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α-Synuclein (aSyn) toxicity is associated with cell cycle alterations, activation of DNA damage responses (DDR), and deregulation of autophagy. However, the relationships between these phenomena remain largely unknown. Here, we demonstrate that in a yeast model of aSyn toxicity and aging, aSyn expression induces Ras2-dependent growth signaling, cell cycle re-entry, DDR activation, autophagy, and autophagic degradation of ribonucleotide reductase 1 (Rnr1), a protein required for the activity of ribonucleotide reductase and dNTP synthesis. These events lead to cell death and aging, which are abrogated by deleting RAS2, inhibiting DDR or autophagy, or overexpressing RNR1. aSyn expression in human H4 neuroglioma cells also induces cell cycle re-entry and S-phase arrest, autophagy, and degradation of RRM1, the human homologue of RNR1, and inhibiting autophagic degradation of RRM1 rescues cells from cell death. Our findings represent a model for aSyn toxicity that has important implications for understanding synucleinopathies and other age-related neurodegenerative diseases., (© 2019 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Ageing is a complex and multifactorial process driven by genetic, environmental and stochastic factors that lead to the progressive decline of biological systems. Mechanisms of ageing have been extensively investigated in various model organisms and systems generating fundamental advances. Notably, studies on yeast ageing models have made numerous and relevant contributions to the progress in the field. Different longevity factors and pathways identified in yeast have then been shown to regulate molecular ageing in invertebrate and mammalian models. Currently the best candidates for anti-ageing drugs such as spermidine and resveratrol or anti-ageing interventions such as caloric restriction were first identified and explored in yeast. Yeasts have also been instrumental as models to study the cellular and molecular effects of proteins associated with age-related diseases such as Parkinson's, Huntington's or Alzheimer's diseases. In this chapter, a review of the advances on ageing and age-related diseases research in yeast models will be made. Particular focus will be placed on key longevity factors, ageing hallmarks and interventions that slow ageing, both yeast-specific and those that seem to be conserved in multicellular organisms. Their impact on the pathogenesis of age-related diseases will be also discussed.

In many organisms, attenuation of growth signaling by caloric restriction or mutational inactivation of growth signaling pathways extends lifespan and protects against cancer and other age-related diseases. The focus of many efforts to understand these effects has been on the induction of oxidative stress defenses that inhibit cellular senescence and cell death. Here we show that in the model organism S. cerevisiae, growth signaling induces entry of cells in stationary phase into S phase in parallel with loss of reproductive capacity, which is enhanced by elevated concentrations of glucose. Overexpression of RNR1 encoding a ribonucleotide reductase subunit required for the synthesis of deoxynucleotide triphosphates and DNA replication suppresses the accelerated loss of reproductive capacity of cells cultured in high glucose. The reduced reproductive capacity of these cells is also suppressed by excess threonine, which buffers dNTP pools when ribonucleotide reductase activity is limiting. Caloric restriction or inactivation of the AKT homolog Sch9p inhibits senescence and death in stationary phase cells caused by the DNA replication inhibitor hydroxyurea or by inactivation of the DNA replication and repair proteins Sgs1p or Rad27p. Inhibition of DNA replication stress represents a novel mechanism by which caloric restriction promotes longevity in S. cerevisiae. A similar mechanism may promote longevity and inhibit cancer and other age-related diseases in humans.

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.

Genetic damage through mutations and genome rearrangements has been hypothesized to contribute to aging. The specific mechanisms responsible for age-induced increases in mutation and chromosome rearrangement frequencies and a potential causative role for DNA damage in aging are under active investigation. Retrotransposons are mobile genetic elements that cause insertion mutations and contribute to genome rearrangements through nonallelic recombination events in humans and other organisms. We have investigated the role of endogenous Ty1 retrotransposons in aging-associated increases in genome instability using the Saccharomyces cerevisiae chronological aging model. We show that age-induced increases in loss of heterozygosity and chromosome loss events are consistently diminished by mutations or treatments that reduce Ty1 retrotransposition. Ty1 mobility is elevated in very old yeast populations, and new retromobility events are often associated with chromosome rearrangements. These results reveal a correlation between retrotransposition and genome instability during yeast aging. Retrotransposition may contribute to genetic damage during aging in diverse organisms and provides a useful tool for studying whether genetic damage is a causative factor for aging.

