, C24- and C26-ceramide were higher in patients with neuropathy than those without neuropathy (12.3±3.7 vs 10.6±4.1, 3184.6±762.7 vs 2709.5±921.8, & 131.1±39.7 vs 104.6±35.6 nM, p<0.05). Cysteine was higher in patients with neuropathy (2.0±1.0 vs 1.4±0.6 μM, p<0.01). No differences in other sphingolipids or amino acids were detected. The Odds Ratio of positive neuropathy diagnosis was associated with increased levels of cysteine (p<0.01), deoxy-C22:1-, deoxy-C24-, and deoxy-C24:1-ceramide (p<0.05), C22-, C24-, and C26-ceramide (p<0.05). The Odds Ratio of negative neuropathy was associated with increased sphingosine (p<0.05).
Conclusion: high plasma levels of select deoxy-ceramide and ceramide species may be involved with neuropathy in Type-1 diabetes.

P1: Sphingosine kinase 1 mediates tumour monocyte interaction and cancer chemoresistance.

Joao NUNES¹, Heba ALSHAKER², Charlotte BEVAN¹, Jonathan WAXMAN¹, Dmitri PCHEJETSKI3,
1Imperial College, 2University Of Petra, 3University Of East Anglia

Objective: Tumour-associated macrophages promote solid tumour growth through secretion of cytokines and chemokines. Sphingosine kinase 1 (SK1) is a known mediator of inflammation and is critically implicated in cancer progression, resistance and poor prognosis. Here we have investigated the role of SK1 as signal transduction component during the tumour-monocyte cellular interaction.
Methods: Boyden chambers were used for macrophage-tumour cells co-culturing. Cytokine production was measured by proteome profiler and ELISA assays and gene expression was measured by qRT-PCR. Cell viability was measured using MTT assay. Human prostate tumours were established in Nude mice.
Results: During co-culture both monocytes and cancer cells showed a transient increase in SK1 activity and mRNA expression together with an increase in MCP-1 and IL-6 secretion. Silencing of SK1 in cancer cells abrogated co-culture-induced phosphorylation of AKT, ERK1/2, NF-kB and SK1 in monocytes and monocyte-induced cancer cell proliferation. Furthermore, monocytes induced cancer cell chemoprotection via a SK1/Akt/ERK1/2-dependent mechanism, reducing the inhibitory effect of docetaxel on cancer cell proliferation. Selective inhibition of SK1 in tumour cells significantly reduced the cytokine secretion from both cancer cells and monocytes affecting IL-6, MCP-1, GROα, IL-32 and ICAM-1. Our data show that STAT1 binds to SK1 promoter and may be involved in SK1 transcriptional regulation in cancer cells upon monocyte stimulation. Pharmacological targeting of SK1 in mouse prostate cancer model reduced tumour volume, TAM infiltration and cytokine secretion.
Conclusion: Our results suggest a novel SK1/ERK1/2/IL-6/MCP-1 – dependent mechanism of tumour inflammation and monocyte-induced cancer chemoresistance.

P2: Investigation of epigenetic markers in the sphingosine kinase locus for a prognostic blood test to predict prostate cancer risk.

Heba ALSHAKER¹, Philip JORDAN², Alexandre AKOULITCHEV², Dmitri PCHEJETSKI3
1University Of Petra, 2Oxford Biodynamics, 3University Of East Anglia

Objective: Sphingosine kinase 1 (SPHK1) is a proto-oncogene and has been reported to be elevated in many cancers including the prostate, but its regulation is poorly understood. Chromosome conformation patterns represent early changes in epigenetic regulation during tumourigenesis.
Methods: Chromosomal conformation studies were performed in the locus of SPHK1 gene using PCR.
Results: Epigenetic regulation of SPHK1 was investigated in 80 treatment naive patients, which were separated into low and high risk groups according to the NCCN criteria. Relevance vector machine based algorithm was used to predict the potential chromosomal conformations, which were then identified using PCR. 20 Chromosmal conformations were identified in the SPHK1 locus. Of these, an inhibitory conformation SPHK1-5/17 was consistently associated with prostate cancer patients in a low risk group. Conversely, SPHK1-1/15 conformation was shown to be associated with high risk prostate cancer. When united with other chromosomal conformation markers, in the loci of several PCa-related genes, including MYC, PSMA, PTEN, SMAD4, and SAM68, SPHK1 formed a part of a final epigenetic test that was trained on 64 samples and cross-validated on 16 independent samples. The final EpiSwitchTM test can accurately predict PCa aggressiveness status in both training and validation sets, with a combined accuracy of 98.6% (95% CI, 93% – 99%).
Conclusion: In Addition to stratification of PCa patients this prognostic, non-invasive test has the potential to be used for making treatment decisions, monitoring and predicating for PCa mortality. The EpiSwitchTM platform technology can be extended to address other clinical questions in diagnostics and theranostics.

P3: Membrane lipids regulate ganglioside gm2 catabolism and the activity of lysosomal lipid binding and transfer proteins.

Breiden BERNADETTE¹, Susi ANHEUSER², Guenter SCHWARZMANN², Konrad SANDHOFF²
1Lymes Institut, University Bonn, 2Lymes Institut, University Bonn

Objective: During endocytosis, luminal endolysosomal vesicles are formed which serve as platforms of lipid and membrane digestion. Ongoing in vitro studies on the catabolism of radiolabeled liposomal sphingolipids (SL) and glycosphingolipids (GSL) indicated that stabilizing lipids of the plasma membrane, i.e. sphingomyelin (SM) and cholesterol, inhibit several steps of lysosomal SL and GSL catabolism, and also the lipid mobilization and transfer activities of several sphingolipid activator proteins (SAPs) and NPC2. We reconstituted the catabolic proteins on liposomal surfaces, mimicking luminal vesicles of the lysosomes as platforms for SL degradation. Liposomes with no net surface charge generated only negligible catabolic rates. However, incorporation of various anionic lipids into the liposomal membranes had a strong stimulatory impact on GM2 catabolism and on mobilization and intervesicular transfer of membrane lipids by GM2AP, whereas cholesterol exerted a strong inhibitory effect. The intervesicular transfer of 2-NBD-labeled GM1 from donor to acceptor liposomes was stimulated by BMP, ionic strength and low pH values, whereas both, cholesterol and SM, were inhibitory. The current work demonstrates the importance of electrostatic interaction between anionic SL-carrying vesicles and cationic proteins for the stimulation of SL hydrolysis. It also raises major concerns about the usage of His-tagged GM2AP and other lipid binding proteins to investigate the properties of the natural GM2AP. His-tagged GM2AP binds more strongly to anionic SL-carrying liposomal surfaces due to its positively charged His6-tag at low pH values. Its presence increases GM2 hydrolysis, allows transfer of 2-NBD-GM1 even at low ionic strength but prevents mobilization of membrane lipids.

P4: Ceramide mediated lethal mitophagy: a novel cell death mechanism in FLT3 targeted therapy of acute myeloid leukemia.

Mohammed DANY¹, Besim OGRETMEN¹
1Medical Universiy Of South Carolina

Objective: Mutations in FLT3 receptor tyrosine kinase are common in Acute Myeloid Leukemia (AML) and confer a worse prognosis. Ceramide, a bioactive sphingolipid, is synthesized de novo by Ceramide Synthases (CerS) and mediates cancer cell death in response to various chemotherapeutic agents. This study investigates the biological role of ceramide in FLT3-positive AML pathogenesis. We show that AML cell lines and patient samples expressing FLT3 have suppressed CerS1 expression and lower levels of its product C18-ceramide compared with FLT3 negative AML cells. Silenced FLT3 expression or its pharmacological inhibition increased CerS1 and C18-ceramide levels while FLT3 overexpression suppressed them. The increase in C18-ceramide after FLT3 inhibition is required for cell death as silencing CerS1 expression or inhibiting its enzymatic activity protected from FLT3 inhibitors-induced cell death. Targeting FLT3 resulted in C18-ceramide dependent mitophagy, as determined by increased LC3B-II levels and formation of autophagosomes around mitochondria. Mechanistically, C18-ceramide accumulated in the mitochondria to bind directly to LC3B-II recruiting autophagosomal membranes for the execution of mitophagy. This process was accompanied by mitochondrial depolarization, decreased ATP generation, and DRP-1 oligomerization. In summary, our novel study is the first to highlight the importance of ceramide metabolism in AML oncogenesis by showing that FLT3 suppresses CerS1 expression and ceramide generation while its inhibition reactivates CerS1/C18-ceramide axis leading to lethal mitophagy and AML cell death.

P5: Acid ceramidase expression in normal and neoplastic melanocytes and its role in melanoma progression.

Natalia REALINI¹, Francesca PALESE¹, Daniela PIZZIRANI¹, Silvia PONTIS¹, Abdul BASIT¹, Anders BACH¹, Anand GANESAN², Daniele PIOMELLI3
1Fondazione Istituto Italiano Di Tecnologia, Genova, Italy, 2University Of California, Irvine, USA, 3Fondazione Istituto Italiano Di Tecnologia, Genova, Italy AND University Of California, Irvine, USA

Objective: In the present study, we used a combination of lipidomics, morphology, pharmacology and genetics to investigate the expression and functions of acid ceramidase (AC) in normal melanocytes and melanoma cells. We then evaluated AC inhibition as a possible strategy for melanoma therapy.
Results: We first measured AC levels in various human cancer cell lines, discovering a greater AC expression in melanoma than in other cancers. To determine whether AC overexpression distinguishes melanoma cells from non-malignant melanocytes, we measured AC gene transcription, protein concentration and enzyme activity in melanocytes and melanoma cells. Unexpectedly, normal melanocytes and melanoma expressed AC at similar levels. However, confocal microscopy experiments revealed that melanocytes and melanoma cells differ in the intracellular localization of this enzyme. Based on their similar AC levels, we predicted that ceramide content should be comparable in normal and malignant cells. In striking contrast with that prediction, lipidomic analysis showed significantly lower ceramide levels in melanoma, due to a deregulation of enzymes involved in the novo ceramide biosynthesis. Lastly, we examined whether AC inhibition influences melanoma cell viability. Our new AC inhibitor ARN14988, with nanomolar potency and good stability, inhibits AC and elevates ceramide levels in a concentration and time dependent manner. This compound alters the balance between ceramide and sphingosine-1-P and synergizes with different antitumoral agents in proliferative melanomas.
Conclusıon: In conclusion, we demonstrated a major involvement of AC and ceramide metabolism in melanoma and normal melanocytes and we propose the use of AC inhibitors for melanomas with a proliferative phenotype.

P6: The role of sphingosine kinase-1 in VHL mutant clear cell renal cell carcinoma.Mohamed SALAMA1,

Brittany CARROLL¹, Mohamad ADADA¹, Michael PULKOSKI-GROSS¹, Yusuf HANNUN¹, Lina OBEID¹,
1Stony Brook University

Objectıve: Sphingosine kinase 1 (SK1), the enzyme responsible for sphingosine 1-phosphate (S1P) production, is overexpressed in many human solid tumors. However, its role in clear cell renal cell carcinoma (ccRCC) has not previously been described.
Methods: VHL defective ccRCC 786-0 cells were used in this study. SK1 down-regulation was achieved by short-hairpin RNA (shRNA) to study the role of SK1 in ccRCC. Proliferation, invasion, and in vivo angiogenesis assays were conducted.
Results: We showed that SK1 is over-expressed in 786-0 cells lacking functional VHL with high S1P levels. By using TCGA RNA seq database, we showed that SK1 expression was higher in solid tumor tissue from ccRCC patients that was associated with less survival. Knocking-down of SK1 in ccRCC cells had no effect on cell proliferation; whereas, it was associated with less invasion, less phosphorylation of focal adhesion kinase (FAK), and less angiogenesis. Additionally, S1P treatment of SK1-kncock-down cells resulted in S1PR2-mediated phosphorylation of FAK and invasion.
Conclusıons: These results suggest that higher SK1 and S1P levels in VHL-defective ccRCC could induce invasion in an autocrine manner and angiogenesis in a paracrine manner. Accordingly, targeting SK1 could reduce both the invasion and angiogenesis of ccRCC and therefore improve the survival rate of patients.

P7: Untargeted lipidomic analysis of endothelial to mesenchymal transition in prostate cancer cells.

Núria DALMAU¹, Joaquim JAUMOT¹, Romà TAULER¹, Carmen BEDIA¹
1Institute Of Environmental Assessment And Water Research (IDAEA-CSIC)

Objectıve: Epithelial to mesenchymal transition (EMT) is a biological process that plays a crucial role in cancer metastasis in several human malignancies, including prostate cancer. Little is known about the involvement of lipids in EMT. In this work, an untargeted lipidomic analysis has been performed to investigate which are the important lipids and sphingolipids involved in this process.
Methods: EMT was induced in DU145 prostate cancer cells under TNF treatment. Sphingolipid and total lipid extracts of induced and non-induced EMT cells were analyzed through UPLC-TOF. Then, the chemometric tools Partial Least Squares-Discriminant Analysis (PLS-DA) and Multivariate Curve Resolution–Alternating Least Squares (MCR-ALS) were applied to data in order to decipher which were the lipids (sphingolipids and other lipids) that presented significant changes after EMT induction.
Results: A significant increase of 14 unsaturated triacylglycerides (TAGs) was observed under EMT induction. According to this, cell presented an accumulation of lipid droplets, as well as a concomitant up-regulation of fatty acid synthase (FASN), a gene that has been related to cancer progression and metastasis. Among other changes in sphingolipid levels, a decrease of C16:0 ceramide was observed under EMT induction.
Conclusıons: The application of chemometric tools for the study of EMT induction enabled the detection of increased levels of TAGs and changes in specific sphingolipid compounds. These results could be helpful for a better understanding of the process in the research of therapeutic targets to prevent metastasis initiation.

P8: Overcoming nilotinib resistance by specific inhibition of sphingosine-1-phosphate receptor 2/Gq/phospholipase C axis in chronic myeloid leukemia.

Aysun ADAN¹, Besim OGRETMEN², Yusuf BARAN¹
1Izmir Institute Of Technology, 2Medical University Of South Carolina

Objective: The changes in sphingolipid metabolism are considered a significant BCR-ABL1 dependent resistance mechanism as well as BCR-ABL mutations. Sphingosine kinase-1 (SK-1)/sphingosine-1 phosphate (S1P)-mediated drug resistance is related to sphingosine-1 phosphate receptor 2 (S1P2) signaling through the inhibition of protein phosphatase 2A (PP2A), resulting in increased stability of BCR-ABL1. However, specific signaling cascade involved in this process remain unkown.
Methods: The antiproliferative effects of nilotinib, SK-1 inhibitor (PF-543), S1P2 inhibitor (JTE-013), phospholipase C inhibitor (U-73122) and nilotinib/PF-543 and nilotinib/JTE-013 combinations on 32D-p210Bcr-Abl(wt) and 32D-p210Bcr-Abl (T315I) cells were determined by MTT assay. Isobologram analysis was performed using CompuSyn program. The mRNA and protein levels of BCR-ABL1, SK-1 and S1P2 were checked by qRT-PCR and western blotting. Resistant cells were also transfected with Gq peptide.
Results: IC50 values of nilotinib were 8 and >500 nm for wt and resistant cells, respectively. IC50 values for JTE-013 were calculated as 20 and 40 μM while that for PF-543 were 8 and 30 μM, respectively. Combination treatments displayed strong synergistic effects on both cell types. Although there is no significant changes in BCR-ABL1 levels for both cells, SK-1 and S1P2 levels increased in resistant cells. Combination studies decreased BCR-ABL1 protein levels in resistant cells comparing to untreated control, PF-543 or JTE-013 treatments. Although U-73122 and Gq peptide treatments decreased BCR-ABL1 protein, their combination with okadaic acid restored BCR-ABL1 protein levels.
Conclusion: BCR-ABL1 levels decreased by activating PP2A via Gq and phopholipase C inhibition, a novel mechanism, which could be a potential therapeutic target to overcome nilotinib resistance.

P9: Correlations between expression levels of bioactive sphingolipid genes and drug-sensitivity and -resistance in chronic myeloid leukemia (CML) patients.

Melis KARTAL YANDIM¹, Ilknur KOZANOGLU², Hakan OZDOGU², Ozden PISKIN3, Mehmet Ali OZCAN², Guray SAYDAM4, Fahri SAHIN4, Ali Ugur URAL5, Ali UNAL6, Yusuf BARAN¹
1Izmir Institute Of Technology, Department Of Molecular Biology And Genetics, 2Baskent University, Department Of Hematology, 3Dokuz Eylul University, Department Of Hematology, 4Ege University, Department Of Hematology, 5Bayindir Hospital, Department Of Hematology, 6Erciyes University, Department Of Hematology

Objective: CML is characterized by Philadelphia chromosome encoding BCR/ABL oncoprotein with tyrosine kinase function. Tyrosine kinase inhibitors (TKIs) are used for CML therapy. In this study, we aimed to examine the correlation between expression levels of bioactive sphingolipid genes and the response of newly diagnosed (ND), TKI-treated and shown hematological response (HR), or TKI-resistant CML patients against therapy.
Methods: Bone marrow samples of 66 CML patients were harvested, and mononuclear cells were isolated by lysis buffer. cDNAs were synthesized via reverse transcription following the RNA isolation. Expression levels of ceramide synthase (CERS1-6), sphingosine kinase-1 (SK1), glucosylceramide synthase (GCS), and BCR/ABL genes were determined by qRT-PCR.
Results: Patients treated with TKIs and shown HR expressed significantly higher levels of CERS1-6 genes than ND or TKI-resistant patients, whereas TKI-resistant patients expressed higher levels of GCS and SK1 genes compared to ND or TKI-treated and shown HR. Comparing the expression levels in different samples acquired once in six months of the same patient revealed that expression levels of CERS1-6 genes increased, whereas GCS and SK1 decreased in patients treated with TKIs and shown HR. While the patients were developing resistance to the therapy, their GCS and SK1 expressions increased.
Conclusion: We showed a correlation between disease progression and bioactive sphingolipid gene expression levels in CML patients. Particularly, the results revealed that besides being alternative targets for more efficient therapy, bioactive sphingolipids might be crucial markers for anticipating drug resistance. This study was supported by The Scientific and Technological Research Council of Turkey with 111S392 project number.

P10: CerS4 and CerS5 transcription is GPER1-mediated and AP-1-dependent regulated in human breast cancer cells.

