Peter Laun

Induction of autophagy by spermidine promotes longevity

Ageing results from complex genetically and epigenetically programmed processes that are elicited in part by noxious or stressful events that cause programmed cell death. Here, we report that administration of spermidine, a natural polyamine whose intracellular concentration declines during human ageing, markedly extended the lifespan of yeast, flies and worms, and human immune cells. In addition, spermidine administration potently inhibited oxidative stress in ageing mice. In ageing yeast, spermidine treatment triggered epigenetic deacetylation of histone H3 through inhibition of histone acetyltransferases (HAT), suppressing oxidative stress and necrosis. Conversely, depletion of endogenous polyamines led to hyperacetylation, generation of reactive oxygen species, early necrotic death and decreased lifespan. The altered acetylation status of the chromatin led to significant upregulation of various autophagy-related transcripts, triggering autophagy in yeast, flies, worms and human cells. Finally, we found that enhanced autophagy is crucial for polyamine-induced suppression of necrosis and enhanced longevity.

Aged mother cells of Saccharomyces cerevisiae show markers of oxidative stress and apoptosis

Recently, we and others have shown that genetic and environmental changes that increase the load of yeast cells with reactive oxygen species (ROS) lead to a shortening of the life span of yeast mother cells. Deletions of yeast genes coding for the superoxide dismutases or the catalases, as well as changes in atmospheric oxygen concentration, considerably shortened the life span. The presence of the physiological antioxidant glutathione, on the other hand, increased the life span of yeast cells. Taken together, these results pointed to a role for oxygen in the yeast ageing process. Here, we show by staining with dihydrorhodamine that old yeast mother cells isolated by elutriation, but not young cells, contain ROS that are localized in the mitochondria. A relatively large proportion of the old mother cells shows phenotypic markers of yeast apoptosis, i.e. TUNEL (TdT‐mediated dUTP nick end labelling) and annexin V staining. Although it has been shown previously that apoptosis in yeast can be induced by a cdc48 allele, by expressing pro‐apoptotic human cDNAs or by stressing the cells with hydrogen peroxide, we are now showing a physiological role for apoptosis in unstressed but aged wild‐type yeast mother cells.

A yeast BH3-only protein mediates the mitochondrial pathway of apoptosis

Mitochondrial outer membrane permeabilization is a watershed event in the process of apoptosis, which is tightly regulated by a series of pro‐ and anti‐apoptotic proteins belonging to the BCL‐2 family, each characteristically possessing a BCL‐2 homology domain 3 (BH3). Here, we identify a yeast protein (Ybh3p) that interacts with BCL‐XL and harbours a functional BH3 domain. Upon lethal insult, Ybh3p translocates to mitochondria and triggers BH3 domain‐dependent apoptosis. Ybh3p induces cell death and disruption of the mitochondrial transmembrane potential via the mitochondrial phosphate carrier Mir1p. Deletion of Mir1p and depletion of its human orthologue (SLC25A3/PHC) abolish stress‐induced mitochondrial targeting of Ybh3p in yeast and that of BAX in human cells, respectively. Yeast cells lacking YBH3 display prolonged chronological and replicative lifespans and resistance to apoptosis induction. Thus, the yeast genome encodes a functional BH3 domain that induces cell death through phylogenetically conserved mechanisms.

Yno1p/Aim14p, a NADPH-oxidase ortholog, controls extramitochondrial reactive oxygen species generation, apoptosis, and actin cable formation in yeast

