α-Synuclein toxicity in yeast and human cells is caused by cell cycle re-entry and autophagy degradation of ribonucleotide reductase 1.

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.

Yeast at the Forefront of Research on Ageing and Age-Related Diseases.

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.

Guidelines and recommendations on yeast cell death nomenclature.

Elucidating the biology of yeast in its full complexity has major implications for science, medicine and industry. One of the most critical processes determining yeast life and physiology is cel-lular demise. However, the investigation of yeast cell death is a relatively young field, and a widely accepted set of concepts and terms is still missing. Here, we propose unified criteria for the defi-nition of accidental, regulated, and programmed forms of cell death in yeast based on a series of morphological and biochemical criteria. Specifically, we provide consensus guidelines on the differ-ential definition of terms including apoptosis, regulated necrosis, and autophagic cell death, as we refer to additional cell death rou-tines that are relevant for the biology of (at least some species of) yeast. As this area of investigation advances rapidly, changes and extensions to this set of recommendations will be implemented in the years to come. Nonetheless, we strongly encourage the au-thors, reviewers and editors of scientific articles to adopt these collective standards in order to establish an accurate framework for yeast cell death research and, ultimately, to accelerate the pro-gress of this vibrant field of research.

Reactive oxygen species, ageing and the hormesis police.

For more than 50 years, the free radical theory served as the paradigm guiding most investigations of ageing. However, recent studies in a variety of organisms have identified conceptual and practical limitations to this theory. Some of these limitations are related to the recent discovery that caloric restriction and other experimental manipulations promote longevity by inducing hormesis effects in association with increased reactive oxygen species (ROS). The beneficial role of ROS in lifespan extension is consistent with the essential role of these molecules in cell signalling. However, the identity of specific forms of ROS that promote longevity remains unclear. In this article, we argue that in several model systems, hydrogen peroxide plays a crucial role in the induction of hormesis.

Longevity pathways and maintenance of the proteome: the role of autophagy and mitophagy during yeast ageing.

Ageing is a complex and multi-factorial process that results in the progressive accumulation of molecular alterations that disrupt different cellular functions. The budding yeast Saccharomyces cerevisiae is an important model organism that has significantly contributed to the identification of conserved molecular and cellular determinants of ageing. The nutrient-sensing pathways are well-recognized modulators of longevity from yeast to mammals, but their downstream effectors and outcomes on different features of ageing process are still poorly understood. A hypothesis that is attracting increased interest is that one of the major functions of these "longevity pathways" is to contribute to the maintenance of the proteome during ageing. In support of this hypothesis, evidence shows that TOR/Sch9 and Ras/PKA pathways are important regulators of autophagy that in turn are essential for healthy cellular ageing. It is also well known that mitochondria homeostasis and function regulate lifespan, but how mitochondrial dynamics, mitophagy and biogenesis are regulated during ageing remains to be elucidated. This review describes recent findings that shed light on how longevity pathways and metabolic status impact maintenance of the proteome in both yeast ageing paradigms. These findings demonstrate that yeast remain a powerful model system for elucidating these relationships and their influence on ageing regulation.

DNA replication stress-induced loss of reproductive capacity in S. cerevisiae and its inhibition by caloric restriction.

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.

Depletion of deoxyribonucleotide pools is an endogenous source of DNA damage in cells undergoing oncogene-induced senescence.

In normal human cells, oncogene-induced senescence (OIS) depends on induction of DNA damage response. Oxidative stress and hyperreplication of genomic DNA have been proposed as major causes of DNA damage in OIS cells. Here, we report that down-regulation of deoxyribonucleoside pools is another endogenous source of DNA damage in normal human fibroblasts (NHFs) undergoing HRAS(G12V)-induced senescence. NHF-HRAS(G12V) cells underexpressed thymidylate synthase (TS) and ribonucleotide reductase (RR), two enzymes required for the entire de novo deoxyribonucleotide biosynthesis, and possessed low dNTP levels. Chromatin at the promoters of the genes encoding TS and RR was enriched with retinoblastoma tumor suppressor protein and histone H3 tri-methylated at lysine 9. Importantly, ectopic coexpression of TS and RR or addition of deoxyribonucleosides substantially suppressed DNA damage, senescence-associated phenotypes, and proliferation arrest in two types of NHF-expressing HRAS(G12V). Reciprocally, short hairpin RNA-mediated suppression of TS and RR caused DNA damage and senescence in NHFs, although less efficiently than HRAS(G12V). However, overexpression of TS and RR in quiescent NHFs did not overcome proliferation arrest, suggesting that unlike quiescence, OIS requires depletion of dNTP pools and activated DNA replication. Our data identify a previously unknown role of deoxyribonucleotides in regulation of OIS.

