Sci. Aging Knowl. Environ., 18 May 2005
Vol. 2005, Issue 20, p. pe14
[DOI: 10.1126/sageke.2005.20.pe14]

PERSPECTIVES

MiMage: A Pan-European Project on the Role of Mitochondria in Aging

C. Scheckhuber, and H. D. Osiewacz

The authors are at the Department of Molecular and Developmental Biology and Biotechnology, Botanical Institute, J. W. Goethe University, Marie-Curie-Strasse 9, 60439 Frankfurt, Germany. E-mail: scheckhuber{at}em.uni-frankfurt.de

http://sageke.sciencemag.org/cgi/content/full/2005/20/pe14

Key Words: calorie restriction • DNA repair • heat shock proteins • mitochondria • oxidative stress • respiratory chain

Introduction

Aging is the progressive and irreversible decline of physiological function, leading ultimately to an increase in morbidity and mortality. Although the aging process itself has not been halted, in industrialized countries the mean life span of humans has increased significantly in the last century owing to improved standards of medical treatment and public health (see Tuljapurkar Perspective). Birth rates have also decreased in these countries, leading to a "graying" of the population, and the resulting increase in the proportion of dependent individuals threatens to pose serious problems for the prevailing health and social systems. Dealing with this dramatic situation will require political initiatives aimed at changing the existing systems for social welfare and health care, and in the scientific arena there will be a need for fundamental research seeking to elucidate the complex mechanisms involved in aging so as to provide a basis for the development of effective interventions into various age-related diseases.

Toward this scientific aim, the aging process has been extensively studied in a number of model organisms and systems. Even today, though, the mechanisms of aging are far from being unraveled completely even in relatively simple organisms. However, it is clear that aging is controlled by a complex network of defined molecular pathways, some of which are conserved from model systems like fungi to higher organisms, including mammals. Consequently, experimental modulation of these fundamental mechanisms in any system will generate meaningful and valuable general results.

Mitochondria are likely to play a vital role in aging in all eukaryotic systems (see "Power Cut"). According to the mitochondrial theory of aging formulated by D. Harman (see Harman Classic Paper), the generation of reactive oxygen species (ROS) during electron transport in the inner mitochondrial membrane (IMM) leads to a progressive accumulation of damaged proteins, lipids, and nucleic acids and ultimately to cell death (see "The Two Faces of Oxygen"). However, more recent data indicate that aging cannot be attributed solely to the devastating action of ROS and that the role of mitochondria in aging therefore appears to be more complex. How mitochondrial functions are linked to various age-related defects that were previously thought to be "non-mitochondrial" remains a major unsolved problem.

MiMage, the "integrated project on the role of mitochondria in conserved mechanisms in aging" (Fig. 1), is a new venture agreed upon in December 2004 by the European Commission and the coordinating institution, the J. W. Goethe University in Frankfurt, Germany. The research project will be supported within the Sixth Framework for Research and Technological Development of the European Community for a period of 5 years beginning 1 January 2005. Twelve research teams from seven European countries, together with one associated team from Canada and the United States, will contribute complementary expertise in different research areas, including biochemistry, cell biology, genetics, molecular biology, and physiology.



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Fig. 1. Logo of the MiMage project.

 
The Objectives of MiMage

The overall aim of the MiMage project is to elaborate a complete view of the mechanistic role of mitochondria in aging. Of special interest is the discovery and experimental manipulation of evolutionarily conserved mechanisms shared between invertebrate and mammalian model systems (for an evolutionary biologist's view on mechanisms of aging, see Reznick Perspective). A range of experimental organisms (Saccharomyces cerevisiae, Podospora anserina, Caenorhabditis elegans, Drosophila melanogaster, mouse, and rat) and cell culture systems is being studied. Specific age-related issues will be systematically raised and addressed experimentally. These include (i) the effect on aging of modulating the amount of mitochondrial ROS, (ii) the role of molecular and cellular pathways involved in maintaining a "healthy" population of mitochondria, (iii) the nature and impact of age-related signaling pathways on mitochondrial functions, (iv) the effect of dietary restriction on mitochondrial activity, and (v) the impact of hitherto unknown age-related mitochondrial functions, which will include the use of proteomic techniques to reveal and characterize proteins that might be important in the aging process.

Some of the experimental organisms in use are characterized by a short life span; thus, the effect on aging of experimental perturbations in the aforementioned biological pathways can be studied in a relatively short period of time. The relevance of signaling pathways and protein-protein interactions identified by experimentation can subsequently be tested in cultured human cells, which links MiMage to other European programs devoted to research on human aging.

