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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.
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.
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.
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.
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.
Respirasomes are macromolecular assemblies of the respiratory chain complexes I, III and IV in the inner mitochondrial membrane. We determined the structure of supercomplex I1III2IV1 from bovine heart mitochondria by cryo-EM at 9 Å resolution. Most protein-protein contacts between complex I, III and IV in the membrane are mediated by supernumerary subunits. Of the two Rieske iron-sulfur cluster domains in the complex III dimer, one is resolved, indicating that this domain is immobile and unable to transfer electrons. The central position of the active complex III monomer between complex I and IV in the respirasome is optimal for accepting reduced quinone from complex I over a short diffusion distance of 11 nm, and delivering reduced cytochrome c to complex IV. The functional asymmetry of complex III provides strong evidence for directed electron flow from complex I to complex IV through the active complex III monomer in the mammalian supercomplex.
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.
Downstream effects of plectin mutations in epidermolysis bullosa simplex with muscular dystrophy
(2016)
Mutations of the human plectin gene (PLEC) on chromosome 8q24 cause autosomal recessive epidermolysis bullosa simplex with muscular dystrophy (EBS-MD). In the present study we analyzed the downstream effects of PLEC mutations on plectin protein expression and localization, the structure of the extrasarcomeric desmin cytoskeleton, protein aggregate formation and mitochondrial distribution in skeletal muscle tissue from three EBS-MD patients.
PLEC gene analysis in a not previously reported 35-year-old EBS-MD patient with additional disease features of cardiomyopathy and malignant arrhythmias revealed novel compound heterozygous (p.(Phe755del) and p.(Lys1040Argfs*139)) mutations resulting in complete abolition of plectin protein expression. In contrast, the other two patients with different homozygous PLEC mutations showed preserved plectin protein expression with one only expressing rodless plectin variants, and the other markedly reduced protein levels. Analysis of skeletal muscle tissue from all three patients revealed severe disruption of the extrasarcomeric intermediate filament cytoskeleton, protein aggregates positive for desmin, syncoilin, and synemin, degenerative myofibrillar changes, and mitochondrial abnormalities comprising respiratory chain dysfunction and an altered organelle distribution and amount.
Our study demonstrates that EBS-MD causing PLEC mutations universally result in a desmin protein aggregate myopathy phenotype despite marked differences in individual plectin protein expression patterns. Since plectin is the key cytolinker protein that regulates the structural and functional organization of desmin filaments, the defective anchorage and spacing of assembled desmin filaments is the key pathogenetic event that triggers the formation of desmin protein aggregates as well as secondary mitochondrial pathology.
In this meeting report, particularly addressing the topic of protection of the cardiovascular system from ischemia/reperfusion injury, highlights are presented that relate to conditioning strategies of the heart with respect to molecular mechanisms and outcome in patients’ cohorts, the influence of co-morbidities and medications, as well as the contribution of innate immune reactions in cardioprotection. Moreover, developmental or systems biology approaches bear great potential in systematically uncovering unexpected components involved in ischemia–reperfusion injury or heart regeneration. Based on the characterization of particular platelet integrins, mitochondrial redox-linked proteins, or lipid-diol compounds in cardiovascular diseases, their targeting by newly developed theranostics and technologies opens new avenues for diagnosis and therapy of myocardial infarction to improve the patients’ outcome.
Mitochondrial respiratory supercomplexes (mtRSCs) are stoichiometric assemblies of electron transport chain (ETC) complexes in the inner mitochondrial membrane. They are hypothesized to regulate electron flow, the generation of reactive oxygen species (ROS) and to stabilize ETC complexes. Using the fungal ageing model Podospora anserina, we investigated the impact of homologues of the Saccharomyces cerevisiae respiratory supercomplex factors 1 and 2 (termed PaRCF1 and PaRCF2) on mtRSC formation, fitness and lifespan. Whereas PaRCF2’s role seems negligible, ablation of PaRCF1 alters size of monomeric complex IV, reduces the abundance of complex IV-containing supercomplexes, negatively affects vital functions and shortens lifespan. PaRcf1 overexpression slightly prolongs lifespan, though without appreciably influencing ETC organization. Overall, our results identify PaRCF1 as necessary yet not sufficient for mtRSC formation and demonstrate that PaRCF1-dependent stability of complex IV and associated supercomplexes is highly relevant for maintenance of the healthy lifespan in a eukaryotic model organism.