Marthe-Susanna WEGNER¹, Stephanie Beatrice OERTEL¹, Rolf MARSCHALEK², Gerd GEISSLINGER¹, Sabine GRÖSCH¹
1Pharmazentrum Frankfurt/ZAFES, 2Institute For Pharmaceutical Biology

Objective: Ceramide synthases (CerS) catalyze the synthesis of ceramides, which are beside an element of membranes, important signal molecules in the cell. Only little is known about the transcriptional and posttranscriptional regulation of CerS. We wanted to investigate whether the transcriptional regulation of the CerS in human breast cancer cells is G-protein coupled estrogen receptor (GPER) 1-dependent and what role does the activator protein (AP) -1 play.
Methods: MCF-7 cells were transfected with the CerS2, -4, -5, or -6 promoter and CerS4 and -5 promoter AP-1 deletion constructs, cloned into luciferase reporter gene plasmid, and an GPER1 expression plasmid. Subsequently, cells were treated with estrogen and luciferase activity was determined. An AP-1 transcription factor activation ELISA was performed after GPER1 transfection. The expression of GPER1 was determined by quantitative Real-Time PCR and Western Blot analysis. Activation of GPER1 by different ligands was determined by a cAMP-Assay.
Results: Co-transfection of CerSx promoter and GPER1 results in an increased CerS2, -4 and 6 promoter and a reduced CerS5 promoter activity in MCF-7- cells. Deletion of an AP-1 binding site in the CerS4 and -5 promoter as well as an AP-1 transcription factor activation ELISA revealed that GPER1 mediates its effect on the CerS4 and -5 promoter via the AP-1 site. Endogenous GPER1 expression is decreased after estrogen treatment in a concentration dependent manner.
Conclusion: The GPER1-effect on CerS4 and -5 promoter in MCF-7 cells is AP-1 mediated. The complex consists of cFos and Fra-1 and can be activated by diverse ligands.

P11: The lysosomal lipid storage disease Niemann Pick type A disease sensitizes to acetaminophen hepatotoxicity by impairing mitophagy.

Jose C FERNANDEZ-CHECA¹, Anna BAULIES¹, Vicente RIBAS¹, Susana NUÑEZ¹, Sandra TORRES¹, Laura MARTINEZ¹, Jo SUDA2, Maria YBANEZ², Neil KAPLOWITZ², Carmen GARCIA-RUIZ¹
1IIBB-CSIC-IDIBAPS, 2Keck School Of Medicine, USC University, Los Angeles, CA

Objective: The role of lysosomes in acetaminophen (APAP) hepatotoxicity is poorly understood. Niemann-Pick type A (NPA) disease caused by acid sphingomyelinase (ASMase) deficiency is a lysosomal storage disease (LSD) characterized by increased lysosomal sphingomyelin (SM) and cholesterol accumulation.
Methods and Results: We show that ASMase-/- mice exhibit higher liver damage and mortality after APAP overdose than ASMase+/+ mice. APAP metabolism and predominant toxic mechanisms, including mitochondrial GSH depletion and JNK activation, are independent of ASMase. ASMase-/- hepatocytes exhibit lower threshold for APAP-induced cell death and defective fusion of mitochondria-containing autophagosomes with lysosomes compared to ASMase+/+ hepatocytes, which translates in decreased mitochondrial quality control. Lysosomal cholesterol (LC) accumulation in ASMase+/+ hepatocytes by the cationic amphiphilic drug U18666A reproduces the susceptibility of ASMase-/- hepatocytes to APAP. LC extraction by 25-hydroxycholesterol (25-HC) protects ASMase-/- hepatocytes against APAP hepatotoxicity by improving formation of mitochondria-containing autolysosomes. 25-HC treatment rescues ASMase-/- mice against APAP-induced liver injury. The regulation of LC by U18666A or 25-HC does not affect total cellular SM content or lysosomal distribution. LC accumulation and subsequent sensitization to APAP was also observed in hepatocytes treated with amitriptyline, a widely used tricyclic antidepressant that inhibits ASMase or conduritol B-epoxide, a chemical inducer of Gaucher disease (GD), the most common LSD, due to the irreversible inhibition of glucocerebrosidase.
Conclusion: These findings indicate that LC accumulation determines APAP hepatotoxicity by impairing mitophagy, implying that patients with NPA or GD may be at risk of developing APAP-induced acute liver failure.

P12: Structure and regulation of human neutral sphingomyelinase-2.

Prajna SHANBHOGUE¹, Mıchael AIROLA¹, Kıp GUJA¹, Rohan MAINI¹, Mıguel GARCIA-DIAZ¹, Yusuf HANNUN¹
1Stony Brook University

Objective: Neutral sphingomyelinase 2 (nSMase2) is a magnesium-dependent phophodiesterase that generates the bioactive sphingolipid ceramide. Ceramide is a critically important secondary messenger that acts in a variety of cellular pathways such as apoptosis, inflammation, cell growth and differentiation. Accordingly, mutations in nSMase2 can affect ceramide production and have been implicated in cancers of the breast, urinary bladder and colon. Obtaining a detailed understanding of the structure and function of this enzyme is of significant interest and has the potential to uncover new avenues for cancer therapeutics. We have obtained the first high-resolution X-ray crystallographic structure of human nSMase2 and developed a yeast two-hybrid system to analyze its inter-domain interactions, which likely play a role in activation of the enzyme by anionic phospholipids (APLs). Previous work in our laboratory identified amino acid residues in the N-terminal hydrophobic domain of nSMase2 that interact with APLs in the inner membrane. Our current results suggest that these residues also interact with the cytoplasmic C-terminal domain of nSMase2 and may help regulate activation of the enzyme by APLs. Furthermore, we have identified additional amino acid residues in the juxtamembrane region of nSMase2 that may also contribute to regulation of enzyme activity. In sum, our results provide a model for activation of nSMase2, and reveal a novel structure that paves the way for rational drug design endeavors.

P13: Cellular and molecular effects of acid ceramidase deficiency in MEFs derived from Farber mice.

Mustafa KAMANI¹, Fabian YU², Tonny HUANG², Jeffrey MEDIN¹
1University Health Network, 2University Of Toronto

Background: Farber disease is a severe LSD that results from reduced activity of acid ceramidase (ACDase), a catabolic enzyme required for the lysosomal breakdown of ceramide into sphingosine and a free fatty acid. We recently developed the first viable mouse model of ACDase deficiency and Farber disease by introducing a clinically relevant single nucleotide mutation into the Asah1 gene. The resulting Asah1P361R/P361R Farber mice accumulate ceramides in all organs, suffer from multiple abnormalities, and die by 9-13 weeks of age.
Objective: To examine the effects of mutant ACDase on enzyme processing, cell growth and potential activation of compensatory mechanisms to deal with the ceramide load. Methods: Using mouse embryonic fibroblasts (MEFs) derived from WT and Farber mice, we analyzed ACDase expression and trafficking by Western blot and immunofluorescence. Transcript levels of sphingolipid metabolic enzymes were assessed by qPCR.
Results: We show herein differential processing of WT and mutant ACDase. We also show activation of compensatory mechanisms in Farber MEFs that address the ceramide accumulation: upregulation of genes encoding ceramide-utilizing enzymes, including alkaline ceramidases, sphingomyelin synthases, and ceramide kinase, along with downregulation of genes encoding ceramide-producing enzymes, including glucocerebrosidase and ceramide synthases. Lastly, Farber MEFs show reduced proliferation and increased susceptibility to apoptosis.
Conclusions: Work from our mouse model will pave the way for understanding potential alternative functions of ACDase, identifying the particular ceramide species responsible for affecting specific cell signaling pathways, and elucidating the mechanism(s) by which lysosomal sphingolipid accumulations elicit changes in gene expression.

P14: Bibliometric evaluation of the evolution of the ceramide research landscape.

Jihad OBEID¹
1Medical University Of South Carolina

Objective: Research in lipidomics has been rapidly evolving in the post genomic era with the realization that many cell and organismal functions are mediated by lipids. Several years ago, work on ceramides, sphingosine phosphate, and many other bioactive lipids, has focused on the study of basic roles of these molecules in living systems, for example, as key regulators of important cellular and intercellular functions such as cell signaling, growth, senescence, cell death, and inflammation. We have analyzed bibliometric data collected at the Medical University of South Carolina (MUSC) to assess the evolution of lipidomics research over the several phases of Centers of Biomedical Research Excellence funding by the National Institutes of Health. Relevant PubMed MeSH terms (e.g. Ceramides and Sphingolipids) were used to identify a cohort of publications and authors in the field. Analysis of the interrelationship of MeSH terms shows increased emphasis in recent years on the translational aspects of the field and more focus on human diseases than in previous years. Network analysis using research networking software data reveals a steady rise in the average number of co-authorship consistent with a trend towards increased team science. This transition into translational science signifies a relative maturation of the lipidomics field as a whole.

P15: Involvement of S1P signalling in FGF-induced neurogenesis and proliferation in inner ear sensory cells.

Ricardo ROMERO-GUEVARA¹, Ilaria RIZZ0¹, Marina BRUNO¹, Francesca CENCETTI¹, Chiara DONATI¹, Paola BRUNI¹
1University Of Florence

Objective: According to the world health organization (WHO) 360 million people in the world is affected by debilitating hearing loss. Currently the only available treatment is the cochlear implant, but this device is not suitable in many cases. Thus, it is necessary to explore new molecular targets that could prevent and/or restore hearing loss. Sphingosine 1-phosphate (S1P) signalling was recently shown to be essential for the maintenance of the auditory epithelium in postnatal mice. Nonetheless, the information about S1P signalling pathway in the context of inner ear is still scarce. Our aim is to understand in more detail the role of S1P signalling during the development of the inner ear sensory cells. For this purpose we are currently using a mouse otocyst-derived cell line (VOT33) suitable to study neurogenesis in vitro. As reported previously, FGF2 induced the formation of neuroblasts in VOT33 cells. Moreover, FGF2 induced proliferation and prevented apoptosis induced by serum-starvation and staurosporine treatment. Interestingly, pharmacological inhibition or knockdown of both isoforms of S1P-generating enzyme sphingosine kinase (SK)-1 and -2, and the S1P receptors S1P₁ and S1P₂ affected FGF-induced proliferation, indicating that S1P signalling axis is necessary in FGF biological action. In addition to the up-regulation of the sensory neuron markers Islet1/2 and β-Tubulin-III, FGF2 treatment induced the up-regulation of S1P₁, SK1 and SK2, and down-regulation of S1P lyase, suggesting a positive involvement of S1P in FGF-induced differentiation. Currently, we are developing new strategies to improve neural maturation, as well as the establishment of new complementary in vitro models of inner ear development to gain a more comprehensive insight of S1P signalling in this biological system.

P16: The impact of ceramide-synthases on colitis ulcerosa.

Stephanie OERTEL¹, Marthe-Susanna WEGNER¹, Nerea FERREIRÓS BOUZAS¹, Gerd GEISSLINGER¹, Sabine GROESCH¹
1Clinical Pharmaology, Goethe University

Background: We could show, that ceramides of various chain length have different impacts on colon cancer cell proliferation. Therefore, we want to investigate how ceramides of different chain length influence the development of colorectal carcinoma in the colitis ulcerosa mouse model by using ceramide synthase (CerS) knockout mice.
Methods: Colitis Ulcerosa was induced by Azoxymethane/Dextrane Sodium Sulfate in CerS knockout and wild type mice. CerS expression in different mouse tissues was assessed by RT-PCR and ceramide concentrations were determined by LC-MS/MS. The impact of CerS expression on different immune cell populations were investigated by FACS analysis. The protein levels of CerS in lamina propria cells of colon tissue from wild type and knockout mice were compared by immunohistochemistry after chronic inflammation.
Results: Inflammatory symptoms as well as tumor development were increased in CerS 4 knockout mice and reduced in CerS 2 knockout mice. Significant alterations in CerS expression and ceramide levels were detected in colon cells of wild type and knockout mice. CerS 2 and CerS 4 deficiency impacted various immune cell populations, like regulatory T-cells, in Blood, Lymph nodes and Spleen.
Conclusion: Ceramides of different chain lengths influence the progression and outcome of colon cancer development induced by chronic inflammation in a colitis ulcerosa mouse model.

P17: Sphingosine 1-phosphate metabolism: a novel way to sensitize melanoma cells to anticancer treatments.

David GARANDEAU¹, Marie-Lise BATS¹, Virginie GARCIA¹, Virginie ALBINET2, Thierry LEVADE¹, Nathalie ANDRIEU-ABADIE¹
1Inserm UMR1037, 2Imavita

Objective: The treatment of metastatic melanoma has changed considerably with the development of targeted therapies against the frequently mutated serine-threonine kinase BRAF. The survival rate increases by 6-8 months but relapses occur in a median of 6 months. This resistance is partly due to changes in the expression of several pro- and anti-apoptotic members of the Bcl-2 family. Our group has documented significant changes in ceramide metabolism in melanoma cells compared to healthy melanocytes (Albinet et al, Oncogene 2014). In particular, S1P lyase (SPL), which degrades sphingosine 1-phosphate (S1P), is under-expressed (Colié et al, Cancer Res 2009), conversely to sphingosine kinase 1 (SK1) which produces S1P. These alterations lead to increased production of this oncometabolite. Here, we show that SPL overexpression in metastatic melanoma which carries mutated BRAF resulted in sensitization of these cells to apoptosis induced by dacarbazine (DTIC) or vemurafenib (PLX4032). This phenomenon was associated with decreased expression of MITF, a major regulator in melanocyte differentiation and survival of melanoma, and a strong decrease in Bcl-2/Bim ratio that promoted caspase activation. In addition, the pharmacological inhibition of SK1 by SKi-1 or FTY720 induced a synergistic cytotoxic effect with PLX4032 on mutated BRAF melanoma cells which were sensitive or resistant to this agent. While sphingosine and ceramide levels increased in sensitive cells treated with PLX4032, they did not change in resistant cells. Thus, by controlling the expression of key proteins in cell survival, S1P metabolism could represent a new therapeutic approach to enhance the effectiveness of targeted therapies.

P18: The oncogenic lipid kinase SK1 promotes the migration and polarization of macrophages in melanoma tumors.

Marguerite MRAD¹, Celine COLACIOS¹, Claire DAVID¹, Nicole THERVILLE¹, Stephane CARPENTIER¹, Thierry LEVADE¹, Rania AZAR², Mona DIAB-ASSAF², Nathalie ANDRIEU-ABADIE¹
1Inserm Umr 1037, 2Lebanese Univeristy

Objective: Tumor infiltration by tumor-associated macrophages (TAM) is often correlated with poor prognosis in melanoma. However, the mechanisms by which TAM mediate melanoma growth are still poorly understood. Recent studies suggest a role for Sphingosine Kinase 1 (SK1), the enzyme that catalyzes the formation of the oncogenic lipid sphingosine-1-phosphate (S1P), in melanoma progression. The aim of this study was to investigate the role of SK1 in the interaction between melanoma cells and TAM. In vitro migration assays of human or murine monocytes, treated with S1P or melanoma cell-conditioned media, demonstrated that exogenous S1P as well as overexpression of SK1 in melanoma cells are able to amplify monocyte migration. This migration was disrupted in the presence of S1P receptor antagonists. On the other hand, macrophage polarization towards M1 antitumor phenotype was enhanced upon incubation with the medium of SK1-silenced melanoma cells. Intradermal injections of murine melanoma cells (B16F10), either knocked-down or not for SK1, into wild-type or SK1-deficient mice showed that the inhibition of SK1 in the host and/or in the tumor reduces melanoma growth. It also decreased tumor infiltration by macrophages. Furthermore, SK1 inhibition in melanoma cells significantly increased the expression of antitumor cytokines in the tumor microenvironment. Accordingly, preliminary flow cytometry studies indicated that, after SK1 inhibition macrophage polarization was reoriented toward an M1 antitumor profile. These findings suggest a key role of melanoma SK1 in macrophage recruitment and polarization within the tumor microenvironment, thereby enhancing the aggressiveness of this cancer.

P19: Down-regulation of ceramide synthase-6 during epithelial-to-mesenchymal transition enhances plasma membrane fluidity and breast cancer cell motility triggered by FAS/CD95.

Seguı BRUNO¹, Edmond VALERIE², Dufour FLORENT3, Poıroux GUILLAUME¹, Shojı KENJI², Levade THIERRY¹, Mıcheau OLIVIER3, Legembre PATRICK²
1Inserm UMR1037 / Paul Sabatier University, Toulouse III, 2Inserm UMR1085 / Rennes 1 University, 3Inserm UMR866 / Burgundy University

Objective: Epithelial-to-mesenchymal transition (EMT) promotes cell motility, which is important for the metastasis of malignant cells, and blocks CD95-mediated apoptotic signaling triggered by immune cells and chemotherapeutic regimens. CD95L, the cognate ligand of CD95, can be cleaved by metalloproteases and released as a soluble molecule (cl-CD95L). Unlike transmembrane CD95L, cl-CD95L does not induce apoptosis but triggers cell motility. Electron paramagnetic resonance was used to show that EMT and cl-CD95L treatment both led to augmentation of plasma membrane fluidity that was instrumental in inducing cell migration. Compaction of the plasma membrane is modulated, among other factors, by the ratio of certain lipids such as sphingolipids in the membrane. An integrative analysis of gene expression in NCI tumor cell lines revealed that expression of ceramide synthase-6 (CerS6) decreased during EMT. Furthermore, pharmacological and genetic approaches established that modulation of CerS6 expression/activity in breast cancer cells altered the level of C16- ceramide, which in turn influenced plasma membrane fluidity and cell motility. Therefore, this study identifies CerS6 as a novel EMT-regulated gene that plays a pivotal role in the regulation of cell migration.

P20: Discovery of novel S1PL inhibitors against multiple sclerosis disease.P20: Discovery of novel S1PL inhibitors against multiple sclerosis disease.

Utku DENIZ¹, Kutlu O. ULGEN¹, Elif O. OZKIRIMLI¹,
1Bogazici University, Chemical Engineering Department

Background: Sphingosine 1-phosphate lyase, a significant polar sphingolipid metabolite, serves as a contributory component in the regulation of cell migration, vascular stability and differentiation [1]. It carries out communication within the cell as a second messenger [2]. Multiple sclerosis (MS), a progressive inflammatory disorder of central nervous system (CNS), is mediated by the decreased intracellular S1P concentrations which induces migration of pathogenic T cells to the blood stream and disrupts the CNS [3]. The microsomal enzyme sphingosine 1-phosphate lyase (S1PL) degrades the intracellular S1P concentration and thus, S1PL is a promising drug target against MS disease.
Results: Virtual screening of ZINC database of 500,000 compounds via ligand-based and structure-based pharmacophore models retrieved 10,000 hits. After multistep docking and substructure search of common scaffolds, the compounds based on high docking scores, fitness values to the hypothesis and binding efficiency index (BEI), low molecular weight and high percentage of human oral absorption (HOA) were selected and further analyzed via induced fit docking (IFD) and molecular dynamic (MD) simulations in order to gain an insight into the binding modes and the key residues in binding. As a final outcome, the molecules based on having more drug-like properties were proposed as potential inhibitors of S1PL and some of the proposed compounds have also been proven effective against specific cancer cells and malarial parasites (antiviral action).
Conclusion: These potential inhibitors arouse future medicinal chemistry efforts to find out new compounds against destructive actions of pathogenic T cells.
References: 1. Bartke, N., and Y. A. Hannun, “Bioactive sphingolipids: metabolism and function”, Journal of Lipid Research, 50(Suppl.), 91-96, 2009. 2. Hait, N. C., et al. “Regulation of histone acetylation in the nucleus by sphingosine-1-phosphate”, Science, 325, 1254-1257, 2009. 3. Matloubian, M., et al. “Lymphocyte egress from thymus and peripheral lymphoid organs is dependent on S1P receptor 1”, Nature, 427(6972), 355-360, 2004

P21: Computer-aided discovery of sphingosine kinase 1 (SphK1) inhibitors.