MiMage: The Participants

Here we provide a short overview of research in the participating laboratories constituting the MiMage consortium, in ascending order of complexity of the model organism being studied.

M. Breitenbach, based at the University of Salzburg in Austria, investigates the role of oxidative stress in replicative aging of S. cerevisiae. In one approach, an analysis of genes that are differentially expressed between young and senescent yeast cells is carried out, with the aim of identifying new components of the machinery involved in regulation of the aging process. T. Nyström (Göteborg University College, Sweden) also investigates different aspects of yeast aging, including defense against oxidative damage in aging cells, global regulatory circuits activated to protect the cell from macromolecular deterioration, links between mitochondrial activity and oxidative damage to proteins, and links between translational fidelity and oxidation of aberrant proteins.

R. F. Hoekstra's team, at Wageningen University in The Netherlands, specializes in genetic analysis in fungal model organisms, and one research project involves the investigation of altered mitochondrial function during calorie restriction (CR) (see Masoro Review) in the filamentous fungus P. anserina. A. Sainsard-Chanet (Centre de Génétique Moléculaire, Gif-sur-Yvette, France) has made important contributions to understanding the effect of mitochondrial respiration on aging in P. anserina, and current research involves characterization of long-lived mutants with impaired respiratory chains. H. D. Osiewacz (one of the authors of this report), at the J. W. Goethe University, is coordinator of the MiMage project, and his group is devoted to unraveling the molecular network of pathways controlling aging in P. anserina (1). For example, mitochondrial-nuclear interactions, stability of mitochondrial DNA (mtDNA), the dynamics of "healthy" mitochondria, and ROS generation are investigated using appropriate experimental tools in long-lived mutants and in specific transgenic strains. N. A. Dencher (University of Technology, Darmstadt, Germany) focuses on the study of biological membranes, especially those involved in energy and signal transduction. The structure and function of native and reconstituted mitochondrial membranes and their building blocks are studied at the molecular and atomic level using physical, chemical, and cell biological techniques. For example, age-related changes in the composition of mitochondrial membranes from model organisms like P. anserina (2) and rats are investigated using native polyacrylamide gel electrophoresis, which permits a detailed analysis of native proteins, protein complexes, and protein-protein interactions, including posttranslational modifications.

J. R. Vanfleteren, a biochemist at Ghent University in Belgium, works on aging in C. elegans. His recent research has focused on the biochemical and metabolic alterations occurring in the wild type and in long-lived mutants of C. elegans.

R. M. Tanguay (Laval University, Canada) investigates the aging process in D. melanogaster. Major research topics include structure/function analysis of the small heat shock proteins (Hsp) Hsp22, Hsp23, Hsp26, and Hsp27 and their impact on development, differentiation, and life span; the identification of proteins and signaling pathways involved in the aging process; and the importance of chaperones in protecting mitochondria during aging.

J. Bereiter-Hahn leads the kinematic cell research group at J. W. Goethe University, focusing on studies of mitochondrial behavior and physiology in living cells; the interaction of mitochondria with other organelles, such as lysosomes, which serve to maintain a population of functional mitochondria during aging; and the role of the cytoskeleton in compartmentalizing cellular energy metabolism. P. Jansen-Dürr, at the Institute for Biomedical Aging Research (Austrian Academy of Sciences, Innsbruck, Austria) is pursuing research on molecular and genetic studies of aging processes in cultured human cells; the role of candidate genes that may be involved in aging in vivo, employing biopsies of tissues obtained from human donors of varying age; and functional analysis of selected molecules that are known to play a role in age-related processes.

T. Stevnsner at Aarhus University in Denmark, in collaboration with V. A. Bohr (National Institute on Aging, Baltimore, USA), is carrying out research on topics including genomic instability in mammalian cells, DNA repair processes in mammalian nuclei and mitochondria, oxidative DNA damage and repair in relation to aging, and structure-function analysis of the Cockayne Syndrome group B protein, which is involved in keeping genomic DNA free of oxidatively modified bases such as 8-oxoguanine and 8-oxoadenine. Patients suffering from Cockayne Syndrome display some of the hallmarks of normal aging abnormally early in life, including cataracts and neurological disorders.

ProteoSys is a company based around proteomics and cell biology, located in Mainz, Germany. Areas of expertise include toxicology, stem cell culture and differentiation, protein profiling, stable isotope analysis, mass spectrometry, and bioinformatics. In the MiMage project, high sensitivity multichannel radiolabeling and detection methods are used to identify differences in the abundance of specific proteins in various cell types.