Inhibition of growth signaling pathways protects against aging and age-related diseases in parallel with reduced oxidative stress. The relationships between growth signaling, oxidative stress and aging remain unclear. Here we report that in Saccharomyces cerevisiae, alterations in growth signaling pathways impact levels of superoxide anions that promote chronological aging and inhibit growth arrest of stationary phase cells in G0/G1. Factors that decrease intracellular superoxide anions in parallel with enhanced longevity and more efficient G0/G1 arrest include genetic inactivation of growth signaling pathways that inhibit Rim15p, which activates oxidative stress responses, and downregulation of these pathways by caloric restriction. Caloric restriction also reduces superoxide anions independently of Rim15p by elevating levels of H₂O₂, which activates superoxide dismutases. In contrast, high glucose or mutations that activate growth signaling accelerate chronological aging in parallel with increased superoxide anions and reduced efficiency of stationary phase G0/G1 arrest. High glucose also activates DNA damage responses and preferentially kills stationary phase cells that fail to arrest growth in G0/G1. These findings suggest that growth signaling promotes chronological aging in budding yeast by elevating superoxide anions that inhibit quiescence and induce DNA replication stress. A similar mechanism likely contributes to aging and age-related diseases in complex eukaryotes.

The free radical theory of aging posits oxidative damage to macromolecules as a primary determinant of lifespan. Recent studies challenge this theory by demonstrating that in some cases, longevity is enhanced by inactivation of oxidative stress defenses or is correlated with increased, rather than decreased reactive oxygen species and oxidative damage. Here we show that, in Saccharomyces cerevisiae, caloric restriction or inactivation of catalases extends chronological lifespan by inducing elevated levels of the reactive oxygen species hydrogen peroxide, which activate superoxide dismutases that inhibit the accumulation of superoxide anions. Increased hydrogen peroxide in catalase-deficient cells extends chronological lifespan despite parallel increases in oxidative damage. These findings establish a role for hormesis effects of hydrogen peroxide in promoting longevity that have broad implications for understanding aging and age-related diseases.

Werner and Bloom syndromes are human diseases characterized by premature age-related defects including elevated cancer incidence. Using a novel Saccharomyces cerevisiae model system for aging and cancer, we show that cells lacking the RecQ helicase SGS1 (WRN and BLM homologue) undergo premature age-related changes, including reduced life span under stress and calorie restriction (CR), G1 arrest defects, dedifferentiation, elevated recombination errors, and age-dependent increase in DNA mutations. Lack of SGS1 results in a 110-fold increase in gross chromosomal rearrangement frequency during aging of nondividing cells compared with that generated during the initial population expansion. This underscores the central role of aging in genomic instability. The deletion of SCH9 (homologous to AKT and S6K), but not CR, protects against the age-dependent defects in sgs1Delta by inhibiting error-prone recombination and preventing DNA damage and dedifferentiation. The conserved function of Akt/S6k homologues in lifespan regulation raises the possibility that modulation of the IGF-I-Akt-56K pathway can protect against premature aging syndromes in mammals.

The chronological lifespan of eukaryotic organisms is extended by the mutational inactivation of conserved growth-signaling pathways that regulate progression into and through the cell cycle. Here we show that in the budding yeast S. cerevisiae, these and other lifespan-extending conditions, including caloric restriction and osmotic stress, increase the efficiency with which nutrient-depleted cells establish or maintain a cell cycle arrest in G1. Proteins required for efficient G1 arrest and longevity when nutrients are limiting include the DNA replication stress response proteins Mec1 and Rad53. Ectopic expression of CLN3 encoding a G1 cyclin downregulated during nutrient depletion increases the frequency with which nutrient depleted cells arrest growth in S phase instead of G1. Ectopic expression of CLN3 also shortens chronological lifespan in concert with age-dependent increases in genome instability and apoptosis. These findings indicate that replication stress is an important determinant of chronological lifespan in budding yeast. Protection from replication stress by growth-inhibitory effects of caloric restriction, osmotic and other stresses may contribute to hormesis effects on lifespan. Replication stress also likely impacts the longevity of higher eukaryotes, including humans.

