Sphingosine kinase 1 (SK1) converts sphingosine to the bioactive lipid sphingosine 1-phosphate (S1P). S1P binds to G-protein-coupled receptors (S1PR1–5) to regulate cellular events, including Ca2+ signaling. The SK1/S1P axis and Ca2+ signaling both play important roles in health and disease. In this respect, Ca2+ microdomains at the mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) are of importance in oncogenesis. Mitofusin 2 (MFN2) modulates ER-mitochondria contacts, and dysregulation of MFN2 is associated with malignancies. We show that overexpression of SK1 augments agonist-induced Ca2+ release from the ER resulting in increased mitochondrial matrix Ca2+. Also, overexpression of SK1 induces MFN2 fragmentation, likely through increased calpain activity. Further, expressing putative calpain-cleaved MFN2 N- and C-terminal fragments increases mitochondrial matrix Ca2+ during agonist stimulation, mimicking the SK1 overexpression in cells. Moreover, SK1 overexpression enhances cellular respiration and cell migration. Thus, SK1 regulates MFN2 fragmentation resulting in increased mitochondrial Ca2+ and downstream cellular effects.

not received Plenary IV. Mitochondria and cancer Metabolic Management of Glioblastoma Thomas Seyfried Department of Biology, Boston College, Chestnut Hill, Massachusetts, United States Glioblastoma multiforme (GBM) remains among the most aggressive and difficult to manage primary brain tumours in humans. Glucose and glutamine are recognized as the major fuels that drive GBM growth and invasion through glycolysis and glutaminolysis, respectively. The glutamine antagonist, 6-diazo-5-oxo-L-norleucine (DON), was administered together with a calorically restricted ketogenic diet (KD-R) to treat late-stage orthotopic growth in two syngeneic mouse models of GBM; the highly invasive mesenchymal tumour, VM-M3, and the high-grade stem cell glioma, CT-2A. DON targets glutaminolysis while the KD-R reduces glucose and, at the same time, elevates neuroprotective and non-fermentable ketone bodies. The diet/drug therapeutic strategy caused massive tumour cell death or mitotic arrest, while reversing disease symptoms and improving overall survival without toxicity. The therapeutic strategy also reduced edema, hemorrhage, and inflammation associated with rapid tumour growth. Moreover, the KD-R diet facilitated DON delivery to the brain and allowed a lower nontoxic dosage to achieve therapeutic effect. Data from human case reports will also be presented. These findings support the importance of glucose and glutamine in driving GBM growth and provide a plausible therapeutic strategy for the non-toxic metabolic management of GBM.

A challenge to protein based therapies is the ability to produce biologically active proteins and their ensured delivery. Various approaches have been utilised including fusion of protein transduction domains with a protein or biomolecule of interest. A compounding issue is lack of specificity, efficiency and indeed whether the protein fusions are actually translocated into the cell and not merely an artefact of the fixation process. Here we present a novel platform, allowing the inducible export and uptake of a protein of interest. The system utilises a combination of the Tetracyline repressor system, combined with a fusion protein containing the N-terminal signal peptide from human chorionic gonadotropin beta-subunit, and a C-terminal poly-arginine domain for efficient uptake by target cells. This novel platform was validated using enhanced green fluorescent protein as the gene of interest. Doxycycline efficiently induced expression of the fusion protein. The human chorionic gonadotropin beta-subunit facilitated the export of the fusion protein into the cell culture media. Finally, the fusion protein was able to efficiently enter into neighbouring cells (target cells), mediated by the poly-arginine cell penetrating peptide. Importantly we have addressed the issue of whether the observed uptake is an artefact of the fixation process or indeed genuine translocation. In addition this platform provides a number of potential applications in diverse areas such as stem cell biology, immune therapy and cancer targeting therapies.

