2nd PhD STUDENTS CONTEST

Johannes Morstein

Optical Control of Sphingosine-1-Phosphate Formation and Function

Johannes Morstein, Rose Z. Hill, Alexander J. E. Novak, Suihan Feng, Derek D. Norman, Prashant C. Donthamsetti, James A. Frank, Takeshi Harayama, Benjamin M. Williams, Abby L. Parrill, Gabor J. Tigyi, Howard Riezman, Ehud Y. Isacoff, Diana M. Bautista, Dirk Trauner

Department of Chemistry, New York University, New York, New York 10003, United States.
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States.
Department of Biochemistry, University of Geneva, Geneva, Switzerland.
National Centre of Competence in Research (NCCR) in Chemical Biology, University of Geneva, Geneva, Switzerland.
Department of Physiology, College of Medicine, University of Tennessee Health Science Center (UTHSC), Memphis, Tennessee 39163.
Department of Chemistry and Center for Integrated Protein Science, Ludwig Maximilians University Munich, 81377 Munich, Germany.
Department of Chemistry, University of Memphis, Memphis, Tennessee 38152, United States.
Computational Research on Materials Institute, University of Memphis, Memphis, Tennessee 38152, United States.
Helen Wills Neuroscience Institute, University of California, Berkeley, Berkeley, United States.

Sphingosine-1-phosphate (S1P) plays important roles as a signaling lipid in a variety of physiological and pathophysiological processes. S1P regulates angiogenesis, cell proliferation and migration, immunity, and pain. S1P signals via a family of G Protein Coupled Receptors (S1P1-5 receptors) as well as a number of intracellular proteins including HDAC, TRAF2, and PKC. Here we report on photoswitchable analogs of S1P and its precursor sphingosine, respectively termed PhotoS1P and PhotoSph. PhotoS1P enables optical control of S1P1-5 receptor activity in vitro, shown through its ability to rapidly and reversibly control S1P-sensitive currents and S1P-evoked increases in intracellular Ca2+ via engagement of S1P receptors in cultured cells. We evaluated PhotoS1P in vivo, where it reversibly controlled S1P Receptor 3-dependent pain hypersensitivity in mice via activation of nociceptive somatosensory neurons. The pain hypersensitivity induced by PhotoS1P is comparable to that induced by S1P. PhotoS1P is uniquely suited for the study of S1P biology in cultured cells and in vivo because it exhibits prolonged metabolic stability compared to rapidly metabolized S1P. Using lipidomic analysis, we constructed a comprehensive metabolic map of PhotoS1P and PhotoSph. The formation these photoswitchable lipids was found to be light-dependent, providing a novel tool to optically probe sphingolipid biology.

Briana James

Roles of ceramide in allergic asthma exacerbation

Briana N. James, Clement Oyeniran, Jamie L. Sturgill, Jason Newton, Rebecca Martin, Erhard Bieberich, Cynthia Weigel, Melissa A. Maczis, Elisa N. D. Palladino PhD1, Joseph C. Lownik, John B. Trudeau, Joan M. Cook-Mills, Sally Wenzel, Sheldon Milstien, and Sarah Spiegel

Departments of Biochemistry and Molecular Biology and Microbiology and Immunology, Virginia Commonwealth University School of Medicine, Richmond, VA, USA

Despite growing evidence supporting the involvement of sphingolipids in asthma, little is known about the mechanism by which increased ceramide contributes to allergic responses. The aim of our study was to investigate the role of ceramide in allergic asthma by utilizing mouse models that recapitulate key symptoms of human allergic asthma. Mice were sensitized and challenged intranasally with house dust mite or the fungal allergen Alternaria alternata to induce an allergic asthma response. Allergen challenge caused significant lung cell death, increased levels of reactive oxygen species, and neutrophil infiltration. Concomitantly, ceramide levels in lung and bronchoalveolar lavage were significantly increased. Moreover, specific ceramide species were increased in bronchoalveolar lavage fluid from patients with severe asthma and correlated with airway neutrophilia. Interestingly, suppression of lung ceramide levels protected against allergen-induced apoptosis, reactive oxygen species, and neutrophil infiltration. However, decreasing lung reactive oxygen species by dietary supplementation of antioxidant α-tocopherol did not affect levels of ceramide or apoptosis, suggesting that both are independent of oxidative stress. Our results suggest that ceramide elevation after allergen challenge contributes to apoptosis, reactive oxygen species generation, and neutrophilic infiltrate that characterize the severe asthmatic phenotype. Ceramide could be a biomarker to optimize diagnosis and to monitor and improve clinical outcomes in this disease.

