Didac Carmona-Gutierrez

Induction of autophagy by spermidine promotes longevity

Ageing results from complex genetically and epigenetically programmed processes that are elicited in part by noxious or stressful events that cause programmed cell death. Here, we report that administration of spermidine, a natural polyamine whose intracellular concentration declines during human ageing, markedly extended the lifespan of yeast, flies and worms, and human immune cells. In addition, spermidine administration potently inhibited oxidative stress in ageing mice. In ageing yeast, spermidine treatment triggered epigenetic deacetylation of histone H3 through inhibition of histone acetyltransferases (HAT), suppressing oxidative stress and necrosis. Conversely, depletion of endogenous polyamines led to hyperacetylation, generation of reactive oxygen species, early necrotic death and decreased lifespan. The altered acetylation status of the chromatin led to significant upregulation of various autophagy-related transcripts, triggering autophagy in yeast, flies, worms and human cells. Finally, we found that enhanced autophagy is crucial for polyamine-induced suppression of necrosis and enhanced longevity.

Apoptosis in yeast: triggers, pathways, subroutines

A cells decision to die is controlled by a sophisticated network whose deregulation contributes to the pathogenesis of multiple diseases including neoplastic and neurodegenerative disorders. The finding, more than a decade ago, that bakers yeast (Saccharomyces cerevisiae) can undergo apoptosis uncovered the possibility to investigate this mode of programmed cell death (PCD) in a model organism that combines both technical advantages and a eukaryotic ‘cell room.’ Since then, numerous exogenous and endogenous triggers have been found to induce yeast apoptosis and multiple yeast orthologs of crucial metazoan apoptotic regulators have been identified and characterized at the molecular level. Such apoptosis-relevant orthologs include proteases such as the yeast caspase as well as several mitochondrial and nuclear proteins that contribute to the execution of apoptosis in a caspase-independent manner. Additionally, physiological scenarios such as aging and failed mating have been discovered to trigger apoptosis in yeast, providing a teleological interpretation of PCD affecting a unicellular organism. Due to its methodological and logistic simplicity, yeast constitutes an ideal model organism that is efficiently helping to decipher the cell death regulatory network of higher organisms, including the switches between apoptotic, autophagic, and necrotic pathways of cellular catabolism. Here, we provide an overview of the current knowledge about the apoptotic subroutine of yeast PCD and its regulation.

An immunosurveillance mechanism controls cancer cell ploidy

Keeping Cancer Cells At Bay Cancer cells are often aneuploid; that is, they have an abnormal number of chromosomes. But to what extent this contributes to the tumorigenic phenotype is not clear. Senovilla et al. (p. 1678; see the Perspective by Zanetti and Mahadevan) found that tetraploidization of cancer cells can cause them to become immunogenic and thus aid in their clearance from the body by the immune system. Cells with excess chromosomes put stress on the endoplasmic reticulum, which leads to movement of the protein calreticulin to the cell surface. Calreticulin exposure in turn caused recognition of cancer cells in mice by the host immune system. Thus, the immune system appears to serve a protective role in eliminating hyperploid cells that must be overcome to allow unrestricted growth of cancer cells. Polyploid cancer cells trigger an immune response owing to proteins aberrantly exposed on their outer surfaces. Cancer cells accommodate multiple genetic and epigenetic alterations that initially activate intrinsic (cell-autonomous) and extrinsic (immune-mediated) oncosuppressive mechanisms. Only once these barriers to oncogenesis have been overcome can malignant growth proceed unrestrained. Tetraploidization can contribute to oncogenesis because hyperploid cells are genomically unstable. We report that hyperploid cancer cells become immunogenic because of a constitutive endoplasmic reticulum stress response resulting in the aberrant cell surface exposure of calreticulin. Hyperploid, calreticulin-exposing cancer cells readily proliferated in immunodeficient mice and conserved their increased DNA content. In contrast, hyperploid cells injected into immunocompetent mice generated tumors only after a delay, and such tumors exhibited reduced DNA content, endoplasmic reticulum stress, and calreticulin exposure. Our results unveil an immunosurveillance system that imposes immunoselection against hyperploidy in carcinogen- and oncogene-induced cancers.

