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Senescence and apoptosis in yeast mother cell-specific aging and in higher cells: A short review
Biochimica et Biophysica Acta 1783 (2008) 1328–1334
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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a m c r
Review
Senescence and apoptosis in yeast mother cell-specific aging and in higher cells: A short review Peter Laun, Gino Heeren, Mark Rinnerthaler, Raphaela Rid, Sonja Kössler, Lore Koller, Michael Breitenbach ⁎ Department of Cell Biology, Division of Genetics, University of Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg, Austria
a r t i c l e
i n f o
Article history: Received 4 January 2008 Received in revised form 12 February 2008 Accepted 13 February 2008 Available online 4 March 2008 Keywords: Yeast apoptosis Replicative lifespan Chronological lifespan S. cerevisiae Translational control Eukaryotic ribosome AIF YCA1 NUC1 NMA111
a b s t r a c t 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. © 2008 Elsevier B.V. All rights reserved.
Modern apoptosis research starts with the seminal paper of Kerr [1] which drew the attention of the scientific world to the previously ignored fact that the ordered and programmed removal of cells of multicellular organisms under a variety of physiological conditions is as important as the production of these cells. In this early paper a clear distinction was made between apoptosis, a smooth and mostly unnoticed process which occurs without inflammation, and necrosis, a process usually accompanied with inflammation. At least three rather different biological purposes of the apoptotic process of cellular suicide seem to exist. First, cells commit suicide during the process of morphogenesis early in life. Secondly, cells that have accumulated damage beyond a certain threshold are removed by apoptosis and are replaced thereby ensuring proper functioning of organs in adult life. Thirdly, senescent cells also undergo apoptosis, a process that up to now has been studied mainly in cultured mammalian cells at the Hayflick limit (replicative senescence). A prominent example for morphogenic apoptosis in the embryo which has found its way into numerous textbooks is the formation of digits of the mouse paw during embryogenesis [2]. In this work, apoptotic cells were recognized through acridine orange staining and by electron microscopy analyzing apoptotic bodies which were either ⁎ Corresponding author. E-mail address: [email protected] (M. Breitenbach). 0167-4889/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamcr.2008.02.008
free or engulfed by professional phagocytes. Mouse embryos completely devoid of professional phagocytes as a consequence of a transcription factor mutation in homozygous PU.1 null mice showed a defect in the process of engulfment which was partly taken over by mesenchymal cells. Later [3] apoptotic markers like DNA fragmentation shown by the TUNEL test were shown to occur in the system, and the signaling function of externalized phosphatidyl serine and other possible surface markers was studied in detail [4,5], revealing that externalized phosphatidyl serine signals both to macrophages which subsequently engulf the apoptotic cells, and to down-regulate the inflammatory response in the tissue [6]. Mutants in known “apoptotic genes” that were studied revealed mostly a mild phenotype but occasionally an embryonic or perinatal lethal phenotype as shown by the DNAse II alpha homozygous knock-out mutant [7–9]. The embryonic lethal phenotype in this case is probably not due to lack of apoptotic functions but due to a defect in the essential “day-job” function of the DNAse II alpha, for instance in haematopoiesis. It is remarkable that the signals and the mechanism of removal of apoptosed cells seem to have been conserved between widely divergent animal species, like Drosophila melanogaster, nematodes and vertebrates. What is also conserved to a certain degree are the genes and proteins that are needed for the execution of the programmed cell death process. Many of the CED (cell death) genes of Caenorhabditis elegans have been shown to fulfill biochemical
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functions conserved in mammals and also show a certain degree of sequence conservation [10]. On the other hand, the multiple stimuli (pathways) converging into apoptotic processes and the presumed multiplicity of apoptotic mechanisms is less well known and poses a challenge to current research. For instance, little is known about the signals (intracellular and extracellular) leading into the embryonic morphogenic apoptosis just described and about the conservation of pathways between the embryonic and other forms of apoptosis. An interesting hint can be seen in the fact that exposing C. elegans to oxidative stress during development leads to an increase in the number of cells that undergo apoptosis. Although a correlation of apoptosis with senescence and aging has been found in some systems (see below), in C. elegans the genetic analysis of both processes, apoptosis and aging, has revealed little overlap between the two processes so far [10]. This is to say that compromising the developmental apoptosis through mutation has, surprisingly, not diminished the fitness and fecundity of the organism [11,12], but has also not led to a change in lifespan [13]. Conversely, and interestingly, prohibiting developmental apoptosis through null mutations in pro-apoptotic genes leads to embryonic lethality in D. melanogaster and in the mouse [14,15]. The worm system has arguably the best “genetics of aging” in the form of a large number of mutants that can be ordered in a signaling pathway, the IGFR pathway, which also governs the formation of dauer larvae. However, these mutations have no influence on developmental apoptosis. Although apoptosis occurs in more than 10% of all cells during development, many of them in the nervous system, and apoptosis can be induced through external oxidative stress, apoptotic markers are reportedly absent from senescent worms. Apoptosis plays a major role in several distinct processes of immune regulation. To give an example, the down-regulation of expanded clones of B- and T-cells when a challenge with microbial pathogens is no longer present; and the elimination of anti-self clones from the immune repertoire of the organism. These processes work through activation-induced cell death [16] and involve binding of a relevant ligand to the surface receptor of immune cells, Fas. This system is obviously independent of external oxidative stress and it is
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not completely clear if the well known mitochondrial branch of the apoptotic mechanism is a necessary component of this form of apoptosis [16]. Apoptosis in multicellular animals is well documented as an antiaging mechanism, because it can remove damaged cells from tissues, inducing the replacement of these cells by homeostatic mechanisms, and thereby leads to tissue repair [17]. Many different forms of molecular damage can lead cells into this process. The most generally accepted mechanism of damage that is at work here is the oxidative damage exerted primarily by reactive oxygen species (ROS) produced as by-products of the mitochondrial respiratory chain. This kind of oxidative damage is found in aged and apoptotic cells in all model systems known, including yeast. A huge body of published evidence exists describing the origin (mainly from complex I and III), chemical reactivity, reaction products and forms of cellular damage (genotoxic and cytotoxic) which the primary oxygen radical, superoxide, can induce directly or indirectly in the living cell (summarized in [18]). These processes can be mimicked by exposing the cell to oxidants, like hydrogen peroxide or to a large number of other damaging agents. Much less is known about the possible role in apoptosis of extramitochondrial sources of ROS (for instance, NADPH oxidases; [19]). However, an additional large number of primary triggers different from the mitochondrial oxidative damage just described and leading ultimately also into the apoptotic process have been published. Frequently, as a consequence of these “other” defects, the cell secondarily creates oxidative stress. One example is replication stress [20,21]. The relationship of cell division cycle checkpoints and cell division cycle regulation with the entry into apoptotic pathways is an interesting and unfinished field of apoptosis research. Cell cycle arrest at a checkpoint (often studied at the DNA damage checkpoint in G2) in principle can lead to two scenarios depending on the duration of cell cycle arrest: i) The cell can return into a normal cell cycle after DNA repair; or ii) the cell can switch on the apoptosis program [22,23]. However, another avenue into apoptosis is opened by loss of a checkpoint function [24,25]. This is
Fig. 1. The picture shows in part: (A) exponentially growing young yeast cells of strain W303, a commonly used haploid MATa strain, which are included here to show the size and morphology difference to old cells. (B) A typical lifespan determination of the same wild-type strain. The number of budding cycles that each of a set of 50 “virgin” cells undergoes before it stops dividing was determined by micromanipulation and counting of budding cycles. (C) M is the terminal mother cell after 15 cell cycles. Note the enormous size as compared to young cells and the surface changes. D14 is the second but last daughter that did not completely separate from the mother and did not give rise to new living cells. Also, the surface of D14 is folded or wrinkled. The budding of the “granddaughter” D14-1 stopped at an early stage of the cell cycle. The budding of last daughter cell, D15, also stopped at an early stage of the cell cycle. Reprinted from [28], with permission from Elsevier.