Stable isotope analysis (SIA) of fish otoliths shows great potential for exploring the ecology of fish, but this method has not been applied to the study of lacustrine fish communities. Both sequential and whole-otolith SIAs were performed on six fish species of Lake Annecy and their results compared to muscle SIA. The first purpose of these investigations was to test the use of δ18Ooto values for reconstructing fish thermal history and delimiting spatial distribution in a stratified lake. Comparison of species-specific fractionation equations and the general equation developed for freshwater fishes showed that the general one was the best suited for thermal estimation of Lake Annecy fishes and suggested that inter-specific differences or specific “vital effects” are not the only reason for apparent difference in fractionation. Thermal estimations based on SIA were consistent with descriptions of thermal habitats in the literature, except in the case of roach (Rutilus rutilus). Based on the current results, roach appears to live in a colder habitat than do perch (Perca fluviatilis). The high water transparency and thermal stratification of Lake Annecy could explain this distribution. Moreover, perch juveniles were found to live in two different thermal niches. This finding highlights the great plasticity of the species. Second, the potential use of δ13Coto values to reconstruct variation in diet and metabolism was assessed. The proportion of metabolic carbon (M) contributing to otolith carbon varies markedly among species. Comparison with δ13Cmuscle values shows no direct relationship between δ13Coto and diet at either the intra-individual or the inter-specific level. A strong linear relationship between either M or the isotopic offset between otolith and muscle (Δδ13Coto - muscle) and δ18Ooto values was found; this relationship reveals the dependence of M on the ambient temperature at which the species occurs. This relationship might be general and could be used to explore variation in fish metabolism in the future.

The filamentous fungus Podospora anserina has a limited lifespan. In this organism, aging is systematically associated to mitochondrial DNA instability. We recently provided evidence that the respiratory function is a key determinant of its lifespan. Loss of function of the cytochrome pathway leads to the compensatory induction of an alternative oxidase, to a decreased production of reactive oxygen species and to a striking increase in lifespan. These changes are associated to the stabilization of the mitochondrial DNA. Here we review and discuss the links between these different parameters and their implication in the control of lifespan. Since we demonstrated the central role of mitochondrial metabolism in aging, the same relationship has been evidenced in several model systems from yeast to mice, confirming the usefulness of simple organisms as P. anserina for studying lifespan regulation.

We have created P1 artificial chromosome transgenic mice expressing the human mitochondrial superoxide dismutase 2 (SOD2) and thus generated mice with a physiologically controlled augmentation of SOD2 expression leading to increased SOD2 enzyme activities and lowered superoxide levels. In the transgenic mice, effects on mitochondrial function such as enhanced oxidative capacity and greater resistance against inducers of mitochondrial permeability were observed. Superoxide in the mitochondrial matrix has been proposed to activate uncoupling proteins (UCPs), thus providing a feedback mechanism that will lower respiratory chain superoxide production by increasing a proton leak across the inner mitochondrial membrane. However, UCP1 and UCP3 activities and mitochondrial ATP production rates were not altered in isolated mitochondria from SOD2 transgenic mice, despite lowered superoxide levels. Globally, the transgenic mice displayed normal resting metabolic rates, indicating an absence of effect on any UCP activities, and normal oxygen consumption responses after norepinephrine injection. These results strongly suggest that endogenously generated matrix superoxide does not regulate UCP activity and in vivo energy expenditure.

Several lines of evidence have implicated reactive oxygen species (ROS) in the pathogenesis of various degenerative diseases and in organismal ageing. Furthermore, it has been shown recently that the alternative pathway respiration present in plants lowers ROS mitochondrial production. An alternative oxidase (AOXp) also occurs in the filamentous fungus Podospora anserina. We show here that overexpression of this oxidase does not decrease ROS production and has no effect on longevity, mitochondrial stability or ageing in this fungus. In the same way, inactivation of the gene has no effect on these parameters. In contrast, overexpression of the alternative oxidase in the long-lived cox5::BLE mutant, deficient in cytochrome c oxidase, considerably increases ROS production of the mutant. It rescues slow growth rate and female sterility, indicating an improved energy level. This overexpression also restores senescence and mitochondrial DNA instability, demonstrating that these parameters are controlled by the energy level and not by the expression level of the alternative oxidase. We also suggest that expression of this oxidase in organisms naturally devoid of it could rescue respiratory defects resulting from cytochrome pathway dysfunctions.

Senescence, a progressive degenerative process leading to age-related increase in mortality, is found in most eukaryotes. However, the molecular events underlying aging remain largely unknown. Understanding how longevity is regulated is a fundamental problem. Here we demonstrate that the respiratory function is a key factor that contributes to shortening lifespan of the filamentous fungus Podospora anserina . In this organism, senescence is systematically associated with mitochondrial DNA instabilities. We show that inactivation of the nuclear COX5 gene encoding subunit V of the cytochrome c oxidase complex leads to the exclusive use of the alternative respiratory pathway and to a decrease in production of reactive oxygen species. This inactivation results in a striking increase of longevity associated with stabilization of the mitochondrial chromosome. Moreover, accumulation of several senescence-specific mitochondrial DNA molecules is prevented in this nuclear mutant. These findings provide direct evidence of a causal link between mitochondrial metabolism and longevity in Podospora anserina .