Why yeast cells can undergo apoptosis : death in times of peace, love, and war

The purpose of apoptosis in multicellular organisms is obvious: single cells die for the benefit of the whole organism (for example, during tissue development or embryogenesis). Although apoptosis has also been shown in various microorganisms, the reason for this cell death program has remained unexplained. Recently published studies have now described yeast apoptosis during aging, mating, or exposure to killer toxins (Fabrizio, P., L. Battistella, R. Vardavas, C. Gattazzo, L.L. Liou, A. Diaspro, J.W. Dossen, E.B. Gralla, and V.D. Longo. 2004. J. Cell Biol. 166:1055–1067; Herker, E., H. Jungwirth, K.A. Lehmann, C. Maldener, K.U. Frohlich, S. Wissing, S. Buttner, M. Fehr, S. Sigrist, and F. Madeo. 2004. J. Cell Biol. 164:501–507, underscoring the evolutionary benefit of a cell suicide program in yeast and, thus, giving a unicellular organism causes to die for.

Nucleocytosolic Depletion of the Energy Metabolite Acetyl-Coenzyme A Stimulates Autophagy and Prolongs Lifespan

Summary Healthy aging depends on removal of damaged cellular material that is in part mediated by autophagy. The nutritional status of cells affects both aging and autophagy through as-yet-elusive metabolic circuitries. Here, we show that nucleocytosolic acetyl-coenzyme A (AcCoA) production is a metabolic repressor of autophagy during aging in yeast. Blocking the mitochondrial route to AcCoA by deletion of the CoA-transferase ACH1 caused cytosolic accumulation of the AcCoA precursor acetate. This led to hyperactivation of nucleocytosolic AcCoA-synthetase Acs2p, triggering histone acetylation, repression of autophagy genes, and an age-dependent defect in autophagic flux, culminating in a reduced lifespan. Inhibition of nutrient signaling failed to restore, while simultaneous knockdown of ACS2 reinstated, autophagy and survival of ach1 mutant. Brain-specific knockdown of Drosophila AcCoA synthetase was sufficient to enhance autophagic protein clearance and prolong lifespan. Since AcCoA integrates various nutrition pathways, our findings may explain diet-dependent lifespan and autophagy regulation.

The Search for Antiaging Interventions: From Elixirs to Fasting Regimens

The phenomenon of aging is an intrinsic feature of life. Accordingly, the possibility to manipulate it has fascinated humans likely since time immemorial. Recent evidence is shaping a picture where low caloric regimes and exercise may improve healthy senescence, and several pharmacological strategies have been suggested to counteract aging. Surprisingly, the most effective interventions proposed to date converge on only a few cellular processes, in particular nutrient signaling, mitochondrial efficiency, proteostasis, and autophagy. Here, we critically examine drugs and behaviors to which life- or healthspan-extending properties have been ascribed and discuss the underlying molecular mechanisms.

Cardioprotection and lifespan extension by the natural polyamine spermidine

Aging is associated with an increased risk of cardiovascular disease and death. Here we show that oral supplementation of the natural polyamine spermidine extends the lifespan of mice and exerts cardioprotective effects, reducing cardiac hypertrophy and preserving diastolic function in old mice. Spermidine feeding enhanced cardiac autophagy, mitophagy and mitochondrial respiration, and it also improved the mechano-elastical properties of cardiomyocytes in vivo, coinciding with increased titin phosphorylation and suppressed subclinical inflammation. Spermidine feeding failed to provide cardioprotection in mice that lack the autophagy-related protein Atg5 in cardiomyocytes. In Dahl salt-sensitive rats that were fed a high-salt diet, a model for hypertension-induced congestive heart failure, spermidine feeding reduced systemic blood pressure, increased titin phosphorylation and prevented cardiac hypertrophy and a decline in diastolic function, thus delaying the progression to heart failure. In humans, high levels of dietary spermidine, as assessed from food questionnaires, correlated with reduced blood pressure and a lower incidence of cardiovascular disease. Our results suggest a new and feasible strategy for protection against cardiovascular disease.