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intriguing, because the apoptotic final stage senescent yeast mother cells observed by us [26] often die in the middle of an ongoing cell cycle [27] as judged by their still attached daughter cell (of variable size) or by the initiation of a new cell cycle while the “old” cell cycle is still not finished [28] (Fig. 1). Although not studied or published in detail up to now, the occurrence of such processes in very old yeast mother cells logically would lead to aneuploid cells among the daughters of these mother cells. This is a striking parallel to many human cancer cells which also have lost cell cycle controls and which also are often aneuploids. It is remarkable that the correlation of apoptosis with oxidative stress seems to be general. Even in those cases where the primary cause of apoptosis is unrelated to oxidative stress (for instance during faulty response to DNA damage), the destruction program which is turned on, includes production of ROS [21,29]. Apoptosis can act as a quality control in germ cells. Both in the nematode and in mammals, a very high proportion of the female germ cells undergo apoptosis and only a tiny fraction survive [30]. It has been speculated that this surprising fact serves the purpose of quality control eliminating any haploids with errors due to premeiotic DNA synthesis, faulty segregation at the meiotic divisions and, perhaps also heteroplasmic mitochondrial mutations [31]. A similar quality control was observed in mammalian male meiosis [32]. We now want to give an overview of the key topic of this short review article, which is the correlation of cellular senescence with apoptosis. Very few papers dealing explicitly with the relation of aging and apoptosis have been published so far and most of them are speculative. Their main point is twofold: i) caloric restriction which is the only method of lifespan elongation that works in all known model systems of aging, likewise influences lifespan and apoptosis (compare [33] and the literature references therein); ii) genes in the insulin like growth factor receptor pathway have a strong influence on lifespan in C. elegans, are conserved in mammals, and are in the mammalian system known for their control of apoptosis [33]. More recently, an elongation of the lifespan has been shown for mouse mutants in the same pathway [34]. Papa and Skulachev [35] discuss the mitochondrial origin of oxygen radicals, and their influence on
both processes, aging and apoptosis. However, as mentioned elsewhere in this short review article, in many cases manipulating an “apoptotic gene” has not resulted in the expected lifespan elongation and, vice versa, manipulating a longevity gene has not resulted in the expected effect on apoptosis (compare for instance, [13] and Table 1). Currently available evidence shows that both in replicative aging of yeast mother cells [26] and in the replicative in vitro (Hayflick-type) aging of primary human cells (HUVEC; [36]), these cells in their final stage display markers of apoptosis: inversion of the plasma membrane as analyzed by binding of annexinV without general perforation of the plasma membrane, an increase in ROS as detected by dihydrorhodamine, and fragmentation of nuclear DNA as analyzed by the TUNEL method. Not all human cell cultures show this phenotype, as established lines of fibroblasts do not [36]. In the field of aging research of higher organisms it is an open question to what degree Hayflick-type aging of cell culture cells in vitro might be relevant for the aging process of the organism as a whole. The discrepancy between the in vivo and the in vitro situation might be summarized in three points: i) the cell culture medium does not exactly reproduce the in vivo situation and the neighboring cells of the niche are absent; ii) the cells studied are not stem cells which arguably may be the most relevant cells to study; iii) the lifespan of postmitotic cells of some organs (for instance of the central nervous system) might be limiting for the survival and function of the respective organ, a situation which is not mimicked in the cell culture system. It is highly relevant to answer the questions whether apoptotic cells can be found in healthy centenarians, whether an exhaustion of a stem cell population is indeed found, and whether the exhaustion of a stem cell population takes place (if it takes place) by way of cellular senescence and apoptosis. These questions are presently mostly answered in a negative way. To give an example, there seems to be no exhaustion of the satellite cells of skeletal muscle and no apoptotic markers are observed in biopsies. However, in certain diseases, like Duchenne muscular dystrophy, the stage of exhaustion of stem cells is indeed reached. Clearly, further progress of stem cell research is needed to answer these questions and it is to be expected that in the future the role of stem cell exhaustion and apoptosis in organismic aging will be clarified.
Table 1 Genes involved in the yeast apoptotic pathway with a short description according to Incyte BioBase [63] and the change in replicative and chronological lifespan of the corresponding deletion mutant compared to wild type strain Name
ORF
Proapoptotic YCA1[64,65]
YOR197W
NDI1[66,67]
AIF1 [53,64] NUC1[64,68] NMA111 [69,70] CPR3 [71] DNM1[72,73]
FIS1[72–74]
Antiapoptotic BIR1 [70]
Description [63]
Putative cysteine protease similar to mammalian caspases, involved in regulation of apoptosis upon hydrogen peroxide treatment YML120C NADH:ubiquinone oxidoreductase, transfers electrons from NADH to ubiquinone in the respiratory chain but does not pump protons, in contrast to the higher eukaryotic multisubunit respiratory complex I; phosphorylated; homolog of human AMID YNR074C Mitochondrial cell death effector that translocates to the nucleus in response to apoptotic stimuli, homolog of mammalian Apoptosis-Inducing Factor, putative reductase YJL208C Major mitochondrial nuclease, has RNAse and DNA endo- and exonucleolytic activities; has roles in mitochondrial recombination, apoptosis and maintenance of polyploidy YNL123W Protein of unknown function which may contribute to lipid homeostasis and/or apoptosis; sequence similarity to the mammalian Omi/HtrA2 family of serine proteases YML078W Mitochondrial peptidyl-prolyl cis-trans isomerase (cyclophilin), catalyzes the cis-trans isomerization of peptide bonds N-terminal to proline residues; involved in protein refolding after import into mitochondria YLL001W Dynamin-related GTPase required for mitochondrial fission and the maintenance of mitochondrial morphology, assembles on the cytoplasmic face of mitochondrial tubules at sites at which division will occur; also participates in endocytosis YIL065C Mitochondrial outer membrane protein involved in membrane fission, required for localization of Dnm1p and Mdv1p during mitochondrial division; mediates ethanol-induced apoptosis and ethanol-induced mitochondrial fragmentation
YJR089W
Essential chromosomal passenger protein involved in coordinating cell cycle events for proper chromosome segregation; C-terminal region binds Sli15p, and the middle region, upon phosphorylation, localizes Cbf2p to the spindle at anaphase
Chronological Replicative lifespan [60] lifespan Increased
n. a.
