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In order to investigate whether and how a modification of mitochondrial metabolism can affect yeast sensitivity to programmed cell death (PCD) induced by acetic acid (AA-PCD), yeast cells were grown on raffinose, as a sole carbon source, which, differently from glucose, favours mitochondrial respiration. We found that, differently from glucose-grown cells, raffinose-grown cells were mostly resistant to AA-PCD and that this was due to the activation of mitochondrial retrograde (RTG) response, which increased with time, as revealed by the up-regulation of the peroxisomal isoform of citrate synthase and isocitrate dehydrogenase isoform 1, RTG pathway target genes. Accordingly, the deletion of RTG2 and RTG3, a positive regulator and a transcription factor of the RTG pathway, resulted in AA-PCD, as shown by TUNEL assay. Neither deletion in raffinose-grown cells of HAP4, encoding the positive regulatory subunit of the Hap2,3,4,5 complex nor constitutive activation of the RTG pathway in glucose-grown cells due to deletion of MKS1, a negative regulator of RTG pathway, had effect on yeast AA-PCD. The RTG pathway was found to be activated in yeast cells containing mitochondria, in which membrane potential was measured, capable to consume oxygen in a manner stimulated by the uncoupler CCCP and inhibited by the respiratory chain inhibitor antimycin A. AA-PCD resistance in raffinose-grown cells occurs with a decrease in both ROS production and cytochrome c release as compared to glucose-grown cells en route to AA-PCD., (© 2013.)

The use of non-mammalian model organisms, including yeast Saccharomyces cerevisiae, can provide new insights into eukaryotic PCD (programmed cell death) pathways. In the present paper, we report recent achievements in the elucidation of the events leading to PCD that occur as a response to yeast treatment with AA (acetic acid). In particular, ROS (reactive oxygen species) generation, cyt c (cytochrome c) release and mitochondrial function and proteolytic activity will be dealt with as they vary along the AA-PCD time course by using both wild-type and mutant yeast cells. Two AA-PCD pathways are described sharing common features, but distinct from one another with respect to the role of ROS and mitochondria, the former in which YCA1 acts upstream of cyt c release and caspase-like activation in a ROS-dependent manner and the latter in which cyt c release does not occur, but caspase-like activity increases, in a ROS-independent manner.

To gain further insight into yeast acetic acid-induced programmed cell death (AA-PCD) we analyzed the effects of the antioxidant N-acetyl-L-cysteine (NAC) on cell viability, hydrogen peroxide (H(2)O(2)) production, DNA fragmentation, cytochrome c (cyt c) release and caspase-like activation in wild type (wt) and metacaspase and/or cyt c-lacking cells. We found that NAC prevents AA-PCD in wt cells, by scavenging H(2)O(2) and by inhibiting both cyt c release and caspase-like activation. This shows the occurrence of a reactive oxygen species (ROS)-dependent AA-PCD. Contrarily no NAC dependent change in AA-PCD of mutant cells was detectable, showing that a ROS-independent AA-PCD can also occur., (Copyright 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

To investigate the role of cytochrome c (cyt c) release in yeast acetic acid-induced programmed cell death (AA-PCD), wild type (wt) and cells lacking metacaspase (Deltayca1), cytochrome c (Deltacyc1,7) and both (Deltacyc1,7Deltayca1) were compared for AA-PCD occurrence, hydrogen peroxide (H(2)O(2)) production and caspase activity. AA-PCD occurs in Deltacyc1,7 and Deltacyc1,7Deltayca1 cells slower than in wt, but similar to that in Deltayca1 cells, in which no cytochrome c release occurs. Both H(2)O(2) production and caspase activation occur in these cells with early and extra-activation in Deltacyc1,7 cells. We conclude that alternative death pathways can be activated in yeast AA-PCD, one dependent on cyt c release, which requires YCA1, and the other(s) independent on it.

To gain further insight into the mechanism by which yeast programmed cell death (PCD) occurs, we investigated whether and how proteasome activity changes in Saccharomyces cerevisiae cells undergoing PCD as a result of treatment with acetic acid (AA-PCD). We show that proteasome activation starts 60 min after AA-PCD induction, with a maximum at 90 min, and decreases at 150 min. Moreover, cell survival measurements carried out in the absence or presence of MG132, which inhibits proteasome function, show that the inhibition of proteasome activity partially prevents AA-PCD, thus indicating that a transient proteasome activation is needed for AA-PCD to occur.

To gain insight into the processes by which acetic acid-induced programmed cell death (AA-PCD) takes place in yeast, we investigated both cytochrome c release from yeast mitochondria and mitochondrial coupling over the time course of AA-PCD. We show that the majority of cytochrome c release occurs early in AA-PCD from intact coupled mitochondria which undergo only gradual impairment. The released cytochrome c can be reduced both by ascorbate and by superoxide anion and in turn be oxidized via cytochrome c oxidase, thus working both as a ROS scavenger and a respiratory substrate. Late in AA-PCD, the released cytochrome c is degraded.

To investigate the role of catalase and superoxide dismutase (SOD) in the acetic acid (AA) induced yeast programmed cell death (AA-PCD), we compared Saccharomyces cerevisiae cells (C-Y) and cells individually over-expressing catalase T (CTT1-Y) and Cu,Zn-SOD (SOD1-Y) with respect to cell survival, hydrogen peroxide (H2O2) levels and enzyme activity as measured up to 200 min after AA treatment. AA-PCD does not occur in CTT1-Y, where H2O2 levels were lower than in C-Y and the over-expressed catalase activity decreased with time. In SOD1-Y, AA-PCD was exacerbated; high H2O2 levels were found, SOD activity increased early, remaining constant en route to AA-PCD, but catalase activity was strongly reduced.