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

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

DNA damage and DNA replication stress in yeast models of aging.

DNA damage DNA damage is an important factor in aging in all eukaryotes. Although connections between DNA damage DNA damage and aging have been extensively investigated in complex organisms, only a relatively few studies have investigated DNA damage DNA damage as an aging factor in the model organism S. cerevisiae. Several of these studies point to DNA replication stress DNA replication stress as a cause of age-dependent DNA damage DNA damage in the replicative model of aging, which measures how many times budding yeast cells divide before they senesce and die. Even fewer studies have investigated how DNA damage DNA damage contributes to aging in the chronological aging chronological aging model, which measures how long cells in stationary phase cultures retain reproductive capacity. DNA replication stress DNA replication stress also has been implicated as a factor in chronological aging chronological aging . Since cells in stationary phase are generally considered to be "post-mitotic" and to reside in a quiescent G0/G1 state, the notion that defects in DNA replication might contribute to chronological aging chronological aging appears to be somewhat paradoxical. However, the results of recent studies suggest that a significant fraction of cells in stationary phase cultures are not quiescent, especially in experiments that employ defined medium, which is frequently employed to assess chronological lifespan. Most cells that fail to achieve quiescence remain in a viable, but non-dividing state until they eventually die, similar to the senescent state in mammalian cells. In this chapter we discuss the role of DNA damage DNA damage and DNA replication stress DNA replication stress in both replicative and chronological aging chronological aging in S. cerevisiae. We also discuss the relevance of these findings to the emerging view that DNA damage DNA damage and DNA replication stress DNA replication stress are important components of the senescent state that occurs at early stages of cancer.

Yeast aging and apoptosis.

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

Retrotransposition is associated with genome instability during chronological aging.

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.

Growth signaling promotes chronological aging in budding yeast by inducing superoxide anions that inhibit quiescence.

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.

Caloric restriction or catalase inactivation extends yeast chronological lifespan by inducing H2O2 and superoxide dismutase activity.

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.

The cell cycle is a redox cycle: linking phase-specific targets to cell fate.

Reactive oxygen species (ROS) regulate the strength and duration of signaling through redox-dependent signal transduction pathways via the cyclic oxidation/reduction of cysteine residues in kinases, phosphatases, and other regulatory factors. Signaling circuits may be segregated in organelles or other subcellular domains with distinct redox states, permitting them to respond independently to changes in the oxidation state of two major thiol reductants, glutathione and thioredoxin. Studies in yeast, and in complex eukaryotes, show that oscillations in oxygen consumption, energy metabolism, and redox state are intimately integrated with cell cycle progression. Because signaling pathways play specific roles in different phases of the cell cycle and the hierarchy of redox-dependent regulatory checkpoints changes during cell cycle progression, the effects of ROS on cell fate vary during the cell cycle. In G1, ROS stimulate mitogenic pathways that control the activity of cyclin-dependent kinases (CDKs) and phosphorylation of the retinoblastoma protein (pRB), thereby regulating S-phase entry. In response to oxidative stress, Nrf2 and Foxo3a promote cell survival by inducing the expression of antioxidant enzymes and factors involved in cell cycle withdrawal, such as the cyclin-dependent kinase inhibitor (CKI) p27. In S phase, ROS induce S-phase arrest via PP2A-dependent dephosphorylation of pRB. In precancerous cells, unconstrained mitogenic signaling by activated oncogenes induces replication stress in S phase, which activates the DNA-damage response and induces cell senescence. A number of studies suggest that interactions of ROS with the G1 CDK/CKI network play a fundamental role in senescence, which is considered a barrier to tumorigenesis. Adaptive responses and loss of checkpoint proteins such as p53 and p16(INK4a) allow tumor cells to tolerate constitutive mitogenic signaling and enhanced production of ROS, leading to altered redox status in many fully transformed cells. Alterations in oxidant and energy metabolism of cancer cells have emerged as fertile ground for new therapeutic targets. The present challenge is to identify redox-dependent targets relevant to each cell cycle phase, to understand how these targets control fate decisions, and to describe the mechanisms that link metabolism to cell cycle progression.

Longevity mutation in SCH9 prevents recombination errors and premature genomic instability in a Werner/Bloom model system.

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.