The large protein superfamily of NADPH oxidases (NOX enzymes) is found in members of all eukaryotic kingdoms: animals, plants, fungi, and protists. The physiological functions of these NOX enzymes range from defense to specialized oxidative biosynthesis and to signaling. In filamentous fungi, NOX enzymes are involved in signaling cell differentiation, in particular in the formation of fruiting bodies. On the basis of bioinformatics analysis, until now it was believed that the genomes of unicellular fungi like Saccharomyces cerevisiae and Schizosaccharomyces pombe do not harbor genes coding for NOX enzymes. Nevertheless, the genome of S. cerevisiae contains nine ORFs showing sequence similarity to the catalytic subunits of mammalian NOX enzymes, only some of which have been functionally assigned as ferric reductases involved in iron ion transport. Here we show that one of the nine ORFs (YGL160W, AIM14) encodes a genuine NADPH oxidase, which is located in the endoplasmic reticulum (ER) and produces superoxide in a NADPH-dependent fashion. We renamed this ORF YNO1 (yeast NADPH oxidase 1). Overexpression of YNO1 causes YCA1-dependent apoptosis, whereas deletion of the gene makes cells less sensitive to apoptotic stimuli. Several independent lines of evidence point to regulation of the actin cytoskeleton by reactive oxygen species (ROS) produced by Yno1p.

The influence of oxygen toxicity on yeast mother cell-specific aging

The effect of deleting both catalase genes and of increased oxygen as well as paraquat (a pro-oxidant) on the replicative life span of yeast mother cells has been investigated to test the so-called oxygen theory of aging. This is well established in higher organisms, but has not been extensively tested in the unicellular yeast model system. Life span determinations were performed in ambient air or in a controlled atmosphere (55% oxygen) and an isogenic series of strains deleted for one or both yeast catalases was used and compared with wild type. In the absence of cellular catalase, increased oxygen caused a marked decrease in life span that could be completely reversed by adding 1 mM GSH, a physiological antioxidant, to the yeast growth medium. In a second unrelated strain, the effects were similar although even the wild type showed a decrease in life span when oxygen was increased. The effect could again be compensated by addition of extracellular GSH. Our results show that manipulating the detoxification of reactive oxygen species has a profound effect on yeast aging. These findings are discussed in the light of recent results relating to oxygen toxicity in the aging process of higher organisms.

Quantitation of (a)symmetric inheritance of functional and of oxidatively damaged mitochondrial aconitase in the cell division of old yeast mother cells.

Asymmetric segregation of oxidatively damaged proteins is discussed in the literature as a mechanism in cell division cycles which at the same time causes rejuvenation of the daughter cell and aging of the mother cell. This process must be viewed as cooperating with the cellular degradation processes like autophagy, proteasomal degradation and others. Together, these two mechanisms guarantee survival of the species and prevent clonal senescence of unicellular organisms, like yeast. It is widely believed that oxidative damage to proteins is primarily caused by oxygen radicals and their follow-up products produced in the mitochondria. As we have shown previously, old yeast mother cells in contrast to young cells contain reactive oxygen species and undergo programmed cell death. Here we show that aconitase of the mitochondrial matrix is readily inactivated by oxidative stress, but even in its inactive form is relatively long-lived and retains fluorescence in the Aco1p-eGFP form. The fluorescent protein is distributed between old mothers and their daughters approximately corresponding to the different sizes of mother and daughter cells. However, the remaining active enzyme is primarily inherited by the daughter cells. This indicates that asymmetric distribution of the still active enzyme takes place and a mechanism for discrimination between active and inactive enzyme must exist. As the aconitase remains mitochondrial during aging and cell division, our findings could indicate discrimination between active and no longer active mitochondria during the process.

Aging research in yeast

1. Preface and Introduction to Aging research in Yeast.- 2.Oxidative stresses and ageing.- 3.The role of Mitochondria in the aging processes of yeast.- 4.The Retrograde Response and Other Pathways of Interorganelle Communication in Yeast Replicative Aging.- 5. Chronological Aging in Saccharomyces cerevisiae.- 6. Still waters run deep: Aging and the survival of quiescent and non-quiescent cells in yeast stationary-phase cultures.- 7. Maximising the yeast chronological lifespan.- 8.Amino Acid Homeostasis and Chronological Longevity in Saccharomyces cerevisiae.- 9. DNA damage and DNA replication stress in yeast models of aging.- 10.Yeast aging and apoptosis.-11. Cellular homeostasis in fungi: Impact on the aging process.- 12.Genome-wide analysis of yeast aging.- 13. Genetic Approaches to Aging in Budding and Fission Yeasts: New Connections and New Opportunities.- 14. Evolution of asymmetric damage segregation: a modelling approach.-15. Cellular ageing and the actin cytoskeleton.