Functional Mitochondria Are Required for α-Synuclein Toxicity in Aging Yeast

α-Synuclein is one of the principal toxic triggers of Parkinson disease, an age-associated neurodegeneration. Using old yeast as a model of α-synuclein expression in post-mitotic cells, we show that α-synuclein toxicity depends on chronological aging and results in apoptosis as well as necrosis. Neither disruption of key components of the unfolded protein response nor deletion of proapoptotic key players (including the yeast caspase YCA1, the apoptosis-inducing factor AIF1, or the serine protease OMI) did prevent α-synuclein-induced cell killing. However, abrogation of mitochondrial DNA (rho0) inhibited α-synuclein-induced reactive oxygen species formation and subsequent apoptotic cell death. Thus, introducing an aging yeast model of α-synuclein toxicity, we demonstrate a strict requirement of functional mitochondria.

Caspase-dependent and caspase-independent cell death pathways in yeast.

2009 Elsevier Inc. All rights reserved. Yeast (Saccharomyces cerevisiae) can undergo cell death accompanied by diagnostic features of apoptosis, such as phosphatidylserine externalization, DNA fragmentation, chromatin condensation, cytochrome c release from mitochondria, and dissipation of the mitochondrial transmembrane potential. Both caspase-dependent and caspase-independent cell death executors participate in yeast cell death. On one hand, a yeast caspase-like protease appears to be essential for approximately 40% of the investigated cell death scenarios. On the other hand, factors like the mitochondrially located nucleases AIF (apoptosis-inducing factor) and endonuclease G, can execute caspase-independent cell death in yeast. Furthermore, complex correlates of mammalian cell death are observed in yeast. This applies to cell death-associated cytochrome c release, mitochondrial fragmentation, cytoskeletal alterations, generation of reactive oxygen species and epigenetic histone modifications. Hence, yeast constitutes an excellent model organism to delineate phylogenetically conserved pathways leading to apoptotic or necrotic cell death. Moreover, yeast can be used to identify pharmacological and genetic modulators of cell death pathways that are relevant for human disease. ll rights reserved. . Madeo), +33 1 42 11 60 47 Madeo), kroemer@orange.fr Twelve years ago, apoptotic markers were described in yeast for the first time [1,2], challenging the idea that apoptosis would be exclusively executed in multicellular organisms. Ever since, convincing evidence has been added to that initial description of yeast apoptosis. Thus, several yeast counterparts of crucial mammalian apoptosis regulators have been characterized. Conserved proteasomal, mitochondrial, nuclear and epigenetically-regulated cell death pathways have been profiled. Physiological death scenarios such as viral infections, chronological and replicative aging have been described in yeast [3–7]. Moreover, assays for apoptotic and/or necrotic cell death such as viability, DNA fragmentation, exposition of phosphatidylserine, cell integrity or ROS accumulation have been established and are routinely used in the field of yeast programmed cell death (PCD), both on the qualitative (microscopical) and the quantitative (cytofluorometric) levels [1,2,8]. What is the need for yet another model system for cell death research in view of the existence of established animal systems for the exploration of lethal signaling pathways, including rodents (Mus musculus and Ratus norvegicus), nematodes (Caenorhabditis elegans) and flies (Drosophila melanogaster)? The answer resides in the exclusive advantages offered by yeast for apoptosis research. For instance, the genetic tractability of yeast is unique and allows, for example, the simultaneous overexpression of one gene and the knockout of another gene, helping to establish hierarchies among cell death executors. In addition, yeast avoids one of the technical problems that affect mammalian cell death research: the detection of cell death mostly relies on apoptotic markers, which however 228 F. Madeo et al. / Biochemical and Biophysical Research Communications 382 (2009) 227–231 can yield false positive results. For example, neither caspase activity nor phosphatidylserine externalization is a sufficient criterion for mammalian cell death, because caspase activation can occur without cell death and cell death can occur without caspase activation [9]. In yeast, the precise measurement of actual cell death, i.e. the number of dead versus living cells, is easily and routinely accomplished by the use of plating assays. The combination of such a clonogenic cell death assay with the analysis of apoptotic markers allows for the discrimination between apoptotic and non-apoptotic forms of cell death. Moreover, there is no other organism where mitochondrial functions can be more easily manipulated than