In a recent issue of Molecular Cell, Büttner et al. (2007) described dual roles for the conserved mitochondrial protein endonuclease G in budding yeast apoptosis and proliferation that illuminate some of the contradictory and confusing findings regarding this protein in mammalian cells.

Background: Coordinately regulated genes often physically cluster in eukaryotic genomes, for reasons that remain unclear., Results: Here we provide evidence that many S. cerevisiae genes induced by starvation and other stresses reside in non-random clusters, where transcription of these genes is repressed in the absence of stress. Most genes essential for growth or for rapid, post-transcriptional responses to stress in cycling cells map between these gene clusters. Genes that are transcriptionally induced by stresses include a large fraction of rapidly evolving paralogues of duplicated genes that arose during an ancient whole genome duplication event. Many of these rapidly evolving paralogues have acquired new or more specialized functions that are less essential for growth. The slowly evolving paralogues of these genes are less likely to be transcriptionally repressed in the absence of stress, and are frequently essential for growth or for rapid stress responses that may require constitutive expression of these genes in cycling cells., Conclusion: Our findings suggest that a fundamental organizing principle during evolution of the S. cerevisiae genome has been clustering of starvation and other stress-induced genes in chromosome regions that are transcriptionally repressed in the absence of stress, from which most genes essential for growth or rapid stress responses have been excluded. Chromatin-mediated repression of many stress-induced genes may have evolved since the whole genome duplication in parallel with functions for proteins encoded by these genes that are incompatible with growth. These functions likely provide fitness effects that escape detection in assays of reproductive capacity routinely employed to assess evolutionary fitness, or to identify genes that confer stress-resistance in cycling cells.

In this paper, we present the results of global transcript analysis by the microarray technique of senescent and apoptotic yeast cells. We compared young daughter and old mother cells isolated by elutriation centrifugation, and non-apoptotic and apoptotic cells induced either by a temperature shift of the cdc48(S565G) temperature-sensitive mutant or of the orc2-1 temperature-sensitive mutant. The majority of all genes found to be differentially regulated in these three physiological situations was upregulated, indicating that a cellular death process was initiated rather than an unspecific shut-down of gene expression due to immediate killing. The functional classes of genes upregulated in all three conditions were largely the same, although individual genes were in many cases not identical. The largest group of genes involved were nuclear genes coding for mitochondrial components or functions, which is understandable given the fact that apoptosis can be triggered by mitochondrially generated oxygen radicals and that mitochondria play an important role in the execution of apoptosis. Other functional classes consisted of genes involved in DNA damage response, in cell cycle regulation and checkpoints, in DNA repair, and in membrane lipid and cell wall synthesis. These functional classes represent the response of the cell to the known cellular insults, which occur during aging and apoptosis. As we have shown previously, final-stage senescent yeast mother cells (of the wild-type) are apoptotic.

Apoptosis in metazoans is often accompanied by the destruction of DNA replication initiation proteins, inactivation of checkpoints and activation of cyclin-dependent kinases, which are inhibited by checkpoints that directly or indirectly require initiation proteins. Here we show that, in the budding yeast Saccharomyces cerevisiae, mutations in initiation proteins that attenuate both the initiation of DNA replication and checkpoints also induce features of apoptosis similar to those observed in metazoans. The apoptosis-like phenotype of initiation mutants includes the production of reactive oxygen species (ROS) and activation of the budding-yeast metacaspase Yca1p. In contrast to a recent report that activation of Yca1p only occurs in lysed cells and does not contribute to cell death, we found that, in at least one initiation mutant, Yca1p activation occurs at an early stage of cell death (before cell lysis) and contributes to the lethal effects of the mutation harbored by this strain. Apoptosis in initiation mutants is probably caused by DNA damage associated with the combined effects of insufficient DNA replication forks to completely replicate the genome and defective checkpoints that depend on initiation proteins and/or replication forks to restrain subsequent cell-cycle events until DNA replication is complete. A similar mechanism might underlie the proapoptotic effects associated with the destruction of initiation and checkpoint proteins during apoptosis in mammals, as well as genome instability in initiation mutants of budding yeast.

