they also could be directed to the design of new antifungal drugs. A new generation of antifungal drugs is required for many reasons, including the limitations associated with those currently in use and the increasing number of invasive fungal infections in immunocompromised patients. Thus the exploration of yeast PCD processes to identify molecules that trigger yeast cell death without causing serious side effects in human cells is extremely important.

4. THE GENETICS OF YEAST APOPTOSIS

Although a variety of toxins can promote apoptosis in S. cerevisiae, features of PCD were first described in yeast cells carrying the S565G substitution in the second ATPase domain (D2) of the CDC48 gene, which codes for the AAA-ATPase and plays a role in cell division, ubiquitin-dependent endoplasmic reticulum– associated protein degradation (ERAD), and vesicle trafficking. Later on, it was observed that mutations in the VCP (valosin-containing protein), the metazoan homolog of the yeast CDC48, gave rise to apoptotic phenotypes in mammalian cell culture, in trypanosomes, Drosophila, and zebra fish. Moreover, mutations in VPC also trigger the human multisystem disorder called inclusion body myopathy associated with Paget’s disease of bone and frontotemporal dementia (IBMPFD). More recently, other mutants in numerous cellular

fundamental processes, such as DNA replication, mitochondrial function, RNA, and protein stability, showed apoptotic phenotypes. These findings support the value of yeast as a model organism to study evolutionarily conserved mechanisms of apoptotic regulation.

5. PROGRAMMED AND ALTRUISTIC AGING

Much of the skepticism for the existence of apoptosis in yeast was supported by the apparent lack of an evolutionary base for the suicide of an organism. Perhaps one of the most convincing types of apoptosis in S. cerevisiae is that consistently observed during aging of yeast populations in a medium similar to that encountered in natural environments. Wild-type yeast aging chronologically in glucose/ethanol medium show features of apoptotic death such as generation of superoxide, nuclear condensation/fragmentation, and phosphatidylserine exposure; meta-caspase activation is also seen in some contents. ROS formation is enhanced in old cells, in agreement with a crucial role for superoxide in the mediation of yeast apoptosis during aging (see the remaining text in this section and Figure 33-2). Almeida et al. (2007) have recently provided evidence for the production of nitric oxide (NO) in aging yeast. According to their studies, NO contributes to superoxide production, and its removal by treatment with oxyhemoglobin, an

NO scavenger, extends the CLS (chronological life span) of wild-type cells. A number of genetic interventions that delay chronological aging and the appearance of the apoptotic features associated with it have been described. Among these are the disruption of the yeast meta-caspase YCA1 gene and of NDI1 (coding for the yeast homolog of the AIF-homologous mitochondrion associated inducer of death, AMID) and the overexpression of the stress-dependent transcription factor Yap1, although the most effective mutations known to block age-dependent apoptosis are the deletion of either RAS2 or SCH9 or both, which prolong the mean CLS by up to fivefold (Fabrizio et al., 2003; Fabrizio et al., 2001).

A nongenetic intervention that delays PCD and extends survival in wild-type yeast is calorie restriction/starvation obtained by switching yeast to water after growth in glucose medium (SDC [synthetic dextrose complete]). Starvation, as well as the reduction of the glucose concentration in the medium, can double the survival of S. cerevisiae, which suggests that yeast apoptosis depends on the type of environment encountered by the organism. Similar phenotypes including stress resistance and extended survival are shared between starved or ethanol-depleted cells and most of the long-lived mutants, including those lacking SCH9 or RAS2. When aging wild-type cells incubated in glucose medium (SDC) are compared with long-lived sch9 and ras2 mutants, besides the apoptotic markers, major differences in stress response are observed. Both sch9 and ras2 mutants are less susceptible to oxidative stress than wild-type cells. Their resistance to oxidants depends in part on the expression of SOD2.

The presence of apoptotic markers and the role of nutrients and signal transduction pathways involved in nutrient signaling led to our hypothesis that aging S. cerevisiae activates a program that blocks cell protection and accelerates the death of the cells. The existence of such a program would be unlikely based on evolutionary theories, which rule out that a unicellular organism can activate a program that benefits other organisms. However, several lines of evidence have indicated that, for a population of millions or billions of yeast that have encountered an environment in which extracellular nutrients are scarce, cellular “suicide” represents a “group-level survival strategy.” In fact, in approximately 50% of the aging wild-type cultures studied, cellular “regrowth” is observed (colony-forming units increase by up to 100 folds) after the great majority of the population has died. The percent of regrowth reaches more than 80% for cultures of mutants lacking SOD1, which codes for the cytosolic superoxide dismutase, suggesting a direct correlation between intracellular superoxide and frequency of regrowth. The regrowth phenotype, which