Ozge BAYRAKTAR¹, Elif O. OZKIRIMLI¹, Kutlu O. ULGEN2
1Department of Computational Science and Eng., Bogazici University – 2Department of Chemical Eng., Bogazici University, 34342 Bebek, Istanbul, Turkey

Objective: Sphingosine-1-phosphate (S1P) signaling pathway plays an important role in controlling cell survival, cell proliferation, lymphocyte trafficking, angiogenesis, and cell differentiation. Sphingosine kinases (SphKs) catalyze the phosphorylation of sphingosine that leads to the production of S1P. Elevated expression of SphK1 is observed in many different cancers, including breast, colon, and prostate [1,2]. Thus, SphK1 inhibitors have been considered as effective chemotherapeutic agents for the treatment of cancers. The recently revealed crystal structure of SphK1 has two binding pockets for ATP-binding and sphingosine-binding. Calculations based on the X-ray crystal structure to compare the druggability of two pockets suggested that sphingosine-binding pocket is druggable [3,4]. In this work, it is aimed to identify potential SphK1 inhibitors by means of virtual screening and docking simulations. Using Schrödinger’s software, 500,000 compounds from the ZINC database were screened to find their ability to interact with the SphK1 sphingosine-binding pocket. Different ligand-based pharmacophore hypotheses were developed by QSAR studies from 28 heterocycle derivatives containing oxadiazole and 54 heterocycle derivatives containing thiazole using ChEMBL database. The validity of these hypotheses was tested by their ability to predict the known activity (pIC50) data reported in literature. The compounds that matched with pharmacophore hypotheses were docked and scored. The pharmacophore hypotheses were further analyzed according to ligand efficiency indices. After strain energy corrections, top hits will be proposed as potential inhibitors of SphK1 after final elimination based on pharmacokinetic and druglikeness properties. This work will provide a guide for future development of inhibitors targeting SphK1.
References: [1] Alshaker, H., Sauer, L., Monteil, D., Ottaviani, S., Srivats, S., Böhler, T., Pchejetski, D. Adv. Cancer Res. 2013, 117, 143–200 [2] Heffernan-Stroud, L.A., and Obeid, L.M. Adv. Cancer Res. 2013, 117, 201–235 [3] Cheng, A. C., Coleman, R. G., Smyth, K. T., Cao, Q., Soulard, P., Caffrey, D. R., Salzberg, A. C., Huang, E. S. Nat. Biotechnol. 2007, 25, 71

P22: Targeting ceramide transfer protein CERT to mitochondria triggers ceramide-dependent apoptosis.

Amrıta JAIN¹, Olıver BEUTEL¹, Sergey KORNEEV¹, Joost HOLTHUIS¹
1Unyversyti Of Osnabrueck

Objective: Ceramides are central intermediates of sphingolipid metabolism with critical functions in cell organization and survival. They are synthesized on the cytosolic surface of the endoplasmic reticulum (ER) and then transported by ceramide transfer protein CERT to the Golgi for conversion into sphingomyelin (SM) by SM synthase SMS1. Our lab previously identified SMS-related protein SMSr (SAMD8), an ER-resident ceramide phosphoethanolamine (CPE) synthase, as suppressor of ceramide-mediated cell death. Disruption of SMSr catalytic activity in HeLa cells causes a rise in ER ceramides and their mislocalization to mitochondria, triggering a mitochondrial pathway of apoptosis. Targeting a bacterial ceramidase to mitochondria rescued SMSr-deficient cells from apoptosis, arguing that ER ceramides exert their apoptogenic activity in mitochondria (Tafesse et al., 2014). To verify this concept, we set out to investigate the consequences of redirecting the biosynthetic ceramide flow from the Golgi to mitochondria on cell viability. To this end, the Golgi-directed PH domain of CERT was swapped for an outer mitochondrial membrane-targeting signal, yielding MitoCERT. MitoCERT accumulates at ER-mitochondrial contact sites owing to its interactions with the VAP-A receptor in the ER. Photo-affinity labeling experiments with a bifunctional ceramide analogue revealed that the protein binds ceramide through its START domain. Heterologous expression of MitoCERT in HeLa cells triggers mitochondrial apoptosis. Removal of the START domain did not affect the ability of MitoCERT to accumulate at ER-mitochondrial contact sites but completely abrogated its apoptogenic activity. Moreover, MitoCERT-induced apoptosis is suppressed by targeting bacterial ceramidase to mitochondria or treatment with myriocin. Our results indicate that mistargeting ER ceramides to mitochondria suffices to trigger a mitochondrial pathway of apoptosis.

P23: A kinome-wide siRNA screening combined with targeted lipidomic analysis reveals potential new regulators of sphingolipid metabolism in human cells.

Charlotte GEHIN¹, Noemi JIMÉNEZ-ROJO¹, Isabelle RIEZMAN¹, Howard RIEZMAN¹,
1Department Of Biochemistry, Univeristy Of Geneva

Objective: The control of membrane lipid homeostasis is an essential process that allows cells to maintain both their energetic balance and the structural integrity of their different membrane systems. Eukaryotic cells invest substantial resources in generating thousands of different lipids, so there must be evolutionary advantages that are dependent on a complex lipid repertoire. If most enzymes involved in lipid metabolism are now characterized, some aspects regarding their genetic control are still outstanding. The methodology used in this work allows the monitoring of lipid changes in cells using a large-scale RNAi screening of the human kinome combined with targeted lipidomic analysis by mass spectrometry. This strategy makes possible to observe precisely the function of many genes on a large range of lipids species and it is a very promising strategy to discover aspects of lipid metabolism that remain unknown. Statistical analysis of the screening highlights some genes whose knockdown induces changes in sphingolipid levels. Among them some members of the TRIM (tripartite motif), MAPK (mitogen-activated protein kinases) and BRD (bromodomain kinase) family proteins were found to induce changes in ceramide, glucosylceramide and/or sphingomyelin levels. Validations of these results are presented here, which would be the first step to understand the mechanism by which these proteins regulate sphingolipid metabolism.

P24: Sphingosine-1-phosphate lyase deficiency in the brain promotes cognitive deficits.

Daniel N. MITROI¹, Dan EHNINGER2, Julie SABA3, Konstantin GLEBOV¹, Markus GRÄLER4, Gerhild VAN ECHTEN-DECKERT¹,
1University Bonn, 2German Centre For Neurodegenerative Diseases (DZNE), 3University Of California San Francisco, 4Univesity Hospital Jena

Objective: Sphingosine‐1‐phosphate (S1P), a bioactive signaling molecule, has been shown to modulate a wide range of cellular processes including proliferation, differentiation, motility, cytoskeleton rearrangements and calcium homeostasis. It is generated by the phosphorylation of sphingosine, a catabolic intermediate of all sphingolipids. S1P is a rather short‐lived molecule that is either recycled back to sphingosine via dephosphorylation by S1P phosphatases (SPP1 and SPP2) or irreversibly cleaved by S1P lyase (S1PL). Systemic deletion of S1PL leads to a severe phenotype and early postnatal death. Since our main goal was to investigate the impact of S1PL in the central nervous system we generated a mouse model with neural‐restricted deletion of S1PL. The phenotype of these mice is rather unremarkable despite a considerable increase of sphingosine and of S1P that persists throughout life and a decrease of phosphatidylethanolamine (PE), detectable only in older animals. These changes in lipid composition of the brain were accompanied by biochemical and physiological changes including impaired autophagy and altered processing of the amyloid precursor protein (APP), increased proteasomal activity and decreased expression of presynaptic proteins. Finally, ablation of S1PL in neural tissue resulted in profound deficits of motor coordination, spatial learning and memory of the respective animals.

P25: Ligation of Gq-coupled GPCRs elicits phosphorylation independent membrane translocation of sphingosine kinase-1 in murine myoblasts.

Gennaro BRUNO¹, Kira BLANKENBACH2, Dagmar MEYER ZU HERINGDORF3, Paola BRUNI¹,
1Università Degli Studi Di Firenze, Dipartimento Di Scienze Biomediche Sperimentali E Cliniche, Italy, 2Institut Für Allgemeine Pharmakologie Und Toxikologie, Klinikum Der Goethe-Universität Frankfurt Am Main, Germany. 3Institut für Allgemeine Pharmakologie und Toxikologie, Klinikum der Goethe-Universität Frankfurt am Main, Germany

Objective: Sphingosine 1-phosphate (S1P) is synthesized by the enzyme sphingosine kinase (SphK)-1 and -2 isoforms. Regulation of the activity and intracellular localization of these enzymes is known to be crucial for all those biological processes which depend on the S1P signaling. It has been shown in HEK-293 cells that Gq-coupled receptors induce a rapid plasma membrane translocation of SphK1. Aim of the present study is to check if the translocation of SphK1 is a general mechanism related to Gq-coupled receptor activation, and if the phosphorylation of the enzyme is involved. We have performed studies in C2C12 myoblasts, a well recognized cellular model for studying physiological processes in vitro.
Methods: C2C12 cells were co-transfected with a vector encoding for SphK1 tagged with GFP (SphK1-GFP) or the phosphorylation-defective mutant of SphK1 (SphK1S225A -GFP), in the presence of different plasmids encoding for Gq-coupled GPCRs, separately. SphK1 translocation was analyzed using a confocal microscope (Zeiss LSM 510 Meta). Results: Activation of Gq-coupled GPCRs such as bradykinin B2, thromboxane TxA2, or histamine H1 induces a rapid translocation (translocation half life = 4-7s) of the SphK1 from cytosol to the plasma membrane. The translocation of the SphK1 following Gq-coupled receptor activation is an event independent from the phosphorylation of the enzyme, as proven by the observed translocation of the SphK1S225A -GFP following bradykinin treatment.
Conclusion: The Gq-mediated SphK1 translocation appears to be a general mechanism of rapid regulation of SphK1, as proven in HEK-293 and C2C12 cells; moreover, this event is independent from enzyme phosphorylation.

P26: Discovery of two potent and selective inhibitors of the neutral ceramidase.

Josefina CASAS¹, José Luís ABAD¹, Daniel CANALS¹, Yusuf H. HANNUN¹, Antonio DELGADO¹, Gemma FABRIAS¹,
1CSYC, 2STATE UNIVERSITY OF NEW YORK AT STONY BROOK, 3CSYC AND UNIVERSITY OF BARCELONA

Objective: Although neutral ceramidase (NC) has been reported to be a therapeutic target in colon cancer, potent and selective inhibitors have not been identified. The aim of this study was to discover NC inhibitors by high throughput screening (HTS) of several libraries.
Experimental Procedures: Ceramidase activities were determined using a fluorogenic HTS method[1] in 96 well-plates using the appropriate buffer, different concentrations of test compound and pure human neutral ceramidase (rhNC). Ceramide analysis in lipid extracts was performed by LC/MS.
Results: Two compounds able to inhibit NC were identified in one of the screened libraries. Kinetic studies showed that both compounds are non-competitive inhibitors with the substrate with Ki values of 0.77 and 2 μM. No inhibitory activity occurred for acid and alkaline ceramidases. When the effect of detergents was examined, we found that NC inhibition was dependent on the presence of sodium cholate in the reaction mixture with no inhibition occurring when other detergents were used (i. e. Triton X100). Preliminary studies in cell culture indicate that the two compounds provoke an accumulation of ceramides.
Conclusions: Two potent and selective inhibitors of NC have been discovered by library screening. They probably act by impairing the activation of NC by bile acids. [1] Bedia, C. et al. ChemBioChem 2007, 8, 642–648.

P27: Inhibition of dihydroceramide desaturase and autophagy induction in glioblastoma cell lines.

Yadira ORDONEZ¹, Mireia CASASAMPERE¹, Josefina CASAS¹, Gemma FABRIAS¹
1Csyc

Objective: The occurrence of a relationship between dihydroceramides (dhCer) increase and autophagy has been reported. The aim of this study is to determine the implication of dhCer in autophagy induced by the dihydroceramide desaturase (Des1) inhibitors gamma-tocotrienol (gTE), celecoxib (CCX), phenoxodiol (PXD) and resveratrol (RV) in glioblastoma cell lines.
Experimental Procedures: U87 and T98 cell lines were used. Cell viability (MTT or Sulforhodamine B) was determined 24 h after treatments. Autophagy (WB analysis of LC3-II) and sphingolipid composition (LC/MS) were measured in cells treated with the maximum non-toxic concentrations of compounds for 24 h. Gene silencing of Beclin 1 and acid sphingomyelinase (ASMase, SMPD1 gene) was achieved by transfection with siRNA using lipofectamine (24 h). Myriocin (5 μM, 24 h) was used to inhibit serine palmitoyltransferase.
Results: All compounds, but not gamma-tocopherol (gT), affected cell viability. gTE, CCX, PXD and RV induced autophagy, while gT did not. gTE, CCX, PXD and RV, but not gT, produced an accumulation of dhCer. Induction of LC3-II by the test compounds was decreased by both myriocin-treatment and knocking down of SMPD1. Beclin 1 silencing impaired autophagy induction by the test compounds without modifying dhCer levels.
Conclusion: Autophagy induction by the Des1 inhibitors gTE, CCX, PXD and RV in T98 and U87 cells involves both the de novo ceramide synthesis pathway and ASMase activity. The lack of effect of Beclin 1 silencing on dhCer levels supports that, in T98 and U87 cells, dhCer increase occurs before autophagy activation by gTE, CCX, PXD and RV.

P28: Modulation of acid sphingomyelinase in melanoma reprogrammes the tumor immune microenvironment.

Cristiana PERROTTA¹, Emma ASSI2, Davide CERVIA3, Laura BIZZOZERO4, Clara DE PALMA¹, Annalisa CAPOBIANCO5, Emilio CLEMENTI6
1Department Of Biomedical And Clinical Sciences “L. Sacco” University Of Milan, 2Division Of Molecular Oncology, San Raffaele Scientific Institute, Milan, 33Department For Innovation In Biological, Agro-food And Forest Systems (DIBAF), Università Della Tuscia,, 4Department Of Oncology, Università Di Torino And Laboratory Of Neurovascular Biology, Candiolo Cancer Institute,5Division Of Regenerative Medicine, San Raffaele Scientific Institute, Milan , 6Department Of Biomedical And Clinical Sciences “Luigi Sacco” University Of Milan

Objective: The inflammatory microenvironment induces tumors to acquire an aggressive and immunosuppressive behavior. Since acid sphingomyelinase (A-SMase) downregulation in melanoma was shown to determine a malignant phenotype, we aimed here to elucidate the role of A-SMase in the regulation of tumor immunogenic microenvironment.
Methods: We analysed two in vivo murine melanoma models in which A-SMase was either downregulated or maintained at constitutively high levels. In order to investigate the dependence of immune cell infiltration by A-SMase expression in melanoma we analysed by flow cytometry, the following cell populations: myeloid-derived suppressor cells (MDSCs), CD8+ and CD4+ T lymphocytes, regulatory T lymphocytes (Tregs) and dentritic cells (DCs).
Results: We found high levels of inflammatory factors in low A-SMase expressing tumors which also displayed an immunosuppressive/pro-tumoral microenvironment: high levels of MDSCs and Tregs, and low levels of DCs. In contrast, the restoration of A-SMase in melanoma cells not only reduced tumor growth and immunosuppression, but induced an high recruitment at tumor site of effector immune cells with an anti-tumoral function. Indeed, we observed a poor homing of MDSCs and Tregs and the increased recruitment of CD8+ and CD4+ T lymphocytes as well as the infiltration of DCs and CD8+/CD44high T lymphocytes.
Conclusions: This study demonstrates that changes of A-SMase expression in cancer cells is sufficient per se to tune in vivo melanoma growth and that A-SMase levels modulate immune cells at tumor site. This A-SMase-dependent modulation of microenvironment events may have a therapeutic outcome in terms of tumor growth.

P29: Ceramide in CF lung infection and inflammation: complex analyses for enigmatic sphingolipids.

Anna CARETTI¹, Michele VASSO2, Josefina CASAS3, Gemma FABRIAS3, Alessandra BRAGONZI4, GASCO5, Ceciclia GELFI6, Riccardo GHIDONI¹, Paola SIGNORELLI¹
1University Of Milan, 22Lita Institute, Segrate, University Of Milan, 34Research Unit On Bioactive Molecules, Department Of Biomedicinal Chemistry, Catalan Institute Of Advanced Chemistry (IQAC/CSIC), Barcelona, Spain, 4 Raffaele Scientific Institute, Milan, Italy, 5Nanovector, Turin , Italy, 6Lita Institute, Segrate, University Of Milan

Objective: The sphingolipid ceramide is a known inflammatory mediator and its accumulation in lung inflammation has been reported in different models of emphysema, Chronic Obstructive Pulmonary disease and Cystic Fibrosis (CF). This latter is caused by mutation of chloride channel and associated with hyper-inflammation of respiratory airways and with high susceptibility to un-resolving infections. We previously demonstrated that ceramide de novo synthesis is enhanced in lung inflammation and sustains P.aeruginosa pulmonary infection in a CF murine model (Caretti et al. BBA 2014). Our present aim was to take advantage of quantitative and qualitative analyses to obtain further insight in the role of ceramide in CF. We concluded that the integrated use of Liquid Chromatography and MALDI Imaging coupled to Mass Spectrometry, Confocal Laser Scan Microscopy, histology analyses, is able to unmask information that is not decipherable by any single approach: i) ceramide up regulated synthesis in the alveoli is strictly related to alveolar infection and inflammation; ii) alveolar ceramide can be specifically targeted by nanocarriers delivery of the ceramide synthesis inhibitor Myr; iii) Myr is able to down-modulate pro-inflammatory Lyso-PC, favoring the increase of the anti-inflammatory PCs.

P30: Ceramide and inflammation in labor.

