C. Role of distinct ceramide species in the development of atherosclerosis

Professor Jens Brüning

Rationale and aims

Ceramides have been classically viewed as a homogeneous group of sphingolipids that have also been linked to metabolic and vascular disease, respectively. However, ectopic accumulation of ceramides has evolved a major risk factor for the development of obesity-associated insulin resistance and has been also linked to vascular inflammatory disease such as atherosclerosis.

Current state of research and own preliminary work

Figure 1: SILAC based chemoproteomic screen reveals CERS5 and CERS6-derived sphingolipid binding-proteins. (A) Experimental design of proteomic screen to identify specifically CERS5- and CERS6-derived sphingolipid interacting-proteins. Theoretical MS1 SILAC spectra that indicate the loss of sphingolipid/protein interaction in CERS5- or CERS6- deficient cells. SILAC channel color code: light: green, medium: orange, heavy: red. (B) Volcano plot showing the log2 fold change of SGPL1Δ (- UV) versus SGPL1Δ (+ UV) against the –log10 p value of a one-sided t-test. Significance was considered for Benjamini-Hochberg corrected P-values < 0.05 and log2 fold change > 0.58. Proteins that are annotated by ‘ceramide’ in the GOBP (Biological Process) term are annotated by red color. (C) Volcano plot of log2 fold change of CERS6Δ/SGPL1Δ (+ UV) versus CERS5Δ/SGPL1Δ (+) UV against the –log10 P-value of a two-sided t-test. FDR < 0.05 and log2 fold change > 0.58 was used as the significance cut off.

Interestingly, C16-containing ceramides, whose synthesis is specifically catalyzed by ceramide synthase enzymes (CerS)-5 and -6, play a pivotal role in the development of metabolic disorders. We demonstrated that by specifically reducing CerS6-derived C16 ceramides protection against obesity and obesity associated insulin resistance is conferred (Turpin et al., Cell Metab. 2014). However and in striking contrast ablation of CerS5-dependent C16 ceramide formation fails to protect from the adverse metabolic consequences of high fat diet-induced obesity (Hammerschmidt et al., under revision). We also revealed that CerS5- and CerS6-dependently generated C16-ceramides differentially localize to the ER and mitochondria, respectively (Hammerschmidt et al., under revision). In order to test, whether CerS5- or CerS6-dependently formed C16 ceramides according to their distinct subcellular localization might also interact with distinct effector proteins, we performed immunoprecipitations with photoactivatable ceramide precursors, which were incubated in control cells, CerS5-deficient, as well as CerS6-deficient cell lines. Through the comparative analyses of ceramide-interacting partners as identified by SILAC-based quantitative mass-spectrometry approaches, we indeed successfully identified proteins that specifically interact with C16 ceramides dependent on whether they were generated by CerS5 or CerS6 (Figure 1). Strikingly, CerS6-dependently-formed C16 ceramides specifically interact with the mitochondrial fission factor (Mff), a key regulator of mitochondrial dynamics and function. Collectively, we have defined a novel effector mechanism, how distinct acyl-chain lengths ceramides dependent on the subcellular localisation of their specific synthetizing enzymes determines specific metabolic functions.

Experimental approach and work program

In the proposed study, we aim to delineate the specific underlying molecular and cellular mechanisms through which CerS6-derived C16-ceramides promote atherosclerotic lesion formation. To this end, we will identify the subcellular localization of CerS5- and CerS6-dependently generated C16 ceramides in endothelial cells and atherosclerotic plaque using LDLR-/- mice. Finally, we will study the specific contribution of the thereby identified signaling network(s) in control of lipid-induced ER-stress activation in endothelial and smooth muscle cells – key events during dysregulated stress signaling in vascular dysfunction and functionally validate the contribution of CerS6-dependently formed C16 ceramides in the progression of atherosclerosis through analysis of conventional as well as myeloid lineage, vascular smooth muscle as well as endothelial-specific CerS6-deficient mice. Collectively, these experiments will unravel novel molecular effector networks and pathways of critical importance for the pathogenesis of atherosclerosis associated with obesity.

Potential therapeutic implications

This project will further promote the development of specific CerS6 inhibitors, which may have the potential to emerge as pharmacological tools to treat vascular inflammatory diseases such as atherosclerosis.

Added value through collaborations within the CCRC

The project ideally integrates into the framework of the proposed graduate program since it aims to unravel a novel regulatory principle of stress-response alterations in metabolism and vascular dysfunction. Close collaborations with the Trifunovic and Pasparakis groups will allow us to actively pursue studies on mitochondrial dynamics as well as interactions. Moreover, the core facility of mass spectrometry based proteomics will be utilized (C. Frese / M. Krüger). Ultimately, this research project will allow us to define the link between C16-ceramide-induced dysregulation of ER-stress and the activation of inflammatory signaling cascades. In addition, the analysis of atheroclerosis progression in the different CerS6-deficient mouse models will be pursued in close collaboration with the group of S. Rosenkranz.

General research interests

The research fields of Prof. Brüning include the study of central and peripheral regulation of energy homeostasis using modern mouse genetics, the generation and characterization of mouse models of insulin resistance in obesity, diabetes mellitus, inflammation and atherosclerosis and of mouse models of aging-related diseases. The Brüning lab has access to the most advanced technology for metabolic characterization of mice, including equipment for behavior tests, phenomaster, NMR analyzer, and a mouse CT. In addition, euglycemic, hyperinsulinemic clamps in mice for complete in vivo profiling of glucose metabolism in mice is provided.

Jens Brüning’s profile

Jens Brüning is Professor of Genetics at the Institute for Genetics, where he heads the Department of Mouse Genetics and Metabolism since 2003. At the same time, he is Director of the Center for Endocrinology, Diabetes and Preventive Medicine, University Hospital Cologne and, since 2011, Director of the Max Planck-Institute for Neurological Research in Cologne. He is Coordinator of the Cologne Excellence Cluster “Cellular stress responses in aging-associated diseases (CECAD)” at the University of Cologne. He received his education at the Medical School, University Hospital of Cologne and was a postdoctoral fellow at the Joslin Diabetes Center, Harvard Medical School, in the laboratory of Professor C. Ronald Kahn. Prof. Brüning is the recipient of several awards, among them the Outstanding Scientific Achievement Award of the American Diabetes Association, the Ernst Jung-Award for Medicine, the Minkowski Prize of the European Association for the Study of Diabetes (EASD), the Gottfried Wilhelm Leibniz-Award of the Deutsche Forschungsgemeinschaft, the Lesser-Loewe-Award, the Wilhelm-Vaillant-Award, the Ferdinand-Bertram-Award of the German Diabetes-Society, and the Ernst and Berta Scharrer Award of the German Society of Endocrinology.