MiMage: First Symposium on The Role of Mitochondria in Conserved Mechanisms of Aging

The inaugural MiMage symposium was held in February 2005 at J. W. Goethe University. Investigators who will be involved in MiMage gave presentations on their research, and these are outlined here.

S. cerevisiae has been widely studied as a model for "replicative aging," with emphasis on respiration, mitochondrially generated ROS, and mitochondrial inheritance. With each division, a mother cell becomes older but continues to produce offspring exhibiting full replicative potential, continuing on average for 20 to 30 divisions up until death. It has been proposed that a hypothetical senescence factor accumulates in the mother cell but is prevented in some way from being transferred to daughter cells. Extrachromosomal rDNA circles and dysfunctional mitochondria have both been suggested to be possible senescence factors, although conclusive evidence on either of these two candidates is still lacking. T. Nyström gave a presentation on the elucidation of a mechanism that prevents the transmission of damaged cellular components to a daughter cell. It became clear that oxidatively modified proteins represent a further candidate for a senescence factor in yeast, as suggested by the accumulation of carbonylated proteins during replicative aging (3) (see "Protective Parents"). M. Breitenbach reported on altered mitochondrial function during yeast aging: A genome-wide transcript analysis employing DNA microarrays allowed the identification of about 600 genes that are differentially expressed in senescent as compared with younger cells. In addition, experimental data indicate that yeast mother cell-specific aging involves an apoptotic process and that mitochondria play a functional role in it.

P. anserina has been used as a model system to study aging for more than half a century (see "Copper Stopper"), and three talks were devoted to the investigation of mechanisms of aging in this organism. A. J. Debets (University of Wageningen, The Netherlands) discussed the ability of linear mitochondrial plasmids to reduce the extension of life span produced by CR in P. anserina (4). S. Lorin, from A. Sainsard-Chanet's laboratory, gave a presentation on the impact of respiratory activity on the aging process in P. anserina. After discussion of a link between respiration and longevity in a long-lived mutant mold with an altered respiratory chain (cox5::ble), the function of the P. anserina oxa1 gene--which encodes an essential mitochondrial membrane protein conserved from bacteria to mitochondria--was detailed. Oxa1 is necessary for the assembly of electron transport complexes I and IV in the IMM (see Fig.1 in Nicholls Perspective), as has also been shown in Neurospora crassa and yeast. Oxa1 seems to modulate longevity through a genetic interaction with the rmp1 gene (5), although the function of rmp1 has not been clearly established (6). Finally, the effect of modulating expression of the SIR2 gene, encoding a histone deacetylase linked to life-span determination in several model organisms (see Kaeberlin Perspective), was outlined. Interestingly, neither disruption nor overexpression of SIR2 had an effect on the life span of P. anserina under standard growth conditions. Whether SIR2 is required for the increase of longevity observed in strains subjected to CR is currently under investigation. C. Q. Scheckhuber (one of the authors of this report), from H. D. Osiewacz's group, presented data concerning changes of mitochondrial morphology during aging in P. anserina. Senescent isolates of the wild-type fungus display mitochondrial fragmentation, and if this process is inhibited by the disruption of a gene involved in mitochondrial dynamics, a marked increase in life span occurs. One reason for this effect could be suppressed reorganization of mtDNA, which is otherwise observed systematically in P. anserina wild-type strains. Further parameters such as adenosine triphosphate (ATP) concentration and oxidative stress are currently being investigated in the long-lived strain of fungus where mitochondrial fragmentation is disrupted.

K. Brys, from Vanfleteren's group, detailed the isolation and characterization of mitochondria from the nematode C. elegans. Comparison of citrate synthase enzyme activity in mitochondria and in whole worms suggests that the novel isolation procedure did not selectively damage the organelle. Examination of mitochondrial function indicates that the activity of isolated wild-type mitochondria declines with age (see Nicholls Perspective). In contrast, mitochondria isolated from long-lived worms lacking a functional daf-2 gene exhibited only a slight reduction in coupling efficiency--the conversion of redox energy stored as a proton gradient across the IMM to ATP synthesis--and capacity for ATP generation with age.

R. M. Tanguay reported on the ability of small Hsp (such as Hsp16 and Hsp27) to extend life span in the fruit fly D. melanogaster. Hsp ensure the proper folding of nascent or misfolded proteins and allow cells to survive under conditions of increased stress, and recent reports on Hsp expression in long-lived mutants of model organisms have established a link between these proteins and longevity (see Longo Perspective and "Stress for Success"). Mutants enjoying an increased life span tend to be more resistant to different kinds of stress, including heat shock, and in certain cases this stress resistance has been ascribed to up-regulation of genes encoding Hsp proteins (7).