Paola SIGNORELLI¹, Laura AVAGLIANO¹, Nadia TOPPI¹, Gemma FABRIAS2, Fina CASAS2, REFORGIATO¹, Anna CARETTI¹
1University Of Milan, 24Research Unit On Bioactive Molecules, Department Of Biomedicinal Chemistry, Catalan Institute Of Advanced Chemistry (IQAC/CSIC),Barcelona, Spain

Objective: Gestation is a unique mechanism involving a pro-inflammatory phase that allows blastocyst implanting into the uterus, a second anti-inflammatory phase allowing fetus growth and a final acute inflammatory phase that triggers contraction of the uterus and parturition. The comprehension of the regulation of inflammatory switches represents an important acquisition for the identification of therapeutic targets in pregnancy defects. Sphingolipids are a broad class of structural membrane components endowed with important signaling activities. Among sphingolipids, ceramide is a well-known mediator of stress signals and pro-inflammatory responses. Ceramide is a known activator of NF-kB and this transcription factor controls the expression of many labor-associated genes. Placenta accumulation of ceramide was shown to be responsible for PGE2 induction in acute inflammation of labor and parturition. Our group recently demonstrated that de novo synthesis of ceramide is not only enhanced in acute lung inflammation but also supports it and that. pharmacological inhibition of this activity down regulates inflammatory response. In this project we evaluated the content of ceramide (by LC-MS analysis) and the expression of Serine Palmytoil Transferase (by Western blotting and RT-PCR techniques) in placenta from cesarean delivery versus spontaneous or induced parturition. We observed a significant co-relation between ceramide increased synthesis and inflammation. These observations are particularly interesting when considering that fast labor is desirable for both mother and fetus and that pre-term labor is initiated by inflammatory cascades. Thus ceramide metabolism can be implicated in controlling important process and may become a therapeutic target in parturition complications.

P31: The oncogene GOLPH3 at the crossroad between sphingolipid metabolism, membrane trafficking, and DNA damage.

Riccardo RIZZO¹, Daniela MONTARIELLO¹, Domenico SUPINO¹, Gaelle BONCOMPAIN2, Serena CAPASSO¹, Gabriele TURACCHIO¹, Seetharaman PARASHURAMAN¹, Frank PEREZ2, Giovanni D’ANGELO¹, Alberto LUINI¹
1Institute Of Protein Biochemistry, National Research Council Of Italy, Naples, 2Institut Curie, Paris France

Objective: The GOLPH3 (Golgi phosphoprotein 3) gene is located in a human chromosome region (5p13), which is frequently amplified in several solid tumours. The precise molecular mechanism of GOLPH3 mediated oncogenesis is so far unknown, but its role in the positive control of cell proliferation via the Akt/mTOR pathway, is widely accepted. In mammalian cells GOLPH3 has been shown to contribute to vesicular trafficking and Golgi architecture, retention of Golgi enzymes and response to DNA Damage. The yeast homologue of GOLPH3, named VPS74, has essential roles in maintaining the localisation of some Golgi enzymes and in the regulation of sphingolipid homeostasis. Manipulating the expression levels of GOLPH3 in mammalian cells, we found that GOLPH3 positively regulates glycosphingolipid (GSL) synthesis by controlling the localization and stability of GSL synthesizing enzymes. Specifically we found that GOLPH3 interacts with a specific glycosphingolipid (GSL) metabolic enzyme and regulates its sub-cellular localization counteracting lysosomal degradation. The change in sphingolipid metabolism induced by the manipulation of GOLPH3 levels has an impact on p53 activation and DNA damage response following genotoxic insults. Our data suggest that GOLPH3 oncogenic activity involves the regulation of the key cancer modulator p53 through the control of sphingolipid metabolism

P32: Inhibition of ceramide synthesis as post-ischemic therapy for myocardial reperfusion injury.

Marta REFORGIATO¹, Giuseppina MILANO2, Josephina CASAS3, Gemma FABRIAS3, Paolo GASCO4, CAMPISI¹, Michele SAMAJA¹, Riccardo GHIDONI¹, Anna CARETTI¹, Paola SIGNORELLI¹
1University Of Milan, 2University Hospital Centre Vaudois (chuv), Service De Chirurgie Cardio-vasculaire (ccv), Lausanne, Switzerland, 3Cardiovascular Research Center (csyc-yccc), Biomedicinal Chemistry, Barcelona, Spain, 4Nanovector, Turin, Italy

Objective: Therapeutical interventions aimed at reducing post-ischemic injury may have enormous potential to improve short and long-term morbidity and mortality. Besides an array of collateral effects, reperfusion after ischemia triggers a pathological inflammatory reaction caused by several mediators including lipotoxins such as the sphingolipid ceramide. Ischemia-reperfusion (I/R) injury was shown to increase myocardial ceramide content (Beresewicz A. 2002) and pharmacological inhibition of ceramide formation to ameliorate cardiac dysfunction (Gundewar S. 2008). We recently proved that pharmacological inhibition of ceramide synthesis significantly reduces acute inflammation in lung infection (Caretti A. BBA 2014). Our present aim was to evaluate the therapeutic potential of ceramide synthesis inhibition in I/R myocardial injury in mice. After 30 minutes of left anterior descending coronary artery ligation (LAD), we performed intra-myocardial injection of the ceramide synthesis inhibitor just at the beginning of 3 hours reperfusion. The treatment reduced infarct size (36% decrease), decreased ceramide content, expression of inflammatory cytokines, formation of hydroperoxides within the risk area. Finally, the treatment enhanced Nrf2 activated transcription of HO1. We concluded that inhibition of I/R induced accumulation of the stress lipid mediator ceramide during reperfusion of infarcted myocardium allows i) a decrease in tissue infarct, ii) a significant reduction in inflammatory and oxidative factors production, iii) enhanced myocardial pro-survival adaptive response.

P33: Activity of neutral and alkaline ceramidases on fluorogenic N-acylated coumarine-containing aminodiols.

Mireia CASASAMPERE¹, Luz CAMACHO¹, Francesca CINGOLANI¹, José Luís ABAD¹, Carmen BEDIA¹, Ruijuan XU2, Kai WANG2, Cungui MAO2, Daniel CANALS2, Yusuf HANNUN2
1Csyc, 2State University Of New York At Stony Brook

Objectıve: In previous articles[1,2] we reported on the use of compounds RBM14, coumarinic analogues of ceramide with N-acyl chains of different length (RBM14-Cn), to determine acid ceramidase (AC) activity. Here we aim at determining the ability of other ceramidases to hydrolyze these substrates.
Experımental Procedures: Ceramidase activities were determined as reported[1,2] in 96 well-plates using the appropriate buffer for each ceramidase, different concentrations of test substrate and either recombinant bacterial or human neutral ceramidase (rhNC) or microsomes from cell lysates.
Results: In rhNC, the substrate affinity increases directly with increasing the N-acyl chain length; RBM14-C16 is the best substrate. All the RBM14 substrates, mainly RBM14-C14 and RBM14-C16, were hydrolyzed in both intact mouse embryonic fibroblasts defective in the ASAH2 gene and lysed cells at basic pH, suggesting that the compounds were hydrolyzed by at least one alkaline ceramidase (ACER). Lower fluorescence levels were released from RBM14-C12, RBM14-C14 and RBM14-C16 in microsomes from stable ACER3 knockdown HCT 116 cells than from their controls. However, microsomes from both HeLa-based cell lines ACER1-TET-ON and ACER2-TET-ON cultured in the presence of tetracycline had no activity on any RBM14 substrate.
Conclusıons: Compounds RBM14 are hydrolyzed by NC and ACER3, but not by ACER1 and ACER2. [1] Bedia, C. et al. ChemBioChem 2007, 8, 642–648. [2] Bedia, C. et al. J Lipid Res 2010, 51, 3542–3547.

P34: Regulation of vascular calcification by bioactive sphingolipids .

Thomas G MORRIS¹, Christopher J CLARKE2, Yusuf A HANNUN2, Vasken OHANIAN¹, Ann E CANFIELD¹, Jacqueline OHANIAN¹,
1University Of Manchester, 2Stony Brook University

Objective: Vascular calcification (VC) is associated with cardiovascular mortality and involves the osteogenic differentiation of vascular smooth muscle cells (VSMCs) and matrix mineralisation. It has similarities to bone formation, a process in which ceramide and sphingosine-1-phosphate (S1P) are implicated. Ceramide can be generated by lysosomal acid-sphingomyelinase (L-SMase) and be converted to sphingosine and S1P by acid ceramidase (ACDase) and sphingosine kinases (SK) respectively. We investigated whether ceramide and S1P regulate VC. VSMCs were cultured with β-glycerophosphate to induce differentiation and mineralisation. Controls were cultured without β-glycerophosphate. Changes in L-SMase and SK activity were determined using an in vitro assay and mRNA levels with Q-PCR. Ceramide, sphingosine and S1P were measured by mass spectroscopy. Inhibition of L-SMase and ACDase was achieved using desipramine (1-10 μM). When mineralisation was widespread; C18 and C20-ceramide increased 75%, sphingosine decreased 33%, S1P increased three-fold, L-SMase activity decreased 30% but mRNA levels were unchanged, SK activity increased 66%, SK2 mRNA increased but SK1 was unchanged; all compared to control VSMCs. Desipramine dose-dependently inhibited mineralisation, increased total ceramide two-fold, decreased sphingosine 33% and prevented the increase of S1P, when compared to VSMCs cultured with β-glycerophosphate alone. Exogenous C2-ceramide decreased, whereas S1P increased, mineralisation. All changes significant, n=3 (p<0.05). In conclusion, SK activity and S1P levels are increased during VSMC mineralisation. Inhibition of L-SMase/ACDase leads to ceramide accumulation, prevents S1P production and attenuates mineralisation; identifying ceramide as an inhibitor and S1P as a promoter of matrix mineralisation. These findings identify sphingolipids as potential therapeutic targets for the treatment of VC.

P35: Effective synthesis and testing of ceramide analogues with antichlamydial activity.

Essa M. SAIED¹, Christoph ARENZ¹,
1 Institute For Chemistry, Humboldt Universität Zu Berlin

Objective: There is ample evidence for a role of sphingolipids in regulation of cellular signaling and for a therapeutic potential of sphingolipid inhibitors in the treatment of human disease. The quest for highly potent mediators of sphingolipid metabolism not only relies on the development of efficient synthetic routes but also on the discovery of homogenous high-throughput-screening approaches. In this study, we present two highly efficient and versatile approaches for the synthesis of ceramide analogues with anti-chlamydial activity. Parallely, we have developed robust, fast, and simple assays for assessment of biological activity in vivo and in vitro. In one project we have identified a number of ceramide analogues as a novel class of anti-Chlamydia compound that efficiently inhibits C. trachomatis growth without affecting the viability of the host cell. The potency of some compounds was higher than that of the sphingomyelin synthase inhibitor D609 or that of the antibiotic chloramphenicol. A notable result of our study is the identification of the first sub-micromolar inhibitors of chlamydial growth. Although the molecular targets of this compound class is yet unknown, the potency of the compounds, as well as the acquired knowledge about structure-activity relationship study of this compound class defines an important step for target identification soon .

P36: Structural and functional studies of neutral sphingomyelinase 2 provide new insight into regulation and inhibition.

Michael AIROLA¹, Prajna SHANBHOGUE¹, Achraf SHAMSEDDINE¹, Kip GUJA¹, Rohan MAINI¹, Nana BARTKE2, Chris CLARKE¹, Bill WU2, Miguel GARCIA-DIAZ¹, Yusuf HANNUN¹
1Stony Brook University, 2MUSC

Objective: Neutral sphingomyelinase 2 (nSMase2) hydrolyzes the relatively insert signaling lipid sphingomyelin to generate the bioactive lipid ceramide. NSMase2-derived ceramide has emerged as a critical regulator of exosome-mediated intercellular communication and has been proposed as a potential therapeutic target for breast cancer and Alzheimer’s disease. To illustrate the mechanism of ceramide generation and aid in therapeutic development, we have conducted a detailed structural and functional analysis of nSMase2. We present the 2.0 Å crystal structure of the catalytic domain of human nSMase2 that provides insight into the critical residues involved in catalysis and sphingomyelin recognition. In addition, we delineate a key mechanistic step in modulating nSMase2 activity that mediates activation by anionic phospholipids and appears to be the target of the widely used, non-competitive nSMase2-specific inhibitor GW4869. Overall, this work provides fundamental insight into the action of this novel enzyme that may facilitate development of more potent nSMase2-specific inhibitors.

P37: Reverse modulation of Gq-coupled receptor-induced [Ca2+]i increases and sphingosine kinase-1 translocation by a WD repeat protein.

Kira BLANKENBACH¹, Gennaro BRUNO2, Hans VIENKEN¹, Paola BRUNI2, Dagmar MEYER ZU HERINGDORF¹,
1Institut Für Allgemeine Pharmakologie Und Toxikologie, Klinikum Der Goethe-Universität Frankfurt Am Main, Germany, 2Dipartimento Di Scienze Biomediche, Sperimentali E Cliniche, Università Degli Studi Di Firenze, Italy,

Objective: Sphingosine kinases (SphK), which catalyze the formation of sphingosine-1-phosphate, are activated by diverse signaling pathways according to the wide variety of their roles. We have shown before that Gq-coupled receptors induce a rapid plasma membrane translocation of SphK1, independently of phospholipase C (PLC) downstream signaling such as protein kinase C and [Ca2+]i. Recently, the WD repeat protein, WDR36, has been identified as a scaffold promoting the interaction of the thromboxane A2 receptor-β (TPβ), Gαq and PLCβ, thereby enhancing TPβ-induced PLC activation. Aim of the present study was to analyze the influence of WDR36 on Gq-mediated SphK1 translocation.
Methods: SphK1 translocation was analyzed by confocal laser scanning microscopy using a Zeiss LSM 510 Meta microscope. [Ca2+]i increases were analyzed in single cells using a Till Photonics Calcium Imaging system.
Results: [Ca2+]i increases induced by a maximal stimulation of the M3 muscarinic acetylcholine receptor stably expressed in HEK-293 cells were significantly enhanced in cells overexpressing GFP-WDR36 compared to vector-transfected cells. In contrast, M3 receptor-induced SphK1 translocation was significantly delayed by overexpression of WDR36. This was shown in three series of experiments with 1. non-tagged WDR36 and GFP-SphK1, 2. GFP-tagged WDR36 and CFP-SphK1 and 3. GFP-tagged WDR36 and mCherry-SphK1. Furthermore, bradykinin B2 receptor-induced CFP-SphK1 translocation was significantly delayed by GFP-WDR36 in C2C12 myoblasts.
Conclusion: We conclude that WDR36 may function as a molecular switch promoting the coupling of Gq to PLC-mediated [Ca2+]i increases while impeding Gq-mediated SphK1 targeting. The data support the hypothesis that SphK1 and PLC are independent effectors of Gαq.

P38: Sphingosine inhibits no-mediated vasorelaxation of mouse thoracic aorta via binding to calmodulin.

Tünde JUHASZ¹, Eva RUISANCHEZ2, Veronika HARMAT3, Jozsef KARDOS4, Monika KABAI¹, Zoltan BENYO2, Karoly LILIOM¹
1Institute Of Enzymology, Research Centre For Natural Sciences, Hungarian Academy Of Sciences, Budapest, 2Instutite Of Human Physiology And Clinical Experimental Research, Semmelweis University, Budapest, 3Laboratory Of Structural Chemistry And Biology, Institute Of Chemistry, Eötvös Loránd University, Budapest, 4Department Of Biochemistry, Institute Of Biology, Eötvös Loránd University, Budapest

Objective: Calmodulin-dependent endothelial nitric oxide synthase (eNOS) is a main determinant of the vascular tone and permeability. The sphingolipid mediator sphingosine is suggested to have vasoactive properties. We investigated the effect and mechanism of action of sphingosine on the NO-dependent vasorelaxation.
Methods: To test the ability of sphingosine to alter eNOS activity in intact vessels, myography experiments were performed on mouse thoracic aorta ex vivo. We compared the acetylcholine-induced eNOS-mediated relaxation before and during incubation of the vessels with sphingosine. The binding of sphingosine to calmodulin and its effect on calmodulin’s function in vitro were characterized by fluorescence spectroscopy, isothermal titration calorimetry, and crystallography.
Results: Treatment of the intact vessels with sphingosine induced significant rightward shift of the acetylcholine dose-response curve. Sphingosine exhibited no significant effect when vasodilation was elicited by the direct NO-donor sodium nitroprusside. We characterized in vitro the inhibition by sphingosine of calmodulin-dependent activity of eNOS, phosphodiesterase, and calcineurin. Calmodulin was found to bind sphingosine in vitro with low nanomolar affinity when the lipid was clustered. The crystal structure of their complex showed a few lipid molecules wrapped around by the protein in its inhibitory conformation.
Conclusions: No under physiologic conditions is a vasorelaxant and attenuates inflammation, while its increased production leads to the initiation and progression of inflammation. We have shown here, that the vascular effects of sphingosine in inflammation might be due to its influence on the activity of Ca2+-calmodulin-dependent eNOS enzyme via binding to calmodulin thereby preventing eNOS activation.

P39: Sphingolipidomics in gangliosidoses and metachromatic leukodystrophy: identification of differentiated sphingolipids.

Adem ÖZKAN¹, Funda YILDIRIM2, Mehmet KAYILI3, Ömür ÇELIKBIÇAK3, Erdal COŞGUN¹, Fatma Müjgan SÖNMEZ4, Meral TOPÇU¹, Bekir SALIH3, Hatice Asuman ÖZKARA¹
1Hacettepe University Faculty Of Medicine, 2Hacettepe University Faculty Of Science, 3Hacettepe University Faculty Of Science, 4Fatih University Faculty Of Medicine

Objective: Sphingolipids are ubiquitous elements of the plasma membrane. They are synthesized in ER and Golgi. Their degradation are made by lysosomal enzymes in lysosome. Defective lysosomal enzymes cause lysosomal storage diseases. Accumulation of undegraded substrates in the nervous system leads to severe impairment of neurological function with a fatal outcome. The aim of this study is to identify sphingolipid/s for predicting clinical phenotype and rate of disease progression. In this study, sphingolipids were extracted from serums of patients with gangliosidoses and Metachromatic Leukodystrophy which are the most common sphingolipid storage diseases in Turkey. Their mass spectrums are taken by MALDI-ToF-MS. Sphingolipids that make difference between groups and clinical findings were determined by cluster analysis. Internal standards were used for quantitative analysis by LC-ESI-MS/MS. After evaluation by cluster analysis of the obtained sphingolipid profiles, sphingosine and sphinganine 1-P in gangliosidoses group were found as differentiated molecules. Results of quantitative analysis indicated increased ceramide and ceramide 1-P levels in gangliosidoses group. Our preliminary data supports sphingolipidomes of the serum may show difference between diseases, difference between diseases and the controls in sphingolipid storage diseases. MALDI-ToF-MS and LC-ESI-MS/MS are appropriate methods for detection of this differences.

P40: The sphingolipid rheostat is associated with function and viability of dendritic cells.