in yeast. Given the fact that mitochondria are decisive for cell death execution [10,11], such manipulations are of special importance. Deletion of mitochondrial DNA or growth on fermentable carbon sources can suppress mitochondrial functions in yeast, whereas the switch to non-fermentable carbon sources strongly boosts respiration, increases mitochondrial mass and enhances the propensity to undergo mitochondria-mediated cell death. Finally, yeast is an ideal tool for high-throughput screenings. The availability of a complete collection of yeast knockout strains render genome-wide yeast screens fast and inexpensive, at least in comparison to other model organisms. Altogether, these factors have led to the recent explosion of research on yeast apoptosis— the topic of this minireview. ROS: a major cause of yeast apoptosis A simple and experimental convenient way to induce yeast apoptosis is the induction of reactive oxygen species (ROS). The first evidence that ROS can trigger unicellular programmed death came from experiments exposing yeast (which were grown at a low cell density) to mild doses of H2O2. While high doses of H2O2 lead to a necrotic phenotype, low doses induced apoptosis, which could be genetically mimicked by deletion of glutathione-generatFig. 1. The molecular machinery of yeast apoptosis. Exogenous and endogenous inductio death, which is configured by conserved apoptotic key players such as the yeast caspase inducing factor Aif1p. Furthermore it involves complex processes like histone mo perturbations. ing enzymes [2]. Subsequent studies revealed several core apoptotic executioners that are involved in ROS-mediated cell death, including the yeast caspase YCA1 [12] or the apoptosis-inducing factor AIF1 [13] (see Fig. 1). Caspase-dependent yeast apoptosis Apart from ROS-mediated cell death, deletion of the yeast metacaspase YCA1 can protect yeast cells against multiple distinct forms of lethal insult [12] (see Table 1). For instance, yeast cells exposed to salt (NaCl) [14] or low doses of valproic acid, a short chained fatty acid with anti-tumor activity, undergo YCA1-dependent apoptosis [15,16]. Excessive iron causes YCA1-dependent cell death [17– 19], presumably through a pathway in which cardioliping activates the neutral sphingomyelinase ISC1, which in turn generates ceramide. However, deletion of ISC1 has been shown to shorten chronological lifespan and to enhance H2O2 sensitivity, which is YCA1-dependent and can be suppressed by iron chelation. Of note, ISC1 deletion is connected to an upregulation of the iron regulon that increases iron levels, which are known to catalyze the production of the highly reactive hydroxyl radicals (Fenton reaction) [19]. Besides iron, further metals like manganese and cadmium have been shown to induce YCA1-dependent apoptosis in yeast [20,21]. Exposure to toxins produced by virus-carrying killer strains also leads to PCD in yeast [3,22,23]. Deletion of YCA1 in the attacked strain leads to reduced toxin sensitivity [3]. Similarly, heterologous expression of expanded polyglutamine domains, which cause protein aggregation and neurodegeneration in human Huntington’s disease, leads to PCD in yeast [24], and this is again inhibited by YCA1 deletion [25]. Finally, yeast death triggered by defects in ubiquitination, defective DNA replication initiation, reduced mRNA stability, mitochondrial fragmentation or aging can occur at least partly in a caspase-dependent fashion [6,26–31]. During chronological aging (see below), deletion of YCA1, initially ameliorates the survival in the n of yeast apoptosis leads to the activation of the basic molecular machinery of cell Yca1p, the yeast homolog of mammalian HtrA2/OMI (Nma111p) or the apoptosisdification, mitochondrial fragmentation, cytochrome c release, and cytoskeletal

A yeast BH3-only protein mediates the mitochondrial pathway of apoptosis

Mitochondrial outer membrane permeabilization is a watershed event in the process of apoptosis, which is tightly regulated by a series of pro‐ and anti‐apoptotic proteins belonging to the BCL‐2 family, each characteristically possessing a BCL‐2 homology domain 3 (BH3). Here, we identify a yeast protein (Ybh3p) that interacts with BCL‐XL and harbours a functional BH3 domain. Upon lethal insult, Ybh3p translocates to mitochondria and triggers BH3 domain‐dependent apoptosis. Ybh3p induces cell death and disruption of the mitochondrial transmembrane potential via the mitochondrial phosphate carrier Mir1p. Deletion of Mir1p and depletion of its human orthologue (SLC25A3/PHC) abolish stress‐induced mitochondrial targeting of Ybh3p in yeast and that of BAX in human cells, respectively. Yeast cells lacking YBH3 display prolonged chronological and replicative lifespans and resistance to apoptosis induction. Thus, the yeast genome encodes a functional BH3 domain that induces cell death through phylogenetically conserved mechanisms.