There is as yet no feasible experimental approach to study aging at the organismal level in humans using invasive experimentation, so in vitro cell cultures have long been used as systems to investigate basic mechanisms of human aging. E. Hütter and H. Unterluggauer, from Jansen-Dürr's group, presented results concerning mitochondrial activity and the generation of ROS in cultured cells from different human tissues as a function of senescence. Using high-resolution respirometry, partial uncoupling of the mitochondrial respiratory chain (i.e., proton flow across the IMM without ATP synthesis) was shown to contribute to fibroblast senescence, possibly due to a reduction of oxidative phosporylation in these cells. By using a different cell system, human umbilical vascular endothelial cells (HUVECs), an increased load of ROS could be detected during senescence in culture, although age-associated impairments of mitochondrial function could not be identified. This suggests that the ROS observed in HUVECs might be derived from extramitochondrial sources.

Mitochondrial dynamics in HUVECs were addressed by M. Jendrach from J. Bereiter-Hahn's lab. Fusion and fission of mitochondria are frequent events in most cultured cells (8). Fusion of mitochondria can result in the mixing of mtDNA and mitochondrial proteins, and this process could represent a repair mechanism for defective mitochondria. The effects of aging on mitochondrial functions such as fusion and fission, mtDNA content, and morphology were investigated in HUVECs as a model for mitochondrial activity and dysfunction. Cells that had been "aged" in culture showed a strong reduction in both mitochondrial fusion and fission activity as compared with "younger" cells. Furthermore, in postmitotic as compared with dividing cells, a significant reduction in the abundance of intact mtDNA was observed.

Respiratory competence and the integrity of mtDNA are characteristics that are intimately linked in most systems, and these topics were discussed in two talks concerning the characterization of mammalian mitochondria. In their talk "Towards the mitochondrial proteome and the supramolecular architecture of OXPHOS complexes: Evaluation of age and ROS-stress related modifications," N. A. Dencher and F. Krause reported findings regarding the composition of the respiratory chain in bovine heart mitochondria. For the first time, proof for the existence of the protein supercomplex I1III2IV1 from the IMM of bovine heart mitochondria was gained by single-particle electron microscopic analysis. According to the "respirasome" theory, this supercomplex is one of the key functional components of the mitochondrial respiratory chain (9), and investigation of possible age-dependent variations in its architecture, as well as in the observed oligomeric state of active mammalian ATP synthases (10), is under way in various model organisms. T. Stevnsner gave a talk on DNA repair in mammalian mitochondria and its relation to aging. MtDNA is particularly vulnerable to oxidative damage because it is not protected by nucleosomes as nuclear DNA is, and of course mtDNA exists close to the site of the oxidative phosphorylation process, where hazardous ROS are believed to be produced. Several lines of evidence support the notion that accumulation of DNA damage and mutations in the mitochondrial genome leads to mitochondrial dysfunction and consequent cell death, which eventually leads to aging.

An important objective of MiMage is the characterization of novel age-related mitochondrial functions. With the experimental goal of identifying changes in the mitochondrial proteome (see Gibson Perspective) and subsequently investigating the cause of these changes at the molecular level, investigators at ProteoSys contribute high-sensitivity methods for proteome analysis and mass spectrometry. A. Schrattenholz gave a presentation on reducing the complexity of protein samples by differential and quantitative proteomics. Roughly 30,000 human genes are transcribed and translated to give several million highly dynamic protein isoforms, which moreover can vary in abundance over 8 to 15 orders of magnitude. To cope with this level of complexity, quantitative and differential displays are absolutely required to reduce complexity, allowing statistical methods to be used successfully to analyze the data. In both the separation and identification of proteins, reliable quantitative and differential detection remains the major challenge.

Outlook

From the meeting, it became clear that MiMage brings together researchers applying complementary expertise to address the same scientific problems pertaining to the aging process in different experimental organisms and systems. The highly integrated research that is being planned and pursued under the MiMage project will allow members of the consortium to go considerably beyond the state of the art in the field of biogerontology. It can be expected that the collaborative efforts of the scientists participating in MiMage will provide important contributions toward a complete view of the mechanistic role of mitochondria in aging.


May 18, 2005
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Citation: C. Scheckhuber, H. D. Osiewacz, MiMage: A Pan-European Project on the Role of Mitochondria in Aging. Sci. Aging Knowl. Environ. 2005 (20), pe14 (2005).








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