Increased
n. a.
Increased
n. a.
Increased
no change
Increased
n. a.
Increased
n. a.
Increased
increased
Decreased
increased
n. a.
n. a.
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The situation of aging yeast mother cells is simpler than the scenario just described. Büttner et al. [37] have given a nice overview of the physiological situations in which yeast cells display apoptotic phenotypes. Both of the aging processes of yeast cells, chronological aging and mother cell-specific aging, have been described to lead to apoptosis of the terminally senescent cells. This is remarkable because the physiological situation of terminal mother cells is very different from chronologically aged cells. The aging of yeast mother cells depends on the number of cell generations that a mother cell has undergone and not on calendar time (Fig. 2). The distribution function of lifespans of a cohort of yeast cells is described by the Gompertz law. The aging process occurs in the presence of nutrients and has nothing to do with starvation. The old mother cells, like the senescent HUVEC, are much larger than young cells, display a longer cell cycle time and a slowed down translational activity of the ribosomes, their nuclei appear fuzzy (not compact like in young cells), DHR staining indicates that the cells have produced an oxidizing internal milieu even if external oxidative stress is absent, and their actin cytoskeleton looks collapsed and appears clumpy [26,38]. On the other hand, chronologically aged (stationary) yeast cells are reproductively still young, but undergo a process of senescence due to the lack of nutrients. From stationary phase cultures two quite different cell populations can be isolated on density gradients [39]. One population of cells in a stationary culture acquires a long chronological lifespan through a process of differentiation involving changes of the cell wall, synthesis of reserve carbohydrates like trehalose and glycogen and quiescence of the genome, resembling a true G0 state. This cell population consists of the daughter cells resulting from the last cell division cycle
Fig. 2. Yeast mother cell specific aging is characterized by a limited capacity to produce daughter cells. The lifespan is determined by the number of cell cycles a mother cell has undergone, not by calendar time, and in a population of cells the lifespan distribution follows the Gompertz law. Daughter cells reset their clock to zero and enjoy the full lifespan characteristic for the strain. Reprinted from [62] with permission from Elsevier.
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which the culture has performed. The other population of cells in the stationary cultures shows adherent daughter cells in unfinished cell cycles, is much shorter lived than the first one, creates oxygen radicals and dies much earlier than the first one. Eventually, both populations at very different times reach apoptosis. It has been speculated that the postmitotic aging just described and resulting in apoptotic death might be an adaptive trait that was positively selected for during evolution [37,40] because it supports the survival of the yeast strain in times of lack of nutrients. This was further elaborated by Longo and Skulachev who hypothesized that most aging processes (also those that occur in higher organisms) have an adaptive value and are “altruistic” [41]. However this and similar hypotheses concerning multicellular organisms have been challenged on the grounds of evolutionary theory, in our opinion with convincing arguments [42– 44], two of which are as follows: i) The vast majority of individuals in a wild population never reach senescence; therefore, the process of aging cannot have been selected for; and ii) the force of biological selection is at work until progeny has been procreated and can survive on their own. Turning to yeast mother cell-specific aging, we can say that the final demise of the senescent mother cells is almost certainly of no positive value for the growing yeast strain or for the growing yeast colony. Wild type laboratory yeast strains display a median replicative lifespan of about 20–30 generations and a maximum lifespan of about 40–60 generations [38]. The fraction of cells which is senescent in a population of yeast cells is exceedingly small (1/2n) where n is the number of generations after which the cell dies. The nutrients supplied by the death and lysis of the old mother cell are therefore negligible. We have to look for other more convincing reasons why those old mother cells undergo apoptosis. The key observation was that cell division cycles of budding yeast are asymmetric, producing one daughter that is rejuvenated and has reset the clock to zero, and one mother cell which in every cell cycle acquires not only an additional bud scar, but also a gradual change in the parameters mentioned above (cell size etc.). This cell division asymmetry is similar to the asymmetric division of a stem cell in the human body, which produces one new stem cell and one cell that has undergone the first step to cell differentiation. A further key observation is that life and metabolism inevitably produce waste and the waste must be either cleanly removed or at least asymmetrically segregated between mother and daughter cell if the species is to survive over evolutionary time spans. Waste in the case of the yeast cell (but also in higher cells) is not exclusively but predominantly the oxidation products of cellular components. There are a myriad of compounds that occur in vivo and could be monitored because they are waste products that do not occur in newborn cells. One method that has been used frequently for quantitation and for fluorescent in situ microscopy is the following: The carbonyl groups which exist on proteins due to oxidative reactions (“protein carbonyls”) are conjugated with p-nitro phenylhydrazine and then the hydrazones are decorated with standardized antibodies directed to p-nitro phenylhydrazone (the “oxy-blot” technique) [45,46]. In dividing yeast cells it is found that both soluble cytoplasmic proteins and mitochondrial proteins are oxidized during aging and under stress that can induce apoptosis [47]. Oxidation is not generally found on every protein but is observed on a small number of specific proteins and with a strong bias in the mother cell, not in the daughter cell [47–49]. The mechanism of this asymmetric segregation or asymmetric scavenging which occurs also in E. coli and in higher cells [49] is presently not known. However, manipulating the asymmetric segregation through mutation of the yeast SIR2 gene, leads to the expected shortening of the lifespan [48]. In one scenario that looks plausible to us, survival and evolutionary success of a (single-celled) species primarily depends on the ability to asymmetrically segregate damage in cell division cycles. A side effect of this phenomenon is the gradual accumulation of damage in mother cells, leading to the activation of programmed cell death once a
