Sci. Aging Knowl. Environ., 28 June 2006
Vol. 2006, Issue 10, p. pe15
[DOI: 10.1126/sageke.2006.10.pe15]

PERSPECTIVES

The Role of Mitochondria in Conserved Mechanisms of Aging

Christian Scheckhuber, and Heinz D. Osiewacz

The authors are at the Department of Molecular Developmental Biology, Institute of Molecular Biosciences, J. W. Goethe University, Max-von-Laue-Strasse 9, 60438 Frankfurt, Germany. E-mail: scheckhuber{at}em.uni-frankfurt.de (C.S.)

http://sageke.sciencemag.org/cgi/content/full/2006/10/pe15

Key Words: mitochondria • reactive oxygen species • retrograde response • electron transport chain • daf-2 • PARP-1

Introduction

MiMage is an integrated project on the role of mitochondria in conserved mechanisms of aging, which is funded by the European Commission (see also Scheckhuber Perspective). The overall aim of this project is to elaborate a complete view of the mechanistic role of mitochondria in aging. Therefore, different experimental organisms (Saccharomyces cerevisiae, Podospora anserina, Caenorhabditis elegans, Drosophila melanogaster, mouse, and rat), as well as cell culture systems, are being studied.

At the second MiMage symposium held in April 2006 in Frankfurt, Germany, members from participating laboratories as well as invited speakers gave presentations on their research, and some of these are outlined here.

Studies on S. cerevisiae

S. cerevisiae has been widely studied as a model for "replicative aging" (see Kaeberlein Perspective), with emphasis on respiration, mitochondrial inheritance, and mitochondrially generated reactive oxygen species (ROS). 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.

In his talk, S. M. Jazwinski (Lousiana State University, New Orleans, USA) detailed the link between the retrograde response (RR) and longevity. The RR is a signaling pathway from the mitochondrion to the nucleus that provides information about mitochondrial dysfunction and leads to adjustments in metabolism. In yeast, mitochondrial activity becomes progressively impaired with age, which leads to the induction of an RR. This mechanism is also induced in petite mutants, which bear dysfunctional mitochondria. The RR compensates for the loss of mitochondrial Krebs cycle activity by providing an alternate source of biosynthetic precursors through the glyoxylate cycle. However, induction of the RR also leads to an enhanced production of extrachromosomal rDNA circles, the accumulation of which leads to yeast death. The retrograde signal transducer protein Rtg2 is involved in both the transduction of the retrograde signal and the suppression of rDNA circle formation and, thus, genome instability (1). Rtg2 is not able to carry out both functions at the same time, which explains the concomitant increase of rDNA circle formation when RR is induced. Thus, activation of RR has to be carefully regulated, although some of the activities induced by RR are able to counteract the negative effects of rDNA circle accumulation on life span, as demonstrated by certain petite mutants that live longer than normal strains even though they produce more rDNA circles. A protein that is able to potentiate RR and, moreover, is necessary for life-span extension by this mechanism, is the small guanine nucleotide-binding protein Ras2.

L. Hlavata from T. Nyström's group (Göteborg University College, Sweden) described the characterization of slowly growing and prematurely aging yeast cells containing an overactive RAS2 allele, RAS2val19 (2). Interestingly, RAS2val19 mutants display an abrogated activity of the mitochondrial adenine nucleotide translocator (ANT), which is a result of proteolysis-dependent fragmentation caused by highly elevated ROS production. This extensive oxidative stress is derived from mitochondrial nonphosphorylating respiration--a form of respiration that is prone to generating ROS--and down-regulation of major antioxidant defense mechanisms. Overexpression of the gene encoding the yeast ANT (AAC2) was shown to restore ANT activity in RAS2val19 cells, although phosphorylating respiration was not recovered. Moreover, AAC2-overexpressing cells displayed a restored growth fitness, suggesting that AAC2 plays an important role during yeast growth.

E. Bogengruber and P. Laun from the group of M. Breitenbach (University of Salzburg, Austria) reported on the characterization of two novel yeast genes differentially expressed in senescent versus exponentially growing wild-type cells, YGR076C (encoding a mitochondrial ribosomal protein) and YKL056C (encoding the yeast ortholog of the mammalian translationally controlled tumor protein TCTP). Deletion of YGR076C leads to respiratory deficiency, but the life span of such mutants is about 50 % longer than that of the congenic wild-type strain. Experimental data show that YGR076C plays a role in the TOR (target of rapamycin) pathway, which affects life span in other model organisms like C. elegans and D. melanogaster (see "More Without TOR" and Kapahi Perspective). The protein encoded by YKL056C, also known as Mmi1, is needed for the proper assembly of microtubules. Cells that lack this protein ({Delta}mmi1 cells) are very sensitive to the microtubule-destabilizing drug benomyl. Interestingly, in comparison with wild-type cells, the mutant cells are also more resistant to hydrogen peroxide and longer lived. Bogengruber and Laun hypothesize that destabilization of microtubules can lead to oxidative stress resistance, although at present the mechanism responsible for this effect is not clear.