Initiation of DNA replication in eukaryotes requires the origin recognition complex (ORC) and other proteins that interact with DNA at origins of replication. In budding yeast, the temperature-sensitive orc2-1 mutation alters these interactions in parallel with defects in initiation of DNA replication and in checkpoints that depend on DNA replication forks. Here we show that DNA-damaging drugs modify protein-DNA interactions at budding yeast replication origins in association with lethal effects that are enhanced by the orc2-1 mutation or suppressed by a different mutation in ORC. A dosage suppressor screen identified the budding yeast co-chaperone protein Mge1p as a high copy suppressor of the orc2-1-specific lethal effects of adozelesin, a DNA-alkylating drug. Ectopic expression of Mge1p also suppressed the temperature sensitivity and initiation defect conferred by the orc2-1 mutation. In wild type cells, ectopic expression of Mge1p also suppressed the lethal effects of adozelesin in parallel with the suppression of adozelesin-induced alterations in protein-DNA interactions at origins, stimulation of initiation of DNA replication, and binding of the precursor form of Mge1p to nuclear chromatin. Mge1p is the budding yeast homologue of the Escherichia coli co-chaperone protein GrpE, which stimulates initiation at bacterial origins of replication by promoting interactions of initiator proteins with origin sequences. Our results reveal a novel, proliferation-dependent cytotoxic mechanism for DNA-damaging drugs that involves alterations in the function of initiation proteins and their interactions with DNA.

In budding (Saccharomyces cerevisiae) and fission (Schizosaccharomyces pombe) yeast and other unicellular organisms, DNA damage and other stimuli can induce cell death resembling apoptosis in metazoans, including the activation of a recently discovered caspase-like molecule in budding yeast. Induction of apoptotic-like cell death in yeasts requires homologues of cell cycle checkpoint proteins that are often required for apoptosis in metazoan cells. Here, we summarize these findings and our unpublished results which show that an important component of metazoan apoptosis recently detected in budding yeast-reactive oxygen species (ROS)-can also be detected in fission yeast undergoing an apoptotic-like cell death. ROS were detected in fission and budding yeast cells bearing conditional mutations in genes encoding DNA replication initiation proteins and in fission yeast cells with mutations that deregulate cyclin-dependent kinases (CDKs). These mutations may cause DNA damage by permitting entry of cells into S phase with a reduced number of replication forks and/or passage through mitosis with incompletely replicated chromosomes. This may be relevant to the frequent requirement for elevated CDK activity in mammalian apoptosis, and to the recent discovery that the initiation protein Cdc6 is destroyed during apoptosis in mammals and in budding yeast cells exposed to lethal levels of DNA damage. Our data indicate that connections between apoptosis-like cell death and DNA replication or CDK activity are complex. Some apoptosis-like pathways require checkpoint proteins, others are inhibited by them, and others are independent of them. This complexity resembles that of apoptotic pathways in mammalian cells, which are frequently deregulated in cancer. The greater genetic tractability of yeasts should help to delineate these complex pathways and their relationships to cancer and to the effects of apoptosis-inducing drugs that inhibit DNA replication.

The free radical theory of aging posits oxidative damage to macromolecules as a primary determinant of lifespan. Recent studies challenge this theory by demonstrating that in some cases, longevity is enhanced by inactivation of oxidative stress defenses or is correlated with increased, rather than decreased reactive oxygen species and oxidative damage. Here we show that, in Saccharomyces cerevisiae, caloric restriction or inactivation of catalases extends chronological lifespan by inducing elevated levels of the reactive oxygen species hydrogen peroxide, which activate superoxide dismutases that inhibit the accumulation of superoxide anions. Increased hydrogen peroxide in catalase-deficient cells extends chronological lifespan despite parallel increases in oxidative damage. These findings establish a role for hormesis effects of hydrogen peroxide in promoting longevity that have broad implications for understanding aging and age-related diseases. [ABSTRACT FROM AUTHOR]