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threshold is reached and finally to senescence and cell death. This kind of cell death clearly is evolutionarily neutral and was therefore not selected against; however it is not “altruistic”. What appears as a genetic program of aging is in reality the genetic program(s) of stress response, because the accumulation of damage means that the aging cells build up stress, most importantly, oxidative stress. Part of this stress response program is identical with the apoptotic program which in a rudimentary form existed in evolution already before the first multicellular organisms appeared and which still exists today in many single celled organisms. However, not every aspect of apoptosis exists in monocellular organisms. To give and example, the highly important “engulfment” part of the apoptotic program [50] and the Fas-receptor are not found, but extrinsic pathways (alpha factor; [51]) as well as the intrinsic mitochondrial pathway into apoptosis are surprisingly well conserved. This includes mitochondrial components that are translocated to the nucleus (AIF, endo-G) and a growing number of genes and gene functions which were previously thought to be specific for the apoptotic process in metazoan eukaryotes, are now known to exist in yeast too. In addition, the genetic system of yeast which is probably the most highly advanced genetic system existing, enabled the researchers to discover new “apoptotic” genes, which are highly conserved but previously unknown as “apoptotic” in higher cells. One prominent example is CDC48 [52]. Another one is AIF, which was found in both systems [53]. A list of yeast genes which directly or indirectly are involved in the apoptotic mechanism is given in other review articles in this volume. Another example is “ER stress” related to the unfolded protein response and, closely related, the stress created by defects in the ribosome and protein synthesis machinery. There is also indirect evidence for a switch in ribosome specificity associated with the switch of a cell to the apoptotic mode, which includes a change in gene expression at the transcriptional but also at the translational level. As this “translational” road into apoptosis has not been reviewed in detail before, we will now briefly elaborate on this emerging topic. Recently, modulation of ribosome function during the apoptotic response was described [54]. The authors show that apoptosis is dependent on synthesis of apoptosis-specific proteins through a switch in the protein synthesis machinery. Similar “switching” of the ribosome is observed from yeast to mammals in recent reports for the aging process [55,56] and for apoptosis [54,57]. The translational switch in the course of the unfolded protein response in mammalian cells leads to differential translation of ATF4 and Bip/GRP78 mRNAs, both encoding anti-apototic proteins, via PERK kinase and eIF2 phosphorylation. The kinases that mediate translational response to stress (starvation stress and ER stress) are counteracted by kinases that mediate nutrient signaling and growth signals. The TOR (Target of Rapamycin) kinase is a highly conserved, central controller of cell growth. In yeast and human cells, TOR is found in two distinct multisubunit complexes, the rapamycin sensitive TORC1 completely and the rapamycin insensitive TORC2 complex. In regulation of translation, TORC1 controls protein synthesis and ribsosome biogenesis. Via phosphorylation of S6 kinase and eIF-4B binding proteins (4E-BP), two key regulators of translation initiation, TORC1 orchestrates increased translation of mRNAs encoding growth control genes, while repressing genes encoding components of the apoptotic machinery. Thus, active TORC1 favors translation of mRNAs that drive cellular growth, which is supported by the timely expression of antiapoptotic proteins, to ameliorate the cellular stress derived from increased protein synthesis, i.e. activation of the UPR pathway. Aberrant activation of the TOR pathway induces apoptosis [57] which is a crucial event in the early response to malignant development. Ribosomal proteins (RP) have been shown to regulate activity of the prototypic apoptotic protein p53. Ribosomal proteins L23, L11, and L5 regulate P53 activity by abrogating Mdm2-induced degradation and RPL26 modulates p53 translation [58]. A variant ribosmal protein, S27L, is pro-apoptotic and biosynthesized under the direct control by
p53 [58]. We have recently shown in yeast that mutants of RPL10 and RPS6 mediate differential translation as shown in 2D gel analysis (unpublished results). It will be interesting to learn whether these RPs are involved in a translational switch between growth and stress induced apoptosis. We return now to the questions asked in the beginning of this review article. i) Is apoptosis a cause or a consequence of senescence? ii) Is the apoptotic killing of yeast cells an adaptive trait? One simple approach to start answering the first question is to determine the mother cell specific lifespan of deletion mutants (available through the EUROSCARF deletion collection) corresponding to the “apoptotic” yeast genes identified so far. A research program is under way to systematically determine the mother cell-specific lifespans of all 4800 mutants, and about 1/9 of these mutants are published [59]. All 4800 mutants have been studied for their influence on chronological aging [60]. There is in general very little concordance between the mother cell-specific and chronological lifespan data obtained with the 4800 deletion mutants [61]. We have now reexamined these published data and compared them with the data obtained in our laboratory for mutants in apoptotic genes. There is one important caveat: practically all apoptotic genes of yeast have a second function (we like to call this the “dayjob”) in growing cells. This second function in yeast is in many cases not essential for life. However, even if it is not, we cannot separate the two functions through specific mutations (certainly not when dealing with deletion mutations). The final phenotype, in particular if it shortens the lifespan, could be due to the loss of the apoptotic function or, alternatively, due to the loss of the dayjob function. In Table 1 we are presenting a short list of genes that were found to be directly involved in the apoptotic process and their effect on mother cell specific as well as chronological aging. One essential yeast gene that has been shown to be involved in apoptosis is included in the list but the somewhat more labor-intensive lifespan experiment with this mutant has not been done. Another way to gain information related to question i) is to look at the apoptotic phenotypes of long-lived mutants. For example, a mutation of YGR076C leading to a 60% (and statistically significant) elongation of the mother cell-specific lifespan (unpublished work) does not prevent yeast apoptosis to occur; the apoptotic phenotypes just appear at a later cell generation, according to the longer lifespan. Taken together, these data rather seem to support the notion that apoptosis is a consequence (not a cause) of mother cell specific aging, however, more systematic data are needed before this question can be definitely answered. The answer to the second question posed above (is apoptotic killing of yeast cells an adaptive trait?) seems to be: Yes, for chronological aging. The argument is that a substantial part of a stationary population dies and lyses either in spent medium or in water and the nutrients liberated can be used by surviving cells. However, the answer is “no” for mother cell-specific (replicative) aging, because senescence occurs in spite of adequate food supply and the fraction of cells dying is very small. Acknowledgements We are grateful to the Austrian Science Fund FWF (Vienna, Austria) for grants S9302-B05 (to M.B.) and to the EC (Brussels, Europe) for project MIMAGE (contract no. 512020; to M.B.). References [1] J.F. Kerr, A.H. Wyllie, A.R. Currie, Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics, Br. J. Cancer 26 (1972) 239–257. [2] W. Wood, M. Turmaine, R. Weber, V. Camp, R.A. Maki, S.R. McKercher, P. Martin, Mesenchymal cells engulf and clear apoptotic footplate cells in macrophageless PU.1 null mouse embryos, Development 127 (2000) 5245–5252.