Studies on Podospora anserina

The filamentous fungus Podospora anserina has been a model system for the study of aging for more than 50 years. Mitochondria play a key role in the aging process in this organism [for a review, see (3)]. For example, inactivation of the respiratory chain complex IV (cytochrome-c-oxidase) has been shown to lead to a marked increase of life span and to stabilization of the mitochondrial DNA (mtDNA), which is dramatically reorganized in wild-type strains during aging. A. Sainsard-Chanet (Centre National de la Recherche Scientifique, Paris, France) presented data on the characterization of a complex III (cytochrome-c-reductase) loss-of-function mutant. The phenotype of the cyc1 mutant is similar to that of complex IV-deficient strains (in that it displays a slow growth rate, female sterility, etc.). Other characteristic features are lowered ROS production and a pronounced increase of life span as compared with wild type.

A. D. van Diepeningen (Wageningen University, The Netherlands) from R. Hoekstra's group gave a presentation on calorie restriction and life-span extension in P. anserina. Life span was shown to increase if the amount of carbon or nitrogen in the growth medium was reduced. Possibly the response to calorie restriction has some adaptive value to the fungus, because its self-fertile period is also extended. Interestingly, large-scale reorganizations of the mtDNA that appear systematically in P. anserina wild-type strains during aging are impaired under low-caloric conditions.

J. Grief (Johann Wolfgang Goethe University, Frankfurt, Germany) from the group of H. D. Osiewacz detailed the characterization of the P. anserina electron transport chain (ETC). Applying measurements on isolated mitochondria, which display tight coupling between electron transport and oxidative phosphorylation and are bioenergetically fully competent, revealed that the ETC in P. anserina is a fine-tuned mixture of standard and alternative pathways (4). The latter are composed of alternative NADH (nicotinamide adenine dinucleotide) dehydrogenases and a cyanide-insensitive AOX (alternative oxidase). AOX is induced in several long-lived P. anserina mutants as a result of complex IV deficiency. Respiration in these mutants (for example, grisea and PaCox17::ble) is increased but less effective as compared with that in wild type. Moreover, the mutant grisea is characterized by a four-fold reduction of mitochondrial superoxide generation as compared with a wild-type strain.

Studies on C. elegans

T. E. Johnson (University of Colorado, Boulder, USA) reported on differences in individual life spans of the nematode C. elegans that cannot be attributed to either measurable genetic or environmental causes (see "How Long Do I Have, Doc?"). At least 50% of these stochastic differences can be ascribed to variations in the physiological state that can be elegantly measured by using transgenic worms that express green fluorescent protein (GFP) under the control of a heat-shock inducible promoter (5). Chance variation in the level of GFP induction measured on the first day of adult life allows researchers to separate worms into distinct populations that are destined to have a three- to five-fold difference in subsequent longevity. The molecular and physiological concomitants of these observations are currently under investigation in the Johnson group.

K. Brys (Ghent University, Belgium) from the group of J. Vanfleteren spoke on the physiological comparison of mitochondria isolated from wild type and long-lived daf-2 (e1370) mutants, in which an insulin/insulin-like growth factor receptor is defective (see Sonntag Perspective). In both cases, mitochondrial function decreases with age. There is, however, some attenuation of this age-dependent decline in daf-2(e1370) versus wild-type mitochondria, as could be shown by measuring oxygen consumption, ATP synthesis capacity, and citrate synthase activity, for example, as measures of mitochondrial activity. Intriguingly, production of the ROS H2O2 is enhanced in daf-2(e1370) mutants and decreases more gradually with age than in wild-type animals, suggesting that mitochondrial H2O2 production in daf-2(e1370) reflects enhanced (contrary to deregulated) mitochondrial function. These data are seemingly difficult to bring in line with the free radical theory of aging, which links aging to oxidative damage by ROS (see Harman Classic Paper and Dugan Perspective). K. Brys hypothesized the existence of some hitherto unknown mechanism that probably is able to reduce or prevent mitochondrial damage in the mutant.

