TY - JOUR A1 - Kunz, Tobias C. A1 - Götz, Ralph A1 - Gao, Shiqiang A1 - Sauer, Markus A1 - Kozjak-Pavlovic, Vera T1 - Using Expansion Microscopy to Visualize and Characterize the Morphology of Mitochondrial Cristae JF - Frontiers in Cell and Developmental Biology N2 - Mitochondria are double membrane bound organelles indispensable for biological processes such as apoptosis, cell signaling, and the production of many important metabolites, which includes ATP that is generated during the process known as oxidative phosphorylation (OXPHOS). The inner membrane contains folds called cristae, which increase the membrane surface and thus the amount of membrane-bound proteins necessary for the OXPHOS. These folds have been of great interest not only because of their importance for energy conversion, but also because changes in morphology have been linked to a broad range of diseases from cancer, diabetes, neurodegenerative diseases, to aging and infection. With a distance between opposing cristae membranes often below 100 nm, conventional fluorescence imaging cannot provide a resolution sufficient for resolving these structures. For this reason, various highly specialized super-resolution methods including dSTORM, PALM, STED, and SIM have been applied for cristae visualization. Expansion Microscopy (ExM) offers the possibility to perform super-resolution microscopy on conventional confocal microscopes by embedding the sample into a swellable hydrogel that is isotropically expanded by a factor of 4–4.5, improving the resolution to 60–70 nm on conventional confocal microscopes, which can be further increased to ∼ 30 nm laterally using SIM. Here, we demonstrate that the expression of the mitochondrial creatine kinase MtCK linked to marker protein GFP (MtCK-GFP), which localizes to the space between the outer and the inner mitochondrial membrane, can be used as a cristae marker. Applying ExM on mitochondria labeled with this construct enables visualization of morphological changes of cristae and localization studies of mitochondrial proteins relative to cristae without the need for specialized setups. For the first time we present the combination of specific mitochondrial intermembrane space labeling and ExM as a tool for studying internal structure of mitochondria. KW - Expansion microscopy KW - mitochondria KW - cristae KW - structured illumination microscope KW - ultrastructure Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-208296 SN - 2296-634X VL - 8 ER - TY - THES A1 - De Lira, Maria Nathalia T1 - The regulation of T cell metabolism by neutral sphingomyelinase 2 T1 - Die Regulation des T-Zell-Metabolismus durch Neutrale Sphingomyelinase 2 N2 - T cells play an essential role in the immune system. Engaging the T cell receptor (TCR) initiates a cascade of signaling events that activates the T cells. Neutral sphingomyelinase (NSM) is a member of a superfamily of enzymes responsible for the hydrolysis of sphingomyelin into phosphocholine and ceramide. Sphingolipids are essential mediators in signaling cascades involved in apoptosis, proliferation, stress responses, necrosis, inflammation, autophagy, senescence, and differentiation. Upon specific ablation of NSM2, T cells proved to be hyper-responsive to CD3/CD28 co-stimulation, indicating that the enzyme acts to dampen early overshooting activation of these cells. It remained unclear whether a deregulated metabolic activity supports the hyper-reactivity of NSM2 deficient T cells. This work demonstrates that the ablation of NSM2 activity affects the metabolism of the quiescent CD4+ T cells. These accumulate ATP in mitochondria and increase basal glycolytic activity by increasing the basal glucose uptake and GLUT1 receptor expression, which, altogether, raises intracellular ATP levels and boosts cellular respiration. The increased basal metabolic activity is associated with rapid phosphorylation of S6, a mTORC1 target, as well as enhanced elevation total ATP levels within the first hour after CD3/CD28 costimulation. Increased metabolic activity in resting NSM2 deficient T cells does, however, not support sustained stimulated responses. While elevated under steady-state conditions and elevated early after co-stimulation in NSM2 deficient CD4+ T cells, the mTORC1 pathway regulating mitochondria size, oxidative phosphorylation, and ATP production is impaired after 24 hours of stimulation. Taken together, the absence of NSM2 promotes a hyperactive metabolic state in unstimulated CD4+ T cells yet fails to support sustained T cell responses upon antigenic stimulation