Activation of Mitochondrial complex II-dependent respiration is beneficial for α-Synucleinopathies
(2015)
Parkinson’s disease and dementia with Lewy bodies are major challenges in research and clinical medicine world-wide and contribute to the most common neurodegenerative disorders. Previously, specific mitochondrial polymorphisms have been found to enhance clearance of amyloid-β from the brain of APP-transgenic mice leading to beneficial clinical outcome. It has been discussed whether specific mitochondrial alterations contribute to disease progression or even prevent toxic peptide deposition, as seen in many neurodegenerative diseases. Here, we investigated α-synuclein-transgenic C57BL/6J mice with the A30P mutation, and a novel A30P C57BL/6J mouse model with three mitochondrial DNA polymorphisms in the ND3, COX3 and mtRNAArg genes, as found in the inbred NOD/LtJ mouse strain. We were able to detect that the new model has increased mitochondrial complex II-respiration which occurs in parallel to neuronal loss and improved motor performance, although it exhibits higher amounts of high molecular weight species of α-synuclein. High molecular weight aggregates of different peptides are controversially discussed in the light of neurodegeneration. A favourable hypothesis states that high molecular weight species are protective and of minor importance for the pathogenesis of neurodegenerative disorders as compared to the extreme neurotoxic monomers and oligomers. Summarising, our results point to a potentially protective and beneficial effect of specific mitochondrial polymorphisms which cause improved mitochondrial complex II-respiration in α-synucleinopathies, an effect that could be exploited further for pharmaceutical interventions.
Recent advances in basic cardiovascular research as well as their translation into the clinical situation were the focus at the last "New Frontiers in Cardiovascular Research meeting". Major topics included the characterization of new targets and procedures in cardioprotection, deciphering new players and inflammatory mechanisms in ischemic heart disease as well as uncovering microRNAs and other biomarkers as versatile and possibly causal factors in cardiovascular pathogenesis. Although a number of pathological situations such as ischemia-reperfusion injury or atherosclerosis can be simulated and manipulated in diverse animal models, also to challenge new drugs for intervention, patient studies are the ultimate litmus test to obtain unequivocal information about the validity of biomedical concepts and their application in the clinics. Thus, the open and bidirectional exchange between bench and bedside is crucial to advance the field of ischemic heart disease with a particular emphasis of understanding long-lasting approaches in cardioprotection.
Background: Protein translocation across membranes is a central process in all cells. In the past decades the molecular composition of the translocation systems in the membranes of the endoplasmic reticulum, peroxisomes, mitochondria and chloroplasts have been established based on the analysis of model organisms. Today, these results have to be transferred to other plant species. We bioinformatically determined the inventory of putative translocation factors in tomato (Solanum lycopersicum) by orthologue search and domain architecture analyses. In addition, we investigated the diversity of such systems by comparing our findings to the model organisms Saccharomyces cerevisiae, Arabidopsis thaliana and 12 other plant species.
Results: The literature search end up in a total of 130 translocation components in yeast and A. thaliana, which are either experimentally confirmed or homologous to experimentally confirmed factors. From our bioinformatic analysis (PGAP and OrthoMCL), we identified (co-)orthologues in plants, which in combination yielded 148 and 143 orthologues in A. thaliana and S. lycopersicum, respectively. Interestingly, we traced 82% overlap in findings from both approaches though we did not find any orthologues for 27% of the factors by either procedure. In turn, 29% of the factors displayed the presence of more than one (co-)orthologue in tomato. Moreover, our analysis revealed that the genomic composition of the translocation machineries in the bryophyte Physcomitrella patens resemble more to higher plants than to single celled green algae. The monocots (Z. mays and O. sativa) follow more or less a similar conservation pattern for encoding the translocon components. In contrast, a diverse pattern was observed in different eudicots.
Conclusions: The orthologue search shows in most cases a clear conservation of components of the translocation pathways/machineries. Only the Get-dependent integration of tail-anchored proteins seems to be distinct. Further, the complexity of the translocation pathway in terms of existing orthologues seems to vary among plant species. This might be the consequence of palaeoploidisation during evolution in plants; lineage specific whole genome duplications in Arabidopsis thaliana and triplications in Solanum lycopersicum.