Anja SCHWIEBS¹, Olga ARLT¹, Nerea FERREIRÓS BOUZAS2, Yannıck SCHREIBER2, Lukasz JAPTOK3, Burkhard KLEUSER3, JOsef M PFEILSCHIFTER¹, Heınfrıed H RADEKE¹
1Unyversyti Of Frankfurt, Instytute Of General Pharmacologi And Toxycologi, 2Unyversyti Of Frankfurt, Instytute Of Clynycal Pharmacologi And Toxycologi, 3Unyversyti Of Potsdam, Instytute Of Nutrytyonal Scyence

Objective: Regulation of the complex functions of the immune system has excitingly been proven to be sensitive to alterations in the sphingosine-1-phosphate (S1P) receptor-ligand interaction. Dendritic cells (DCs) mediate immune responses in inflammation, cancerogenesis and autoimmunity, however it is unclear whether DC-activation, function and life cycle are affected by the sphingolipid rheostat. Thus, here we investigated primary bone marrow-derived DCs upon toll-like-receptor (TLR) stimulation by RNA-, protein- and activity-measurements as well as LC-MS. Shortly upon stimulation of distinct TLRs, we found a rapid induction of IL12 secretion. In parallel we observed a transient increase in Sphingosine kinase (SK) 1 expression and total SK activity. However, upon long-term TLR-stimulation a much stronger up-regulation of SK1 mRNA levels was present in parallel to a down-regulation of SK2 levels. Interestingly, the absence of SK1 or SK2 in knockout DCs strongly augmented IL-12 induction but was dampened upon exogenous S1P supplementation. In addition, a pronounced decrease of mRNA, protein levels and enzyme activity of S1P-lyase was monitored in a time-dependent manner. Moreover, we discovered the induction of an intrinsic apoptotic pathway indicated by annexin V and caspase 3-activation and a time-dependent decrease of intracellular S1P and pronounced increase of sphingosine and ceramide levels in activated, not in resting DCs. Obviously, the ability of DCs to modulate immune responses by secreting specific cytokines but also determined by their balance between survival or cell death, is associated with a highly diverse regulation of S1P-enzyme expression and thus complex modulation of the sphingolipid rheostat.

P41: regulation of ceramide-mediated mitophagy by hpv oncoproteins and Rb/E2F axis in head and neck cancer.

Raquela THOMAS¹, Besim OGRETMEN¹
1Medical University Of South Carolina

Background: The five-year survival rate of Head and Neck Squamous Cell Carcinoma (HNSCC) is only 50% due, in part, to a lack of effective, targeted therapies. Human papillomavirus (HPV) is the causative agent of approximately 60% of HNSCC cases. Cellular transformation is achieved by the inhibitory actions of HPV E6 and E7 oncoproteins on p53 and Rb, respectively. Interestingly, HPV-associated HNSCC patients respond better to treatment, demonstrating a 54% higher survival rate compared to HPV-negative HNSCC patients. However, the underlying molecular mechanism for this has not been well-defined. We have identified a novel form of lethal mitophagy that is mediated by ceramide and dependent on DRP1 signaling as an important mechanism of cell death in HNSCC.
Objective: As no studies have examined sphingolipid signaling or mitophagy in HPV-associated HNSCC, we asked whether this form of mitophagy may be involved in the improved response to treatment. Methods: HPV-positive or HPV-negative HNSCC cells were treated with radiation or C18-pyridinium-ceramide. Overexpression or knockdown of relevant genes was performed then cell number, mitophagy, and binding assays performed.
Results: Radiation of HPV-positive cells resulted in increased levels of CerS1/ C18-ceramide. CerS1 overexpression or C18-pyridinium-ceramide treatment resulted in lethal mitophagy, dependent on DRP1 oligomerization. This response was attenuated by knockdown of E2F5 and enhanced by Rb knockdown. Additionally, E2F5/DRP1 association was detected in response to ceramide.
Conclusion: We have identified ceramide-mediated lethal mitophagy as an important cell death mechanism in HPV-associated HNSCC, regulated by the E7/Rb/E2F5 axis.

P42: Insights into the effects of the mutations of arylsulfatase A on its structure using molecular dynamic simulations.

Ayşe EREN¹, Maral BUDAK2, Asuman ÖZKARA3, Kutlu ÜLGEN¹, Elif ÖZKIRIMLI¹
1Department Of Chemical Engineering, Bogazici University, 34342 Bebek, Istanbul, Turkey, 2Department Of Molecular Biology And Genetics, Bogazici University, 34342 Bebek, Istanbul, Turkey,3Department Of Medical Biochemistry, Faculty Of Medicine, Hacettepe University, Ankara, Turkey

Objective: Arylsulfatase A (ASA) is a lysosomal enzyme catalyzing the hydrolysis of sulfate ester bonds. Its major substrate is cerebroside-3 sulphate, and the product of the reaction, cerebroside, is the major constituent of myelin sheats. In case of the deficiency of this enzyme, demyelination of neurons occurs causing Metachromatic Leukodystrophy Disease (MLD). In this work, we focus on the effect of previously identified mutations on the activity and functionality of ASA [1]. We first checked for conservation of these residues, which are away from the active site, by comparison with their homologs and no significant conservation was found. Next, calculation of the free energies of the wild type and mutated types of ASA using FoldX showed that the mutant form has higher energy in the monomeric, dimeric and octameric forms. Then, the W318C mutant of ASA was generated using VMD and molecular dynamics (MD) simulations of 10 ns were performed for monomers at pH~7. The results of these MD simulations and the free energy calculations will be discussed. It is predicted that these mutations decrease the enzyme stability, which would result in loss of enzyme activity. In order to test this hypothesis, MD simulations for both pH values (5 and 7) of the wild type and mutants are performed. [1] Önder,E.,Sinici,I.,Sönmez,M.,Topçu,M.,Özkara,H.A.(2009) Identification of two noval arylsulfatase A mutations with a polymorphism as a cause of metachromatic leukodystrophy, in Neurological Research 31,pp 60-66

P43: Investigating the role of ORMDL proteins as regulators of the mammalian serine palmitoyltransferase.

Assem ZHAKUPOVA¹, Arnold VON ECKARDSTEIN¹, Thorsten HORNEMANN¹
1Institute Of Clinical Chemistry, University Hospital Zurich

Objective: Serine Palmitoyltransferase (SPT) typically catalyzes the first step in de novo sphingolipid synthesis. Studies in yeast demonstrated that SPT activity is tightly regulated by a metabolic feedback mechanism, mediated by Orm1 and Orm2 proteins. In contrast to yeast, mammalian cells express three Orm isoforms (ORMDL1-3). However, the role of ORMDL proteins in regulating SPT activity in mammalian cells is not yet understood. Several SNP variants in ORMDL3 were found to be closely associated with the risk for early childhood asthma. A role of SPT and de-novo sphingolipid metabolism in asthma is discussed. Methods. The role of ORMDL proteins on SPT activity was tested in HEK293 cells individually overexpressing ORMDL1, 2 or 3 and in MEF cells which are devoid of ORMDL3 expression. Furthermore we analyzed the association of ORMDL SNPs and plasma sphingoid base levels in a comprehensive population based cohort with 1200 individuals. Results. Neither the individual overexpression of ORMDL proteins nor the absence of ORMDL3 had a significant effect cellular SPT activity. None of the annotated SNPs in ORMDL genes including those that were reported to be associated with childhood asthma showed a significant association with plasma sphingoid base levels. However, increasing intracellular Ceramide levels by the addition of C6-Ceramide led to a significant suppression of SPT. Conclusion. Increasing levels of C6-Ceramides inhibit SPT activity, which indicates a regulatory mechanism of the mammalian SPT similar to yeast. However, a role of individual ORMDL isoforms including ORMDL3 in this regulatory process could not be confirmed.

P44: The use of hair samples to monitor 1-deoxy-sphingolipids in HSAN1.

Thorsten HORNEMANN¹, Heiko BODE¹, Saranya SURIYANARAYANAN¹
1University Zurich

Background: 1-Deoxysphingolipids (1-deoxySL) are formed by the enzyme serine palmitoyltransferase due to an alternative use of L-alanine over its canonical substrate L-serine. Pathologically elevated 1-deoxSL levels are formed in the context of the inherited neuropathy HSAN1 which is caused by missense mutations in SPT. HSAN1 is a progressive ulcerating axonopathy associated with a progressive peripheral sensory loss (pain, temperature, vibration), neuropathic pain and ulcers (slow healing wounds). Often, HSAN1 patients suffer also from anhydrosis, reduced hair growth and a fine and delicate skin structure. An oral L-serine supplementation was shown to suppress 1-deoxySL formation in transgenic HSAN1 mice and HSAN1 patients. Patients under L-serine therapy reported consistently improvements in skin structure, wound healing and a faster growth of hair and finger nails.
Method: The sphingoid base profile was analyzed in hair samples from healthy controls and HSAN1 patients before and during therapy. The extracted lipids were analyzed by high resolution mass spectrometry.
Results: We observed a variety of sphingoid bases including 1-deoxySL present in the analyzed hair sample. Canonical sphingoid bases were comparable between control and HSAN1 samples whereas 1-deoxySLs were significantly higher in HSAN1^samples. After extraction the HSAN1 hair appeared more brittle and showed structural abnormalities. Longitudinal, 1-deoxySL levels correlated with the history of L-serine supplementation in the patient.
Conclusion: Significant 1-deoxySL levels were present in hair of HSAN1 patients. The longitudinal profile reflected changes in 1-deoxySL production over time. Hair samples might therefore be a non-invasive alternative for the diagnosis and therapy monitoring in HSAN1.

P45: Glucosylceramide as a modulator of the properties of lipid raft-like membrane domains.

Ana Raquel PINTO VARELA¹, André SÁ COUTO2, Anthony FUTERMAN3, Manuel PRIETO4, Liana C. SILVA2,
1Imed. Ulisboa –research Institute For Medicines, Faculty Of Pharmacy, Universidade De Lisboa, Av. Professor Gama Pinto, 1649-003 Lisbon, Portugal; Department Of Biological Chemistry, Weizmann Institute Of Science, Rehovot 76100, Israel; Centro De Química-física Molecular & In – Institute Of Nanoscience And Nanotechnology, Instituto Superior Técnico, Universidade De Lisboa, Lisboa, Av. Rovisco Pais, 1049-001 Lisbon, Portugal, 2Ymed. Ulisboa –research Institute For Medicines, Faculty Of Pharmacy, Universidade De Lisboa, Av. Professor Gama Pinto, 1649-003 Lisbon, Portugal, 3Department Of Biological Chemistry, Weizmann Institute Of Science, Rehovot 76100, Israel, 4Centro De Química-física Molecular & In – Institute Of Nanoscience And Nanotechnology, Instituto Superior Técnico, Universidade De Lisboa, Lisboa, Av. Rovisco Pais, 1049-001 Lisbon, Portugal

Objective: Glucosylceramide (GlcCer) is a glycosphingolipid involved in different physiological and pathological events. It is widely accepted that GlcCer is an active participant in lipid domain formation, particularly in lipid rafts. However, the interplay between GlcCer and lipid raft components is still poorly studied. In the present study, we used fluorescence spectroscopy and microscopy to investigate the interaction between GlcCer and cholesterol (Chol) in models of biological membranes. To better understand the interplay between these lipids, the results were rationalized by a ternary phospholipid/sphingolipid/chol phase diagram. Our results showed that ternary POPC/GlcCer/Chol mixtures containing low GlcCer content display properties typical of raft-domains, i.e., a liquid-ordered (lo)/liquid-disordered (ld) phase coexistence. However, compared to the canonical POPC/Sphingomyelin/Chol raft-like mixtures, this phase coexistence region is smaller, mainly due to the higher ability of GlcCer to segregate into gel-enriched domains. This results in an extensive 3-phase coexistence region of ld(POPC-enriched)/ lo(Chol-enriched) and gel(GlcCer-enriched). Moreover, the effect of acidification in the properties of these mixtures, was also analyzed. Our data showed that upon acidification GlcCer solubility in the lo phase increases, leading to a larger raft-like coexistence region. Quantitative analyses of the data allowed determination of the phase’s composition at neutral and acidic pH. These results enable to predict GlcCer impact in domain formation and membrane organization in complex biological membranes, and provide background to unravel the role of GlcCer-biophysical properties in its biological action. Acknowledgments: This work was supported by Fundação para a Ciência e Tecnologia (FCT), Portugal: PTDC/BBB-BQB/0506/2012, SFRH/BD/69982/2010 to ARV and Investigador FCT 2014 to L.C.S.

P46: Funcional lipidomics during oriented mitosis in zebrafish.

Thomas HANNICH¹, Howard RIEZMAN¹, Marcos GONZALEZ-GAITAN¹, Irinka CASTANON¹
Biochemistry/University Of Geneva1

Objective: In zebrafish, dorsal epiblast cells are an excellent system to study oriented cell division in a developing tissue. Throughout gastrulation, these cells display divisions that are robustly oriented along the anterior-posterior axis. In these cells, division orientation is under the control of the Wnt/Planar Cell Polarity (PCP) pathway. How the PCP pathway specifies the division plane had been elusive until recently, when we found that the PCP pathway induces the formation of an asymmetrically polarized actin cap. This cortical actin cap triggers the spatial redistribution of the transmembrane protein Anthrax Toxin Receptor 2a (Antxr2a), which becomes enriched at the actin cap and is required to exert torque on the spindle to position it along the embryonic axis. Using a novel interdisciplinary approach combining system-level lipid analysis, high-resolution imaging, and quantitative cellular biology, we found that the PCP pathway controls oriented divisions by regulating lipid homoestasis. We have identified specific lipid species that are modified in Wnt/PCP mutants, where divisions are randomized. Indeed, interfering with the first step in de novo sphingolipid synthesis results in randomized divisions of epiblast cells. In these embryos, epiblast cells still exhibit a normal, asymmetrically polarized actin cap, whereas the asymmetric distribution of Antxr2a is lost. By imaging membrane-order sensing dyes in epiblast cells using fluorescence lifetime imaging microscopy (FLIM), we will evaluate whether epiblast cell membranes have a homogenous lipid order or are a mixture of different lipid-order microenvironments. We will investigate whether the existence and abundance of ordered lipid domains modulate Antxr2a behaviour.

P47: Hsp70 based treatment of lysosomal storage disorders.

Thomas KIRKEGAARD JENSEN¹, Nikolaj H. T. PETERSEN¹
1Orphazyme ApS

Objective: Lysosomal storage diseases (LSDs) often manifest with severe systemic and central nervous system (CNS) symptoms. The existing treatment options are sparse and none of them are effective against the devastating neurological manifestations. We have demonstrated proof-of-concept for heat shock protein 70 (Hsp70)-based strategies as potential pan-LSD therapies. Recombinant Hsp70 improves the binding of several sphingolipid-degrading enzymes to their essential co-factor, bis(monoacylglycero)phosphate, in vitro and reverts lysosomal pathology in primary fibroblasts from 14 patients with eight different LSDs. It penetrates effectively to murine tissues including CNS, inhibits glycosphingolipid accumulation in murine models of Fabry (GLA-/- ), Sandhoff (HEXB-/- ) and Niemann-Pick type C (NPC1-/-) diseases, and effectively alleviates a wide spectrum a disease-associated neurological symptoms in HEXB-/- and NPC1-/- mice. Importantly, oral administration of a clinically enabled small molecule co-inducer of Hsp70, recapitulates the pan-LSD and neurological potential of recombinant Hsp70 encouraging the development of Hsp70-based therapies for LSDs.

P48: Endothelial-derived sphingolipids preserve systemic vascular functions.

Annarita DI LORENZO¹,Anna CANTALUPO¹, Yi ZHANG¹, Hideru OBINATA¹, Sylvain GALVAIN¹, Mariarosaria BUCCI2, Xiang-Cheng JIANG3, Frank GIORDANO4, Timothy HLA¹,
1Weill Cornell Medical College, 2University Of Naples Federico II, 3SUNY Downstate Medical Center, 4Yale Medical School

Objective: Endothelial dysfunction is a critical event in many cardiovascular diseases including hypertension. Although lipid signaling is implicated in endothelial dysfunction and cardiovascular diseases, specific molecular mechanisms are poorly understood. The goal of our study was to define the role of locally produced sphingolipids on vascular tone regulation. To this aim, we employed genetic mouse models and assessed blood pressure in vivo by using tail-cuff system, in normotensive and angiotensin-II-hypertensive mice. Vascular reactivity of resistance arteries was evaluated ex-vivo by using the pressure myograph system. We identified endothelial-derived sphingosine 1-phosphate (S1P) as critical regulator of vascular tone and flow-mediated vasodilation through the activation S1P1-receptor-eNOS signaling axis. We discovered a novel regulation of endothelial sphingolipid de novo synthesis by Nogo-B, membrane protein of the endoplasmic reticulum that modulates local sphingolipid production with direct effects on vascular function and blood pressure. Nogo-B inhibits serine palmitoyltransferase, rate-limiting enzyme of the sphingolipid de novo synthesis, controlling endothelial S1P production and its autocrine G-protein-coupled receptor-dependent signaling actions. Mice lacking Nogo-B are hypotensive and resistant to Ang-II-induced hypertension (150.4±2.5 vs. 108.4±1.5 mmHg, compared to WT mice, 24 days after AngII infusion), and preserve endothelial function and nitric oxide release. Pharmacological inhibition of serine palmitoyltransferase with myriocin in mice that lack Nogo-B reinstated endothelial dysfunction and Ang-II-induced hypertension (143.9±1.5 vs. 90.1±1.6 mmHg, myriocin vs. vehicle treated Nogo-A/B-/- mice). Our study identifies Nogo-B as a key inhibitor of local sphingolipid synthesis and indicates that autocrine sphingolipids signaling within the endothelium are critical for vascular function and blood pressure homeostasis.

P49: Effect of sphingosine on the biophysical properties and permeability of raft-mimicking mixtures.

Ana CARREIRA¹, Eva ZUPANCIC¹, Rodrigo DE ALMEIDA2, Liana C. SILVA¹
1Faculty Of Pharmacy, University Of Lisbon, 2Faculty Of Science, University Of Lisbon

Objective: In addition to its several roles as a bioactive lipid, when abnormally accumulated in the lysosomes and late endosomes, sphingosine (Sph) leads to Niemann Pick type C1 (NPC1), one of the most complex storage diseases in terms of molecular phenotype. At the moment little is known about the interaction of Sph with other membrane lipids. It is our aim to characterize the effect of Sph on the biophysical properties of model membranes composed of different lipids, so that new insights into its mode of action might arise. Using an established fluorescence spectroscopy approach we evaluated the effect of Sph in membrane organization and permeability of different 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/Sphingomyelin/Cholesterol raft-mimicking mixtures. Our results showed that Sph accumulation leads to the formation of Sph-enriched gel domains in raft mimicking mixtures. These domains are more easily formed at neutral pH mimicking the plasma membrane environment (PM) as compared to the acidic lysosomal membrane environment (LM), where higher Sph concentrations are required. The determination of ζ-potential further revealed that in PM-raft models Sph is mainly neutral, whereas in LM-raft models the positive charge of Sph leads to electrostatic repulsion, reducing Sph ability to form gel domains. It was also shown that the external addition of Sph affects the membrane permeability, being the effect more noticeable in acidic environment. These results support the hypothesis that Sph biological actions, including those related to NPC1, could be exerted through biophysical changes in cellular membranes.