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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a m c r
Review
Senescence and apoptosis in yeast mother cell-specific aging and in higher cells: A short review Peter Laun, Gino Heeren, Mark Rinnerthaler, Raphaela Rid, Sonja Kössler, Lore Koller, Michael Breitenbach ⁎ Department of Cell Biology, Division of Genetics, University of Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg, Austria
a r t i c l e
i n f o
Article history: Received 4 January 2008 Received in revised form 12 February 2008 Accepted 13 February 2008 Available online 4 March 2008 Keywords: Yeast apoptosis Replicative lifespan Chronological lifespan S. cerevisiae Translational control Eukaryotic ribosome AIF YCA1 NUC1 NMA111
a b s t r a c t 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. © 2008 Elsevier B.V. All rights reserved.
Modern apoptosis research starts with the seminal paper of Kerr [1] which drew the attention of the scientific world to the previously ignored fact that the ordered and programmed removal of cells of multicellular organisms under a variety of physiological conditions is as important as the production of these cells. In this early paper a clear distinction was made between apoptosis, a smooth and mostly unnoticed process which occurs without inflammation, and necrosis, a process usually accompanied with inflammation. At least three rather different biological purposes of the apoptotic process of cellular suicide seem to exist. First, cells commit suicide during the process of morphogenesis early in life. Secondly, cells that have accumulated damage beyond a certain threshold are removed by apoptosis and are replaced thereby ensuring proper functioning of organs in adult life. Thirdly, senescent cells also undergo apoptosis, a process that up to now has been studied mainly in cultured mammalian cells at the Hayflick limit (replicative senescence). A prominent example for morphogenic apoptosis in the embryo which has found its way into numerous textbooks is the formation of digits of the mouse paw during embryogenesis [2]. In this work, apoptotic cells were recognized through acridine orange staining and by electron microscopy analyzing apoptotic bodies which were either ⁎ Corresponding author. E-mail address: [email protected] (M. Breitenbach). 0167-4889/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamcr.2008.02.008
free or engulfed by professional phagocytes. Mouse embryos completely devoid of professional phagocytes as a consequence of a transcription factor mutation in homozygous PU.1 null mice showed a defect in the process of engulfment which was partly taken over by mesenchymal cells. Later [3] apoptotic markers like DNA fragmentation shown by the TUNEL test were shown to occur in the system, and the signaling function of externalized phosphatidyl serine and other possible surface markers was studied in detail [4,5], revealing that externalized phosphatidyl serine signals both to macrophages which subsequently engulf the apoptotic cells, and to down-regulate the inflammatory response in the tissue [6]. Mutants in known “apoptotic genes” that were studied revealed mostly a mild phenotype but occasionally an embryonic or perinatal lethal phenotype as shown by the DNAse II alpha homozygous knock-out mutant [7–9]. The embryonic lethal phenotype in this case is probably not due to lack of apoptotic functions but due to a defect in the essential “day-job” function of the DNAse II alpha, for instance in haematopoiesis. It is remarkable that the signals and the mechanism of removal of apoptosed cells seem to have been conserved between widely divergent animal species, like Drosophila melanogaster, nematodes and vertebrates. What is also conserved to a certain degree are the genes and proteins that are needed for the execution of the programmed cell death process. Many of the CED (cell death) genes of Caenorhabditis elegans have been shown to fulfill biochemical
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functions conserved in mammals and also show a certain degree of sequence conservation [10]. On the other hand, the multiple stimuli (pathways) converging into apoptotic processes and the presumed multiplicity of apoptotic mechanisms is less well known and poses a challenge to current research. For instance, little is known about the signals (intracellular and extracellular) leading into the embryonic morphogenic apoptosis just described and about the conservation of pathways between the embryonic and other forms of apoptosis. An interesting hint can be seen in the fact that exposing C. elegans to oxidative stress during development leads to an increase in the number of cells that undergo apoptosis. Although a correlation of apoptosis with senescence and aging has been found in some systems (see below), in C. elegans the genetic analysis of both processes, apoptosis and aging, has revealed little overlap between the two processes so far [10]. This is to say that compromising the developmental apoptosis through mutation has, surprisingly, not diminished the fitness and fecundity of the organism [11,12], but has also not led to a change in lifespan [13]. Conversely, and interestingly, prohibiting developmental apoptosis through null mutations in pro-apoptotic genes leads to embryonic lethality in D. melanogaster and in the mouse [14,15]. The worm system has arguably the best “genetics of aging” in the form of a large number of mutants that can be ordered in a signaling pathway, the IGFR pathway, which also governs the formation of dauer larvae. However, these mutations have no influence on developmental apoptosis. Although apoptosis occurs in more than 10% of all cells during development, many of them in the nervous system, and apoptosis can be induced through external oxidative stress, apoptotic markers are reportedly absent from senescent worms. Apoptosis plays a major role in several distinct processes of immune regulation. To give an example, the down-regulation of expanded clones of B- and T-cells when a challenge with microbial pathogens is no longer present; and the elimination of anti-self clones from the immune repertoire of the organism. These processes work through activation-induced cell death [16] and involve binding of a relevant ligand to the surface receptor of immune cells, Fas. This system is obviously independent of external oxidative stress and it is
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not completely clear if the well known mitochondrial branch of the apoptotic mechanism is a necessary component of this form of apoptosis [16]. Apoptosis in multicellular animals is well documented as an antiaging mechanism, because it can remove damaged cells from tissues, inducing the replacement of these cells by homeostatic mechanisms, and thereby leads to tissue repair [17]. Many different forms of molecular damage can lead cells into this process. The most generally accepted mechanism of damage that is at work here is the oxidative damage exerted primarily by reactive oxygen species (ROS) produced as by-products of the mitochondrial respiratory chain. This kind of oxidative damage is found in aged and apoptotic cells in all model systems known, including yeast. A huge body of published evidence exists describing the origin (mainly from complex I and III), chemical reactivity, reaction products and forms of cellular damage (genotoxic and cytotoxic) which the primary oxygen radical, superoxide, can induce directly or indirectly in the living cell (summarized in [18]). These processes can be mimicked by exposing the cell to oxidants, like hydrogen peroxide or to a large number of other damaging agents. Much less is known about the possible role in apoptosis of extramitochondrial sources of ROS (for instance, NADPH oxidases; [19]). However, an additional large number of primary triggers different from the mitochondrial oxidative damage just described and leading ultimately also into the apoptotic process have been published. Frequently, as a consequence of these “other” defects, the cell secondarily creates oxidative stress. One example is replication stress [20,21]. The relationship of cell division cycle checkpoints and cell division cycle regulation with the entry into apoptotic pathways is an interesting and unfinished field of apoptosis research. Cell cycle arrest at a checkpoint (often studied at the DNA damage checkpoint in G2) in principle can lead to two scenarios depending on the duration of cell cycle arrest: i) The cell can return into a normal cell cycle after DNA repair; or ii) the cell can switch on the apoptosis program [22,23]. However, another avenue into apoptosis is opened by loss of a checkpoint function [24,25]. This is
Fig. 1. The picture shows in part: (A) exponentially growing young yeast cells of strain W303, a commonly used haploid MATa strain, which are included here to show the size and morphology difference to old cells. (B) A typical lifespan determination of the same wild-type strain. The number of budding cycles that each of a set of 50 “virgin” cells undergoes before it stops dividing was determined by micromanipulation and counting of budding cycles. (C) M is the terminal mother cell after 15 cell cycles. Note the enormous size as compared to young cells and the surface changes. D14 is the second but last daughter that did not completely separate from the mother and did not give rise to new living cells. Also, the surface of D14 is folded or wrinkled. The budding of the “granddaughter” D14-1 stopped at an early stage of the cell cycle. The budding of last daughter cell, D15, also stopped at an early stage of the cell cycle. Reprinted from [28], with permission from Elsevier.
