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The filamentous ascomycete Podospora anserina is a well-established model system to study organismic aging. Its senescence syndrome has been investigated for more than fifty years and turned out to have a strong mitochondrial etiology. Several different mitochondrial pathways were demonstrated to affect aging and lifespan. Here, we present an update of the literature focusing on the cooperative interplay between different processes.
Ferroptosis is an iron-dependent form of cell death, which is triggered by disturbed membrane integrity due to an overproduction of lipid peroxides. Induction of ferroptosis comprises several alterations, i.e. altered iron metabolism, response to oxidative stress, or lipid peroxide production. At the physiological level transcription, translation, and microRNAs add to the appearance and/or activity of building blocks that negatively or positively balance ferroptosis. Ferroptosis contributes to tissue damage in the case of, e.g., brain and heart injury but may be desirable to overcome chemotherapy resistance. For a more complete picture, it is crucial to also consider the cellular microenvironment, which during inflammation and in the tumor context is dominated by hypoxia. This graphical review visualizes basic mechanisms of ferroptosis, categorizes general inducers and inhibitors of ferroptosis, and puts a focus on microRNAs, iron homeostasis, and hypoxia as regulatory components.
Background: Alzheimer’s disease (AD) is the most common form of dementia, and it affects more women than men. Mitochondrial dysfunction (MD) plays a key role in AD, and it is detectable at an early stage of the degenerative process in peripheral tissues, such as peripheral mononuclear blood cells (PBMCs). However, whether these changes are also reflected in cerebral energy metabolism and whether sex-specific differences in mitochondrial function occur are not clear. Therefore, we estimated the correlation between mitochondrial function in PBMCs and brain energy metabolites and examined sex-specific differences in healthy participants to elucidate these issues.
Methods: The current pilot study included 9 male and 15 female healthy adults (mean age 30.8 ± 7.1 years). Respiration and activity of mitochondrial respiratory complexes were measured using a Clarke-electrode (Oxygraph-2k system), and adenosine triphosphate (ATP) levels were determined using a bioluminescence-based assay in isolated PBMCs. Citrate synthase activity as a mitochondrial marker was measured using a photometric assay. Concentrations of brain energy metabolites were quantified in the same individuals using 1H-magnetic resonance spectroscopy (MRS).
Results: We detected sex-associated differences in mitochondrial function. Mitochondrial complexes I, I+II, and IV and uncoupled respiration and electron transport system (ETS) capacity in PBMCs isolated from blood samples of females were significantly (p < 0.05; p < 0.01) higher compared to males. ATP levels in the PBMCs of female participants were approximately 10% higher compared to males. Citrate synthase (CS) activity, a marker of mitochondrial content, was significantly (p < 0.05) higher in females compared to males. Sex-associated differences were also found for brain metabolites. The N-acetylaspartate (NAA) concentration was significantly higher in female participants compared to males in targeted regions. This difference was observed in white matter (WM) and an area with a high percentage (> 50%) of gray matter (GM) (p < 0.05; p < 0.01). The effect sizes indicated a strong influence of sex on these parameters. Sex-associated differences were found in PBMCs and brain, but the determined parameters were not significantly correlated.
Conclusions: Our study revealed sex-associated differences in mitochondrial function in healthy participants. The underlying mechanisms must be elucidated in more detail, but our study suggests that mitochondrial function in PBMCs is a feasible surrogate marker to detect differences in mitochondrial function and energy metabolism in humans and it underscores the necessity of sex-specific approaches in therapies that target mitochondrial dysfunction.
Hypoxia triggers several mechanisms to adapt cells to a low oxygen environment. Mitochondria are major consumers of oxygen and a potential source of reactive oxygen species (ROS). In response to hypoxia they exchange or modify distinct subunits of the respiratory chain and adjust their metabolism, especially lowering the citric acid cycle. Intermediates of the citric acid cycle participate in regulating hypoxia inducible factors (HIF), the key mediators of adaptation to hypoxia. Here we summarize how hypoxia conditions mitochondria with consequences for ROS-production and the HIF-pathway.
Broad AOX expression in a genetically tractable mouse model does not disturb normal physiology
(2017)
Plants and many lower organisms, but not mammals, express alternative oxidases (AOXs) that branch the mitochondrial respiratory chain, transferring electrons directly from ubiquinol to oxygen without proton pumping. Thus, they maintain electron flow under conditions when the classical respiratory chain is impaired, limiting excess production of oxygen radicals and supporting redox and metabolic homeostasis. AOX from Ciona intestinalis has been used to study and mitigate mitochondrial impairments in mammalian cell lines, Drosophila disease models and, most recently, in the mouse, where multiple lentivector-AOX transgenes conferred substantial expression in specific tissues. Here, we describe a genetically tractable mouse model in which Ciona AOX has been targeted to the Rosa26 locus for ubiquitous expression. The AOXRosa26 mouse exhibited only subtle phenotypic effects on respiratory complex formation, oxygen consumption or the global metabolome, and showed an essentially normal physiology. AOX conferred robust resistance to inhibitors of the respiratory chain in organello; moreover, animals exposed to a systemically applied LD50 dose of cyanide did not succumb. The AOXRosa26 mouse is a useful tool to investigate respiratory control mechanisms and to decipher mitochondrial disease aetiology in vivo.