without affecting T cell survival. N2 - T-Zellen spielen eine wesentliche Rolle im Immunsystem. Die Aktivierung des T-Zell-Rezeptors (TCR) löst eine Kaskade von Signalereignissen aus, die die T-Zellen aktivieren. Neutrale Sphingomyelinase (NSM) gehört zu einer Superfamilie von Enzymen, die für die Aufspaltung von Sphingomyelin in Phosphocholin und Ceramid verantwortlich sind. Sphingolipide sind wesentliche Mediatoren in Signalkaskaden, die an Apoptose, Proliferation, Stressreaktionen, Nekrose, Entzündung, Autophagie, Seneszenz und Differenzierung beteiligt sind. NSM2-depletierte T-Zellen erwiesen sich als hyper-reaktiv gegenüber CD3/CD28-Kostimulation, was darauf hinweist, dass das Enzym eine überschießende Aktivierung dieser Zellen dämpft. Es blieb unklar, ob die Hyperreaktivität NSM2-defizienter T-Zellen durch eine deregulierte Stoffwechselaktivität unterstützt wird. Diese Arbeit zeigt, dass NSM2-Insuffizienz den Metabolismus ruhender CD4+-T-Zellen beeinflusst: Diese akkumulieren ATP in Mitochondrien und zeigen eine erhöhte basale glykolytische Aktivität, die auf einer erhöhten Glukoseaufnahme und Expression des GLUT1-Rezeptors beruht und mit einer Erhöhung intrazellulärer ATP-Werte und gesteigerten Zellrespiration einhergeht. Aufgrund ihrer bereits erhöhten basalen metabolische Aktivität zeigen NSM2 defiziente T Zellen eine im Vergleich zu Kontrollzellen schnellere, effizientere Aktivierung nach Kostimulation, die sich in Phosphorylierung von S6, eines mTORC1 Targets, sowie erhöhtem ATP Spiegel manifestiert. Dies kann jedoch nicht aufrechterhalten werden:Die mTORC1-Aktivierung, die die Größe der Mitochondrien, die oxidative Phosphorylierung und die ATP-Produktion reguliert, unter stationären Bedingungen in NSM2-defizienten CD4+-T-Zellen erhöht ist, ist nach 24-stündiger Kostimulation beeinträchtigt. Insgesamt scheint die NSM2-Aktivität wesentlich für die Regulation der basalen metabolischen Aktivität ruhender T-Zellen und der Vermeidung überschiessender Antworten nach Kostimulation zu sein, jedoch ebenso wichtig für die dauerhafte Aufrechterhaltung des Aktivierungssignals zu sein. KW - T zellen KW - metabolism KW - t cell KW - NSM2 KW - mitochondria Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-215673 ER - TY - JOUR A1 - Wagner, Michael A1 - Bertero, Edoardo A1 - Nickel, Alexander A1 - Kohlhaas, Michael A1 - Gibson, Gary E. A1 - Heggermont, Ward A1 - Heymans, Stephane A1 - Maack, Christoph T1 - Selective NADH communication from α-ketoglutarate dehydrogenase to mitochondrial transhydrogenase prevents reactive oxygen species formation under reducing conditions in the heart JF - Basic Research in Cardiology N2 - In heart failure, a functional block of complex I of the respiratory chain provokes superoxide generation, which is transformed to H\(_2\)O\(_2\) by dismutation. The Krebs cycle produces NADH, which delivers electrons to complex I, and NADPH for H\(_2\)O\(_2\) elimination via isocitrate dehydrogenase and nicotinamide nucleotide transhydrogenase (NNT). At high NADH levels, α-ketoglutarate dehydrogenase (α-KGDH) is a major source of superoxide in skeletal muscle mitochondria with low NNT activity. Here, we analyzed how α-KGDH and NNT control H\(_2\)O\(_2\) emission in cardiac mitochondria. In cardiac mitochondria from NNT-competent BL/6N mice, H\(_2\)O\(_2\) emission is equally low with pyruvate/malate (P/M) or α-ketoglutarate (α-KG) as substrates. Complex I inhibition with rotenone increases H2O2 emission from P/M, but not α-KG respiring mitochondria, which is potentiated by depleting H\(_2\)O\(_2\)-eliminating capacity. Conversely, in NNT-deficient BL/6J mitochondria, H2O2 emission is higher with α-KG than with P/M as substrate, and further potentiated by complex I blockade. Prior depletion of H\(_2\)O\(_2\)-eliminating capacity increases H\(_2\)O\(_2\) emission from P/M, but not α-KG respiring mitochondria. In cardiac myocytes, downregulation of α-KGDH activity impaired dynamic mitochondrial redox adaptation during workload transitions, without increasing H\(_2\)O\(_2\) emission. In conclusion, NADH from α-KGDH selectively shuttles to NNT for NADPH formation rather than to complex I of the respiratory chain for ATP production. Therefore, α-KGDH plays a key role for H\(_2\)O\(_2\) elimination, but is not a relevant source of superoxide in heart. In heart failure, α-KGDH/NNT-dependent NADPH formation ameliorates oxidative stress imposed by complex I blockade. Downregulation of α-KGDH may, therefore, predispose to oxidative stress in heart failure. KW - mitochondria KW - α-Ketoglutarate