Membrane-embedded β-barrel proteins are found in the outer membranes (OM) of Gram-negative bacteria, mitochondria and chloroplasts. In eukaryotic cells, precursors of these proteins are synthesized in the cytosol and have to be sorted to their corresponding organelle. Currently, the signal that ensures their specific targeting to either mitochondria or chloroplasts is ill-defined. To address this issue, we studied targeting of the chloroplast β-barrel proteins Oep37 and Oep24. We found that both proteins can be integrated in vitro into isolated plant mitochondria. Furthermore, upon their expression in yeast cells Oep37 and Oep24 were exclusively located in the mitochondrial OM. Oep37 partially complemented the growth phenotype of yeast cells lacking Porin, the general metabolite transporter of this membrane. Similarly to mitochondrial β-barrel proteins, Oep37 and Oep24 expressed in yeast cells were assembled into the mitochondrial OM in a pathway dependent on the TOM and TOB complexes. Taken together, this study demonstrates that the central mitochondrial components that mediate the import of yeast β-barrel proteins can deal with precursors of chloroplast β-barrel proteins. This implies that the mitochondrial import machinery does not recognize signals that are unique to mitochondrial β-barrel proteins. Our results further suggest that dedicated targeting factors had to evolve in plant cells to prevent mis-sorting of chloroplast β-barrel proteins to mitochondria.
We proposed previously that closure of voltage-dependent anion channels (VDAC) in the mitochondrial outer membrane after ethanol exposure leads to suppression of mitochondrial metabolite exchange. Because ureagenesis requires extensive mitochondrial metabolite exchange, we characterized the effect of ethanol and its metabolite, acetaldehyde (AcAld), on total and ureagenic respiration in cultured rat hepatocytes. Ureagenic substrates increased cellular respiration from 15.8 ± 0.9 nmol O(2)/min/10(6) cells (base line) to 29.4 ± 1.7 nmol O(2)/min/10(6) cells in about 30 min. Ethanol (0-200 mM) suppressed extra respiration after ureagenic substrates (ureagenic respiration) by up to 51% but not base line respiration. Urea formation also declined proportionately. Inhibition of alcohol dehydrogenase, cytochrome P450 2E1, and catalase with 4-methylpyrazole, trans-1,2-dichloroethylene, and 3-amino-1,2,3-triazole restored ethanol-suppressed ureagenic respiration by 46, 37, and 66%, respectively. By contrast, inhibition of aldehyde dehydrogenase with phenethyl isothiocyanate increased the inhibitory effect of ethanol on ureagenic respiration by an additional 60%. AcAld, an intermediate product of ethanol oxidation, suppressed ureagenic respiration with an apparent IC(50) of 125 μM. AcAld also inhibited entry of 3-kDa rhodamine-conjugated dextran in the mitochondrial intermembrane space of digitonin-permeabilized hepatocytes, indicative of VDAC closure. In conclusion, AcAld, derived from ethanol metabolism, suppresses ureagenesis in hepatocytes mediated by closure of VDAC.
Mitochondrial dysfunction is well documented in presymptomatic brain tissue with Parkinson's disease (PD). Identification of the autosomal recessive variant PARK6 caused by loss-of-function mutations in the mitochondrial kinase PINK1 provides an opportunity to dissect pathogenesis. Although PARK6 shows clinical differences to PD, the induction of alpha-synuclein "Lewy" pathology by PINK1-deficiency proves that mitochondrial pathomechanisms are relevant for old-age PD. Mitochondrial dysfunction is induced by PINK1 deficiency even in peripheral tissues unaffected by disease, consistent with the ubiquitous expression of PINK1. It remains unclear whether this dysfunction is due to PINK1-mediated phosphorylation of proteins inside or outside mitochondria. Although PINK1 deficiency affects the mitochondrial fission/fusion balance, cell stress is required in mammals to alter mitochondrial dynamics and provoke apoptosis. Clearance of damaged mitochondria depends on pathways including PINK1 and Parkin and is critical for postmitotic neurons with high energy demand and cumulative stress, providing a mechanistic concept for the tissue specificity of disease.