Tobias Kunz

Sphingolipid expansion microscopy

Tobias C. Kunz, Ralph Götz, Julian Fink, Franziska Solger, Jan Schlegel, Jürgen Seibel, Vera Kozjak-Pavlovic, Thomas Rudel & Markus Sauer

Julius-Maximilians University Würzburg, Department of Microbiology, Germany

Superresolution microscopy enables to bypass the diffraction limit of fluorescence light microscopy of around 200-250nm. However, such approaches enabling a resolution <100 nm require expensive setups as well as expert knowledge and are therefore limited to highly specialized laboratories. Recently, Boyden and colleagues introduced an alternative tool, the so-called expansion microscopy (ExM). ExM enables superresolution microscopy on a conventional confocal setup by embedding the sample into a swellable hydrogel which gets isotropically expanded. Since its introduction in 2015, ExM developed rapidly with various different protocols for 4x, 10x or even 20x expansion of proteins and RNA in cells, clinical specimens and tissues. As part of my PhD, we demonstrated the applicability of ExM to study host-pathogen interactions of bacterial pathogens, such as Chlamydia trachomatis, Simkania negevensis, Neisseria gonorrhoeae and Staphylococcus aureus. Since the importance of sphingolipids during infection of various pathogens is well established, we introduced an approach to link functionalized lipids into the hydrogel for expansion, which we termed sphingolipid expansion microscopy, and were therefore the first to enable lipid expansion. Our novel tool enabled the nanoscale visualization of ω-N3-sphingosine in the membrane of Neisseria gonorrhoeae and α-NH2-ω-N3-C6-ceramide in the membrane of Chlamydia trachomatis and Simkania negevensis. The efficient accumulation of our functionalized lipids enables in combination with tenfold expansion to study interactions between proteins and plasma membrane, organelles and bacteria. In addition, we could show the incorporation of α-NH2-ω-N3-C6-ceramide in the inner and outer membrane of individual chlamydial particles and determine their distance to 27.6 ± 7.7 nm.

References:
Kunz TC, Ralph G, Sauer M, Rudel T; Detection of Chlamydia developmental forms and secreted effectors by expansion microscopy, Front Cell Infect Microbiol, 2019, 9:276
Ralph G, Kunz TC, Fink J, Solger F, Schlegel J, Seibel J, Kozjak-Pavlovic V, Rudel T, Sauer M; Nanoscale imaging of cellular and bacterial membranes by sphingolipid expansion microscopy, Nat commun, 2020, 11:6173
Solger F, Kunz TC, Fink J, Paprotka K, Pfister P, Hagen F, Schumacher F, Kleuser B, Seibel J, Rudel T; A Role of sphingosine in the intracellular survival of Neisseria gonorrhoeae, Front Cell Infect Microbiol, 2020, 10:215
Kunz TC, Kozjak-Pavlovic V; Diverse facets of sphingolipid involvement in bacterial infections, Front Cell Dev Biol, 2019, 7:203
Kunz TC, Ruehling M, Moldovan A, Paprotka K, Kozjak-Pavlovic V, Rudel T, Fraunholz M; The expandables: Cracking the staphylococcal cell wall for expansion microscopy, Front Cell Infect Microbiol, in print

Jeanne Guitton

Involvement of hypothalamic de novo ceramide synthesis in resistin induced neuronal inflammation, insulin resistance and glucose intolerance.

Jeanne Guitton, Mohammed Taouis, Yacir Benomar, Hervé Le Stunff.

Paris-Saclay Institute of Neurosciences, CNRS UMR 9197, Paris-Saclay University, France

During obesity, the adipokine resistin, like saturated fatty acids, lead to an impairment of glucose homeostasis control by the hypothalamus, a risk factor for type 2 diabetes (T2D). We investigate the involvement of hypothalamic de novo ceramide synthesis in resistin-induced neuronal inflammation and insulin resistance which lead, to glucose intolerance. Using the mHypoA mouse hypothalamic cell line, we analyzed the impact of resistin overexposure on expression levels of enzymes driving ceramide biosynthesis. Intracellular ceramide contents were quantified by lipidomic analysis. Myriocin, a pharmacological inhibitor was used to evaluate de novo ceramide synthesis involvement in resistin-induced neuronal inflammation and defect of insulin signaling. In C57BL6J mice we evaluated the impact of resistin intracerebroventricular (ICV) infusion on hypothalamic expression of enzymes involved in ceramide biosynthesis. We studied the impact of serine palmitoyl-transferase 1 (SPT1) hypothalamic invalidation by adenoviral shRNA strategy on neuronal inflammation and glucose intolerance induced by resistin ICV infusion. In mHypoA cells, we show that resistin treatment increases ceramide contents and expression levels of enzymes driving de novo ceramide synthesis. Resistin overexposure induces inflammation and inhibits insulin signaling in a de novo ceramide synthesis-dependent manner. In mice, resistin ICV infusion upregulates hypothalamic gene expression of enzymes driving de novo ceramide biosynthesis. In vivo invalidation of hypothalamic SPT1 counteracts resistin-induced inflammation and prevents glucose intolerance. These findings reveal de novo ceramide synthesis as a new regulatory pathway of neuronal inflammation and insulin resistance that drive resistin-induced glucose intolerance. This pathway may constitute a breakthrough to overcome obesity and T2D occurrence.

©2020 by Sphingolipid Biology: Sphingolipids in Physiology and Pathology