Studies on D. melanogaster

R. M. Tanguay (Laval University, Québec, Canada) presented novel data on the characterization of transgenic D. melanogaster strains in which the gene encoding the small mitochondrial heat-shock protein Hsp22 is overexpressed. Previous studies performed by Tanguay's team showed that overexpression of wild-type Hsp22 in motor neurons increases the flies' life span and resistance to stress and results in the extended maintenance of locomotor activity (6, 7). Microarray analysis showed that transcripts of genes involved in (i) protein and DNA processing, (ii) control of the cell cycle, and (iii) cell defense are the most up-regulated in Hsp22-overexpressing versus wild-type flies. Furthermore, these experiments have revealed that a transcription factor involved in the insulin pathway signaling, dFoxo, might play a role in the age-dependent expression of Hsp22.

Studies on Human Cells

The analysis of cellular and molecular determinants underlying the senescence response of human umbilical vascular endothelial cells (HUVECs) was the central topic of the talk given by E. Hütter (from the group of P. Jansen-Dürr, Austrian Academy of Sciences, Innsbruck, Austria). Upon reaching senescence, HUVEC cells die by an apoptotic mechanism. Moreover, oxidative stress accelerates this senescence-associated apoptosis in this cell culture system. One source of ROS in old cells is, in addition to dysfunctional mitochondria, the calcium-dependent NADPH oxidase (NOX) isoform 5. This protein was found to be more abundant alongside elevated calcium concentrations in senescent HUVECs, demonstrating a close relation between oxidative stress, calcium concentrations, and NOX 5 during aging of HUVEC cells.

M. Jendrach from the group of J. Bereiter-Hahn (Johann Wolfgang Goethe University, Frankfurt, Germany) presented data on the dynamics of mitochondrial inner membrane proteins in human HeLa cells (see Scheckhuber Perspective for a discussion of mitochondrial dynamics). Two transgenic HeLa cell lines were created, each expressing a differently labeled subunit of respiratory complex I. In one case, the protein was tagged with GFP, and in the other with the red fluorescent protein DsRed. Representative cells were then fused, and efficient mixing of inner mitochondrial proteins (derived from different mitochondria) by mitochondrial fusion and fission was assessed using fluorescence microscopy. The presence of yellow-colored mitochondria indicated that such mixing had occurred. Analysis of these hybrid mitochondria by high-resolution fluorescence microscopy and electron microscopy indicated that in fact not only proteins but whole cristae are being transferred between mitochondria. Mitochondrial fission and fusion activity is regarded as a rescue or repair mechanism, and mitochondrial dynamics are significantly decreased in senescent versus actively dividing HUVEC cells (8), implying a role for the mixing of mitochondrial components in the aging process of these cells.

Additional Studies

F. Krause from the group of N. A. Dencher (University of Technology, Darmstadt, Germany) gave a talk on biochemical techniques to investigate age-triggered alterations of the mitochondrial proteome. Especially powerful tools are blue and colorless native polyacrylamide gel electrophoresis to separate functional respiratory complexes and ATP synthase oligomers from mitochondria treated with the detergent digitonin (9, 10). Currently, mitochondrial proteomes from various models used in aging-related research (for example, P. anserina and rat) are being analyzed in the Dencher laboratory with a focus on understanding the assembly and stability of respiratory proteins during the course of senescence.

V. A. Bohr (National Institute on Aging, Baltimore, USA, and Aarhus University, Denmark) discussed mtDNA repair mechanisms. In contrast to the elaborate repair pathways of nuclear DNA, relatively little is known about how mitochondria keep their mtDNA free from damage. For example, it seems that the nucleotide excision repair found in the nucleus is not present in mitochondria. However, these organelles possess a base excision repair (BER) mechanism that is capable of removing oxidative mtDNA modifications (11). The functionality of BER is essential for mtDNA stability and cellular survival. Moreover, BER activity appears to decline with human age and is also likely to be associated with neurodegeneration.

In his talk, A. Bürkle (University of Konstanz, Germany) reported on the ability of poly(ADP-ribose) polymerase 1 (PARP-1) to preserve genomic stability (see Beneke Review). Instability of the genome is regarded as an important factor involved in diseases (such as cancer) as well as aging [for a review, see (12)]. The increased poly(ADP-ribosylation) capacity identified in long-lived species might contribute to a retarded accumulation of DNA damage and thus slow down the rate of aging more efficiently than in short-lived species.

Conclusion

The second symposium on the role of mitochondria in conserved mechanisms of aging succeeded in bringing together researchers working on a wide range of model organisms and systems in the field of aging-related research. It became clear that just 1 year after the start of the MiMage project, several important interim results have been obtained by the participating laboratories. Their collaborative efforts in different research areas, including biochemistry, cellular and molecular biology, and physiology, will considerably expand the knowledge on how mitochondria control aging.


June 28, 2006
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Citation: C. Scheckhuber, H. D. Osiewacz, The Role of Mitochondria in Conserved Mechanisms of Aging. Sci. Aging Knowl. Environ. 2006 (10), pe15 (2006).








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