@article{ZoicasSchumacherKleuseretal.2020, author = {Zoicas, Iulia and Schumacher, Fabian and Kleuser, Burkhard and Reichel, Martin and Gulbins, Erich and Fejtova, Anna and Kornhuber, Johannes and Rhein, Cosima}, title = {The forebrain-specific overexpression of acid sphingomyelinase induces depressive-like symptoms in mice}, series = {Cells}, volume = {9}, journal = {Cells}, number = {5}, publisher = {MDPI}, address = {Basel}, pages = {12}, year = {2020}, abstract = {Human and murine studies identified the lysosomal enzyme acid sphingomyelinase (ASM) as a target for antidepressant therapy and revealed its role in the pathophysiology of major depression. In this study, we generated a mouse model with overexpression of Asm (Asm-tg(fb)) that is restricted to the forebrain to rule out any systemic effects of Asm overexpression on depressive-like symptoms. The increase in Asm activity was higher in male Asm-tg(fb) mice than in female Asm-tg(fb) mice due to the breeding strategy, which allows for the generation of wild-type littermates as appropriate controls. Asm overexpression in the forebrain of male mice resulted in a depressive-like phenotype, whereas in female mice, Asm overexpression resulted in a social anxiogenic-like phenotype. Ceramides in male Asm-tg(fb) mice were elevated specifically in the dorsal hippocampus. mRNA expression analyses indicated that the increase in Asm activity affected other ceramide-generating pathways, which might help to balance ceramide levels in cortical brain regions. This forebrain-specific mouse model offers a novel tool for dissecting the molecular mechanisms that play a role in the pathophysiology of major depression.}, language = {en} } @article{SeitzSchumacherBakeretal.2019, author = {Seitz, Aaron P. and Schumacher, Fabian and Baker, Jennifer and Soddemann, Matthias and Wilker, Barbara and Caldwell, Charles C. and Gobble, Ryan M. and Kamler, Markus and Becker, Katrin Anne and Beck, Sascha and Kleuser, Burkhard and Edwards, Michael J. and Gulbins, Erich}, title = {Sphingosine-coating of plastic surfaces prevents ventilator-associated pneumonia}, series = {Journal of molecular medicine}, volume = {97}, journal = {Journal of molecular medicine}, number = {8}, publisher = {Springer}, address = {Heidelberg}, issn = {0946-2716}, doi = {10.1007/s00109-019-01800-1}, pages = {1195 -- 1211}, year = {2019}, abstract = {Ventilator-associated pneumonia (VAP) is a major cause of morbidity and mortality in critically ill patients. Here, we employed the broad antibacterial effects of sphingosine to prevent VAP by developing a novel method of coating surfaces of endotracheal tubes with sphingosine and sphingosine analogs. Sphingosine and phytosphingosine coatings of endotracheal tubes prevent adherence and mediate killing of Pseudomonas aeruginosa, Acinetobacter baumannii, and Staphylococcus aureus, even in biofilms. Most importantly, sphingosine-coating of endotracheal tubes also prevented P. aeruginosa and S. aureus pneumonia in vivo. Coating of the tubes with sphingosine was stable, without obvious side effects on tracheal epithelial cells and did not induce inflammation. In summary, we describe a novel method to coat plastic surfaces and provide evidence for the application of sphingosine and phytosphingosine as novel antimicrobial coatings to prevent bacterial adherence and induce killing of pathogens on the surface of endotracheal tubes with potential to prevent biofilm formation and VAP.Key messagesNovel dip-coating method to coat plastic surfaces with lipids.Sphingosine and phytosphingosine as novel antimicrobial coatings on plastic surface.Sphingosine coatings of endotracheal tubes prevent bacterial adherence and biofilms.Sphingosine coatings of endotracheal tubes induce killing of pathogens.Sphingosine coatings of endotracheal tubes ventilator-associated pneumonia.