dehydrogenase KW - reactive oxygen species KW - nicotinamide nucleotide transhydrogenase Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-234907 SN - 0300-8428 VL - 115 ER - TY - JOUR A1 - Wasmus, Christina A1 - Dudek, Jan T1 - Metabolic Alterations Caused by Defective Cardiolipin Remodeling in Inherited Cardiomyopathies JF - Life N2 - The heart is the most energy-consuming organ in the human body. In heart failure, the homeostasis of energy supply and demand is endangered by an increase in cardiomyocyte workload, or by an insufficiency in energy-providing processes. Energy metabolism is directly associated with mitochondrial redox homeostasis. The production of toxic reactive oxygen species (ROS) may overwhelm mitochondrial and cellular ROS defense mechanisms in case of heart failure. Mitochondria are essential cell organelles and provide 95% of the required energy in the heart. Metabolic remodeling, changes in mitochondrial structure or function, and alterations in mitochondrial calcium signaling diminish mitochondrial energy provision in many forms of cardiomyopathy. The mitochondrial respiratory chain creates a proton gradient across the inner mitochondrial membrane, which couples respiration with oxidative phosphorylation and the preservation of energy in the chemical bonds of ATP. Akin to other mitochondrial enzymes, the respiratory chain is integrated into the inner mitochondrial membrane. The tight association with the mitochondrial phospholipid cardiolipin (CL) ensures its structural integrity and coordinates enzymatic activity. This review focuses on how changes in mitochondrial CL may be associated with heart failure. Dysfunctional CL has been found in diabetic cardiomyopathy, ischemia reperfusion injury and the aging heart. Barth syndrome (BTHS) is caused by an inherited defect in the biosynthesis of cardiolipin. Moreover, a dysfunctional CL pool causes other types of rare inherited cardiomyopathies, such as Sengers syndrome and Dilated Cardiomyopathy with Ataxia (DCMA). Here we review the impact of cardiolipin deficiency on mitochondrial functions in cellular and animal models. We describe the molecular mechanisms concerning mitochondrial dysfunction as an incitement of cardiomyopathy and discuss potential therapeutic strategies. KW - cardiolipin KW - mitochondria KW - Barth syndrome KW - Sengers syndrome KW - respiratory chain KW - Dilated Cardiomyopathy with Ataxia KW - cardiomyopathy Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-219286 SN - 2075-1729 VL - 10 IS - 11 ER - TY - JOUR A1 - Palladino, Viola Stella A1 - Chiocchetti, Andreas G. A1 - Frank, Lukas A1 - Haslinger, Denise A1 - McNeill, Rhiannon A1 - Radtke, Franziska A1 - Till, Andreas A1 - Haupt, Simone A1 - Brüstle, Oliver A1 - Günther, Katharina A1 - Edenhofer, Frank A1 - Hoffmann, Per A1 - Reif, Andreas A1 - Kittel-Schneider, Sarah T1 - Energy metabolism disturbances in cell models of PARK2 CNV carriers with ADHD JF - Journal of Clinical Medicine N2 - The main goal of the present study was the identification of cellular phenotypes in attention-deficit-/hyperactivity disorder (ADHD) patient-derived cellular models from carriers of rare copy number variants (CNVs) in the PARK2 locus that have been previously associated with ADHD. Human-derived fibroblasts (HDF) were cultured and human-induced pluripotent stem cells (hiPSC) were reprogrammed and differentiated into dopaminergic neuronal cells (mDANs). A series of assays in baseline condition and in different stress paradigms (nutrient deprivation, carbonyl cyanide m-chlorophenyl hydrazine (CCCP)) focusing on mitochondrial function and energy metabolism (ATP production, basal oxygen consumption rates, reactive oxygen species (ROS) abundance) were performed and changes in mitochondrial network morphology evaluated. We found changes in PARK2 CNV deletion and duplication carriers with ADHD in PARK2 gene and protein expression, ATP production and basal oxygen consumption rates compared to healthy and ADHD wildtype control cell lines, partly differing between HDF and mDANs and to some extent enhanced in stress paradigms. The generation of ROS was not influenced by the genotype. Our preliminary work suggests an energy impairment in HDF and mDAN cells of PARK2 CNV deletion and duplication carriers with ADHD. The energy impairment could be associated with the role of PARK2 dysregulation in mitochondrial dynamics. KW - ADHD KW - hiPSC KW - PARK2 KW - mitochondria KW - disease modelling Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-220074 SN - 2077-0383 VL - 9 IS - 12 ER -