Hydrogen peroxide production in yeast cells undergoing programmed cell death in response to acetic acid occurred in the majority of live cells 15 min after death induction and was no longer detectable after 60 min. Superoxide anion production was found later, 60 and 90 min after death induction when cells viability was 60 and 30%, respectively. In cells protected from death due to acid stress adaptation neither hydrogen peroxide nor superoxide anion could be observed after acetic acid treatment. The early production of hydrogen peroxide in cells in which survival was 100% could play a major role in acetic acid-induced programmed cell death signaling. Superoxide anion is assumed to be generated in cells already en route to acetic acid-induced programmed cell death.

Yeast cells lacking the metacaspase-encoding gene YCA1 (Deltayca1) were compared with wild-type (WT) cells with respect to the occurrence, nature and time course of acetic-acid triggered death. We show that Deltayca1 cells undergo programmed cell death (PCD) with a rate lower than that of the WT and that PCD in WT cells is caused at least in part by the caspase activity of Yca1p. Since in Deltayca1 cells this effect is lost, but z-VAD-fmk does not prevent both WT and Deltayca1 cell death, PCD in WT cells occurs via a Yca1p caspase and a non-caspase route with similar characteristics.

In this work evidence is presented that acid stress adaptation protects Saccharomyces cerevisiae from acetic acid-mediated programmed cell death. Exponential-phase yeast cells, non-adapted or adapted to acid stress by 30 min incubation in rich medium set at pH 3.0 with HCl, have been exposed to increasing concentrations of acetic acid and time course changes of cell viability have been assessed. Adapted cells, in contrast to non-adapted cells, when exposed to 80 mM acetic acid for 200 min did not display loss of cell viability associated to morphological alterations typical of apoptosis. Thus, 80 mM acetic acid death-inducing conditions were selected to further characterize the early molecular events leading to such active cell death process. Catalase was specifically activated during acid stress adaptation and protection against acetic acid-induced death was associated with maintenance of its activity during treatment with 80 mM acetic acid. On the other hand, intracellular superoxide dismutase activity was found present at comparable levels both in adapted and in dying yeast cells, excepting in non-adapted cells which displayed a maximum activity value after 15 min acetic acid exposure, corresponding to more than 80% cell viability. This study gives first experimental evidence that H2O2, rather than superoxide, detoxification may have a major role in preventing yeast cell death in response to acetic acid. The results, as a whole, suggest that commitment of S. cerevisiae to a programmed cell death process in response to acetic acid is mediated through a ROS-dependent apoptotic pathway.

Although it is recognized that ATP plays a part in apoptosis, whether and how its level changes en route to apoptosis as well as how ATP is synthesized has not been fully investigated. We have addressed these questions using cultured cerebellar granule cells. In particular, we measured the content of ATP, ADP, AMP, IMP, inosine, adenosine and L-lactate in cells undergoing apoptosis during the commitment phase (0-8 h) in the absence or presence of oligomycin or/and of citrate, which can inhibit totally the mitochondrial oxidative phosphorylation and largely the substrate-level phosphorylation in glycolysis, respectively. In the absence of inhibitors, apoptosis was accompanied by an increase in ATP and a decrease in ADP with 1:1 stoichiometry, with maximum ATP level found at 3 h apoptosis, but with no change in levels of AMP and its breakdown products and with a relatively low level of L-lactate production. Consistently, there was an increase in the cell energy charge and in the ratio ([ATP][AMP])/[ADP](2). When the oxidative phosphorylation was completely blocked by oligomycin, a decrease of the ATP content was found both in control cells and in cells undergoing apoptosis, but nonetheless cells still died by apoptosis, as shown by checking DNA laddering and by death prevention due to actinomycin D. In this case, ATP was provided by anaerobic glycolysis, as suggested by the large increase of L-lactate production. On the other hand, citrate itself caused a small decrease in ATP level together with a huge decrease in L-lactate production, but it had no effect on cell survival. When ATP level was further decreased due to the presence of both oligomycin and citrate, death occurred via necrosis at 8 h, as shown by the lack of DNA laddering and by death prevention found due to the NMDA receptor antagonist MK801. However, at a longer time, when ATP level was further decreased, cells died neither via apoptosis nor via glutamate-dependent necrosis, in a manner similar to something like to energy catastrophe. Our results shows that cellular ATP content increases in cerebellar granule cell apoptosis, that the role of oxidative phosphorylation is facultative, i.e. ATP can also derive from anaerobic glycolysis, and that the type of cell death depends on the ATP availability.

Cytochrome c (cyt c) release was investigated in cerebellar granule cells used as an in vitro neuronal model of apoptosis. We have found that cyt c is released into the cytoplasm as an intact, functionally active protein, that this event occurs early, in the commitment phase of the apoptotic process, and that after accumulation, this protein is progressively degraded. Degradation, but not release, is fully blocked by benzyloxycarbonyl-Val-Ala-Asp-fluoromethylchetone (z-VAD-fmk). On the basis of previous findings obtained in the same neuronal population undergoing excitotoxic death, it is hypothesized that release of cyt c may be part of a cellular attempt to maintain production of ATP via cytochrome oxidase, which is reduced by cytosolic NADH in a cytochrome b5-soluble cyt c-mediated fashion.