}, language = {en} } @misc{BeckerRiethmuellerSeitzetal.2018, author = {Becker, Katrin Anne and Riethmueller, Joachim and Seitz, Aaron P. and Gardner, Aaron and Boudreau, Ryan and Kamler, Markus and Kleuser, Burkhard and Schuchman, Edward and Caldwell, Charles C. and Edwards, Michael J. and Grassme, Heike and Brodlie, Malcolm and Gulbins, Erich}, title = {Sphingolipids as targets for inhalation treatment of cystic fibrosis}, series = {Advanced drug delivery reviews}, volume = {133}, journal = {Advanced drug delivery reviews}, publisher = {Elsevier}, address = {Amsterdam}, issn = {0169-409X}, doi = {10.1016/j.addr.2018.04.015}, pages = {66 -- 75}, year = {2018}, abstract = {Studies over the past several years have demonstrated the important role of sphingolipids in cystic fibrosis (CF), chronic obstructive pulmonary disease and acute lung injury. Ceramide is increased in airway epithelial cells and alveolar macrophages of CF mice and humans, while sphingosine is dramatically decreased. This increase in ceramide results in chronic inflammation, increased death of epithelial cells, release of DNA into the bronchial lumen and thereby an impairment of mucociliary clearance; while the lack of sphingosine in airway epithelial cells causes high infection susceptibility in CF mice and possibly patients. The increase in ceramide mediates an ectopic expression of beta 1-integrins in the luminal membrane of CF epithelial cells, which results, via an unknown mechanism, in a down-regulation of acid ceramidase. It is predominantly this down-regulation of acid ceramidase that results in the imbalance of ceramide and sphingosine in CF cells. Correction of ceramide and sphingosine levels can be achieved by inhalation of functional acid sphingomyelinase inhibitors, recombinant acid ceramidase or by normalization of beta 1-integrin expression and subsequent re-expression of endogenous acid ceramidase. These treatments correct pulmonary inflammation and prevent or treat, respectively, acute and chronic pulmonary infections in CF mice with Staphylococcus aureus and mucoid or non-mucoid Pseudomonas aeruginosa. Inhalation of sphingosine corrects sphingosine levels only and seems to mainly act against the infection. Many antidepressants are functional inhibitors of the acid sphingomyelinase and were designed for systemic treatment of major depression. These drugs could be repurposed to treat CF by inhalation.}, language = {en} } @article{PewznerJungTabazavarehGrassmeetal.2014, author = {Pewzner-Jung, Yael and Tabazavareh, Shaghayegh Tavakoli and Grassme, Heike and Becker, Katrin Anne and Japtok, Lukasz and Steinmann, Joerg and Joseph, Tammar and Lang, Stephan and Tuemmler, Burkhard and Schuchman, Edward H. and Lentsch, Alex B. and Kleuser, Burkhard and Edwards, Michael J. and Futerman, Anthony H. and Gulbins, Erich}, title = {Sphingoid long chain bases prevent lung infection by Pseudomonas aeruginosa}, series = {EMBO molecular medicine}, volume = {6}, journal = {EMBO molecular medicine}, number = {9}, publisher = {Wiley-Blackwell}, address = {Hoboken}, issn = {1757-4676}, doi = {10.15252/emmm.201404075}, pages = {1205 -- 1214}, year = {2014}, abstract = {Cystic fibrosis patients and patients with chronic obstructive pulmonary disease, trauma, burn wound, or patients requiring ventilation are susceptible to severe pulmonary infection by Pseudomonas aeruginosa. Physiological innate defense mechanisms against this pathogen, and their alterations in lung diseases, are for the most part unknown. We now demonstrate a role for the sphingoid long chain base, sphingosine, in determining susceptibility to lung infection by P.aeruginosa. Tracheal and bronchial sphingosine levels were significantly reduced in tissues from cystic fibrosis patients and from cystic fibrosis mouse models due to reduced activity of acid ceramidase, which generates sphingosine from ceramide. Inhalation of mice with sphingosine, with a sphingosine analog, FTY720, or with acid ceramidase rescued susceptible mice from infection. Our data suggest that luminal sphingosine in tracheal and bronchial epithelial cells prevents pulmonary P.aeruginosa infection in normal individuals, paving the way for novel therapeutic paradigms based on inhalation of acid ceramidase or of sphingoid long chain bases in lung infection.