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intriguing, because the apoptotic final stage senescent yeast mother cells observed by us [26] often die in the middle of an ongoing cell cycle [27] as judged by their still attached daughter cell (of variable size) or by the initiation of a new cell cycle while the “old” cell cycle is still not finished [28] (Fig. 1). Although not studied or published in detail up to now, the occurrence of such processes in very old yeast mother cells logically would lead to aneuploid cells among the daughters of these mother cells. This is a striking parallel to many human cancer cells which also have lost cell cycle controls and which also are often aneuploids. It is remarkable that the correlation of apoptosis with oxidative stress seems to be general. Even in those cases where the primary cause of apoptosis is unrelated to oxidative stress (for instance during faulty response to DNA damage), the destruction program which is turned on, includes production of ROS [21,29]. Apoptosis can act as a quality control in germ cells. Both in the nematode and in mammals, a very high proportion of the female germ cells undergo apoptosis and only a tiny fraction survive [30]. It has been speculated that this surprising fact serves the purpose of quality control eliminating any haploids with errors due to premeiotic DNA synthesis, faulty segregation at the meiotic divisions and, perhaps also heteroplasmic mitochondrial mutations [31]. A similar quality control was observed in mammalian male meiosis [32]. We now want to give an overview of the key topic of this short review article, which is the correlation of cellular senescence with apoptosis. Very few papers dealing explicitly with the relation of aging and apoptosis have been published so far and most of them are speculative. Their main point is twofold: i) caloric restriction which is the only method of lifespan elongation that works in all known model systems of aging, likewise influences lifespan and apoptosis (compare [33] and the literature references therein); ii) genes in the insulin like growth factor receptor pathway have a strong influence on lifespan in C. elegans, are conserved in mammals, and are in the mammalian system known for their control of apoptosis [33]. More recently, an elongation of the lifespan has been shown for mouse mutants in the same pathway [34]. Papa and Skulachev [35] discuss the mitochondrial origin of oxygen radicals, and their influence on
both processes, aging and apoptosis. However, as mentioned elsewhere in this short review article, in many cases manipulating an “apoptotic gene” has not resulted in the expected lifespan elongation and, vice versa, manipulating a longevity gene has not resulted in the expected effect on apoptosis (compare for instance, [13] and Table 1). Currently available evidence shows that both in replicative aging of yeast mother cells [26] and in the replicative in vitro (Hayflick-type) aging of primary human cells (HUVEC; [36]), these cells in their final stage display markers of apoptosis: inversion of the plasma membrane as analyzed by binding of annexinV without general perforation of the plasma membrane, an increase in ROS as detected by dihydrorhodamine, and fragmentation of nuclear DNA as analyzed by the TUNEL method. Not all human cell cultures show this phenotype, as established lines of fibroblasts do not [36]. In the field of aging research of higher organisms it is an open question to what degree Hayflick-type aging of cell culture cells in vitro might be relevant for the aging process of the organism as a whole. The discrepancy between the in vivo and the in vitro situation might be summarized in three points: i) the cell culture medium does not exactly reproduce the in vivo situation and the neighboring cells of the niche are absent; ii) the cells studied are not stem cells which arguably may be the most relevant cells to study; iii) the lifespan of postmitotic cells of some organs (for instance of the central nervous system) might be limiting for the survival and function of the respective organ, a situation which is not mimicked in the cell culture system. It is highly relevant to answer the questions whether apoptotic cells can be found in healthy centenarians, whether an exhaustion of a stem cell population is indeed found, and whether the exhaustion of a stem cell population takes place (if it takes place) by way of cellular senescence and apoptosis. These questions are presently mostly answered in a negative way. To give an example, there seems to be no exhaustion of the satellite cells of skeletal muscle and no apoptotic markers are observed in biopsies. However, in certain diseases, like Duchenne muscular dystrophy, the stage of exhaustion of stem cells is indeed reached. Clearly, further progress of stem cell research is needed to answer these questions and it is to be expected that in the future the role of stem cell exhaustion and apoptosis in organismic aging will be clarified.
Table 1 Genes involved in the yeast apoptotic pathway with a short description according to Incyte BioBase [63] and the change in replicative and chronological lifespan of the corresponding deletion mutant compared to wild type strain Name
ORF
Proapoptotic YCA1[64,65]
YOR197W
NDI1[66,67]
AIF1 [53,64] NUC1[64,68] NMA111 [69,70] CPR3 [71] DNM1[72,73]
FIS1[72–74]
Antiapoptotic BIR1 [70]
Description [63]
Putative cysteine protease similar to mammalian caspases, involved in regulation of apoptosis upon hydrogen peroxide treatment YML120C NADH:ubiquinone oxidoreductase, transfers electrons from NADH to ubiquinone in the respiratory chain but does not pump protons, in contrast to the higher eukaryotic multisubunit respiratory complex I; phosphorylated; homolog of human AMID YNR074C Mitochondrial cell death effector that translocates to the nucleus in response to apoptotic stimuli, homolog of mammalian Apoptosis-Inducing Factor, putative reductase YJL208C Major mitochondrial nuclease, has RNAse and DNA endo- and exonucleolytic activities; has roles in mitochondrial recombination, apoptosis and maintenance of polyploidy YNL123W Protein of unknown function which may contribute to lipid homeostasis and/or apoptosis; sequence similarity to the mammalian Omi/HtrA2 family of serine proteases YML078W Mitochondrial peptidyl-prolyl cis-trans isomerase (cyclophilin), catalyzes the cis-trans isomerization of peptide bonds N-terminal to proline residues; involved in protein refolding after import into mitochondria YLL001W Dynamin-related GTPase required for mitochondrial fission and the maintenance of mitochondrial morphology, assembles on the cytoplasmic face of mitochondrial tubules at sites at which division will occur; also participates in endocytosis YIL065C Mitochondrial outer membrane protein involved in membrane fission, required for localization of Dnm1p and Mdv1p during mitochondrial division; mediates ethanol-induced apoptosis and ethanol-induced mitochondrial fragmentation
YJR089W
Essential chromosomal passenger protein involved in coordinating cell cycle events for proper chromosome segregation; C-terminal region binds Sli15p, and the middle region, upon phosphorylation, localizes Cbf2p to the spindle at anaphase
Chronological Replicative lifespan [60] lifespan Increased
n. a.