P50: Effect of ceramide-acyl chain structure on the biophysical properties of living cells.

Sandra PINTO¹, Alfred MERRILL, JR.2, Manuel PRIETO¹, Anthony FUTERMAN3, Liana SILVA4
1Instituto Superior Técnico, 2Georgia Institute Of Technology, 3Weizmann Institute Of Science, 4Faculdade De Farmácia, Universidade De Lisboa

Objective: Ceramide (Cer) is involved in the regulation of several cellular processes by mechanisms that depend on Cer-induced changes on membrane biophysical properties. Accumulating evidence shows that ceramides with different N-acyl chain composition differentially impact cell physiology, which may in part be due to specific alterations in membrane biophysical properties. We now address how the sphingolipid (SL) N-acyl chain affects membrane properties in cultured human embryonic kidney cells by overexpressing different Cer synthases (CerS). Our results show an increase in the order of cellular membranes in CerS2-transfected cells caused by the enrichment in very long acyl chain SLs. Formation of Cer upon treatment of cells with bacterial sphingomyelinase promoted sequential changes in the properties of the membranes: after an initial increase in the order of the fluid plasma membrane, reorganization into domains with gel-like properties whose characteristics are dependent on the acyl chain structure of the Cer was observed. Moreover, the extent of alterations of membrane properties correlates with the amount of Cer formed. These data reinforce the significance of Cer-induced changes on membrane biophysical properties as a likely molecular mechanism by which different acyl chain ceramides exert their specific biological actions.
Acknowledgments: This work was supported by Fundação para a Ciência e Tecnologia (FCT), Portugal grants PTDC/BBB-BQB/0506/2012 and Investigador FCT 2014 to LCS.

P51: Sphingosine 1-phosphate and renal fibrosis.

Koch ALEXANDER¹, Wünsche CHRISTIN¹, Huwiler ANDREA2, Pfeilschifter JOSEF¹
1Department Of General Pharmacology And Toxicology, Goethe University Hospital, Frankfurt Am Main, Germany, 2Institute Of Pharmacology, University Of Bern, Bern, Switzerland

Objective: Fibrosis is a common end-point of nearly all forms of acute and chronic inflammatory kidney diseases. Here, we summarize our findings about the effect of sphingosine 1-phosphate (S1P) on the expression of connective tissue growth factor (CTGF) as an important mediator of renal fibrosis.
Methods: Using several pharmacological and genetic approaches, we investigated the effect of S1P on the expression of pro-fibrotic CTGF in glomerular cells (podocytes and mesangial cells) and in kidney glomeruli from sphingosine kinase (SK)-1 deficient mice (SK-1-/-) suffering from streptozotocin-induced diabetic nephropathy.
Results: The alteration of CTGF expression in renal cells strongly depends on the localization of S1P. In human mesangial cells endogenous S1P enhances CTGF expression by activation of S1P2, whereas S1P5 seems to be involved in the pro-fibrotic action of phospho-FTY720. Moreover, recent data show that S1P5 is directly involved in the induction CTGF expression by transforming growth factor beta 2 (TGF-b2). In contrast, activation of SK-1 by TGF-b2 reduces CTGF expression in renal glomerular cells. Our results using SK-1 siRNA, the SK inhibitor SKI II, and “caged” S1P demonstrate that the CTGF lowering effect of SK-1 activation is mediated by intracellularly formed S1P. Additionally, SK-1-/- mice develop a more severe diabetic nephropathy and express higher amounts of CTGF in glomeruli, further demonstrating the anti-fibrotic capacity of SK-1 activation and subsequent formation of intracellular S1P.
Conclusion: Overall, we suggest that the contribution of S1P to renal fibrosis is of Janus-faced nature as S1P exhibits both pro- and anti-fibrotic effects depending on its site of action.

P52: Synovial fluid sphingolipids across species and in human osteoarthritis.

Kosinska M.K.¹, Liebisch G.2, Wilhelm J.3, Kaesser U.4, Schmitz G.2, Steinmeyer J.¹
1Orthopedic Research Laboratories, Dept. Orthopedics, University Giessen, Germany, 2Dept. Clinical Chemistry & Laboratory Medicine, University Hospital Regensburg, Germany, 3Medical Clinic II/IV, University Giessen, Germany, 4Internistisches Praxiszentrum Am Krankenhaus Balserische Stiftung, Giessen, Germany

Objectives: Sphingolipids within synovial fluid (SF) of articular joints can be involved in osteoarthritis (OA) and rheumatoid arthritis (RA). We quantified for the first time the composition of sphingolipids in the SF of knee joints from unaffected controls, patients with early (eOA) and late (lOA) stages of OA, and from patients with RA. Furthermore, sphingolipids of human, canine and equine SF were compared to identify a large animal particularly suited as a model of human lipid metabolism.
Methods: Lipids were extracted from cell and cellular debris free SF of 9 postmortem donors used as healthy controls, 17 eOA, 13 lOA, 18 RA patients as well as from 9 dogs and 14 horses with healthy joints. Sphingolipids were quantified using ESI-MS/MS directly or coupled with HILIC. This study was approved by the ethics committee of our university. The Kruskal-Wallis test, Benjamini-Hochberg adjustment and the Steel-Dwass statistical test were applied.
Results: We provide a novel overview of 35 sphingolipids present in human SF. Based on the fatty acids, 19 sphingomyelin, 6 ceramide, 5 hexosylceramide and 5 dihexosylceramide species were identified in SF. Compared to control SF, all sphingolipids were found to be elevated in OA and RA SF. Across species similar numbers of sphingolipids were observed whereas the ceramide levels appears to be species dependent
Conclusions: Compared to control SF the concentrations of sphingolipids are elevated in eOA, lOA and RA SF which may be used to develop biomarkers. Both large animal species partly reflect the normal human sphingolipid metabolism.

P53: Unravelling the biophysical properties of sphingolipids: from model to cell membranes.

Ester VENTURA¹, Raquel VARELA2, Sandra PINTO3, Amelia GONCALVES DA SILVA4, Anthony FUTERMAN5, Liana SILVA6, Manuel PRIETO3
1IMed.UL – Research Institute For Medicines And Pharmaceutical Sciences, Faculdade De Farmácia;Centro De Química-Física Molecular, Instituto Superior Tecnico, Universidade De Lisboa, Lisboa, Portugal, 2IMed.UL – Research Institute For Medicines And Pharmaceutical Sciences, Faculdade De Farmácia, Universidade De Lisboa, Portugal; Weizmann Institute Of Sciences, Dept Of Biological Chemistry, Rehovot, Israel; Centro De Química-Física Molecular, Instituto Superior Tecnico, Universidade De Lisboa, Lisboa, Portugal, 3Centro De Química-Física Molecular, Instituto Superior Tecnico, Universidade De Lisboa, Lisboa, Portugal, 4Centro De Química Estrutural, Instituto Superior Tecnico, Universidade De Lisboa, Lisboa, Portugal, 5 Weizmann Institute Of Science, Dept Of Biological Chemistry, Rehovot, Israel, 6IMed.UL – Research Institute For Medicines And Pharmaceutical Sciences, Faculdade De Farmácia Universidade De Lisboa, Lisboa, Portugal

Objective: Sphingolipids (SLs) have emerged as an important class of lipids due to their bioactive role in several cellular events and in disease. The evidence that several SL species participate in the formation of lipid domains, and that this might underlie their biological mechanism of action has fostered research in the biophysical aspects of bioactive SLs. This work will focus on two important SLs – ceramide and glucosylceramide – and their interplay with other lipid components in simple and complex membrane models.
Methods: A combination of biophysical methodologies that include fluorescence spectroscopy, confocal and two-photon microscopy, surface pressure-area measurements, were used to elucidate the effects of these lipids on the biophysical properties of membranes with different lipidic components and displaying different phase properties.
Results: Our results showed that lipid-lipid interactions are modulated by alterations in the membrane environment, such as changes in pH. Moreover, small structural differences of these lipids influence their packing properties, membrane shaping and lateral organization. The importance of the headgroup, acyl chain length and unsaturation, on the modulation of membrane properties will be discussed in the framework of results obtained for cellular membranes.
Conclusion: Model membrane systems allow to predict the biophysical and biological implications of these lipids in cellular membranes. Supported by FCT (Portugal) grants PTDC/BBB-BQB/0506/2012 and RECI/CTM-POL/0342/2012, SFRH/BD/69982/2010 to ARV, Investigador FCT 2014 to LCS.

P54: Detection and quantitation of novel sphingolipids by mass spectrometry.

Nadıa RANA¹, Yusuf HANNUN¹
1Stony Brook University

Objective: Serine Palmitoyl Transferase (SPT) is the enzyme that carries out the condensation reaction between Serine and Palmitoyl Co-A that is known as the committed step of sphingolipid biosynthesis. Dysregulation of this important pathway through its downstream sphingolipid metabolites has been implicated in a variety of human diseases, including diabetes, metabolic syndrome, neuropathy, and cancer, as well as several key regulatory pathways such as apoptosis, inflammatory responses, and autophagy. Recent discoveries from several laboratories have demonstrated SPT exhibits substrate promiscuity, both with other non-canonical amino acids as well as fatty acids, which has led to the uncovering of novel sphingolipid metabolite by-products of this promiscuity such as deoxysphinganine and deoxydemethylsphinganine. We have now developed novel mass spectral-based methods to allow for high sensitivity and accuracy detection and quantitation of this emerging new class of sphingolipids. Preliminary data suggest there may be a regulatory relationship between sphingolipids and amino acid availability that extends beyond the canonical Serine and Palmitoyl Co-A to other amino acids such as Alanine and Glycine, and other Fatty Acids including Stearate. We have employed Multiple Reaction Monitoring on a triple quad mass spectrometer in conjunction with high quality authentic SL standards to identify and measure relative levels of novel SL metabolites, in the absence of derivatization. Development of this quantitative methodology will serve to define novel SLs and their potential roles in signaling, and to define their biological functions in yeast.

P55: Lipid oversupply to cardiomyocytes induces sphingolipid-dependent oxidative stress and induction of mitophagy through ceramide synthase 2.

Brittany A LAW¹, L. Ashley COWART2
1MUSC, 2MUSC, VA

Objective: Diabetic cardiomyopathy (DbCM) contributes to the high risk of heart failure (HF) in diabetics, but mechanisms underlying DbCM remain unclear. We previously showed that high saturated fat feeding in mice altered cardiomyocyte sphingolipid profiles leading to DbCM and that some of these maladaptations were dependent on autophagy and ceramide synthase 5 (CerS5). In the present study, we sought to further understand the cellular processes in which lipid overload leads to DbCM in in the context of sphingolipids. Mice fed an obesogenic diet and in vitro studies using H9c2 cardiomyocytes were utilized in this study. Increased oxidative stress and apoptosis were identified in the hearts of animals subjected to lipid overload, while animals treated with the sphingolipid synthesis inhibitor myriocin were protected. Similarly, cardiomyocytes treated with palmitate showed a sphingolipid-dependent increase of reactive oxygen species (ROS) and mitophagy. Treatment with mitochondria-targeted ceramide analogs revealed that very long chain ceramides, but not long-chain ceramides, induced cardiomyocyte cell death, which was exacerbated by inhibiting mitophagy. This suggested that lipotoxicity to cardiomyocytes occurs in part through oxidative stress in a sphingolipid-dependent manner, and mitophagy may occur to prevent further damage. In the observation that only very long chain species led to these outcomes suggested involvement of CerS2. Overexpression of CerS2 showed increased mitophagy in cardiomyocytes and knockdown of CerS2 by CRISPR-CAS9 technology decreased mitophagy. Taken together, our data suggest that lipid overload induces mitophagy as a protective measure in defense from CerS2-induced mitochondrial damage, oxidative stress, and cell death in DbCM.

P56: Caspase 2 is required for sphingosine kinase 1 proteolysis in response to doxorubicin in breast cancer cells: implications to the CHK1-supressed pathway.

Brittany CARROLL¹, Achraf SHEMSEDDINE¹, Yusuf HANNUN¹, Lina OBEID¹,
1Stony Brook University

Objective: Sphingosine Kinase 1 (SK1) is a lipid kinase whose activity produces the potent bioactive lipid sphingosine 1-phosphate. Sphingosine 1- phosphate is a pro-survival lipid associated with proliferation, angiogenesis and invasion; subsequently SK1 overexpression has been observed in numerous cancers. Recent studies have demonstrated SK1 proteolysis downstream of the tumor suppressor p53 in response to several DNA damaging agents. Moreover, loss of SK1 in p53 knockout mice resulted in complete protection from thymic lymphoma providing evidence that regulation of SK1 constitutes a major tumor suppresser function of p53. Given this profound phenotype, this study aims to investigate the mechanism by which wild type p53 regulates proteolysis of SK1 by doxorubicin in breast cancer cells. We find that p53-mediated activation of Caspase 2 was required for SK1 proteolysis and that Caspase 2 activity significantly alters the levels of endogenous sphingolipids. As p53 is mutated in 50% of all cancers, we extended our studies to investigate whether SK1 is deregulated in the context of triple negative breast cancer cells (TNBC) harboring a mutation in p53. Indeed Caspase 2 was not activated in these cells and SK1 was not degraded. Moreover, Caspase 2 activation was recently shown to be downstream of the CHK1- Suppressed pathway in mutant p53 cells, whereby inhibition of the cell cycle kinase CHK1 leads to Caspase 2 activation and apoptosis. Indeed knock-down and inhibition of CHK1 led to loss of SK1 in p53 mutant TNBC cells, providing evidence that SK1 maybe the first identified effector of the CHK1-Suppressed pathway

P57: Ceramide limits cell motility in ovarian cancer: potential of ceramide as a metastasis suppressor lipid.

Kazuyuki KITATANI¹, Toshinori USUI¹, Shravan SRIRAMAN2, Masafumi TOYOSHIMA¹, Shogo SHIGETA¹, Hideo OGISO3, Toshiro OKAZAKI3, Vladimir TORCHILIN2, Yusuf HANNUN4, Nobuo YAEGASHI¹
1Tohoku University, 2Northeastern University, 3Kanazawa Medical University, 4Stony Brook University

Objective: Targeting cell motility, which is required for dissemination and metastasis, has a therapeutic potential for treating ovarian cancer metastasis, and regulatory mechanisms of cell motility needs to be uncovered for developing novel therapeutics. Invasive ovarian cancer cells spontaneously formed protrusions, such as lamellipodia, required for generating locomotive force in cell motility. Small interfering RNA screening identified class II phosphatidylinositol 3-kinase C2β (PI3KC2β) as the predominant isoform of PI3K involved in lamellipodia formation of ovarian cancer cells. Ceramide, one of bioactive sphingolipids, has been emerging as an antitumorigenic lipid, and treatment with short-chain C6-ceramide decreased the number of ovarian cancer cells with PI3KC2β-driven lamellipodia. Pharmacological analysis demonstrated that long-chain ceramide regenerated from C6-ceramide through the salvage/recycling pathway, at least in part, mediates the action of C6-ceramide. Mechanistically, ceramide was revealed to interact with PI3KC2β and affect its compartmentalization, thereby suppressing PI3KC2β activation and its-driven cell motility. Moreover, ceramide treatment suppressed cell motility promoted by epithelial growth factor known as a prometastatic factor. To examine role for ceramide in ovarian cancer metastasis, ceramide liposomes were employed and confirmed to suppress cell motility in vitro. Those ceramide liposomes displayed an inhibitory effect on peritoneal metastasis in a murine xenograft model of human ovarian cancer. Taken together, our studies identified ceramide as a bioactive lipid that limits PI3KC2β-governed cell motility, and ceramide is proposed to serve as a metastasis suppressor lipid in ovarian cancer. Those could be translated into developing ceramide-based therapy for metastatic diseases.

P58: Signaling pathways of sphingosine-1-phosphate in vascular smooth muscle contraction.

Peter Tibor DANCS¹, Dorottya MÓRɹ, Éva RUISANCHEZ¹, Margit KERÉK¹, Cecília Rita PANTA¹, Henrique Martins Aranda CALDEIRA2, Stefan OFFERMANNS3, Zoltán BENYÓ¹
1Institution Of Human Physiology And Clinical Experimental Research, Semmelweis University, Budapest, Hungary, 2Science Without Borders, CAPES, Brasil, 3Max Planck Institute For Heart And Lung Research, Bad Nauheim, Germany

Objectives: We aimed to examine the direct effect of sphingosine-1-phosphate (S1P) on vascular smooth muscle (VSM) contractility and to elucidate the underlying signaling pathways.
Methods: Isometric tension of endothelium-denuded thoracic aorta segments isolated from male wild type (WT) and knockout (KO) mice deficient in S1P2, S1P3 receptor or Gα12/13 was measured in myographs. Vasoactive effect of 10 μM S1P was detected at physiological (4 mM) and elevated (6-124 mM) extracellular K+-concentration [K+]e.
Results: At physiological [K+]e S1P had negligible vasoactive effect. Slightly increased [K+]e (6 mM) failed to influence the vascular tone by itself, but addition of S1P induced marked vasoconstriction that was further intensified if [K+]e was increased to 8 and 10 mM. At higher [K+]e (20-80 mM), K+ increased the vascular tone progressively and attenuated the additional vasoconstriction by S1P. Further experiments on the signaling pathways were performed with 8 mM [K+]e, which enhanced the action of S1P markedly without significantly influencing the resting vascular tone by itself. The vasoconstrictor effect of S1P disappeared in S1P2-KO and Gα12/13-KO vessels, whereas remained unchanged in S1P3-KO mice. S1P-induced vasoconstriction was also abolished by the Rho-kinase inhibitor Y-27632 (10 μM) in WT vessels.
Conclusion: S1P induces vasoconstriction in case of moderately increased [K+]e. The effect of S1P is mediated by S1P2-receptor, Gα12/13, and Rho-kinase. This phenomenon may lead to increased vascular tone under conditions of systemic or local elevation of [K+]e surrounding the VSM, like in hyperkalemia or tissue ischemia. Grant support: OTKA K-101775 and MedInProt Protein Science Research Synergy Program.

P59: Regulation of inflammatory cytokine signaling by acid sphingomyelinase.