}, language = {en} } @article{CarpinteiroBeckerJaptoketal.2015, author = {Carpinteiro, Alexander and Becker, Katrin Anne and Japtok, Lukasz and Hessler, Gabriele and Keitsch, Simone and Pozgajova, Miroslava and Schmid, Kurt W. and Adams, Constantin and M{\"u}ller, Stefan and Kleuser, Burkhard and Edwards, Michael J. and Grassme, Heike and Helfrich, Iris and Gulbins, Erich}, title = {Regulation of hematogenous tumor metastasis by acid sphingomyelinase}, series = {EMBO molecular medicine}, volume = {7}, journal = {EMBO molecular medicine}, number = {6}, publisher = {Wiley-Blackwell}, address = {Hoboken}, issn = {1757-4676}, pages = {714 -- 734}, year = {2015}, abstract = {Metastatic dissemination of cancer cells is the ultimate hallmark of malignancy and accounts for approximately 90\% of human cancer deaths. We investigated the role of acid sphingomyelinase (Asm) in the hematogenous metastasis of melanoma cells. Intravenous injection of B16F10 melanoma cells into wild-type mice resulted in multiple lung metastases, while Asm-deficient mice (Smpd1(-/-) mice) were protected from pulmonary tumor spread. Transplanting wild-type platelets into Asm-deficient mice reinstated tumor metastasis. Likewise, Asm-deficient mice were protected from hematogenous MT/ret melanoma metastasis to the spleen in a mouse model of spontaneous tumor metastasis. Human and mouse melanoma cells triggered activation and release of platelet secretory Asm, in turn leading to ceramide formation, clustering, and activation of 51 integrins on melanoma cells finally leading to adhesion of the tumor cells. Clustering of integrins by applying purified Asm or C-16 ceramide to B16F10 melanoma cells before intravenous injection restored trapping of tumor cells in the lung in Asm-deficient mice. This effect was revertable by arginine-glycine-aspartic acid peptides, which are known inhibitors of integrins, and by antibodies neutralizing 1 integrins. These findings indicate that melanoma cells employ platelet-derived Asm for adhesion and metastasis.}, language = {en} } @misc{HollmannReuterWerneretal.2016, author = {Hollmann, C. and Reuter, D. and Werner, S. and Avota, Elita and Mueller, N. and Japtok, Lukasz and Kleuser, Burkhard and Becker, Katrin Anne and Gulbins, Erich and Schneider-Schaulies, J{\"u}rgen and Beyersdorf, Niklas}, title = {Pharmacological inhibition of acid sphingomyelinase or genetic ablation enhances CD4(+) Foxp3(+) regulatory T cell activity}, series = {European journal of immunology}, volume = {46}, journal = {European journal of immunology}, publisher = {Wiley-Blackwell}, address = {Hoboken}, issn = {0014-2980}, pages = {14 -- 14}, year = {2016}, language = {en} } @article{BeckmannKadowSchumacheretal.2018, author = {Beckmann, Nadine and Kadow, Stephanie and Schumacher, Fabian and Goethert, Joachim R. and Kesper, Stefanie and Draeger, Annette and Schulz-Schaeffer, Walter J. and Wang, Jiang and Becker, Jan U. and Kramer, Melanie and Kuehn, Claudine and Kleuser, Burkhard and Becker, Katrin Anne and Gulbins, Erich and Carpinteiro, Alexander}, title = {Pathological manifestations of Farber disease in a new mouse model}, series = {Biological chemistry}, volume = {399}, journal = {Biological chemistry}, number = {10}, publisher = {De Gruyter}, address = {Berlin}, issn = {1431-6730}, doi = {10.1515/hsz-2018-0170}, pages = {1183 -- 1202}, year = {2018}, abstract = {Farber disease (FD) is a rare lysosomal storage disorder resulting from acid ceramidase deficiency and subsequent ceramide accumulation. No treatments are clinically available and affected patients have a severely shortened lifespan. Due to the low incidence, the pathogenesis of FD is still poorly understood. Here, we report a novel acid ceramidase mutant mouse model that enables the study of pathogenic mechanisms of FD and ceramide accumulation. Asah1(tmEx1) mice were generated by deletion of the acid ceramidase signal peptide sequence. The effects on lysosomal targeting and activity of the enzyme were assessed. Ceramide and sphingomyelin levels were quantified by liquid chromatography tandem-mass spectrometry (LC-MS/MS) and disease manifestations in several organ systems were analyzed by histology and biochemistry. We show that deletion of the signal peptide sequence disrupts lysosomal targeting and enzyme activity, resulting in ceramide and sphingomyelin accumulation. The affected mice fail to thrive and die early. Histiocytic infiltrations were observed in many tissues, as well as lung inflammation, liver fibrosis, muscular disease manifestations and mild kidney injury. Our new mouse model mirrors human FD and thus offers further insights into the pathogenesis of this disease. In the future, it may also facilitate the development of urgently needed therapies.