Microstructure, mineralogy and elemental and isotopic compositions of recent and fossil otoliths have been investigated. Fossil otoliths come from marine Pliocene localities of southeastern France. The study of the different parameters show that diagenetic changes are weak. Microstructural features specific to otoliths, such as needle-like crystals and growth increments, are usually well preserved. When present, the microstructural modifications remain limited inside each otolith. Except a few pyritic specimens, the only mineral found is aragonite like in recent specimens. The chemical compositions of fossil and recent otoliths are not significantly different, and any element is preferentially changed by diagenesis. The isotopic composition does not show strong alterations. However, the difference in O-18 content between recent fishes having different ecological habits is not found with the same magnitude in fossil specimens. Finally, when elemental and isotopic compositions are compared, the specimens are sorted according to taxonomy and not according to their geologic age. This suggests that biogenic signals have been preserved.

ABSTRACT Podospora anserina is a filamentous fungus with a limited life span. It expresses a degenerative syndrome called senescence, which is always associated with the accumulation of circular molecules (senDNAs) containing specific regions of the mitochondrial chromosome. A mobile group II intron (α) has been thought to play a prominent role in this syndrome. Intron α is the first intron of the cytochrome c oxidase subunit I gene (COX1). Mitochondrial mutants that escape the senescence process are missing this intron, as well as the first exon of theCOX1 gene. We describe here the first mutant of P. anserina that has the α sequence precisely deleted and whose cytochrome c oxidase activity is identical to that of wild-type cells. The integration site of the intron is slightly modified, and this change prevents efficient homing of intron α. We show here that this mutant displays a senescence syndrome similar to that of the wild type and that its life span is increased about twofold. The introduction of a related group II intron into the mitochondrial genome of the mutant does not restore the wild-type life span. These data clearly demonstrate that intron α is not the specific senescence factor but rather an accelerator or amplifier of the senescence process. They emphasize the role that intron α plays in the instability of the mitochondrial chromosome and the link between this instability and longevity. Our results strongly support the idea that in Podospora, “immortality” can be acquired not by the absence of intron α but rather by the lack of active cytochromec oxidase.

Biology of the CellVolume 91, Issue 7 p. 553-553 Absence of cytochrome C oxidase leads to a striking increase of life span in podospora anserina E. Dufour, E. Dufour Centre de Génétique Moléculaire-Centre National de la Recherche Scientifique, Allée de la terrasse, 91198 Gif sur Yvette Cedex, FranceSearch for more papers by this authorJ. Boulay, J. Boulay Centre de Génétique Moléculaire-Centre National de la Recherche Scientifique, Allée de la terrasse, 91198 Gif sur Yvette Cedex, FranceSearch for more papers by this authorA. Sainsard-Chanet, A. Sainsard-Chanet Centre de Génétique Moléculaire-Centre National de la Recherche Scientifique, Allée de la terrasse, 91198 Gif sur Yvette Cedex, FranceSearch for more papers by this author E. Dufour, E. Dufour Centre de Génétique Moléculaire-Centre National de la Recherche Scientifique, Allée de la terrasse, 91198 Gif sur Yvette Cedex, FranceSearch for more papers by this authorJ. Boulay, J. Boulay Centre de Génétique Moléculaire-Centre National de la Recherche Scientifique, Allée de la terrasse, 91198 Gif sur Yvette Cedex, FranceSearch for more papers by this authorA. Sainsard-Chanet, A. Sainsard-Chanet Centre de Génétique Moléculaire-Centre National de la Recherche Scientifique, Allée de la terrasse, 91198 Gif sur Yvette Cedex, FranceSearch for more papers by this author First published: 01 February 2012 https://doi.org/10.1016/S0248-4900(99)90254-XAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume91, Issue7September 1999Pages 553-553 RelatedInformation

Dietary differences in two forms of Lake Constance Coregonids have been tested using measurements of stable isotopic ratios of nitrogen and carbon in muscle tissue. No significant difference was found between the two forms. Moreover, the standard deviation for the whole population of specimens ranging between 3 to 5 years of age was small (0.9 parts per thousand for delta(13)C and 1.3 parts per thousand for delta(15)N) and indicated the same food resources. The only significant difference measured was a slight depletion of female delta(13)C values relative to male delta(13)C values. This difference was related to C/N and lipid content in muscle and not to ecological features. indeed, C/N values were low for both sexes, but were lower for females than males, suggesting that muscle fat reserves had been mobilized especially, in female muscle, at spawning time.