Benjamin J NEWCOMB¹, David PERRY2, Yusuf A HANNUN3
1Suny Stony Brook, 2Medical University Of South Carolina

Objective: Acid sphingomyelinase (ASM), which hydrolyzes sphingomyelin to produce ceramide, regulates a key step in the sphingolipid salvage pathway. Ceramides are biologically active lipids that have been linked to cellular stress responses and cytokine production in several model systems, but the mechanism by which ASM regulates cytokine production has not been established. In this work we define a mechanism by which ASM regulates IL-6 production in breast cancer, and demonstrate a role for ASM in tumor progression. We found that down regulation of ASM in two distinct genetic models, reduced IL-6 production in response to the tumor promoter, 4-phorbol 12-myristate 13-acetate (PMA). Down-regulation of another ceramide producing enzyme, acid β-glucosidase (GBA), potentiated IL-6 production in response to PMA, whereas knock down of the sphingosine producing enzyme, acid ceramidase (ACD), had no effect. From these data we conclude that spatiotemporal regulation of ceramide production plays a role in its function as a second messenger within the cell, suggesting specific roles for GBA and for ASM derived ceramides. In addition, indirect pharmacologic inhibition of ASM with desipramine blocked both the PMA and TNF-α induced production of IL-6. Inhibition of ASM correlated with a reduction in p38 MAP Kinase phosphorylation, suggesting that ASM is regulating IL-6 production through modulation of MAP Kinase activity. Lastly, we found that inhibition of ASM or of p38 reduced tumor cell migration in matrigel transwell migration assays. Taken together, we have defined a role for ASM in pro-tumor signaling events, and identified a role for ASM in tumor progression.

P60: Neutral sphingomyelinase 2 and hepatic acute phase response.

Mariana NIKOLOVA-KARAKASHIAN¹, Daipayan BANERJEE¹, Gergana DEEVSKA¹, Alexander KARAKASHIAN¹
1University Of Kentucky

Objective: Hepatic acute phase response (APR) plays a central role in host response to infection and it is exacerbated in aged animals and humans. In vitro studies indicate that neutral sphingomyelinase 2 (nSMase-2) is essential component of APR. Objectives: The goal of this study is to investigate the role of nSMase-2 in a rat model of inflammation.
Methods: Four- and 21- months old rats were administered with adenovirus encoding a GFP-tagged shRNAi against nSMase-2 or scrambled control and, 6 days later, with LPS (5.6 mg/kg) or saline. Hepatic samples were analyzed 4 hours later for efficiency of infection, nSMase activity, and levels of expression of nSMase-2 and several acute phase proteins.
Results: GFP fluorescence was observed in up to 30% of the cells in the liver but not in other tissues. Livers of LPS-treated animals exhibited 2-to 3-fold higher nSMase-2 mRNA levels than saline-treated animals without significant ageing-associated differences. The mRNA levels of IGFBP1 increased, while those of PEPCK1 decreased in response to LPS in an age-specific manner. These differences were attenuated (by approximately 20-25%) in animals treated with shRNA against nSMase-2.
Conclusions: (i) LPS stimulates hepatic expression of nSMase2 mRNA. (ii) Liver-specific silencing of nSMase-2 is achievable in vivo; (iii) in spite of a relatively moderate efficacy of silencing, a statistically significant reversal of LPS effects on IGFBP1 and PEPCK1 can be detected, confirming the role of NSMase2 in APR and indicating a possible therapeutic use of targeting nSMase-

P61: Comparison of serum sphingosine-1-phosphate levels among euthymic, manic and depressive patients with bipolar disorder.

Yesim OZTAS¹, Burcu ESER2, Serdar ATIK2, Ahmet YALCINKAYA¹, Murat SEMIZ2, Murat ERDEM2, Husamettin GUL2
1Hacettepe University, 2Gulhane Military Medical Academy

Objective: Bipolar disorder (BD) is a chronic and overwhelming psychiatric condition with manic-depressive episodes. Sphingolipids are biologically active lipids ubiquitously expressed in the CNS. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid acting through G-protein-coupled receptors. It has regulatory roles in endothelial injury, inflammation, and thrombosis in vascular biology. Increase of serum S1P levels was reported to have a role in stress-induced anxiety. In a recent study S1P receptor gene is reported as one of the susceptibility genes in BD. We hypothesized that serum levels of S1P might have been different among patients with manic, depressive or euthymic forms of BD.
Methods: There were three groups in the study involving euthymic (N=14), manic (N=10) and depressive (N=7) patients. Plasma samples (100 μl) were mixed with 500 μl of methanol containing an internal standard (IS) colchicine. Supernatants were separated after centrifugation. An Agilent 1260 Infinity UPLC system was used for chromatography. Total run time was 10 minutes; the flow rate was 0.5 ml/min with solvent A, water (10 mM ammonium acetate, 0.1% formic acid) and solvent B, methanol (0.1% formic acid). The eluent was directly introduced into a mass spectrometer by electrospray. An Agilent 6420 triple quadrupole mass spectrometer was used. S1P and IS were analyzed in positive ion mode and multiple reaction monitoring with ion pairs 380.2/264.3 and 400.2/358.2 respectively.
Results: There was no statistically significant difference between S1P levels among groups.
Conclusion: This is a preliminary study and S1P levels should be investigated in larger number of patients with BD.

P62: A drosophila neurodegenerative autophagy mutant perturbs sphingolipid metabolism, autophagic clearance, and stress signalling.

Rachel KRAUT¹, Ishtapran SAHOO2, Sarita HEBBAR3, Kate OSBORNE4, Artur MATYSIK4, Irene ARGUDO¹, Dominik Schwudke5
1Tu Dresden, 2Ncbs, Bangalore, 3Mpy-cbg, Dresden, 4Ntu, Singapore, 5Research Cr. Borstel

Objective: We propose that imbalances in ceramide metabolism are a key factor in determining the neurodegenerative severity of a Drosophila autophagy mutant, blue cheese. Shotgun lipidomics on blue cheese mutant brains reveals increased ceramide levels, as well as expected sphingolipidomic changes in n-Ceramidase and n-Sphingomyelinase processing mutants that modify the degeneration in blue cheese. Mutant neurons show defects in autophagic trafficking and clearance of sphingolipids, an imbalance in de novo vs. recycled sources of sphingolipids, and alterations in growth vs. stress signaling by members of the MAPK and insulin cascades. Surprisingly, although total ceramides are higher in blue cheese mutant brains and RNAi-S2 cells, genetic backgrounds that increase the available ceramide pool rescue all phenotypes observed, including neuronal death, ceramide accumulation in autophagosomes, autophagic vesicular trafficking, MAPK signaling defects, and ER stress. Increasing autophagic flux by rapamycin treatment similarly rescues blue cheese degeneration, but also reverses the signaling defects in blue cheese RNAi cells.

P63: Molecular mechanisms of Atg7 and p53 in regulation of sphingolipid-dependent autophagy.

Abigail WASHISPACK¹, Sarah RUSSO¹, Ashley COWART¹
1Medical University Of South Carolina

Objective: Diabetic cardiomyopathy (DbCM) is defined as significant changes in the structure and function of the heart in the absence of coronary artery disease and hypertension. Recently our lab found that cardiomyocyte hypertrophy and autophagy play a role in the development of DbCM in a mouse model and that inhibiting de novo sphingolipid synthesis attenuated this process. In vitro studies of fatty acid oversupply revealed that hypertrophy and autophagy are dependent on ceramide synthase 5 (CerS5) and we identified various autophagy-associated genes, including Atg7, that were upregulated in DbCM in a sphingolipid-dependent manner. Interestingly many of these genes are p53 targets. Further cells studies revealed that sphingolipids induced p53, and loss of p53 prevented sphingolipid-dependent autophagy. This indicated that p53, at least in part, mediates sphingolipid-dependent autophagy. Studies from other labs suggest that direct binding of Atg7 to p53 may regulate p53 function under some conditions. Therefore we tested whether Atg7-p53 association is sphingolipid-dependent and/or required for autophagy induction. Using proximity ligation assay technology and confocal imaging we found that Atg7 and p53 association is increased under autophagy-inducing conditions, and that this association is decreased with knockdown of CerS5. Furthermore, we found that nuclear levels of p53 are regulated by sphingolipids. From these results we conclude that Atg7 and p53 are important mediators of sphingolipid-induced autophagy and that their association may be regulated by specific sphingolipid species and in turn regulate sphingolipid-induced autophagy.

P64: Defining regulation of the sphingolipid network in response to doxorubicin.

Justin Snider¹, Achraf Shamseddine¹, Brittany Carroll¹, Christopher Clarke¹, Lina Obeid¹, Darryl Pappin2, Yusuf Hannun¹
1Stony Brook University, 2Cold Spring Harbor

Objective: Sphingolipids have been implicated in numerous cellular biologies, both pro-and anti-growth; however, the study of the enzymes responsible for their metabolism has been hindered by difficulties in detection. Furthermore, these enzymes represent a dynamic network in which a “single” signal results in diverse responses leading to multiple potential biologies, specifically in cancer progression. Our lab is exploring the effects of both lethal and sub-lethal doses of doxorubicin to mimic the decreasing clinical concentrations of chemotherapeutics over time. In MCF7 cells sub-lethal and lethal doses of doxorubicin result in biphasic ceramide generation paralleling two distinct biologies; growth arrest and apoptosis, respectively. While antibodies are available for some of the sphingolipid enzymes, there is a need for a systems approach to examine the role of sphingolipid enzymes, their metabolic products, and the resulting biologies in response to doxorubicin treatment. To this end, LC/MS/MS methods were developed to quantify 32 enzymes central to sphingolipid metabolism and monitor flux in sphingolipid content generated by this enzymatic regulation. The studies presented here will determine the role of sphingolipids and their metabolizing enzymes in response to chemotherapeutics.

P65: Characterization of cholesterol homeostasis in sphingosine-1-phosphate lyase-deficient fibroblasts.

Hans VIENKEN¹, Agnes RUDOWSKI¹, Nathalie MABROUKI¹, Alexander KOCH¹, Josef PFEILSCHIFTER¹, Dieter LÜTJOHANN2, Gerhild VAN ECHTEN-DECKERT3, Dagmar MEYER ZU HERINGDORF¹
1Institut Für Allgemeine Pharmakologie Und Toxikologie, Klinikum Der Goethe-Universität Frankfurt Am Main, Germany, 2Institut Für Klinische Chemie Und Klinische Pharmakologie, Universitätsklinikum Bonn, Germany, 3Membranbiologie Und Lipidbiochemie Einheit Des Life And Medical Sciences (LIMES) Instituts, Universität Bonn, Germany

Objective: Mice lacking sphingosine-1-phosphate (S1P) lyase have a strongly reduced life span and suffer from multiple organ defects, immunosuppression, and accumulation of S1P and sphingosine in cells and tissues. Interestingly, these mice have enhanced plasma levels of cholesterol and triglycerides while suffering from strongly reduced body fat. Aim of the study was to further analyze the link between S1P lyase and cholesterol homeostasis using embryonic fibroblasts from S1P lyase-deficient mice (Sgpl1-/–MEFs).
Results and Conclusions: Since we had observed that ABCA1, which transports both S1P and cholesterol, was upregulated in Sgpl1-/–MEFs, we initially speculated that accumulation of S1P triggered reverse cholesterol transport. However, total cholesterol content was not altered in Sgpl1-/–MEFs grown in serum-free medium, and it was even enhanced in the presence of 10 % FCS. In agreement, the uptake of [3H]cholesterol was enhanced while [3H]cholesterol release was unaltered, and the low-density lipoprotein (LDL) receptor was upregulated in the knockout MEFs.These alterations were in line with an upregulation and enhanced proteolytic activation of the transcription factor, sterol regulatory element-binding protein (SREBP)-2. Interestingly, the expression of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was decreased, leading to reduced formation of biosynthetic precursors of cholesterol in Sgpl1-/–MEFs. Expression and phosphorylation of AMP-activated protein kinase was not altered. We conclude that decreased HMG-CoA reductase expression and activity leads to activation of SREBP-2 which in turn induces ABCA1 and LDL receptor and increases cholesterol uptake in Sgpl1-/–MEFs.

P66: Sphingosine kinase 1 regulates adiponectin expression.

Andrea ANDERSON¹, Johana LAMBERT¹, Ashley COWART¹
1Medical University Of South Carolina

Objective: Obesity affects more than 1/3 of the United States population and is associated with various pathophysiological conditions, which may arise in part from increased plasma lipids. Previous studies from our lab showed that palmitate induced sphingosine kinase 1 (SK1), leading to such outcomes as Il-6 production from skeletal muscle. Adipose tissue secretes cytokines and hormones and this is affected by obesity; thus we sought to test a role of SK1 in adipocyte function. We used a combination of methods including: in vitro models of primary adipose-derived stem cells (ADSCs) isolated from wild type and whole-body SK1-/- mice, in vivo models of adipose-specific SK1 deletion and obesogenic diets, quantification of lipids by LC/LC/MS, qRT-PCR, and western blotting. The adipocyte-specific cytokine adiponectin is a potent regulator of metabolic homeostasis, and we observed an increase in adiponectin mRNA expression in undifferentiated ADSCs isolated from SK1-/- mice compared to wild type, suggesting that SK1 may suppress adiponectin expression. Furthermore, we observed over the course of differentiation, S1P-mediated suppression of adiponectin mRNA compared to control. We then performed an S1P-dose response experiment, and found that S1P effectively suppresses adiponectin mRNA expression at a maximum concentration of 40 nM. This specificity indicated to us that the effects on adiponectin may be mediated through an S1P receptor. S1P receptor expression changed over the course of differentiation. These outcomes are under current investigation in mice with adipocyte-specific deletion of SK1. The involvement of the SK1/S1P axis in adiponectin regulation may present a novel target for obesity-related therapies.

P67: Roles for sphingolipid metabolism in nonalcoholic fatty liver disease.

Michael HARLAND¹, L. Ashley COWART¹
1Medical University Of South Carolina

Objective: Nonalcoholic fatty liver disease (NAFLD) is a progressive disease in which triacylglycerols first accumulate in the liver, and in 10-20% of cases, this progresses to an inflammatory state referred to as nonalcoholic steatohepatitis (NASH). Factors that promote this progression remain unknown. We developed a mouse model of progressive NAFLD by feeding a high saturated fat diet, which induced profound hepatic steatosis, inflammation, and pro-fibrotic signaling. Previous studies from our lab demonstrated that saturated fatty acids upregulated Sphingosine Kinase 1 (SphK1), and we found elevated SphK1 message in liver in this model and in human NASH samples. Consistent with a role for SphK1 in NAFLD progression, SphK1-/- mice fed a high fat diet were protected from inflammation. In addition to proinflammatory signaling, sphingosine 1-phophate induced ER stress in cultured hepatocytes, and ER stress is increasingly appreciated as a component of NASH. During the course of these studies we found that Ceramide Synthase 6 (CerS6) expression was also increased in the mouse model of NAFLD, yet remained unchanged in SphK1-/- mice. CerS6 expression was also increased following treatment of hepatocytes with sphingosine 1-phosphate, suggesting a possible cross-talk between different sphingolipid metabolic enzymes in NAFLD. We observed regulation of ER stress by manipulating CerS6. We speculate that this novel cross-talk between branches of sphingolipid metabolism may at least in part underlie the relationship between inflammation and ER stress in NAFLD, and regulation of this pathway by saturated fatty acids may promote progression of hepatic steatosis to NASH.

P68: Intracellular SK2-derived S1P mediates EGF-induced ERM phosphorylation and cancer invasion.

Mohamad ADADA¹, Daniel CANALS¹, Yusuf HANNUN¹, Lina OBEID¹
1Stony Brook University

Objective: ERM (ezrin, radixin, and moesin) proteins are a group of adaptor molecules linking the cortical actin cytoskeleton to the plasma membrane, and are emerging as critical regulators of cancer metastasis and progression via regulation of cell morphology and motility. Recently, our lab has identified S1P as an acute and potent ERM activator (via phosphorylation) through its action on its receptor S1PR2. We have also demonstrated that S1P-mediated filopodia formation, a first step in cell invasion, is through ERM activation. Growth factors are known activators of ERM proteins; however, it is not known if this involves the newly related S1P/S1PR2 axis as well as upstream metabolites of the sphingolipid pathway. Using pharmacological inhibitors, siRNA technology as well as genetic approaches, we have demonstrated that SK2 is not only essential but also sufficient in EGF-mediated ERM phosphorylation. Surprisingly, and for the first time, we proved that this event, although dependent on S1PR2 activation, does not require extracellular S1P secretion. Finally, we identified SK2 and S1PR2 as two novel and potent targets in the pathway of EGF-driven invasion. In fact, the inhibition of SK2 or S1PR2 eradicated EGF-mediated lamellipodia formation, and subsequent adhesion and extracellular matrix invasion. We also showed that SK2 overexpression increases EGF-mediated adhesion and invasion in an erin dependent manner. In conclusion, this body of work does not only uncover new mechanistic insights for EGF-mediated invasion, it also set the stage for two novel alternative therapeutic targets that could be of utmost importance especially in patients that become resistant to current EGFR-tyrosine kinase inhibitors.

P69: Neutral sphingomyelinase-2 mediates a protective role by mediating S phase arrest in reponse to doxorubicin.

Achraf SHAMSEDDINE¹, Christopher CLARKE¹, Michael AIROLA¹, Brittany CARROL¹, Lina OBEID¹, Yusuf HANNUN¹
1Suny Stony Brook

Objective: p53 is a tumor suppressor involved in mediating responses such as cell cycle arrest, DNA repair, and apoptosis in response to stress. While mutations in the p53 gene are associated with aggressive cancers, a shift in thought suggests that the presence of mutant p53 in cancer cells leads to better clinical outcomes after chemotherapy. This is based on the rationale that chemotherapeutics are DNA damaging agents and hence mutant p53 impairs endogenous defense mechanisms of cancer cells resulting in death without repair of the damage. However, synergistic inhibition of p53 with chemotherapy is not a viable option, as it would result in side effects. As such, targeting effectors of specific arms of p53 biology would be one avenue to explore. Neutral sphingomyelinase-2 (nSMase2) generates the bioactive lipid ceramide by hydrolyzing sphingomyelin. It has been implicated in biologies such as cell cycle arrest and apoptosis.
Methods: We use molecular biology techniques of siRNA silencing, flow cytometry, qRT PCR and immunoblotting Results: Here we show that, in MCF7 breast cancer cells, nSMase2 is activated in a dose-dependent manner by doxorubicin to generate ceramide. Moreover, this is concomitant with a p53-dependent S phase arrest that is abolished by knockdown of nSMase2. Finally, this inhibition of S arrest in nSMase2 knockdown cells results in failure to resume growth in these cancer cells following doxorubicin treatment.
Conclusion: Taken together, our data suggest that inhibition of nSMase2 could have synergistic benefits if combined with DNA damaging chemotherapeutics in the context of non-mutant p53 breast cancer.

P70: Deoxysphingolipids as neurotoxic intermediates of systemic taxane treatment.