}, language = {en} } @article{WiggerGulbinsKleuseretal.2019, author = {Wigger, Dominik and Gulbins, Erich and Kleuser, Burkhard and Schumacher, Fabian}, title = {Monitoring the Sphingolipid de novo Synthesis by Stable-Isotope Labeling and Liquid Chromatography-Mass Spectrometry}, series = {Frontiers in Cell and Developmental Biology}, volume = {7}, journal = {Frontiers in Cell and Developmental Biology}, publisher = {Frontiers Media}, address = {Lausanne}, issn = {2296-634X}, doi = {10.3389/fcell.2019.00210}, pages = {16}, year = {2019}, abstract = {Sphingolipids are a class of lipids that share a sphingoid base backbone. They exert various effects in eukaryotes, ranging from structural roles in plasma membranes to cellular signaling. De novo sphingolipid synthesis takes place in the endoplasmic reticulum (ER), where the condensation of the activated C₁₆ fatty acid palmitoyl-CoA and the amino acid L-serine is catalyzed by serine palmitoyltransferase (SPT). The product, 3-ketosphinganine, is then converted into more complex sphingolipids by additional ER-bound enzymes, resulting in the formation of ceramides. Since sphingolipid homeostasis is crucial to numerous cellular functions, improved assessment of sphingolipid metabolism will be key to better understanding several human diseases. To date, no assay exists capable of monitoring de novo synthesis sphingolipid in its entirety. Here, we have established a cell-free assay utilizing rat liver microsomes containing all the enzymes necessary for bottom-up synthesis of ceramides. Following lipid extraction, we were able to track the different intermediates of the sphingolipid metabolism pathway, namely 3-ketosphinganine, sphinganine, dihydroceramide, and ceramide. This was achieved by chromatographic separation of sphingolipid metabolites followed by detection of their accurate mass and characteristic fragmentations through high-resolution mass spectrometry and tandem-mass spectrometry. We were able to distinguish, unequivocally, between de novo synthesized sphingolipids and intrinsic species, inevitably present in the microsome preparations, through the addition of stable isotope-labeled palmitate-d₃ and L-serine-d₃. To the best of our knowledge, this is the first demonstration of a method monitoring the entirety of ER-associated sphingolipid biosynthesis. Proof-of-concept data was provided by modulating the levels of supplied cofactors (e.g., NADPH) or the addition of specific enzyme inhibitors (e.g., fumonisin B₁). The presented microsomal assay may serve as a useful tool for monitoring alterations in sphingolipid de novo synthesis in cells or tissues. Additionally, our methodology may be used for metabolism studies of atypical substrates - naturally occurring or chemically tailored - as well as novel inhibitors of enzymes involved in sphingolipid de novo synthesis.}, language = {en} } @article{NeuberSchumacherGulbinsetal.2014, author = {Neuber, Corinna and Schumacher, Fabian and Gulbins, Erich and Kleuser, Burkhard}, title = {Method to simultaneously determine the sphingosine 1-phosphate breakdown product (2E)-hexadecenal and its fatty acid derivatives using isotope-dilution HPLC-electrospray ionization-quadrupole/time-of-flight mass spectrometry}, series = {Analytical chemistry}, volume = {86}, journal = {Analytical chemistry}, number = {18}, publisher = {American Chemical Society}, address = {Washington}, issn = {0003-2700}, doi = {10.1021/ac501677y}, pages = {9065 -- 9073}, year = {2014}, abstract = {Sphingosine 1-phosphate (S1P), a bioactive lipid involved in various physiological processes, can be irreversibly degraded by the membrane-bound S1P lyase (S1PL) yielding (2E)-hexadecenal and phosphoethanolamine. It is discussed that (2E)-hexadecenal is further oxidized to (2E)-hexadecenoic acid by the long-chain fatty aldehyde dehydrogenase ALDH3A2 (also known as FALDH) prior to activation via coupling to coenzyme A (CoA). Inhibition or defects in these enzymes, S1PL or FALDH, result in severe immunological disorders or the Sjogren-Larsson syndrome, respectively. Hence, it is of enormous importance to simultaneously determine the S1P breakdown product (2E)-hexadecenal and its fatty acid metabolites in biological samples. However, no method is available so far. Here, we present a sensitive and selective isotope-dilution high performance liquid chromatographyelectrospray ionizationquadrupole/time-of-flight mass spectrometry method for simultaneous quantification of (2E)-hexadecenal and its fatty acid metabolites following derivatization with 2-diphenylacetyl-1,3-indandione-1-hydrazone and 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide. Optimized conditions for sample derivatization, chromatographic separation, and MS/MS detection are presented as well as an extensive method validation. Finally, our method was successfully applied to biological samples. We found that (2E)-hexadecenal is almost quantitatively oxidized to (2E)-hexadecenoic acid, that is further activated as verified by cotreatment of HepG2 cell lysates with (2E)-hexadecenal and the acyl-CoA synthetase inhibitor triacsin C. Moreover, incubations of cell lysates with deuterated (2E)-hexadecenal revealed that no hexadecanoic acid is formed from the aldehyde. Thus, our method provides new insights into the sphingolipid metabolism and will be useful to investigate diseases known for abnormalities in long-chain fatty acid metabolism, e.g., the Sjogren-Larsson syndrome, in more detail.}, language = {en} } @article{NeuberSchumacherGulbinsetal.2017, author = {Neuber, Corinna and Schumacher, Fabian and Gulbins, Erich and Kleuser, Burkhard}, title = {Mass Spectrometric Determination of Fatty Aldehydes Exemplified by Monitoring the Oxidative Degradation of (2E)-Hexadecenal in HepG2 Cell Lysates}, series = {Lipidomics}, volume = {125}, journal = {Lipidomics}, publisher = {Humana Press}, address = {Totowa}, isbn = {978-1-4939-6946-3}, issn = {0893-2336}, doi = {10.1007/978-1-4939-6946-3_10}, pages = {147 -- 158}, year = {2017}, abstract = {Within the last few decades, liquid chromatography-mass spectrometry (LC-MS) has become a preferred method for manifold issues in analytical biosciences, given its high selectivity and sensitivity. However, the analysis of fatty aldehydes, which are important components of cell metabolism, remains challenging. Usually, chemical derivatization prior to MS detection is required to enhance ionization efficiency. In this regard, the coupling of fatty aldehydes to hydrazines like 2,4-dinitrophenylhydrazine (DNPH) is a common approach. Additionally, hydrazones readily react with fatty aldehydes to form stable derivatives, which can be easily separated using high-performance liquid chromatography (HPLC) and subsequently detected by MS. Here, we exemplarily present the quantification of the long-chain fatty aldehyde (2E)-hexadecenal, a break-down product of the bioactive lipid sphingosine 1-phosphate (S1P), after derivatization with 2-diphenylacetyl-1,3-indandione-1-hydrazone (DAIH) via isotope-dilution HPLC-electrospray ionization-quadrupole/time-of-flight (ESI-QTOF) MS. Moreover, we show that the addition of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC hydrochloride) as a coupling agent allows for simultaneous determination of fatty aldehydes and fatty acids as DAIH derivatives. Taking advantage of this, we describe in detail how to monitor the degradation of (2E)-hexadecenal and the concurrent formation of its oxidation product (2E)-hexadecenoic acid in lysates of human hepatoblastoma (HepG2) cells within this chapter.}, language = {en} }