PhD STUDENTS CONTEST

Jeanne Guitton

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22 Mar 2021

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.

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Tobias Kunz

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22 Mar 2021

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

Briana James

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22 Mar 2021

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.

Johannes Morstein

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22 Mar 2021

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.

Patrick Niekamp

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21 Sep 2020

Sphingomyelin serves a critical role in the repair of damaged organelles



Johannes Patrick Niekamp, Joost Holthuis



Department of Chemistry, New York University, New York, New York 10003, United Molecular Cell Biology Division, University of Osnabrück, 49076 Osnabrück, Germany



Sphingomyelin (SM) displays a strict asymmetric distribution across cellular membranes, with the bulk localized to the exoplasmic leaflet of late secretory and endocytic organelles. Using an engineered SM-binding probe, we and others (Ellison et al., 2020, Curr Biol 30, 1-10) found that SM is readily exposed to the cytosolic surface of these organelles upon membrane damage inflicted by pathogenic bacteria, lysosomotropic drugs or a two-photon laser. Remarkably, cytosolic exposure of SM at sites of membrane damage precedes the recruitment of galectins, ESCRT proteins and other components of the membrane repair machinery, suggesting that a break in SM asymmetry may serve a fundamental role in the mechanism by which cells detect and repair damaged organelles. By monitoring the recovery of LysoTracker fluorescence in lysosomes transiently exposed to lysosomotropic drugs, we found that cells lacking SM displayed a significant defect in the repair of damaged lysosomal membranes in comparison to control cells. Moreover, SM-deficient cells showed an enhanced sensitivity to lysosomotropic drugs and a prolonged retention of ESCRT-III components on their damaged lysosomes. These phenotypes could be suppressed by restoring SM biosynthesis. Analogous to the role of phosphatidylserine displayed on the surface of damaged cells, we postulate that cytosolic SM serves as a key indicator of damaged organelles and actively participates in their repair. In this talk, I will present our latest findings regarding the mechanism by which SM contributes to the mending of damaged organelles.

Hana Matuskova

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21 Sep 2020

Sphingosine-1-phoshate signaling in astrocytes is a critical modulator of stroke outcome



Matuskova H. , Matthes F., Petzold G. C. and Meissner A.



Department of Neurology, University Hospital Bonn, Germany

German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany

Department of Experimental Medical Sciences, Lund University, Sweden

Wallenberg Centre for Molecular Medicine, Lund University, Sweden



Stroke remains a leading cause of long-term disability worldwide. Due to its complexity, treatment options are sparse. The bioactive phospholipid sphingosine-1-phosphate (S1P) is involved in variety of physiological processes particularly, in vascular and immune cell responses. Moreover, altered S1P levels have been reported in several cardiovascular and inflammation-associated diseases, including stroke. As astrocytes play a critical role in the regulation of both vascular and immune responses in the injured brain, we sought to investigate astrocytic S1P signaling and its contribution to stroke. In a mouse model of transient middle cerebral artery occlusion (MCAo), we discovered significantly increased expression of one of S1P’s receptors (S1PR3) 24 hrs post-ischemia in the ipsilateral hemisphere. Vessel-parenchyma fractionation of brain tissue revealed the majority of S1PR3 protein associated to cerebral vessels. Astrocyte-specific RiboTag analysis confirmed an augmentation of ipsilateral S1PR3 expression 24 and 72 hrs post-stroke. This was further supported by colocalization of Gfap, a marker of reactive astrocytes, and S1PR3 in the ischemic hemisphere by RNA scope technique. Moreover, single administration of an S1PR3 antagonist 4 hrs after permanent MCAo revealed significant improvements of regional cerebral blood flow in the ipsilateral hemisphere 24 hrs that persisted after 72 hrs. Consequently, infarct size was markedly reduced in mice treated with S1PR3 antagonist. In conclusion, our findings point to an important involvement of the S1P/S1PR3 signaling axis during stroke, and a potential contribution of astrocytes. Modulating S1PR3-mediated vascular and inflammatory responses may emerge as viable target to improving stroke outcome.

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Gabriel Matos

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21 Sep 2020

Reciprocal regulation of cell cycle and sphingolipid metabolism



Gabriel Soares Matos, Mónica Montero Lomeli



Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro



Sphingolipids are central bioactive lipids that have important functions in cell division. However, the control sphingolipid metabolism occurs during cell cycle is not well understood. To answer this question, we used S. cerevisiae as a model. We first studied the gene expression profile of key enzymes involved in the ceramide synthesis pathway and found, by qRT-PCR experiments, that genes involved in the synthesis of long chain bases (LCBs) and ceramides are periodically expressed during the mitotic cell cycle, having a peak at G1/S. This transcription peak coincides the canonical targets of the SBF complex, which is formed by the transcription factor SWI4 and its regulator SWI6, and which play a crucial role in G1/S transition. The transcription of sphingolipid genes was decreased in SBF mutants furthermore, SBF mutants were sensitive to myriocin, which inhibits the first step of sphingolipid synthesis. In addition, HPLC- MS/MS data indicated that the swi4Δ mutant had decreased levels of dihydrosphingosine, phytosphingosine (PHS) and total ceramides. A decrease in the length of ceramide fatty acids and in the hydroxylation of its long chain bases was also observed. The reduced sphingolipid content in swi4Δ strain was mimicked in WT cells by myriocin treatment and resulted in cell cycle arrest at the G2/M phase, which was reversed by addition of PHS to the media. Our results, overall suggests that the SWI4 transcription factor is important to coordinate transcription and synthesis of sphingolipids, which may affect not just membrane architecture, but also the biological activity of ceramides which are reported to control a myriad of phenomena including cell cycle progression.

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Ahmed Elsherbini

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21 Sep 2020

Association of Aβ with ceramide-enriched astrosomes mediates Aβ neurotoxicity



Ahmed Elsherbini, Haiyan Qin, Zhihui Zhu, Priyanka Tripathi, Simone M. Crivelli, and Erhard Bieberich



Department of Physiology, University of Kentucky, Lexington, KY



Amyloid beta (Aβ) is a pathologic hallmark of Alzheimer’s disease (AD), however, the mechanism of Aβ neurotoxicity is not fully understood. Exosomes associate with Aβ, but it is not clear how this association would affect Aβ neurotoxicity. We report that the sphingolipid ceramide mediates neurotoxicity of Aβ. We show that sera and brains from AD transgenic mouse model (5xFAD) and sera from AD patients, but not the WT or healthy controls, contain a subpopulation of astrocyte-derived exosomes that are enriched with ceramide and are prone to aggregation (termed astrosomes) as confirmed by nanoparticle tracking and cluster analyses. When taken up by Neuro2A cells and human iPS cell-derived neurons, these astrosomes are shuttled to mitochondria where they induce mitochondria clustering, evident by elevation of expression of the fission protein dynamin related protein1 (Drp1). Using proximity ligation assays (PLA), we show that Aβ associates with voltage dependent anion channel 1 (VDAC1), a key protein in mitochondria-mediated apoptosis. PLA signals colocalized with ceramide cotransported with Aβ by astrosomes. The interaction between Aβ and VDAC1 leads to caspase3 activation and subsequently apoptosis. This effect was mitigated by removal of the ceramide enriched exosomes from the exosomes pool. Interestingly, the novel ceramide analog N-oleoyl serinol (S18) prevented the aggregation of exosomes, and Aβ association with astrosomes, and reduced Aβ interaction with VDAC1. Our data suggests that association of Aβ with ceramide in astrosomes enhances Aβ interaction with VDAC1 and mediates Aβ neurotoxicity in AD, which can be prevented by novel ceramide analogs.