Katrin Anne BECKER¹, Anne-Kathrin UERSCHELS¹, Joan COLGLAZIER2, Jacek BIELAWSKI2, Erhard BIEBERICH3, Stefka SPASSIEVA2,
1University Of Essen, Germany, 2Medical University Of South Carolina, USA, 3Georgia Regents University, USA

Objective: Taxanes are chemotherapy drugs widely used for treatment of variety of cancers, such as breast, ovarian, lung, prostate, bladder, Kaposi sarcoma. In some cases taxanes are used as the last line of treatment. A major dose limiting side effect of taxane treatment is peripheral neuropathy compromising its effectiveness. With our study we tested weather the levels of a neurotoxic class of lipids, the deoxysphingolipids, are elevated in the dorsal root ganglia of mice treated with a taxane. We also compared in vitro in neurons, the toxic effect of taxanes and deoxysphingolipids.
Methods: Lipids were extracted from ganglia isolated from mice intraperitoneally injected (three times with four weeks in between intervals) with a taxane (docetaxel) and subjected to quantitative mass spectrometry analyses of sphingolipids. We used immunocytochemistry analyses to compare the effect of taxanes and deoxysphingolipids in neurons.
Results: We observed significant elevation of deoxysphingolipid levels (i.e. deoxydihydroceramide, deoxyceramide, deoxysphinganine, and deoxysphingosine) in the mouse ganglia after the administration of the third taxane injection. Not all deoxysphingolipid species were affected the same way. Interestingly, deoxydihydroceramide and deoxyceramide species with unsaturated fatty acid moieties were not elevated. While ceramide levels were elevated, too, the levels of sphingosine and sphingosine-1-phosphate were not. Importantly, our in vitro data showed that only deoxysphingosine treatment resulted in morphological changes in the neurons; taxane and the sphingosine treatments did not.
Conclusion: taken together our data suggest that in the dorsal root ganglia, deoxysphingolipids are the likely toxic intermediates of the systemic taxane treatment.

P71: Associations between vitamin d and lpa receptor expressions in pregnant women and their infants living in Yozgat region, Turkey.

Ayse Yesim GOCMEN¹, Emel KIYAK CAGLAYAN2, Namik DELIBAS3
1Bozok University, Medical Faculty, Biochemistry Dept., 2Bozok University, Medical Faculty, Obstetrics ADept.nd Gynecology , 3Hacettepe University, Medical Faculty, Biochemistry Dept.

Objective: Vitamin D (Vit D) induced phospholipase D (PLD) activity results in the production of diacylglycerol (DAG), which stimulates protein kinase C (PKC). Another consequence of PLD activation is the production of lysophosphatidic acid (LPA), a bioactive lysophospholipid that has recently been implicated in the regulation of bone and cartilage. These findings implicated LPA as a second messenger in Vit-D-directed signaling. In this study we aimed to study associations between lysophosphatidic acid(LPAR) expressions in pregnant women and their infants at delivery living in Yozgat region, Turkey. We studied 50 pregnant women within a prospective longitudinal study of maternal nutrition and lifestyle before and during pregnancy. Using high-resolution ultrasound, we measured fetal femur length along with head and neck circumference. Vitamin D, Vit D receptor, LPA and LPA receptors levels were measured in serum, placental fluid and tissues samples with ELISA. Pregnant women with serum vitamin D levels less than 5 ng/ml were classified as Vitamin D deficient group (n=14), the rest were controls (n=36). In placental tissue, the levels of Vit D, Vit D receptor, LPA and LPA receptors were all low in deficiency. In placental fluid, the levels of Vit D, Vit D receptor and LPA were all low in deficiency and in serum samples only LPAR levels were decreased in deficiency. In linear regression analysis serum and placental vitamin D and PLA levels were associated with femoral size. Correlation revealed the strength of LPA levels observed associations and their statistical significance in pregnant women. Our results demonstrated that levels of Vit D, LPA and their receptor were reduced in placental tissues in Vitamin D deficiency. Additionally, these reductions in placental tissues might also be associated with the rapid removal of the possible complexes formed by Lpa and autoantibodies by various cells. Further studies should be performed to investigate the role LPA acting in placenta during bone development.

P72: Selective sphingosine-1-phosphate receptor 5 agonists can modulate lipid content in the brain and thereby potentially treat neurodegenerative disorders.

Elizabeth L VAN DER KAM¹, Sean C TURNER¹, Michael OCHSE¹, Jeroen VAN BERGEIJK¹, Reinhold MUELLER¹, Mario MEZLER¹, Katja HEMPEL¹, Adrian HOBSON2, Christopher M HARRIS2, Alfred HAHN¹, Anton BESPALOV¹, Beatrice RENDENBACH-MUELLER¹
1AbbVie Deutschland GmbH & CO KG, 2AbbVie Bioresearch Center

Objective: Sphingosine-1-phosphate (S1P) plays an important role as a regulator of signal transduction, cellular plasticity, cell proliferation, membrane stability/BBB integrity, and is proposed to modulate the ceramide-S1P and cholesterol homeostasis. S1P exerts these actions through G protein-coupled receptors, such as the brain-preferred S1P5 receptor. Given these actions, S1P5 agonism could potentially be a beneficial treatment for neurodegenerative disorders.
Methods: AbbVie has developed highly selective S1P5 receptor agonists, such as A-971432. A-971432 has an EC50 of 10 nM on the hS1P5 receptor with a large selectivity window. As the molecule shows excellent pharmacokinetic properties and good brain penentration, it was examined in models to test the hypothesis that selective S1P5 agonists will a) reduce CNS, but not peripheral lipid content, b) improve cognition, and c) change disease progression as assessed in a model mimicking key features of AD.
Results: Sub-acute treatment with A-971432 (7 days) in either T-maze (0.03 mg/kg – 3 mg/kg) or MWM/ORT (0.1 and 0.5 mg/kg) fully reversed the age-related cognitive deficits with a minimale effective dose of 0.1 mg/kg (40 ng/mL). Concomitantly, A-971432 normalizes the age-related CNS sphingolipid imbalance without affecting plasma levels. In a murine model of Niemann Pick C disease (NPC), A-971432 was able to improve the behavioral phenotype (dystonia, motor impairment), promote survival, normalize CNS sphingolipid content, and normalized Amyloid-β levels in the CSF.
Conclusion: These data indicate that S1P5 agonism provides an innovative mechanism for the potential treatment of neurodegenerative disorders such as AD and lysosomal storage disorders such as Niemann Pick C.

P73: Ceranib-2, a novel ceramidase inhibitor, induces apoptosis of prostate cancer cells.

Gokhan KUS¹*, Selda KABADERE2, Ruhi UYAR2, Hatice Mehtap KUTLU3
1Department of Health Programme, Open Faculty, Anadolu University, 2Department of Physiology, Faculty of Medicine, Eskisehir Osmangazi University, 3Department of Biology, Faculty of Science, Anadolu University, Eskisehir, Turkey

Objective: Ceramide has been identified as an important second messenger that plays important roles in various aspects of inducing apoptosis. We questioned ceranib-2, a novel ceramidase inhibitor, affects the survival of prostate cancer cells (LnCaP and DU145) in vitro.
Methods: The cell viability was determined with MTT and apoptosis with flow cytometry. We examined structural changes both with confocal and transmission electron microscopy.
Results: Comparing to the control 0.1, 1, 5, 10, 25 and 50 μM ceranib-2 reduced the percentage of viable LNCaP cells to 84, 80, 64, 56, 40 and 15 after 24 hr and 81, 74, 60, 55, 27 and 11 % after 48 hr, respectively. Treatment of DU145 cells with the same six doses of ceranib-2 lowered numbers of living cell by 84, 82, 63, 50, 41 and 18 % after 24 hr; 64, 42, 30, 20, 8 and 5 % in 48 hr, respectively. Observed early apoptotic rate of LNCaP cells were 5 and 36 % after 24 hr and 15 and 60 % after 48 hr treatments with 25 and 50 μM ceranib-2, respectively. The signs of apoptosis were detected as fragmented nuclei, chromatin condensations and cytoskeleton laceration in the cells.
Conclusions: Ceranib-2 posseses a strong dose and time dependent survival decreasing effect on both prostate cancer cell lines.

P74: Evaluation of an inhibitor of human ceramidases, ceranib-2 induced apoptosis cytotoxicity on MCF7 cells.

H. Mehtap Kutlu¹, Djanan Vejselova1, Gokhan KUS¹*
1Anadolu University, Faculty of Science, Department of Biology, Eskişehir, Turkey, Department of Health Programme, Open Faculty, Anadolu University

Objective: The aim of the study was to investigate the potential cytotoxic effects of ceranib-2 on MCF7 cells and its effects on MCF7 cell structure.

Methods: Cytotoxic effects of ceranib-2 on MCF7 cells was detected via MTT test system. Dilutions prepared from the stock solution (in DMSO) of ceranib-2 was applied on MCF7 cells (1×104 cells/well) for 24 hours at 37 °C and 5% CO2 in air. The plates were read on ELISA reader (ELx808), at wavelength of 540 nm (n=3). For detecting the structural alterations IC50 concentration of ceranib-2was applied on MCF7 cells for 24 hours. Treated cells were stained with Alexa fluor-488 phalloidine and acridine orange and observed under confocal microscope.
Results: Viability percentages and IC50 (13μM) value were determined. Morphological alterations detected on our confocal micrographs were damaged cytoskeleton as hole formation, shrinked cells and fragmented and condensed nuclei as apoptotic hallmarks.
Conclusion: According to our results, ceranib-2 caused structural changes in MCF7 cells morphology. We can conclude that ceranib-2 showed high cytotoxicity on MCF7 cancer cells in low concentrations and may be encouraging in designing of pharmaceutical products helpful in cancer treatment.

P75: Yeast sphingolipid protein intraction network: function annotation for YDL222C.

F. Betul KAVUN OZBAYRAKTAR¹
1Bogazici University Department Of Chemical Engineering

Objective: Sphingolipids are both structural and regulatory components of the cell, where they control processes decisive in cell’s fate. The first effort of constructing the protein-protein interaction network of sphingolipids in Saccharomyces cerevisiae enabled us to understand the details of the topological properties of the newly constructed network as well as to assign functions to some of the uncharacterized proteins involving in the network of sphingolipids. The topological analysis of sphingolipid related proteins, especially those under clinical trials for cancer therapy, yielded novel potential drug targets. Novel interactions are predicted using a newly developed integrated methodology employing sequence and structure based computational interaction prediction tools, orthology, expression profiles, co-localization information and Gene Ontology (GO) terms. The sphingolipid network shows topological properties of a scale-free, small-world, and modular structure, as it is the case for biological networks. The function annotation of uncharacterized proteins of the network is performed using a multi-dimensional hybrid method which combines the results from modules and neighbors, and examines them by information gathered from genetic interactions, expression profiles, and sequence similarity. The here constructed sphingolipid network coupled with the newly developed hybrid function annotation method constitutes an efficient platform for function annotation and drug target identification.

Very long chain ceramides interfere with c16- ceramide-induced channel formation: a plausible mechanism for regulating the initiation of intrinsic apoptosis.

Johnny STIBAN¹, Meenu PERERA2,
1Birzeit University, 2University Of Maryland

Objective: Mitochondria mediate both cell survival and death. The intrinsic apoptotic pathway is initiated by the permeabilization of the mitochondrial outer membrane to pro-apoptotic intermembrane space proteins. A number of pathways are known to cause the egress of IMS proteins. Of particular interest is the ability of ceramide to self-assemble into dynamic water-filled channels. The formation of ceramide channels is regulated extensively by Bcl-2 family proteins as well as by dihydroceramide, the immediate precursor in the de novo biosynthetic pathway. Here, we present evidence that the chain length of biologically active ceramides serve as an important regulatory factor. Ceramides are synthesized by a family of six mammalian ceramide synthases (CerS) each of which produces a subset of ceramides that differ in their fatty acyl chain length. Interestingly, the presence of very long chain ceramides reduces the potency of C16-mediated permeabilization of mitochondria indicating that the intercalation of the lipids in the dynamic channel is destabilizing. Moreover, cells overexpressing the ceramide synthase responsible for the production of C16-ceramide (CerS5) are more vulnerable to etoposide compared to cells over expressing CerS2 (very long chain fatty acyl ceramides). We also find that co-overexpression of CerS2 and CerS5 reduces the fraction of dead cells upon etoposide treatment, indicating that the product of CerS2 inhibits C16-channel formation in vivo. This interplay between different ceramide metabolic enzymes and their products adds a new dimension to the complexity of mitochondrial-mediated apoptosis, and emphasizes its importance as a key regulatory step that commits cells to life or death.

Ceramide-induced biophysical alterations in membranes of live cells.

Ana Ester VENTURA¹, Sandra PINTO¹, Ana Raquel VARELA¹, Anthony FUTERMAN2, Manuel PRIETO¹, Liana SILVA3,
1Instituto Superior Tecnico, 2Weizmann Institute Of Science, 3Faculdade De Farmácia

Objective: Ceramides are involved in the regulation of several cellular events and implicated in a number of diseases. It has been hypothesized that their mechanism of action is related to the changes promoted by these lipids in the biophysical properties of cell membranes. The main goal of this study was to evaluate ceramide-induced alterations in membrane biophysical properties of live cells. Formation of ceramide upon treating HEK, HeLa or SW620 cells with TNF-α or bacterial sphingomyelinase (bSMase) led to a marked increase in the formation of intracellular vesicles compared to untreated cells. Analysis of membrane order through the variation of Laurdan generalized polarization (GP) showed that these vesicles are more ordered than the plasma membrane or intracellular membranes, displaying GP values typical of a gel phase. Both the extent and biophysical properties of the vesicles was cell-type dependent. Inhibition of neutral sphingomyelinase yielded a clear decrease in the number of vesicles and in the global order of the membrane. These results further suggest that alterations in membrane biophysical properties are driven by ceramide formation. To further evaluate the biological significance of ceramide-induced vesicles, studies were performed to address whether these vesicles would traffic along the endosomal-lysosomal pathway. A time-dependent increase in the colocalization of those vesicles with the endosomal and lysosomal markers, Rab5 and Lamp1, respectively, was observed. This was accompanied by a time-dependent increase in the order of the vesicles, suggesting that ceramide might be a modulator of the endo-lysosomal pathway by changing the biophysical properties of endocytic vesicles.

Unravelling the biophysical properties of sphingolipids: from model to cell membranes.

Ester VENTURA¹, Raquel VARELA2, Sandra PINTO3, Amelia GONCALVES DA SILVA4, Anthony FUTERMAN5, Liana SILVA6, Manuel PRIETO3,
1IMed.UL – Research Institute For Medicines And Pharmaceutical Sciences, Faculdade De Farmácia;Centro De Química-Física Molecular, Instituto Superior Tecnico, Universidade De Lisboa, Lisboa, Portugal, .- 2IMed.UL – Research Institute For Medicines And Pharmaceutical Sciences, Faculdade De Farmácia, Universidade De Lisboa, Portugal; Weizmann Institute Of Sciences, Dept Of Biological Chemistry, Rehovot, Israel; Centro De Química-Física Molecular, Instituto Superior Tecnico, Universidade De Lisboa, Lisboa, Portugal, – 3Centro De Química-Física Molecular, Instituto Superior Tecnico, Universidade De Lisboa, Lisboa, Portugal, – 4Centro De Química Estrutural, Instituto Superior Tecnico, Universidade De Lisboa, Lisboa, Portugal, – 5 Weizmann Institute Of Science, Dept Of Biological Chemistry, Rehovot, Israel, – 6IMed.UL – Research Institute For Medicines And Pharmaceutical Sciences, Faculdade De Farmácia Universidade De Lisboa, Lisboa, Portugal

Objective: Sphingolipids (SLs) have emerged as an important class of lipids due to their bioactive role in several cellular events and in disease. The evidence that several SL species participate in the formation of lipid domains, and that this might underlie their biological mechanism of action has fostered research in the biophysical aspects of bioactive SLs. This work will focus on two important SLs – ceramide and glucosylceramide – and their interplay with other lipid components in simple and complex membrane models.
Methods: A combination of biophysical methodologies that include fluorescence spectroscopy, confocal and two-photon microscopy, surface pressure-area measurements, were used to elucidate the effects of these lipids on the biophysical properties of membranes with different lipidic components and displaying different phase properties.
Results: Our results showed that lipid-lipid interactions are modulated by alterations in the membrane environment, such as changes in pH. Moreover, small structural differences of these lipids influence their packing properties, membrane shaping and lateral organization. The importance of the headgroup, acyl chain length and unsaturation, on the modulation of membrane properties will be discussed in the framework of results obtained for cellular membranes.
Conclusion: Model membrane systems allow to predict the biophysical and biological implications of these lipids in cellular membranes. Supported by FCT (Portugal) grants PTDC/BBB-BQB/0506/2012 and RECI/CTM-POL/0342/2012, SFRH/BD/69982/2010 to ARV, Investigador FCT 2014 to LCS.

Ceramide synthase 4 is involved in the regulation of adult hair follicle stem cell populations.

Franziska PETERS¹, Susanne VORHAGEN¹, Susanne BRODESSER2, Kristin JAKOBSHAGEN3, Jens C. BRÜNING4, Carien M. NIESSEN¹, Martin KRÖNKE3,
1Department Of Dermatology, 2Cologne Excellence Cluster On Cellular Stress Responses In Aging Associated Diseases (CECAD) , 3Institute For Medical Microbiologie, Immunology And Hygiene,4Institute For Genetics

Objective: Ceramides are crucial for skin barrier function but little is known whether stem cell populations that control epidermal regeneration depend on specific ceramide species. Our study shows that ceramide synthase 4 (CerS4) is highly expressed in adult murine epidermis where it is localized in the interfollicular epidermis and specific compartments of the hair follicle where CerS4+ cells co-localize with bulge hair follicle stem cell markers. Inactivation of CerS4 led to precocious activation of hair follicle bulge stem cells. This was manifested in a loss of quiescent label retaining cells and in a continuous growth state of CerS4-/- hair follicles at a time point where CerS4+/+ hair follicles had entered the resting phase. This ultimately led to an almost complete depletion of bulge stem cells in one-year old mice. At the time point where CerS4+/+ hair follicles had entered the resting phase a reduction in BMP target gene mRNA expression was identified in CerS4-/- epidermis, indicating a decrease in BMP signaling. As BMP activity promotes entry into the resting phase and quiescence of bulge stem cells this may explain the inability of CerS4-/- hair follicle stem cells to properly enter the resting phase. Further the reduction in BMP activity likely promoted enhanced Wnt target gene mRNA expression in CerS4-deficient mice. Our data reveal an essential role of CerS4 in the regulation of hair follicle stem and progenitor cell activation and dynamics. In conclusion our data suggest a novel mechanism to hair follicle stem cell activation.