we called “adaptive regrowth,” has been characterized extensively. Its characteristics are (1) correlation with frequency of nuclear mutations, which accumulate during aging, and (2) requirement for the nutrients released by the dead cells into the medium. Both features appear to be promoted by both cytoplasmic and mitochondrial superoxide, which, on the one hand, promotes accelerated cell death and consequently the release of nutrients and, on the other hand, causes DNA damage that facilitates the appearance of mutations with the ability to confer reentry into the cell cycle under conditions that normally prevent growth. In fact, the sod1 deletion mutant is one of the mutants from the yeast knockout collection with the highest spontaneous mutation frequency. Importantly, the long-lived mutants, which are better protected against superoxide damage (including DNA damage), generally do not show adaptive regrowth, in agreement with a role for superoxide and protective systems in preventing the conditions necessary to achieve regrowth. Further analysis of yeast apoptosis during chronological aging has revealed that the pH of the medium affects cell survival. By day 3 the pH of the culture for cells grown in SDC medium of yeast cultures reaches 3 to 3.5. After adjustment to 6 to 7, the survival of the culture is significantly extended. In mammals the release of cytochrome c that triggers the caspase cascade activation in apoptotic cells is preceded by mitochondrial alkalinization and cytosol acidification. Cytochrome c release has also been observed in yeast treated with acetic acid, alpha factor, or overexpressing mammalian BAX, although its function in apoptosis activation has not been established yet. A possibility that needs to be investigated is whether the extracellular and consequently intracellular acidification observed in yeast aging cultures triggers apoptosis via the release of cytochrome c. In agreement with the role for a switch to water in doubling the survival, we determined that ethanol is an important promoter of agedependent PCD in yeast. This carbon source is normally accumulated during fermentative growth but is not rapidly depleted in chronologically aging cultures of wild-type cells, at least in certain genetic backgrounds such as DBY746. When ethanol is removed from the incubation medium by evaporation, a significant life span extension is observed. Moreover, ethanol addition to cultures switched to water rapidly kills the yeast, suggesting an active role of ethanol in the activation of the death program in nondividing cultures, but also in the absence of all the nutrients required for growth. Recent work by Allen et al. (2006) has characterized yeast stationary phase cultures grown in rich medium by using an equilibrium density-gradient centrifugation method. After 24 hours from the inoculation, they could

separate the cells in two fractions. The density of the lower fraction (high-density) increased steadily until day 7. The characterization of the two fractions revealed the following: (1) the high-density cells were mostly unbudded and showed several features of quiescent cells, whereas the low-density cells were both budded and unbudded and morphologically more similar to dividing cells (nonquiescent); (2) the low-density cells lost viability more rapidly than the others, were more sensitive to heat-shock, and produced more ROS than the quiescent cells; and (3) quiescent cells showed higher levels of apoptotic markers (DNA fragmentation and phosphatidylserine exposure) than non-quiescent cells.

It will be very important to continue to study the dynamics of the two types of cells to establish whether their ratio changes over time and whether quiescent cells become non-quiescent once the apoptotic program is activated. The apoptotic features described previously for the non-quiescent cells resemble those observed in wild-type cells aging chronologically in glucose/ethanol, suggesting the presence of non-quiescent cells in these cultures as well.

Adaptive regrowth therefore could represent a form of kin selection or group selection, with clear implication in relation to the aging theory. Thus an adaptive death program in the context of microbial populations is plausible and should be considered in parallel with classical evolutionary theories. Although natural selection primarily acts to increase the fitness of an individual, a multilevel selection process may more accurately reflect the complexity of the selection process, which must account for a population-based death program that appears to play a central role in the fitness of microbial populations. Notably, programmed aging/adaptive regrowth is only one of the strategies available to yeast populations to overcome periods of starvation. As mentioned earlier, incubation in water promotes life span extension and high levels of stress resistance, in agreement with the activation of a “survival program” that benefits all the individual yeast cells. Adaptive regrowth is observed also in budding yeast isolated from the natural environment, indicating that it is unlikely that the studies described above reflect only laboratory genotypes. Moreover, when laboratory strains are grown in grape extract (a medium that better reflects the natural environment for yeast), the survival curves and regrowth pattern are very similar to those obtained in glucose medium, strongly suggesting that adaptive regrowth is not an artifact owing to the use of synthetic medium.

Schizosaccharomyces pombe shares several similarities with S. cerevisiae in terms of chronological aging and its regulation. Although fewer studies have been

performed on PCD in S. pombe, apoptotic markers have been detected in cells expressing BAX/BAK and in mutants lacking the enzymes responsible for the biosynthesis of triacylglycerol. Furthermore, a homolog of the budding yeast meta-caspase Yca1 has been identified in the S. pombe genome. With respect to chronological aging, fission yeast show activation of meta-caspase activity, which is reduced in the long-lived pka1 and sck2 mutants (PKA and SCK2 are homologs of S. cerevisiae PKA and SCH9, respectively). Although still preliminary, these results and the conservation of the life regulatory pathways in the two yeast species suggests that an aging program and possibly adaptive regrowth also may be common among many yeast species.

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