Background: Ischemia-reperfusion injury (IRI) is a major challenge in liver transplantation. The mitochondrial pathway plays a pivotal role in hepatic IRI. Levosimendan, a calcium channel sensitizer, was shown to attenuate apoptosis after IRI in animal livers. The aim of this study was to investigate the effect of levosimendan on apoptosis in human hepatocytes.
Methods: Primary human hepatocytes were either exposed to hypoxia or cultured under normoxic conditions. After the hypoxic phase, reoxygenation was implemented and cells were treated with different concentrations of levosimendan (10ng/ml, 100ng/ml, 1000ng/ml). The overall metabolic activity of the cells was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and aspartate aminotransferase (AST) levels were determined in order to quantify hepatic injury. Fluorescence-activated cell sorting (FACS) analysis was applied to measure necrosis and apoptosis. Finally, Western blotting was performed to analyze apoptotic pathway proteins.
Results: Administration of levosimendan during reperfusion increases the metabolic activity of human hepatocytes and decreases AST levels. Moreover, apoptosis after IRI is reduced in treated vs. untreated hepatocytes, and levosimendan prevents down-regulation of the anti-apoptotic protein Bcl-2 as well as up-regulation of the pro-apoptotic protein BAX.
Conclusion: The present study suggests a protective effect of levosimendan on human hepatocytes. Our findings suggest that treatment with levosimendan during reperfusion attenuates apoptosis of human hepatocytes by influencing BAX and Bcl-2 levels.
Mitochondrial complex I is a 1MDa membrane protein complex with a central role in aerobic energy metabolism. The bioenergetic core functions are executed by 14 central subunits that are conserved from bacteria to man. Despite recent progress in structure determination, our understanding of the function of the ~30 accessory subunits associated with the mitochondrial complex is still limited. We have investigated the structure of complex I from the aerobic yeast Yarrowia lipolytica by cryo-electron microscopy. Our density map at 7.9Å resolution closely matches the 3.6-3.9Å X-ray structure of the Yarrowia lipolytica complex. However, the cryo-EM map indicated an additional subunit on the side of the matrix arm above the membrane surface, pointing away from the membrane arm. The density, which is not present in any previously described complex I structure and occurs in about 20 % of the particles, was identified as the accessory sulfur transferase subunit ST1. The Yarrowia lipolytica complex I preparation is active in generating H2S from the cysteine derivative 3-mercaptopyruvate, catalyzed by ST1. We thus provide evidence for a link between respiratory complex I and mitochondrial sulfur metabolism.
Background: NH exchangers (NHEs) play a crucial role in regulating intra/extracellular pH, which is altered in cancer cells, and are therefore suitable targets to alter cancer cell metabolism in order to inhibit cell survival and proliferation. Among NHE inhibitors, amiloride family members are commonly used in clinical practice as diuretics; we focused on the amiloride HMA, reporting a net cytotoxic effect on a panel of human cancer cell lines; now we aim to provide new insights into the molecular events leading to cell death by HMA.
Methods: Colon cancer cell lines were treated with HMA and analysed with: morphological and cellular assays for cell viability and death, and autophagy; biochemical approaches to evaluate mitochondrial function and ROS production; in situ detection of DNA damage; molecular tools to silence crucial autophagy/necroptosis factors.
Results: HMA affects cellular morphology, alters mitochondrial structure and function, causes an increase in ROS, which is detrimental to DNA integrity, stimulates poly(ADP-ribose) synthesis, activates RIPK3-dependent death and triggers autophagy, which is unable to rescue cell survival. These features are hot points of an intricate network of processes, including necroptosis and autophagy, regulating the homeostasis between survival and death.
Conclusion: Our results allow the identification of multiple events leading to cell death in cancer cells treated with HMA. The here-defined intricate network activated by HMA could be instrumental to selectively target the key players of each pathway in the attempt to improve the global response to HMA. Our data could be the starting point for developing a newly designed targeted therapy.
The endoplasmic reticulum–mitochondria encounter structure (ERMES) connects the mitochondrial outer membrane with the ER. Multiple functions have been linked to ERMES, including maintenance of mitochondrial morphology, protein assembly and phospholipid homeostasis. Since the mitochondrial distribution and morphology protein Mdm10 is present in both ERMES and the mitochondrial sorting and assembly machinery (SAM), it is unknown how the ERMES functions are connected on a molecular level. Here we report that conserved surface areas on opposite sides of the Mdm10 β-barrel interact with SAM and ERMES, respectively. We generated point mutants to separate protein assembly (SAM) from morphology and phospholipid homeostasis (ERMES). Our study reveals that the β-barrel channel of Mdm10 serves different functions. Mdm10 promotes the biogenesis of α-helical and β-barrel proteins at SAM and functions as integral membrane anchor of ERMES, demonstrating that SAM-mediated protein assembly is distinct from ER-mitochondria contact sites.
Fusion of mitochondrial outer membranes is crucial for proper organelle function and involves large GTPases called mitofusins. The discrete steps that allow mitochondria to attach to one another and merge their outer membranes are unknown. By combining an in vitro mitochondrial fusion assay with electron cryo-tomography (cryo-ET), we visualize the junction between attached mitochondria isolated from Saccharomyces cerevisiae and observe complexes that mediate this attachment. We find that cycles of GTP hydrolysis induce progressive formation of a docking ring structure around extended areas of contact. Further GTP hydrolysis triggers local outer membrane fusion at the periphery of the contact region. These findings unravel key features of mitofusin-dependent fusion of outer membranes and constitute an important advance in our understanding of how mitochondria connect and merge.