Mitochondria in yeast aging

Yeast mother cell-specific aging is being studied intensively by a growing number of research groups in Europe and the USA. The idea behind this revival of yeast aging research is, of course, that the simple and versatile yeast genetic system might reveal mechanisms of aging which are common (so-called "public mechanisms of aging") to both yeast cells and higher cells. In our recent research, we have shown that highly oxidative (but still not identified) molecules accumulate in mitochondria of senescent yeast mother cells. These molecules are presumably reactive oxygen species (ROS). Senescent cells show biochemical markers of yeast apoptosis (programmed cell death). Antioxidants and pro- oxidants have the expected physiological effects on the lifespan of yeast mother cells. Involvement of oxygen toxicity in aging has also been shown through genetic studies in C. elegans, in D. melanogaster, and in the mouse. However, more detailed work is needed to clarify the origin and role of ROS and of mitochondrial physiology in aging and to test whether the role of ROS is causative in the process. Therefore, the major aims of the current project are: I. To determine the role in the aging process of reactive oxygen species accumulating in mitochondria. As we showed that ROS or highly oxidizing molecules are primarily located in the mitochondria of senescent yeast cells, we will biochemically and structurally analyze the mitochondria of senescent yeast cells and study the effect of mitochondrial mutations (located both on the nuclear and mitochondrial genome) on the aging process. For instance, respiratorydeficient pet- and mit- mutants as well as the "petite-negative" mutant lacking the mitochondrial adenine nucleotide translocator will be tested. ESR measurements and spin trap experiments are aimed at identifiying the oxidizing molecular species occurring in old cell mitochondria. II. To identify yeast mutants which are either hypersensitive or resistant to oxidative stress. The Euroscarf collection of 4627 viable deletion mutants corresponding to nearly all non-essential yeast genes is at our disposal. Candidate mutants recognized through this screen will be tested for their influence on replicative lifespan. A new mutant screening system will be developed and also point mutations (which frequently are more revealing than the deletions) will be isolated which change the oxidative stress resistance of yeast cells. A simple but efficient screening system will be used to test lifespan in these mutants. The system is based on a conditional mutant strain in which only the mother cell lives, but all daughter cells die. III. To test the involvement of the protein kinase A (RAS/cAMP) signaling system of the yeast cells in the creation of oxidative stress and in senescence. This is based on our earlier work showing that constitutively active yeast RAS2 greatly reduces lifespan and on recent work showing that oxidative stress and apoptosis are induced by ras activation in primary human cells.

Studies of yeast mother cell-specific aging are performed by a growing number of research groups in Europe and in the U.S.A. The idea behind this revival of yeast aging research is that the simple and versatile genetic system of yeast might reveal mechanisms of aging which are common to all eukaryotic cells and organisms, so-called "public mechanisms of aging". Despite an enormous increase in life expectancy of humans over the last 50 years, very little is known about the basic intrinsic mechanism of aging which also explains the renewed interest in aging research. Results obtained and genes identified in yeast can be tested in other model systems of aging like the mouse or human cell cultures. The most important results obtained in this project are: i) a novel assay for measuring the replicative lifespan of yeast cells was developed which enables us to test large numbers of candidate genes and mutants which are suggested to be involved in the intrinsic aging process; ii) we showed that the redox metabolism of the cells plays a central role in the aging process. Reactive oxygen species (ROS) originating from mitochondria and the cellular defense systems against ROS have a strong influence on the lifespan; iii) A mutation in the yeast RAS2 gene that induces apoptosis and premature aging (very similar to the homologous mutation in human cells) does so by inducing a large amount of oxygen radicals as measured by electron spin resonance. These radicals arise by influencing mitochondrial physiology and the extramitochondrial production of superoxide; iv) DNA damage caused by a defect of the origin recognition complex strongly induces ROS and apoptosis and activates a battery of genes which are silent in growing cells under non-stressed conditions. Stress physiology in a broad sense plays a central role in aging. v) The asymmetric distribution of mitochondria between mother and daughter cells during a cell division cycle was tested both with and without applying oxidative stress. Contrary to expectation, no asymmetric distribution of mitochondria was observed. However, as found by others, some cytoplasmic oxidized proteins are indeed distributed asymmetrically; vi) Genes and mutants were identified using global transcriptome analysis on microarrays and screening of the yeast deletion collection which substantially increase the yeast lifespan on deletion. Both of these genes (YKL056c and YGR076c) code for proteins that are located in or on mitochondria. The detailed function of these genes is studied in the subsequent FWF project S9303-B05 and in an international joint project of the EU (MIMAGE). In general, we have shown that yeast mother cell-specific aging to a large degree is governed by oxidative stress generated in the mitochondria in accordance with one of the most generally accepted aging theories, the so-called oxygen theory of aging. However, aging is clearly a multifactorial process and we are now studying the relationship between our results and other aging theories, for instance the influence of specific dietary restriction on the aging process, and the cross-reactivity with a non-general yeast aging mechanism related to extrachromosomal minicircles of DNA.