Increased
n. a.
Increased
n. a.
Increased
no change
Increased
n. a.
Increased
n. a.
Increased
increased
Decreased
increased
n. a.
n. a.
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The situation of aging yeast mother cells is simpler than the scenario just described. Büttner et al. [37] have given a nice overview of the physiological situations in which yeast cells display apoptotic phenotypes. Both of the aging processes of yeast cells, chronological aging and mother cell-specific aging, have been described to lead to apoptosis of the terminally senescent cells. This is remarkable because the physiological situation of terminal mother cells is very different from chronologically aged cells. The aging of yeast mother cells depends on the number of cell generations that a mother cell has undergone and not on calendar time (Fig. 2). The distribution function of lifespans of a cohort of yeast cells is described by the Gompertz law. The aging process occurs in the presence of nutrients and has nothing to do with starvation. The old mother cells, like the senescent HUVEC, are much larger than young cells, display a longer cell cycle time and a slowed down translational activity of the ribosomes, their nuclei appear fuzzy (not compact like in young cells), DHR staining indicates that the cells have produced an oxidizing internal milieu even if external oxidative stress is absent, and their actin cytoskeleton looks collapsed and appears clumpy [26,38]. On the other hand, chronologically aged (stationary) yeast cells are reproductively still young, but undergo a process of senescence due to the lack of nutrients. From stationary phase cultures two quite different cell populations can be isolated on density gradients [39]. One population of cells in a stationary culture acquires a long chronological lifespan through a process of differentiation involving changes of the cell wall, synthesis of reserve carbohydrates like trehalose and glycogen and quiescence of the genome, resembling a true G0 state. This cell population consists of the daughter cells resulting from the last cell division cycle
Fig. 2. Yeast mother cell specific aging is characterized by a limited capacity to produce daughter cells. The lifespan is determined by the number of cell cycles a mother cell has undergone, not by calendar time, and in a population of cells the lifespan distribution follows the Gompertz law. Daughter cells reset their clock to zero and enjoy the full lifespan characteristic for the strain. Reprinted from [62] with permission from Elsevier.
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which the culture has performed. The other population of cells in the stationary cultures shows adherent daughter cells in unfinished cell cycles, is much shorter lived than the first one, creates oxygen radicals and dies much earlier than the first one. Eventually, both populations at very different times reach apoptosis. It has been speculated that the postmitotic aging just described and resulting in apoptotic death might be an adaptive trait that was positively selected for during evolution [37,40] because it supports the survival of the yeast strain in times of lack of nutrients. This was further elaborated by Longo and Skulachev who hypothesized that most aging processes (also those that occur in higher organisms) have an adaptive value and are “altruistic” [41]. However this and similar hypotheses concerning multicellular organisms have been challenged on the grounds of evolutionary theory, in our opinion with convincing arguments [42– 44], two of which are as follows: i) The vast majority of individuals in a wild population never reach senescence; therefore, the process of aging cannot have been selected for; and ii) the force of biological selection is at work until progeny has been procreated and can survive on their own. Turning to yeast mother cell-specific aging, we can say that the final demise of the senescent mother cells is almost certainly of no positive value for the growing yeast strain or for the growing yeast colony. Wild type laboratory yeast strains display a median replicative lifespan of about 20–30 generations and a maximum lifespan of about 40–60 generations [38]. The fraction of cells which is senescent in a population of yeast cells is exceedingly small (1/2n) where n is the number of generations after which the cell dies. The nutrients supplied by the death and lysis of the old mother cell are therefore negligible. We have to look for other more convincing reasons why those old mother cells undergo apoptosis. The key observation was that cell division cycles of budding yeast are asymmetric, producing one daughter that is rejuvenated and has reset the clock to zero, and one mother cell which in every cell cycle acquires not only an additional bud scar, but also a gradual change in the parameters mentioned above (cell size etc.). This cell division asymmetry is similar to the asymmetric division of a stem cell in the human body, which produces one new stem cell and one cell that has undergone the first step to cell differentiation. A further key observation is that life and metabolism inevitably produce waste and the waste must be either cleanly removed or at least asymmetrically segregated between mother and daughter cell if the species is to survive over evolutionary time spans. Waste in the case of the yeast cell (but also in higher cells) is not exclusively but predominantly the oxidation products of cellular components. There are a myriad of compounds that occur in vivo and could be monitored because they are waste products that do not occur in newborn cells. One method that has been used frequently for quantitation and for fluorescent in situ microscopy is the following: The carbonyl groups which exist on proteins due to oxidative reactions (“protein carbonyls”) are conjugated with p-nitro phenylhydrazine and then the hydrazones are decorated with standardized antibodies directed to p-nitro phenylhydrazone (the “oxy-blot” technique) [45,46]. In dividing yeast cells it is found that both soluble cytoplasmic proteins and mitochondrial proteins are oxidized during aging and under stress that can induce apoptosis [47]. Oxidation is not generally found on every protein but is observed on a small number of specific proteins and with a strong bias in the mother cell, not in the daughter cell [47–49]. The mechanism of this asymmetric segregation or asymmetric scavenging which occurs also in E. coli and in higher cells [49] is presently not known. However, manipulating the asymmetric segregation through mutation of the yeast SIR2 gene, leads to the expected shortening of the lifespan [48]. In one scenario that looks plausible to us, survival and evolutionary success of a (single-celled) species primarily depends on the ability to asymmetrically segregate damage in cell division cycles. A side effect of this phenomenon is the gradual accumulation of damage in mother cells, leading to the activation of programmed cell death once a