Senescence and apoptosis in yeast mother cell-specific aging and in higher cells: a short review.

It is our intention to give the reader a short overview of the relationship between apoptosis and senescence in yeast mother cell-specific aging. We are studying yeast as an aging model because we want to learn something of the basic biology of senescence and apoptosis even from a unicellular eukaryotic model system, using its unrivalled ease of genetic analysis. Consequently, we will discuss also some aspects of apoptosis in metazoa and the relevance of yeast apoptosis and aging research for cellular (Hayflick type) and organismic aging of multicellular higher organisms. In particular, we will discuss the occurrence and relevance of apoptotic phenotypes for the aging process. We want to ask the question whether apoptosis (or parts of the apoptotic process) are a possible cause of aging or vice versa and want to investigate the role of the cellular stress response system in both of these processes. Studying the current literature, it appears that little is known for sure in this field and our review will therefore be, for a large part, more like a memorandum or a program for future research.

Yeast mother cell-specific ageing, genetic (in)stability, and the somatic mutation theory of ageing.

Yeast mother cell-specific ageing is characterized by a limited capacity to produce daughter cells. The replicative lifespan is determined by the number of cell cycles a mother cell has undergone, not by calendar time, and in a population of cells its distribution follows the Gompertz law. Daughter cells reset their clock to zero and enjoy the full lifespan characteristic for the strain. This kind of replicative ageing of a cell population based on asymmetric cell divisions is investigated as a model for the ageing of a stem cell population in higher organisms. The simple fact that the daughter cells can reset their clock to zero precludes the accumulation of chromosomal mutations as the cause of ageing, because semiconservative replication would lead to the same mutations in the daughters. However, nature is more complicated than that because, (i) the very last daughters of old mothers do not reset the clock; and (ii) mutations in mitochondrial DNA could play a role in ageing due to the large copy number in the cell and a possible asymmetric distribution of damaged mitochondrial DNA between mother and daughter cell. Investigation of the loss of heterozygosity in diploid cells at the end of their mother cell-specific lifespan has shown that genomic rearrangements do occur in old mother cells. However, it is not clear if this kind of genomic instability is causative for the ageing process. Damaged material other than DNA, for instance misfolded, oxidized or otherwise damaged proteins, seem to play a major role in ageing, depending on the balance between production and removal through various repair processes, for instance several kinds of proteolysis and autophagy. We are reviewing here the evidence for genetic change and its causality in the mother cell-specific ageing process of yeast.

Yeast Aging and Apoptosis

A concerted balance between proliferation and apoptosis is essential to the survival of multicellular organisms. Thus, apoptosis per se, although it is a destructive process leading to the death of single cells, also serves as a pro-survival mechanism pro-survival mechanism that ensures healthy organismal development and acts as a life-prolonging or anti-aging anti-aging program. The discovery that yeast also possess a functional and, in many cases, highly conserved apoptotic machinery has made it possible to study the relationships between aging and apoptosis in depth using a well-established genetic system and the powerful tools available to yeast researchers for investigating complex physiological and cytological interactions. The aging process of yeast, be it replicative replicative or chronological chronological aging, is closely related to apoptosis, although it remains unclear whether apoptosis is a causal feature of the aging process or vice versa. Nevertheless, experimental results obtained during the past several years clearly demonstrate that yeast serve as a powerful and versatile experimental system for understanding the interconnections between these two fundamentally important cellular and physiological pathways.