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threshold is reached and finally to senescence and cell death. This kind of cell death clearly is evolutionarily neutral and was therefore not selected against; however it is not “altruistic”. What appears as a genetic program of aging is in reality the genetic program(s) of stress response, because the accumulation of damage means that the aging cells build up stress, most importantly, oxidative stress. Part of this stress response program is identical with the apoptotic program which in a rudimentary form existed in evolution already before the first multicellular organisms appeared and which still exists today in many single celled organisms. However, not every aspect of apoptosis exists in monocellular organisms. To give and example, the highly important “engulfment” part of the apoptotic program [50] and the Fas-receptor are not found, but extrinsic pathways (alpha factor; [51]) as well as the intrinsic mitochondrial pathway into apoptosis are surprisingly well conserved. This includes mitochondrial components that are translocated to the nucleus (AIF, endo-G) and a growing number of genes and gene functions which were previously thought to be specific for the apoptotic process in metazoan eukaryotes, are now known to exist in yeast too. In addition, the genetic system of yeast which is probably the most highly advanced genetic system existing, enabled the researchers to discover new “apoptotic” genes, which are highly conserved but previously unknown as “apoptotic” in higher cells. One prominent example is CDC48 [52]. Another one is AIF, which was found in both systems [53]. A list of yeast genes which directly or indirectly are involved in the apoptotic mechanism is given in other review articles in this volume. Another example is “ER stress” related to the unfolded protein response and, closely related, the stress created by defects in the ribosome and protein synthesis machinery. There is also indirect evidence for a switch in ribosome specificity associated with the switch of a cell to the apoptotic mode, which includes a change in gene expression at the transcriptional but also at the translational level. As this “translational” road into apoptosis has not been reviewed in detail before, we will now briefly elaborate on this emerging topic. Recently, modulation of ribosome function during the apoptotic response was described [54]. The authors show that apoptosis is dependent on synthesis of apoptosis-specific proteins through a switch in the protein synthesis machinery. Similar “switching” of the ribosome is observed from yeast to mammals in recent reports for the aging process [55,56] and for apoptosis [54,57]. The translational switch in the course of the unfolded protein response in mammalian cells leads to differential translation of ATF4 and Bip/GRP78 mRNAs, both encoding anti-apototic proteins, via PERK kinase and eIF2 phosphorylation. The kinases that mediate translational response to stress (starvation stress and ER stress) are counteracted by kinases that mediate nutrient signaling and growth signals. The TOR (Target of Rapamycin) kinase is a highly conserved, central controller of cell growth. In yeast and human cells, TOR is found in two distinct multisubunit complexes, the rapamycin sensitive TORC1 completely and the rapamycin insensitive TORC2 complex. In regulation of translation, TORC1 controls protein synthesis and ribsosome biogenesis. Via phosphorylation of S6 kinase and eIF-4B binding proteins (4E-BP), two key regulators of translation initiation, TORC1 orchestrates increased translation of mRNAs encoding growth control genes, while repressing genes encoding components of the apoptotic machinery. Thus, active TORC1 favors translation of mRNAs that drive cellular growth, which is supported by the timely expression of antiapoptotic proteins, to ameliorate the cellular stress derived from increased protein synthesis, i.e. activation of the UPR pathway. Aberrant activation of the TOR pathway induces apoptosis [57] which is a crucial event in the early response to malignant development. Ribosomal proteins (RP) have been shown to regulate activity of the prototypic apoptotic protein p53. Ribosomal proteins L23, L11, and L5 regulate P53 activity by abrogating Mdm2-induced degradation and RPL26 modulates p53 translation [58]. A variant ribosmal protein, S27L, is pro-apoptotic and biosynthesized under the direct control by
p53 [58]. We have recently shown in yeast that mutants of RPL10 and RPS6 mediate differential translation as shown in 2D gel analysis (unpublished results). It will be interesting to learn whether these RPs are involved in a translational switch between growth and stress induced apoptosis. We return now to the questions asked in the beginning of this review article. i) Is apoptosis a cause or a consequence of senescence? ii) Is the apoptotic killing of yeast cells an adaptive trait? One simple approach to start answering the first question is to determine the mother cell specific lifespan of deletion mutants (available through the EUROSCARF deletion collection) corresponding to the “apoptotic” yeast genes identified so far. A research program is under way to systematically determine the mother cell-specific lifespans of all 4800 mutants, and about 1/9 of these mutants are published [59]. All 4800 mutants have been studied for their influence on chronological aging [60]. There is in general very little concordance between the mother cell-specific and chronological lifespan data obtained with the 4800 deletion mutants [61]. We have now reexamined these published data and compared them with the data obtained in our laboratory for mutants in apoptotic genes. There is one important caveat: practically all apoptotic genes of yeast have a second function (we like to call this the “dayjob”) in growing cells. This second function in yeast is in many cases not essential for life. However, even if it is not, we cannot separate the two functions through specific mutations (certainly not when dealing with deletion mutations). The final phenotype, in particular if it shortens the lifespan, could be due to the loss of the apoptotic function or, alternatively, due to the loss of the dayjob function. In Table 1 we are presenting a short list of genes that were found to be directly involved in the apoptotic process and their effect on mother cell specific as well as chronological aging. One essential yeast gene that has been shown to be involved in apoptosis is included in the list but the somewhat more labor-intensive lifespan experiment with this mutant has not been done. Another way to gain information related to question i) is to look at the apoptotic phenotypes of long-lived mutants. For example, a mutation of YGR076C leading to a 60% (and statistically significant) elongation of the mother cell-specific lifespan (unpublished work) does not prevent yeast apoptosis to occur; the apoptotic phenotypes just appear at a later cell generation, according to the longer lifespan. Taken together, these data rather seem to support the notion that apoptosis is a consequence (not a cause) of mother cell specific aging, however, more systematic data are needed before this question can be definitely answered. The answer to the second question posed above (is apoptotic killing of yeast cells an adaptive trait?) seems to be: Yes, for chronological aging. The argument is that a substantial part of a stationary population dies and lyses either in spent medium or in water and the nutrients liberated can be used by surviving cells. However, the answer is “no” for mother cell-specific (replicative) aging, because senescence occurs in spite of adequate food supply and the fraction of cells dying is very small. Acknowledgements We are grateful to the Austrian Science Fund FWF (Vienna, Austria) for grants S9302-B05 (to M.B.) and to the EC (Brussels, Europe) for project MIMAGE (contract no. 512020; to M.B.). References [1] J.F. Kerr, A.H. Wyllie, A.R. Currie, Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics, Br. J. Cancer 26 (1972) 239–257. [2] W. Wood, M. Turmaine, R. Weber, V. Camp, R.A. Maki, S.R. McKercher, P. Martin, Mesenchymal cells engulf and clear apoptotic footplate cells in macrophageless PU.1 null mouse embryos, Development 127 (2000) 5245–5252.
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